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GENERAL Monday, PROGRAM September 11:25 A.M. PRIZE PAPERS 27, 1999 O-001 Spindle Observation and Its Relationship with in Living Human Oocytes. W. ...

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GENERAL Monday,

PROGRAM September 11:25 A.M.

PRIZE PAPERS 27, 1999

O-001 Spindle Observation and Its Relationship with in Living Human Oocytes. W. H. Wang, R. J. Keefe. Department of Obstetrics and Gynecology, pital of Rhode Island, Brown University School RI.

Fertilization after ICSI Hackett, L. Meng, D. L. Women & Infants’ Hosof Medicine, Providence

Objective: The LC polscope allows non-destructive imaging of macromolecular structures within cells based on their birefringence. The spindle in oocytes is highly birefringent and its integrity essential for normal chromosome segregation during meiosis. Viewing the spindle in living human oocytes is valuable to study spindle dynamics and oocyte viability. In this study our objectives were 1) to image spindles in normal and aged human oocytes and 2) to examine the relationship between spindle structure and fertilization after intracytoplasm sperm injection (RX). Design: The LC polscope (CRI, Cambridge, MA) was used to examine spindles in living oocytes before ICSI or in aged, unfertilized oocytes l-4 days after ICSI or IVF. Oocytes with or without spindles were separated after ICSI to examine subsequent fertilization. Aged oocytes were further examined by immunocytochemical staining and confocal microscopy. Materials and Methods: 1) Oocytes were recovered from patients in an academic IVF clinic. After retrieval, cumulus-oocyte complexes were cultured for 5-6 h in HTF containing 10% synthetic serum substitute. Cumulus was removed from oocytes before examination. All oocytes with visible polar body (Pb) in the perivitelline space were examined before ICSI and the temperature was kept at 37°C during imaging and ICSI. After ICSI, oocytes with or without visible spindles under the polscope were cultured separately. Fertilization rates were examined 14-16 h after ICSI. Size of spindle and distance between spindle and Pb were measured with a computerized image analysis system. 2) Discarded oocytes (l-4 day old) that remained unfertilized after IVF or ICSI were examined by Polscope, and then stained by anti-tubulin antibody and examined by laser confocal microscopy to compare the two imaging modalities. Results: 1) 162 oocytes from 16 women were examined and spindles were visible in 59.9% of oocytes. The size of spindles and distance between spindles and Pb’s were 15.24 +/2.33 Frn and 36.42 +I18.19 pm, respectively. The spindle was close to Pb in most day 1 oocytes. After ICSI, more (63.9%; P < .05) oocytes with spindles were fertilized than oocytes without spindles (46.2%). Most fertilized oocytes (88%) had 2 pronuclei, with no difference being observed between oocytes with or without spindle. 2) only 2 oocytes on day 1 had intact spindles while in 30 oocytes the spindles had depolymerized with only a few microtubules remaining around the chromosomes or dispersed in the cytoplasm. In most aged oocytes, chromosomes were scattered throughout the cytoplasm. Confocal images of immunostained spindles were almost identical to polscope images of spindle birefringence. Conclusions: This study indicates that spindles in living human oocytes can be imaged with the LC Polscope. This technology can be used to monitor spindle position in human ICSI and to study spindle dynamics in living human and other mammalian oocytes. The presence of a birefringent spindle predicts increased fertilization rate after ICSI. The LC Polscope may help select structurally normal oocytes for selective rescue ICSI.

Monday,

September 11:40 A.M.

27, 1999

O-002 Progesterone and Estradiol Regulate the Transcription of Two AbdomB Hoxu Genes Critical for Implantation. ‘M. Yao, *L. Ma, *Y. Ding, ‘R. L. Maas. ‘Department of Obstetrics and Gynecology, and ‘Genetics Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.

inal

FERTILITY

& [email protected]

Objectives: Hoxa-IO and Hoxa-11 are Abdominal B (Abd B) class homeobox genes which are expressed in the developmental patterning along the anteroposterior axis of the female upper genital tract. In addition, these genes have been shown to function critically in implantation since mutant Hoxa-10 and Hoxa-II mice each exhibit severe implantation defects. The uterine expression of these genes has been shown to be regulated by progesterone and estrogen in viva. We hypothesized that progesterone and estrogen, via their respective receptors, directly regulate the transcription of Hoxa-IO and Hoxa-11 by binding to progesterone (P4) and estrogen (E2) response elements (PRE’s and ERE’s) in these genes. Our objective was to test this hypothesis at a molecular level. Design: Molecular cloning, site-directed mutagenesis, and transient transfection experiments were employed. Materials and Methods: Using restriction enzymes, the Hon.IO and -II sequences were cut into DNA fragments. Each fragment was cloned into a vector containing an SV40 promoter located upstream of the luciferase gene. These constructs were transiently transfected into P19 embryonal cancer cells. Estrogen or progesterone receptor constructs and an independent construct containing /3-galactosidase to control for transfection efficiency were co-transfected. E2, P4 or control were added to cell lines 6 hours after transfection and the cells were harvested at 28 hours. Luciferase activity was assayed and normalized against S-galactosidase activity. DNA sequences which showed increased activity with either P4 or E2 were further divided into smaller sequences and retested to locate functional PRE’s or ERE’s. Site-directed mutagenesis was used to mutate the presumptive PRE’s or ERE’s and these mutated constructs were tested in transfection experiments. The transfection experiments were also repeated in the AN3 CA cell line (human uterine cancer cells) to confirm the findings in a cell line of mullerian origin. Results: One functional PRE (PREl) and one functional ERE (EREl) were identified. In addition, a second, novel sequence (PRE2), was found to respond to P4 in a non-classical manner. PREl, PRE2, and EREl showed absolute luciferase to P-galactosidase (L/B) ratios which were significantly higher than those of controls (P < ,001). The mean increases in L/B activity were 3.7 2 0.5 and 13.4 2 4.4, for PREl and PRE2, respectively, each in 5 sets of triplicate P19 cell transfection experiments. The EREl showed a 2-fold increase in L/B activity compared to control. The increased activity was confirmed in the AN3 CA cells. Mutated constructs of PREI showed significantly decreased activation compared to the non-mutated PREl (P < ,001) and no increase in activity compared to control. Further, a dose-response relationship was demonstrated between the concentration of progesterone and the luciferaselp-galactosidase activity. Conclusion: Progesterone and estradiol, via their respective receptors, regulate the gene expression of Hoxa-10 and Hoxu-I1 through functional PRE and ERE in these genes. These results demonstrate that the expression of the Hoxa-IO and Hoxa-11 implantation genes are regulated by progesterone and estradiol on a molecular basis.

ASSISTED

REPRODUCTIVE TECHNOLOGY CRYOPRESERVATION Monday,

September

2:00

27, 1999

P.M.

O-003 A Model to Test the Safety of Human Ovarian Tissue Transplantation After Cryopreservation: Xenografts of Ovarian Tissues from Cancer Patients Into NOD/LtSz-Scid Mice. ‘S. S. Kim, ‘R. G. Gosden, ‘J. A. Radford, *M. Harris, 3A. Jox, ‘A. J. Rutherford. ‘Centre for Reproduction, Growth, & Development, University of Leeds, Leeds, United Kingdom, ‘Christie Hospital, Manchester, United Kingdom, 3University of Cologne, Cologne, Germany. Objectives: Ovarian cryopreservation and autografting is an attractive option for cancer patients who desire to preserve their fertility. However, clinical application should be cautious because of the potential risk of harboring malignant cells in stored ovarian tissues which could be transferred to patients after autografting. Our primary aim was to test the safety of cryopreserved human ovarian tissue from cancer patients for transplantation using in vivo NOD/scid mouse xenograft model. Design: Prospective, controlled, animal study.

Sl

Materials and Methods: Ovarian tissues were collected by laparoscopy or laparotomy after informed consent from patients with advanced or relapsed cancers before chemotherapy and radiotherapy (Hodgkin’s disease [HD], n = 13: Non-Hodgkin’s lvmphoma INHL], n = 5; ALL, n = 2; AML, n = 2). This study wasapprovedunder a Home Office (UK) license. Thin slices (l-2 mm thickness) of ovarian cortex were cryopreserved by slow freezing in 1.5 M dimethyl sulfoxide. Stored ovarian tissues were thawed rapidly, washed, held in fresh medium for 1 hour at 37°C before grafting them into female NOD/scid mice. Histopathologic examination with light microscopy as well as transmission electron microscopy [TEM] was performed on fresh and frozen-thawed surplus tissue. Under halothane anesthesia, slices of ovarian cortex were grafted subcutaneously into 50 NODlscid mice using 6-O prolene suture and matrigel. 11 animals were inoculated with human malignant cells as positive controls (5 with HD cell line L1236; 3 with AML cells; 3 with NHL lymph nodes). The health status of animals was monitored until sacrifice when specimens including lymph nodes, spleen, liver, bone marrow were recovered. Specimens containing malignant cells were further evaluated using histopathology and immunophenotypic analysis to identify the derivation of these malignant cells. Results: 11 weeks after surgery, 96% of animals were still alive and healthy, 2% having died acutely (< 5 days) of transplant-unrelated causes. Histopathologic analysis of cryopreserved tissues before transplantation revealed no evidence of residual disease, and these results were compared with tissues after xenografting. Analysis of frozen-thawed ovarian tissues by TEM demonstrated that majority of cells were well preserved without ultrastructure changes, confirming tissue viability. Conclusions: Our data suggest that future clinical application of cryopreserved ovarian tissue might be safe for selected groups of cancer patients.

Monday, September 27, 1999 2:15

P.M.

O-004 Cycles of Human Oocyte Cryopreservation and Intracytoplasmic Sperm Injection: Results of 112 Cycles. E. Porcu, R. Fabbri, P. M. Ciotti, T. Marsella, B. Balicchia, G. Damiano, D. Caracciolo, S. Giunchi, R. De Cesare, C. Flamigni. Infertility and IVF Centre, Department of Obstetrics and Gynecology, University of Bologna, Bologna, Italy. Objective: Oocytes cryopreservation is an attractive solution to legal and moral problems of embryo storage and give an alternative for patients at risk of losing ovarian function because of antineoplastic treatments. However, oocyte storage has faced technical difficulties compared with sperm or embryo cryopreservation, as documented by the low number of births achieved after oocyte cryopreservation. The advantage of intracytoplasmic sperm injection (ICSI) of cryopreserved oocytes has been recently described in terms of higher fertilization and cleavage rate. Our group obtained the first birth of an healthy female with the combination of oocytes cryopreservation and ICSI. Present study reports the results of high number of cycles of ICSI in cryopreserved oocytes in patients undergoing IVF. Materials and Methods: 96 women underwent 112 IVF cycles with oocyte cryopreservation. Multifollicular development was induced with GnRH analogue and purified FSH. Vaginal ultrasound-guided eggs retrieval was performed 34 hours after the administration of hCG 10.000 IU. After decoronization with jaluronidase and assessment of nuclear maturity, oocytes were cryopreserved using a low freeze-rapid thaw protocol and subsequent storing in liquid nitrogen. After thawing, oocytes were incubated at 37°C in an atmosphere of 5% CO2 until ICSI. Sperm selection was done by percoll technique. The sperm suspension was kept in the 37°C incubator until intracytoplasmic injection of the oocytes. Results: 1769 oocytes were frozen and 1502 were thawed. The survival rate was 54.1%, the fertilization rate 57.7% and the cleavage rate 9 1.2%. 16 pregnancies were achieved. Nine pregnancies (seven singletons and two twin) ended with the birth of eleven healthy children, eight females and three males. Conclusions: Doubts have been raised about the safety and the efficiency of human oocytes cryopreservation. Recent pre-clinical studies

s2

Abstracts

were reassuring concerning the risk of chromosomal abnormalities. Our investigation gives the first clinical confirmation of the safety of the technique with the birth of several healthy children. Until recently, oocyte cryopreservation was considered a low efficiency procedure because of low survival, fertilization and cleavage rates, With the introduction of ICSI, the results, in terms of fertilization, embryo cleavage and implantation, approach those obtained with fresh oocyte. The only limiting step seems to be oocyte survival which should be improved further.

Monday, September 27, 1999 2:30

P.M.

O-005 Vitrification of Mouse and Human Blastocysts Using Novel Containerless Cryo-loop Method. M. Lane, W. B. Schoolcraft, D. K. Gardner. Colorado Center for Reproductive Medicine, Englewood, Colorado. Objectives: With the increasing number of human embryos being grown to the blastocyst stage, there is an increased need for a reliable and successful method to cryopreserve blastocysts. Recently, a novel vitrification procedure which suspends embryos on a cryo-loop before plunging into liquid nitrogen has successfully cryopreserved cleavage stage hamster embryos. The aim of this study was to examine the success of cryo-loop vitrification on both mouse and human blastocysts. Design: Biastocyst development and viability following a novel vitrification procedure was determined. Materials and Methods: One-cell Fl mouse embryos were cultured to the blastocyst stage. Human blastocysts not deemed suitable for freezing or human triploid embryos were donated. All embryos were cultured in media Cl.2 & 2.2 and subsequently vitrified in a thin film of medium containing cryoprotectants suspended on a nylon loop, which was plunged directly into liquid nitrogen. Blastocyst re-expansion, hatching and outgrowth on gelatin coated plates were compared to control noncryopreserved embryos. Additionally, some mouse blastocysts that underwent vitrification were transferred to recipient females and their viability determined. Results: Mouse blastocysts that were vitrified (n = 126), hatched (87.5%) and attached (78.1%) at rates not different to the control (n = 112) blastocysts (95.5% and 85.9% respectively). Similarly, the implantation rate (78.3%), fetal development rate (55.0%) and fetal weight (0.24 g) of vitrified blastocysts (n = 60) were not different to control (n = 48) blastocysts (85.4%, 56.0%, and 0.25 g). Furthermore, all fetuses were morphologically normal. Eighteen human blastocysts were vitrified and 15 re-expanded (83.3%). Of these 11 hatched (73.3%) and 9 (60.0%) attached and formed outgrowths in culture. Of the control blastocysts, 63.6% hatched and 36% formed an outgrowth in culture. Conclusions: Vitrification using a cryo-loop can be used to successfully vitrify both mouse and human blastocysts, resulting in very high survivability. More significantly, the vitrified mouse blastocysts maintained their viability resulting in equivalent fetal development and normality, as noncryopreserved control blastocysts. As blastocyst culture and transfer becomes an accepted procedure in human ART, such vitrification procedures should be evaluated clinically.

Monday, September 27, 1999 2:45

P.M.

O-006 Pregnancy and Implantation From Vitrified Oocytes Following In Vitro Fertilization (IVF) and In Vitro Culture (IVC). K. Y. Cha, S. W. Hong, H. M. Chung, D. H. Choi, J. J. Ko, T. K. Yoon. Infertility Medical Center of CHA General Hospital and College of Medicine, Pochon CHA Universitv. ’ Objective: We previously reported (Chung et al., ASRM 1998; Hong et al., ASRM 1998) that vitrification method is available for cryopreservation of human oocytes, which yields improved development after thawing. In this special case, we applied our vitrification method, which used 5.5 M EG

Vol. 72, No. 3, Suppl. 1, September 1999

and 1.0 M sucrose as a cryoprotectant and employed a 4-step thawing protocol with 2.5 min interval, for a 30-years-old patient with repeated IVF failure and male infertility disorder. Design: Case study at university affiliated hospital, Pochon CHA University. Materials and Methods: The patient was stimulated with GnRH/FSH/ hMG and oocytes were retrieved by an ultrasound-guided oocyte aspiration technique. After being classified oocytes based on the maturational status, each stage of oocytes were vitrified by our standard protocol. After thawing, morphology of oocytes were evaluated under the stereo microscope and survived oocytes at the immature (CV) and maturing (GVBD/MI) stages were additionally cultured in TCM-199 with FBS, PMSG and hCG for 24 and 18 h, respectively. ICSI was then conducted and normally fertilized oocytes were cultured in Pl medium. Cleaved embryos were transferred to the patient at 3 days after ICSI. Pregnancy was detected by serum hCG concentration, and implantation and fetus viability was confirmed by ultrasonogram. Results: A total of 34 oocytes were retrieved from the patients. Twenty oocytes were provided for routine IVF program (no pregnancy was established) and the remaining 14 oocytes (4 at the GV, 3 at the GVBD/MI and 7 at the MI1 stages) were cryopreserved for 10 months. All vitrified oocytes survived after thawing, but oocytes vitrified at the GV or GVBD/MI stage did not mature following culture. Total 6 mature oocytes were normally fertilized and all of them were cleaved. These embryos developing to the 2to &cell stages were transferred to the uterus of the patient. Pregnancy was detected by 260.8 mIU/mL p-hCG level on 14 days after ET. One fetal sac and fetal heart beat were detected by ultrasonogram. The patient is in ongoing pregnancy (18 weeks). Post-vitrified Oocytes Retrieved

14

Stages at retrieval

Survival

GV GVBD/MI MI1

Maturation

4 3 7

Fertilization

0 (0) 0 (0)

were collected and cultured in modified P-l/blastocyst medium up to 6 days after IVF. Embryos developed into the blastocyst stage were cryopreserved by our standard vitrification method. After being stored in liquid nitrogen for 30 to 42 days, blastocysts were thawed by a 4-step method with 2.5 minutes intervals. Survived blastocysts were then transferred to the patients who failed to get pregnant in the previous cycle and ET was scheduled on either 5 days after ovulation or day 18 to 19 of artificial cycle with estradiol valerate and progesterone. Pregnancy was detected by serum hCG concentration, and implantation and fetal viability was confirmed by ultrasonogram. Results: A total of 30 2- to g-cell stage embryos were obtained from 5 patients for this study. Thirteen of them (13/30 = 43%) developed to the blastocyst stage and 7 out of 13 blastocysts (54%) survived following vitrification and thawing. When these blastocysts were transferred to 3 patients, one of these patients established triplet pregnancy. This patient is in ongoing pregnancy with normal fetal development. No. (%) of blastocysts Patients 1 2 3 4 5

7 6 5 3

6 (67) 2 (29) 2 (33) 2 (40) 1 (33)

Survived

Transferred

4 (67) 2(100) 1 (50) 0 (0) 0 (0)

3 2 1 0 0

Implanted 3 0 0

-

ASSISTED REPRODUCTIVE TECHNOLOGY FROZEN EMBRYO TRANSFER

6 (86)

Cleaved

9

Blastocysts WJ)

Conclusions: Pregnancy was established for the first time by transferring vitrified blastocysts. This result indicated that vitrification program could be applicable for cryopreservation of blastocysts, as well as oocytes.

Embryo Oocytes Retrieved

No. of embryos cultured

Monday, September 27, 1999 3:45 P.M.

Stages at ET O-008

14

6 (100)

2-cell

(1), 4-cell

(4). S-cell (1)

Conclusions: Pregnancy was established for the first time in the patients who received vitrified oocyte-derived embryos. This case report suggested that vitrification could be used for various purposes in human reproductive medicine.

Monday, September 27, 1999 3:00 P.M. O-007 Clinical Application of Vitrification Method for Cryopreservation of Human Blastocysts for Further Transfer. D. H. Choi, W. S. Lee, E. A. Park, N. Y. Yoon, W. S. Park, K. Y. Cha. Infertility Medical Center of CHA General Hospital and College of Medicine, Pochon CHA University. Objective: We previously reported that a vitrification method using ethylene glycol and an electron microscope copper grid was successfully applicable for cryopreservation of human oocytes at various maturational stages (Chung et al., 1998; Hong et al., 1998). In this study, we evaluated whether this vitrification method could be used for cryopreservation of blastocysts derived from IVF program. Design: Case study at university affiliated hospital, Pochon CHA University. Materials and Methods: Embryos were obtained from the consented patients undertaken our standard IVF program. Surplus embryos after ET

FERTILITY

& [email protected]

Frozen Embryo Transfer (FET) Implantation and Ongoing Pregnancy Rates Resulting From Day 3 Multi-Cell Embryos Compared With Day 3 Multi-Cell Embryos Cultured to Day 5 and Day 5 Blastocyst Embryos. D. Marek, M. Langley, N. Confer, L. Cram, L. Underwood, K. M. Doody, K. J. Doody. Center for Assisted Reproduction, Bedford, TX. Objective: Pregnancy rates following FET might be influenced by the stage of embryonic development at cryopreservation/thaw and at transfer. This study was undertaken to compare the implantation and ongoing pregnancy rates obtained with three different protocols for timing of cryopreservatiomthaw and transfer: 1) cryopreservation/thaw and transfer of Day 3 multi-cell embryos, 2) CryopreservationHhaw of Day 3 multi-cell embryos with subsequent culture to and transfer on Day 5 and 3) cryopreservation/ thaw and transfer of Day 5 blastocyst embryos. Design: A retrospective analysis of frozen embryo transfer results from January 1, 1998 through December 31, 1998. Materials and Methods: Embryos thawed for transfer at the multi-cell stage on Day 3 or blastocyst stage on Day 5 were placed into S-2 Media (IVF Science) and cultured approximately four hours prior to transfer. Multi-cell Day 3 embryos selected for culture to Day 5 were placed into 100 ~1 micro-drops of S-2 Media under oil and cultured two days prior to transfer. All embryos were cultured in 5% CO, and air at 37°C. Blastocyst and multi-cell embryos were frozen and thawed using respective glycerol and propanediol protocols (Testart et al. 1986, Tucker et al. 1997, Menezo et al. 1992, Menezo et al. 1996). In preparation for embryo transfer, estrogen and progesterone supplementation was administered to all patients. Transfers were performed with an abdominal ultrasound (5 MHz) to aid intrauterine placement of the embryo transfer catheter (Wallace).

s3

Results:

P

Control

Mean patient age Thaws/transfers Pregnancies (+ hCG)/ ongoing Biochemical/miscarriages Pregnancy rate (+ hCG) per thaw/transfer Ongoing pregnancy rate per thaw Total embryos thawed/ transferred Average embryos transferred No. clinical sacs (1 Sac/ 2 Sacs/3 Sacs) Implantation rate Total blastocyst development (%)

Day 3 Only

Day 3 Culture to Day 5

Day 5 Only

35.8 48/48 16/10

37.0 919 413

35.5 35135 21/l 1

313 33.3%/33.3%

011 44.4%/44.4%

Patients with embryo transfer Age (?S.D.) Total number of embryos thawed/survived (% survived) Total number of embryos transferred (per patient) Partially damaged embryos (W of transferred) N” positive HCG (% per transfer) N” ongoing pregnancy (% per transfer) Ongoing implantation rate

614 60.0%/60.0%

20.8%

33.3%

31.4%

l98/129

59122

102/83

2.7

2.4

2.4

1 l/l/l

3/l/O

9/5/o

12.4% NA

22.7% 10 (16.9)

22.9% NA

Monday, September 27, 1999 P.M.

O-009 Laser-Assisted Hatching and Removal of Degenerated Blastomere(s) Frozen-Thawed Embryos Improves Pregnancy Rate. 2. P. Nagy, Rienzi, M. Iacobelli, F. Morgia, F. Ubaldi, M. Schimberni, C. Aragona, Greco. Reproductive Medicine, European Hospital, Rome, Italy.

of L. E.

Objectives: Pregnancy and implantation rates are usually lower when frozen-thawed embryos are transferred compared to fresh embryo transfer. The decreased viability of the frozen-thawed embryos maybe related in part to the alterations of the zona pellucida and to the presence of damaged blastomeres caused by the cryopreservation. Therefore, our objective was to study the effect of assisted hatching and the removal of damaged blastomeres, when present, on the pregnancy and implantation rates. Design: A randomized, controlled study involving 40 patients with embryo cryopreservation. Materials and Methods: Supernumerary embryos from ICSI-IVF cycles that had less than 20% fragmentation were cryopreserved at the 4 to 8 cell stage using the propanediol slow freezing protocol. At thawing, only those embryos were considered to be eligible for transfer where at least 50% of the blastomeres remained intact. In half of the patients, no any treatment of the embryos were performed (control group) prior to transfer. In the other half of the patients, laser-assisted hatching was performed on all embryos (study group). Additionally, in embryos of the study group that had one or more degenerated blastomeres after thawing these damaged blastomeres were removed by aspiration into a microneedle. Results: A total of 18 of the 20 patients in the control group and 20 out of the 20 patients in the study group had embryos eligible for transfer after freezing-thawing. Patients’ and embryo parameters were similar in the two groups (Mann-Whitney-U test). Total and ongoing pregnancy rates and ongoing implantation rate was significantly higher (chi-squared test) in the group of patients where laser-assisted hatching and removal of degenerated blastomeres were performed.

s4

Abstracts

Study group

value

31.4 (Z.7) 73/54 (74.0%)

32.0 (2.0) 93/70 (75.3%)

NS NS

52 (2.9)

65 (3.2)

NS

19 (36.5%)

16 (24.6%)

NS

4 (22.2%)

12 (60.0%)

0.025

2 (11.1%)

10 (50.0%)

0.015

16.9%

0.036

3.8%

Conclusions: The results of the present study suggest that opening zona pellucida and removal of degenerated blastomeres of frozen-thawed human embryos increases implantation and pregnancy rates. It may hypothesized that impaired embryo viability after freezing-thawing is lated, at least in part, to the altered features of the zona pellucida and to presence of damaged blastomeres.

Conclusion: These results indicate that implantation and ongoing pregnancy rates increase despite transfer of fewer Day 5 blastocyst embryos compared with Day 3 embryos. Many Day 3 embryos that survive thaw, do not develop to the blastocyst stage by Day 5, which suggests that extended culture may select out those normal appearing Day 3 embryos which have a reduced potential for implantation.

4:00

group

the be rethe

Monday, September 27, 1999 4:15

P.M.

O-010 Biochemical Assisted Hatching (BAH) Increased the Implantation and Pregnancy Rate in Human Cryopreserved Embryo Transfer. J. E. Lee, D. R. Lee, H. R. Paik, H. N. Shim, J. H. Cho, S. I. Roh, H. S. Yoon. Infertility Research Center, Jeil Women’s Hospital, Seoul, Korea. Objective: Although cryopreservation of supernumerary embryos has increased the cumulative success of human IVT-ET, the implantation rate after frozen-thawed embryo transfer is lower than fresh cycle. This may be caused by hardening of zona pellucida occurred during cryopreservation. In many cases of human IVF-ET, assisted hatching has improved the implantation rate. Recently, we reported that biochemical assisted hatching (BAH) using pronase was a simple and safe technique. In this study, we evaluated the effects of assisted hatching, especially zona drilling and BAH, on the implantation and pregnancy in cryopreserved embryo transfer. Design: A randomized clinical study. Materials and Methods: From January 1995 to December 1998, 417 cycles undergoing frozen-thawed embryo transfer in Jeil Women’s Hospital were studied. The pronucleus and 2-4 cell stage embryos in excess of these transferred in fresh cycle were cryopreserved using a slow freezing/rapid thawing protocol with 1,2-propanediol and sucrose. Surviving embryos were cultured in HTF containing 10% synthetic serum substitute (Irvine) or patient’s serum for 24 or 48 hours, and transferred to patients. In experimental groups, surviving embryos were assistedhatched using acid tyrode solution (AHA) or cultured in HTF containing 0.5% human serum albumin and 1.0 pg/ml pronase (Sigma) for 24 hours (BAH) prior to embryo transfer. The implantation and clinical pregnancy rates were compared among three groups by ,$-square test. Results: Patient’s age, survival rate of cryopreserved embryos and number of transferred embryos were not different among three groups. But, the clinical pregnancy and implantation rates of BAH group were significantly higher than these of control group. Although not significantly different between BAH and AHA group, implantation of embryos in BAH group was increased. AHA No. of cases Age (years)* No. of survived embryos No. of transferred embryos*

No. of clinical pregnancy? No. of implanted embryos

(%)

BAH

(%)

Control

(%)

80 32.2 2 3.9 41 l/477 (86.2%)

89 32.2 2 4.2 500/545 (91.7)

248 31.0 2 3.2 117111379 (84.9%)

3.4 + 1.7

3.5 T 1.6 35 (39.3%)

3.0 2 1.7 55 (22.2%)

24 (30%) 35/270 (13.0%)

59/312 (18.9%) 90/754 (11.9%)

Vol. 72, No. 3, Suppl. 1, September 1999

AHA vs. BAH

AHA vs. con.

Monday, September 27, 1999

No. of csscs Age (years)* No. of survived embryos No. of transferred embryos* No. of clinical pregnancy7 No. of implanted embryos

o-012

P = ,205 P = .052

P = .155 P = A59

P = 50027 P = .0028

*Clinical pregnancy was defined as the presence of an intrauterine sac confirmed by ultrasonography. t Values are mean ? SE. Conclusions: From these results, we obtained that assisted hatching, especially BAH using pronase, improved the implantation and pregnancy in cryopreserved embryo transfer. Therefore, BAH can be used for cryopreserved embryo transfer as a simple and effective assisted hatching tech-

Monday, September 27, 1999 4:30 P.M. 0.011 Assisted Hatching (AH) Causes Beneficial Effects on the Outcome of Subsequent Frozen Embryo Transfers of Donor Oocyte Cycles. M. A. Cohen, S. R. Lindheim, M. V. Sauer. Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University, New York, NY. Objectives: AH is typically reserved for poor prognosis IVF cycles. We assessed the efficacy of AH on cryopreserved embryos generated from donor oocyte cycles. Design: Retrospective review of a university based practice. Materials and Methods: From lo/95 to 12/98 women (n = 97) undergoing FET cycles with embryos generated from donor oocytes were evaluated. All oocyte donors were under 35 years old‘and underwent GnRH-agonist downregulation followed by controlled ovarian hyperstimulation. All recipients were synchronized to the donors with GnRH-agonist downregulation followed by estrogen and progesterone supplementation. Recipient ages were similar in the two groups (42.3 2 1.0 years no AH vs. 43.4 + 0.7 years (SEM) AH). Patients in the AH group were given methylprednisolone for immunosuppression. AH was performed with micromanipulation and acidic Tyrode’s solution. Outcome measures included implantation, pregnancy, and miscarriage rates. Statistical analysis was done with the SPSS statistical package. Results: The results of FET cycles are tallied below: No AH (n = 49) No. of embryos transferred FET cycle Implantationrate embryo Pregnancy rate FET cycle Miscarriage rate PET cycle Ongoing pregnancyrate PET cycle

445 P.M.

BAH vs. Con.

(n iH48)

value ’

4.08 ” 0.20 (SEM) 6.0% (12/200) 16.3% (8/49) 37.5% (318)

4.08 2 0.12 @EM) 23.0% (45/196) 60.4% (29148) 24.1% (7129)

NS co.01 CO.01 NS

10.2% (S/49)

45.8% (22/48)


Assisted Hatchhtg Does Not Affect the Outcome of Cryopreservation of Mouse 4-Cell Embrvos. X. Z. Zhena, U. Ulun,* R. A. Yaziai, T. A. Baramki, G. Compton, E. Katz, J. Liu. The Greater Baltimore-Medical Center Fertility Center, The Greater Baltimore Medical Center, Baltimore, MD. *Izmit Buyukeshir Belediyesi, Izmit Kocaeli, Turkey. Objective: Assisted hatching is often used to help embryo implantation. Sometimes, it is necessary to cryopreserve assisted hatched embryos. The aim of this study was to assessthe influence of assisted hatching with zona. drilling on the outcome of cryopreservation using mouse 4-cell embryos as a model. Design: Mouse 4-cell embryos were randomly divided into two groups; group 1 had zona drilling prior to cryopreservation. In group 2, 4-cell embryos were cryopreserved without assisted hatching. The rates of survival after thawing and in vitro development to blastocvsts were compared between group 1 and 2. Materials and Methods: Mouse embryos were collected from 7-week old Fl female mice (C57Bl X CBA/Ca) superovulated with an i.p. injection of 5 IU pregnant mare’s serum gonadotrophin followed by an injection of 5 IU human chorionic gonadotrophin given 48 h later. Mouse embryos were cultured to the 4-cell stage in human tube fluid medium (H-HTF) with 3% synthesis serum substitute (SSS) in a 37°C incubator with 5% CO, in air. For group 1, zona pellucida of 4-cell mouse embryos had been opened using an acid Tyrode solution (pH 2.5). About 30 pm hole was made in the zona pellucida. Embryos of groups 1 and 2 were cryopreserved in a cryo-nunc using a standard slow freezing procedure with 1.5M propanadial + O.lM sucrose. Briefly, embryos were incubated 10 min at room temperature in the H-HTF containing 1.5M propanadial and O.lM sucrose, and they were transferred into a tube containing 300 PL of the same solution. Seeding was done at -7°C. Freezing was stopped at -35’C and the straw was plunged into liquid nitrogen. For thawing, the nuncs were taken out of liquid nitrogen, and left in a 30°C waterbath for 2 min, the embryos were then taken out and incubated in 1M PROH, 0.2M sucrose, H-HTF for 5 min, respectively, in each solution. Survival was determined when all blastomeres of a 4-cell embryo were intact and had normal morphology. After thawing, embryos were cultured in HTF with 3%SSS for another three days. Blastocyst formation was recorded. Results: 87 out of 92 embryos from group 1 and 90 out of 93 embryos from group 2 were recovered after freezing and thawing; survival rates of 91% (79/87) in group 1 and 92% (83/90) in group 2 were observed, respectively. 90% from group 1 and 91% from group 2 of surviving embryos developed to the blastocyst stage. There was no statistically significant difference in the rates of embryo survival and blastocyst formation between group 1 and group 2 (P>O.O5, ,$ test). Conclusion: Assisted hatched mouse 4-cell embryos can be successfully cryopreserved. Assisted hatching procedure prior to cryopreservation has no adverse effect on the survival rate and further development even if there is a hole in the zona pellucida. This study was supported by a grant from The Greater Baltimore Medical Center. ASSISTED

REPRODUCTIVE TECHNOLOGY MALE FACTOR

Monday, September 27, I999 200 P.M.

NS = not significant. SEM = standard error of the mean,

o-013

Conclusion: Our data demonstrate that AH significantly improves implantation and pregnancy rates on cryopreserved embryos generated from donor oocytes, probably by ameliorating deleterious alterations in the zona pelucida which arise during the freeze/thaw process. We recommend that all cryopreserved embryos undergo AH.

Somatic Cell Contribution to Cytoplasmic Maturatlon and Developmental Competence In Vitro. ‘*‘D. El Guiziry, ‘M. Kamel, ‘*3A. Abdel Rahman, 31. Bagdady, ‘I. Ismail, la3HassanAly Hassan. ‘Department of Obstetrics & Gynecology Faculty of Medicine Alexandria University, ‘Department of Clinical Pathology Faculty of Medicine Alexandria University, Alexandria IVF & ICSI Center.

FERTILITY

& [email protected]

s5

Objective: Occasional dissociation of somatic and germ cell component has been recognized particularly in relation to controlled ovarian hyperstimulation. Moreover during germ cell maturation in vivo and in vitro, nuclear and cytoplasmic responses to maturational signals are reportedly variant. Cytoplasmic capacitation or acquisition of competence during zygote and subsequent preembryo development, is related to maternal transcripts and corona, cumulus cell contribution. This study investigates the effect of oocyte incubation in the absence and presence of surrounding somatic cells on ICSI outcome. Design: Prospective randomized. Materials and Methods: The study was performed on 441 M2 oocytes retrieved from 67 ICSI women. For denudation and injection, oocytes were randomly allocated to 3 protocols: Protocol A (immediate denudation, immediate injection, n= 152 oocytes), Protocol B (delayed denudation, delayed injection, n= 149 oocytes), Protocol C (early denudation, delayed injection, n=150 oocytes). ICSI outcome was also compared between 2 techniques of sperm ejection from the injecting pipette: 1 - immediate withdrawal 2 - slow withdrawal of the pipette after sperm ejection. Results:

Number Fertilization Good embryo Pregnancy

Technique 1 Technique 2

Protocol A

Protocol B

Protocol C

152 43.4%” 27.3%” 9.5%

149 72.5%b 60.2%b 25%

150 46% 31.9% 13.6%

Protocol A

Protocol B

Protocol c

37.8% 48.7%

62.5%” 81.8%b

52.7% 39.5%

Fertilization rate after

Overall (n = 103) Tubal (n = 37) Endometriosis (n = 17) Unexplained (n = 19)

2:15

P.M.

O-014 Comparison of Fertilization and Embryonic Development Between Conventional Insemination and ICSI Treatment in the Sibling Oocytes of Non-Male Factor Infertility. ‘J. H. Jun, ‘C. K. Lim, ‘J. W. Kim, ‘I. P. Son, ‘M. K. Koong, ‘I. 0. Song, 2J. H. Song, ‘K. J. Yoo, ‘I. S. Kang. ‘Infertility Research Laboratory, Department of Obstetrics and Gynecology, College of Medicine, Sungkyunkwan University, Samsung Cheil Hospital, Seoul, Korea. Objective: The purpose of this study was to evaluate whether or not the fertilization and embryonic development after ICSI is better than those of conventional insemination in human IVF-ET program of the non-male factor infertility. Design: Prospective clinical study. Comparing the outcome after conventional insemination or ICSI. Materials and Methods: The half-ICSI procedure, combining conventional insemination and ICSI were performed on the sibling oocytes in the same cycle, were carried out 103 cycles from January 1995 through December 1998. The oocytes were randomly divided. The subjects had normal sperm parameters and they were considered non-male factor infertility. According to the infertility etiology, the subjects were classified into tubal (37 cycles), endometriosis (17 cycles) and unexplained (19 cycles) group. The fertilization rate and quality of embryos were compared. Results: The results are summarized in the table.

S6

Abstracts

52.5 2 32.3” 58.4 ? 30.1 51.2 + 37.9 51.2 + 31.0

65.6 t 21.1b 63.3 If: 23.7 67.3 2 17.1 62.5 k 20.5

Insemination

ICSI

Overall (n = 103) 49.2% (150/350) 59.2% (270/456)b Tubal (n = 37) 49.6% (68/137) 51.0% (75/147) Endometrlosis (n = 17) 35.3% (12/34) 66.7% (48/72)b Unexplained (n = 19) 60.7% (34/56) 72.4% (55176) a*bValues with different superscripts in the same category differ significantly (P<.Ol). Total fertilization failure occurred in 7 (6.8%) of 103 cycles after conventional insemination but none after ICSI. The incidence of abnormal fertilization, poly pronucleus (3-PN), after conventional insemination was 5.5% (52/943), it is significantly higher than that after ICSI (0.7%; 7/971). Conclusions: The ICSI treatments offer better outcome of fertilization and embryonic development than those of conventional insemination in the sibling oocytes of non-male factor infertility. Although total fertilization failure occurred in conventional insemination, half-ICSI procedure provides the chance of embryo transfer in all IVF-ET cycles. Further studies are needed to determine whether or not half-ICSI beneficial in subgroup of infertility. Monday, September 27, 1999 2:30

Monday, September 27, 1999

ICSI

Proportion of good and fair embryos after

“.bSignificantly different Pc.05. The second table shows fertilization rate in relation to technique of sperm ejection. Conclusion: Beneficial effect of oocyte incubation is mediated through the surrounding somatic cell. After sperm injection slow withdrawal of the pipette could decrease the incidence of sperm displacement and thus maintains good fertilization rate.

Insemination

P.M.

o-01.5 Blastocyst Transfer in Couples Undergoing Testicular Sperm Extraction for Nonobstructive Azoospermia. B. Balaban, A. Isiklar, R. Mercan, S. Aksoy. C. Alatas, A. Nuhoglu, B. Urman. Assisted Reproduction and Fertility Unit, American Hospital of Istanbul, Turkey. Objectives: Blastocyst transfer (ST) is becoming more common in assisted reproduction centers. It is unknown whether BT is feasible in nonobstructive azoospermic couples where spermatozoa are retrieved surgically. The aim of this study was to determine the progression of cleavage stage embryos to blastocysts in couples where testicular spermatozoa are used for intracytoplasmic injection. Design: Prospective case series. Material and Methods: BT on day 5 or 6 was undertaken in 31 couples with nonobstructive azoospermia. Women with three or more embryos available on day 2 were eligible for BT. Testicular sperm extraction and intracytoplasmic sperm injection were performed as previously described. Blastocyst culture was performed in sequential media (Sl +S2; Scandinavian Science). Three to four blastocysts were transferred according to their morphology. Results: A total of 503 oocytes were retrieved of which 416 were MII. Of these 271 fertilized and 266 cleaved. On day 3, 162 embryos were either Gl or G2 and 104 embryos were G3 or G4. Of the 266 cleavage stage embryos, 115 progressed to the blastocyst stage on day 5 or 6. According to morphologic criteria 32 blastocysts were grade 1 (27.8%), 51 were Grade 2 (44.3%). and 20 were grade 3 (17.3%). On the day of transfer 28 (24.3%) blastocyst were hatching spontaneously, 38 (33%) were in the expanded stage, 37 (32.1%) were in the early blastocyst stage, and 12 (10.4%) were in the cavitating morula stage. A total of 102 blastocysts were transferred (average: 3.5; min: 1, max: 4). Of the 31 patients, 29 were transferred at the blastocyst stage. The rate of positive beta hCG 10 days after transfer was 62% (18/29). There were 2 biochemical and 5 clinical pregnancy losses resulting in an ongoing pregnancy rate of 37.9% (11129). Of the pregnancies obtained 8 were multiples (all twins) yielding a multiple pregnancy rate of 72.2%. Implantation rate per embryo was 34.3%. Vol. 72, No. 3, Suppl. 1, September 1999

Discussion: This study demonstrates that embryos obtained from intracytoplasmic injection of testicular spermatozoa progress to the blastocyst stage and transfer at this stage yields satisfactory implantation and pregnancy rates. However, the high rate of multiple pregnancy obtained in this study makes a case for restricting the number of embryos transferred in these patients. Monday, September 27, 1999

2:45 P.M. O-016 A Prospective Study of 206 Babies Born After Intracytoplasmic Sperm Injection (ICSI). M. A. Aboulghar, R. T. Mansour, G. I. Serour, Y. Amin. The Egyptian IVF-ET Center, Cairo, Egypt. Objectives: Concern has been expressed about the potential and longterm hazards faced by offspring resulting from ICSI. Our objective is to evaluate the safety of ICSI by studying the outcome of babies born after the procedure. Design: A prospective study assessedthe incidence of multiple pregnancy, prematurity, congenital malformations and chromosomal abnormalities in ICSI babies and compared it with the incidence in the general population. Materials and Methods: Of the 934 successful ICSI pregnancies at our center till the end of 1997, only 161 patients were available for follow up. The age of the mothers was 31 2 5.04 years (mean 2 SD). The duration of infertility was 7.9 t 5.1 years (mean?SD). 124 procedures used ejaculated sperm and 37 used surgically retrieved sperm. All babies were examined by a senior pediatrician in the first week after delivery to detect congenital malformations. Appropriate investigations were performed to confirm the diagnosis. A blood sample was taken from all babies for karyotyping. The results were compared with the corresponding incidence in the general population as reported in the literature. Results: The study included 161 patients who delivered 206 babies including 119 singletons, 42 sets of twins (26.7%) and one set of triplets (0.6%). Premature delivery (before 37 completed weeks gestation) occurred in 18 patients (11.1%). A total of 13 babies (6.3%) were born with a major congenital malformation, defined as a congenital abnormality causing functional impairment or requiring surgical correction. Ten babies (4.8%) had an abnormal karyotype including two babies with 47XY + 21, three babies with 47XXY, two babies with 46xX/45X0 and one with 47XYY. One baby was chimeric with two cell lines (46XY/46XX), and one baby with 47XXYl 46XY. The percentage of babies with major congenital malformations and abnormal karyotype did not differ between the two groups using surgically retrieved sperm or ejaculated sperm for ICSI. Conclusions: In babies resulting from ICSI the incidence of multiple pregnancy and premature delivery was very high compared to that of the general population. The congenital malformation rate was slightly higher than that reported in the literature. The incidence of chromosomal abnormality (4.8%) was significantly higher than in the general population (0.5%). A larger study is underway to confirm these findings and to elucidate the possible causes of the increased incidence of abnormal karyotype in ICSI babies. We are also investigating the incidence of these abnormalities in a group of babies resulting from conventional IVF. Monday, September 27, 1999

3:00 P.M. O-017 Chromosome Abnormalities in Couples and Male Partner Undergoing Intracytoplasmic Sperm Injection (ICSI)-Prevalence, Types and Correlation with Sperm Concentration. ‘E. Borges, ‘A. Iaconelli, 2*3L. M. Farah, ‘C. C. Rocha, ‘M. E. Vieira, ‘A. R. Medeiros, *R. Joffe, 2M. E. Sportello-Sanchez. ‘Fertility - Centro de Fertiliza9Bo Assistida, Slo Paulo, SP, Brasil, ‘Clinica e Laboratdrio de Genetica, Szo Paulo, SP, Brasil and ‘Disciplina de Genttica. Departamento de Morfologia, Escola Paulista de MedicinaiUNIFESP, Slo Paulo, SP, Brasil. Objectives: Chromosome abnormalities are known as a major contributor to the genetic risks of infertility treatment by ICSI. Even so, cytogenetic

FERTILITY & [email protected]

analysis has been only recently introduced in our country in the laboratory protocol used for the selection of subfertile patient’s candidates to this reproductive technique. Our purpose was to determine the incidence of cytogenetic abnormalities among these patients and to verify which are the types more frequently found and its correlation with the sperm concentration of men undergoing ICSI. Design: Prospective study. Materials and Methods: The sample was composed of 96 individual presenting subfertility, being 31 couples and 34 male partners. The main reason for the infertility was male factor. All had sperm analysis before the procedure. In all patients, male or female, cultured peripheric lymuhocvtes were initiallv . . * were G-banded with triosin. and 15 metaohases 1 analyzed. If any chromosome alteration was detected, 40 metaphases were analyzed. Results: A chromosome abnormality was detected in 16,6% from the whole sample. Among the males, 16,9% had aberrations in all the metaphasedstudied (93% autosomal and the other 90,7% were numerical or structural sex chromosomes abnormalities). Mosaicism for sex chromosomes appeared in 6,15% of all the male patients studied. The female partners studied presented 16,15% of chromosomal aberrations, 3,2% of which were balanced structural autosomic and the other 129% were sex chromosome mosaicisms. Both males and females had a chromosomic polymorphism level (4,16%) higher than it is normally observed in the general Brazilian population (2,5%). Regarding the correlation between sperm counting and chromosomal aberrations, 82% were found in the azoospermic group, 9% among the oligospermic patients and 9% in the normal sperm count group. The alterations detected in these last two groups were sex chromosome mosaicism. Conclusions: The prevalence of chromosomal disorders in considerable high in the tested group. The incidence of sex chromosome mosaicism is important and the more expressive results are found among the azoospermic men group. The reason for higher frequencies of cytogenetic abnormalities in our sample is the more rigid selection of the cases studied.

ASSISTED REPRODUCTIVE TECHNOLOGY GENETICS Monday, September 27, 1999

3:45 P.M. O-018 Inheritance of Y-Linked Deletions in Infertile Males: Cryptic Mosaicism as a New Etiology of Idiopathic Male Infertility. ‘,3M. G. KentFirst, ‘A. Muallem, ‘E. Memilli, ‘T. Osredkar, ‘5. Pryor. ‘K. Roberts, 3L. Miesner, 3W. Nolten, 4A. Chandley, ‘J. Itskovitz, ?I. Kol. ‘Promega Corporation, Madison, WI, ‘University of Minnesota, Minneapolis, 3University of Wisconsin, Madison, WI, 4MRC-Edinburgh UK-retired, 5Rambam Medical Center, Haifa Israel. Objectives: Deletions of Azoopermia Factor (AZF) regions of the Ychromosome have been detected in blood derived DNA (mesoderm) and sperm of infertile men pursuing ICSI. Approximately 6% of male children derived from ICSI manifest infertility-associated deletions that are not detected in the blood of their fathers. We hypothesize that these children inherit their deletions from their fathers who are mosaic for intact and cryptic deleted Y-chromosomes. The propositus phenotype is dependent on the timing of the mutation event in the developing embryo. Objectives: 1. confirm the existence of Y-chromosome deletions in infertile males who have fathered sons carrying deleted Y-chromosomes by ICSI 2. assessthe impact of mosaicism on gonadal development and function by comparing the mutation distribution and phenotypes of individuals carrying AZF deletions. Design: Incidence and tissue distribution of Y-linked deletions were determined in samples derived from individuals, suspected of having cryptic mutations among intact Y-chromosomes. Models for heritability were developed following phenotype evaluation. Materials and Methods: IRB approval was obtained for DNA analysis of Y-linked deletions. DNA from sperm and/or blood and/or buccal cells was screened for up to 92 Y-linked loci by per in: Experimental Group (EG)l:

s7

2 infertile males who had previously been shown to have an intact Ychromosomes in blood derived DNA, but produced sons by ICSI who carry deleted Y-chromosomes. EG2: 1 subfertile father and adult son. EG3: 3 fertile fathers of sons with mutations EG4: 5 infertile males, suspected of carrying cryptic Y-linked mutations in replicated amplifications. 5: 3 fertile male controls. Single lymphocytes and/or sperm were screened in EPl, EP4, and EPS individuals. Single cells were amplified by PEP and subsequently amplified with STS deleted in male relatives, in tandem with flanking positive STS followed by statistical analysis of data. Results: EPl: 2 individuals carried the respective mutations diagnosed in their ICSI derived sons (AZFd and AZFc) cryptically among cells bearing intact Y-chromosomes in single lymphocytes (63% and 17%, respectively) and single sperm (40% and 26% respectively). EP2: father and son carried similar AZFb deletions in all tissues. EP3: No microdeletions detected in mesoderm DNA of 3 fertile fathers. Deletions of 4, 2, and 1 AZF regions were detected in DNA derived from at least two tissue sources in all three respective offspring. The mutation in 1 of 3 resulted in an unstable isodicentric Y-chromosome that was lost by blastocyst stage resulting in a mosaic (X0/X isodicentric Y-chromosome). EP4: Cryptic deleted Y-chromosomes were detected in sperm and/or blood of 5 men in tandem with intact Y-chromosomes. EP5: Comparable deletions were detected in <2% of cells of control fertile males, Conclusions: I. Mosaiscism involving AZF is confirmed in infertile males II. Mutations arise by two mechanisms: 1. Randomly as de novo meiotic (or mitotic) mutations in the germ cell lineage of fertile males who may transmit the mutation to offspring and 2., as mitotic mutations prior to testis differentiation resulting in an infertile mosaic. III. The mosaic patient who carries intact and deleted Y-chromosomes in their germ cell lineage are candidates for preimplantation genetic diagnosis.

Monday, September 27, 1999 4:00 P.M. o-019 Improvement in FISH Sensitivity Using Tyramide Signal AmpliRcation System for Preimplantation Diagnosis. J. L. Gong, K. P. Xu, Z. Rosenwaks. Center for Reproductive Medicine & Infertility, Weill Medical College of Cornell University, New York, NY Objectives: Fluorescent in situ hybridization (FISH) is a powerful technique to determine gender, numerical and structural chromosomal statusfor preimplantation genetic diagnosis (PGD). Due mainly to the small size of DNA probes, weak signals are often seen and consequently, false negative results may be obtained. We describe here a new detecting method for FISH using tyrarnide signal amplification (TSA) system. Design: Two small and one large size DNA probes were chosen, and blastomeres and lymphocyte cells were used as target cells. FISH signal intensities prior to and after TSA were compared to determine the effect of TSA. Materials and Methods: Two small size telomeric probes (<200 kb) were kindly provided by Dr. Ning (George Washington University Hospital), one labeled with biotin and the other with digoxigenin. A centromeric probe for Y chromosome (Vysis, IL) was also included for comparison. A standard FISH protocol, provided by the manufacture (Oncor, Gaithersburg, MD), was used. The FISH signals were recorded by an imaging system with a grading method used previously (from Grade 5: strongest signal, to Grade 0: no signal; Xu et al, JARG, 1999, 15:570). Slides were then treated with TSA (NEN Life Science, MA). Briefly, slides were washed with TNT solution and blocked by TNB solution, and were incubated with streptavidin-peroxidase (NEN) or anti-digoxigenin-peroxidase (Boehringer Mannheim). Subsequently, fluorophore tyramide (FT) substrate solution was applied on to the slides and incubated for 10 minutes. Finally, amplified signals were viewed and recorded as the same manner described above. Results: Average signal intensities and numbers of nuclei examined (in parentheses) are tabulated below.

St3

Abstracts

Average Signal Intensities Probe lo-pter(biotin) 4-qter (digoxigenin) Y-CEP (biotin)

Lymphocyte (TSA)

Lymphocyte (control)

4.83 (240) 4.75 (200) 5.00 (100)

1.50 (200) 1.65 (186) 3.00 (80)

Average Signal Intensities Probe lo-pter(biotin) 4-qter (digoxigenin) Y-CEP (biotin)

Blastomere (TSA)

Blastomere (control)

4.83 (30) 4.85 (20) 4.88 (17)

1.63 (32) 1.57 (23) 3.74 (19)

The FISH signal intensity was improved dramatically when small probes (2 telomeric probes) were used, whereas slight improvement was seen when CEP probe was used. Conclusions: Our results demonstrate that this tyramide-based detection method can significantly improve the signal intensities. Because of its sensitivity, TSA detection system can increase the accuracy in certain applications of PGD, particularly, when small size DNA probes are used. Monday, September 21, 1999 4:15 P.M. O-020 Blastocyst Transfer: A New Therapeutic Approach to Infertility In Carriers of Chromosomal Translocatlons. ‘*‘Y. MhCzo, 2E. J. Servy. ‘Insa, Laboratoire de Biologie Villeubanne, France, 2Augusta Reproductive Biology Associates, Augusta, GA. Blastocyst transfers (BT) after in vitro fertilisation is a promising assisted reproduction technique that may benefit chromosomal tmnslocation-carrier couples who suffer multiple miscarriages or are unable to achieve pregnancy following classical ART techniques. Gonadotropin hyperstimulation of the ovary increasesthe number of oocytesavailable for fertilization, increasing the probability of creating healthy embryos. In vitro culture applies an additional selection pressure, so that those embryos which achieve blastocystformation have higher survival probability as healthy balanced ttanslocation carriers or unaffectedembryos. In sevencouples to whom this strategyhas been applied, 4 pregnancies havebeen initiated leading to the bii of 2 singletons (one male carrier, one unaffectedfemale). One twin pregnancy is ongoing, with amniocentesis at 25 weeks revealing one unaffected male and one healthy female carrier. One pregnancy is currently at 10 weeks’ gestation. In 4 patients no or only poor quality blastocystswere obtained. We propose that this strategymay be used initially as an alternative to preimplantation genetic diagnosis, and to apply the forces of natural selection in vitro for female and male patients carrying cbromosomal translocations.This technology can be used asa strategy for other chromosomal abnormalities: an ongoing pregnancy was initiated after blastocysttransfer 15 weeks ago in a sterile couple where the male partner was mosaic with a fragment of chromosome 21. Carrier CA Female t(l;22)(pl3.6;qll.2) NL Female t( l;lO)(p?;q?) BE Male der(13;14)(qlO;qlO) Ch Female tGmq35;q32) BM Male t(l;?(q22;pl5) PL Female t(8-17)(q21;pll) SE Female t(9-17)(qlo;plo)

History

Blastocysts

Miscarriages 3 failed IVF Primary (6~) 6 yrs infertility 5 Miscarriages 9-12 weeks Primary 12 yrs infertility Ilary

4116 (2 attempts) 3 normal timing 3/10 (2 attempts) Delayed (poor) 3/l 1 (1 attempts) 2 normal timing No blastocyst (2 attempts) No blastocyst

Primary 4 yrs infertility Primary 10 yrs infertility

No blastocyst 2/l 1 (1 attempt)

Vol. 72, No. 3, Suppl. 1, September 1999

Carrier

Outcome 1 Male carrier (born) 1 Female unaffected None

CA Female t(1;22)(p13.6;ql1.2) NL Female t( l;lO)(p?;q?) BE Male der(l3;14)(qlO;qlO) Ch Female t(2;7)(q35;q32) BM Male t(l;7)(q22;P15) Carrier PL Female t(8-17)(q2l;pll) SE Female t(9-17)([email protected],plO)

1 Male noncarrier 1 Healthy female carrier None None Outcome None Pregnant pending (10 wks)

Monday,

September

4:30

27, 1999

P.M.

Design: Embryos generated during an ICSI-IVF cycle were biopsied on day 3 to obtain one or two blastomeres for genetic analysis using either polymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH). Embryos were transferred to the uterus on day 4. Materials and Methods: PGD was performed during 48 ICSI-IVF cycles from a total of 28 patient couples (maternal age: 25-40 years) presenting either with a genetically transmittable condition (e.g., Marfan’s syndrome, Turner’s syndrome, retinitis pigmentosa, DAZ deletion, translocation) or as a carrier(s) for a like disorder (e.g., epidermolysis bullosa, cystic fibrosis, hemophilia, FG syndrome). In most patients the disorder was X-linked. Embryos were biopsied on day 3 and PCR or FISH was completed that day; embryo transfer was on day 4. All affected and discarded embryos were subsequently re-tested to confirm the original diagnosis. Results: Embryo transfer was performed in 45/48 cycles. Of these cycles, 19 (40%) resulted in pregnancy with an implantation rate of 26% (30/l 19). Eighteen of the 28 couples have become pregnant (64%) and 10 have delivered healthy children including 5 twins. One pregnancy ended with a first trimester miscarriage and a second couple lost two separate pregnancies during the second trimester due spontaneous membrane rupture unrelated to the PGD procedure. In all cases subsequent amniocentesis testing has confirmed the original preimplantation diagnosis. Moreover, we have also reconfirmed the original diagnosis for all the discarded affected embryos.

O-021 Delivery of a Healthy Baby Following Preimplantation Genetic Diagnosis for Reciprocal Translocation. K. P. Xu, P. Chung, T. H. Huang, L. L. Veeck, Z. Rosenwaks. The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY. Objectives: While aneuploidy analysis in polar bodies and blastomeres for woman of advanced maternal age has routinely been used in several IVF centers, few studies have been reported on FISH for chromosome analysis of structural changes. We wish to explore the potentials of FISH in detecting structural abnormalities in preimplantation embryos. Design: A novel strategy, combining whole chromosome painting probes (WCP) for the polar body chromosomes and telomeric probes for interphase blastomeres, was used to determine the feasibility of FISH for diagnosis of reciprocal translocation. Materials and Methods: A 27 year-old female patient, who had a history of five miscarriages and one ectopic pregnancy was diagnosed as a carrier of balanced reciprocal translocation [46, XX, t(4;l l)(q2l;q13)]. Two types of FISH probes were utilized to detect structural changes in the metaphase of the first polar bodies and in the interphase of blastomeres. Following a standard IVF procedure, 13 oocytes were retrieved from the patient. The first polar body was removed from 9 mature oocytes. Six polar bodies were successfully fixed and FISH with WCP probes for chromosomes 4 and 11 were applied. Of the three informative polar bodies, one showed that both affected chromosomes were presented in the polar body, and thus reflecting the corresponding oocyte was normal. Oocytes were fertilized by ICSI, and three embryos were obtained. Blastomere biopsy was performed on day 3. Second FISH using a telomeric probe (1 lqter) was applied on blastomeres. Results: A single embryo was predicted to be normal based on information from the polar body and the blastomere, and was transferred on day 4. Pregnancy was established and ultrasound showed a fetal heart. Amniocentesis was performed by an independent laboratory and the fetus was diagnosed as having a normal karyotype (46, XX). The patient underwent spontaneous labor at 36.5 weeks and delivered a healthy female infant. Conclusion: The successful diagnosis for the reciprocal translocation case confirms the feasibility of this new strategy. More importantly, this technique may be applicable for the majority of female translocation patients. Monday,

September

4:45

27, 1999

P.M.

O-022

ASSISTED REPRODUCTIVE TECHNOLOGY PREDICTORS OF OUTCOME AND CYCLE PREPARATION Monday,

2:00

embryology and pregnancy genetic diagnosis program

9195-2199. FERTILITY

& [email protected]

outcomes associconducted from

27,

1999

P.M.

O-023 Day 3 Serum Inbibin B and Estradiol Are the Best Predictive Factors of Success in Assisted Reproductive Technologies. ‘.*A. Hazout, ‘R. Frydman, ‘R. Fanchin, 2,3M. Dumont-Hassan, 2,3P. Cohen-Bacrie. ‘Antoine Beclere Hospital, Clamart, France, ‘Clinique de La Muette, ART Unit, Paris, France, 3Laboratoire d’ Eylau, Paris, France. Objectives: To determine the prognosis of day 3 inhibin B (INHB) >45 pg/mL (conversion factor to SI unit, 1.00) alone and correlated to day 3 FSH and estradiol (E,) levels, before assisted reproductive technologies (ART). Design: Analysis of INHB, FSH and E, concentrations in day 3 serum samples. INHB was measured by ELISA (Serotec) and E, and FSH by chemiluminescence system (ACS 180). Patients: Five hundred and fifty-five (555) women who underwent 545 assisted reproductive technology cycles with luteal phase agonist suppression plus recombinant FSH stimulation were analyzed. Main Outcome Measures: Serum Ez on day of hCG, number of oocytes retrieved per patient, fertilization rate, cleavage rate, clinical pregnancy rate and cancellation rate per initiated cycle, were determined. Results: 1) Group one: women with a basal serum day 3 FSH 5 10 IU/L (N= 426) demonstrated the best pregnancy rate when day 3 INHB was >45 pg/mL (36.9% versus 4% for women whose INHB was 545pg/mL, P<.OO5). 2) Group two: women with a basal serum day 3 FSH >lO IUiL (N= 119) obtained a pregnancy rate of 17.6%, when day 3 INHB was > 45pg/mL compared to 6.7% when INHB was 5 45pg/mL (PC.05). 3) Whatever the FSH level the correlation with day 3 E, level permit to improve the predictive value of INHB in those two groups of women. If day 3 E, is >50 pg/mL the pregnancy rate is very low: 7.7% versus 2.5% and 1.7% versus 0.5% respectively if INHB was > or 5 45pg/mL. Conclusions: The results suggest that within a normal range of FSH (4-12 IU/L), the day 3 E, and INHB levels in a previous cycle are the best predictive factors of success in ART.

A Summary of Embryology and Pregnancy Outcomes Associated With Preimplantation Genetic Diagnosis (PGD). J. A. Grifo, Y.-X. Tang, A. Adler, L. C. Krey. Program for In Vitro Fertilization, Reproductive Surgery and Infertility, New York University School of Medicine, New York, NY. Objective: To summarize ated with a preimplantation

September

Monday,

September

2:15

27, 1999

P.M.

O-024 Levels of Inhibin A and B in GnRH Prior to Gonadotropin Stimulation

Agonist Down for In Vitro

Regulated Women Fertilization Are

s9

Highly Predictive of Pregnancy Outcome. D. Smith, B. Carr, .I. O’Neil, G. Ackerman, W. Byrd. Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX. Objectives: Circulating levels of inhibin A and B fluctuate during the menstrual cycle. Inhibin A being higher during the luteal phase and inhibin B during the follicular phase. Previous work has established that the levels of inhibin A, but not inhibin B, on cycle day 21 of pregnancy in women undergoing IVF-ET were predictive of pregnancy outcome. In this study, we examined the levels of inhibin A and B in down regulated women before gonadotropin stimulation as a possible predictor of pregnancy outcome. Design: Retrospective study in a university based IVF-ET program. Materials and Methods: Sixty couples undergoing IVF-ET were randomly chosen for the initial study. These comprised 30 couples with live born and 30 nonpregnant couples. Patients were stimulated by gonadotropins after down regulation by GnRH. Patient’s FSH levels were determined in a cycle prior to stimulation. Circulating levels of inhibin A, B and estradiol were measured after GnRH down regulation for at least 10 days, prior to the initiation of gonadotropins. Embryos were transferred at the 4-8 cell stage. The outcomes measured was pregnancy with a live delivery. Statistical analysis was performed using the student t test, Results: Women with a live birth following IVF-ET had statistically higher levels (Mean t SEM) of inhibin A (169.2 t 75.7 pg/mL) and inhibin B (99.5 5 25.5 pg/mL) than women who did not become pregnant. Non pregnant women had levels of 18.9 2 11.5 pg/mL of inhibin A and 18.7 5 4.5 pg/mL of inhibin B. There was no significant difference in day 3 FSH levels in these women. Down regulated estradiol levels, the number of follicles greater than 10 mm in diameter or the number of follicles greater than 14 mm on the day of hCG injection were not different in the two groups. The same number of oocytes was retrieved in both groups. Conclusions: The data suggests that elevated circulating levels of inhibin A and B in down regulated women are predictive of which women who will deliver a live born. Inhibin A and B levels may be beneficial in selecting which patients will conceive prior to the start of stimulatory medications.

Monday,

September 2:30 P.M.

27, 1999

O-025 Evaluation of the Reproductive Predictive Value of High Day 3 Serum Estradiol and Follicle Stimulating Hormone Prior to Ovarian Stimulation. Y. Khalaf, A. Taylor, P. Braude. The Assisted Conception Unit of Guys’ & St. Thomas’ Hospitals, London, UK. Objective: To compare the reproductive predictive value of high day 3 serum estradiol with high day 3 serum FSH in subfertile women undergoing in-vitro fertilization and intracytoplasmic sperm injection (ICSI). Design: Retrospective clinical comparative study. Materials and Methods: From a computerised database of ovarian stimulation cycles for IVFlICSI (n= 1785) performed between January 96 and November 98 we identified 185 patients with diminished ovarian reserve as indicated by high basal serum e&radio1 (>60 pg/mL, n=52) or high basal serum FSH (>lO iu/L, n= 133). Multifollicular stimulation was carried out using either urinary or recombinant FSH. Buserelin acetate was used for down regulation in the long (n=87) and short (n=98) protocol. The study evaluated number of days of FSH stimulation, total amount of FSH used per cycle, cycle cancellation rates, number of preovulatory follicles, number of oocytes obtained, normal fertilization rates, number of embryos available for replacement, those replaced, implantation and pregnancy rates. A difference was significant when Pc.05. Results: The two groups were matched for age, number of previous cycles and indication for treatment. The high estradiol group required significantly lower number of FSH ampoules (48.62 14.2 vs. 62.82 14.7, P<.OOOl) and shorter duration of stimulation (11.4L2.1 vs. 10.6Z2.1, P 1.03). The cycle cancellation rate for poor response was significantly higher in the high estradiol group (28.8 vs. 14.3, P<.O3). The numbers of follicles (13.558.2 vs. 10.925.2, P<.O2), oocytes (9.2k6.9 vs. 7.254.9) obtained and embryos available (5.3k4.5 vs. 3.623.0, Pc.01)) were higher in the high estradiol group. An average of 2.1 (with a maximum of 3) embryos were replaced in the two groups. No significant differences were found between the two groups in clinical pregnancy rate per cycle (15.4 vs.

SlO

Abstracts

15.0%) or implantation rate (17.0 vs. 14.0%) for the high estradiol and high FSH groups respectively. Conclusions: This study suggests that high serum day 3 estradiol is an indicator of diminished ovarian reserve and predicts higher cycle cancellation and poor reproductive outcome. High basal estradiol level on cycle day 3 is a useful prognosticator of response to stimulation in IVF patients with normal basal FSH levels.

Monday,

September 2:45 P.M.

27, 1999

O-026 Evaluation of Endometrial Thickness and Vaginal Bleeding as Predictors of Pituitary Desensitisation Using Long Protocol GonadotrophinReleasing Hormone Agonists (GnRH-a). K. Sharif, M. Afnan, I. Yassen, A. Awonuga. Birmingham Women’s Hospital, Birmingham, UK. Objectives: GnRH-a are commonly used for pituitary desensitisation in IVF. The usual practice in monitoring for desensitisation is by measuring serum oestradiol (Ez) levels. We aimed to determine if pituitary desensitisation can be accurately predicted by transvaginal scan measurement of endometrial thickness and/or history of vaginal bleeding. Design: Prospective study. Materials and Methods: 250 patients had 274 IVF cycles using long protocol GnRH-a, using Nafarelin (Synarel, Searle, Bucks, UK) 200 wg nasal spray thrice daily starting in the mid-luteal phase. Two weeks later serum E, levels were measured, the occurrence of vaginal bleeding since the start of taking the Nafarelin was inquired about and recorded, and a transvaginal ultrasound scan was performed and the endometrial thickness was recorded as 5 4 mm or > 4 mm. Pituitary desensitisation was assessed at 2 levels of E,; 550 pg/mL (virtual hypo-oestrogenism) and 5 100 pg/mL (relative hypo-oestrogenism). These levels have been chosen as they have been shown in previous reports to be associated with similar pregnancy rate. The tests evaluated were 1) endometrial thickness (positive 5 4 mm, negative > 4 mm), 2) history of vaginal bleeding (positive: bleeding, negative: no bleeding), and 3) either of these tests (and/or). For each level of desensitisation, the sensitivity (Sn), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) were calculated from 2 X 2 tables. Sn was defined as the probability of the test being positive if desensitisation has occurred; Sp as the probability of the test being negative if desensitisation has not occurred; PPV as the probability of desensitisation if the test is positive; and NPV as the probability of no desensitisation if the test is negative. Results: After 2 weeks of GnRH-a, history of vaginal bleeding was present in 254 (92.7%) patients. In 205 (80.7%) of these the E, was 5 50 pg/mL and in 246 (96.8%) it was 5 100 pg/mL. Endometrial thickness of 5 4 mm was present in 239 (87.2%) patients. In 195 (8 1.6%) of these the E2 was 5 50 pg/ml and in 234 (97.9%) it was 5 100 pg/ml. A history of vaginal bleeding and/or endometrial thickness of 5 4 mm were present in 258 (94.2%) patients. In 211 (81.7%) of these the E, was % 50 pg/mL and in 252 (97.4%) it was 5 100 pg/mL. The best combination for Sn, Sp, PPV and NPV was for the test ‘endometrial thickness and/or vaginal bleeding’; for desensitisation at 50 pg/mL level the Sn, Sp, PPV and NPV were lOO%, 28%, 81% and lOO%, respectively. At a level of 100 pg/ml the corresponding values were 98%, 66%, 97% and 72%, respectively. Conclusions: Pituitary desensitisation after long-protocol GnRH-a, as indicated by absolute or relative hypo-oestrogenism, can be predicted with a high degree of accuracy from a history of vaginal bleeding and/or endometrial thickness of 5 4 mm. These criteria could be used instead of measuring serum E, levels.

Monday,

September 3:00 P.M.

27, 1999

O-027 A Prospective Randomized Study to Assess the Effect of Progestogen Therapy During Pituitary Desensitization With GnRH Agonist in the Prevention of Functional Ovarian Cyst Formation. L. Engmann, N. Maconochie, J. Bekir, S. L. Tan. McGill University Reproductive Center,

Vol.

72, No.

3, Suppl.

1, September

1999

Royal Clinic,

Victoria London,

Hospital, UK.

Montreal,

Canada

and The

London

Women’s

Objectives: The development of functional ovarian cysts as a result of gonadotropin releasing hormone (GnRH) agonist therapy during IVF treatment, may unduly prolong the duration of pituitary suppression and exert a detrimental effect on outcome of the cycle. The aim of this study was to assess whether the use of norethisterone and GnRH agonist therapy in the early follicular phase reduces the occurrence of functional ovarian cysts and shortens the duration of pituitary desensitization. We also assessed whether the use of norethisterone impairs implantation rates after IVF treatment. Study Design: Prospective randomized single-blind study of women undergoing IVF treatment using the long treatment Buserelin (LTB) protocol. Materials and Methods: 117 patients were randomized to receive norethisterone commenced 24 hours before GnRH agonist therapy (n = 63, Treatment Group) or GnRH agonist alone (n = 54, Control group) for pituitary desensitization. Patients recruited into the treatment group commenced norethisterone from the first day of the menses for five days (10 mg start dose and 5 mg twice daily for 4 days). All patients in the study commenced GnRH agonist therapy on day 2 of the menstrual cycle. Pituitary suppression was defined as the absence of follicular activity (ovarian cyst < lOmm), endometrial thickness < 5mm and serum estradiol (E,) concentration 5 150 pmol/L. Ovarian stimulation with gonadotropin was commenced once pituitary suppression was achieved. The main outcome variables were the incidence of functional ovarian cysts (presence of ovarian cysts 2 1Omm and serum E, 2 150 pmoliL), duration of pituitary suppression and implantation rates. The chi-squared tests and the t-tests were used for categorical and continuous variables respectively. The analyses were then controlled for age using logistic and linear regression as appropriate. Results: The incidence of functional ovarian cyst formation after 7 days of GnRH agonist therapy was significantly lower in the treatment group compared to the control group (5% versus 39%, P<.OOOl). None of the patients in the treatment group had functional ovarian cyst after 14 days of GnRH agonist therapy compared with 20 patients (37%) in the control group. Furthermore, the duration of pituitary suppression was significantly shorter in the treatment group compared to the control group. There were no significant differences between the two groups in the follicular response and embryo quality. Adjusted for age, the implantation rate (23% versus lo%, P = .02) and clinical pregnancy rate (34% versus 18%, P = .04) were significantly higher in the treatment group compared to the control group. Conclusions: A combination of norethisterone and GnRH agonist therapy is, therefore, more effective in achieving prompt pituitary suppression without impairing implantation rates and should be considered for routine use during IVF cycles.

ASSISTED

REPRODUCTIVE TECHNOLOGY OVARIAN STIMULATION Monday, September 27, 1999 3:45

P.M.

O-028 Results of a Prospective, Randomized, Multicenter Study to Assess the Efficacy and Safety of a Gonadotropin Releasing Hormone (GnRH) Antagonist-Org 37462 (Ganirelix Acetate) Treatment in Women Undergoing Controlled Ovarian Hyperstimulation (COH). North American Ganirehx Study Group. Objectives: The objective of this study was to assess efficacy of Org 37462 (Org), a potent GnRH antagonist in terms of number of oocytes, number of good quality embryos, ongoing pregnancy rate (PR), and safety and tolerability, in women undergoing COH. Leuprolide acetate (LA), a GnRH agonist was used as a reference drug. Design: A prospective, multicenter [ 11 IVF centers from US and Canada] randomized study. Randomization to Org or LA group was done by remote access telephone randomization system in a ratio of 2: 1. Materials and Methods: Women between the age of 18 and 39 years, with normal menstrual cycle (24-35 days) and normal hormone values were randomized to Org or LA groups. In Org group, recFSH (Follistim) treatment was started on day 2 or 3 of menstrual cycle. The dose of Follistim was

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fixed for the first 5 days (225 IU, SC, per day). On day 6 of Follistim treatment, Org treatment (0.25 mg, SC, per day) was initiated. A midluteal phase protocol was followed for LA group with a fixed Follistim starting dose of 225 IU for first 5 days. The dose of Follistim could be adjusted from day 6 onwards in both groups. 10,000 U of hCG (Pregnyl) were injected to trigger ovulation and final maturation of oocytes after 3 follicles of 2 17 mm were observed. Org or LA treatment was continued until the day of Pregnyl. Blood samples were collected at specified time points for hormone determinations. Routine IVF or ICSI was performed. An ultrasound scan was performed 12-16 weeks after embryo transfer (ET) for confirmation of ongoing pregnancy. Results: A total of 313 subjects were randomized to Org (208) or LA treatments (105). In Org group, 198 were treated of whom 178 had ET. In LA group, 99 subjects were treated of whom 91 had ET. A median (range) total number of oocytes collected was 11 (O-3.5) and 13 (O-38) in Org and LA groups. The mean total number of good quality embryos was 4.3 and 4.8 in Org and LA groups. The ongoing PR estimated using Cochran-MantelHaenszel approach were 3 1.3% and 35.3% per attempt and per transfer in Org group and were 36.1% and 39.7% in the LA group. The median (range) duration of GnRH analog treatment was 4 (2-14) days in Org group versus 22 (16-28) days in LA group. The median (range) total dose of recFSH was 1800 (1200-4275) and 2025 (1350-5100) IU in the Org and LA groups, None of the subjects discontinued the study due to an adverse event (AE). Conclusions: Org treatment regimen is short and safe for COH and results in good clinical outcomes in terms of number of oocytes, number of good quality embryos, and ongoing pregnancy rate. This study was sponsored by Organon Inc.

Monday, September 27, 1999 4:00

P.M.

O-029 Impact

of a GnRH Antagonist on the Endometrium in Women UnderIVF. ‘A. Agameya, *MM. Fhiker, ‘A. Leader, ‘P. Claman, ‘A. Yuzpe. ‘Division of Reproductive Medicine, Department of Obstetrics and Gynecology, University of Ottawa, ONT, Canada, ‘Genesis Fertility Centre, Vancouver, BC, Canada.

going

Objective: Accumulating experience with the GnRH antagonists cetrorelix and ganirelix in IVF suggests that pregnancy rates may be lower despite the transfer of similar numbers of good quality embryos. To evaluate whether GnRH antagonists might have a less favourable effect on implantation, we compared the effect of leuprolide acetate (a GnRH agonist) versus ganirehx on the endometrial profile of women undergoing IVF. Design: As part of a multicenter, open-labeled, randomized, prospective study, 89 patients were randomized in our centers to treatment with either ganirelix or leuprolide acetate for the prevention of premature LH surges during ovarian stimulation with rFSH (Puregon). Materials and Methods: A total of 89 patients were assigned in a 2: 1 ratio to group 1 (n=59) receiving ganirelix from day 6 of stimulation with rFSH and to group 2 (n=30) receiving leuprolide acetate in a long protocol for pituitary desensitization. Endometrial pattern and thickness were assessed by transvaginal real time ultrasound on the day of hCG. An independent observer reviewed the film records and assessed the pattern and thickness of the endometrium. A Mann-Whitney Rank Sum test and a Chi-square test were used to determine statistical significance. Results: There was no difference in endometrial thickness between the groups [mean; range] (Group 1: 10.6mm; 7.2-18.lmm; Group 2: 1 l.Omm; 8- 15mm). There was a non-significant trend towards a homogeneous endometrial pattern in the Group 1 compared to Group 2 (triple line pattern: 63% vs. 83%; P=O.O54). There was a similar non-significant trend towards lower pregnancy rates in Group 1 compared to Group 2 (18.8% vs 33.3%) in this small series. In contrast, the overall ongoing pregnancy rate of the multicentre study was 31.3% and 35.3% per attempt and 36.1% and 39.7% per transfer in the ganirelix group (n=198) and the leuprolide acetate group (n=99), respectively. Conclusions: Ganirelix and leuprolide acetate were comparable when endometrial thickness was assessed on the day of hCG. Nevertheless, the lower pregnancy rate when using GnRH antagonists might be related to an altered endometrial pattern in certain patients. This finding will require confirmation in a larger series.

Sll

Monday,

September 4:15 P.M.

27, 1999

O-030 Ultra-Low Dose Lupron Flare Offers Improved Outcome for Poor Responders. K. M. Silverberg, R. A. Ormand, L. J. Hansard, T. Turner, J. Slapak, B. Williamson, T. C. Vaughn. Texas Fertility Center, Austin, TX. Objectives: The poor responder represents a significant challenge to the contemporary ART program. Many alterations in stimulation protocols have been proposed for these patients, including high dose gonadotropin, low dose GnRH analogs, stimulation without GnRH analogs, and the addition of growth hormone. None, however, have proven to be reproducibly successful. Preliminary data have suggested that the ultra-low dose Lupron flare (ULDLF) protocol may offer such patients an improved prognosis. This study was designed to prospectively evaluate this protocol in the poor responder. Design: Prospective, sequential trial of the ULDLF in poor responders undergoing controlled ovarian hyperstimulation for IVF. Materials and Methods: Fifty-three poor responders were treated with both late luteal Lupron (LLL) and ULDLF stimulation protocols. The LLL protocol was initiated with late luteal Lupron (0.5 mg QD). Once downregulation was assured, the Lupron dosage was decreased to 0.25 mg QD and gonadotropin stimulation was begun. Patients on the ULDLF protocol were given 21 days of oral contraceptives (OCP). Three days following the last OCP, Lupron (4Opg BID) was initiated. Two days later, gonadotropin therapy was initiated. In both protocols, Lupron and gonadotropins were continued until there were at least 2 follicles > 18 mm. Human chorionic gonadotropin (hCG) (10,000 IU) was then administered. Oocyte retrieval was performed 36 hours later, and the intrauterine transfer of hatched embryos was performed 75 hours following retrieval. Results: Of those patients who completed both LLL and ULDLF cycles (n=23), there were no statistically significant differences between patients’ responses in terms of days of stimulation, total dose of gonadotropin received, peak estradiol level, or number of oocytes retrieved. The clinical pregnancy rate in ULDLF cycles was significantly greater than that in LLL cycles (50% vs. 24%, p< 0.001). The delivery rate was also significantly greater in ULDLF cycles (37.5% vs. 8.3%, p< 0.001). The overall LLL cycle cancellation rate for poor responders was 22/59 (37.3%) The overall ULDLF cycle cancellation rate was 6/53 (11.3%). Thirty-five LLL patients whose cycles were cancelled prior to oocyte retrieval subsequently attempted ULDLF cycles. Twenty-one of these successfully completed their ULDLF cycle (60%), twelve achieved pregnancy (34.3%) and eight successfully delivered (22.9%). Conclusions: The ULDLF affords the poor responder several potential advantages. First, patients who fail to get to oocyte retrieval with the LLL protocol have a significant likelihood not only of successfully completing a ULDLF cycle, but also of achieving pregnancy. More importantly, even poor responders who can successfully complete a LLL cycle may benefit from the significantly higher delivery rate afforded by the ULDLF protocol.

Monday,

September 4:30 P.M.

27, 1999

O-031 Metformin Treatment of Patients with Polycystic Ovarian Syndrome Undergoing IVF Increases the Number of Mature Oocytes, the Fertilization Rate and the Number of Embryos with Changes in the Levels of Insulin-Like Growth Factors. L. A. Stadtmauer, R. M. Riehl, S. K. Toma, S. Huang, S. Barker, L. M. Talbert. North Carolina Center for Reproductive Medicine, Gary, NC. Objectives: Patients with polycystic ovarian syndrome (PCOS) undergoing IVF have a high number of oocytes retrieved. However, the percentage of high quality of oocytes and fertilization rates are significantly lower than patients with tubal infertility. This is presumably due to the high androgen environment in the ovary. We hypothesized that metformin, an agent that lowers insulin secretion in patients with PCOS, may have an effect on increasing the number of mature oocytes. We also chose to compare levels of preovulatory follicular fluid insulin-like growth factors (IGFs) and bind-

s12

Abstracts

ing proteins (Bps) obtained from patients with or without metformin treatment. Design: A prospective controlled analysis of the preovulatory follicular fluid and outcome of patients with PCOS undergoing IVF-ET with or without metformin treatment. Materials and Methods: Patients (n=26) ages 25-34 with PCOS and male factor infertility undergoing gonadotropin stimulated IVF-ET and ICSI were treated with or without metformin. A dose of 500 mg b.i.d.. was started day 1 of the cycle prior to leuprolide acetate suppression and continued to the day of the pregnancy test. In half of the cases, patients acted as their own control, where metformin was used in the subsequent cycle. Follicular fluid was aspirated from 1 mature follicle and analyzed for IGF-I, IGF-II, IGFBP-1 and IGFBP-3 using two-site immunoradiometric assays. Only patients requiring ICSI were studied so that oocyte maturity could be accurately assessed by the presence of a polar body. Differences between the two groups were compared using the Student’s t-test. PcO.05 was statistically significant. Results: As shown in Table I, metformin treatment significantly increased number of mature oocytes, fertilization rates and number of embryos produced, without changes in total number of oocytes, and peak estradiol levels (data not shown). Metformin treatment also increased follicular fluid levels of IGF-1 and IGF-II and decreased levels of IGFBP-1. IGFBP-3 levels were not changed (Table 2). Table I Embryology Patient

(n=26)

- metformin + metformin

Age

#oocyte

3122 29.522

1922 2322

#mature oocyte 1022 1723*

fertilization rate 47% 77%+

#embryos 4.527 12-+3*

* PCO.5 Table 2 Growth Patient (n=26) -metformin +metfonnin

factors

Summary

IGF-I (rig/ml) 166218 2lOrtl8*

IGF-II (@ml) 502+22 548?27

IGFBP-1 (rig/ml) 1682 14 123?15*

* PCO.5 Conclusions: Metformin treatment of PCOS patients led to significantly increased number of mature oocytes retrieved during IVF-ET, with improvement in fertilization rates and number of embryos obtained. We speculate that this is through modulation of insulin-like growth factor levels.

Monday,

September 4:45 P.M.

27,

1999

O-032 A Multi-Center, Randomized, Comparative, Open-Label Trial to Assess the Safety and Efficacy of Gonal-F (r-hFSH) versus Gonal-F and Recombinant Human Lutenizing Hormone (r-hLH) in Patients Undergoing ICSI: Preliminary Data. ‘The U.S. Gonal Fa Multi-Center Clinical Trial Group, ‘L. Werlin, ‘E. E. Kelly, ‘P. Weathersbee, *L. Nebiolo, ‘L. Ferrande. ‘Reproductive and Women’s Health SBU, Serono Laboratories Inc. Norwell, MA, USA. Objectives: To evaluate the benefit of ICSI in a well controlled, multicenter trial and to evaluate in a prospective, randomized manner if the addition of LH provides a benefit to outcomes in pituitary desensitized patients. Preliminary data from 208 patients is evaluated. Design: A multi-center, randomized, open-label, comparative trial involving 43 US ART centers. Materials and Methods: Only ICSI patients with male factor infertility were included. All patients were treated with GnRHa (Lupron) starting 7-8 days post ovulation at the dose of 0.5 mg/daily (SC) until down regulation was achieved. Lupron dose was decreased to 0.25 mg/daily thereafter until the day of hCG (Profasi). Gonadotropin stimulation was initiated on day l-2 after down regulation. Group A received Gonal-F 225 IU for a minimum of five days. On day 6 the dose was adjusted according to patient’s response. Group B received Gonal-F as Group A, with the addition of r-hLH at a fixed dose of 150 IU (SC) commencing on day 6 of stimulation.

Vol.

72, No.

3, Suppl.

1, September

1999

Gonadotropin treatment continued until hCG administration. Serum LH, estradiol and ultrasound were used to monitor the patient response. 10,000 U hCG were administered when one follicle was 2 18 mm with two other 2 16 mm. OPU was performed and oocytes were classified. ICSI was performed according to each laboratory’s protocol. Transfer was limited to 3 embryos. Results: In group A, 105 (95.5%) and in group B, 95 (96.9%) patients received hCG. The number of treatment days (9.6 + 2.3 vs 9.5 -C 1.7) and the number of r-hFSH ampules used (31.2 IT 12.1 vs. 30.4 t 10.0) were NS between the groups (P>O.O5). Estradiol at d/hCG was NS between groups (2170 2 1580 vs 2331 ? 1366 pg/mL). The total number of follicles at d/hCG was similar in both groups (15.0 5 8.4 vs. 14.9 + 6.3). The percentage of MI1 oocytes was similar in group A, 79.2 ? 17.3 vs. group B, 81.6 -C 18.4 (p>O.O5). No differences in fertilization rates were found (58 vs. 57%). The number of grade A embryos transferred were 1.27 t 1.24 vs. 1.20 ? 1.46 (NS). Implantation rate was higher in group A than group B (27 2 34% vs 21 2 27%) (NS). The clinical pregnancy rates per cycle and per transfer were higher in group A than group B, 43.8 and 45.5% vs. 40.0 and 41.3% respectively (NS). Conclusions: No difference was obtained in % of MI1 oocytes after the treatment with Gonal-F alone or r-hFSH + r-hLH. The similar number of r-hFSH ampules utilized in both groups showed that the addition of r-hLH did not change the amount of r-hFSH needed for achieving follicular maturation. The estradiol levels obtained at d/hCG indicate that no additional exogenous LH was required for appropriate steroidogenesis. The difference observed in clinical pregnancy rate and implantation rate in favor of the Gonal F group needs to be confirmed with the analysis of higher number of patients. These results indicate that the addition of exogenous LH provided no additional benefit to the outcome of patients undergoing ovarian stimulation for ART.

REPRODUCTIVE LABORATORY TECHNOLOGY PROFESSIONAL GROUP Monday,

September 2:00 P.M.

27, 1999

O-033 Microsurgical Fragment Removal on Day Two Enhances Preembryo Quality and Increases Pregnancy Rate in Poor Prognostic Patients. N. Zaninovic, L. L. Veeck, K. Xu, Z. Rosenwaks. CUMC, Cornell University Medical College, New York, NY. Objectives: Human preembryos (PE) frequently exhibit cytoplasmic fragments during early development and their implantation ability decreases with increased fragmentation. Previous studies from our group showed that early fragmentation, before the first cleavage, subsequently leads to poor PE development. Routine fragment removal during the assisted hatching (AHA) procedure is usually performed on these PEs on day 3 (D3-AHA), and before intrauterine transfer. To date, no report has clearly shown a beneficial effect of fragment removal on PE development and implantation. This clinical study was undertaken to test the potential beneficial effect of early fragment removal (day 2) on subsequent PE development (day 3) and implantation. Design: Only cycles exhibiting excessive fragmentation (220%) on all PE on day 2 (48h after insemination or ICSI) were included in study. PEs from each patient were randomly split into control and fragment removal groups on day 2. The best-looking PE were selected for transfer from both groups. Pregnancy rate from the day 2 fragment removal group was compared to a group of patients with similar PE characteristics on day 2, where only day 3 AHA was performed. Material and Methods: Individually cultured PEs that exhibited excessive fragmentation were subjected to extensive fragment removal on day 2. A single hole was made in the zona pellucida was made using acidic Tyrode’s medium (pH=2.35) and fragment removal was performed using controlled suction; an effort was made to remove a maximum number of cytoplasmic fragments. Cell number and fragment development were recorded the next day (day 3). Results: One hundred and five PEs from 53 patients with excessive fragmentation of all PEs were manipulated on day 2 (avg. cell # and fragmentation: 3.3 and 30.2%), and 91 PE were used as controls (avg. cell 2.84, avg. fragmentation 32.3%). Significantly higher average cell numbers

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(5.24 vs. 4.2; pcO.05) and more rapid development to 26 cells (47.6% vs. 17.6%. pcO.05) on day 3 was observed in the D2-AHA group compared to the control group. Also, significantly lower cytoplasmic fragmentation was observed (avg. fragmentation 18.2% vs. 34.5%; p30% fragments. Analysis showed a highly beneficial effect on PE with <4 cells on D2, while PE with more than 24 cells demonstrated a better developmental capability in both groups. Significantly higher clinical pregnancy rates and lower miscarriage rates were obtained in the D2-AHA group compared to D3-AHA group of patients (42% vs. 15.6% and 2% vs. 14.1%: pCO.05). Conclusions: 1) Excessively fragmented PE (220%) benefit from early fragment removal. 2) This beneficial effect leads to greater cell division and a reduction in further fragment development. 3) Using this technique, additional PE may become available for transfer on day 3 (>6 cells and less than 20% fragmented) and a higher pregnancy rate may be realized.

Monday,

September 2:15 P.M.

27, 1999

O-034 Outcome of Fresh and Cryopreserved Embryos Derived from RescueICSI in Cases of IVF Fertilization Failure. Z. Liu, A. A. Yuzpe, M. R. Fluker. Genesis Fertility Centre, Vancouver, British Columbia, Canada. Objectives: There are few reports of successful pregnancies following the use of delayed or rescue-ICSI to salvage IVF cycles with fertilization failure. We wished to evaluate the outcome of a rigorous protocol of rescue ICSI performed 18-20 hours after initial insemination for cases of IVF fertilization failure. Design: Retrospective comparison of results of rescue-ICSI versus standard ICSI between August 1, 1997 and January 31, 1999. Materials and Methods: Rescue-ICSI was performed in 31 of 481 IVF cycles with complete failure of fertilization and in two additional cycles with 525% fertilization. Fertilization failure was documented 18 hours after oocyte insemination (0730-08OOh), and rescue-ICSI was performed on 233 metaphase II oocytes within the next two hours using sperm from the initial insemination samples. Fertilization was reassessed 10 hours after rescueICSI. Embryo transfer occurred on the third day following oocyte retrieval, approximately 50 hours following rescue-ICSI. Results: Successful rescue-ICSI fertilization (2 pronuclei) occurred in 26/31 cases with complete fertilization failure and in 2/2 cases with partial fertilization failure. Among 233 metaphase II oocytes injected, 143 (61%) showed normal fertilization compared with 82% (2607/3 192) from standard ICSI in the same period at our clinic (P(O.05). An average of 3.1 (range l-5) embryos was replaced. In the fertilization failure group, the clinical pregnancy rate was 19% (5/26) per embryo transfer with an implantation rate of 7.5%. Corresponding pregnancy rates of 35% (137/396) and implantation rates of 19% were obtained with standard ICSI during the same period (P
Monday,

September 2:30 P.M.

27, 1999

O-035 Embryo Developmental Delay on Day 5 Results Fertilization Success. .I. L. Frattarelli, R. Alvero,

in Decreased B. T. Miller,

In Vitro L. Scott,

s13

J. H. Segars. Combined Federal Program of Reproductive WRAMC, NNMC, USUHS, and NIH, Bethesda, MD.

Endocrinology

2145 P.M.

Objectives: Published data reports a blastocyst rate of 60-80% per patient and 40-60% per embryo, which have resulted in excellent pregnancy rates. The goal of blastocyst stage transfers is to lower multiple birth rates while maintaining pregnancy rates. However, not all patients will have embryos that develop to the blastocyst stage by day 5 post fertilization. Thus, these patients may have only morulas available for transfer on day 5. Some groups have advocated embryo transfer on day 6 for embryos that have not yet reached the blastocyst stage on day 5. Little has been published regarding the pregnancy results for those embryos demonstrating developmental delay at the morula stage on day 5. To address this, we examined all patients undergoing day 5 embryo transfers. Patients were split into three groups based on the type of embryos transferred (blastocyst-only, combined blastocyst and morula, and morula-only) and evaluated them with respect to: implantation rate, pregnancy rate, age, number of embryos transferred, number of follicles retrieved, number of mature follicles, fertilization rate, number of embryos cultured, embryos reaching the blastocyst or morula stage on day 5, and day 3 embryo grades. Design: An analysis of 84 patients undergoing day 5 transfers at a university-based assisted reproductive center was performed on patients entered serially during a six month period from July 1998 to December 1998. Materials and Methods: In total, 84 patients underwent embryo transfer on day 5. All patients undergoing day 5 transfers were included in the study regardless of infertility diagnosis. Blastocyst-only (BO) embryos were transferred in 68 patients, combination blastocyst and morual-stage (BM) embryos were transferred in 10 patients, and morula-only (MO) embryos were transferred in 6 patients. A single embryologist evaluated all embryos. Statistical analysis was performed using a one-way Analysis of Variance, Wilcoxon-Mann-Whitney Rank Sum test, t-test, and Fisher’s Exact test where appropriate. An alpha error of 0.05 was considered significant for all calculations. Results: The mean age of the patients in all groups was similar. The overall clinical pregnancy rate was 75% (63/84). Moreover, 68 patients (81%) who had embryos cultured to day 5 had blastocyst-stage transfers, 10 patients (12%) had blastocysts and morula-stage embryos transferred, and 6 patients (7%) had morula-only embryos transferred. There was no difference in number of oocytes retrieved or number of mature oocytes between the groups. The patients receiving blastocyst-stage embryos had a significantly higher number of oocytes fertilized and cultured compared to those receiving morula-only transfers. Likewise, the BO group had significantly more high-grade embryos on day 3 embryo grading, higher pregnancy rates, and higher implantation rates compared to the other two groups. Of note, no excess morula-stage embryos in the BM or MO group reached the blastocyst stage on day 6. Conclusions: The day 5 transfer of blastocyst-stage embryos is associated with a significantly higher pregnancy rate compared to morulastage embryos. This suggests that those embryos not reaching the blastocyst stage on day 5 demonstrate a developmental delay, which negatively impacts cycle outcome. The patients with morula-only embryo transfers had significantly fewer oocytes fertilized and subsequently cultured. Based on this, patients with < 10 oocytes fertilized and <3 high grade embryos on day 3 may have a better prognosis with a conventional day 3 transfer. BO

BM

MO

p-value

Ongoing Pregnancy Rate Implantation Rate Oocytes Retrieved Oocytes Fertilized High Grade Embryos Embryo # Cultured Embryo # Transferred

32.5 ? 4.0 75% 56.3% 22.3 lr 9.6 14.1 ? 7.4 4.6 k 3.3 13.9 + 6.0 2.0 k 0.3

32.8 f 3.7 60% 29.2% 21.0 k 7.2 12.9 2 5.7 2.8 -c 2.6 12.7 2 3.1 2.4 5 0.7

34.3 + 4.0 16.7% 5.9% 20.3 k 10.0 9.2 2 3.1 2.5 k 2.1 9.0 2 3.8 2.8 k 0.8

0.56 0.008*
* BO values are significant t BO values are significant

compared compared

Age

s14

Abstracts

to MO. to BM and MO.

Monday, September 27, 1999

at

O-036 Successful Delayed Analysis for a Second Gene Following a Cycle of Preimplantation Genetic Diagnosis. S. A. Gitlin, W. E. Gibbons. The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA. Objectives: Couples carrying X-linked recessive diseases can undergo Preimplantation Genetic Diagnosis (PGD) for sex determination of their embryos to transfer only female embryos if the specific mutation for the affected gene is not known. Statistically half of the male embryos will have the affected gene thus some couples elect to have those male embryos cryopreserved for later analysis. This study’s objective was to establish a non-invasive method for determining if previously sexed male embryos could be eligible for transfer for a woman who carries X-linked Duchenne Muscular Dystrophy (DMD). Design: Blastomeres were reanalyzed by polymerase chain reaction (PCR) and compared to base pair size fragments against known controls. Materials and Methods: A woman carrying a deletion in the dystrophin gene responsible for DMD had previously undergone two cycles of embryo biopsy and PGD by nested PCR for sex determination since specific dystrophin gene testing was unavailable at the time. After the transfer of female embryos, embryos diagnosed as males were cryopreserved. The original PCR reaction tubes were stored frozen at -20°C. It was later determined that PGD testing for the specific dystrophin deletion could be offered following control PCR experiments using lymphoblasts from a male affected with the same deletion for DMD. The risks and benefits were explained to the couple who elected to proceed. The stored PCR reaction tubes containing the blastomeres from seven previously determined male embryos were reanalyzed by nested PCR, this time for the dystrophin gene. Primers for the known deleted exon, along with primers for an intact region of the dystrophin gene were used. Base pair sizes after polyacrylamide gel electrophoresis were compared to known controls and the diagnosis was determined. Results: Of the seven male embryos that were reanalyzed by PCR, two were diagnosed as having both regions of the dystrophin gene (normal), three showed only one region of the dystrophin gene (thus affected with the known muscular dystrophy deletion), and no amplification signal was seen for two of the embryos. Conclusions: Stored PCR reaction tubes from previously analyzed single cells can be reused to evaluate for a second gene: the most obvious reason being for determining a specific mutation after sex determination for an X-linked disease. This study demonstrated our ability to obtain a diagnosis of a second gene in five of seven cryopreserved male embryos. As more mutations are identified, this may allow couples who have cryopreserved embryos previously diagnosed as males by PCR to have the opportunity to thaw and transfer these embryos, thus avoiding the need to rebiopsy.

Monday, September 27, 1999 3:00

P.M.

O-037 Impact of Chromosome Abnormalities in Human Embryo Development. ‘C. Rubio, ‘,‘A. Pellicer, 3F. Vidal, ‘Y. Mfnguez, ‘J. Ll Romero, 3C. Gimenez, i,*J. Remohf, ‘.*C . Simon. ‘Institute Valenciano de Infertilidad and 2Department of Pediatrics, Obstetrics and Gynecology, Valencia University School of Medicine, Valencia Spain, ‘Department of Cell Biology, Universidad Autonoma de Barcelona, Bellatetra, Spain. Objective: To assess the influence of chromosome abnormalities in preimplantation embryo development. Design: This is a prospective study assessing the developmental potential of chromosomally normal and abnormal embryos. Material and Methods: Preimplantation diagnosis was performed in 12 patients because of each related aneuploidy and recurrent miscarriage. Embryo biopsy was performed on day 3 and one or two blastomeres were removed from 6-8 cells embryos. A total of 89 embryos were analyzed by fluorescent in situ hybridization (FISH) using centromeric and locus specific probes for chromosomes 13,16,18,21,22,X and Y (Vysis, Inc., France).

Vol. 72, No. 3, Suppl. 1, September 1999

Embryos were cocultured with endometrial epithelial cells after biopsy procedure and their development was checked each 24 hours. IRB approval for preimplantation diagnosis was obtained. Statistical analysis was carried out using the chi-square test (p
No. of embryos NORMAL ABNORMAL Monosomy Trisomy Mon/Tris Haploidy Triploidy Tetraploidy

43 46 21 8 4 7 3 3

No. of arrested embryos on day 3 (%)

No. of arrested embryo on day 4 (%)

No. of morula (%)

No. of blastocyst 6)

4 (9.3) 18 (39.1) 4 (19.0) 2 (25.0) l(25.0) 5 (71.4) 2 (66.7) 3(100)

5 (11.6) 2 (4.3) 2 (9.5) 0 0 0 0 0

13 (30.2) 15 (32.6) 12 (57.2) 1 (12.5) 1 (25.0) l(14.2) 0 0

21 (48X)* 11 (23.9)* 3 (14.3)? 5 (62.5)t 2 (50.0) 1 (14.3) 1 (33.3) 0

Conclusions: Our results show that the presence of chromosome abnormalities in preimplantation embryos reduces significantly their developmental potential in early stages, although a low percentage of chromosome abnormalities can be found among blastocyst. There is a significant decrease in the percentage of monosomic embryos that reach blastocyst stage compared to trisomic embryos, suggesting that monosomies have a more lethal effect on embryo development than trisomies.

Monday,

September

3:45

27, 1999

P.M.

O-038 Lack of Effect of Shortened Sperm-Oocyte Exposure Time on In Vitro Fertilization (IVF) and Embryo Quality. M. C. Graham, W. R. Phipps, A. B. Partridge, L. A. Paulhamus. Department of Obstetrics and Gynecology, University of Rochester, Rochester, NY. Objective: To determine whether shortening the time of sperm-oocyte exposure in human IVF affects fertilization rates or embryo quality as assessed by morphologic grading. Design: We conducted a prospective, randomized, blinded study involving 30 IVF cycles in which at least eight oocytes were retrieved. Male factor cases were included if intracytoplasmic sperm injection was judged unlikely to be beneficial on the basis of prior semen analyses and history. Oocytes from each patient were assigned in equal numbers to the two study groups, A and B, randomly, essentially allowing each patient to serve as her own control. Group A oocytes were inseminated four hours after retrieval, and removed from the sperm 14-16 hours later, a standard practice in many IVF programs. Group B oocytes were also inseminated four hours after retrieval, but removed from the sperm two hours later. Fertilization rates and embryo quality parameters for the two groups were compared. Materials and Methods: For both groups, oocytes in 100.PL drops of Human Tubal Fluid/human serum albumin (HTF/HSA) under oil were inseminated with sperm processed in HTF/HSA by either a mini-Percoll or swim-up technique, using an insemination concentration of 18,000 to 50,000 motile sperm per 100 pL. Group A oocytes were denuded after 14-16 hours of incubation to assess for occurrence of fertilization. Group B oocytes were incubated with sperm for two hours and then transferred to drops of Human Preimplantation Medium, Stage I/Synthetic Serum Substitute (Pl/SSS) under oil, and denuded 12-14 hours later for fertilization assessment. Excess zygotes were frozen, according to individual circumstances, without regard to study group. All zygotes not frozen at the pronuclear stage were cultured in drops of PllSSS under oil until three days after retrieval. At that point each embryo was assigned a quality score, in a blinded fashion, representing the product of cell number and grade on a scale of 1 to 4 (poorest to best; Steer et al. Hum Reprod 1992;7:117-9).

FERTILITY

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Embryos for transfer were chosen also in a blinded fashion by a single technician, and 2-5 embryos transferred per patient. Statistical comparisons were made using &i-square tests or analysis of variance, as appropriate. Results: There were no statistically significant differences between the two groups. Overall fertilization rates for groups A and B were 126/187 (67%) and 116/182 (63%), respectively. In three cycles, fertilization occurred in group A but not group B (fertilization rates of l/5 vs O/5, 5/6 vs O/6, and 2/5 vs O/4, for group A vs group B, respectively). Mean (2 SEM) embryo quality scores for groups A and B were 20.5 ? 0.9 and 22.4 ? 1.0, respectively. For group A, the proportion of available embryos selected for transfer was 54/101 (53%), compared to the corresponding group B proportion of 47/94 (50%). Conclusions: In our center, shortening the time of sperm-oocyte exposure in cases not involving an apparently clinically important male factor did not significantly improve fertilization rate or embryo quality. Our design did not allow for demonstration of an effect on pregnancy rate.

Monday,

September

4:00

27, 1999

P.M.

O-039 Comparison of Short and Long Sperm-Oocyte Coincubation in Organ Culture Dishes (OCD) and Tubes (TU). t,‘D. G. Hammitt, ‘K. M. Barud, ‘T. M. Galanits, ‘M. A. Wentworth, *D. L. Walker, ‘M. A. Damario, ‘A. P. Singh, ‘D. A. Dumesic. Department of Obstetrics and Gynecology, Mayo Clinic, ‘Rochester, MN and ‘Scottsdale, AZ, USA. Objective: Recent studies suggest shorter sperm-oocyte coincubation times may increase embryo quality by decreasing exposure of the oocyte to oxygen-free radicals produced by sperm. OCD and TU have been used for insemination of oocytes for IVF. Fertilization rates may be increased with the TU method due to sperm concentrating around the oocyte at the bottom of the TU. It is presently unknown whether sperm concentrations need to be lowered when using TU insemination due to this concentration phenomenon. The purpose of this study was to compare our standard long-interval sperm-oocyte coincubation in OCD to short-interval coincubation in OCD and TU and to evaluate if different insemination concentrations are reauired for the OCD versus TU insemination methods. Design: Oocytes from 58 patients were assigned to l-3 treatments based on the number available (1 treatment if < 6 oocytes; 2 if 6-8 oocytes; 3 if 2 9 oocytes): 1 g-hour insemination in OCD (1 8-hr-OC), 1.5.hour insemination in OCD (1.5-hr-OC), and 1.5-hour insemination in TU (1.5-hr-TU). Insemination concentrations were determined on the basis of a previous semen analysis with Strict Criteria and World Health Organization morphology. Full, and one-half, of our standard concentrations were used in Studies I and II, respectively, for TU. In Study I, fewer oocytes were assigned to the 1.5~hr treatments until good fertilization results were ascertained. Materials and Methods: Oocytes were inseminated in 1 ml of media 5 hours following retrieval in groups of l-4. Oocytes assigned to l&hr-OC were left with sperm until the time of the fertilization check. Oocytes assigned to 1.5-hr-OCD or 1.5~hr-TU were removed 1.5 hours after insemination with a 600 u StripperTM and placed in a microdrop under oil without rinsing or removing adherent coronal or sperm cells. Coronal cells were removed and oocytes were examined for fertilization 16-18 hours following insemination. Results: During the preliminary study (Study I), increased polyploidy was observed with the 1.5-hr-TU method using our standard insemination concentrations. Insemination concentrations were therefore decreased by onehalf for the 1.5-hr-TU treatment for Study II. There were no significant differences among the three treatments in 2PN, polyploidy or 1PN rates in Study II. Study I-TIJ at Full Concentration

Study

Treatment

Inseminated

2PN

2 3PN

1PN

Insetnnated

18.hr-OC 1.5.hr-OC 1.5-hr-TU

136 56 30

64.7% 76.8% 66.7%

11.0% 10.7% 20.0%

8.1% 7.1% 6.7%

209 173 180

II-TU at One-Half Concentration 2PN

2 3PN

1PN

66.5% 60.7% 62.2%

1.6% 9.8% 8.9%

7.6% 5.8% 3.3%

s15

Conclusion: Shortening the sperm-oocyte coincubation period from 16-18 hours to 1.5 hours does not decrease the rate of normal fertilization and may enhance embryo quality by reducing exposure of the oocyte to oxygen-free radicals produced by sperm. Insemination in TU may prove superior to OCD insemination because only one-half the number of motile sperm are required to obtain equal rates of fertilization. This may permit the use of standard insemination, as opposed to ICSI, in some cases of male infertility.

Table 2. SPICSI,

% Fertilization N 6

September 4:15 P.M.

denuded

(range)

InseminatedCohort 78 (71-86)

Monday, Monday,

only ICSI oocytes

27, 1999

September 4:30 P.M.

ICSICohort 86 (60-100)

27, 1999

O-041 o-040 Cumulus Cell Removal Hinders Fertilization in Split-ICSI Cases. ‘D. E. Battaglia, ‘C. Rainer, ‘A. Khabani, ‘V. Y. Fujimoto. ‘Division of Reproductive Endocrinology, Department of Obstettics and Gynecology, University of Washington, Seattle, WA. Objective: Determining whether a couple requires intracytoplasmic sperm injection (ICSI) can be problematic, particularly with cases of unexplained infertility. With the aim of identifying couples with fertilization dysfunction in IVF we developed a protocol for dividing mature oocytes into inseminated and ICSI cohorts with the ICSI cohort serving as insurance against fertilization failure. The fertilization rate of the inseminated cohort would determine the need for ICSI intervention in subsequent IVF cycles. Design and Methods: The split-ICSI protocol (SPICSI) was reserved entirely for couples with unexplained infertility who were undergoing their first IVF cycle. After oocyte retrieval, all oocytes were stripped of their cumulus cells by short-term hyaluronidase treatment (751U for 30-60 set) followed by passage through narrow bore pipettes to permit the identification of mature (metaphase II) stage oocytes. If there were ~8 mature oocytes they were equally divided into an inseminated cohort (0.25 X IO6 sperm/ml) and an ICSI cohort. Fertilization rates of <50% were considered the basis to recommend ICSI for future IVF cycles. Results: The results of 43 cycles are summarized in Table 1. There were 22 cases of fertilization failure in the inseminated cohorts, which far exceeded our expectations of fertilization dysfunction in the unexplained infertility population. We suspected that the process of cumulus cell removal may have been the cause of much of this fertilization failure since the failures nearly always occurred in cohorts of oocytes that had been very thoroughly denuded of cumulus (i.e. more vigorous stripping techniques). To address this possible etiology we performed additional SPICSI cases in which the inseminated cohort was not denuded of cumulus cells. For each of these cases P 10 oocytes were required. The oocytes were progressively stripped of cumulus until at least 4 mature oocytes were obtained. Cumulus cell removal proceeded with additional oocytes until half of the expected mature oocytes were denuded for each case. All other oocytes remained untreated and were inseminated with the cumulus cells intact. The results of 6 cycles are summarized in Table 2. As can be seen, the fertilization rates of the oocytes returned to expected levels if the cumulus mass was left intact. Conclusions: From these data we speculate that cumulus cell removal can interfere with fertilization either through disruption of direct influence of the cumulus cells on fertilization, or perhaps through disturbances to the zona pellucida. It seems likely that cumulus cell removal could affect sperm receptors on the zona surface directly or induce the release of some of the cortical granules by the vigorous action of denuding pipettes. For SPICSI cases we recommend denuding only the oocytes destined for the ICSI cohort. Determining the need for ICSI is important and we believe that SPICSI may be a useful tool for cases of unexplained infertility if cumulus cell removal is not done for the inseminated oocytes. Table

1. SPICSI

with all oocytes

% Fertilization N 43

S16

InseminatedCohort 21 (o-88)

Abstracts

denuded

Culture of Embryos in an Environment Shielded from Exposure to Electra-Magnetic Fields has no Effect on IVF Outcome. ‘C. J. Turczynski, ‘C. Chang, ‘W. Hovis, *A. Marino, ‘S. L. London. ‘The Center for Fertility and Reproductive Health, Department of Obstetrics and Gynecology and ‘Department of Orthopedic Surgery, LSU Medical Center-Shreveport, LA. Objectives: Incubators and other electrical equipment used in IVF centers produce low intensity electro-magnetic fields (EMF) in the range reported to have cellular effects. To determine if the levels of EMF found in our laboratory were exhibiting a detrimental effect, we evaluated the outcome of IVF when insemination and embryo culture were shielded from EMF. Design: By placing a chamber constructed of Mu metal directly into a standard incubator, we shielded gametes and embryos from EMF, and then prospectively randomized patients to standard or EMF shielded culture. Multiple IVF outcome variables where evaluated for statistical differences between the two culture methods. Materials and Methods: Measurements of EMF were taken in and around the incubators and inside of the Mu metal chamber using a Gauss meter. Treatment and control data collected from January 1997-December 1998 (n= 122 cycles) were analyzed by T-test or Chi-square analysis using the statistical software package GB-STAT. Significance was determined to be p< .05. Embryo transfers were performed on day 3 and supernumerary embryos were cultured to blastocysts before freezing. Results: Our measurements estimated that the standard cultures were exposed to magnetic fields in the range of 13.4-3.50 milli-gauss (mG), mean 81.7 mG and the shielded cultures 5 7.6 mG. The patient and cycle data, specititally, primary diagnosis, primary/secondary infertility, years of infertility, age, days stimulated, number of eggs collected, type of insemination or number of embryos transferred did not differ significandy between the two groups. The chemical, clinical and ongoing pregnancy rate per retrieval for EMF shielded vs standard culture respectively were 52% vs 44%, 46% vs 37% and 34% vs 33%. The implantation rate was 23% vs 20%. These differences were not statistically different. In addition, the fertilization rate, mean embryo grade, grade of lead embryo and supernumerary embryo blastocyst rate did not differ significantly between standard and Eh4F-shielded culture. Conclusions: Although the chemical and clinical pregnancy rate was higher in the EMF shielded culture group, these data were not statistically different and the differences were not reflected in the ongoing pregnancy or implantation rate. These data indicate that shielding embryos from EMF, when cultured in routine in-vitro conditions, provides no beneficial effect and levels found in a standard IVF lab are probably not harmful. Knowledge of the levels found in individual IVF laboratories should be considered a quality control issue, however, because there is considerable variation between and within instrumentation used and therefore these data cannot be extrapolated to all laboratories.

Monday,

September 4:45 P.M.

27, 1999

O-042

(range) ICSICohort 73 (42-100)

The Anti-Estrogen ICI 182,780 Abolishes Developmental Injury for Murine Embryos Exposed In Vitro to the Estrogenic Pesticide o,p’DDT. ‘A. R. Greenlee, ‘C. A. Quail, ‘R. L. Berg. ‘Rural Health Research Department, ‘Department of Clinical Research, Marshfield Medical Research Foundation, Marshfield, WI.

Vol. 72, No. 3, Suppl. 1, September 1999

Objectives: In vitro exposure of murine preimplantation embryos to xenobiotics is a potentially useful tool for screening developmental toxicants. Previously, we reported that in comparison to control and isomer treatments, in vitro exposure of murine preimplantation embryos to 0.1-10 pg/ml o,p’-DDT (an estrogenic organochlorine pesticide) reduced development to blastocyst, reduced mean cell number per embryo and increased programmed cell death (apoptosis) at 114 h of culture. The objective of the present study was to determine if developmental injury induced by o,p’DDT resulted from estrogenic, anti-estrogenic or unrelated adverse biological mechanisms. Design: Pronuclear embryos from CD-l mice were cultured 114 h in protein-free Earle’s balanced salt solution (EMG) supplemented with ethanol (control), or one of the following compounds: o,p’-DDT, [email protected], ICI 182,780 or pair-wise combinations of the compounds. Percent development to blastocyst, mean cell number per embryo and percent apoptosis were evaluated at 114 h. Materials and Methods: Pronuclear embryos were obtained from superovulated CD-l female mice mated to CD-l males. Embryos were collected 16-17 h after hCG injection from the oviducts of females with vaginal plugs. Cumulus cells were removed by incubation in hyaluronidase. Embryos were rinsed in 3 changes of modified EMG before transferring groups of 20-25 embryos to 25 ~1 drops of EMG under mineral oil containing 1 pi/ml ethanol or 0.1 pg/ml of o,p’-DDT, 17gestradio1, ICI 182,780 or pair-wise combinations of the compounds at 0.1 pg/ml. Percent blastocyst development was determined 114 h after hCG injection. Zona pellucidaintact embryos were stained for apoptosis and cell counts by incubation in fluorescein-conjugated dUTP, TdT and propidium iodide. A factorial design and analysis of variance were used to perform a weighted analysis of group means. Results: In comparison to control values, percent development to blastocyst, mean cell number and percent apoptosis were significantly reduced for embryos cultured 114 h in the presence of o,p’-DDT or ICI 182,780 (all p 50.019) and percent development to blastocyst was significantly different for embryos cultured in the presence of l’lfi-estradiol (p=O.O04). For percent development to blastocyst and mean cell number per embryo, significant interaction occurred when ICI 182,780 was combined with o,p’-DDT or 17P-estradiol (all p 50.012). Significant interaction was not observed when o,p’-DDT was combined with 17P-estradiol (p 20.175). Conclusions: The results indicate that developmental injury due to the estrogenic pesticide o,p’-DDT was abolished by the addition of the antiestrogen ICI 182,780 and not by the addition of the estrogen 17P-estradiol. The findings underscore the utility of the model for uncovering mechanisms of developmental injury that may go unrecognized if only the parents are exposed. Financial support for this study was provided by the Connor Trust and Marshfield Medical Research Foundation.

NURSES PROFESSIONAL Monday,

September 290 P.M.

GROUP 27, 1999

O-043 Evaluation of Implantation Rates in the Recipients of Donor Oocytes. M. L. Slate, Costa, T. J. Smith, R. T. Scott. The Institute Science of Saint Barnabas Medical Center,

Presence of Leiomyomata in S. D. Ernst, M. M. Jackson, D. for Reproductive Medicine and Livingston, NJ.

Objective: To investigate the effect of uterine myomata on embryo implantation rates in recipients of donor oocytes. Design: A retrospective comparative study of implantation rates in donor oocyte recipient cycles with and without the presence of uterine myomata. Materials and Methods: Data was compiled and analyzed from all oocyte recipient cycles between September 1995 and December 1998. Recipients are categorized as to whether or not they had uterine myomata at the time of their cycle. Examined are the size of myomas where present, the number of embryos transferred, the mean percentage of fragmentation and cell numbers of all embryos transferred, pregnancy rates, implantation rates and deliveries. Results are analyzed with ANOVA’s and contingency tables, and ROC curves are drawn to evaluate possible threshold values for myoma size. Results: A total of 355 recipient cycles were evaluated. Forty seven cycles were completed in women with sonographically identifiable myo-

FERTILITY

& [email protected]

mas. The myomas ranged in size from 6mm to 47mm with none being judged to impinge on the cavity. Overall pregnancy, ongoing pregnancy, and implantation rates were 70.1%, 63X%, and 40.6%, respectively. Women with and without myomas had equivalent numbers of embryos transferred (2.6 vs 2.8). These embryos had cleaved to equivalent blastomere number (8.2 vs 8.3) and had similarly low fragmentation rates (8.9% vs 7.1%). They also had equivalent endometrial thickness and patterns. Most importantly, the ongoing pregnancy rates (64.1% vs 62.9%) and implantation rates (41.0% vs 39.9%) in women with and without sonographically evident myomas were not different. Conclusion: The oocyte donation recipient model is a useful one because it allows for control of the variable of embryo quality and better isolation of the impact of uterine leiomyomata on implantation rates. Uterine myomata which do not impinge on the uterine cavity do not appear to impair implantation in recipients of donor oocytes.

Monday,

September 2:15 P.M.

27, 1999

O-044 Do Anonymous Oocyte Donors Want to Know the Outcome of the Cycle and Meet Any Offspring? M. M. Jackson, M. L. Slate, S. D. Ernst, D. Costa, R. T. Scott. The Institute for Reproductive Medicine and Science of Saint Barnabas Medical Center, Livingston, NJ. Objectives: Most anonymous programs do not currently share cycle outcomes with anonymous donors nor do they encourage meetings with the offspring from their donations. Do donors want to know the outcome of the cycle and would they be willing to meet the offspring? Should this nondisclosure policy be reconsidered based on the wishes of the donors? Design: Women being considered for acceptance in an anonymous oocyte donation program were asked to complete surveys. Materials and Methods: The study group consisted of 110 women being considered for acceptance in an anonymous oocyte donation program and responses reflect their pre-cycle attitudes. Three questions were posed concerning their desire to meet the offspring: 1.) Did they desire to know the outcome of the oocyte donation cycle? 2.) Would they be willing to meet the child in the future if the child desired such a meeting? 3.) Would they like to meet any offspring that result from their donation cycle? Results: The responses to the questionnaire were commonly incomplete. Ninety-nine of the donors noted their opinion on being informed of the cycle outcome, while 44 addressed their interest in meeting any offspring and 75 addressed their opinion about their willingness to meet the offspring if the child desired to meet them. The overall response rate to the various questions were significantly different (PcO.05) (knowing cycle outcome > willingness to meet at child’s request > their own desire to meet the child). Fifty donors wished to know the cycle outcome while 29 did not desire to know the outcome with the last 20 stating they were undecided. The substantial majority of potential donors said they would be willing to meet the child if the child desired such a meeting (n=70), with only 3 expressing an unwillingness and 2 remaining undecided on the issue. In contrast, the proportion of potential donors who desired to meet the offspring (n=27) compared to those who would like to meet (n=27) were more evenly distributed (undecided=2). Conclusions: The women in this study appeared to be somewhat ambivalent regarding their desire to know if pregnancy occurred since approximately half expressed the desire to know while the other half were either not interested or undecided. These findings differ from those in studies by Power et al (1990), Schover et al (1991) and SoderstromAntilla (1995) who found that the majority of women who had donated oocytes preferred to know the outcome. This difference may be indicative of when they were surveyed (pre versus post donation) and that prior to the actual donation they are more interested in the process itself rather than the actual outcome. These data indicate mixed results regarding the donor’s desire to meet any children in the future which favors maintenance of the policy of non-disclosure at this time. Should the policy change in the future and these children desire a meeting, most of these women would be amenable.

s17

Monday,

September

2:30

27,

1999

P.M.

O-045 Effect of Micronized Vaginal Progesterone Gel on Gamete Intrafallopian Transfer Pregnancy Rates. K. R. Hammond, M. P. Steinkampf, S. W. Fraley. Department of Obstetrics/Gynecology, University of Alabama at Birmingham, Birmingham, AL. Objective: Hormonal supplementation of the luteal phase after oocyte retrieval is a standard protocol for assisted reproduction treatments. Intramuscular (IM) injections of progesterone are most commonly given, but these injections are painful and inconvenient. The recent availability of a progesterone gel (Crinone) approved for vaginal administration to support pregnancy is an attractive alternative for luteal supplementation, but the efficacy of Crinone relative to progesterone injections has not yet been determined. The purpose of this study is to examine the effect of Crinone use on pregnancy rates in women undergoing gamete intrafallopian transfer (GIFT). These patients provide an excellent opportunity to explore the effects of ovarian stimulation/supplementation regimens since the technique does not rely on the vagaries of in vitro embryo culture, and the patient population is relatively homogeneous. Design: Retrospective study of women undergoing GIFT using either Crinone 8% once daily or progesterone 50 mg/day IM for luteal supplementation beginning three days after oocyte retrieval, Materials and Methods: In our program, all patients undergoing GIFT between May 1, 1998 and December 31,1998 were prescribed Crinone. The results of this series were compared to patients undergoing GIFT from January 1, 1997 to April 30, 1998, during which progesterone IM was administered to all patients. Patient selection, ovarian stimulation protocols, and the technical aspects of the GIFT procedure stayed the same over the study period. Results: Twenty-six women were treated with C&one, and 57 women received IM progesterone. There were no differences between the two groups in patient age [mean?SD] (Crinone 33.1? 10.9 yrs, IM 35.6+ 11.3 yrs; P=O.34), number of eggs retrieved (Crinone 14.128.2, IM 14.9k6.9; P=O.65), or number of eggs transferred (Crinone 7522.4, IM 8.Ok2.1; P=O.39). However, only 5 (19.2%) women achieved a clinical pregnancy after GIFT using Crinone, while 26 (45.6%) became pregnant after using IM progesterone (P=O.O39). Conclusion: Our results using Crinone for assisted reproduction are not encouraging. We recommend that this drug be subjected to well-designed randomized controlled trials before incorporating it into routine clinical use.

Monday,

September

2:45

27, 1999

P.M.

O-046 Predictive Value of hCG Level 14 Days After Embryo Transfer of Day 3 Embryos With Assisted Hatching. B. Guth, E. S. Critser, M. A. Henry, W. L. Gentry. Advanced Fertility Institute, Indianapolis, IN. Objectives: It has been reported that the quantitative serum hCG level 14 days after embryo transfer (ET) correlated with pregnancy outcome as well as multiple gestation pregnancy. These studies have reported outcomes with day 2 embryos. This study was designed to assess the predictive value of a 14 day post-ET hCG level of day 3 embryos that were assisted hatched. Design: To compare 14 day post-ET serum quantitative hCG levels to ongoing pregnancies as well as multiple gestation pregnancies associated with day 3 assisted-hatched embryos. Materials and Methods: Patients undergoing in vitro fertilization (IVF) and embryo transfer (ET) with day 3 assisted-hatched embryos were monitored by serum quantitative hCG levels 14 days after ET. If positive, serial values of hCG were obtained and transvaginal ultrasound was performed 3 weeks after ET, and weekly until fetal activity was seen. Results: One hundred fifty-eight (158) patients had positive hCG level 14 days post ET; 1301158 (82%) had ongoing pregnancies. The spontaneous abortion rate was 18%. If hCG > 300, ongoing pregnancy rate was 92%. However, 41/80 pregnancies (51%) were multiples, If hCG was > 600, the ongoing pregnancy rate was 97%, but the multiple rate was 70%.

S18

Abstracts

hCG Level

Ongoing Pregnancies

<300 300-600 >600

56/78 (72%) 41/46 (89%) 33134 (97%)

Twins

Triplets

18 13 16

Quadruplets

0 6 6

0 0 1

Conclusions: These data support the hypothesis that higher hCG levels 14 days post-ET of day 3 assisted-hatched embryos are more likely to have ongoing pregnancies and have a higher incidence of multiple gestation pregnancies. This is consistent with previous reported studies with day 2 embryos.

Monday,

September

3:00

27, 1999

P.M.

O-047 Chlamydia IgG and IgM Serology Screening Associated with Poor Pregnancy Prognosis in an In Vitro Fertilization Program. T. D. Nichtberger, T. L. Viancos, S. K. Frederick, K. A. Petermann, V. L. Schnell. Center of Reproductive Medicine, Webster, TX. Objective: Cervical chlamydia DNA probe, culture or PCR screening has been the standard for IVF patients. A cervical swab screen is frequently negative in patients with acute pelvic inflammatory disease and routinely negative in patients with extensive clinical evidence of chronic pelvic inflammatory disease. Many patients are now entering IVF programs without hysterosalpingograms or diagnostic laparoscopies. Chronic pelvic inflammatory disease may be associated with a normal baseline pelvic ultrasound but after gonadotropin therapy a hydrosalpinx and/or endometrial fluid collection will appear. To evaluate other diagnostic criteria for chronic pelvic inflammatory disease (PID), all patients entering our IVF program were screened with Chlamydia IgG and IgM serology. Design: Retrospective review of charts for 140 couples who underwent l-6 cycles of IVF and/or ICSI between January, 1997 and December 31, 1998 were screened with serum Chlamydia IgG and IgM in addition to other standard screening parameters. Materials and Methods: Prior to entering the IVF program both female and male partners were screened with Indirect fluorescent antibody Chlamydia IgG and IgM serology. These screens were repeated yearly. Other microbiologic screens included: cervical ureaplasma and mycoplasma cultures. Results: Females with positive Chlamydia IgG titers had a higher incidence of first trimester pregnancy loss than those with negative Chlamydia IgG titers: [9/31 vs. 4/31, P
Vol.

72, No.

3, Suppl.

1, September

1999

Monday,

September 3:45 P.M.

27, 1999

O-048 Intrauterine Insemination is a Successful and Cost Effective Alternative to Assisted Reproductive Technologies. E. Formentini, T. Abozaid, M. Sasy, H. Salem, S. Benford-Loyer, A. Copes, C. Bowman, M. I. Abuzeid. The Center for Reproductive Medicine, Hurley Medical Center, Flint, MI. Objectives: Couples faced with infertility often do not have insurance coverage and are faced with many out of pocket expenses. Intrauterine insemination (IUI) is often recommended as a cost effective first line treatment prior to proceeding with assisted reproductive technologies (ART). The success rate for IUI that is reported in the literature is low, ranging from 10 to 20% per cycle. This low success rate coupled with costly alternatives leads many couples to abandon treatment. We have analyzed data from our program to determine if the unique technique for IUI that we utilize results in higher pregnancy rates, and therefore can be recommended as an effective first line treatment for cases where there is not severe male factor or bilateral tubal factors. Design: Retrospective review of all cases of IUI. Materials and Methods: Nine hundred and thirteen cycles of ovulation induction for IUI from 6/90 to 2/3/99 were reviewed. Thirty three cycles were excluded from this analysis as ART was originally intended. Eight hundred and sixty four cycles progressed to IUI, and data from these cycles was analyzed. Diagnoses included: idiopathic, ovulatory disorder, endometriosis, tubal factors, uterine factors, and mild male factor. Ages ranged from 23-43 years old. Two ovarian hyperstimulation protocols were used: clomiphene citrate plus injectable gonadotropins; or a GnRH agonist with injectable gonadotropins. Ovulation was induced with HCG when there were two to four 16mm follicles, or if the LH surge was detected. IUI was carried out 42 hours later using a washed prepared fresh semen sample from the partner. Prior to the IUI, a transvaginal follicular ultrasound was performed in order to assess follicular collapse as well as the presence of fluid in the cul de sac. The IUI was abandoned if excessive ovulation was noted (3 cases), and delayed by four hours if there were no signs of ovulation (16 cases). Results: The overall pregnancy rate was 31.7%. with a 27.3% delivery/ ongoing pregnancy rate per cycle. The miscarriage rate was 12%, with a 1.8% ectopic pregnancy rate. The cycle cancellation rate prior to IUI was 5.7%, with 61.5% of those cases cancelled due to poor ovarian response, and another 21.2% were cancelled to avoid ovarian hyperstimulation syndrome (OHSS). The remainder of the cycles were cancelled due to social reasons. Of those patients proceeding to IUI, 1.3% developed OHSS. The data was further analyzed in terms of stimulation protocols, age and underlying etiologies. Conclusions: IUI as performed in our center is a successful and cost effective treatment modality. The pregnancy and delivery rates are similar to those achieved nationwide through ART without the operative risks. This method of ovulation induction and timing of IUI as performed in our center is easily reproducible, and can become a cost effective alternative for patients at various fertility centers. IUI following this protocol should be recommended as a first line treatment for patients in cases where there is not severe male factor or bilateral tubal factors prior to proceeding with costly ART procedures.

Monday,

September

4:00

27, 1999

P.M.

O-049 Pregnancy Outcomes in Women with Unexplained Infertility. ‘E. Formentini, 2A. Amanze, ‘A. Arthur, ‘M. Sasy, ‘M. I. Abuzeid. ‘The Center for Reproductive Medicine, Hurley Medical Center, Flint, MI and ‘Michigan State University College of Human Medicine, Lansing, MI. Objective: Experts are beginning to focus greater attention on a unique subgroup of patients whose infertility remains unexplained following all diagnostic testing. From up to 10% of infertile couples fall into the category of unexplained infertility. Although couples may become pregnant on their own, a benefit from treatment has been demonstrated for those with prolonged infertility. Currently, no research compares pregnancy outcomes

FERTILITY

& STERILITYa

among the various techniques employed, particularly gamete intrafallopian transfer (GIFT) and intrauterine insemination (IUI) after controlled ovarian hyperstimulation (COH). The purpose of this study is to compare the results of IUI with COH and GIFT in the treatment of unexplained infertility. Design: Retrospective analysis. Materials and Methods: Treatment cycles for GIFT and IUI with COH between June 1990 and April 1998 were reviewed. Included in the analysis were all cases with a diagnosis of unexplained infertility and a female age of less than 40 years. The stimulation protocol consisted of mid-luteal GnRH agonist and injectable gonadotropins for both treatment groups. A total of 99 cycles were reviewed, and comparison between GIFT and IUI treatment cycles were made. Results: There were 30 cycles treated by GIFT and 69 cycles by IUI with COH. The groups were similar in age, duration of infertility, and estradiol levels. Total pregnancy rate was 53% per cycle. The pregnancy rate for GIFT was 66.7% and for IUI with COH was 46.4%. This difference approached significance ($=3.4, p=O.O6). There was no difference in miscarriage rates (p=O.7) or multiple births (p=O.3). Conclusion: Women with unexplained infertility present unique challenges. This data suggest that both GIFT and IUI with COH are very effective treatment modalities for unexplained infertility. GIFT may be more effective than IUI in each treatment cycle. However, in view of simplicity and cost containment, similar cumulative pregnancy rates may be achieved with IUI and COH.

Monday,

September

4:15

27,

1999

P.M.

O-050 The Nurse’s Role in Office Transvaginal Drainage (TVD) of Ascites in Ovarian Hyperstimulation Syndrome (OHSS): A Method of Avoiding Hospitalization. J. E. Copeland, M. R. Fluker, A. A. Yuzpe. Genesis Fertility Centre, Vancouver, British Columbia, Canada. Objectives: (1) to assess the safety and efficacy of drainage of ascites in cases of moderate to severe OHSS in an office setting and (2) to establish a nursing protocol for the care of these patients. Setting: A private infertility clinic. Materials and Methods: Patients at risk for OHSS (>20 follicles at oocyte retrieval) were given written and verbal instructions about signs and symptoms of OHSS and asked to maintain periodic telephone contact with the nursing staff. An increase in symptoms of abdominal distension or shortness of breath (SOB) prompted a standardized nursing-initiated protocol to assess hematologic and renal function parameters. If symptoms were worsening or blood tests abnormal, a transvaginal ultrasound was performed to assess ovarian size and the presence of ascitic fluid. Results: Nine patients (average age 35 years) had tense ascites requiring transvaginal drainage between October 1997 and January 1999. Eight had undergone in vitro fertilization and one had undergone controlled ovarian hyperstimulation. At the time of TVD, they had a mean Hbg of 149 g/L (range 120-169), Hct of 43.7 (range 36-51), serum albumin of 29.6 g/L (range 23-37). and total protein of 57.4 g/L (range 54-64). All had normal coagulation studies. Transvaginal drainage was performed 15.5 days (range 13-19) following the midcycle hCG injection, and all 9 were known to be pregnant at the time their symptoms escalated. Patients were prepped and draped as for oocyte retrieval, receiving an IV of ringer’s lactate, Ativan 1 mg sl and Atropine 0.4 mg IV. Fentanyl (lpg/kg) and diphenhydramine (25 mg) were given intravenously as needed for analgesia and nausea. Following a paracervical block with 1% Xylocaine, transvaginal ultrasound drainage of ascitic fluid from the cul-de-sac occurred in the same manner as follicular fluid collection. An average of 1780 cc (range 1000-2500 cc) of fluid was collected over 20-30 minutes, associated with marked relief of SOB. Vital signs and 02 saturation were monitored every 5 minutes during the procedure and for at least 1 hour after. Follow-up bloodwork was repeated at 2-4 day intervals until acute symptoms subsided and serum albumin normalized. Six patients required a second TVD, while 3 patients required a third drainage over the ensuing 8 days. Five women also received 250 cc of 25% albumin following six of the procedures because of subnormal serum albumin concentrations. The average time to resolution of symptoms was 12 days (range 5-25 days). None required hospitalization for OHSS and non developed post-procedure complications, although one patient was subsequently found to have an ectopic pregnancy.

s19

Conclusions: TVD of ascites is a safe, effective and economical method of treating moderate to severe OHSS in an office setting. A comprehensive nursing protocol to identify patients in the early stages of OHSS is imperative for timely diagnosis and treatment.

Monday,

September 4:30 P.M.

27,

1999

O-051 A Cost and Time Effective Patient Management Program Which Maximizes Patient Compliance and Minimizes Patient Clinic Visits and Work Load While Maintaining High Pregnancy Rates. D. Materia, C. Needham. Reproductive Science Center, Walter Reed Army Medical Center, Washington, DC. Objectives: Human In Vitro Fertilization (IVF) is extremely time consuming for both the patient and clinic staff. The program at Walter Reed Army Medical Center has a patient pool that is both national and international and was designed to minimize clinic visits using strict control and timing of cycles. This study analyses the results of three years of patient intake in terms of satisfaction and outcome and balances this with patient compliance. Design: The data was analyzed according to intake, compliance, satisfaction, outcome. Methods: Patients are members or dependents of the Department of Defense of the USA. They are referred by their own physician with a diagnosis, duration of infertility, age and basic male semen parameters. Patients obtain a check list for completion at home base (pap smear, physical, day 3 FSH/LH/EZ). The first visit to the clinic includes a mandatory orientation, a 3 hour lecture informing the patient of the program operation and their responsibilities and a consultation with a program physician. On completion of the check list the patient is allocated a start day in the next available series (run quarterly) and placed on continuous low dose oral contraceptive pills (OCP’s) without the use of the placebos. The next visit is the base-line (BL) appointment. At BL OCP’s are discontinued and a microdose GnRH flare followed by gonadotropins commences. There are 4-6 subsequent visits until oocyte retrieval, estimated to occur 15 days after BL and one further visit for embryo transfer. Outcome Measures: The percent of patients entering the program, the drop out rate, compliance with the program was measured against outcome and satisfaction. Results: The results are presented in the table below: Overall there was a high rate of compliance, a high approval rating and satisfactory outcome for minimum nursing intervention and minimal office visits.

Referrals received from outside physicians Patients started (% accepted after referral screening) Non-complaint/nonclinical drop out (%) Clinical Cancellations for poor stimulation (%a) Oocyte Retrievals (OR) Clinical Pregnancies (% of OR) Full Time Nurses (Patients/nurse) Positive approval rating obtained from satisfaction surveys

1996

1997

1998

Total

309

551

593

1453

430 (78%)

451 (76%)

1132 (78%)

3 (0.7%)

2 (0.4%)

5 (0.4%)

57 (23%) 194

90 (21%) 340

91 (20%) 360

238 (21%) 894

78 (40%)

126 (37%)

177 (49%)

381 (43%)

1 (251)

1.5 (287)

2 (223)

NA

85%

82%

0

Abstracts

V. Wilkie.

Fertility

Clinical

Unit,

Purpose & Objectives: Patient education is an integral part of nursing responsibilities. Since 1989, the nurses in our IVF unit have conducted the initial patient education portion of the IVF visit which is actually called “Initial Visit.” Initial visits were conducted in a structured manner on a 1: 1 basis with good results, in that our patients expressed satisfaction. In 1997, expectations, compounded by financial constraints to improve efficient use of nurses time, forced our unit to examine other possible methods of patient teaching and teaching options. We decided to undertake “group initial visits,” which were designed to teach four couples at a time versus 1: 1. To evaluate this method of teaching we conducted a qualitative prospective survey using a convenience sample one group design. Each patient scheduled to a group initial visit, received a patient satisfaction survey entitled “Quality of Care”. The summary of results are presented here. Materials and Methods: The survey questions were extrapolated from several different nursing-patient satisfaction surveys. We paid particular attention to questions that were pertinent to our IVF patients, It was imperative to this survey that the questions were easy to read and comprehendible by the patient prior to the initiation of the full survey. Therefore, a pilot analysis of the first 10 surveys was done to test the feasibility of the questions. Following the pilot analysis, the survey was handed out to 102 patients spanning a six month period. The survey included 13 questions using a rating scale (l-5; 1 meaning dissatisfaction and 5 meaning high satisfaction) and 2 questions that asked for narrative response. In the first of these two questions, the patients were asked to recount the next step in their treatment program. The second question asked for their suggestions for improvement. Results: Of the 102 surveys, 50 were returned, i.e. a return rate of 49%. 90% (45150) were complete, 10% (5/50) were incomplete. 64% (29/45) had overall good to excellent rating (all questions were rated 3-5). 35% (16/45) had a lower rating (l-2) to some questions. 80% (34/45) of the patients demonstrated understanding of next step in their treatment program. Conclusions: Group teaching was rated favourably by the majority of the patients. In addition, the results suggest that knowledge was imparted to a large proportion of the patients and therefore has become a standard of practice in our unit. This part of patient education is a valuable, costeffective method. However, during our review of the lower rating surveys, we discovered that most of these patients gave a rating of l-2 with respect to waiting times only (questions 1 and 2). Based on this finding we have decided to take the opportunity to explore corrective measures. In conclusion, further nursing research should be conducted into the area of group teaching with fertility patients. This type of research could promote more efficient nurses teaching time while imparting knowledge to the patients.

RESEARCH

Monday,

September 2:00 P.M.

NEWS

27, 1999

o-o.53

75%

87%

Conclusions: It is feasible to run a cost and time effective IVF program in series with minimal nursing support without significant drop out or compromising patient care, satisfaction or pregnancy outcome. This helps to reduce the amount of time patients require away from job and home, reduces cost to both the patient and the program and allows the nurses breaks to organize for the subsequent cycles.

s20

Patient Teaching. M. Morrison, Hospital--Civic Campus.

LATE-BREAKING 251 (81%)

27, 1999

O-052 Group Ottawa

Monday,

September 414.5 P.M.

Enhanced Implantation of Day-6 Blastocysts Following Assisted Hatching With Acidic Tyrode’s Medium. M. Tucker, J. Graham, T. Han, M. Levy, A. Sagoskin, R. Stillman. Shady Grove Fertility Center, Rockville, Maryland. Objective: Blastocyst implantation relative to transfer on day-5 or day-6 seems worse for the “slower” embryos. To counter this, we undertook assisted hatching (AH) of all day-6 blastocysts to observe if this was beneficial. Design: Implantation rate [IR], clinical and ongoing pregnancy rates [CPR; OPR] were retrospectively compared after the transfer of day-6 blastocysts either with intact zonae or following zona drilling. Materials and Methods: All normal zygotes were maintained individually in microdroplets of stage-appropriate medium until day-6 of development, when blastocysts were selected according to morphology and degree of expansion. An initial group of women (n=41; avrg. age 34.3 yrs) had

Vol.

72, No.

3, Suppl.

1, September

1999

blastocysts transferred with zonae intact with day-6 [control group]. A subsequent group of day-6 transfer women (n=39; avrg. age 36.1 k3.5 yrs) were offered AH prior to transfer. In this test group all blastocysts were exposed to O.lM sucrose in mHTF for 1 min. to shrink the embryo, and a hole (approx. 35wm) was drilled with acidic Tyrode’s medium in the zona away from the shrunken embryo. After washing, such AH blastocysts were allowed to re-expand prior to embryo transfer (ET; 1 to 4 hrs later). Results: Following ET of an average of 2.6 blastocysts in the control group, CPR, OPR and IR were 29%, 22% and 14% respectively. This compared with the test group, in which all blastocysts underwent AH (avrg. 2.4 for ET), where CPR, OPR and IR were 46%, 38.5% and 22.5% respectively. Chi’ analysis AH vs. Control: CPR, p
Monday,

September

2:1.5

27, 1999

P.M.

O-054 Ovarian Function After Autologous Transplantation of Frozen-Banked Human Ovarian Tissue. K. Oktay, G. Karlikaya, R. Gosden, R. Schwarz. Department of Obstetrics and Gynecology, New York Methodist Hospital and Cornell University Weill Medical College, New York, New York. Objectives: Animal studies have shown that cryopreserved ovarian tissue can be transplanted successfully. We designed the current study to develop a technique to transplant frozen-banked human ovarian tissue and to demonstrate that the auto-transplanted tissue can resume its functions. Design: An experimental laparoscopic procedure followed by ultrasound and hormonal monitoring. The study was approved by the Institutional Review Board and the State Department of Health. Material & Method: M.H. is a 29 year-old Caucasian female who had had a right salpingo-oophorectomy and left cystectomy with wedge resection at the age of 17, to remove dermoid cysts. Later, the patient developed exercise-induced hypothalamic amenorrhea. At age 28, she received a left salpingo-oophorectomy. After the salpingo-oophorectomy, 2X5 to 5X 10 mm. ovarian cortical strips were cryopreserved, using the slow freeze protocol, as described previously. 1.5 M DMSO was used as the cryoprotcctant. Six months later, the patient contacted us via the Internet. After eight weeks of Internet and telephone consultations, the tissues were shipped on a plane in a dry-shipper. Preoperatively, on 0.75 mg of transdermal estradiol, her FSH and LH levels were l-3 mIU/mL. One of the ovarian pieces was thawed and half of it was serially sectioned. The other half was cultured for 1 week, and hormone production was measured daily. Histology revealed few I-2-layer follicles. 50% of the stroma appeared to be healthy. Ovarian pieces produced increasing amounts of E,, (baseline vs. inculture: 21.6? 1.2 pg/mL vs. 222.6233.0, P
FERTILITY

& STERILITY@

gonadotropins. After 11 days of stimulation, a dominant follicle emerged in the ovarian graft and hormone replacement was discontinued. Post-stimulation T levels were significantly higher than the pre-stimulation levels (18.323 na/dL vs. 35.622, P=O.O002). Serial ultrasound exams demonstrated cominual follicle growth. The E, levels peaked at 93 pg/mL and 10,000 IU of hCG was given when the largest follicle diameter reached 16.5 mm. Thirty-six hours after the hCG injection, serum P4 levels rose from 0.7kO.06 ng/mL to 1.9 ng/mL, on the day of this report. Conclusions: We have reported follicle development after the first case of laparoscopic auto-transplantation of frozen-banked human ovarian tissue. This procedure promises a new alternative for reproductive age women who are facing loss of ovarian function due to chemo/radiotherapy, surgery or age-related factors.

Monday,

September

2:30

27, 1999

P.M.

O-055 Detection of Paternal Mitochondrial DNA (mtDNA) in Normal, Multicellular Human Embryos. i?l.C. St. John, ‘.2,3D. Sakkas, 3K. Dimitriadi, 4A.M. Barnes, ‘,2.3C.L.R. Barratt, 4,5C.J. De Jonge. ‘Assisted Conception Unit, 2Department of Medicine, 3Department of Obstetrics & Gynaecology, Birmingham Women’s Hospital, Birmingham, UK. 4Center for Reproductive Medicine, University of Nebraska Medical Center, Omaha, NE. ‘The Reproductive Medicine Center, Department of Obstetrics & Gynecology and Women’s Health, University of Minnesota, Minneapolis, Minnesota. Objectives: It is generally agreed that mtDNA is maternally inherited in humans. In interspecific crosses of mice, paternal transmission has been detected in the offspring. In contrast, in intraspecific crosses, paternal mtDNA is eliminated at the pronuclear stage in mice and by the 8 cell stage in bovine. The use of assisted reproductive technologies (ART) to produce human offspring reopens the dispute of whether human mtDNA is strictly maternally inherited. Two studies have reported that paternal mtDNA is not present in babies produced by ICSI. However, the detection techniques used in those investigations lacked sufficient sensitivity. Using a highly sensitive, paternal-specific nested PCR technique, we recently reported the presence of paternal mtDNA in 16-cell stage abnormal human (3PN) embryos generated by ART. It can be argued that detection may have resulted due to embryo abnormality. This study was conducted to determine if paternal mtDNA is detectable in normal human embryos produced by ART. Design: mtDNA isolated from embryos at various stages of development was analysed with a PCR technique designed specifically to detect paternal molecules to determine if leakage of paternal mtDNA occurs in humans. Materials and Methods: Couples (n=5) seeking treatment at the Center for Reproductive Medicine donated embryos for research (University of Nebraska IRB approved). To eliminate extraneous paternal mtDNA (zonabound sperm), acidified tyrode’s solution was used to solubilize zonae. Zona-free embryos were washed in medium and stored at -20°C until PCR analysis. For each couple, the D-loop and hypervariable region 1 of the human mtDNA genome from sperm and embryo were amplified using nested PCR. Products of nested PCR amplification were sequenced to identify heteroplasmic restriction sites. Products of the first round of PCR were digested with a maternal-specific restriction enzyme. Subsequent second round PCR used a paternal-specific primer to identify paternal product. The products from paternal specific amplification were resolved on 4% agarose gels. Paternal products were confirmed by restriction enzyme digest. Results: For each couple (n=5), molecules of paternal mtDNA were detected in embryos from the 2-6 cell stage. However, for 3 couples paternal molecules were also detected in g-cell stage embryos. Conclusions: This study demonstrates the presence of paternal mtDNA molecules in normal human embryos. Previously, it was shown that sperm mitochondria are eliminated by the g-cell stage. However, our results demonstrate the presence of paternal mfDNA up to the g-cell stage. Based on our previous study using polypronuclear embryos, we can predict that human mtDNA will persist in >8-cell stage normal embryos, and we are currently testing this hypothesis. Many maternally inherited mtDNA disorders are life-threatening and fatal. The results herein stimulate the question of whether paternal mtDNA transmission might lead to, as yet, undiscovered health disorders. Funded privately.

s21

Monday,

September 245 P.M.

27, 1999

O-056 Anti-Flkl Antibodies Block Corpus Luteum Formation in Immature Mouse Treated with PMSG/bCG. ‘,‘R.C. Zimmerman, ‘T. Vo, 3L. Leung, 4P. Bohlen, ‘M.V. Sauer. ‘Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and ‘Department of Psychiatry, Columbia University, New York, New York, ‘University of Southern California, Los Angelos, California, 41mClone, New York, New York. Objectives: Adult mammalian angiogenesis occurs predominantly in female reproductive organs: ovary and uterus. Abnormal regulation of angiogenesis might contribute to pathophysiologic conditions encountered in obstetrics and gynecology: Ovarian hyperstimulation syndrome, luteal phase defects, dysfunctional uterine bleeding, implantation failures, and gynecological cancers. To develop new treatment modalities for these illnesses a complete understanding of the regulation of angiogenesis in reproductive organs is prerequisite. Ferrara et al. (Nature Medicine 4:336-340, 1998) demonstrated that blocking substances against VEGF interfere with functioning of endothelial specific receptors Flk and Fltl, and block corpus luteum formation. The aim of this study was to expand on these findings by studying the effect of anti-Flkl antibodies on corpus luteum formation. Design: Randomized, placebo controlled study. Material and Methods: Immature (22 day old) female Swiss Webster mice were divided into 3 study groups (10 animals per group): control group 1 (Cl): no treatment; control group 2 (C2): intraperitoneal injection (ipi) of rat IgG (800 pglanimal) q 2 days (d) starting on day 22 of life; treatment group: ipi of rat anti-Flkl antibody (800 pg/animal) (ImClone, New York, NY) q 2 d starting on day 22. Follicular development and ovulation were induced with PMSG (d25) and hCG (d27) in C2 and treatment group. All animals were sacrificed on day 30 of life. Outcome measures included ovarian weight, number of corpora lutea per largest cross section and histological appearance using eosin hematoxilyn staining, and immunohistochemistry using the blood vessel specific antigene PECAM (PharMingen, San Diego, CA) together with the VECTASTAIN ABC kit (Vector Laboratories, Burlington, Ca). Analysis of variance was used for statistical evaluation with significance of p
Monday,

September 3:00 P.M.

27,

1999

O-057 Localization of the Prolactin Receptor in Lutenized Human Granulosa Cells. N.P. Vlahos, EM. Bugg, M.j. Shamblott, J.Y. Phelps, J.E. Garcia, J.D. Gearhart, H.A. Zacur. Division of Reproductive Endocrinology the Johns Hopkins Medical Institutions, Baltimore, Maryland. Objectives: Pituitary prolactin is a 198 amino acid protein primarily involved in the process of lactation. Prolactin is also produced in a variety of extrapituitary tissues such as the myometrium, endometrium, cells of the immune system and the mammary gland and is present in the follicular fluid of antral and mature oocytes. Prolactin receptors are also present in the above tissues as well as the ovary, indicating a paracrine or autocrine effect.

s22

Abstracts

In the rat and mouse ovary, prolactin receptors have been localized in the follicular granulosa cells. In the human, however, the exact localization of the prolactin receptor within the follicular apparatus is unknown. The purpose of this study is to investigate the presence of prolactin receptors in human luteinized granulosa cells. Design: Immunohistochemical staining of human luteinized granulosa cells obtained at the time of oocyte retrieval. Material and Methods: We obtained human granulosa cells at the time of follicular aspiration from women undergoing controlled ovarian hyperstimulation and follicular aspiration for IVF at the Johns Hopkins Hospital Fertility Center. We retrieved the follicular cells from the aspirate under direct vision with an inverted microscope. Following serial washings with Phosphate Buffered Saline (PBS) and centrifugation to remove the red blood cells, the granulosa cells were embedded in 1% low melting point agarose and pipetted into molds. Solidified molds were fixed in 10% buffered formalin and embedded in paraffin. Six-micron paraffin block sections were stained using an antibody specific for the human prolactin receptor. Additionally subsequent sections from each block were stained with human common leucocyte antigen (CLA) to evaluate the presence of white blood cells that also express prolactin receptors. Sections stained with secondary antibody alone were used as negative controls. Results: The presence of prolactin receptor was confirmed by immunohistochemical localization, within the cytoplasm and cell membrane of luteinized granulosa cells. The absence of CLA staining excluded significant white blood cell contamination. Conclusion: Immunohistochemical staining identified prolactin receptors in human luteinized granulosa cells. Our findings in association with the presence of prolactin in the follicular fluid, provide suggestive evidence that prolactin may play a paracrine or autocrine role in the follicular physiology. Final identification of prolactin receptor mRNA expression from these cells by RT-PCR is underway to further support our findings.

SOCIETY

FOR MALE REPRODUCTION ANDUROLOGY

Monday,

September 2:00 P.M.

27, 1999

0-0.58 Sperm Cellular Maturity and The Treatment Choice of IVF or ICSI: The Contributions of Sperm Creatine Kinase M Isoform (CK-M) Ratio. A. Dokras, J. L. Giraldo, A. Habana, T. Erel, G. Huszar. Section of Reproductive Medicine and Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT. Objective: The presence of CK-M in human spermatozoa indicates the completion of the events of spermiogenetic maturation, including cytoplasmic extrusion, sperm plasma membrane remodeling and development of the sperm-zona binding site. These properties facilitate oocyte fertilization and hence, the presence of CK-M in sperm reflects their fertilizing ability. The objectives were (1) to determine if sperm CK-M ratio in male partners of infertility patients, irrespective of etiology are predictive for IVF fertilization and occurrence of pregnancies (2) to evaluate the role of sperm maturity determinations in the current era when male infertility is increasingly treated with ICSI without adequate sperm assessment. Design: Retrospective study of 194 patients treated with IVF. Materials and Methods: All IVF cycles performed between Jan97-June98 were included except when ICSI was performed. The couples were assigned according to their sperm CK-M ratio [%CK-M/ (CK-M+CK-B)] of the male partner into 2 groups: lO%M. Parameters compared included sperm concentration and motility, CK-M ratio and activity, oocytes retrieved, fertilization rate, occurrence of pregnancy and associated female data including womens age, cause of infertility, day 3 FSH levels and ampules of gonadotropins used. Statistical analysis was performed by T-test and Wilcoxan ranked test. Results: There were 15 men in the < lO%M group and 179 men in > lO%M group (median sperm concentrations: 22.7 X 106/ml versus 62.3 X 106/ml, plO%M group (p20 X lo6 sperm/ml), thus had unexplained male

Vol.

72, No.

3, Suppl.

1, September

1999

infertility, whereas in the >lO%M group 13 of 179 men (7.2%) were oligospermic. The percentage of oocytes fertilized per patient in the lO%M group (50% versus 80%, plO%M. None of the associated female factors investigated showed differences in the two patient groups. When we excluded factors that may affect oocyte quality such as age >40 years of FSH>l2, the differences in fertilization and pregnancy rates in the two groups remained unchanged. Conclusion: Irrespective of female factors, the sperm CK-M ratio predicts failure of IVF cycles. The present data suggests, in line with our previous study (Huszar et al, F&S 57:882, 1992), that both oligospermic and normospermic men with less than 10% sperm CK-M ratio should be treated with ICSI. Monday,

September

2:15

27, 1999

P.M.

O-059 Sperm Morphology is an Important Prognostic Factor in Intrauterine Insemination Cycles. C. Sonmez, K. Ozgur, G. Abban, M. Uner, 0. Erman. IVF Unit, Department of Obstetrics and Gynecology, Akdeniz University Medical School, Antalya, Turkey. Objective: In recent years sperm morphology assessed by Kruger’s strict criteria has been reported to be a very important prognostic factor in in-vitro fertilization (IVF) cycles in various centers and this classification system has been used extensively worldwide. It is widely accepted that teratozoospermia has a poor prognostic value in patients with severe oligoasthenozoospermia in controlled ovarian hyperstimulation and intrauterine insemination (COH+IUI) cycles. However, in isolated teratozoospermic patients without concentration and motility problems contradictory reports are present. The aim of this study is to evaluate the role of sperm morphology on COH+IUI success in patients with normal motility and concentration. Design: Prospective cohort study in a university based infertility unit. Materials and Methods: Inclusion criteriae were determined as follows: female age less than 40, day 3 serum FSH < 10 IU/L, at least one patent tube at hysterosalpingography or laparoscopy, sperm concentration of at least 20 million/ml and motility of at least 50%, presence of at least 10 million progressive motile sperm after swim-up. For ovarian stimulation human menopausal gonadotropin and/or pure follicle stimulating hormone were used. Sperm morphology assessment by Kruger’s strict criteria was performed with the semen used for the insemination. All women were inseminated with 10 million progressive motile spermatozoa of their husbands. Main outcome measure was the presence of pregnancy. Receiver operating characteristic (ROC) curve was constructed for assessment of effectiveness of sperm morphology in predicting pregnancy. Student’s t or Mann-Whitney U test and Chi-square or Fisher’s exact test were used for statistical analysis where appropriate. Results: One hundred and sixteen treatment cycles were performed on 88 couples. Twenty one pregnancies were achieved (pregnancy rate per cycle 18.1%). ROC curve analysis demonstrated two distinct cut-off values of normal sperm morphology percentage. The pregnancy rate per cycle was 3.8% (l/26) and 26.3% (20/76) in patients whose normal sperm morphology percentage was less than 2 and between 2-7 respectively (p=O.Ol). Interestingly no pregnancies were achieved in 14 cycles in which the normal sperm morphology percentage was greater than 7. This was significantly lower as compared to 2-7 group (p=O.O3). Conclusion: This prospective study suggests that sperm morphology assessed by Kruger’s strict criteria has prognostic significance on COH+IUI outcome in patients with normal sperm concentration and motility. Severe teratozoospermia (~2%) as well as normal sperm morphology (>7%) may have a negative impact on COH+IUI success. The patients with teratozoospermia, but not severe, (2-7%) may be the ones that will benefit from the increase of gamete density at the fertilization site which is the rationale of the COH+IUI treatment. Monday,

September

2:30

27, 1999

P.M.

O-060 Detection by Electron Microscopy of Sperm Tail Axonemal Abnormalities in Infertile Men. ‘,‘N. Bar-Chama, 3M. Goldstein, *A. Copperman,

FERTILITY

& STERILITY@

*T. Mukherjee, ‘W. Hassan, 3R. Gordon. ‘Department of Urology, *Department of Obstetrics and Gynecology, ‘Department of Pathology, The Mount Sinai School of Medicine, New York, NY. Objectives: Electron microscopy (EM) is unique in its ability to accurately assess axonemal abnormalities (AA) of the sperm tail. As a result of multiple genetic defects, a broad spectrum of AA exist, with variable effects on sperm motility. Furthermore, axonemal microtubule integrity an extension of the sperm centrosome apparatus, may play a critical role in human fertilization. In this study we reevaluated the indication for EM to assess for sperm tail defects in the work-up of the infertile male. Design: Retrospective review of 39 infertile men who underwent a semen analysis (SA) as part of an infertility evaluation. EM studies were performed on men with an abnormality noted on SA. Material and Methods: Semen samples were evaluated for volume, concentration, and motility using World Health Organization criteria. Kruger strict morphology was manually performed according to standard protocol. Normal tails were described as uncoiled, uniform, slightly thinner than the midpiece, and measuring approximately 45 pm long. For EM, centrifuged sperm were fixed in a 0.2M cacodylate buffered 2% gluteraldehyde solution. Ultra thin sections (50-60 nm) stained with uranyl acetate and 2% lead citrate were examined and photographed using a JEM 1lCX transmission electron microscope. Statistical analysis was performed using the student t test and Chi square analysis. Results: Of the 39 men studied 9 demonstrated abnormal axonemal structure (AAS) on EM studies (Group 1). Five men demonstrated absence of the central microtubule pair in at least 25% of the sperm and 4 men demonstrated incomplete and degraded axonemal structure in the majority of sperm. The remaining 30 men demonstrated normal axonemal structure (NAS) (Group 2). Total sperm concentration, motility, and overall normal Kruger morphology did not differ between the two groups. The percentage of sperm with tail defects was significantly higher in those men with AAS (36% +/- 30.2) as compared to those with NAS (9.0% +/- 8.5) (p<.OOl). AAS was noted in 7/10 men with > 15% tail defects as compared to only 2/29 men with 515% tail defects (p<.OOl). Conclusions: The high incidence (23%) of infertile men with abnormal axonemal structure (AAS) noted in this preliminary study suggests that EM is informative not only in men with classic immotile cilia syndrome. Infertile men with a Kruger morphology revealing > 15% tail defects may benefit from an EM study to identify unrecognized axonemal defects in the sperm tail. The implication of AAS on assisted reproductive technology outcomes is unclear and warrants further investigation.

Monday,

September

2:45

27, 1999

P.M.

SMRU Prize Paper O-061 Reflexes and Somatic Responses as Predictors of Ejaculation by Penile Vibratory Stimulation (PVS) in Spinal Cord Injured (SCI) men. ‘V. G. Bird, ‘**C. M. Lynne, *T. C. Aballa, *S. M. Ferrell, ‘,‘N. L. Brackett. ‘Department of Urology, ‘The Miami Project to Cure Paralysis, University of Miami School of Medicine, Miami, Florida. Objectives: PVS has been used as a first line treatment for an ejaculation in men with SC1 due to its relative safety, effectiveness, low investment of time and money, and better semen quality compared to electrojaculation. A successful response to PVS is currently not predictable, although there is a relationship to level of injury (J Urol, 1998, 159: 1931-4). The objectives of this study were: (1) to determine if the presence of a bulbocavemosus reflex (BCR), and a hip flexion reflex (HR) were predictive of ejaculation with PVS of SC1 men, (2) to determine if the various somatic responses often observed during PVS could predict ejaculation in these men. Design: A retrospective chart review of all individuals undergoing PVS from 1995 to 1999 at our institution was performed. Included were patients with traumatic SC1 injured 2 years or longer, for whom BCR, HR, somatic response, and ejaculation data had been collected. Materials and Methods: Presence of BCR and HR before PVS, and somatic responses during PVS, were evaluated for their frequency of occurrence on trials with and without ejaculation in men with SCI. Somatic responses included: contraction of abdominal muscles, piloerection, with-

drawal response (bringing knees toward chest with thighs apart), extremity spasms, thigh abduction and thigh adduction. Results: Of all the patients in the study (n= 123), 59% ejaculated with PVS. 60% of all patients showed both BCR and HR. 80% of patients who had both BCR and HR ejaculated. Only 8% with neither reflex ejaculated. Both BCR and HR could be elicited in 73%. 60% and 49% of patients with levels of injury at C3-8, Tl-6, and T7-12, respectively. Of these, 78%, 94% and 67% respectively, ejaculated. One or more somatic responses were observed in 99% of all PVS trials (n=204). Abdominal contraction was the most frequent (94%), followed in frequency by extremity spasms (47%), thigh abduction (44%). piloerection (31%), thigh adduction (26%), and withdrawal (16%). To evaluate the usefulness of a response for predicting ejaculation, its % frequency on ejaculation trials was divided by its % frequency on non-ejaculation trials. The resulting dividends were: withdrawal=3.7, piloerection=2.2, extremity spasms=2.0, thigh abduction= 1.6, thigh adduction= 1.1, abdominal contractions= 1.1. To evaluate the usefulness of no response for predicting no ejaculation, % frequency of no response on trials with no ejaculation, was divided by % frequency of no response on trials with ejaculation. Dividends were: abdominal contractions=3.3, extremity spasms= 1.6, thigh abduction= 1.4, piloerection= 1.3, withdrawal= 1.3, adduction= 1.0. Conclusions: (1) Presence of BCR and HR prior to PVS were highly associated with successful ejaculation. It is especially important to note that 67% of patients with T7-T12 in whom HR and BCR were present had an ejaculation. (2) When present, a withdrawal response, piloerection, and extremity spasms were more useful than other somatic responses for predicting ejaculation. When absent, abdominal contractions, and extremity spasms were more useful than other responses for predicting no ejaculation. This project was supported by the Miami Project to Cure Paralysis and the State of Florida Specific Appropriations.

Seq.# # of Clones Exons: IIIS2 IVS3 Linker IVS3

21 22 31 32 33 34

1 14

2 10

3 7

4 6

5 2

6 22

+ + + +

+ + + +

+ + + +-+

+ +

+ -

+

+

++++--+ -------++--

3:00

P.M.

O-062 Variability in L-type Voltage Dependent Calcium Channel (L-VDCC) Sequence in Human Testis and Sperm. ‘L. 0. Goodwin, ‘N. B. Leeds, *D. S. Karabinus, ‘1. R. Hurley, ‘R. G. Pergolizzi, 3S. Benoff. Departments of ‘Research and 30bstetrics/Gynecology, North Shore University HospitalNYU School of Medicine, Manhasset, NY and ‘Department of Obstetrics/ Gynecology, The University of Arizona, Tucson, AZ. Objective: To examine which splicing events occur together in mRNA transcripts encoding L-VDCC (Y,,= subunits in human testis and sperm. Sperm mannose (Man) receptor (MR) binding to Man-containing zona ligands activates L-VDCC, inducing the acrosome reaction (AR). The consensus sequence of L-type VDCC oic pore-forming subunit expressed in human testis, compiled from 8 initial overlapping clones slkb, differed from cardiac muscle by alternative splicing of the 5’ end and transmembrane segments IS6, IIIS2, IVS3. Each clone encoded only one alternative splice site. Additional (13/84) clones expressed cardiac-specific exons (IS6, IIIS2, IVS3). Design: Pooled human testis RNA (n=20) and individual RNA isolates from ejaculated motile sperm of different normospermic donors (n=9) served as template in RT-PCR amplifications of ollc exons 20 through 35. Materials and Methods: PCR products from human testis mRNA template (Clontech) were cloned and sequenced. Total RNA was extracted from human sperm populations (HS#l-HS#9) using sodium dodecyl sulfate and citric acid followed by alcohol precipitation. PCR products generated from sperm RNA templates were directly sequenced. MacVector 5.0 program was used for sequence alignments. Sperm MR were quantified using FITCconjugated Man-BSA. Man-AR was assessed by RITC-Pisum sarivum labeling. Results: The results from analysis of 69 clones from human testis template appear below.

S24

Abstracts

8 1

9 1

10 1

11 1

12 2

13 1

-

-

+ + + -

+ +

+ + +

+ + + + +

+

Sequence 1 encodes the testis-specific rrIc isoform and sequence 2 is cardiac-specific. The remaining 11 sequences contained deletions and/or additions of the expected 1650 bp product. The most common deletion was in exon 33 which encodes the hinge between IVS3 and IVS4. IVS3 forms salt bridges with IVS4, the voltage sensor, to regulate channel opening and closing. Deletions in IVS4 were unexpected. Testis-specific L-VDCC exons 22 and 31 were amplified from all sperm RNA templates. Two cases (HS#l, HS#4) exhibited deletions in exons 33 and 34. When sperm from HS#6, having consensus testis-specific oic sequence, were compared with sperm from HS#l both exhibited capacitation-associated increases in MR expression equated with fecundity. However, HS#6 exhibited the expected 50% increase in Man-AR following agonist exposure and HS#l only displayed a 6% increase. Conclusions: L-VDCC o,c subunits are polymorphic in human testis and in mature human sperm from different men. Additions and deletions should alter L-VDCC function. Preliminary studies suggest that structure/function relationships exist and may contribute to inter-male differences in AR. (Support: Department of Research, NSUH; NIH Grant No. ES06100 to SB.)

Monday, September 27, 1999 3:45

Monday, September 27, 1999

7 1

P.M.

O-063 Fertility Outcome in Patients Undergoing Intrauterine Insemination After Varicocelectomy. ‘?I. A. Daitch, ‘*3E. B. Pasqualotto, ‘B. N. Hendin, ‘,‘A. J. Thomas, ‘,3T. Falcone, 4D. R. Nelson, ‘.‘A. Agarwal. ‘Center for Advanced Research in Human Reproduction & Infertility, Departments of ‘Urology, 3Gynecology-Obstetrics, and 4Biostatistics and Epidemiology, The Cleveland Clinic Foundation, Cleveland, OH. Objectives: Approximately 30% to 40% of men who are evaluated at an infertility clinic are diagnosed with a varicocele as a possible cause of their infertility. Surgical repair of varicoceles allows approximately 40% to 50% of these men to father children through natural intercourse. At least fifty percent of men remain infertile despite surgical varicocele repair. Some of these men and their partners will undergo intrauterine insemination (IUI) in order to initiate a pregnancy. The objective of our study was to compare the pregnancy rate for couples undergoing intrauterine insemination after varicocele obliteration to the rate of pregnancy for couples in whom the men have untreated varicoceles. Design: Retrospective study. Material and Methods: We reviewed the medical records of patients with varicocele who underwent IUI treatment at the Cleveland Clinic Foundation from 1993 to 1997. Patients were divided into two groups: Group I-patients who underwent varicocele obliteration (n=24), and Group II-patients with varicocele which were not treated (n= 15). Both groups underwent IUI after failing to achieve pregnancy with natural intercourse. Patients in Group I underwent 80 cycles, and those in Group II, 48 cycles of IUI. The relationships between patient characteristics and sperm quality to IUI outcome (pregnancy rate and live birth rate per cycle) were evaluated. Results: There was no difference in either group regarding the male and female ages. Sperm concentration and morphology were similar in both groups. The pre-wash percent motile sperm (50.33 2 2.61 vs 37.64 -C 1.91, p=O.O06) and post-wash total motile sperm counts (15.9 t 3.24 vs 5.66 ? 0.68, p = 0.02) were significantly higher in Group II compared to Group I. Even though untreated varicocele patients had higher sperm motility characteristics, the per cycle pregnancy rates (PR) and live birth rates (LBR) rates were significantly higher in patients whose varicoceles were obliterated prior to IUI than in untreated patients (PR = 11.3% vs 4.2%, p = 0.007; LBR = 11.3% vs 2.1%, p = 0.02).

Vol. 72, No. 3, Suppl. 1, September 1999

Conclusions: While varicocele obliteration may not improve semen parameters in all men, among couples undergoing IUI, varicocele obliteration improves both pregnancy rates and live birth rates. A thorough evaluation of all men is therefore recommended prior to initiating IUI.

Monday,

September 4:00 P.M.

27, 1999

O-064 Increased Testicular Cadmium (Cd2+) Levels Contribute to Infertility with Varicocele. ‘S. Benoff, ‘I. R. Hurley, ‘A. Jacob, ‘G. W. Cooper, ‘B. Napolitano, 3B. R. Gilbert, 4J. L. Marmar. Departments of ‘Obstetrics/ Gynecology, ‘Research and 3Surgery, North Shore University Hospital, Manhasset, NY and 4Division of Urology, Robert Wood Johnson Medical School, Camden, NJ. Objectives: To determine whether [l] intratesticular Cd2+ concentrations are increased in varicocele-associated infertility (VAI), and [2] a relationship exists between testicular Cd2+ and sperm defects. Prior data indicated that men with VA1 exhibit an acrosome reaction (AR) insufficiency and can be divided into three groups based on seminal plasma (SP) Cd2+ levels: Group I (CO.4 /.@L), Group II (>0.4 but ~0.7 Kg/L), Group III (>0.7 kg/L). After varicocele repair, pregnancy by coitus occurred only in Group I and II patients. SP Cd2+ inversely correlated with actin in heads of sperm from men with VAI. Actin polymerization and depolymerization is required for AR. VA1 defects can be mimicked by exposure of fertile donor sperm to high exogenous Cd2+ and are exacerbated with increased temperature. An intact actin cytoskeleton inhibits apoptosis in somatic cells. Design: Cd2+ concentrations in SP and testis biopsies from non-smokers (fertile men with varicocele, men with VA1 and men without varicocele) were assessed. Testicular Cd2+ levels were correlated with expression of actin and apoptosis. Materials and Methods: Varicocele was documented by Doppler flow ultrasound. Testicular tissues (200-300 mg) were obtained by percutaneous needle aspiration biopsy. The Johnsen scoring system (JSS) was used to assess spennatogenesis in H&E stained biopsy sections. After conversion of metals to sulfides, relative amounts of H,O,-soluble Cd2+-specific precipitates were assessed by autometallography (AMG). Cd2+ levels were quantified by graphite furnace atomic absorption spectroscopy (GFAAS). The distribution of actin in testis biopsies was determined by indirect immunofiuorescence cytochemistry. Apoptosis was quantified in situ by terminal deoxynucleotidyl transferase labeling (TUNEL) assay. All assays were performed with the same biopsy specimens. Data were analyzed with two-sample or paired t-tests. Results: SP Cd2+ in fertile men (n = 4; 0.348 % 0.159 FgiL)) with varicocele was similar to that of fertile men without varicocele (n=68; P=O.3, NS) and Group I VA1 men (n=2; P=O.8, NS) but differed significantly from that of Group II or Group III patients (n= 12, P
FERTILITY

& STERILITY@

Monday,

September 4:15 P.M.

27, 1999

O-065 Prostatitis (NIH Category III) is Associated With Seminal Oxidative Stress. F. F. Pasqualotto, R. K. Sharma, J. M. Potts, ‘D. R. Nelson, A. J. Thomas, Jr., A. Agarwal. Center for Advanced Research in Human Reproduction & Infertility, Departments of Urology, ‘Biostatistics & Epidemiology, The Cleveland Clinic Foundation, Cleveland, OH. Objectives: An association between prostatitis and male subfertility has been suspected, yet poorly understood. Although the clinical significance of leukocytic infiltration in semen is controversial, there is substantial evidence that granulocytes are the major source of reactive oxygen species (ROS), which has been proven to be deleterious to male fertility. The initial steps in proving an association between prostatitis and male infertility requires better understanding of the relationship between clinical prostatitis and oxidative stress. Therefore, we compared ROS, total antioxidant capacity (TAC) levels, and a novel index of oxidative stress (ROS-TAC score) in patients with chronic prostatitis (CP), and normal healthy men. Design: Prospective study. Materials and Methods: Semen specimens from 36 subfertile men with CP (NIH category III,), 8 men with prostatodynia (NIH category III,) and 19 donors attending our urologic clinic were examined according to the World Health Organization (WHO) criteria. Leukocytospermia levels were measured by the Endtz test (myeloperoxidase assay). White blood cell levels 2 1 X 106/mL white blood cells were considered positive for leukocytospermia. ROS production was measured by the chemiluminescence assay using luminol as a probe. Total antioxidant capacity (TAC) was measured in the seminal plasma by an enhanced chemiluminescence reaction and expressed as molar Trolox equivalents. ROS-TAC score was formulated to predict oxidative stress in these men. Receiver operating characteristics (ROC) curves were used to examine the diagnostic ability of the ROS, TAC and ROS-TAC score to predict the diagnosis of chronic prostatitis. Results: There were no differences in sperm characteristics (concentration, percentage motility, and morphology) between patients with or without leukocytospermia when compared to the control group. ROS levels were significantly higher in patients with leukocytospermia (3.2 + 0.6) compared to patients without leukocytospermia (1.8 2 0.2) (p = 0.04) and the controls (1.3 + 0.3) (p = 0.01). TAC levels were significantly depressed in patients irrespective of the leukocytospermia status (859.69 2 193.0 and 914.9 ? 65.2), compared to the control group (1653.98 t 93.6, p = 0.001). ROSTAC score differed significantly among donors (50.0 2 4.1), patients with leukocytospermia (9.2 2 9.2) and patients without leukocytospermia (34.2 2 2.9) (P
THE SOCIETY FOR REPRODUCTIVE ENDOCRINOLOGY AND INFERTILITY Monday,

September 2:00 P.M.

27, 1999

O-066 Ovarian Responses to Recombinant Follicle-Stimulating Hormone (FSH) With and Without Luteinizing Hormone (LH) in Ovulatory Women Undergoing Superovulation. A. P. Cheung. Department of Obstetrics/Gynecology, University of Alberta, Edmonton, AB and University of British Columbia, BC. Current address: University of British Columbia, Vancouver, BC, Canada. Objective: To contrast the effects of recombinant FSH (rFSH) and the added effects of recombinant LH (rLH) in a 1: 1 ratio on ovarian secretion of 17cu-hydroxyprogesterone (170HP), androstenedione (Ad), testosterone

s25

(T), estradiol (E2) and progesterone (P) in ovulatory women undergoing superovulation and intrauterine insemination for unexplained infertility. Design: Prospective, controlled study in a University-based fertility centre. Materials and Methods: Forty-eight ovulatory women with unexplained infertility received daily injections of rFSH or rFSH+LH until the day of hCG administration commencing 1) on day 2-3 of menses; 2) 6 weeks of GnRH agonist treatment (nafarelin, 400 pg b.i.d.); or 3) nafarelin in (2) + daily dexamethasone (commencing 4 days before gonadotropin stimulation). After the initial 5-day fixed gonadotropin regimens, the dosage was adjusted according to serum E, levels and follicle size and number on transvaginal ultrasound assessment. hCG was given ~24 hours after the last gonadotropin dose to trigger ovulation when the E, levels were <20002500 pg/ml and at least 1 leading follicle 215 mm (mean diameter) was seen on ultrasound examination. Between- and within-group differences in serum hormone levels at baseline and on the day of hCG were compared by multivariate analysis of variance (MANOVA). Significant overall effects were followed by univariate ANOVA of individual hormones. The Chi square test was used to compare the pregnancy rates between the two main gonadotropin groups. Results: Basal hormone levels were similar between the rFSH and rFSH+rLH groups but were different among the three treatment subgroups in each gonadotropin regimen. There were uniform increases in hormone levels on the day of hCG with higher values observed when LH was added (p
Monday,

September 2:15 P.M.

27, 1999

O-067 A Non Specific Inhibitor of Cytochrome P450 C17, and P450 Aro Improved ICSI Outcome in Insulin Resistant PCO. Hassan Aly Hassan (‘.3) D. El Guiziry (2,3), H. Azab (‘.3), A. Abdel Rahman (‘.‘), I. Bagdady (3), I. Ismail (31, ‘Department of Obstetrics & Gynecology Faculty of Medicine Alexandria University* Department of Clinical Pathology Faculty of Medicine Alexandria University.’ Alexandria IVF&ICSI center. Objective: CYPl la (encoding P45Oscc)‘]’ is the major candidate gene for abnormal steriodogenesis in PCO, yet down regulating CYP17 (encoding cytochrome P450 Cl7 lyase) could theoretically decrease androgen, although it was excluded as a major PC0 gene. “‘Thus molecular basis of hyperandrogenism associated with the characteristic ultrasound stromal hypertrophy 13) is more recognized. The relationship of androgens to insulin resistance (41, and the additional role of the insulin gene (5), underscores the therapeutic potential of anti androgens (6) The present work investigates the potential use of a non specific, non steroidal inhibitor of certain cytochrome P45Os (Ketoconazole) (7) on ICSI outcome of insulin resistant PCO. Design: prospective randomized study. Material and Methods: The study included a discrete group of 34 ICSI women, characterized by ultrasound PC0 and stromal hypertrophy, hyper androgenism, centripetal obesity and BMI ~35 as well as insulin resistance. All had a depot goserline long ICSI protocol and were randomly allocated into two groups; group I received 400mg of ketoconazole / day starting 2 weeks before and continued during suppression phase of GnRh agonist. Group II same agonist protocol without Ketoconazole. Results: *significant p
S26

Abstracts

suppression day

cyst formation

stimulation days

ketoconazole non kitoconazole

13.3-e3.9* 19.6+4.7*

5.9%* 35.3%*

10.6?2.1 12.823.2

groups

final E2

groups

ketoconazole non kitoconazole

9672172 874+201

ampoules required 41.6?10* 51*7.8*

oocytes

fertilization

pregnancy

8.822.1 6.2tl.l

78112%* 48t9%*

29.4% 5.9%

Conclusion: non specific aromatase inhibition could improve ICSI outcome in obese, hyperandrogenemic PC0 women with insulin resistance. Follicular sensitivity was enhanced both for suppression and stimulation. References (1)Gharani N., et al. Hum. Mol. Genet. 6:3972402, 1997. (2) Carey, A. H., et al. Hum. Mol. Genet. 3:1973-6, 1994 (3) Kyei, M., et al. Hum. Reprod. 13(6):1437-41, 1998. (4) Dale P.O., et al. Hum. Reprod. 13(3):567-70, 1998. (5) Water Worth et al. Lancet 349:986-9, 1997. (6) Dalgren E et al. Hum. Reprod 13(10):2706-11, 1998. (7) Barrie et al. Endocrine-Related Cancer 3:41-56. 1996.

Monday,

September 2:30 P.M.

27, 1999

O-068 Metformin Improves Lipid Profile in Hyperinsulinemic Women with Polycystic Ovary Syndrome. ‘B. Kolodziejczyk, ‘A. J. Duleba, ‘R. 2. Spaczynski, ‘L. Pawelczyk. ‘Division of Infertility & Reproductive Endocrinology, Department of Gynecology/Obstetrics, K. Marcinkowski University of Medical Sciences, Poznan, Poland and ‘Department of Obstetrics/ Gynecology, Yale University, New Haven, CT. Objectives: Polycystic ovary syndrome (PCOS) is typically characterized by hyperandrogenism, chronic anovulation, infertility, and insulin resistance with compensatory hyperinsulinemia. Long term sequelae of PCOS include adverse lipid profile: elevated total cholesterol, low density lipoproteins (LDL) and triglycerides, with concomitantly reduced high density lipoproteins (HDL). Insulin sensitizing medications such as metformin have been recently shown to be beneficial in the treatment of PCOS. This study was designed to evaluate the effect of metformin on lipid profile and insulin secretion in women with PCOS. Design: Prospective study assessing baseline and post-treatment levels of lipids, testosterone and fasting insulin. Materials and Methods: Forty three women (25.7kO.7 years of age; BMI=32.8+ l.Okg/m’) with PCOS and elevated fasting insulin levels (>17 PUlml) were treated with metformin (500 mg p.o., t.i.d.) for 12 weeks. Serum samples were collected at the baseline and after 12 weeks of treatment. Insulin, cholesterol, HDL, triglycerides, and glucose were measured by specific assays while LDL was calculated. Statistical analysis was performed using paired t-test. Results: Four of the 43 women became pregnant during metformin treatment and were excluded from the final data analysis. Results are summarized in the table below (mean’SEM): Variable

Before 1.02 26.7 217.0 138.7 48.6 159.2

Testosterone (rig/ml) Insulin-fasting (@J/ml) Total cholesterol (mg/dl) LDL (mg/dl) HDL (mg/dl) Triglycerides (mg/dl)

Treatment ? 2 ? 2 2 2

After

0.05 1.1 7.0 7.0 2.6 10.9

Treatment

0.7 16.3 200.0 124.0 51.7 127.8

i ? 5 i+ 2

0.04 1.0 5.9 5.0 2.2 8.6

p 5 0.001 0.001 0.005 0.05 NS 0.01

Conclusions: Metformin therapy significantly improved lipid profile in hyperinsulinemic women with PCOS. This effect may be due to a decrease of testosterone and/or insulin levels. This work was supported by grant no 4P05E 07512 form Polish State Committee for Scientific Research (KBN).

Vol.

72, No.

3, Suppl.

1, September

1999

Monday, September 27, 1999 2:45

P.M.

O-069 Congenital Rubella Syndrome is Associated with Polycystic Ovarian Syndrome: M. D. Keltz’, E. Perepelyuk’, B. Fedun’. Department of Obstetrics and Gynecology, Department of Pediatrics, St. Lukes-Roosevelt Hospital Center, Columbia College of Physicians and Surgeons, New York, NY. Objective: Congenital rubella has been associated with adult onset diabetes. Additionally, a high rate of symptoms associated with PCOS was noted in patients with congenital rubella cared for at our institution’s Developmental Disabilities Center. We undertook a study of the rate of PCOS, and its relationship with hyperinsulinemia, obesity and androgen excess in these patients. Design: Retrospective analysis of all women with congenital rubella syndrome followed at this institution. Materials and Methods: The charts of 60 female subjects with documented congenital rubella syndrome were reviewed. A combination of irregular menses and clinical or laboratory evidence of androgen excess was used to define PCOS. Hyperinsulinemia was defined by a fasting insulin greater than 33.3% of the fasting glucose. High LH/FSH ratios were defined as greater than a 2 to 1 ratio. Results: Out of 60 subjects with congenital rubella, 19 had PCOS (31.7%). Body mass indexes of subjects with and without PCOS were 31.2 ? 6.7 and 23.9 + 5.0 respectively (p< 0.001). 24 of 42 (57.1%) subjects tested were hyperinsulinemic, and those with PCOS were significantly more likely to be hyperinsulinemic, 14/16 (87.5%) versus lo/26 (38.5%) (p=O.O03). Likewise an elevated BMI was associated with hyperinsulinemia, 28.5 2 5.8 versus 24.1 -C 5.3 (p=O.O16). 24/60 (40.0%) subjects displayed clinical and or laboratory evidence of androgen excess. Among tested subjects, Hyperinsulinemia was associated with androgen excess in 17/20 (85.0%) versus 7/22 (31.8%) subjects without androgen excess (p
Monday, September 27, 1999 3:00

P.M.

O-070 Familiarity and Clinical Features in a Cohort of Patients with Idiopathic Premature Ovarian Failure. ‘P. G. Crosignani, ‘W. Vegetti, zG. Testa, ‘A. Nicolosi, ‘T. Motta, ‘L. De Lauretis, 3M. G. Tibiletti, ‘M. Taborelli, ‘P. F. Bolis, 4A. Marozzi, 4L. Dalpra. ‘I Department of Obstetrics and Gynecology, University of Milan, ‘Obstetrics and Gynecology Unit, Department of Biology and Clinical Science, University of lnsubria, Varese, 3Department of Biology and Clinical Science, University of lnsubria, Ospedale di Circolo, Varese, 4Department of Biology and Genetics for Medical Science, University of Milan, Italy. Objectives: Secondary amenorrhea with elevated gonadotropins occurring under the age of 40, (premature ovarian failure=POF), and at the age between 41 and 44 years (early menopause=EM) respectively affects l-2% and 5% of women in the general population. Familial association for these conditions has been suggested but the prevalence and the pattern of inheritance are still unknown. Objective of this study was to evaluate the prevalence of familial cases of POF and EM and to assess the clinical and genetic characteristics of these patients. Design: Cohort study of 177 patients with secondary idiopathic hypergonadotropic amenorrhea before the age of 45. Materials and Methods: 177 women with secondary amenorrhea before

FERTILITY

& STERILITY@

the age of 45 and serum FSH levels 240 IU/l were included in this study. Tests performed on patients included complete medical history, genetic counselling, clinical and ultrasound pelvic examination, gonadotropins and thyroid assessment, autoantibody detection and chromosomal analysis. Results: Twenty-seven subjects were excluded from the study due to presence of POF or EM related conditions. The remaining 150 patients showed idiopathic POF (n= 120) or EM (n=30). Following pedigree assessment,we were able to identify an incidence of familial cases of 30.0% in the POF group (n=36) and of 50% in the EM group (n= 15). POF and EM condition were often present in the same family. There were no differences between POF and EM patients and between familial and sporadic cases regarding age at menarche, personal history, gynecological history, weight, height and diet habits. There was a statistically significant difference between sporadic and familial cases in age at POF onset: 32.0 ? 7.7 years (12-40) compared to 34.9 ? 5.8 (18-40) respectively (p=O.O26). The POF and EM families identified showed two or more affected females and transmission through either maternal or paternal relatives; in 4 families both maternal and paternal transmission was observed. In families with paternal transmission, normal males transmitted the disease to their girls. A linkage analysis in POF and EM families is actually in progress and preliminary results on haplotype-sharing are in agreement with an X-linked mode of inheritance. Conclusions: This study suggests that idiopathic POF and EM conditions, differing only in age of menopause onset, may represent a variable expression of the same genetic disease. The different age of menopause onset in these patients may be explained by genetic heterogeneity and/or by different environmental factors. Our results indicate a high rate of familial transmission of the condition. Pedigrees and haplotype-sharing analysis suggests an X-linked dominant sex-limited pattern of inheritance for POF and EM.

Monday, September 27, 1999 3:45 SREI

Prize

P.M.

Paper

O-071 Coordinated Regulation of Endometrial Epithelial Apoptosis Induced by the Human Blastocyst as a Crucial Mechanism During Human Implantation. Galan A.‘, Herrer R.‘, Mercader A.‘, Meseguer M.‘, Remohi J.‘, Pellicer A.‘, O’Connor J. E.‘, Simon C.‘. ‘lnstituto Valenciano de lnfertilidad and Department of Pediatrics, Obstetrics and Gynecology and ‘Department of Biochemistry and Molecular Biology, Valencia University, Spain. Objective: The implanting blastocyst has to appose and adhere to the endometrial epithelium and subsequently invade it. Locally regulated uterine epithelial apoptosis induced by the embryo is a crucial step in the epithelial invasion in rodents (Mol Hum Rep 1998). To address the physiological relevance in humans, we have investigated the effect of single human blastocysts on the regulation of apoptosis in cultured human endometrial epithelial cells (hEEC) in the apposition and adhesion phases of implantation. Design: hEEC apoptosis induced by a blastocyst was detected and quantified by TUNEL, confocal microscopy and flow cytometry (FC) in the apposition phase. To study the adhesion phase, in vifro attachment assays assessed by double labeling (P-hCG and TUNEL) were used. Material and Methods: We have developed an in vim model to study interactions between the human embryo and hEEC (Biol Reprod 1996, JCEM 1997) during the implantation process. Individual human embryos were cocultured with hEEC (from day 2 until day 6) and those achieving the blastocyst stage were transferred back to the mother. To measure Annexin-V positive (apoptotic) cells, hEEC previously cultured with blastocysts (n= 13), hEEC with arrested embryos (n= 10) and hEEC without embryos (n=l5) were detached, stained with Annexin V-FITC and propidium iodide (PI), and analyzed by FC. According to their FC pattern, cells were classified as: alive=annexin V/PF; apoptotic=annexin V+/Pl dim; necrotic =annexin V+/Pl brigh’. In situ detection of apoptosis was performed by TdT-enzyme and FITC-dUTP counterstained with PI (n=5 in each category). Confocal microscopy was used to visualize TUNEL experiments. For the study of the adhesion phase an in vitro embryo attachment assay was used. Tridimensional cultures of polarized hEEC with stromal cells seeded beneath them (n=3) where cocultured with spare human blastocysts for 3

S27

days. Attached blastocysts were localized with an anti-B-hCG and apoptotic cells were detected with TUNEL and confocal microscopy. IRB approval was obtained for the use of spare blastocysts. Results: The presence of a human blastocyst induces a reduction of hEEC apoptotic cells (35.2%) compared to hEEC cultured without blastocyst (48.8%) (pcO.05). This embryonic effect occurs early in the apoptotic pathway (phosphatidylserine externalization) since TUNEL or Confocal Microscopy (DNA chopped) can not detect this effect. However, when the human blastocyst adheres to polarized hEEC, we found higher apoptosis occurring in the hEEC around the embryo attachment site compared to cells away from the blastocyst and control cultures. Conclusion: This work demonstrates a coordinated regulation of embryonic induction of hEEC apoptosis crucial for embryonic implantation. In the apposition phase, the presence of a blastocyst rescues hEEC from the apoptotic pathway keeping more hEEC apoptotic resistant. However, when the blastocyst adheres to the hEEC monolayer, it induces a paracrine apoptotic reaction that is maximal in hEEC in close contact with the blastocyst. This study suggests that the induction of hEEC apoptosis may be an important mechanism to invade the luminal epithelium, and the manipulation of this “kiss of death” could have potential clinical implications as an interceptive mechanism.

Monday,

September 4:00 P.M.

27,

1999

O-072 Retinoic Acid Treatments apy in an Experimental Bruner, N. R. Keller, E. search Center, Vanderbilt

Can Mimie the Effects of Progesterone TherModel of Endometriosis. K. G. Osteen, K. L. Eisenberg. Women’s Reproductive Health ReUniversity School of Medicine, Nashville, TN.

Objectives: Most practitioners agree that endometriosis can occur following ectopic attachment, invasion and growth of refluxed menstrual tissue. We previously demonstrated that progesterone (P.,) suppresses matrix metalloproteinase (MMP) expression, preventing establishment of human endometrial lesions in a model of endometriosis. However, regulation of endometrial MMP expression at ectopic sites may be more complex, requiring both gene suppression and blocking stimulatory factors such as interleukin-lcr (IL-la). We find that retinoic acid (RA) synthesis can be induced by P4 during in vitro decidualization and appears to be important for normal MMP regulation. The current study examined the interactive role of RA and P4 in regulating basal and IL-la-stimulated MMP-3 expression in vitro and determined the in viva therapeutic effects of P, or RA in experimental endometriosis. Design: Proliferative phase human endometrial tissue was acquired by biopsy from normal, cycling women. Organ cultures or isolated stromal cells were used to examine MMP-3 expression following P4 or RA treatments. Ectopic human endometrial lesions were established in nude mice to examine the in viva response to P., and RA therapy in experimental endometriosis. Material and Methods: The in vitro secretion of MMP-3 was determined by Western analysis. Ovariectomized nude mice received subcutaneous, slow-release estradiol pellets (1.5 mg; Innovative Research) prior to intraperitoneal injections of human endometrium to establish ectopic lesions, After IO days, mice received P4 (15 mg) or RA (10 mg) therapy via slow-release pellets. Animals were sacrificed after lo-20 days of therapy and the number and appearance of remaining ectopic lesions was determined. Morphological assessment, size analysis and MMP-3 expression (via in siru hybridization) was performed following formalin fixation. Results: Either P4 or RA treatment of endometrial organ cultures reduced basal secretion of MMP-3. In isolated stromal cells, P4 or RA treatment for 4 days suppressed MMP-3 secretion as well as blocked the ability of IL-la to stimulate secretion of this enzyme. In animals with experimental endometriosis, P4 or RA therapy reduced both the number and size of ectopic lesions. The P, therapy was most effective, reducing lesions by 10 days, although RA had a similar effect by 20 days. Finally, MMP-3 mRNA expression was decreased in lesions removed following P, or RA therapy compared to control animals receiving placebo treatment. Conclusions: The results indicate that P, induction of RA synthesis may be an important component of endometrial MMP regulation. Our expe$ mental endometriosis model suggests that P, and RA may act in concert at ectopic sites of endometrial growth to regulate MMPs, including blocking

S28

Abstracts

the stimulatoty action of local cytokines HD28 128 and #HD36707.

Monday,

such as IL-la.

September 4:15 P.M.

Supported

by NIH#

27, 1999

O-073 Regulation of Vascular Endothelial Growth Factor (VEGF) Gene Transcription by Estrogen Receptors “d’ and “p” in Human Endometrial Cells. M. D. Mueller, D. I. Lebovic, J.-L. Vigne, D. C. Leitman, R. N. Taylor. Reproductive Endocrinology Center, University of California, San Francisco, CA Objective: Our group and others have shown that estradiol increases the expression of VEGF mRNA and protein in human endometrial cells. To determine whether the transcription of the VEGF gene is altered by estrogen agonists or antagonists we developed a model system by transiently transfecting a human VEGF promoter-luciferase reporter construct into human endometrial adenocarcinoma cells. Design: In vitro study of the transcriptional regulation of the VEGF gene by estrogen agonists and antagonists. Materials and Methods: Ishikawa cells, derived from a well-differentiated human endometrial adenocarcinoma, were transiently transfected with hciferase fusion vectors containing 2.5 Kb of 5’ flanking DNA relative to the VEGF transcriptional start site by calcium phosphate co-precipitation or electroporation. The VEGF promoter was transfected alone or with an expression vector encoding either the a-form (ERo) or the p-form (ERB) of the human estrogen receptor. After recovery overnight the medium was changed to phenol-red free DME-H21 with 2.5% charcoal dextran filtered fetal bovine serum. Ethanolic vehicle (control), 17@estradiol, raloxifene, tamoxifen, ICI 182780 or phorbol-12,13-dibutyrate (12,13-PDB) were then added for 16-18 hours. The cells were lysed and luciferase activity measured and compared to the controls. Results: Neither 17P-estradiol nor the estrogen antagonists had an effect on luciferase activity in the absence of ERa or -B. 12,13-PDB showed a 5.9-fold increase, indicating successful transfection and a responsive VEGF promoter in these cells. In cells co-transfected with ERcr or ERB expression vectors, 17P-estradiol induced 3.6- and 3.0-fold increases in luciferase activity of the VEGF-construct, respectively. Treatment with ICI 182780, a pure estrogen antagonist, provoked 8.7- and 5.4.fold increases, respectively. Tamoxifen induced 3.2-fold increases when ERo was co-transfected, but only a 1.4-fold increase when ERP was used. Raloxifene induced 2.5-fold and 2.6fold increases, respectively. Conclusion: Our findings indicate that increased endometrial VEGF gene expression is mediated, in part, by ligand-bound ERL~ and ERP through sequences lying upstream from the transcriptional start site. Furthermore, VEGF transcription is activated by both estrogen agonists and antagonists. Estrogenic regulation of VEGF may play a key role in the neovascularization process of endometriosis, in dysfunctional uterine bleeding and may be an essential step in the development of endometrial polyps occuring after treatment with certain antiestrogens. Studies with rimary human endometrial cells are underway to confirm these findings. Supported by a fellowship from the Swiss National Science Foundation (MDM) and ROlHD33238 @NT).

Monday,

September 4:30 P.M.

27, 1999

O-074 Identification and Localization of Gonadotropin Releasing Hormone (GnRH) Receptor on Sperm Plasma Membrane by Specific Monoclonal Antibodies. ‘G. Lee, ‘A. Cheung, ‘E. Wong, ‘C. Schwab, *K. Yasojima, *P. McGeer. ‘Department of Obstetrics and Gynecology and *Department of Psychiatry, University of British Columbia, Vancouver, British Columbia, Canada. Objectives: Gonadotropin releasing hormone strated to enhance human sperm-zona binding ence of GnRH receptor on the sperm has never monoclonal antibodies against GnRH receptor probes to localize if GnRH receptor is indeed

Vol.

72, No.

(GnRH) has been demonin vitro. However, the presbeen confirmed. To this end, were generated and used as present on human sperm.

3, Suppl.

1, September

1999

Design: An improved hybridoma technique was used to generate monoclonal antibodies against four synthetic peptides derived from the extracellular domains of the GnRH receptor. Human pituitary extracts and ovarian cell lines were used as reference for comparison. Materials and Methods: Four peptides corresponding to residues l-29, 182-194,195-206 and 293-205 respectively of the GnRH receptor were custom-synthesized and conjugated to haemocyanin obtained from keyhole limpets. They were used as immunogens to raise antibody-producing hybrid cell lines by employing a proprietary procedure for cell fusion and hybridoma selection. Immunohistochemical procedures were then employed to localize GnRH receptor on human sperm, human pituitary tissue sections, OC3-VGH ovarian cancer cells. The molecular size of the GnRH receptor from each of the tissue extracts was determined by Western blot assay. Results: A total of 13 hybrid cell lines were identified by enzyme-linked immunosorbent assay to secrete monoclonal antibodies specific for the GnRH receptor. Among these monoclonal antibodies, GRX-3 and GRX-6 were shown to have high specificity and affinity for tissue sections of the pituitary gland and glutaraldehyde-fixed human sperm on immunohistochemical assays. In the case of human sperm, GnRH receptor was found to localize on the sperm acrosome with a high concentration on the equatorial/ postacrosomal region. On Western blot analysis, it was clearly demonstrated that the GnRH receptor of human sperm had a molecular weight of 45,000 t 5,000 daltons on sodium dodecyl sulfate polyacrylamide gels similar to those present on membrane extracts of human pituitary gland and OC3-VGH ovarian cancer cells. Conclusion: By employing specific monoclonal antibodies against synthetic peptides derived from extracellular domains of the GnRH receptor, we have confirmed for the first time, the existence of GnRH receptor on human sperm and localized a high receptor concentration in the acrosome region. The presence of human sperm GnRH receptor may be responsible for GnRH-induced enhancement of sperm-zona binding in vitro and likely in viva as well.

Monday, September 27, 1999 4:45

P.M.

O-075 Estrogen Deficiency, Obesity, and Skeletal Abnormalities in Follitropin (FSH) Receptor Knockout (FORKO) Female Mice. N. Danilovich, M. R. Sairam, P. S. Babu, W. Xing, M. Gerdes. Molecular Reproduction Research Laboratory, Clinical Research Institute of Montreal, Montreal, Quebec, Canada. Objectives: Follicle stimulating hormone acting through its receptor system(s) in granulosa cells of the ovary induces follicular development, steroidogenesis, and ovum maturation. Certain mutations of the receptor gene are known to be responsible for ovarian dysgenesis or testicular malfunction. In order to understand the molecular and physiological events underlying the loss of the receptor function, we have inactivated the mouse FSH receptor gene by molecular genetic techniques and analyzed me phenotypes. Design: Reproductive performance and structural changes were compared using wild type, heterozygote (+/-), and homozygote (-/-) mice of different ages. Materials and Methods: Homozygote (-/-) mutant mice were generated by crossing +/animals. Breeding patterns among various crosses were evaluated up to a year or more. Morphological and histological examination of the urogenital system including the vagina was performed. Body weight and adipose tissue mass were indicators of obesity. Skeletal changes were recorded by x-ray and femur weights. Marker genes were analyzed by the polymerase chain reaction and Western blotting using specific antibodies. Results: Heterozygote females showed reduced fertility and early reproductive senescence. Mutant females showed no estrous cycles and were completely sterile. Histological examination of the ovary revealed only primary and secondary follicles with no evidence of ovulation or corpora lutea. Uterus and vagina were completely atrophic. These were a consequence of low estrogen production due to failure of aromatase activation. The estrogen receptor system ((u and &?) in the mutant remains intact. Mutants became obese by 3-4 months of age. Skeletal deformity was also evident in the mutants at an early age and increased in severity. There was no significant effect on longevity of the mutants.

FERTILITY

& STERILITY”

Conclusion: Based on our observations we infer that lack of FSH receptor signaling in the mutants leads to a failure of the steroidogenic machinery in the ovary causing severe estrogen deficiency. As a consequence, signs and symptoms similar to menopausal conditions are induced in these animals. It is tempting to suggest that the mutants may serve to establish the structural and physiological changes associated with ovarian dysfunction. (Supported by MRC of Canada)

ASSISTED REPRODUCTIVE TECHNOLOGY BLASTOCYST DEVELOPMENT Tuesday, September 28, 1999 2:00

P.M.

O-076 Blastocyst Culture in Patients With Limited Numbers of Rapidly Cleaving Embryos. ‘M. Langley, ‘D. Marek, *D. K. Gardner, ‘N. Confer, ‘L. Cram, ‘L. Underwood, ‘K. M. Doody, ‘K. J. Doody. ‘Center for Assisted Reproduction, Bedford, TX and ‘Colorado Center for Reproductive Medicine, Englewood, CO. Objective: Previous studies of blastocyst culture have focussed on patients with high numbers of follicles, oocytes, or embryos. It is possible that limited numbers of rapidly cleaving embryos might affect the outcome of subsequent blastocyst culture. For this reason, currently many centers performing blastocyst culture elect to transfer at the cleavage stage when faced with few rapidly dividing embryos. The objective of this study is to examine blastocyst development, implantation and ongoing pregnancy rates resulting from cycles in which less than five eight cell embryos are present on Day 3. Design: A retrospective analysis of IVF and ICSI embryo transfer (ET) results from January 1, 1998 through December 31, 1998. Materials and Methods: Patients undergoing IVF were selected for blastocyst culture regardless of the number of Day 3 eight-cell embryos (D38~). Pronuclear embryos were cultured in 100 PL micro-drops S-l Media (IVF Science) in groups of two under oil for 48 hours followed by culture in 100 PL micro-drops S-2 Media for 48 to 72 hours to blastocyst stage (Day 5). Results: Age (Mean) Number

Day 3 8-cell:

Transfer Day ~/NO. Transfer Positive hCG/Ongoing BC/SAB/Ectopic Ongoing Pregnancy per ET No. Clinical Sacs (l/2/3) Implantation Rate Total/Average ET Total Blast ET D3-8c: Total/to Blast D3-8c ET: Total/Blast

Day 3 8-cell:

Transfer Day ~/NO. Transfer Positive hCG/Ongoing BCISABIEctopic Ongoing Pregnancy per ET No. Clinical Sacs (l/2/3) Implantation Rate Total/Average ET Total Blast ET D3-8c: Total/to Blast D3-8c ET: Total/Blast

(31.1)

l-2

34

14

65/O 46131 5/4/o 56.9% 2211112 39.4% 15712.4 142 85161 5 l/47

36/O 27119 2/5/l 52.8% 1 l/12/1 43.7% 8712.4 76 120/84 55/48

17/1* 1301 l/l/O 64.7% 51413 52.4% 4212.5 42 116169 27127

Age (Mean) Number

<35

35-39

(37.0)

l-2

34

>4

4711 31125 3/3/O 53.2% 201513 30.5% 12812.7 96 70149 46139

12/O 9/7 2/o/o 58.3% 21213 50.0% 3012.5 28 43136 25125

5/o 3/l l/l/O 20.0% l/O/l 28.6% 1412.8 14 36120 S/8

S29

Age (Mean) Number

Day 3 8-cell:

>39

Tuesday, September 28, 1999

(41.8)

2:30

l-2

3-4

>4

1 l/O 212 o/o/o 18.2% 2/o/o 5.9% 3413.1 17 1716 1217

2/o z/2 o/o/o 100.0% o/2/0 57.1% 713.5 7 716 515

2/o o/o o/o/o 0% o/o/o 0% 613 6 18/15 414

P.M.

O-078 Transfer Day S/No. Transfer Positive hCG/Ongoing BCISABiEctopic Ongoing Pregnancy per ET No. Clinical Sacs (l/2/3) Implantation Rate Total/Average ET Total Blast ET D3-8c: Total/to Blast D3-8c ET: Total/Blast * No transfer

performed

because of risk of hyperstimulation.

Conclusion: This data suggest that regardless of age or number of Day 3 8-cell embryos, ongoing pregnancy rate, implantation rate, and blastocysl development is consistent. In addition, only 45.9% (264/575) of all Day 5 embryos transferred resulted from Day 3 8-cell embryos, however, 84.8% (428/505) of all embryos transferred were blastocyst. This indicates blastocyst formation from multi-cell embryos with initially retarded development. Therefore, it can be suggested number of Day 3 8-cell embryos and age should not be the primary determinant for selection of patients for blastocyst transfer.

Tuesday,

September 2:15 P.M.

28, 1999

O-077 The Management of Patients With Randomized Prospective Trial. D. Nahum, H. Zakut, M. Glezerman. IVF Gynecology, Wolfson Medical Center, icine, Tel Aviv University, Israel.

Repeated Implantation Failure: A Levran, A. Weissman, J. Farhi, H. Unit, Department of Obstetrics and Holon, and Sackler Faculty of Med-

Objectives: Subjects with repeated implantation failure (RIF) after numerous IVF-ET attempts remain an extremely challenging patient group. We have recently demonstrated the beneficial therapeutic effect of zygote intrafallopian transfer (ZIFT) in patients with RIF (Fertil Steril 1998:69; 26-30). Uterine transfer of embryos at the blastocyst stage has also been suggested as a means for improving embryo implantation and pregnancy rates. The objective of our study was to compare between conventional embryo transfer (ET), blastocyst transfer (BT), and ZIFT in patients with RIF. Design: Prospective, randomized study. Materials and Methods: Eighty-three patients, who had a minimum of 4 previous failed conventional IVF-ET cycles were randomized to undergo ZIFT (n = 45; 7.7 2 3.1 previous cycles) or BT (n = 40; 8.3 ? 3.9 previous cycles). The control group consisted of 39 patients who had their fifth embryo transfer at our program and a variable number (8.2 2 5.3) of previous failed attempts (ET group). The three groups were compared for response to stimulation and cycle outcome. Results: The mean age of the patients was similar for all groups (34.8 ? 5.5 years). The number of oocytes retrieved was 8.7 2 6.0, 12.2 2 7.0, and 11.4 2 5.3 for the ET, ZIET, and BT groups, respectively (P<.O5 for ET vs. ZIFT and BT). Fertilization rate was similar in all groups (70 2 24%, 73 2 IS%, and 72 5 19%, for the ET, ZIm, and BT groups, respectively). A mean of 5.5 ? 0.9 pre-embryos were replaced in the ZIFT group, a mean of 2.1 + 1 .O blastocysts in the BT group (excluding 12 patients who had no blastocysts available for transfer) and a mean of 3.7 ? 1.9 embryos were transferred in the ET group. Clinical pregnancy rates were 12.8%. 38%, and 0% for the ET, ZIFT and BT groups, respectively (overall P<.OOOl, and ET vs. ZIFT P<.O5). No complications related to the ZIFT procedure occurred. Conclusions: Our study clearly indicates that BT is not effective in the management of IVF patients with RIF. Compared to uterine transfer, the ZIm procedure requires a higher degree of expertise and is not free from risks and complications. Nevertheless, in our experience, ZIFT remains a powerful clinical tool that should be offered to patients with RIF who are anatomically suitable for the procedure.

s30

Abstracts

Apparent Optimal Inner Cell Mass Size for Successful Blastocyst Transfer. B. S. Shapiro, D. C. Harris, K. S. Richter. Fertility Center of Las Vegas, Las Vegas, NV. Objective: The goal of this study was to determine the predictive value of the size of the inner cell mass (ICM) of a blastocyst on the probability of implantation following embryo transfer (ET). Design: A retrospective clinical study of 119 IVF patients receiving blastocyst transfers on day 5 or 6. Materials and Methods: The largest diameter of the inner cell mass (ICM) was recorded for each blastocyst with a visible ICM before transfer. All patients having at least one identifiable ICM among their transferred embryos were included in the study. The average ICM size of the cohort of embryos transferred for each patient was compared between pregnant and nonpregnant groups using an unpaired r-test. Implantation rates were based on the maximum number of fetal heart rates (FHRs) observed on ultrasound between 6 and 7 weeks gestational age. Multiple regression analysis was used to predict the number of FHRs as a function of the number of embryos transferred and the average ICM size of these embryos. The absolute value of the deviation from the mean ICM size of the pregnant group was calculated for each patient and compared between the pregnant and nonpregnant groups using a Mann-Whitney U test. For the 98 patients for whom ICMs were measured for every blastocyst transferred, blastocysts were sorted into three groups according to ICM size: small (less than 70 pm, n = 51); medium (70-95 pm, n = 110); or large (100 pm or greater, n = 89). The numbers of transferred embryos in these three groups were used as independent variables in a multiple regression analysis predicting the number of FHRs developing. Results: There was no difference in mean ICM size between the patients becoming pregnant and those that did not (83.8 Km versus 86.2 pm, P=.42). The number of transferred embryos (P<.OOOl), but not the average ICM size per patient (P= .56), was found to have a significant effect on the number of FHRs. Patients becoming pregnant were found to have an average ICM length that deviated from the mean less than the average ICM length of patients not becoming pregnant (median = 9.5 pm versus 12.9 pm, P= .04). All three ICM size groupings contributed significantly to the total number of embryos that subsequently implanted (P= ,024, P<.OOOl, P= ,002 for small, medium, and large, respectively). Estimates of implantation rates, base upon this multiple regression analysis, were 25% for the small ICM group, 49% for the medium size ICM group, and 23% for the large ICM group. Conclusion: There is no evidence of a linear relationship between ICM size and implantation rate. However, ICM size does appear to influence the probability of implantation. Patients who become pregnant have average blastocyst ICM sizes closer to the average size of 83.8 pm. Blastocysts with ICM lengths near the average of 83.8 &rn (range 70-95 pm) have estimated implantation rates of 49%, whereas estimates of implantation rates are much lower for blastocysts with smaller ICMs (25%) or larger ICMs (23%).

Tuesday, September 28, 1999 2~45

P.M.

O-079 Reduced Oxygen Tension Increases Blastocyst Development, tiation, and Viability. D. K. Gardner, M. Lane, J. Johnson, J. Stevens, W. B. Schoolcraft. Colorado Center for Reproductive Englewood, CO.

DifferenL. Wagley, Medicine,

Objectives: Within the female reproductive tract the preimplantation mammalian embryo is exposed to an oxygen (0,) tension of between 2% and 10%. Such levels of 0, are in stark contrast to the 20% 0, used routinely in the culture of human embryos conceived through IVF. The aim of this study was to determine the effect of 0, concentration on blastocyst development in the mouse and human. Furthermore, the effect of 0, concentration on mouse blastocyst differentiation and subsequent viability was determined.

Vol. 72, No. 3, Suppl. 1, September 1999

Design:

Embryo

development

was assessed after culture

in 5% and 20%

0,. Materials and Methods: Pronucleate embryos were obtained from outbred CFl mice, which were then cultured in either 5% or 20% 0, for 96 hours in sequential media. Blastocyst development, cell allocation to the inner cell mass (ICM) and trophectoderm (TE), and viability after transfer to recipient females were determined. Fifty-nine frozen-thawed human pronucleate embryos, donated for research, were cultured in either 5% or 20% oxygen for 4 days in the sequential media G 1.2 and 2.2. Embryo development was assessed after 48 and 96 hours. Results were analysed using linear-logistic regression. Results: Mouse embryo development: Oxygen Cont.

Blast (%I

Total Cell No.

KM

20% 5% P value

48.9 66.7 PC.05

57.5 2 2.2 15.3 2 2.2 PC.01

14.6 _’ 0.8 23.5 2 0.1 P<.OOl

Implantation (%I

ICM/Total (%) 20% 5% P value n = 286 embryos

25.4 2 0.8 30.8 r+_0.6 PC.01 cultured

Cell No.

TE Cell No. 42.3 -e 1.8 52.3 t 1.7 PC.05 Fetal Development (%)

30.95 64.29 PC.05

in 20% 0,, and 255 embryos

19.05 50 PC.05 cultured

September

390

28, 1999

P.M.

O-080 Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Promotes Human Blastocyst Development Independently of Culture Media and Source of Recombinant Cytokine. ‘C. Sjoblom, 2S. A. Robertson, iM. W&land. ‘Fertilitetscentrum, Goteborg, Sweden, ‘Department of Obstetrics and Gynaecology and Reproductive Medicine Unit, University of Adelaide, Adelaide SA, Australia. Objective: We have shown previously that addition of recombinant human (rh)GM-CSF to sequential human embryo culture media (IVF-50/ S2) enhances the rate and proportion of embryo development to blastocyst stage and beyond (1). The objective of the current study was to compare the effects on the development of human preimplantation embryos of a pharmaceutical preparation and laboratory-grade source of E. co&derived rhGM-CSF, in two alternative sequential culture systems. Materials and Methods: Frozen 2- to 4-cell embryos were thawed and cultured in 20-/J,L drops of sequential media. Embryos were transferred into fresh media every 48 hours. Culture was performed in IVF-50/S2 or G1.2/G2.2 media (both Scandinavian IVF science) alone, or with 2 ng/mL

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G1.2tG2.2 G1.21G2.2 G1.21G2.2 IVF-50/S2 IVF-50/S2 IVF-50/S2

alone + rhGM-CSF (R&D Systems) + Molgramostim alone + rhGM-CSF (R&D) + Molgramostim

No. B&n

% B$

‘7123 15121 1209 14/38 30/38 13120

30 71 63 31 79 65

P value (vs. media alone)

,007 ,035 .0002 .04

Conclusion: These results confirm that the proportion of embryos reaching blastocyst stage is dramatically improved by the addition of rhGM-CSF to culture media. The effect can be achieved with a pharmaceutical preparation of rhGM-CSF and is exerted irrespective of the type of culture media used. 1. Sjoblom C, Robertson S, Wikland M. Hum Reprod 1998;58:115.

ASSISTED REPRODUCTIVE TECHNOLOGY EMBRYO DEVELOPMENT

in 5% 0,.

Forty-two blastocysts cultured in each 0, concentration were transferred to recipient females. Human embryo development: on day 3 (44 hours post-thaw) cell numbers were 5.3 t 0.38 (20% 0,) vs. 6.7 f 0.31 (5% 0,; P<.Ol). On day 5 blastocyst development was 27% and 31% in 20% and 5% 0,. respectively. Blastocyst cell numbers were 34.5 ? 5.1 (20% 0,) vs. 51.3 f 5.2 (5% 0,; PC.05). Conclusions: Culturing mouse embryos in a reduced 0, concentration significantly increased blastocyst formation and blastocyst cell number. Analysis of cell allocation revealed that a reduced O2 concentration increased the development of the ICM and that this was associated with a significant increase in blastocyst implantation and fetal development. Although culturing human embryos in a 5% 0, environment did not result in an increase in blastocyst development, resultant blastocysts had significantly more cells than those blastocysts in the 20% 0, group. It is most plausible that this will translate clinically into higher implantation rates. Therefore, it is recommended that human embryos should be cultured to the blastocyst stage in a 5% 0, environment.

Tuesday,

rhGM-CSF (R&D Systems) or 2 ng/mL Molgramostim (Schering-Plough). Developmental rate was scored every 8 hours until expanded blastocyst stage. Data were analysed by Fisher’s exact test. Results: The rate and extent of development of 2- to 4-cell embryos to blastocysts (BI$) was significantly increased by the addition of 2 ng/mL rhGM-CSF. The effect was exerted on embryos cultured in the presence of both the R&D Systems product and Molgramostim and was independent of the type of culture media used.

Tuesday,

September 3:45 P.M.

28, 1999

O-081 Mitochondrial Inheritance and the Incidence of Heteroplasmy After Ooplasmic Transplantation. ‘J. A. Barr&, ‘J. Cohen, ‘S. Willadsen, ‘R. T. Scott, ‘C. A. Brenner. ‘Gamete and Embryo Research Laboratory, The Institute for Reproductive Medicine and Science of Saint Barnabas, West Orange, NJ. Objective: Ooplasmic transfer may improve developmental capacity in compromised oocytes from patients with recurrent IVF failures. The mixing of populations of ooplasm has provoked considerable debate especially regarding artificial mitochondrial heteroplasmy. With the current approach, injection of 5%-10% of ooplasm from a donor to a recipient oocyte may entail transfer of mRNAs, proteins, mitochondria, as well as other organelles. All of these may be involved in the cytoplasmic reconstitution of the manipulated egg. The addition of donor ooplasm could alter the maternal inheritance of mitochondria. So far, this laboratory has attempted ooplasmic transfer in 18 cycles in 16 women resulting in nine clinical pregnancies and one first trimester miscarriage. Five apparently healthy babies have been born and three other pregnancies are ongoing. In light of these findings we are investigating the patterns of mitochondrial inheritance and the condition of heteroplasmy in offspring. Design: Only consenting patients were involved in this study. The hypervariable region of the mtDNA was amplified and sequenced from both donor and recipient bloods resulting in mitochondrial fingerprints. Mitochondrial DNA heteroplasmy was determined in single embryos, amniocytes, fetal placenta and cord blood. Materials and Methods: Donor and recipient DNA isolated from blood was amplified by PCR and DNA sequenced to determine differences in the mtDNA hypervariable region. In five ooplasmic transfer cases discarded tissues were collected and analyzed, but in only one were tissues from all phases examined. For parental confirmation, nuclear DNA fingerprinting of fetal placenta and cord blood was performed. Results: A heteroplasmic mixture of both donor and recipient mitochond&l DNA was found in samples with the following frequencies and proportions: embryos (n = 17/30, l%-10% donor), amniocytes (n = l/5, 5%-10% donor), fetal placenta (n = 2/2, lo%-80% donor) and cord blood (n = 2/2, lo%-40% donor). One fetal cord sample positive for donor

s31

mitochondria was not previously verified at amniocentesis. Fingerprinting showed that nuclear DNA was inherited only from the recipient mother and father in both placenta and fetal cord blood of the babies. Conclusion: Ooplasmic transfer can result in mitochondrial heteroplasmy representing both donor and recipient mitochondrial DNA. This was confirmed in embryos, amniocytes, fetal placenta and cord blood. Some donor mitochondrial DNA detected in embryos replicated during fetal developmerit. These results demonstrate, at least in principal, that the transmission of heteroplasmic mtDNA occurs after ooplasmic transfer and biparental nuclear inheritance.

Tuesday,

September 4:00 P.M.

28,

1999

O-082 In Vitro Maturation of Reconstructed Oocytes After Heterologous Germinal Vesicle Transfer-A Model to Study Nuclear Cytoplasmic Interactions. S. R. Gao, W. H. Wang, L. Meng, D. L. Keefe. Women & Infants Hospital of Rhode Island, Brown University School of Medicine, Providence, RI. Objectives: Mitochondria mediate aging and apoptosis in many cell types, and manipulation of oocyte cytoplasm has been proposed for oocyte infertility from mitochondrial dysfunction and mtDNA mutations. However, concerns have been raised about the safety of disrupting nuclear and cytoplasmic interactions. Because laboratory rodent species are so inbred, we sought to develop a heterologous model that might better mimic the mtDNA heteroplasmy of humans, in order to study nuclear-cytoplasmic interactions after nuclear transfer. Design: Germinal vesicles (GV) were removed and exchanged between rat and mouse. Reconstructed oocytes were matured and analyzed by examination of microtubule (MT), cortical granule (CG), and nuclear organization. Materials and Methods: Cumulus enclosed GV oocytes were collected from ovaries of SD rats and CD1 mice in Hepes-CZB medium with IBMX 44 hours after PMSG injection. Oocytes were pre-cultured in MEM with 5% FBS, 0.23 mM Pyruvate and IBMX for 1.5 hours in vitro. Denuded oocytes were transferred to two drops of Hepes-CZB with 7.5 mg/mL cytochalasin B (CB) and IBMX, respectively. GV-karyoplast of rat (Group 1. n= 131) or mouse (Group 2, n= 134) were transferred to PVS of mouse or rat enucleated oocytes, accordingly. Reconstructed GVcytoplasmic pairs were fused by electric pulse (3 DC pulses at 2.6 kv/cm, 20ms, at 30 minutes intervals) following alignment by AC voltage (50 v/cm). Resulting oocytes were matured for 14 hr in vitro. Rat (n=65) and mouse (n=48) GV oocytes were used as control groups. MT localization was confirmed by mouse antitubulin-tubulin antibody labeled with goat anti-mouse IgG-FITC; CG distribution labeled by LCAbiotin and visualized by Texas-Red, and nuclear configuration stained by propidium iodide. Images of microtubule, cortical granule and chromatin distribution were evaluated by a confocal microscope. Results: Reconstructed and control oocytes released their first polar body after 14 hours maturation in vitro. M II spindles associated with CG free areas appeared in matured oocytes, indicating normal morphological maturation after heterolonous nuclear-cvtonlasmic reconstruction. Maturation rates did not differ when compaiing oocytes reconstructed from rat GV’s and mouse cytoplasm to control, homologously reconstructed rat or mouse oocytes (83.7 vs. 81.5 or 79.1%, vs. P>.O5). Interestingly, lower (P<.O5) fusion and maturation rates occurred when mouse GVs were fused with rat cytoplasts than the reverse (51% vs. 84%). Conclusion: Heterologous, reconstructed oocytes can be created by combining karyoplasts and cytoplasts from rat and mouse. Differences existed in fusion and maturation rates, depending on the species of origin of the karyoplasts and cytoplasts. Rat GV’s fused with mouse cytoplasm should prove useful to study interactions between nucleus and cytoplasm in readily available laboratory animals. Funded by National Institutes of Health R21 #RR 12718-02 and K081099 to DLK.

S32

Abstracts

Tuesday,

September 4:15 P.M.

28,

1999

O-083 Co-Expression of HLA-G and P-hCG mRNA in Human Preimplantation Embryos. ‘?I. S. Kriissel, ‘Y. Wen, ‘E. Jeremias, ‘H. Y. Huang, ‘J. Hirchenhain, ‘P. Bielfeld, ‘M. L. Polan. ‘Reproductive Immunology Laboratory, Department of Gynecology/Obstetrics, Stanford University Medical Center, Palo Alto, CA, ‘Department of Obstetrics/Gynecology, Heinrich-Heine-University Medical Center, Diisseldorf, Germany. Objectives: Both, HLA-G and hCG are known to be expressed by the human trophoblast during pregnancy. Both proteins are essential for the maintenance of pregnancy: HLA-G prevents the rejection of the trophoblast by the maternal organism (“self/non-self recognition”), and hCG stimulates the progesterone production by the corpus luteum. Thus far, only little is known about the expression of these proteins during embryonic implantation or preimplantion embryo development. There is evidence for a polarized distribution of proteins (leptin and STAT-3) during the earliest stages of preimplantion development. The ability to detect markers for trophoblast differentiation in single embryos may help to increase the knowledge about cell determination and differentiation during preimplantation embryo development. Design: Expression of /3-hCG and HLA-G in human preimplantation embryos was determined by RT/nested PCR. Materials and Methods: Patients undergoing IVF at Stanford University for different reasons were asked to participate by donating their embryos or unfertilized oocytes. For both, ethical and practical reasons, pathologically fertiIized (3PN) embryos were used. Written consent approved by the “Human Subjects in Medical Research Committee at Stanford University” was obtained in all cases. Single oocytes or embryos were examined by reverse transcription (RT)/nested polymerase chain reaction (PCR) for their expression of p-actin (internal standard), HLA-G, and fl-hCG-mRNA. Primers for HLA-G were designed for maximal sensitivity to detect all isoforms without differentiation. Amplified cDNA was stained with ethidiumbromide and detected by UVdensitometry. Results: So far, a total of 33 preimplantation embryos were examined: 5 hatched blastocysts, 12 expanded blastocysts, 4 morulae, and 12 8- to 16-cell embryos. Only embryos expressing fi-actin were considered viable and were consecutively examined for P-hCG and HLA-G expression. So far, 80% of blastocysts did co-express P-hCG and HLA-G mRNA. P-hCG could be detected as early as in morulae-stage, whereas HLA-G could only be detected in blastocysts. Conclusions: The detection of P-hCG and HLA-G mRNA in human embryos during the time of preimplantation development is possible. Increasing the sensitivity may help to trace the onset of expression for these particular genes and therefore may be used as a tool to examine cell differentiation. It may also serve as a possible marker for preimplantation embryo quality. This work was supported in part by a NATO grant to MLP.

Tuesday, September 28, 1999 4:30

P.M.

O-084 Analysis of Human Leukocyte Antigen-G (HLA-G) in Human Embryo Culture Conditioned Medium. S. M. Yie, H. Balakier, G. Motamedi, C. L. Librach. Departments of Obstetrics and Gynecology, Women’s College Hospital, and The Toronto Hospital, University of Toronto and the S.T.A.R.T. Clinic, Toronto, Ontario, Canada. Objectives: Human leukocyte antigen-G (HLA-G) may play a role in the maternal-fetal immune relationship during pregnancy. We have previously reported that HLA-G is expressed at the mRNA and protein levels by some human preimplantation embryos by using RT-PCR and immunocytochemistry. The objective of this study was further investigate whether human embryos secrete soluble HLA-G and whether the soluble HLA-G concentrations are associated with IVF pregnancy outcome.

Vol. 72, No. 3, Suppl. 1, September 1999

Design: HLA-G concentrations in embryo culture conditioned media were determined by using an ELISA. Materials and Methods: 252 samples of fresh embryo culture conditioned medium were collected from 132 patients undergoing IVF. HLA-G concentrations in the samples were assayed by a specific and sensitive ELISA. In the assay system two different murine monoclonal antibodies against HLA-G were used. The assay had 10 ng/mL detection limit, no crossreaction with other classical HLA class I antigens, good analytical recovery, and less than 10% of within- and between assay CVs. Potential associations between HLA-G levels and pregnancy rate as well as other factors known to influence IVF outcome were statistically analyzed. Results: Approximately 68.2% of the samples had detectable HLA-G ranged from 0.01 to 1.9 pg/mL, with a mean of 0.208 pg/mL. There were no differences in embryo grade and patient age between the two groups. The numbers of blastomeres in the HLA-G positive samples at 72 hours were significantly higher than those in HLA-G negative group (6.71 5 0.10 vs. 5.86 & 0.22, mean C SE, P=.OOOl). In the HLA-G positive group, HLA-G concentrations were significantly correlated with embryo cleav-’ age rate (r=0.184, P=.Ol). More interestingly, pregnancy rates from transferred embryos with positive HLA-G secretion were significantly higher than those with undetectable HLA-G (52% vs. 20%, x2= 14.71, P<.Ol). Conclusion: [1] Embryo secretion of soluble HLA-G protein is variable. [2] Secretion of HLA-G is correlated with an increased embryo cleavage rate, and [3] Embryos secreting soluble HLA-G have a higher IVF pregnancy rate than those lacking HLA-G secretion. This work was supported by an MRC of Canada Grant.

Tuesday,

September 4:45 P.M.

28,

scoring system was based on blastomere number and the observed fragmentation pattern (P=.OO8). The third scoring system utilized both blastomere number and fragmentation pattern but also combined this with five morphologic criteria. Two of the features used in this scoring system, namely “pitting” and compaction are unique to Day 3 embryos. Embryos were scored with the following formula: cell number + 0.4 points for each positive morphologic feature (i.e. presence of pitting, presence of compaction, equal sized cells, good blastomere expansion and absence of vacuoles) and -2 points if the observed FP was greater than II. For example, a transfer with at least two 8-cell embryos (FP=I or II) and all of the above positive morphologic features, would receive an overall embryo quality score of 10. Statistical analysis, showed that with a cutoff point at 6.98, this scoring system had a sensitivity of 0.85 in predicting pregnancy outcome and a specificity of 0.39 with a P value of ,013. Conclusion: Although cell number and fragmentation pattern are certainly predictors of positive pregnancy outcomes, additional parameters specific to Day 3 embryos should be used to further stratify a cohort of embryos, Pitting and compaction may be early indicators of cytoplasmic activity and potential for embryonic activation. Further studies are being conducted to determine if this scoring system is also predictive of blastocyst formation in vitro.

ASSISTED

1999 Tuesday,

O-085 Morphologic Evaluation of Human Embryos and Derivation of an Embryo Quality Scoring System Specific for Day 3 Embryos. N. Desai, J. Goldstein, J. Goldfarb. Department of Reproductive Biology, Case Westem Reserve University, Cleveland, OH. Objective: A scoring system specific for Day 3 embryos has not been extensively looked at. Most IVF laboratories continue to grade embryos solely on the basis of cell number and percent fragmentation as was traditionally done for Day 2 embryos. Additional morphologic features, some unique to Day 3 embryos may be useful in selecting embryos most likely to blastulate and implant. The goal of this study was to derive an embryo scoring system for Day 3 transfers that is predictive of positive pregnancy outcomes. Design: Four observers reached a consensus on representative examples of each morphological parameter. One observer scored embryos were videotaped immediately before fresh embryo transfer after 3 days of culture. Materials and Methods: A total of 3 16 embryos from 93 patients were videotaped and evaluated. The following parameters were used to grade the embryos: cell number, fragmentation pattern, pitting, compaction, equal sized blastomeres, blastomere expansion, and absence of vacuoles. Embryos were classified, I through V according to their fragmentation pattern (FP; Alikani et al., 1997). Type I embryos had minimal fragmentation associated with a single blastomere. In Type II embryos fragmentation was localized within the cleavage cavity. Type III embryos exhibited small cellular fragments throughout the cleavage cavity. In Type IV embryos fragments were difficult to distinguish from blastomeres. Type V embryos consisted entirely of degenerating fragments. A coarse texture and appearance of tiny “pits” in individual blastomeres was often observed in Day 3 embryos and was denoted as “pitting.” Early signs of compaction identified by loss of definition between individual blastomeres was also unique to Day 3 embryos. Three different scoring systems were assessed using an independent two tailed t-test with pregnant and non-pregnant as grouping variables. Results: The mean number of embryos transferred per patient was 3.4. The clinical pregnancy rate was 41.9%. The implantation rate was calculated to be 18%. Three formulas were derived which scored embryo quality in each transfer based on the average of the two highest graded individual embryos transferred. In the first scoring system, cell number alone was used to predict pregnancy outcome (P=.Oll). The second

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REPRODUCTIVE TECHNOLOGY EMBRYO TRANSFER

September 2:00 P.M.

28, 1999

O-086 Comparison Between Embryos Transferred on Day 2 or Day 3 in Human IVF or IVF/ICSI Programs: A Prospective, Randomized Study. .I. .I. Koo, H. J. Chi, M. Y. Kim, J. Y. Joo, J. Y. Kim, H. R. Sung, S. S. Chang. IVF Center, Hanna Women’s Clinic, Seoul, Korea. Objectives: To evaluate whether embryos transfer on Day 2 versus on Day 3 effects the pregnancy rates (PRs) and an implantation rates in human IVF or IVF/ICSI programs. Design: Prospective, randomized study. Materials and Methods: One hundred forty eight patients undergoing IVF or IVF/ICSI programs, from July to December 1998, were included in this study. Patients were stimulated with a combination of GnRHa and gonadotropins. The patients were randomly allocated into two different groups: group A (embryos were transferred on Day 2) and group B (embryos were transferred on Day 3). A and B groups were again subdivided into two groups, respectively: Al and B 1 groups (patients undergoing IVF program) and A2 and B2 groups (patients undergoing IVFlICSI program). Oocytes and embryos were cultured in manual Pl medium containing human follicular fluid. A clinical pregnancy was defined by ultrasonic detection of gestational sac. Results: The results are summarized in table. Differences between groups were analyzed by 2. Group Group (IT Number of cycles Mean age Mean number of Mean number of Fertilization rate Mean number of Implantation rate Clinical oreanancv

Al

63

oocytes aspirated injected oocytes (%) embryos transferred (%) rate (%)

33.8 12.2

-e 3.4 2 5.5

71.3 3.8 i 0.2 15.6 41.2

A (Day

2-ET) Group A2 (IVF/ICSI) 32 33.5 2 4.2 11.8 2 4.7 8.9 2 2.1 90.1 3.9 + 0.1

19.0 40.6

s33

Group Group

B (Day Bl

(IW Number of cycles Mean age Mean number of Mean number of Fertilization rate Mean number of Implantation rate Clinical nreenancv

oocytes aspirated injected oocytes (%) embryos transferred (%) rate (%)

42 33.5 -c 3.6 13.5 + 4.5 74.8 4.4 z 0.1 13.2 54.7

3-ET)

Group B2 (IVF/ICSI) 23 35.5 k 10.1 5 7.1 t 80.0 3.7 2 25.3 60.8

3.8 5.3 4.1 0.3

P value

NS NS NS NS NS NS NS

Conclusions: Embryo transfer on Day 3 showed a higher pregnancy rates in both groups undergoing IVF and IVF/ICSI programs, although the difference did not achieve statistical significance. The beneficial effect of Day 3 ET may be associated with selection of embryos with a higher potential for implantation and synchronization of the endometrium and embryos.

Tuesday, September 28, 1999 2:15 P.M. O-087 To Transfer on Day 3 or Day S? The Number of S-Cell Embryos Is the Key Determinant. C. Racowsky, K. V. Jackson, A. Nurredin, S. Shen, M. J. de 10s Santos, M. Pillar, N. A. Cekleniak, J. H. Fox, M. D. Homstein, E. S. Ginsburg. Department Obstetrics/Gynecology and Reproductive Biology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA. Objectives: Improvements in ART protocols and associated increases in implantation rates have resulted in the concomitant challenge to eliminate the occurrence of high order multiple gestations (>2 fetal hearts [FHs]). Whereas Day 5 embryo transfers (D5 ETs) may afford the advantage of selecting embryos with proven development to the blastocyst stage, the question arises as to whether Day 3 transfers (D3 ETs) may avoid compromised development secondary to in vitro conditions. This study was conducted to identify factors specific to success following transfer on D3 or D5. Design: A retrospective analysis of IVF or ICSI cycles with D3 (n=221) or D5 (n= 148) transfer, performed from January 98 to May 98 and May 98 to December 98, respectively. Materials and Methods: Zygotes were cultured individually in 25 PL drops in IVF-500 from Dl-D3, and in S2 from D3-D5. Patients were accepted for D5 ET when they had ~8 zygotes available. The historical controls were patients who had 28 zygotes with D3 ET. Based on initial analyses revealing a positive correlation between number of 8-cell embryos and blastocysts formed, cycles were stratified into six groups according to the day of transfer (D3 vs. D5) and the number of 8-cell embryos available on D3 (0, 1 or 2, and 23). Implantation rates, ongoing pregnancy rates with documented FH(s), and incidence of gestations with >2FHs were compared by 2, with PC.05 considered statistically significant. Results: Patient age, ART attempt number, and number of embryos available did not differ between D3 ETs and D5 ETs for any of the 8-cell groups. For all groups, significantly fewer embryos were transferred on D5 vs. D3 (mean: 2.1 vs. 4.4; P<.OOOl). When no 8-cell embryos were available on D3, the ongoing pregnancy rate increased with D3 ETs vs. D5 ETs (33.3% vs. 0%; P<.Ol; n=27 and 14), although implantation rates were not different (7.9% vs. 0%). With 1 or 2 8-cell embryos available on D3, ongoing pregnancy rates (36.4% vs. 40.4%), implantation rates (17.6% vs. 23.6%) and incidence of gestations with >2FHs (9.1% vs. 0%) were not different between D3 ETs and D5 ETs (n=77 and 51, respectively). With 23 8-cell embryos on D3, D5 ETs (n=83) resulted in an increased implantation rate (35.9% vs. 24.2%; P<.OO3), and a decrease in the incidence of gestations with >2FHs (4.3% vs. 17.7%; PC.03) but no difference in ongoing pregnancy rate (51.3% vs. 50.6%), compared with D3 ETs (n= 117). Conclusions: When no 8-cell embryos are available on D3, it is preferable to perform a D3 ET because, unlike the culture system used, the uterine environment can “rescue” some of the slower cleaving embryos. With the

s34

Abstracts

availability of 1 or 2 &cell embryos on D3, any benefit of D5 ETs compared with D3 ETs, appears equivocal. When 23 8-cell embryos are present, D5 ETs considerably reduce the risk of gestations with >ZFHS, while still maintaining a high pregnancy rate. Collectively, the results of this analysis show that the number of 8-cell embryos available on D3 provides the key for determining the optimum day for embryo transfer.

Tuesday, September 28, 1999 2:30 P.M. O-088 The Effect During In R. Alvero. WRAMC,

of Embryo Transfer Technique on Clinical Pregnancy Rates Vitro Fertilization. R. M. Hearns, J. Hill, L. Scott, J. H. Segars, Combined Federal Program in Reproductive Endocrinology of NNMC, USUHS, and NIH, Bethesda, MD.

Objectives: Pregnancy rates in patients undergoing in vitro fertilization (IVF) are influenced by embryo quality and embryo transfer technique. Several studies have examined variables that affect embryo transfer success. However, most studies do not control for embryo quality. The objective of this study was to examine the effect of embryo transfer technique on clinical pregnancy rates in IVF patients who had grades I and II embryos, or blastocysts transferred. Design: An analysis of 365 consecutive IVF cycles from l/98 to 1 l/98 at a terti2uy care center was performed. Materials and Methods: A total of 290 IVF patients were studied. Patients having grade I and grade II embryos, or blastocysts transferred were included in the study. The same embryologist graded all embryos using the criteria described by Veeck (Ann NY Acad Sci 1989;541:259). Embryo transfers were performed with the Wallace catheter using ultrasound guidance. The following variables were recorded and assessed for each embryo transfer (ET): failed first attempt at ET; catheter contamination with blood and/or mucous; difficulty or ease of ET; ultrasound visualization of catheter placement and expulsion of media containing the embryos; and retention of embryos in the transfer catheter. These data were compared by Fisher’s exact and x2 tests where appropriate. P values <.05 were considered statistically significant. Ongoing clinical pregnancy was the principal outcome variable. Post-hoc analysis to determine the sample size needed to demonstrate 20% reduction in pregnancy rate, using an a=.05 and 90% power revealed that 125 cycles were needed for each arm, a number achieved by this study. Results: Of 290 embryo transfers, 152 resulted in ongoing pregnancies and 138 did not. Multiple attempts at ET, difficulty of ET, retention of embryos in the transfer catheter, and contamination of the transfer catheter with mucous did not affect the ongoing pregnancy rate. There was a significant association between catheter contamination with blood and decreased ongoing pregnancy rate (PC.05, Fisher’s exact test). Pregnancy rates were 54% (n=265) in the absence of blood versus 28% (n=25) in the presence of blood. Conclusions: Our results suggest that ongoing pregnancy rates are not compromised by retention of embryos in the transfer catheter, number of attempts required to replace embryos, provider difficulty of transfer, or contamination of the transfer catheter with mucous. However, contamination of the transfer catheter with blood appears to significantly reduce pregnancy rates in IVF.

Tuesday,

September

2:4.5

28, 1999

P.M.

O-089 Does the Addition of Blastocyst Embryo Using the Double Transfer Technique Have an Advantage Over the Meticulous Selection of Embryos With Faster Cleavage Rate Regarding IVF Outcome? A Prospective, Randomized Study. Y. Gonen, S. Goldman. IVF Unit, HerzliyaMedical-Centers, Haifa, Israel. Objectives: The choice of the best embryo for transfer crucial role in IVF outcome. Data to date on the replacement

plays a more of blastocysts

Vol. 72, No. 3, Suppl. 1, September 1999

indicate that such embryos have a high implantation rate. It has been suggested that only “hardier” embryos would reach the blastocyst stage and that better embryos will progress faster to more advanced stages of embryonic development. Early identification of those embryos may have beneficial effect on IVF results. The purpose of this study was to investigate whether the addition of blastocyst embryo using the double transfer technique yielded higher pregnancy rates than meticulous selection of embryos with the faster development rate. Design: Prospective, randomized trial. Materials and Methods: During the study period all women (n = 99) undergoing therapeutic IVF with at least five embryos available for transfer were included in the study. Patients were randomized to either day 3 transfer following meticulous selection of the embryos with the faster development rate (Group A) or blastocyst culture with additional transfer on day 5 (Group B). All cleaving embryos were examined more than once a day to identify the faster embryos in order to facilitate selection. Clinical pregnancy rates were determined after the transfer of fast-growing embryos at the g-cell stage on day 3 or at the addition of expanded blastocyst on day 5 or 6. Results: The results are summarized in the table below.

Mean Group Group

A (n=48) B (n=51)

30.0

age 5 0.7

31.3 ? 0.6

No. of embryos available for transfer

No. of oocytes 13.1

t

and placed in Stuart’s transfer medium. Each catheter tip was individually inoculated on five primary culture media within 2 hours of collection. Results: Of the 430 cases studied there were negative microbial culture from the tips of the mock ET catheters in 133 (30.9%) patients; Group 1. The tips of the catheters used at the ET were negative for microbial growth in 233 (78.4%; Group 2) of the 297 patients in whom the mock ET catheters tips had been positive. In 64 patients (14.8%) the tips of the catheters both at mock ET and the ET remained positive for microbial growth; Group 3. None of the 133 patients in Group 1 had a positive microbial growth from the tips of the catheters used for the ET. The fertilisation and embryonic cleavage rates were similar in three groups. The implantation and clinical pregnancy rates though similar in groups 1 and 2 (21.6 vs. 19.8%; 41.7 vs. 38.3%) these were statistically significantly higher when either group 1 or 2 was individually compared with group 3 (Group 1 vs. 3: 21.6 vs. 9.3; 41.7 vs. 18.1; Pc.05); (Group 2 vs. 3: 19.8 vs. 9.3; 38.3 vs. 18.1; P<.O5). Discussion: The routine administration of prophylactic antibiotics at oocyte retrieval led to reduction in the incidence of inoculation of the endometrium with endocervical microbial flora that contaminated the ET catheter during the embryo transfer procedure and a statistically significant higher implantation and clinical pregnancy rates.

ASSISTED REPRODUCTIVE TECHNOLOGY POSTOVULATION MANAGEMENT

Clinical pregnancy rate/transfer

0.8

7.6 5 0.5

27/48

11.7 2 0.6

8.1 -c 0.4

28/51 (54.9%)

Tuesday,

(56.3%)

September 3:45 P.M.

28, 1999

O-091 Conclusions: This study highlights for the first time the importance of meticulous selection of the faster cleaving embryos for transfer. The high pregnancy rates achieved choosing the embryos that progressed faster to more advanced stages of embryonic development indicate that such embryos have an equally high implantation potential as blastocyst embryos. Thus, our results suggest that culturing embryos up to fully expanded blastocysts should be offered selectively and IVF cost can be decreased omitting the routine use of very expensive blastocyst culture media without compromising IVF results. Further research will be required to examine whether the double transfer technique has any direct effects on the endometrium.

Tuesday,

September 3:00 P.M.

28, 1999

O-090

Routine Use of Prophylactic Antibiotics to Prevent Inoculation of the Endometrium With Endocervical Microbial Organisms at Embryo Transfer (ET) in In Vitro Fertilization Treatment Cycles: The Impact on Implantation and Clinical Pregnancy Rates. ‘,3P. E. Egbase, ‘M. Al Sharhan, 2E. Udo, 3G. Grudzinskas. ‘IVF Centre, Maternity Hospital, Kuwait; ‘Department of Microbiology, Kuwait University, Kuwait; 3Academic Department of Obstetrics & Gynecology, Royal London Hospital, London, United Kingdom. Objectives: This study investigates the routine administration of prophylactic antibiotic regimen at oocyte retrieval to prevent the inoculation of the endometrium during embryo transfer and the impact on the implantation and clinical pregnancy rates. Materials and Methods: A total of 430 consecutive patients were studied prospectively at the time of oocyte retrieval and embryo transfer procedures. Prior to the oocyte retrieval procedure the cervix was exposed with a bivalve speculum. cleansed with gauge soaked in Earle’s culture medium and a mock embryo transfer procedure performed using a Wallace embryo replacement catheter. Thereafter, the tip of the catheter (last lcm) was cut off and placed in Stuart’s transfer medium for microbial culture. lima-operative prophylactic antibiotics (Ceftriaxone, 2 gm iv, and Metronidazole, 1 gm iv) were administered to all patients during the oocyte retrieval. Embryo transfers were performed using similar Wallace embryo replacement catheter 48 hours after oocyte retrieval in the same manner as in the mock ET. Up to three embryos were transferred per patient. Once it was confirmed that all embryos were transferred, the tip of the catheter (last 1 cm) was cut off

FERTILITY

& STERILITY@

Women Who Develop Severe Ovarian Hyperstimulation Syndrome After Ovulation Induction Exhibit a Markedly Increased Prevalence of Positive Thromhophilia Markers. M. Dulitzky, S. B. Cohen, D. S. Seidman, A. Inbal, U. Seligson, D. Soriano, S. Mashiach, J. Rabinovici, J. Buchman. Gynecology and Maternity Center and Institute of Thrombosis and Hemostasis, The Chaim Sheba Medical Center, Tel Hashomer, Israel. Objectives: Severe ovarian hyperstimulation syndrome (OHSS) is a complication of ovulation induction regimens that can present with life-threatening thrombo-embolic phenomena. High periovulatory serum estradiol levels and multiple ovarian follicles are regarded as risk factors for the development of severe OHSS. However, only a fraction of women with these risk factors will ultimately develop severe OHSS. Thrombophilia is clinically characterized by the repeated occurrence of thrombo-embolism, sometimes without any apparent cause, Several hereditary and/or acquired disorders present with such an increased risk for the development of thrombo-embolic events. These disorders include deficiencies in blood clotting factors, like protein C, protein S, or anti-thrombin III (AT III), resistance to the anticoagulant action of activated protein C (APCR), hyperhomocysteinemia and autoimmune disorders characterized by increased levels of anticardiolipin antibodies and circulating lupus anticoagulants. This study aimed at determining the prevalence of markers of thrombophilia in women hospitalized with severe OHSS. Design: Case-control study. Materials and Methods: The prevalence of tbrombophilia was assessed in 15 consecutive women hospitalized with established severe OHSS (cases) and in 41 women undergoing induction of ovulation without development of OHSS (controls). Blood samples were analyzed for serum levels of antithrombin-III, protein S, protein C, circulating lupus anticoagulant, and anticardiolipin antibodies. DNA analyses for mutations of the factor-V Leiden (APCR) and the methyl tetrahydro folate reductase (MTHFR) genes were performed. Results: Thirteen of 15 women with severe OHSS (86.6%) and only 8 of 41 control women (19.5%) exhibited positive markers of thrombophilia (P<.OOl): Four women with severe OHSS and none of the controls had more than one positive marker for thrombophilia. In the severe OHSS group five women exhibited a homozygote MTHFR gene mutation, one had a heterozygote factor-V Leiden gene mutation, four had a antithrombin-III deficiency, eight had a protein S deficiency and four had abnormal anticardiolipin antibody levels. In women with severe OHSS mean levels of antithrombin-III (P<.OOl) and protein S (P= ,002) were markedly decreased in comparison to the control group, while mean protein C, circulating lupus anticoagulant and anticardiolipin antibodies were not different.

s35

Conclusions: Our results demonstrate a high prevalence of positive thrombophilic markers in women who develop severe OHSS. These findings serve as new evidence for the blood coagulation dysfunction in the course of severe OHSS. In addition they may shed new light on the underlying pathophysiologic mechanisms of this syndrome.

Tuesday,

September 4:00 P.M.

28,

1999 2

X

0 group

B

‘PC.007

hCG

O-092 Subcutaneous Versus Intramuscular Administration of hCG During an IVF Cycle: Follicular Fluid and Serum Levels of hCG. J. R. Stelling, D. Frankfurter, D. H. Harris, A. S. Penzias, S. P. Oskowitz, M. M. Alper, M. .I. Berger, M. Kamis, M. R. Gray, R. H. Reindollar. Beth Israel Deaconess Medical Center, Harvard Medical School, Boston IVF, Boston, MA. Objectives: Currently, most IVF centers administer hCG, by intramuscular (IM) injection to trigger ovulation. Recently, we reported that hCG may be given by subcutaneous (SC) administration with similar efficacy compared to IM after reviewing 600 cycles. There were no statistically significant differences between groups in the number of oocytes retrieved, number of mature oocytes retrieved, fertilization rate, embryo quality, or pregnancy rate. We sought to confirm that the hCG levels in follicular fluid and serum would be comparable between these two administration routes. Design: Prospective comparison of follicular fluid and serum hCG levels between two routes of hCG administration, SC vs. IM. Materials and Methods: After obtaining local IRB approval, 40 women undergoing oocyte retrieval at a university affiliated IVF program were entered into the study at the time of egg retrieval. There were 17 women in the IM group, and 23 women in the SC group. Route of hCG administration (10,000 IU), IM vs. SC, was determined prior to entry in the study, by the patient’s attending physician. Egg retrieval was scheduled to occur 36 hours after hCG administration in both groups. Serum was collected at the time of IV placement prior to induction of anesthesia. Follicular fluid was collected from the first mature follicle aspirated, using media free collection tubes. hCG was measured using an automated, immunoassay analyzer. Statistical analysis was then performed using unpaired, and paired r-tests, and 2 for comparison. Results: There was a significantly higher (P = .O 14) serum hCG level in the SC group (348.6 2 98 IUiL) vs. the IM group (259.0 t 115 IUiL). There was a also significantly higher follicular fluid hCG level in the SC vs. the IM group (233.5 5 85 vs. 143.4 ? 134 IU/L). There was no statistically significant differences between groups in age, BMI, cycle length, total gonadotropin usage, peak E2, percent normal fertilization, or pregnancy rate. Even the lowest hCG levels measured in serum (66 IUIL) and follicular fluid (34 IU/L) had adequate effectiveness, as measured by the number of oocytes retrieved, percent normal fertilization, and pregnancy. Conclusions: It appears that 36 hours after hCG administration by the SC route both serum and follicular fluid levels are greater then when compared with the IM route. There does not appear to be any major clinical significance of this difference in terms of treatment outcome. There is a likely some threshold (below 66 IU/I. in serum and 34 IU/L in follicular fluid) of hCG necessary to achieve the events involved in oocyte maturation and ovulation. Both routes, IM and SC, are able to reliably achieve this threshold. This study further supports the efficacy of SC administration of hCG to trigger ovulation in ART. Therefore, the SC route should be preferred because of its ease and tolerability of administration,

Tuesday, September 28, 1999 4:15

P.M.

O-093 Transvaginal Administration of Progesterone (P) Started at Oocyte Retrieval Reduces Uterine Contractions at the Time of Embryo Transfer. R. Fanchin, C. Righini, J. M. Ayoubl, F. Olivennes, S. Hammamah, D. de Ziegler, R. Frydman. Department of Obstetrics and Gynecology, and REI, Hapita A BCclere, Clamart, France. Objectives: We have observed that in IVF-ET the higher the uterine contraction (UC) frequency on the day of ET, the lower the pregnancy and

S36

0 group

Abstracts

ET

implantation rates (Fanchin et al., Hum Reprod 1998;13: 1968). This led us to query whether the utero-relaxing properties of P could serve to diminish UC frequency on the day of ET and, in turn, improve IVF-ET outcome. We elected to supplement P vaginally because this route results in high uterine tissue concentrations (Miles at al. Fertil Steril 1994;62:485). Design: Prospective randomized study on the effects of transvaginal P administration on UC assessed by computer-assisted ultrasound (US). Materials and Methods: 84 women, aged 25-38, undergoing 84 GnRH-a and FSWhCG cycles for IVF-ET were studied. Luteal phase support with P (Crinone; Wyeth Ayerst, UK, 90 mg/day, vaginally) was randomly started on the day of oocyte retrieval (group A, n=43) or on the evening of ET (group B, n=41). On the day of hCG administration and just before ET, 2-minute sagital uterine scans were obtained by US with a 7.2 MHz vaginal probe (Siemens Elegra, France) and digitized with an image analysis system (IBDP, Paris, France) that allows the objective assessment of UC frequency. As described previously, UC types, sorted as retrograde, antegrade, antagonistic, and nonpropagating UC, were also assessed. Results: Patients in both groups were comparable in regard to population characteristics, ovarian response to COH, and embryology data. As illustrated, whereas UC frequency was similar in both groups on the day of hCG (4.6 2 0.3 and 4.5 + 0.3 UC/min, mean?SE, respectively), only women having started P on the day of oocyte retrieval (group A) showed decreased UC frequency on the day of ET (group A, 2.8 t 0.2 UC/min, P
Tuesday, September 28, 1999 4:30

P.M.

O-094 In IVF-ET, Uterine Contraction (UC) Frequency Decreases at the Time of Blastocyst Transfer. R. Franchin, C. Righini, J. M. Ayoubi, F. Olivennes, N. Frydman, D. de Ziegler, R. Frydman. Department of Obstetrics Gynecology and REI, HBpital A Beclere, Clamart, France. Objectives: In IVF-ET, we have observed that increased UC frequency at the time of 2-4 cell embryo transfers (ET) is associated with lower pregnancy and implantation rates (Fanchin et al, Hum Reprod, 1998;13:1968). Hence, we wondered whether delaying ET such as done with blastocysts might benefit from finding a more quiescent utems. We elected to supplement progesterone (P) vaginally because this route results in high uterine tissue concentrations (Miles at al, Fertil Steril, 1994;62:485). Design: Prospective study on the possible changes on UC rate during the early luteal phase of COH for IVF-ET. Materials and Methods: 42 women, aged 26-38, undergoing 42 GnRH-a and FSWhCG cycles for IVF-ET were studied. Luteal phase was supported with P (Crinone, Wyeth Ayerst, UK, 90 mg/day, vaginally). On the day of hCG administration (hCG), 2-4 cell ET (hCG+4), and 3 days later (theo-

Vol. 72, No. 3, Suppl. 1, September 1999

retical day of blastocyst transfer, hCG+7), 2-minute sagital uterine scans were obtained by ultrasound with a 7.2 MHz vaginal probe (Siemens Elegra, France) and digitized with an image analysis system (IBDP, Paris, France) that allows assessment of UC frequency. UC types were assessed in both groups. Results: As illustrated, a significant decrease in UC frequency occurred

history of previous spontaneous abortion and ectopic pregnancy in the continue progesterone group. The pregnancy rate (Beta hCG 2 10 mIU/mI) per oocyte retrieval was the same between groups (36.5% in continue vs. 35.9% in discontinue, p= 0.77). Pregnancy outcomes were not different between treatment groups, including the clinical pregnancy rates and abortion rates (p= 0.43). Pregnancy outcome per PhCG 2 10 mIU/ml is summarized in the Table below. Outcome

Continue

Discontinue

P4.cm ANOVA

Biochemical Clinical/Ongoing Spontaneous Abortion Ectopic Therapeutic Abortion Total with PhCG 2 lOmIU/ml

256 649 114 32 5 1056

(24.2%) (61.5%) (10.8%) (3.0%) (0.4%) (100%)

63 164 33 4 3 266

(23.3%) (61.7%) (12.4%) (1.5%) (1.1%) (100%) p=O.43

-\ hiG

h&4

hCG+7

from the day of hCG (4.6 2 0.2 UC/mm, mean?SE) to hCG+4 (3.5 2 0.2 UC/min), with a further decrease to a virtually quiescent contractile status on the normal day of blastocyst transfer (1.5 2 0.2 UC/mm; hCG+7). A progressive increase in the relative prevalence of antagonistic and non propagating UC was observed on days hCG+4 and hCG+7 as compared to day of hCG. Conclusions: 1. We observed a significant decrease in UC frequency from the day of hCG onward, with a marked decline between the days of 2-4 cell embryo and blastocyst transfers, on hCG+4 and hCG+7, respectively. 2. These data further support the effectiveness of progesterone in reducing UC frequency and promoting UC reorganization during peri-implantation. 3. Because high frequency UC at the time of 2-4 cell embryo transfers reduce conventional IVF-ET outcome probably by fostering embryo expulsion from the uterine cavity, the virtual uterine quiescence observed at hCG+7 may represent considerable welfare for transfening blastocysts.

Tuesday,

September

4145

28, 1999

P.M.

O-095 Progesterone Support in Early IVF/GIFT Pregnancies May Not Be Necessary. J. R. Stelling, C. B. Barrett, A. S. Penzias, M. M. Alper, M. .I. Berger, S. P. Oskowitz, M. F. Kamis, M. R. Gray, R. H. Reindollar. BIDMC, Harvard Medical School, Boston IVF, Boston, MA. Objectives: Progesterone is an important factor in the maintenance of early pregnancy. The luteal-placental shift occurs between weeks 5-S of gestation. Follicular aspiration during ART may theoretically interfere with the normal functioning of the corpus lutea, and therefore many IVF centers will continue progesterone support well into the first trimester of pregnancy. However, little data exists studying the optimal duration of progesterone support. We sought to determine if progesterone supplementation was necessary after the initial positive pregnancy test in women undergoing IVF and GIFT procedures. Design: A retrospective cohort study was conducted comparing pregnancy rates and outcomes between continuation versus discontinuation of supplemental progesterone at the time of initial hCG. Materials and Methods: 3631 IVF and GIFT cycles with sufficient data that occurred between 1996 and 1998 were included in the analysis. Two physician’s standard practice is to discontinue use of progesterone at the time of the initial hCG (14 days post egg retrieval). The remaining 10 physicians routinely continue supplementation beyond the luteal-placental shift (8-12 weeks gestation). We compared pregnancy rates per oocyte retrieval (pregnancy = PhCG 2 10 mIU/ml) and pregnancy outcomes between these 2 protocols. Statistical analysis was performed using Chisquares, one-way ANOVA, and t-tests as appropriate. Results: There were 2891 women in the continue group and 740 in the discontinue group. Ages ranged from 20-48. There was no significant difference in cycle cancellation rate, oocytes retrieved, or number of embryos transferred between groups. There was a slightly higher rate of a

FERTILITY

& STERILITY@

Conclusions: In this retrospective analysis there does not appear to be a benefit of continuing supplemental progesterone after the initial ShCG. Given these reassuring results a randomized study of continuing vs. discontinuing progesterone is warranted.

ASSISTED

REPRODUCTIVE TECHNOLOGY ART OUTCOMES

Tuesday,

September

2:00

28, 1999

P.M.

O-096 Risk Factors for Multiple Gestation in Women Undergoing Intrauterine Insemination with Ovarian Stimulation. ‘,‘E. B. Pasqualotto, ‘,*T. Falcone, ‘C. Petrauskis, ?I. Goldberg, 4D. R. Nelson, 1.3A. Agarwal. ‘Center for Advanced Research in Human Reproduction and Infertility, Departments of ‘Gynecology-Obstetrics, sUrology, and ‘Biostatistics and Epidemiology, The Cleveland Clinic Foundation, Cleveland, OH. Objectives: We sought to identify whether post-wash sperm characteristics and/or ovulation induction cycle characteristics can predict the occurrence of multiple conception in patients after ovarian stimulation and intrauterine insemination (IUI). Design: Retrospective study. Materials and Methods: The medical records of 162 pregnant women who underwent ovarian stimulation and IUI with their partner’s sperm at the Cleveland Clinic Foundation from January 1993 through December 1997 were reviewed in this retrospective study. Couples with ectopic pregnancies, incomplete data, who did not achieve pregnancy, or who did not undergo ovarian stimulation with clomiphene citrate or human menopausal gonadotropin (hMG) were excluded from analysis (n = 36). The relationship between patient characteristics, ovarian stimulation, and post-wash sperm characteristics to multiple pregnancy were evaluated. Results: Comparison of the multiple (n = 100) and single (n = 22) conceptions showed that the mean serum estradiol (E,) levels on the day of human menopausal gonadotropins injection were significantly higher in the multiple conception group (p = 0.01). Patients in both groups received similar amounts of hMG for comparable intervals. The post-wash total sperm count, total motile sperm count and sperm motility did not differ between groups. However, patients with multiple pregnancies underwent IUI with significantly higher curvilinear velocity (p = 0.04), sperm linearity (p
s37

than ultrasound monitoring the follicle number and size, 3) the total sperm count, total motile sperm count and sperm motility were not correlated with multiple pregnancy, and 4) the probability of fertilization of more than one oocyte most likely is primarily related to the functional characteristics of the sperm available, once several fertilizable oocytes are released in patients with ovarian stimulation therapy.

Tuesday,

September 2:15 P.M.

28, 1999

O-097 Intrafamilial Oocyte Donation is G. Ndukwe, S. Donovan, S. Ambrick. Treatment Unit in Reproduction Queen’s Medical Centre, Nottingham,

Associated Nottingham (NURTURE), UK.

with Poor Outcome. University Research and University Hospital,

Objectives: Premature ovarian failure (POF) varies from occult ovarian failure when regular menstrual cycles still occur to premature menopause. There is mounting evidence that POF maybe familial. In females, familial conditions such as type 1 autoimmune polyendocrinopathy with defect in T suppressor cells, FRAXA pm-mutation, mutation in FSH receptor gene and unbalanced X; autosome translocation are all associated with POF. Occult ovarian failure is associated with impaired reproductive performance. There is a universal paucity of oocyte donors and often sisters act as oocyte donors for their sisters. The objective of this study is to find out if the pregnancy rate in intrafamilial oocyte donation is significantly different compared with anonymous oocyte donation. Design: Retrospective data analysis. Materials and Methods: At NURTURE, a tertiary referral centre for ART, 166 women with POF (Group A) who received oocytes from anonymous donors were compared with 19 women with POF (Group B) who received oocytes from their younger sisters. All donor superovulation was with the long protocol of Buserelin and 150-2251U of gonadotrophins. Recipients’ endometrial preparation was with sequential oestradiol valerate and progesterone supplements. Results: There was a significantly lower clinical pregnancy rate per cycle in intrafamilial oocyte donation (Group B) (10%) compared with anonymous oocyte donation (Group A) (38%) (P < 0.05). Between the two groups there was no significant differences in mean ages of donors and recipients, donor serum FSH, total amount of gonadotrophins, number of oocytes retrieved and number of embryos transferred, 16 out of the 19 intrafamilial donors had conceived in the past. The mean ages of intrafamilial oocyte donation was 29.8 compared with 30 years for anonymous egg donors. Conclusions: Clinical pregnancy rate when a younger sister donates oocytes t an older one was significantly lower than when the oocyte donor was anonymous. There were no differences between the groups, other than that in the intrafamilial oocyte donation the donor and recipient were sisters. The appalling outcome is despite the fact that most of the younger sisters (16 out of 19) had conceived in the past and had children. This would suggest a temporal element in the ovarian dysfunction. The reproductive impairment in POF seems to be familial with progressive expression within a time frame, making younger sisters of women with POF progressively less fertile with time. Should intrafamilial oocyte donation be discouraged and what advice should be given to younger sisters concerning the possibility of impairment in future reproductive potential? We have started studies of possible genetic determinants of ovarian development in the pursuit of possible candidate genes implicated in premature ovarian failure.

Tuesday,

September 2:30 P.M.

28, 1999

O-098 Effect of Body Mass Index (BMI) on Outcome of In Vitro (IVF). H. D. McClamrock, J. B. Loveland, A. M. Malinow, University of Maryland Medical Center, Baltimore, MD.

Fertilization F. I. Sharara.

Objectives: The association between obesity and infertility is well established, however, few studies have evaluated the impact of body weight on IVF outcome. This study compares IVF outcome based on body mass index.

S38

Abstracts

Design: Retrospective. Materials and Methods: Patients less than 40 years old undergoing IVF cycles with fresh embryos from l/97-8/98 were included in the study. BMI was calculated by the formula: weight (kg)/height (mete?) and patients were grouped into BMI 5 25 and BMI > 25. Parameters were compared with appropriate statistical analyses. Results: Ninety-eight patients underwent 137 IVF cycles, of which 15 were canceled for poor response, leaving 122 cycles included in the analyses. Forty-seven patients had BMI 5 25 and 51 had BMI > 25. No difference between groups was found in age, number of canceled cycles, progesterone on day of hCG, endometrial thickness, number of ampules, or duration of stimulation.

Mean BMI % 25 BMI > 25 Significance

BMI 5 25 BMI > 25 Significance

Mean basal FSH (MIU/ml)

BMI

21.95 (2 1.89) 32.82 (t 7.64) <0.0001

Peak E, @g/ml)

7.13 (2 1.89) 6.22 (” 1.60) p<0.0001

2,653 (k 962) 2,477 (t 1022) N.S.

Mean # oocytes

Mean # embryos

Pregnancy Rate

Implantation Rate

13.65 (” 5.16) 15.53 (” 6.92) N.S.

3.77 (2 1.00) 3.71 (2 1.12) N.S.

58.7% 31.0% p=o.o095

26% 15% p=O.Ol

Conclusions: Overweight patients (BMI >25) have statistically lower pregnancy and implantation rates. The similarity between duration of stimulation, gonadotropin requirements, and number of oocytes obtained suggests that these differences in PR/IR are not related to ovarian response, especially in light of lower FSH levels in the high BMI group. Further studies regarding other parameters, such as oocyte/embryo quality may help explain these differences.

Tuesday,

September 2145 P.M.

28, 1999

O-099 Multiple Pregnancy Outcome in IVF-ET Can Be Predicted by Size of Lead Follicle at the Time of hCG Administration. R. Pyrzak, R. D. Dickey, S. Sartor, P. Y. Lu, S. N. Taylor, P. H. Rye. Fertility Institute of New Orleans, New Orleans, LA. Objective: Size of the lead follicles and the estradiol concentration are used as a major indicator in decision making when to administrater hCG. The proper time of hCG administration may have a significant effect on oocyte maturity, fertilization and embryo morphology. Above factors have been associated with the probability of implantation and successful pregnancy. The objective of this study was to examine the relation between implantation rate and pregnancy outcome by the size of the lead follicle in the cohort on the day of hCG administration. Design: Retrospective analysis of patients’ 540 year old undergoing IVF-ET. Evaluation was performed only on cases in which, on day 2 or 3 post retrieval, more than one embryo were transferred. Material and Methods: Superovulation was achieved by down regulation with GnRHa (Nafarelin nasal spray, Synarel) followed by ovarian stimulation with hMG or FSH. Ultrasound examination and estradiol determination were carried out daily from the eight day of stimulation. On the day of hCG administration (10,000 IU) the mean diameter of each follicle in the cohort was recorded. Morphology of the cumulus-oocyte-complex was assessed for oocyte maturity immediately after retrieval. Patients’ cycles were divided into four groups according to the mean size of the lead follicles on the day of hCG administration. Data are presented (Table) as means, difference’s between groups were analyzed using chi square test, P < 0.05 was considered significant. Results: There were no significant differences in mean age (32.2-34.1), years of infertility (3.2-4.8), percent mature oocyte (71-79), fertilization rate (86-95), embryos quality and number transferred (4.0-4.4), or mean estradiol (1712-1988 pg/ml), on day of hCG administration among the four groups. Pregnancy rate, continued pregnancy and implantation rate were significantly higher (pcO.05) in Group 1 compared to Group 3 and 4. In

Vol.

72, No.

3, Suppl.

1, September

1999

cases with smallest leading follicles (Group 1) multiple significantly higher (p
Group1 15-16 Lead Follicle Size in nun (II=)

(20)

Pregnancy Rate (%) Delivered + Ongoing pregnancies (‘%) Multiple Pregnancies (W) Implantation rate (%) Days of stimulation No. of hMG/FSH ampoules used

75 60 92 35.2 9.9 26.6

pregnancy was day of hCG the in Groups 1, 2, 16.2, 13.8 and

Group 2 Group 3 Group 4 17-18 19-20 >21 (27) (53) (84) 52 41 36 18.1 11.0 33.0

49 34 33 15.9 11.4 32.3

potassium channels participate in altering potassium homeostasis during the initial phases of apoptosis induced by oxidative stress. Conclusion: Embryos experiencing apoptosis induced by oxidative stress exhibit potassium efflux early in the course of apoptosis, so non-invasive measurement of potassium homeostasis may serve as a marker of preimplantation embryo viability. Funded by NIH R21 #RR 12718-02 to DLK & PJSS, KO81099 to DLK and NIH P21 RR01395 to PJSS. [1] Jurisicova et al., 1996 Mol Human Reprod 2:93-98. 121 Bortner et al., 1997 J Biol Chem 272:32436-32442. [3] Smith et al., 1994 Methods Cell Biol 40:115-134.

47 33 43 17.7 12.2 37.5

ASSISTED

REPRODUCTIVE TECHNOLOGY UNIQUE ISSUES IN ART

Tuesday, Conclusion: Smaller lead follicles on the day of hCG administration, predicted higher implantation, pregnancy, and multiple pregnancy rates, even though the number of embryo transferred and age were similar in all groups. The number of oocytes retrieved significantly decreased as the size of the lead follicles in the cohort increased. The pregnancy loss increased as the size of lead follicle increased. This study suggest that size of lead follicles on the day of hCG administration may have an impact on embryo implantation. This may allow us to determine prospectively the number of embryos for transfer to reduce multiple pregnancy rate.

Tuesday,

September 3:00 P.M.

28, 1999

O-100 Potassium Efflux Occurs Early During Apoptosis in Preimplantation Mouse Embryos. ‘J. R. Trimarchi, ‘,3L. Liu, *P. J. S. Smith, ‘,3D. L. Keefe. ‘Laboratory of Reproductive Medicine, Marine Biological Laboratory, Woods Hole, MA. ‘BioCmrents Research Center, Marine Biological Laboratory, Woods Hole, MA, 3Women and Infants Hospital, Brown University, Providence, RI. Objective: Poor quality embryos display morphological features consistent with apoptosis including; shrinkage, membrane blebbing and cellular fragmentation [ 11. Physiological changes undoubtedly precede these morphological transformations and could possibly serve as markers of embryo viability. In particular, altered potassium homeostasis occurs during apoptosis in somatic cells [2]. Therefore, we sought to employ the non-invasive, self-referencing, ion-sensitive electrode 131 to determine whether embryos undergoing apoptosis induced by oxidative stress displayed altered potassium homeostasis. Materials and Methods: Mouse embryos were harvested from B6C3Fl mice and cultured in KSOM using standard techniques. Physiological measurements were conducted in modified KSOM (4-14 KSOM) containing reduced NaHC03 (4 n&l) and elevated HEPES (14 mM). Potassiumselective electrodes were fabricated using K+-ionophore I-cocktail B (Fluka) and operated in self-referencing mode [3] by oscillating the electrode in a square wave between two points 5 or 10 microns apart at 0.3 Hz. Calculating the difference between measurements at these two sites reduces the contribution of electronic drift and allows estimation of tlux across the cells’ membrane. Electrode movements, signal amplification and data acquisition and storage all were conducted using hardware and software developed by the BioCurrents Research Center at the Marine Biological Laboratory, Woods Hole, MA. Results: The potassium concentration of the media surrounding embryos increases during the initial phases of apoptosis. Treatment of zygotes with 200 PM H202 induced a potassium efflux that increased the potassium concentration near the embryo by 1.5 2 0.4 PM over that of the bulk media (2.1-2.3 mM). The peak increase in potassium concentration occurred 22 ? 7 minutes after the application of H202. Embryos treated with H202 displayed shrinkage and membrane blebbing, consistent with apoptosis, and failed to cleave. Embryos became propidium iodide permeant and exhibited DNA fragmentation only 72 hours after H202 application. Pretreatment of embryos with the potassium channel blocker tetraethylammonium chloride (TEA-75mM) prevented H202-induced potassium efflux and suggests that

FERTILITY

& STERILITY@

September 3:45 P.M.

28, 1999

O-101 The Likelihood of Pregnancy with Consecutive Failed Cycles of Intrauterine Insemination (WI). ‘,‘B A Stone, 3J. M. Vargyas, sG. E. Ringler, 3A. L. Stein, ‘,sR. P. Marrs. ‘Institute for Fertility Research, ‘Reproductive Technology Laboratories and 3Califomia Fertility Associates, Santa Monica, CA. Objective: For those couples in which IUI is a viable treatment option, most ART programs currently recommend six (failed) cycles of IUI before progressing to more aggressive ART procedures. The present study examines outcomes of IUI cycles in order to establish whether six IUI cycles appropriately exhausts the likelihood of pregnancy through IUI. Design: Retrospective analysis of outcomes of consecutive IUI cycles in an ART center. Materials and Methods: Between January 1991 and December 1996, 9,963 consecutive cycles of IUI by 116 physicians yielded 1,017 pregnancies. Outcomes of consecutive repeated cycles of IUI are compared by Chi2. Results: The average (?SE) age of patients presenting for IUI increased from 36.1 (20.2) years in 1991 to 39.2 (tO.1) years in 1996. The overall pregnancy rate per IUI cycle was 10.5%, similar to values reported elsewhere (Fertil Steril 48:921-7; 1987). Age-corrected values (18.6%/cycle) were higher. Of all pregnancies, 51.7% occurred in the first cycle of IUI, compared with 19.2% in the second, 12.2% in the third, 6.8% in the fourth, 4.2% in the fifth, 3.3% in the sixth, and 2.8% in ~7 cycles. In those patients who conceived through IUI, the likelihood of pregnancy in the first cycle was therefore 51.7%. If pregnancy did not occur in the first IUI cycle, the likelihood of pregnancy in the second cycle decreased to 28.4% (Chia=26.9; P40 years, none of which were statistically significant. For those patients who conceived at 530 years of age, the average number of IUI cycles for pregnancy was 1.99, compared with 2.19 cycles at 31-40 years, and 2.24 cycles for patients conceiving after age 40 years. Conclusion: Our analysis fails to provide a statistical rationale for attemotine more than 3 consecutive failed cvcles of optimized IUI before more aggressive ART, regardless of patient age. I

Y

Tuesday,

September 4:00 P.M.

28, 1999

O-102 Setting Limits at Assisted Reproductive Technology (ART) Clinics: Results of a Survey on Access to Services. ‘J. E. Stem, 2C. P. Cramer, ‘A. C. Garrod, ‘R. M. Green. ‘Dartmouth-Hitchcock Medical Center, Lebanon NH and ‘Dartmouth College, Hanover, NH.

s39

Objectives: ART clinics are often confronted with complex social and ethical questions concerning whether or not to treat particular patients. Patients who are unmarried, HIV positive, or who exhibit previous violent behaviors are examples of cases that may be problematic. Also at issue are use of gametes from family members or friends and unlimited use of advanced technologies. The goal of the present study was to investigate clinic policy and attitudes of providers toward limiting patient access to services at ART clinics in the USA. Design: A survey was sent to the clinic directors of 324 US ART clinics. Materials and Methods: Approval for this study was obtained from the IRB at Dartmouth College and the Society for Assisted Reproductive Technology (SART) Research Committee. The 16.page surveys were sent to the directors of all of the SART-associated ART clinics in the USA. Respondents were asked to identify clinic policy as well as personal attitudes on these issues. Follow-up letters were sent to nonrespondents. Results: Fifty-seven percent of clinics responded to the survey. Respondents came from all regions of the country and represented the demographics of the original sample. Fewer than half of the clinics, 40%, indicated that they had written policies on access to services issues. 30% of those clinics with policies do not follow their written policies all of the time. Results show a lack of universal agreement on policy for any of the situations posed, although some policies are more widely-held than others. Over 70% of all respondents are willing to treat unmarried heterosexual couples, single women and lesbian couples. Over 70% also limit numbers of procedures by age/FSH and set an age limit on the use of donor eggs. Fewer than 30% of clinics set an age limit on men for use of their own sperm or donor sperm. Fewer than 30% will treat HIV positive women. Greater uncertainty, in terms both of whether or not there was a policy and of what form the policy took, surrounded whether they treat patients known to use excessive alcohol, use illegal drugs, have a mental impairment, or have a history of child abuse. Interestingly, individual attitudes of respondents on these questions were more consistent and indicated a greater reluctance to treat patients with these conditions. Conclusions: These data indicate considerable variability in policy between clinics on most access to services questions. In general, the more complex the cases, the more varied the response: varied responses may be indicative of frequent case-by-case analysis of complex situations. These results highlight the importance of ongoing discussion of access issues and the need to develop consistent methods to evaluate complex cases. This work was supported by a grant from the Hitchcock Foundation at Dartmouth College.

Tuesday,

September

4:15

28, 1999

P.M.

O-103 IVF-ET With Processed Semen of HIV-Positive Males in Infertile HIV-Discordant Couples. A. E. Semprini, A. Vucetich, M. Oneta, V. Savasi, C. Castagna, P. Seralini, A. Bulfoni, and S. Fiore. Department of Obstetrics and Gynaecology, San Paolo Biomedical Institute, University of Milan, Medical School. Objective: Analyse the pregnancy rate of infertile HIV-discordant couples seeking assisted conception by IVF-ET with processed semen of the HIV-infected partner to reduce the risk of infection for the seronegative woman. Design: Prospective observational study. Materials and Methods: Since 1989 we have been giving assistance to HIV-discordant couples by intrauterine insemination with processed semen without any case of HIV transmission to the women or their offspring. Selection criteria for this study were the presence of infertility factors precluding conception by insemination or repeated failure to conceive after multiple insemination attempts. 43 couples were enrolled for an IVF-ET procedure. The semen of the HIV-positive males was processed as for insemination and tested by PCR assay to exclude contamination with HIV RNA before performing a standard IVF-ET procedure. The 43 couples accepted the possibility of a residual risk of infection with HIV RNA in the spermatozoa aliquot below the sensitivity of the detecting method we used (800 copies/ml). All women and

s40

Abstracts

children, delivered after the procedure. Results:

so far, remain

negative

Number of IVF-ET/number of couples Pregnancies after IVF-ET Pregnancy rate per couple Clinical miscarriages Ongoing pregnancies Singleton pregnancies Twins Triplets Women delivered Livebirths Maternal infection or congenital infections

for HIV-Ab

after IVF

and HIV

DNA

48143 13 30 3 6 6 2 1 4 7 0

Conclusions: Semen processing and IVF-ET offer the possibility to achieve pregnancy in infertile HIV-discordant couples who cannot conceive by IUI. The success rate in our program is comparable to that of uninfected couples needing IVF-ET. Infertile HIV-discordant couples can be assisted by IVF-ET if they accept a minimal possibility of infection.

Tuesday,

September

4:30

28, 1999

P.M.

O-104 The Risk of Ovarian, Breast, and Uterine Cancer Associated with Infertility and Infertility Treatments. ‘M. S. Croughan-Minihane, ‘L. Camarano, ‘M. Martin Cadieux, ‘V. Emster, ‘K. Kerlikowske, ‘C. Zaloudek, ‘?S. Feigenbaum, ‘,4P. Nelson, ‘.4D. Adamson, ‘D. Deapen, 3L. Bernstein, ‘D. Black, 4A. Whittemore. ‘University of California at San Francisco, ‘Kaiser Permanente Medical Care Program, 3University of Southern California, and ‘Stanford University, CA, USA. Objectives: The primary aim of this case-cohort study is to determine if infertile women exposed to ovulation-inducing agents (OIAs) are at higher risk for ovarian, breast, or uterine corpus cancer than infertile women not exposed to OIAs. The second aim of this study is to determine if there is a difference in cancer risk between infertile women treated with OIAs, presumed fertile women treated with OIAs for timed conception, and fertile women treated with OIAs for the purposes of ovum donation to determine the possible independent effects of infertility and OIAs on cancer risk. Design: We have assembled a cohort of 56,669 women treated for infertility in 15 infertility centers in California between l/1/65 and l/l/98. Tracing and linkage information was abstracted from medical records for all women comprising the study cohort. Linkages with tumor registries have identified 87 cases of ovarian cancer, 616 cases of breast cancer, and 57 cases of endometrial cancer. Full medical record reviews and interviews are being conducted for cancer cases and 7,600 subcohort members to gather complete information on OIA exposure as well as reproductive history, health status, family history, and infertility treatment-related conditions. Data collection will be completed by 7/l/99. Results: The relationship between OIAs and cancer will be analyzed using the proportional hazards regression model, adjusted for the casecohort design. Analyses will consider differences in risk according to formulation, dose and duration of OIAs as well as among subgroups defined by infertility etiology, parity, anovulation, and demographic characteristics among others. The results of this analysis will be completed by 9/l/99. This work is supported by the National Cancer Institute.

Tuesday, September 28, 1999 4:45 P.M. O-105 Real-Time PCR Using Molecular Y Chromosome in Single Human Sanchez, ‘C. Brenner, ‘5. Barr&t,

Beacons for Accurate Detection of the Cells. ‘L. .I. Wangh, ‘J. E. Rice, ‘J. A. ‘K. E. Pierce. ‘Department of Biology,

Vol. 72, No. 3, Suppl. 1, September 1999

Brandeis Medicine

University, Waltham, MA and ‘The Institute for Reproductive and Science of Saint Bamabus, Livingston, NJ.

Objectives: A highly-accurate method for sexing individual human cells using new PCR technologies is presented. The assay serves as a model for preimplantation genetic diagnosis for cases of X-linked diseases. Design: The highly conserved, Y-chromosome-specific TSPY genes (about 27-40 copies/male cell) and the autosomal U2 genes (about 20-40 copies/cell) were amplified from single lymphocytes and blastomeres. Molecular beacons, a new type of fluorescent oligonucleotide probe, were used to monitor the accumulation of amplicons during PCR (i.e., in “real time”). Materials and Methods: Single lymphocytes from male and female donors and single blastomeres from donated, nonviable human embryos were transferred to optical PCR tubes and lysed using an optimized proteinase K/SDS protocol. A PCR reagent mix that included primer pairs and molecular beacons for TSPY and U2 genes was then added to each tube and amplification was carried out in an ABI Prism 7700 Sequence Detector. Fluorescence was monitored during the annealing step of each cycle of the reaction. The initial point of signal detection (threshold cycle, C,) and fluorescence intensity at cycle 38 (about 5 cycles later) were used to measure the efficiency of each PCR assay. Results: Fluorescent signals above threshold were detected in 232 of 240 samples. As expected, all 118 female samples exhibiting a U2 signal had no TSPY signal. A TSPY signal was observed in 113 of 114 male lymphocyte samples. Thus, 231 of 232 lymphocytes (99.6%) were accurately scored for gender. Test of human embryos and blastomeres using this assay are ongoing in our laboratory. Conclusions: Detection of the Y chromosome in single cells using molecular-beacon-monitored PCR is very accurate. Cell lysis, gene amplification, and real-time analysis of multiple samples is simple and can be completed in under 4 hours. These reactions are carried out in sealed tubes and do not require use of nested-PCR primers or electrophoretic analysis of the resulting amplicons, greatly reducing the risk of contamination within the laboratory. Our findings point the way to a new, convenient, reliable PCR strategy that can be used for preimplantation genetic analysis. This work was supported by a grant from Hamilton Thorne Research, Inc.

ASSISTED REPRODUCTIVE TECHNOLOGY GAMETE EMBRYO ENVIRONMENT

Tuesday,

September 290 P.M.

28,

1999

O-106 Current Status of Computer-Aided Human Sperm Morphology Evaluation Using the IVOS (Dimension System). ‘K. Coetzee, ‘T. F. Kruger, ID. M. Douglas-Hamilton. ‘Reproductive Biology Unit, Department of Obstetrics and Gynecology, University of Stellenbosch, Tygerberg Hospital, Tygerberg, RSA and ‘Hamilton Thome Research, Beverly, MA, USA. Objectives: Progress in the evaluation of human semen ejaculates demands the development of accurate and reliable automated semen analyzers, not only to eliminate the inherent subjectivity and variability associated with the performance of a standard semen analysis, but also to increase its diagnostic value through quantification and discriminating statistical evaluation. We therefore systematically investigated the critical aspects determining the performance of the Hamilton Thome Research semen analyzer (IVOS, dimension system); 1) repeat measures and 2) its predictive value for assisted reproduction. Design: Repeat normal sperm morphology evaluations were analyzed on cell-cell, inter-slide, i&a-slide and inter-laboratory evaluations. Prospectively analyzed normal sperm morphology outcomes were also used to determine their value in predicting fertilization in vitro and pregnancy in a gamete intrafallopian tube transfer (GIFT) program. Materials and Methods: Liquefied semen samples were washed, by centrifugation, once using serum supplemented (human serum albumin, 3 mg/mJ) Earle’s Balanced Salt solution and stained using Diff-Quik stain. Sperm morphology was evaluated according to the strict criteria and the

FERTILITY

& STERILITY@

normal sperm morphology outcomes recorded. The repeat measures performed were, cell-cell (300 cells 3X each), intra-slide (20 slides 3X each), inter-slide (30 samples 3X slides each) and inter-laboratory (30 slides, 5 laboratories). The standard Tygerberg GIFT procedure was followed with all supernumerary oocytes inseminated and cultured. Fertilization in vitro and pregnancy outcomes were recorded. Results: The predictive probabilities for an abnormal or a normal cell given two prior abnormal or two prior normal cell outcomes was 95% and 94% respectively. The average coefficients of variation obtained for the intra-slide trial was 9.73% and for the inter-slide trial it was 15.39%. The average inter-laboratory coefficients of variation obtained ranged between 16.3% and 23.1%. The logistic regression model showed that the normal sperm morphology outcomes recorded were significantly associated with fertilization in vitro and pregnancy. Analyzing the data across the 5% normal sperm morphology cut-point; fertilization rates of 39.4% (~5%) and 62.9% (15%) and pregnancy rates of 15.2% (55%) and 37.4% (>5%) were obtained. Conclusion: The magnitudes of variation produced by the repeat readings performed attained the same level that is obtainable by very experienced manual observers. Adhering to adequate quality control steps a less than 10% coefficient of variation will be attainable. Normal sperm morphology as evaluated by the IVOS was shown to have significant predictive value across the 5% cut-point. The IVOS used has produced a level of repeatability, precision and accuracy acceptable for routine application.

Tuesday,

September 2:15 P.M.

28, 1999

O-107 Mitochondrial Transfer to Recipient Oocytes During Ooplasmic Transplantation. ‘J. A. Bar&t, iR. R. Adler, ‘C. A. Brenner, ‘J. Cohen. ‘Gamete and Embryo Research Laboratory, The Institute for Reproductive Medicine and Science of Saint Barnabas, West Orange, NJ, USA. Objective: It is unknown whether transferred cytoplasm is representative of the donor cell during ooplasmic donation. This study was undertaken to investigate if mitochondria are transferred during ooplasmic transplantation by injection. Design: Confocal fluorescent imaging was used to investigate if mitochondria are transferred during ooplasmic transplantation. Materials and Methods: Discarded oocytes were obtained from patients undergoing ICSI and were cultured overnight. In-vitro matured donor oocytes were stained with MitoTracker*M Green FM (Molecular Probes, Eugene OR) to label the mitochondria. Ooplasmic transfer was simulated using intra-cytoplasmic injection of approximately 5-15% of the volume. Ooplasmic transfer was performed using MitoTrackerTM Green labeled donor oocytes and unlabeled recipient oocytes. Confocal imaging was performed to determine if mitochondria were transferred from the donor to the recipient oocytes at 10 minutes and 3 hours after injection. Results: Confocal microscopy confirmed that control donor oocytes contained fluorescently labeled mitochondria throughout the cytoplasm. Unstained control oocytes contained no fluorescent mitochondria in the cytoplasm. Observations 10 minutes after injection showed recipient oocytes containing MitoTrackerTM Green labeled donor mitochondria in a small localized region. Only the region injected with donor ooplasm was stained. Observation 3 hours after injection showed some diffusion of the labeled mitochondria throughout the recipient cytoplasm. Conclusion: Ooplasm extracted from donor oocytes contains mitochondria which are transferred to recipient oocytes. Thus, the recipient oocyte contains a heteroplasmic population of mitochondria immediately after ooplasmic transfer. The nuclear regulation of heteroplasmic mitochondria in the cytoplasm is currently under investigation as well as the potential transfer of other organelles and proteins.

Tuesday,

September 2:30 P.M.

28, 1999

O-108 Relationship Developmental

Between Pronuclear Quality, Nucleoli Number Potential of the Embryo. ‘C. Conord, ‘N. Frydman,

and ‘M.

s41

Benkalifa, ‘M. Volant, ‘E. Dulioust, ‘F. Olivennes, ‘S. Hamamah. ‘Centre de fecondation in vitro, hopital A. Becltre, 157 rue de la Porte de Trivaux, 92141 Clamart, France and ‘Departement de Gynecologie-Obstetrique, hopital A. B&&e, 157 rue de la Porte de Trivaux, 92141 Clamart, France. Objectives: Some embryos morphological features such as blastomers regularity and anucleated fragments % have previously been shown to be correlated with increased pregnancy rates. Embryos with good morphology are possibly those with the correct spatial arrangements and polarization. In this study, we have attempted to establish a relationship between the zygotes pronuclear morphology regarding pronuclei surface (ps), pronuclei diameter (pd), and number and position of nucleoli for each pronucleus (PN), and embryonic development. Design: Zygotes pronuclear morphology regarding pronuclei surface (ps), pronuclei diameter (pd), number and position of nucleoli for each pronucleus (PN), and embryonic development were determined. Material and Methods: 27 couples unselected for age or infertility criteria constituted a continuous series. A total of 250 oocytes were collected either for IVF or ICSI: 200 were inseminated and resulted in 119 normal pronuclear stage embryos. Whatever IVF or ICSI, all zygotes were checked for pronuclear quality 20-22 h after insemination and recorded and stocked in computer. For each zygote, the studied parameters were: (i) number and localization of nucleoli of each pronucleus (polarized at the pronuclear junction or unpolarized), and (ii) pronuclei surface and diameter. The study has been performed by using Visiolab 200 program (Biocom). 28 transferred embryos (2.5 embryos/ couple, n= 11) leading to pregnancy have been compared with 50 transferred (3. I embryos/couple, n= 16) without pregnancy according above studied parameters. Results: Data according to all studied parameters are summarized in Table. Values are mea&SD. 2 PN polarized

1 PN polarized

2 PN unpolarized

49

26 (53%)

10 (20%)

13 (26.5%)

70

19 (27%)

13 (19%)

38 (54%)

Zygote W) pregnancy no pregnancy

ps bm*) pregnancy no pregnancy

448 529 454 526

2 i i +-

pd (wm)

66 61 106 114

23.8 25.9 23.9 25.8

k 2 + 2

1.8 1.5 3.0 3.0

nucleoli

3.ll”‘PN 4.8 2dPN 3.51”PN 5.42dPN

Tuesday, September 28, 1999 P.M.

O-109 Autologous Endometrial Coculture (AECC) Is Associated with a High Rate of Success in Patients Under 36 Years Old with a History of a Single IVF-ET Failure Secondary to Poor Embryo Development. S. D. Spandorfer, R. Clarke, L. Bovis, H.-C. Liu, L. Veeck, 0. K. Davis, Z. Rosenwaks. Department of Obstetrics/Gynecology. The New York Hospital-Cornell University Medical Center, New York, NY. Objective: AECC has been proposed as a method of improving embryo quality and outcome after IVF-ET. We have previously demonstrated a single failed IVF cycle in patients less than the age of 36 years is negatively associated with outcome in a subsequent attempt (SGI, March 1999).

s42

Abstracts

# blastomeres % Fragmentation Pregnancy Clinical Preenancv

Previous Cycle-@ ET (No AECC)

Treatment Cycle-@ ET (AECC)

P value

6.8 (k 1.6) 20.0 (5 19.1) 0% 0%

7.3 (k 1.3) 10.5 (i 7.4) 89.5% 84.2%

0.16 0.04
Conclusion: We have demonstrated an improvement in embryo quality when utilizing AECC. We are able to demonstrate a clinical pregnancy rate of over 80% in these patients. This suggests that the use of AECC may be beneficial in patients with a history of a single failed IVF-ET cycle.

Tuesday, September 28, 1999

(nb)

Conclusion: This data show that there is a slight difference regarding surface as well as diameter for first and second PN in the same zygote leading or non to pregnancy. We have also observed a significant difference in the total number of nucleoli per zygote (7.9 Vs 8.9; p
2~45

Clinical pregnancies significantly decreased from 64% to 53%. In this pilot study, we analyzed the effectiveness of AECC in improving embryo quality and pregnancy rates in young patients (age 535 years) with a history of a single failed IVF-ET cycle. Design: Embryos from each of 19 patients with a single failed IVF-ET cycle (from our institution) associated with poor embryo quality (Grades 3 or below, Veeck Criteria) were allocated to growth on AECC in a subsequent cycle. Materials and Methods: An endometrial biopsy was performed in the luteal phase (timed by a urinary ovulation predictor kit) in a natural cycle prior to the patient’s IVF-ET treatment cycle. The day of LH surge detection was denoted as cycle day (CD) #14 and all biopsies were performed on/after cycle day #19. Glandular (C) and stromal (S) cells were isolated by enzymatic digestion and separated based on differential sedimentation rates. These ceils were cryopreserved, then plated as a combination of 50% G and 50% S cells prior to embryo exposure. The day after oocyte retrieval (at the pronuclear stage) the zygotes were placed on AECC. Statistics included non-parametric paired t-tests. Results: 19 patients with a mean age of 32.7 (22.5) years and a history of 1 previous failed IVF-ET cycles were enrolled in the study. 58% (1 l/19) of the women were nulligravidas. Table 1 illustrates the mean blastomere number and percent fragmentation (frag) at the time of ET and pregnancy outcome.

3:00

P.M.

O-110 Prospective Randomized Crossover Analysis of the Impact of an IVF Incubator Air Filtration Svstem (Coda. GenX) on Clinical Preanancv Rates. .I. F. Mayer, F. Nehchiri, V. M. Weedon, ‘E. L. Jones, H. LyKalin, S. C. Oehninger, J. P. Toner, W. E. Gibbons. S. J. Muasher. The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, VA. Objective: To investigate the potential effect of new specialized intraincubator chamber air filters (Gen X, Connecticut, USA) on embryo quality and clinical pregnancy rates. Design: Prospective randomized study of 110 IVF cycles from Oct. 1998 to Feb. 1999 at an academic, tertiary care institution. Materials and Methods: Mature oocytes were collected following standard controlled ovarian stimulation protocols. At retrieval, patients were randomly assigned to one of two groups. In Group 1, all gametes and embryos were cultured in Forma water-jacketed CO, incubators (model #3326) with “Coda” (Gen X) intra-incubator air purification units while Group 2 was cultured in identical incubators without filter units. All other culture conditions were identical between the two groups, On day three (-72 hours post retrieval) embryos with the best morphology were chosen for fresh transfer. Embryo morphology was graded on a scale of l-5 with 1 being best. After the first 41 cycles in each group, the filter units were removed from initially filtered incubators and transferred to the previously non-filtered incubator chambers. This crossover procedure was performed to eliminate any variables due to the performance of specific incubator chambers. All transfers were to the uterine cavity on Day 3 and were identical for the two groups. Results: The results (summarized below in Table 1) were analyzed by Chi-square. Table 1. Comparison of Outcomes in Filtered and Non-filtered

Vol. 72, No. 3, Suppl. 1, September 1999

Incubators

Filtered

# Treatment Cycles As % 2pn Fertilization # Embryos Transfer # Good quality embryos % Clinical Pregnancy * + = SEM,

trans.

S = significant;

51 34.4 2 0.7* 73.7% 3.7 k 0.2 2.3 2 0.23 54% (31/57)

Non-Filtered 53 34.0 f 0.6 79.0% 3.5 k 0.2 2.8 t 0.21 29% (16/53)

Statistics

NS NS NS NS S (p < 0.018)

NS = not significant

Conclusions: These results demonstrate for the first time, a statistically significant improvement in pregnancy rates with an incubator air purification system. Interestingly, no improvement in embryo morphology after culture in the filtered incubators was observed. This suggests that any improved embryo potential may not be detectable by our method of embryo assessment.

ASSISTED

REPRODUCTIVE TECHNOLOGY FOLLICLE DYNAMICS Tuesday,

September 3:45 P.M.

28, 1999

o-111 In Vitro Preantral Follicle Maturation with Recombinant Gonadotropins Mimics Ovarian Stimulation In Vivo: A Potential Model for an Alternate Source of Oocytes. H. C. Liu, Z. Y. He, Z. Rosenwaks. The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, NY. Objective: To develop a method to mature preantral follicles isolated from ovarian tissue in vitro as an alternate source of competent oocytes for IVF-ET. Design: Throughout preantral follicle maturation in vitro, somatic cell proliferation, steroidogenesis, antral formation, oocyte maturation and embryogenesis were carefully assessed. Materials and Methods: Early preantral follicles (90-120wM) isolated from ovaries of D14 B6D2-Fl mice were singly cultured in microdroplets (20 ~1) with various culture medium: A, Ham’s FlO (n=40); B, Opti-Mem (containing 10 @g/ml insulin) (n=40); C, MEM medium with rLH/rFSH (IO/l00 mIU/ml) (n=25); D, Opti-MEM with rLH/rFSH (lo/100 mIU/ml) (n= 100); E, Opti-MEM with rLWrFSH (lOO/lOO mIU/ml) for 4 days then changed to Opti-MEM with rLH/rFSH (lOi mIU/ml) for the remaining days (n=lOO); and F, Opti-MEM with hMG (100 mlU/ml) (n=78) with medium change daily or every other day. Oocytes were ovulated by HCG/ EGF on day 12 for IVF. Somatic cell proliferation, steroidogenesis, antral formation, oocyte maturation and embryogenesis were assessed. Results: Follicle survival rates were O%, 77.5%. 8%, SO%, 77% and 80% in Medium A, B, C, D, E and F, respectively when the medium was changed every other day, and were 92%, 90%, 88% in medium D, E, and F, respectively when medium was changed daily. Changing media daily (vs. every other day) enhanced granulosa cell outgrowth rates on day 6 (61% vs 92% in D, 85% vs. 92% in E, and 75% vs. 77% in F), augmented Peak E2 concentrations (22,230 vs. 16,440 in D; 37,690 vs. 28,180 in E; 72950 vs. 24580 in F), increased antral formation rate on day 12 (16% vs. 12% in D; 32% vs. 21% in E; and 50% vs. 24% in F), and most importantly increased MI1 oocyte formation rate among the surviving follicles (47.8% vs. 17.7% in D; 53.3% vs. 16.9% in E; and 26.1% vs. 6.4%). Culture in medium E with daily changes seem to be optimal as 90% (45/50) isolated early preantral follicles survived, 53.3% (24145) of surviving follicles produced MI1 oocytes, and 50% (12/24) blastocyst formation after fertilization and culture in vitro. Conclusion: To obtain competent oocytes, preantral follicles must be cultured with medium containing insulin and gonadotropins with medium change daily. Though high initial rLH/rFSH (lOO/lOO mIU/ml) (i.e., F) facilitates thecal cell attachment, granulosa cell proliferation and steroidogenesis, prolonged culture in high rLH/rFSH trigger early differentiation and luteinization of granulosa cells, which is detrimental as reflected by low

FERTILITY

MI1 oocyte and blastocyst formation. However, this problem can be solved by lowering LH/FSH after 4 day culture (i.e., E) or by administering HCG/EGF one or two days earlier. Preantral follicle maturation in vitro can be applied clinically to obtain competent oocytes while bypassing ovarian stimulation in viva. This may be particularly beneficial for patients with physical conditions where ovarian stimulation is contraindicated and where ovarian tissue biopsies can be obtained or previously frozen. In addition, this method can serve as an excellent model for basic research to gain insight into folliculogenesis, oogenesis, and embryogenesis, and allow further study into the interactions between somatic cells and oocytes.

& STERILITY@

Tuesday,

September 4:00 P.M.

28,

1999

o-112 Follicular Homeostasis: Analysis of Vascular Endothelial Growth Factor (VEGF), Leptin and Nitric Oxide (NO) Levels in Individual Follicles, and Their Relationship with Perifollicular Blood Flow, 02 Content and the Developmental Potential of the Corresponding OocytelEmbryo. ‘G. Barroso, ‘M. Barrionuevo, 3P. Rao, 3L. Graham, 3W. Spellacy, 4D. Danforth, IS. Huey, ‘A. Abuhamad, ‘S. Oehninger. ‘The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, VA; ‘Northwest Center for Infertility and Reproductive Endocrinology, Margate, FL; ‘Department Obstetrics/Gynecology, University South Florida, Tampa, FL; 4Department Obstetrics/Gynecology, The Ohio State University, Columbus, OH. Objective: (1) To measure levels of VEGF, leptin and NO in individual ovarian follicles; and (2) to assess the relationship between those regulatory factors and perifollicular blood flow, follicular ~02, pCO2 and pH, and the quality and developmental capacity of the corresponding oocytelembryo. Design: Prospective study performed at academic, tertiary care institutions. Materials and methods: IVF patients undergoing controlled ovarian hyperstimulation were studied. Blood flow was measured by transvaginal, color pulsed Doppler on day hCG+ 1 in randomly designated-mapped ovarian follicles. Follicular fluid pO2 was measured amperometrically whereas pCO2 and pH were analyzed by ion selective electrode potentiometry using a portable clinical analyzer. Individual follicular fluids were frozen at -70°C until used. Oocytes and corresponding embryos were cultured individually; embryo cleavage and morphology were determined on day 3. Follicular fluid leptin and VEGF levels were measured by radioimmunoassay and ELISA, respectively. The stable products of NO oxidation levels were determined by the enzymatic conversion of nitrate to nitrite (N03/N02) and then measured calorimetrically. Results: Fifty-five follicular fluid samples from 16 patients were studied. Mean follicular fluid levels were: VEGF: 1,046.9?863.7 pg/mL (range: (63-3, 332.7), leptin, 20.1% 12.1 ng/mL (range: 3.3-52.2) and N03/N02, 34.2?12 PM (range: 16.4-76.1). Analysis of covariance was performed following data transformation to achieve normal distributions. VEGF had a negative correlation with embryo morphology (r= -0.28, p= 0.01). Follicular leptin levels correlated with VEGF levels (r= 0.46, p= 0.008) and with N03/N02 levels (r= 0.39, p= 0.006). Leptin demonstrated a negative correlation with follicular pO2 (r= -0.42, p= 0.005) and a positive correlation with follicular pCO2 (r= 0.36, p= 0.02). There were no significant associations between the follicular regulatory factors and age, peak serum estradiol levels, follicular diameter or pH, perifollicular blood flow Doppler indices or fertilization. Conclusions: VEGF, leptin and NO appear to play a role in follicular homeostasis and embryo development. These data support previous studies demonstrating that VEGF and NO are markers of follicular hypoxia and sub-optimal oocyte/embryo competence. Whether fluctuations of these molecules determine or reflect changes in the follicular microenvironment affecting oocyte developmental potential remains to be elucidated.

Tuesday,

September 4:15 P.M.

28, 1999

o-113 Immunosuppressive sequent Conception

Properties of Follicular in In Vitro Fertilization.

Fluid Correlate with Sub‘Z. Wiener-Megnagi, ‘E.

s43

Daniel-Spiegel, ‘M. Dimfeld, *S. Goldman, ‘H. Abramovici, ‘E. Shalev. ‘Division for Reproductive Endocrinology and ART, Department of Obstetrics and Gynecology, Ha’Emek Medical Centre, Afula, Israel. *In Vitro Fertilization Unit, Carmel Medical Centre, Haifa, Israel. Objectives: Pregnancy is assumed to be associated with suppression of a variety of humoral and cellular immunological functions. This is presumed to enable the accommodation of the semiallogenic fetal graft. There is no data regarding possible role for follicular fluid in this suppression. The current study aimed to evaluate immunosuppressive properties of follicular fluid (FF) and media conditioned by zygotes and early embryos, correlate with subsequent conception in assisted reproductive treatment. Design: In vitro evaluation of immunosuppressive properties of FF and zygote/embryo conditioned media in relation to later conception in ART. Materials and Methods: FF and media, conditioned be zygotes and early embryos were obtained from 70 women, who underwent oocyte retrieval for ART. Human lymphocytes from healthy people were isolated and incubated with two mitogens: Concavalin A (Con A) and Phytohemagglutinin (PHA) in combination with either follicular fluid from mature oocytes or culture medium conditioned by zygotes and early embryos. Proliferation was assessed by radioactive thymidine incorporation to the lymphocytes. Proliferation Index (PI) of the lymphocyte culture was defined as the ratio between radioactive labeling of lymphocytes incubated with mitogen to that of lymphocytes incubated without mitogen. Results: Culture media conditioned by zygotes and early embryos in conception cycles were found to have immunosuppressive properties for all concentrations of mitogens examined. Follicular fluids from conception cycles, had higher immunosuppressive activity (reflected by lower PI values) than those from cycles in which conception did not occur. Conclusions: The quality of the follicular apparatus and the oocyte, as reflected by immunosuppressive properties of FF are major determinants of chances for conception in ART. The possible physiological role of follicular fluid, in the establishment of later conception should be the aim of future studies,

Tuesday,

September 4:30 P.M.

28,

1999

o-114 Diffusion of Gonadotropin Into Follicle May Be Important Predictor of In Vitro Fertilization Outcome. ‘Y. Nagata, ‘T. Oki, ‘K. Honjou, *M. Sonoda, ‘K. Shirota, 9. Kawarabayashi. ‘Center for Reproductive Medicine, Nagata Clinic, Fukuoka, Japan. *Department of ObstetrlcdGynecology, School of Medicine, Fukuoka University, Fukuoka City, Fukuoka, Japan. Objective: In view of previous observations, the ovarian blood flow is unquestionably an important factor in the success or failure during IVF treatment. We prefer to assess the ovarian blood flow by evaluating phar macokinetic changes. Endogenous gonadotropins are suppressed during superovulation in IVF treatment cycles involving pituitary down-regulation. Patients undergoing IVF treatment lose the endogenous LH surge, and hCG has to be injected to mimic it. Studying the pharmacokinetics of injected hCG is a useful way of assessing gonadotropin diffusion. The purpose of this pharmacokinetic study was to examine the diffusion of hCG from serum to follicular fluid, and to assess how the diffusion of hCG is related to various parameters associated with the ovarian response to human IVF-ET programs. Design: Retrospective pharmacokinetic study. Material and Methods: Eighty-seven patients underwent 137 cycles of IVF-ET at Fukuoka University Hospital. Oocytes were recovered after hMG-hCG stimulation with GnRH agonist. The serum and follicular fluid were collected at the time of oocyte recovery from 34-37 hours after hCG injection. The serum and follicular hCG concentrations were measured by time-resolved fluoroimmunoassay. The hCG ratio (between follicular hCG and serum hCG concentrations, measured by time-resolved fluoroimmunoassay) was evaluated as an index of the diffusion of exogenous gonadotropin. Relationship between hCG ratio and the results and outcome of the IVF-ET program. Results: At the time of oocyte recovery, the mean serum hCG concentration for the 137 IVF-ET cycles was 198279 mIU/mL (range, 50-449),

6544

Abstracts

the mean follicular hCG concentration was 120269 mIU/mL (range, 17-366) and the mean hCG ratio was 0.5950.17 (range, 0.09-0.97). In a given patient, the follicular hCG concentration was always lower than the serum hCG concentration. The hCG ratio decreased with the total dosage of hMG, and increased with the serum E2 level, the number of oocytes recovered, and the number of oocytes fertilized. The poor responders showed a low hCG ratio. A complete lack of fertilization was associated with a low hCG ratio. The mean hCG ratio in the pregnant cycles were significantly higher than for the non-pregnant cycles. An hCG ratio higher than 0.46 was seen in all pregnant cycles. Conclusion: In conclusion, low diffusion of exogenous gonadotropin into the follicular fluid was found in poor responders to IVF cycles. The diffusion of exogenous gonadotropin into the follicular fluid may be an important predictor of IVF outcome.

Tuesday,

September 4:45 P.M.

28,

1999

o-115 Antral Follicle Assessment as a Tool for Predicting Outcome In Vitro Fertilization (IVF). R. T. Nahum, J. L. Shifren, Y.-C. Chang, L. Leykin, K. Isaacson, T. L. Toth. The Vincent IVF Unit, Department of Obstetrics and Gynecology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. Objective: The ovarian response to gonadotropin stimulation reflects the outcome of IVF. Advanced maternal patient age and elevated basal cycle day 3 follicle stimulating hormone (FSH) are associated with reduced ovarian response to gonadotropin stimulation and decreased IVF outcome. The purpose of this study is to determine if antral follicle (AF) assessment on cycle day 3 may serve as additional information in predicting IVF outcome. Design: Prospective descriptive preliminary study of IVF outcome in an academic in vitro fertilization program. Methods: 126 consecutive patients from July 1998 to December 1998, underwent AF assessment using transvaginal ultrasound (follicle size 2-6 mm diameter) on baseline of the planned stimulated IVF cycle. Outcome measures examined were cycle cancellation rate and clinical pregnancy rate (PR). The outcomes were analyzed with respect to AF assessment (56 or >6), basal cycle day 3 FSH (5 10 or > 10 IU/L) and maternal age (535 or >35 years). Of the 126 patients who participated, 55 had 56 AF, and 71 had >6 AF. Results: The clinical pregnancy rate is significantly higher with baseline AF >6 compared to AF 56 (51% vs. 24% respectively; P= 0.018). Controlling for patient age, the pregnancy rate for patients >35 years is significantly higher with AF was >6 compared to AF 56 (57% vs. 20% respectively; P=O.O14). The clinical pregnancy rate for patients 535 years is similar with AF >6 compared to AF 56 (48% vs. 38% respectively; P=O.4). Controlling for basal FSH level, the pregnancy rate for FSH 6 compared to AF 56 (49% vs. 23% respectively; p=O.O34). The cancellation rate is significantly increased with advancing maternal age, elevated basal FSH levels, and baseline AF 56. Controlling for maternal age and basal FSH, the cancellation rate is 0% with AF >6 compared to 17% with AF 56 (p=O.OOl). Conclusions: Baseline AF assessment is predictive of IVF outcome. In patients >35 years, AF assessment is a better predictor of pregnancy rates than basal FSH levels. AF assessment is a better predictor of cycle cancellation than maternal age or basal FSH.

MENTAL HEALTH PROFESSIONAL GROUP Tuesday,

September 200 P.M.

28, 1999

O-233 The Efficacy of Group Psychological Interventions on Reducing Distress in Infertile Women. ‘A. D. Domar, ‘D. Clapp, ‘E. Slawsby, *B. Kessel, 3J. Orav, ‘M. Freizinger. ‘Mind/Body Center for Women’s Health,

Vol.

72, No.

3, Suppl.

1, September

1999

Mind/Body Medical Institute and ‘Department of Obstetrics ogy, Beth Israel Deaconess Medical Center, 3Department Brigham and Women’s Hospital, Harvard Medical School.

and Gynecolof Medicine,

Objectives: Infertile women report elevated levels of distress when compared to fertile women. Depressive symptoms peak between the second to third year of infertility. This preventive intervention study investigated if either of two group psychological interventions offered between the first and second year of infertility could prevent the surge of depression and other negative psychological symptoms usually seen as infertility treatment continues. Design: The study employed a randomized, controlled, prospective, single-blind design. Materials and Methods: IRB approval was obtained for this study. Women who had been trying to conceive for one to two years and were not currently clinically depressed completed a battery of psychological questionnaires and then randomized to either attend a ten session cognitive behavioral (CB) group, a ten session support (S) group, or a routine care control (C) group. All subjects continued to receive routine medical care. The CB subjects learned relaxation and stress management techniques, while the S subjects spent group time discussing the impact of infertility. Subjects came in for repeat psychological testing six months later (Time 2 visit). Results: A total of 185 women were recruited for the study. Sixty one both did not conceive and agreed to come in for the Time 2 visit. The study sample included 21 CB, 27 S and 13 C subjects. There were no significant differences at intake between groups on any demographic variable (including age) or any psychological measure, with the exception that the CB subjects had the worst score at intake on lifestyle-stress management skills (p=O.Ol). At Time 2, the two intervention groups had significantly greater positive changes in self-actualization (p=O.O28), interpersonal support (p=O.O08), use of stress-management skills (p=O.OOOl), state anxiety (p=O.O14), and marital distress (p=O.O42) than the C subjects, as well as a trend toward less depression (p=O.O7). The CB subjects had greater positive changes in stress management skills (p=O.OOOl) and lifestyle habits (p=O.O008) than the S subjects. There were no significant differences between groups in self-esteem, health responsibility, exercise or nutrition habits, or trait anxiety. On all measurements, the C subjects showed a worsening of symptoms over time, while the CB and S subjects showed moderate improvement, with the CB subjects experiencing the most improvement. Conclusions: Group psychological interventions are efficacious in preventing worsening of most psychological symptoms during infertility, with the CB intervention group receiving the most benefit. This study was supported by a grant from the National Institute of Mental Health.

Tuesday,

September 2:15 P.M.

28, 1999

O-234 The Psychological Status of In Vitro Fertilization (IVF) Patients in the Third Trimester of Pregnancy: A Controlled Study. ‘D. A. Greenfeld, ‘S. C. Klock. ‘Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT. ‘Department of Obstetrics and Gynecology, Northwestern University School of Medicine, Chicago, IL. Objectives: It has been suggested that women pregnant as a result of IVF may be more distressed during pregnancy than women who become pregnant naturally. We have previously demonstrated that pregnant IVF and control subjects do not differ on depression, anxiety and self-esteem during the first trimester. The purpose of this study was to compare the psychological status (marital adjustment, self-esteem, depression, and anxiety) of pregnant IVF patients with fertile pregnant women during the third trimester of pregnancy. Design: Case control study. Materials and Method: Subject inclusion criteria were: 1) no previous live births; 2) between the ages of 18-45; 3) English speaking; and 4) married. 74 IVF women who conceived from two IVF programs met inclusion criteria and were invited to participate. 40 women who conceived spontaneously and met inclusion criteria were recruited from two private practices and were control subjects. 12 IVF women subsequently either miscarried (n=7) or dropped out (n=5) and 7 controls dropped out. 62 IVF and 33

FERTILITY

& STERILITY@

control subjects completed the study questionnaires at the beginning of the third trimester. The questionnaire included demographic and health items; marital adjustment; the rewards and concerns of parenting; a self-esteem scale (SE); the Beck depression inventory (BDI) and the State-Trait (STAI) anxiety inventory. The study was approved by the Human Subject Review Boards. Results: 18 IVF women (29%) were pregnant with twins and 3 (5%) were pregnant with triplets compared to only 1 (3%) control woman pregnant with twins (Chi-square = 11.5, p c.003). There were no significant differences between groups on measures of marital functioning, self-esteem, depression or anxiety. Mean scores on all of these measures were in the normal range for both groups. For rewards of parenting, IVF women were significantly more satisfied with being able to get pregnant and were less concerned with closer family relationships with impending parenting. IVF women were significantly less concerned than control women with changes in weight, sexual function, potential loss of husband’s attention, alienation from husband, restriction of independence, expenses and decreased income due to parenting. Paired t-tests comparing subject’s scores on marital adjustment, self-esteem, depression and anxiety from Time 1 (12 weeks of pregnancy) to Time 2 (beginning of third trimester) indicated significant differences across time with IVF women reporting decreased scores on measures of their marriage as a romantic/sexual relationship (Time 1=5.6, Time 2=5.2, p < .006), improved self-esteem (Time 1=14.6, Time 2=13.7, p < .Ol*) and decreased state anxiety (Time 1=35.2, Time 2=32.0, p < .Ol). Control subjects reported significant decreases in their marriages as a friendship (Time 1=6.8, Time 2=6.6, p < .Ol) and as a romantic/sexual relationship (Time 1=5.9, *Time 2=5.4, p < .03). *lower scores indicate better self-esteem. Conclusions: IVF women differ significantly from controls in several aspects of what they consider to be the rewards and concerns of impending parenting. There were no differences between IVF and control women on levels of marital functioning, self-esteem, depression and anxiety during the third trimester of pregnancy but changes within groups over time were noted with IVF women reporting less anxiety and greater self-esteem as they progressed from the first to the third trimester.

Tuesday,

September 2:30 P.M.

28, 1999

O-235 Oocyte Donation: Parents’ Perspective on the Creation of a National Registry, Parental Satisfaction, and Child Disclosure. S. C. Williams, W. Freije, L. Lundquist, D. Weaver, L. Blankenship, B. R. Carr, S. J. Chantilis. The University of Texas Southwestern Medical Center at Dallas, Dallas, TX. Objectives: The purpose of this study was to evaluate patients’ perspectives regarding the creation of a national registry for gamete donors, the satisfaction of parenting following the delivery of a live infant following oocyte donation, and the issue of child disclosure pertaining to the oocyte donation process. Design: A voluntary, anonymous questionnaire was mailed to patients who successfully delivered a child after completing an anonymous donor oocyte IVF cycle. Materials and Methods: All donor oocyte IVF patients completing their cycles by December 1997 who subsequently delivered a livebom child were surveyed using an IRB-approved questionnaire. Information regarding the patients’ views of the creation of a national gamete donor registry, parental bonding satisfaction, and child disclosure as relating to donor oocytes was inquired. Results: From a total of 42 patients whose donor oocyte pregnancy culminated in a livebom infant, 36 (85.7%) returned completed questionnaires. When asked if there is a need to establish a registry for egg donors, 42% agreed, while 58% disagreed. When asked if a national registry should be created in which the offspring of gamete donation could learn the identity of a donor, only 8% agreed, while 92% disagreed. If only non-identifying information such as health history were accessible, 44% agreed that a registry should be created, and 56% still disagreed. If a governmental registry existed, 77% of patients believed a breach of confidentiality could occur. 42% are worried about the child’s future health with respect to his genetic inheritance, while 58% are not concerned. All patients reported satisfactory maternal bonding with their children, however, 8% would feel

s45

differently, i.e., more satisfied, with a child conceived without a donor gamete. All patients believed their spouse was satisfied with paternal bonding, yet 6% agreed the spouse would feel more satisfied with a naturally conceived child. 42% of patients are planning to disclose the issue of oocyte donation to the child, while 3 1% are not, and 28% are undecided. 53% have informed family or friends of the oocyte donation process, while 47% have remained confidential. Conclusion: A majority of patients object to establishing a national donor gamete registry, especially if donor-identifying information were accessible. Even if only non-identifying data relating to health history were available in a registry, most parents do not support the creation of a national registry. This may be due to the fact that in our population, only 42% of patients were concerned about any future genetic health problems. Over three-fourths of patients believe a breach of confidentiality relating to the donor oocyte process could occur if a registry were created. In general, women are satisfied with maternal-child bonding following oocyte donation, however, a few women believe that bonding would be stronger if the children were genetically related to them. The decision to inform the child about this process remains mixed, and several patients were undecided about disclosing this information. The preparatory psychological counseling session should concentrate on the issue of disclosure, as it appears satisfaction is universal. This information can be useful for mental health professionals who counsel these patients.

Tuesday,

September 28, 1999 ’ 2:45

P.M.

O-236 The Psychological Correlates of Vasectomy. ‘J. I. Sandlow, *J. S. Westefeld, ‘h/I. Maples, ‘K. Scheel. The University of Iowa Departments of ‘Urology and *Psychology, Iowa City, Iowa, USA. Objective: Vasectomy is the most common form of permanent sterilization in use today. The postoperative psychological aspects of vasectomy have been previously studied, and it has been demonstrated that men have a more difficult time adjusting than sterilized women. However, there has not been any attempt to address the concerns of men prior to vasectomy. Therefore, we examined the psychological factors involved prior to the decision to undergo a vasectomy. Design: Questionnaires were distributed to patients prior to their evaluation for vasectomy at a university urology clinic. Materials and Methods: Following IRB approval, 74 men completed a 2-part questionnaire prior to their clinic visit for consideration of a vasectomy. The first part consisted of 20 questions pertaining to the patient’s background, reasons for vasectomy, who participated in the decision, and the patient’s anxiety level. The second part consisted of the Tennessee Self-Concept Scale, the Problem Solving Inventory, and the Partner Relationship Inventory. All responses were confidential, and the treating urologist did not have access to them prior to performing the procedure. Results: 74 questionnaires were completed. The average age was 36 years. Most of the respondents were white males of high socioeconomic status, All but one were married or in a committed relationship. More than half of the men had been thinking about undergoing a vasectomy for 1 year or less, and 70 (95%) stated that their partner was involved in the decision, as well as supportive of it. The main reason for having the procedure was listed as desiring no further children in 31 (42%), female factors in 24 (32%). age in 7 (9%), and financial issues in 5 (7%). Anxiety was rated on a scale of l-10 (lO=most anxious). The majority, 64%, expressed a level of 3 or less, whereas only 9% expressed 6 or higher. Most of the anxiety expressed was related mainly to anticipated pain, as well as the length of the recovery period. Interestingly, only 2 (3%) of men were concerned with the “finality” of vasectomy, despite the fact that most men were aware that it is considered a permanent procedure. When asked about reversibility, 41% stated that they did not think that vasectomy was reversible. When compared to accepted standards of problem-solving and self-concept scales, the men in this study were better problem-solvers, and had better self-concepts than the average male. Conclusion: In our study, most men contemplated vasectomy less than 1 year prior to scheduling an appointment. Their decision was supported by their partner, who often helped in the decision-making process. The major source of anxiety for men prior to vasectomy was fear of pain. There was

S46

Abstracts

also a significant percentage of men that were anxious about potential side effects. Almost one-half of men were unaware of the reversibility of vasectomy. This study demonstrates the need for adequate pre-vasectomy counseling, particularly in the area of postoperative expectations, as well as reversibility.

Tuesday, September 28, 1999 390

P.M.

O-237 Psychological Aspects of the Fertile Female Partner in Cases of Male Infertility. ‘L. Baron, ‘G. Rey Valzacchi, L. Blanco, ‘S. Pasqualini. ‘Halitus Instituto Medico, Buenos Aires, Argentina. Objective: To perform a psychological follow up in order to assess the main emotional issues fertile women undergo when facing the infertility of their partners and to evaluate the importance of holding individual psychological interviews with these women. Design: Prospective. Materials and Method: The first 100 couples with male infertility and women under 35 years old with no evident pathology who entered the ICSI (89/100) and Donor Insemination (DI) (111100) programs of the Department of Assisted Reproduction of Halitus Instituto Medico of Argentina after December 1996 were evaluated. Data were collected during joint psychological interviews (JI) with limited objectives, through an anonymous questionnaire (AQ) and during individual psychological interviews (II). Data from all three sources were compared. When appropriate, the replies given before and after the first treatment were comnared. Results:‘The main emotional issues mentioned were: a) Optimism towards the procedure: 1st attempt: 89% of the patients were optimistic. There were no significant differences between the replies provided in the JI, AQ and II. Before the 2nd attempt: figures decreased to 75%, 49% and 25% respectively. b) Sharing emotions with others: 84% of the patients did not talk to their husbands because they feared they will hurt their feelings, 22% talked to friends and 81% did not talk with their families to protect their partner’s image. c) Aggressive attitude towards partners: 1st attempt: 20% expressed having these feelings during the JI but figures increased to 45% and 89% for the AQ and the II respectively. 2nd attempt: figures were 2.5%, 86% and 92% for JI, AQ and II. d) Self-Perception: (II, 2nd attempt): patients felt more isolation (85%), higher level of anguish (79%), more aggression (72%), depression (56%), concern about their own biological clock (65%); and greater anger about having to expose their bodies to an assisted reproduction procedure (87%). e) Fantasies about being unfaithful to their husbands (11 and 2nd attempt): 79% expressed these fantasies, 82% mentioned dreaming or remembering previous boyfriends. Only 37% acknowledged these fantasies in the AQ. f) Sexual desire: 1st treatment: patients expressed feeling less attracted to their partners (17% in the JI, 5 1% in the AQ; and 60% in the II) and these figures increased to 20%, 60% and 81% at the time of the 2nd attempt. g) Need of individual psychological support: 97% of the patients regarded II as being “highly necessary.” Conclusions: Our results suggests that the feelings of fertile women with infertile partners vary after a first unsuccessful attempt and are hard to express in front of other people, during joint psychological interviews, or through an anonymous questionnaires. It was at the time of individual interviews when women felt more comfortable to fully express their feelings and emotions. Therefore, we conclude that it is necessary to include routine individual interviews as part of the treatment of these couples in order to offer them adequate prophylactic psychological support.

REPRODUCTIVE

BIOLOGY

PROFESSIONAL

GROUP

Tuesday, September 28, 1999 2:00

P.M.

O-116 Derivation Blastocyst. Trounson. Melbourne,

of a Human Embryonic Stem Cell Line from a Human i.‘B. E. Reubinoff, ‘M. F. Pera, 3A. Bongso, sC. Y. Fong, ‘A. ‘Institute of Reproduction & Development, Monash University, Vie, Australia, 2Department of Obstetrics & Gynaecology,

Vol. 72, No. 3, Suppl. 1, September 1999

Hadassah University Hospital, Jerusalem, Israel and 3Department of Obstetrics & Gynaecology, National University of Singapore, Singapore. Objectives: Embryonic stem (ES) cell lines have been derived from the totipotent cells of the early embryo of mice and nonhuman primates. ES cells are karyotypically normal and immortal, and are capable of differentiation into cells representative of all three embryonic germ layers. This study was undertaken in an attempt to derive an ES cell line from human blastocysts. Design: Derivation, propagation and characterisation of a human pluripotent stem cell line. Materials and Methods: Inner cell masses (ICMs) were isolated by immune-surgery from three human blastocysts and plated separately on embryonic fibroblast feeder cell layers. Proliferating cells with the morphological appearance of stem cells were further propagated in clumps of about 100 cells. Conventional techniques of immunohistochemistry, karyotyping and RT-PCR were used for cell characterisation. The cells were injected under the testicular capsule of SCID mice to test for teratoma formation. Results: A cell line (HES-I) was derived from the ICM of one of the blastocysts. The cultures have been propagated six months (> 20 passages) with maintenance of the undifferentiated stem cell population. HES-1 stem cells have retained a normal (46Xx) karyotype, and express alkaline phosphatase activity, mRNA for the transcription factor OCT-4 (specific for pluripotent cells), and the human embryonal carcinoma surface markers SSEA-4, TRA-1-60, and GCTM-2 proteoglycan. The pluripotentiality of the cells was maintained in culture as evidenced by their differentiation in-vitro into trophoblast and endodemr, and by the formation of teratomas in SCID mice with differentiated derivatives of the three germ layers (endoderm, ectoderm and mesoderm). Conclusions: These data indicate that HES-1 is a human ES cell line. ES cells with properties similar to pluripotent human embryonal carcinoma cells and monkey ES cells can be derived from the human blastocyst. Human ES cells may have a profound impact on drug development, human embryology, and transplantation therapy.

Tuesday, September 28, 1999 2:15 P.M. o-117 Expression of Purinergic Receptor (P2UR) in Human Granulosa-Luteal Cells C. .I. Tai, S. K. Kang, K. W. Cheng, K. C. Choi, P. S. Nathwani, P. C. K. Leung. Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada. Objectives: The P2U purinoceptor (P2UR) has been functionally and pharmacologically identified in the ovary. Through this G-protein-coupled purinergic receptor, adenosine trlphosphate has been shown to enhance progesterone production and induce intracellular calcium [Ca2+]i mobilization in human granulosa-luteal cells (hGCs). However, the expression of PWR messenger ribonucleic acid (mRNA) in hGCs is still poorly characterized. The present study was designed to examine the expression of PWR in hGCs. Design: Expression of PWR and uridine triphosphate-induced intracellular calcium oscillations were examined in hGCs. Material and Methods: The expression of the P2UR cDNA in hGCs, obtained from women undergoing In Vitro Fertilization-Embryo Transfer, was detected by reverse transcription-polymerase chain reaction (RT-PCR). Primers were designed based on the reported cDNA sequence from the human airway epithelium. The putative P2UR cDNA was cloned, and chain termination method of sequencing was performed for confirmation. The cloned PCR product was used as a probe to detect the expression of the P2UR mRNA in hGCs using Northern blot analysis. Functionally, the effect of UTP in inducing [Ca2+]i oscillations was examined in Furaloaded HGCs using microspectrofluorimetry. Results: A PCR product corresponding to the expected 599 bp P2UR cDNA was obtained from hGCs. Molecular cloning and sequencing of the PCR product revealed an identical sequence to the reported PWR cDNA. Two mRNA transcripts of 2.0 kb and 4.6 kb were identified in hGCs using Northern blot analysis. In addition, microspectrofluorimetry revealed UTP is able to induce [Ca2+]i mobilization in a dose-dependent manner. Conclusions: (1) P2U purinergic receptor mRNA is expressed in human granulosa-luteal cells. (2) Through P2UR, UTP is functionally capable of

FERTILITY

& STERILITY@

inducing [Ca2+]i mobilization in a dose-dependent manner. These results further support a potential role of this nemotransmitter receptor in the human reproductive system.

Tuesday, September 28, 1999 2:30 P.M. O-118 Uterine Gene Transfection With HOXAlO cDNA Increases Litter Size in Mice. C. Bagot, P. Troy, D. Olive, H. S. Taylor. Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT, USA. Objective: Expression of the HOXAlO gene is necessary for proper implantation in both mice and humans. Mice with a targeted disruption of this gene produce normal embryos which fail to implant. We have previously shown that this gene is expressed in the human endometrium and upregulated at the time of implantation. Additionally patients with implantation defects (i.e. endometriosis) show altered HOXAlO expression. We have recently demonstrated that blocking uterine HOXAlO expression in mice on day 2 postcoitus decreases implantation. In this study we determine the effect of augmenting HOXAlO expression. Design: On day 2 post coitus, mice were transfected with a plasmid that constitutively expresses HOXAlO. Materials and Methods: HoxalO cDNA was cloned into pcDNA-3.1. Plasmid DNA alone was used as a control. A DNAiliposome complex containing one of the above two constructs was used to transfect the uteri of pregnant mice. Surgery was performed on day 2 post coitus, injecting the DNA/liposome complex into the base of each uterine horn. Ten mice received the HOXAIO cDNA construct and an equal number received the control, Results: 100% of the HOXAlO transfected mice produced offspring as opposed to 70% of the control mice. The HOXAlO transfected mice all produced litters of maximal size as opposed to control mice which had more variability in litter size. HOXAlO transfected mice had an average litter size of 12 versus 7 for the controls. (PcO.05). Conclusions: These results provide further evidence that HOXAlO plays an essential role in implantation. High HOXAlO levels allow maximal successful implantation of all available embryos. Gene therapy which increases HOXAlO levels is a potential treatment to optimize successful implantation. Supported by grants from the NICHD and ASRMSerono to Hugh S. Taylor.

Tuesday, September 28, 1999 2~45 P.M. o-119 Heparin-Binding EGF-Like Growth Factor and ErbB4-Mediated Signaling Regulate Peri-Implantation Development of Mouse Tropboblast Cells. Jun Wang, D. Randall Armant. C.S. Mott Center for Human Growth and Development, Departments of Obstetrics and Gynecology and Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI. Objective: Unsatisfactory implantation rates due to suboptimal trophoblast differentiation is a major obstacle in IVF. The impeded differentiation of trophoblast cells partly results from deficiencies in critical growth factors produced by the receptive uterus. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in the receptive mouse uterus solely at sites of blastocysts apposition. Per&implantation blastocysts express EGF receptor isoforms, suggesting that HB-EGF may participate in paracrine or juxtacrine regulation of trophoblast peri-implantation development. Design: This study was designed to investigate the effect of HB-EGF on mouse blastocyst development and delineate the associated intracellular signaling pathways. Acceleration of trophoblast differentiation was detected as a shift in maximal fibronectin binding activity (FBA) on the apical surface of trophoblast cells from Day 7 to Day 6 of pregnancy. We hypothesized that the ability of HB-EGF to accelerate trophoblast differentiation is dependent on the expression of EGF receptor isoforms that bind HB-EGF and initiate intracellular Ca*+ signaling. Materials and Methods: Morulae were collected from oviducts of super-

s47

ovulated mice on Day 3 of pregnancy and cultured in Ham’s FlO medium with or without exposure to 1 nM HB-EGF. Intracellular Ca” levels were estimated in trophoblast cells using the fluorescent probe, fluo-3-AM, and an image analysis system. FBA was measured using fluorescent microspheres coated with a fragment of fibronectin, FN-120. The surface expression of EGF receptor isofonns (EGFR, Neu, ErbB3 and ErbB4) was examined using specific antibodies and fluorescent secondary antibodies visualized by confocal microscopy. Results: The induction of intracellular CaZt signaling and acceleration of blastocyst differentiation by treatment with HB-EGF were only observed after 8 P.M. on Day 4 of pregnancy. At this same time ErbB4 was translocated from cytoplasm to the trophoblast apical surface. The other three EGF receptor isoforms, EGFR, Neu and ErbB3, were resident on the apical surface prior to 3 P.M., suggesting that HB-EGF-trophoblast interaction is dependent upon ErbB4. The shift by HB-EGF in the timing of maximal FBA was attenuated in the presence of genestein, or intracellular (10 PM BAPTA-AM) or extracellular (1 mM BAPTA) CaZt chelators, while the phospholipase C inhibitor U73122 had no influence. Conclusion: Our result suggest that binding of HB-EGF to ErbB4 accelerates the differentiation of trophoblast cells to an adhesion-competent stage through elevation of intracellular Ca *+ levels. The effect of HB-EGF appears to be mediated by protein tyrosine kinase, as expected, but not phospholipase C, suggesting that Ca *+ influx is the cause of intracellular Ca*+ elevation. Our findings indicate that supplementation of cultured embryos with HB-EGF to improve implantation rates is most beneficial after ErbB4 becomes available on the embryo surface. Supported by grant HD36764 from the National Institutes of Health.

Tuesday,

September 3:00 P.M.

that become exposed in vivo. Its direct interaction with the blastocyst may be critical for efficient implantation in utero. Supported by the National Institute of Health grant Hll 36764.

Tuesday,

September 3:45 P.M.

28, 1999

o-121 Distribution of the Platelet-Activating Factor-Receptor Between Normal and Abnormal (Low Motility) Spermatozoa. ‘E. Maguire, ‘K. A. Miller, ‘W. E. Roudehush. ‘Department of Obstetrics and Gynecology, Medical University of South Carolina, Charleston, SC and ‘Southeastern Fertility Center, Mt. Pleasant, SC.

28, 1999

O-120 Supplementation with Calcitonin During Mouse Preimplantation Development Accelerates Trophoblast Differentiation. R. Dinsay, J. Wang, D. R. Armant. C.S. Mott Center for Human Growth and Development, Departments of Obstetrics and Gynecology and Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI. Objectives: Calcitonin, a 32 amino acid polypeptide hormone, is under steroid hormone regulation in the uterine endometrium and accumulates specifically during blastocyst implantation. Mouse blastocysts collected on Day 4 of pregnancy express calcitonin receptors and differentiate to the adhesive stage at an accelerated rate in medium supplemented with calcitonin. To further investigate the utility of calcitonin supplementation during in vitro embryo culture, embryos were exposed to calcitonin during early preimplantation development and at the periimplantation stage. Design: Fibronectin binding activity (FBA) on the apical surface of trophoblast cells can reliably estimate the rate of trophoblast differentiation during in vitro culture. We have examined the ability of calcitonin supplementation during the S-cell to blastocyst stages to shift the time of maximal FBA from Day 7 to Day 6 of pregnancy. Materials and Methods: Embryos were harvested from superovulated mice at the S-cell stage by flushing the oviducts on Day 3 of pregnancy and cultured in Ham’s FlO medium. Some embryos were incubated for 30 min in culture medium containing 0 or 10 nM rat calcitonin and then transferred to calcitonin-free medium, while other embryos were cultured continuously in calcitonin-containing media. FBA was assessed on Day 6 and Day 7 of pregnancy. Prior to assay, the embryos were exposed for 1 h to 50 pg/ml of a fibronectin-derived peptide, FN-120, to upregulate FBA on the apical surface of adhesion-competent trophoblast cells. In the second experiment, embryos were cultured in unsupplemented Ham’s FlO medium until Day 6. Calcitonin was added 30 min prior to assessing FBA without exposure to FN-120. Factorial ANOVA was used to analyze the results and detect temporal shifts in peak FBA. Results: Control embryos maximally express FBA on Day 7; however, maximal FBA was observed on Day 6 in embryos treated with calcitonin. There was no significant difference between calcitonin treatment for 30 min on Day 3 and continuous supplementation. Day 6 blastocysts treated with calcitonin immediately prior to assay, in contrast to controls, expressed elevated FBA without the need for exposure to FN-120. Conclusion: Calcitonin is an important regulatory factor that accelerates preimplantation development even at embryonic stages earlier than those

S48

Abstracts

Introduction: Platelet-activating factor (PAF) plays a significant role in spermatozoa motility, fertilization and subsequent preimplantation embryo development. In other cell types, PAF’s mechanism of action is a receptor mediated event. We have recently reported on the presence and distribution of the PAF-receptor along the human spermatozoa. Objective: To determine the distribution of the PAF-receptor along spermatozoa from patients with low motility. Design: Distribution of PAF-receptors between normal and abnormal (low motility) spermatozoa by immunofluorescence. Methods: Human spermatozoa were exposed to rabbit PAF-receptor antibody for 1 hour at 4”C, followed by FITC-conjugated goat anti-rabbit IgG for 10 minutes at 4°C. Immunostained spermatozoa were exposed to fluorescent microscopy and graded by degree of fluorescent intensity (FI) at six regions: acrosome cap, post-acrosome, neck, midpiece, principal tail and end piece. Data were analyzed by ANOVA and Student’s t-test. Results: ANOVA revealed a significant difference (P
Tuesday,

September 4:00 P.M.

28, 1999

o-122 Characterization of Abnormal Protamines and Histone Replacement Sperm From Infertility Patients and Semen Donors and Its Effect ICSI Outcome. ‘.‘D. T. Carrell, ‘L. Liu, ‘C. M. Peterson. ‘Division

Vol.

72, No. 3, Suppl.

1, September

in On of

1999

Urology and ‘Department of Obstetrics and Gynecology, School of Medicine, Salt Lake City, UT.

University

of Utah

Objectives: Sperm nuclear histones are selectively replaced by protamines during spermiogenesis. Incomplete or abnormal histone replacement, measured as either a lack of protamines or as an abnormal protamine 1 to protamine 2 ratio (Pl/P2 ratio), has been documented in infertility patients. We have previously evaluated sperm from both semen donors and infertility patients and shown that abnormal protamine levels are common in infertility patients, but rare in donors of known fertility. Additionally we have shown that abnormal protamine levels are frequently associated with chromatin decondensation following exposure to low levels of heparin. In this study we evaluate the presence of increased levels of a non-PI/P2 nuclear protein found in high levels in some patients with low P2 levels. Additionally, we have evaluated sperm nuclear histone content in donor and patient semen. Design: Protamine 1, protamine 2, and histones were purified and evaluated from the semen of 50 donors of known fertility and 108 infertility patients. Materials and Methods: Nuclear proteins were extracted from semen samples, then purified by electrophoresis on acetic acid-urea microgel. Imaging densitometry was used to calculate the Pl/P2 ratio. Western blot analysis was performed using standard techniques with an antibody previously shown to bind both Pl and P2. Histone content was measured using the acidic aniline blue staining procedure. The percentage of reacted sperm for the histone stain was reported for each sample. Results: The mean Pl/P2 ratio for semen donors was 0.83 -C 0.05 compared to 1.09 ? 0.06 for infertility patients (p < 0.05). Thirteen patients had undetectable levels of P2 and a predominance of slow migrating protein bands. Western blot analysis showed that the Pl, P2-specific antibody did bind to the slow migrating proteins. Additionally, the percentage of sperm staining positive for histones was significantly (p < 0.01) increased when P2 was severely diminished and the slow bands were visible (25.1 ? 1.91 versus 63.6 2 10.6). Intracytoplasmic sperm injection fertilization and pregnancy rates were not decreased in patients with increased Pl/P2 ratios and high levels of histones. Conclusions: These data, combined with previously reported data, indicate that protamine levels and Pl/P2 ratios are commonly abnormal in infertility patients, but rare in donors of known fertility. In most instances abnormal protamine expression was associated with abnormally high levels of nuclear histones found in the sperm, indicating that histone replacement was altered by abnormal protamine expression. Increased histone levels were found much less frequently in donor sperm, and abnormal protamine levels were not observed in the donor samples analyzed. These data indicate that abnormal protamines are a possible cause of reduced fertility, or are associated with syndromes resulting in reduced fertility, but that ICSI success is not altered by abnormal histone/protamine exchange.

Tuesday,

September 4:15 P.M.

28, 1999

O-123 Role of Sulfogalactosylglycerolipid (SGG) in Mouse and Human Sperm-ZP Binding. ‘W. Weerachatyanukul, ‘,*D. White, ‘M. Rattanachaiyanont, 3B. Gadella, 4N. Kamolvarin, ‘**N. Tanphaichitr. ‘Human IVF Program, Reproductive Biology Unit, Loeb Health Research Institute, Ottawa Hospital Civic Campus, Department of Obstetrics/Gynecology and *Department of Biochemistry, Faculty of Medicine, University of Ottawa, Ontario, Canada; 3Department of Basic Science, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands; 4Departments of Obstetrics/Gynecology and Biochemistry, Faculty of Medicine, Chulalongkhom University, Bangkok, Thailand. Objective: SGG is present primarily in male germ cells. Like other glycolipids, SGG may be involved in cell-cell/extracellular matrix adhesion. The aim of our study was to investigate whether sperm SGG was involved in mouse/human zona pellucida (ZP) binding. Design: SGG localization on capacitated sperm was performed to determine whether it was present on the sperm head, where ZP binding occurs. In vitro mouse/human sperm-ZP binding assay was used to evaluate the role of sperm SGG in ZP binding. Sperm were pretreated with various doses of anti-SGG Fab antibodies to mask SGG before gamete co-incubation. The number of anti-SGG pre-treated sperm bound to ZP was compared to that of

FERTILITY

& STERILITY@

control sperm (treated with pre-immune rabbit serum Fab antibody). Furthermore, an attempt to demonstrate SGG binding to mouse ZP was made using fluorescently labeled SGG liposomes and the fluorescent intensity was compared with that of ZP exposed to fluorescently labeled phosphotidylserine (PS) liposomes (negatively charged like SGG), fluorescently labeled galactosylglycerolipid (GG) liposomes, and fluorescently labeled SGG liposomes plus 100X non-fluorescently labeled SGG liposomes. Materials and Methods: Motile, capacitated human and mouse sperm were prepared by Percoll-gradient centrifugation. Oviductal eggs were retrieved from superovulated female mice, while failed fertilization human eggs were collected from various IVF programs, following the Human Ethics Committee’s guidelines. Rabbit polyclonal anti-SGG IgG antibody was prepared and affinity purified as described (Kamolvarin, et nl., Biol Reprod 54,71a, 1996). Indirect immunofluorescence (IIF) was performed in both fixed and live sperm, using anti-SGG IgG and Cy3 conjugated secondary antibody. Sperm pre-treated with affinity purified anti-SGG Fab antibodies were co-incubated with eggs. Fluorescently labelled SGG/PS/GG liposomes, prepared by including -TRITC-conjugated phosphotidylethylnolamine (PE) with SGG, PS or GG (SGG/PS/GG: PE: - 13: 1, molar ratio) for liposome construction, were incubated with ZP intact eggs. Results: IIF revealed the presence of SGG in the sperm anterior, where sperm bind to ZP. In three replicate experiments, ZP binding ability of sperm pre-treated with anti-SGG Fab was significantly reduced (p < 0.001) i.e., to 60.7 ? 7.4% and 36.3 ? 3.1% of the control value at 0.6 and 6.6 Fg/m.l anti-SGG Fab, respectively, for mouse sperm (total egg number, n = 80 and 86). and to 26.0 + 14.7% of the control value at 50 pg/ml anti-SGG Fab for human sperm (n = 37). Antibody treatment did not affect sperm motility or induce a premature acrosome reaction. The results strongly suggested that sperm SGG was involved in ZP binding. Furthermore, ZP exposed to fluorescently labeled SGG liposomes displayed strong fluorescent staining, where ZP treated with fluorescently labeled PS/GG liposomes, or fluorescently labeled SGG plus non-fluorescently labeled SGG liposomes illustrated negative staining, indicating that SGG bound directly to ZP in a specific manner. Conclusion: Sperm SGG was significant in mouse and human sperm ZP binding. Supported by MRC and the Rockefeller Foundation.

Tuesday,

September 4:30 P.M.

28, 1999

O-124 The Role of Inducible Nitric Oxide Synthase (INOS) in Ovulation. ‘A. Beltsos, ‘A. Jablonka-Shariff, *B. Van Voorhis, ‘L. M. Olson. ‘Department of Obstetrics and Gynecology, Washington University, St. Louis, Missouri and ‘Department of Obstetrics and Gynecology, University of Iowa, Iowa City, Iowa, USA. Objective: The signaling molecule nitric oxide (NO) has been implicated as a key element in the ovulatory process. The purpose of this study was to evaluate the importance of NO synthesized by the inducible form of nitric oxide syntbase (iNOS) in the periovulatory period. Design: Ovaries and serum were collected from superovulated mice at several timepoints around the periovulatory period to determine NO production as well as the relative expression of iNOS mRNA and iNOS protein. Ovulation rate and breeding studies were performed to examine the reproductive performance of wildtype (WT) versus iNOS knock-out (iNOS-KO) mice. Methods and Materials: Immature (27 day old) WT (SvEv 129) and INOS-KO mice were superovulated using injections of PMSG (5 IU ip) followed 48 h later by hCG (5 IU ip). To determine ovarian NO production and iNOS mRNA levels in WT and iNOS-KO mice, ovaries were collected and weighed from unstimulated, 0, 4, 8, 10, and 20 h after PMSG/hCG injection and analyzed utilizing a fluorescent nitrite assay and ribonuclease protection assay, respectively. To examine the changing expression of iNOS versus endothelial NOS (eNOS) protein during this period, Western blots were performed. Ovulation rates of WT versus INOS-KO mice were determined by counting the number of ovulated oocytes recovered from the ampullae of 20 h post-hCG animals. Reproductive performance of WT and iNOS-KO breeders pairs was documented. Statistical analysis was performed using ANOVA on a Systat program. Results: Ovarian nitrite/nitrate production, representing NO levels, was higher in the WT than in the iNOS-KO mice at all timepoints. For both

genotypes of mice, the highest levels of NO were observed in the unstimulated ovaries (p < 0.05) which declined with ovulation induction. In WT mice, iNOS mRNA levels and protein levels were highest in the unstimulated ovaries and decreased by 50% and 64%, respectively, at 8 h post-hCG. In contrast, eNOS protein levels were lowest in unstimulated ovaries and increased by 5-fold at 4 h post-hCG. Ovulation rate in the superovulated iNOS-KO mice was lower as compared to WT mice (30% reduction, p < 0.05) although ovarian weights were similar. However, reproductive performance of breeding pairs was not significantly effected by iNOS deficiency. Conclusions: INOS-KO mice showed a decreased ovulation rate but the normal breeding patterns imply that a lack of iNOS-derived NO is not critical to ovulation. This may indicate that iNOS-derived NO does not play a significant role in ovulation, or that eNOS-derived NO compensates for the iNOS deficiency. Moreover, eNOS expression increases in response to ovulation induction, suggesting that eNOS is more important than iNOS in the ovulatory event.

Tuesday, September 28, 1999 4:45 P.M. o-125 Estrogen Stimulates Vascular Endothelial Growth Factor (VEGF) mRNA Expression and Preantral Follicle Growth in the Rat Ovary. D. R. Danforth, L. K. Arbogast, S. Ghosh, E. A. Kennard, C. I. Friedman. Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, The Ohio State University, Columbus, OH. Objectives: The initiation of follicle growth in mammals is poorly understood. Estrogen stimulates preantral follicle development in rats, but not in monkeys, Recent data suggest that ovarian-derived growth factors may participate in ovarian follicle development. We have recently determined that ovarian administration of VEGF stimulates preantral follicle development in the immature rat. The objective of the present study was to investigate whether the effects of estrogen on follicle development are associated with increases in ovarian VEGF mRNA. Design: Preantral follicle growth and VEGF mRNA levels were quantitated 0, 8, 24, and 48 hours after subcutaneous administration of the synthetic estrogen, diethylstilbestrol (DES). Materials and Methods: Twelve immature (21 day old) female Sprague Dawley rats were injected with 2 mg DES subcutaneously. Rats were euthanitized 0, 8, 24, and 48 hours after DES injection. The right ovary was formalin fixed, paraffin embedded, sectioned (10 IL), and stained (H&E) for histological analysis. Every 10th section was examined for small (<200 pm), medium (200-300 pm) and large (>300 pm) preantral follicles. The left ovary was snap frozen in liquid nitrogen for subsequent reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Total RNA was isolated, reverse transcribed, and subjected to 25 cycles of PCR using VEGF primers and P-Actin primers in the presence of drgoxigenin (DIG) iabelled dUTP. The DIG-labelled PCR products were quantitated using the Boehringer-Mannheim PCR ELISA DIG Detection system. VEGF mRNA levels were corrected for fi-actin expression measured in the same reaction tube. Results: DES significantly increased the number of medium-sized and larger preantral follicles in the rat ovary at 24 h and 48 h post injection (p < 0.05, n = 3 at each time point). Similarly VEGF mRNA expression was increased between 8 and 48 hours after DES administration (p < 0.05, n = 3 at each time point). Conclusion: We conclude that estrogen stimulates preantral follicle growth in immature rats and stimulates ovarian expression of VEGF mRNA. Whether the estrogen-stimulated follicle growth in the ovary is mediated via angiogenic factors such as VEGF awaits further investigation.

CONTRACEPTIVE

SPECIAL

INTEREST

GROUP

Tuesday, September 28, 1999 2:00 P.M. O-126 Rheological Behavior Influence of Dilution

s50

Abstracts

of Topical Contraceptive Formulations with Vaginal Fluid. ‘D. H. Owen,

and the ‘J.

J. Peters,

i,‘D F Katz. Departments of ‘Biomedical Engineering Gynecology, Duke University, Durham, NC.

and ‘Obstetrics

and

Objectives: The spreading and retention (“deployment”) of intravaginal contraceptive formulations are fundamental to their efficacy and are governed, in part, by the rheological properties of the formulations. Upon application, these formulations experience a wide range of shear rates due to movements of the vaginal epithelial surfaces and during coitus. Mixing with ambient vaginal fluids alters the rheological properties, affecting the kinetics and extent of deployment. Current commercially available products differ significantly in chemical composition, suggesting different rheological properties, both whole and diluted with vaginal fluid. The present study contributes to characterization of the rheological behavior of these formulations under biologically relevant shear rates and dilutions. Design: Three current products were studied, whole and after dilution to varying extents with a pH 4.2 vaginal fluid simulant (Owen and Katz, Contraception, 1999). A set of rheological measurements was performed on each fluid. Materials and Methods: Conceptrol and KY Plus (Advanced Care Products) and Advantage (Columbia Labs) were studied: whole and after dilution 1:3, 1: 1 and 3: 1 with vaginal fluid simulant. Viscosity as a function of shear rate (0.01-100 s-l), and residual stress upon cessation of steady shear were measured, using a Rheotnetric Scientific ARES rheometer and a Brookfield Instruments viscometer. The range of shear rates spans the expected in viva values, from passive spreading to active shearing during coitus. Results: All products exhibited shear thinning behavior to approximately the same degree, with the viscosity reduced by a factor of about three as the shear rate increased from 0.025 s-i to 0.1 s-‘. These materials differed greatly in the degree to which the viscosity was reduced upon dilution with vaginal fluid simulant. At a shear rate of 0.1 s-i the viscosities of KY Plus, Conceptrol and Advantage were 7940, 6450 and 3460 P, respectively. The 75% gel dilutions had viscosities of 2320, 2090 and 27 P, i.e., the reduction of viscosity of AdvantageTM due to dilution with vaginal fluid simulant was two orders of magnitude greater than that for the other two products. This effect was even more pronounced at lower shear rates. Upon cessation of steady shear, both KY Plus and Advantage exhibited significant residual stresses, which depend upon prior shearing conditions. After steady shearing at 1 ss’ these residual stresses were 60% (KY Plus) and 50% (Advantage) of the limiting steady shear values. Conceptrol exhibited no residual shear stress. Conclusions: These results suggest differences in deployment behavior in viva of the three products studied. The substantial shear thinning indicates that the products cannot bc characterized by single values of viscosity; they will flow more easily when experiencing higher applied local shear rates (coitus) than lower ones (initial spreading after application). The existence of residual stresses means that KY Plus and Advantage (but not Conceptrol) will tend to stop flowing once shearing forces are reduced below specific minimum values, Importantly, the three products respond differently to mixing with vaginal fluid, resulting in a more pronounced reduction of viscosity by Advantage than by KY Plus and Conceptrol. These results contribute to a more rigorous understanding of the physical and chemical mechanisms of formulation deployment and efficacy. (Supported by the CONRAD Program)

Tuesday, September 28, 1999 2:15 P.M. O-127 Effect of Novel Amino Acid Substitutions on the Spermicidal Potency of the Dual-Function Anti-HIV Agent, Aryl Phosphate Derivative of Bromo-Methoxy Zidovudine. ‘,aO. J. D’Cruz, 1,3T. K. Venkatachalam, ‘,4F. M. Uckun. ‘Drug Discovery Program, Departments of *Reproductive Biology, 3Chemistry and 4Virology, Hughes Institute, St. Paul, MN. Objectives: Aryl phosphate derivatives of bromo-methoxy zidovudine (ZDV) are a new class of microbicides potentially capable of preventing HIV transmission as well as providing fertility control (Biol. Reprod. .59:.503;1998). ZDV is functionalized on the pentose ring by a phosphate linker possessing a alanine (Ala) side chain. To better understand how bromo-methoxy derivatives of ZDV contribute to sperm motility loss, we systematically changed the structure of the parent compound with various amino acid substitutions and examined for spermicidal activity. Design: Comparative sperm immobilizing activity (SIA) and cytotoxicity

Vol. 72, No. 3, Suppl. 1, September 1999

profiles of novel ZDV derivatives against human sperm and normal human endocervical epithelial cells, in comparison to detergent-type spermicide nonoxynol-9 (N-9). Materials and Methods: Twenty-two aryl phosphate derivatives of bromo-methoxy ZDV were synthesized by the phosphochloridate chemistry. The Ala side chain in the phenyl phosphate derivative of ZDV was replaced with either phenylalanine (Phe), tryptophan (Trp), proline (Pro), leucine (Leu), methionine (Met), valine (Val) or glycine (Gly). In addition, the effect of substitution of p-bromo, p-chloro, p-nitro or p-methoxy groups on the aryl moiety were also compared. The structures of these derivatives were confirmed by standard analytical techniques. The effect of each of the 22 ZDV derivatives (O-500 PM) was evaluated for SIA by computer-assisted sperm analysis (CASA). The in vitro cytotoxicity of spermicidal ZDV derivatives versus N-9 against normal human endocervical epithelial cells were analyzed using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) cell viability assay. Results: Replacement of Ala side chain in the aryl phosphate derivative of bromo-methoxy ZDV led to marked alterations in SIA. Replacement with Trp, Pro, Met, and Gly caused 3- to 19-fold decrease in potency. The order of efficacy for these ZDV derivatives was Ala > Trp > Pro > Met > Gly with EC,, values (50% inhibitory concentration) of 6, 20, 61, 71, and 116 PM respectively. The substitution of Ala with Phe, Leu, and Val led to loss of SIA (EC,, values >500 PM). Substitution in the aryl moiety also variably affected the potency of spermicidal ZDV derivatives. The unsubstituted aryl moiety of ZDV with heterocyclic Ttp was the most potent to normal human (EC,, = 10 PM). Unlike N-9 which was cytotoxic endocervical epithelial cells (IC,, = 11 PM) at spermicidal doses (EC,, = 81 FM), none of the novel spermicidal ZDV derivatives were cytotoxic (IC,, values >250 FM). Conclusions: Results of structure-activity relationship studies clearly establish that in addition to the bromo-methoxy functional groups, the aryl phosphate derivatization and the Ala side chain contribute to the SIA of these dual-function phosphoramidate derivatives. Their lack of cytotoxicity holds particular clinical promise for the development of novel, non-detergent-type anti-HIV spermicides. (Supported in part by NIH grant ROlHD37357).

Tuesday,

September 2:30 P.M.

28, 1999

O-128 Enhancement of Spermicidal Activity of Vanadocenes by Methyl Substitution on the Cyclopentadienyl Rings. ‘,*O. .I. D’Cruz, 3P. Ghosh, ‘F. M. Uckun. ‘Drug Discovery Program, Departments of *Reproductive Biology and ?hemistry, Hughes Institute, St. Paul, MN. Objectives: Vanadium (IV)-containing his-cyclopentadiene [Cp] complexes (vanadocenes) are a new class of potent and rapid spermicides (Biol. Reprod. 58:1515;1998; M&c. Hum. Reprod. 4:683;1998). To better understand how vanadocenes induce rapid sperm motility loss, we tested the effect of substitution of electron donating methyl groups on the two Cp rings of a vanadocene dihalide on the kinetics of sperm immobilizing activity [SIA] and known biochemical processes contributory to SIA. Design: SIA of vanadocenes was assessed by computer-assisted sperm analysis (CASA). Fluorescent probes and flow cytometry were used to quantitate changes in net intracellular reactive oxygen species (ROS), thiol status, mitochondrial membrane potential (Alum), and apoptotic DNA fragmentation. Western immunoblotting was used to detect sperm protein phosphorylation. Materials and Methods: The SIA activity of the lead vanadocene with five methyl substitutions on the Cp ring, (bis[pentamethylcyclopentadienyl] vanadium dichloride [VPMDC]) was compared with the unsubstituted analogue, vanadocene dichloride [VDC]. Motile human sperm were exposed to increasing concentrations (O-250 PM) of VDC, VPMDC, and the detergent spermicide, nonoxynol-9 (N-9) and then analyzed for SIA by CASA. SIA was further evaluated for the generation of ROS, loss of ATm, reduction of sulfhydryl groups, and nuclear DNA fragmentation (TUNEL assay) by multiparameter flow cytometry using standard fluorescent probes. Western immunoblotting was performed to detect tyrosine phosphorylated proteins in control and vanadocene-

FERTILITY

& STERILITY@

treated sperm. Redox potentials [E,,a values] of vanadocenes were obtained by cyclic voltammograms. Results: Both vanadocenes elicited potent and dose-dependent SIA at < 10 /.LM concentrations. Vanadocene-induced SIA was rapid (< 15 set) and irreversible. The SIA of the unsubstituted analogue, VDC, was enhanced by an order of magnitude by the substitution of electron donating methyl groups in the Cp rings with EC,, values (50% inhibitory concentration) of 7.5 and 0.74 PM, respectively. VPMDC was IO times more potent than VDC and 1 IO-fold more potent than N-9. The SIA of vanadocenes correlated with reduction in redox potential values [E,,, values of +0.655, and +0.127 mV, respectively]. At lower concentrations, SIA of these vanadocenes was not concomitantly accompanied by ROS generation, loss of Alarm, reduction of sulfbydryl groups, nuclear DNA fragmentation or increased tyrosine phosphorylation of sperm proteins; these effects of vanadocenes were maximal at concentrations above 100 PM. Conclusions: Modifications of the Cp ring by 5 electron donating methyl groups augments the SIA of vanadocenes. Vanadocene-induced SIA was not related to known biochemical processes affecting sperm motility loss. These studies suggest a unique mechanism by which vanadocenes induce SIA. The potent SIA of vanadocenes holds particular clinical promise for the development of novel, non-detergent-type spermicidal contraceptives.

Tuesday,

September 2:45 P.M.

28, 1999

O-129 Novel Derivatives of PhenethyWbromopyridylthiourea (PST) and Diiydroalkoxybenzyloxopyrimklme (DABO) are Dual-Function Spermicides with Potent Anti-HIV Activity. ‘.4F. M. Uckun, ‘,3T. K. Venkatachalam, ‘,‘O J D’Cruz. ‘Drug Discovery Program, Departments of 2Reproductive Biology, ?hemistry and 4Virology, Hughes Institute, St. Paul, MN. Objectives: Sexually active women represent the fastest growing HIV/ AIDS risk group. In an effort to develop a vaginal microbicidal contraceptive potentially capable of preventing HIV transmission as well as providing fertility control, we have synthesized novel non-nucleoside inhibitors (NNIs) of HIV-l reverse transcriptase (RT) and examined them for dualfunction anti-HIV and spermicidal activity. Design: Structure-based drug design by use of computer docking procedure for the NNI binding pocket generated from nine RT-NNI crystal structures was used to synthesize novel NNIs. The lead compounds were tested for anti-HIV activity against drug-sensitive and drug-resistant strains of HIV, sperm immobilizing activity (SIA), and cytotoxicity profiles against normal human ectocervical and endocervical epithelial cells. Materials and Methods: Three novel NNIs: N-[2-(2,5-dimethoxyphenethyl)]-N’-[2-(5-bromopyridyl)]-thiourea (D-PBT), N-[2-(2-fluorophenethyl)]-N’-[2-(5-bromopyridyl)]-thiourea (F-PBT), and 5-isopropyl-2[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(lH)-one (S-DABO) were synthesized based on the computer model for the NNI binding pocket of RT that were predicted to inhibit RT. The anti-HIV activities of these NNIs were compared with standard NNIs, trovirdine, delavirdine and nevirapine by measuring viral RT activity and p24 antigen production as markers of viral replication using HTLV,,,,infected human peripheral blood mononuclear cells (PBMC). The lead compounds were also tested against clinically observed NNI resistant mutated strains of HIV. The effects on sperm motion kinematics and sperm membrane integrity were analyzed by computerassisted sperm analysis and by confocal laser scanning microscopy, respectively. The cytotoxic effects of NNIs against normal human ectocervical and endocervical epithelial cells were tested using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) cell viability assay. Results: All three NNIs were potent inhibitors of purified recombinant HIV RT and abrogated HIV replication in PBMCs at nanomolar concentrations (IC,,
S51

SperF-PBT and S-DAB0 was rapid (t,,, = 7-13 min) and irreversible. micidal activity of F-PBT and S-DAB0 was not accompanied by loss of sperm membrane integrity. Both F-PBT and S-DAB0 were selectively spermicidal without cytotoxicity to normal human ectocervical and endocervical epithelial cells. Conclusions: As potent anti-HIV agents with spermicidal activity, and lack of cytotoxicity, F-PBT and S-DAB0 show unique clinical potential to become the active ingredients of a microbicide for women who are at high risk for acquiring HIV by heterosexual contact.

Tuesday,

September

3:00

28, 1999

P.M.

O-130 Nifedipine Prevents Capacitation by Stimulating Human Sperm Cholesterol Synthesis. ‘A. Jacob, ‘G. M. Centola, ‘I. R. Hurley, ‘B. Napoli‘S. Benoff. Departments of ‘Obstettano, ‘G. W. Cooper, ‘A. Hershlag, rics and Gynecology and ‘Research, North Shore University HospitalNYU School of Medicine, Manhasset, NY and Department of 30bstetrics and Gynecology, University of Rochester Medical Center, Rochester, NY. Objectives: Our aim was to dissect the mechanism by which nifedipine produces reversible male infertility, with the long-term goal of developing male contraceptives. Prior data indicate that nifedipine’s action as a calcium channel blocker, inhibiting influx of extracellular calcium into the sperm head (required for the acrosome reaction), was secondary to inhibition of mannose receptor (MR) expression (required for zona binding). In sperm from fertile donors, the movement of MR from subplasmmalemmal stores to the sperm surface requires plasma membrane cholesterol eftlux during capacitation. Sperm from infertile men chronically medicated with nifedipine possess MR stores but do not express MR on the sperm head surface. Membrane cholesterol content is increased in these patients’ sperm during incubation in capacitating media (Ham’s FlO + 30 mg/ml delipidated HSA). It has been recently demonstrated that sperm possess the necessary enzymes to synthesize cholesterol and that nifedipine stimulates cholesterol synthesis in somatic cells. Design: In vitro effects of nifedipine on human sperm were examined for correlation with MR expression and membrane cholesterol content and synthesis. Materials and Methods: Fertile donor sperm (uncapacitated, capacitated and capacitated in the presence of 5 PM nifedipine) were examined for MR expression using FITC-conjugated mannosylated BSA. Sterols extracted from duplicate aliquots in the presence of an internal standard, 5 alpha-chalestane, were analyzed for non-ester&d cholesterol content by gas chromatography (GC). Membrane cholesterol synthesis in the presence or absence of nifedipine for 5 or 24 hrs was assessed through 2-[14C]-acetic acid labeling and liquid scintillation counting. All data were analyzed for statistical significance using Wilcoxon signed rank tests. Results: Membrane cholesterol content was significantly reduced in aliquots incubated in capacitating media without nifedipine (n = 8, P < 0.04). This decrease in cholesterol content was significantly correlated with increased MR expression (n = 8; P < 0.008). In an important contrast, sperm treated with nifedipine in capacitating media exhibited cholesterol levels that were equivalent to that of uncapacitated sperm (n = 8; P = 0.1, NS). MR expression in nifedipine-treated aliquots was significantly lower than in control incubated aliquots (n = 8; P < 0.008) and was indistinguishable from that of uncapacitated aliquots (n = 8; P = 0.2, NS). A significant increase in 14C-acetate incorporation at 5 hrs was higher than at 24 hrs (n =4, P < 0.003). Conclusions: GC analyses confirm that nifedipine blocks canacitation via regulation of membrane cholesterol. The observed increase in1 14C-acetate incorporation into membrane sterols suaeests that cauacitatinn inhibition is effected by stimulation of membrane cholesterol synthesis, This in vitro data mimics prior observations in patients with iatrogenic infertility. The difference in levels of 14C-acetate incorporation between 5 hrs and 24 hrs suggests that there is competition between cholesterol efflux induced by capacitation media and nifedipine-stimulated cholesterol synthesis. Thus, our observations on the increased cholesterol synthesis suggest that compounds like nifedipine which regulate membrane cholesterol can be develIV

s52

Abstracts

oped as male contraceptives. 06100 to SB).

(Supported

Tuesday,

by NIEHS/NIH

September

3:45

Grant

No. ES

28, 1999

P.M.

o-131 Preclinical Studies of Intravaginal Formulations of Spermicidal nadocenes. ‘.‘O. .I. D’CNZ, ‘F. M. Uckun. ‘Drug Discovery Program, partment of 2Reproductive Biology, Hughes Institute, St. Paul, MN.

VaDe-

Objectives: Vanadium (IV)-containing his-cyclopentadiene complexes (vanadocenes) are potent spermicides (Biol. Reprod. 58:1515;1998;Mo~ec. Hum. Reprod. 4:683;1998). In a systematic search for vanadocenes with spermicidal activity as a new class of contraceptive agents, we identified vanadocene acetylacetonato monotriflate (VDACAC) and vanadocene dithiocarbamate monotriflate (VDDTC) as the most potent and stable spermicidal compounds. This study investigated the effect of repetitive intravaginal application of gel-microemuslions of VDACAC and VDDTC, on the reproductive capability of female mice and vaginal tolerance in rabbits. Design: Preovulatory intravaginal administration of gel-microemulsions of VDACAC and VDDTC on ovulation response and pregnancy outcome in PMSGihCG-primed CD-l mice and on vaginal tolerance test in the NZW rabbit model. Histological assessment of rabbit cervico-vaginal tissues and atomic absorption spectrophotometric quantitation of vanadium in the tissues and body fluids. Materials and Methods: For ovulation rate, sexually mature CD-l female mice (2O/group) received intravaginal administration of 0.1% VDACAC or VDDTC in a gel microemulsion for 5 consecutive preovulatory days. Female mice were injected with PMSG followed by 48 h later by hCG to induce superovulation. Ovulation was assessed by examining the number of ova recovered from the oviducts 15 h post-hCG. For the fertility rates, the treated female mice (201group) were mated (1: 1) with fertile males and sacrificed 8 days after gestation for embryo counts. For vaginal tolerance test, 9 does were allocated into 3 groups (3 does/group). Does in group 1 received a placebo (gel-microemulsion only) intravaginally for 10 days. Does in group 2 and 3 received 0.1% of VDACAC and VDDTC gel-microemulsions, respectively. On the 1 lth day, does were sacrificed and the representative samples of the lower, middle, and cervicovaginal segments were fixed, sectioned, and stained with hematoxylin and eosin. Selected organs (liver, lung, kidney, ovary, spleen and vagina) and body fluids (blood plasma and urine) were analyzed for vanadium content by atomic absorption spectrophotometer. Results: There was no significant difference in the percentage of ovulated mice (100% and 93% versus 100%) or the total number of ova recovered for vanadocene and control groups. The mean number of ovulated ova recovered from the VDACAC and VDDTC-treated groups were not different from the control group (41 and 34 versus 35; p > 0.05). There was no significant difference in the number of fertile female mice (45% and 45% versus 55%; p > 0.05) or the number of embryos (13 and 19 versus 25; p > 0.05) between the vanadocene and control groups. Both VDACAC and VDDTC did not irritate rabbit vaginal epithelium (total score <4). Histological evaluation revealed intact vaginal epithelium, lack of leukocyte infiltration, vascular congestion or edema. Vanadium content in tissues from control and vanadocene groups showed no significant difference among groups; all of the values were background levels (<0.4 ppm). Conclusions: Repetitive intravaginal application of spermicidal vanadocenes, VDACAC and VDDTC had no effect on the reproductive performance of female mice and showed no evidence of vaginal irritation or systemic toxicity in female rabbits at doses lOOO-fold of their spermicidal activity. Therefore, these vanadocenes have unprecedented potential as effective and safe vaginal contraceptives for women,

MALE

REPRODUCTION

AND UROLOGY

I

Tuesday,

September

2:00

28, 1999

P.M.

O-132 The Effect of Age on Sex Chromosome Spermatocytes of Normospermatogenic

Vol.

Aneuploidy in Mitotic Men. ‘W. J. Huang,

72, No.

3, Suppl.

and Meiotic ‘D. J. Lamb,

1, September

1999

*E. D. Kim, ‘W. W. Lin, ‘L. I. Lipshultz, 3F. 2. Bischoff. ‘Urology, Veterans General Hospital, Taipei, Taiwan; Department of *Urology and 30bstetrics/Gynecology, Baylor College of Medicine, Houston, TX. Objectives: The association between increased risk of genetic defect due to advancing paternal age remains undetermined. This study is to test the hypothesis: advancing age has effects on increasing the frequency of sex chromosome aneuploidy in developing germ cells. Design: To compare the sex chromosome aneuploid rats of germ cells during early spermatogenesis of testicular tissue from varying ages of men, i.e.: spermatocyte mitosis and meiosis. Materials and Methods: Fixed testis tissue blocks were obtained from men undergoing autopsy due to accidental death or myocardial infarction. Normal spermatogenesis was confirmed in every sample by histologic staining and analysis. Patients were categorized according to age range: 50-59 (n=3), 70-79 (n=3) and 80-89 (n=2). Five testis biopsy samples from obstructed azoospermic men were used as controls representative of young age (30-39 years). Three-color fluorescence in situ hybridization (FISH) was used to detect sex chromosome aneuploidy. Spermatocyte nuclei were classified as diploid (predominantly primary spermatocytes) or haploid (predominantly secondary spermatocytes) based on the presence of two or one, respectively, FISH signal for a control chromosome 18 probe. Results: No significant difference was observed between aneuploid rates of the diploid spermatocytes among the different age groups. Although the aneuploid rate of haploid nuclei was not significantly different between the control and either of the 50-59 and 70-79 age groups, the aneuploidy rate was significantly increased in men >80 years of age. (see Table) Conclusions: These preliminary results suggest that paternal age may be associated with increased risk of chromosome non-disjunction in meiotic divisions. Diploid + Haploid Germ Cell Age 30-39 50-59 70-79 SO-89

Aneuploid/Total

Diploid %

74012,465 27611,017 46211,345 2981738

30.0 27.1 34.3+ 40.4+

731396 611343 681316 391191

+

30-39 50-59 7&79 8&89

Other

Aneuploid/Total

Germ

%

66712,069 2151674

32.2 31.9 38.3 47.3+

39411,029

2591547

Cell

Aneuploid/Total

Haploid Gem Cell Age

Germ

% 18.4 17.8 21.5 20.4

Cell/All %

7712,542 5611,073 7811,423 120/858

+

3.0 5.2 5.5 14.0+ +

Table. Frequency of the aneuploidy among diploid and haploid germ cells in the seminiferous tubules from the testis obtained from different age groups of normospennatogenic men. +, ++: P
Tuesday,

September

2:15

28,

1999

P.M.

o-133 Evaluation of Semen and Sperm Chromosome Aneuploidy Rates in Couples With Unexplained Recurrent Pregnancy Loss. ‘L. .I. Lowy, ‘H. H. Hatasaka, ‘C. M. Peterson, ‘K. P. Jones, ‘L. C. Udoff, * A. L. Wilcox, ‘.*D. T. Carrell. ‘Department of Obstetrics and Gynecology and *Division of Urology, University of Utah School of Medicine, Salt Lake City, UT. Objectives: Recurrent pregnancy loss (RPL), defined as three or more miscarriages with 5 one live birth, affects approximately 1% of couples. Even following comprehensive evaluation, the etiology often remains unexplained. Prior studies by our laboratory have shown that IVF patients with an increased percentage of morphologically abnormal sperm had a decreased mean embryo quality and increased rates of chemical pregnancies

FERTILITY

& STERILITY@

and spontaneous abortion. In this study, we have analyzed standard semen quality parameters and sperm aneuploidy rates in couples with unexplained recurrent pregancy loss (RPL). Design: Semen analysis and sperm aneuploidy rates for chromosomes were evaluated in men with a history of RPL, general infertility patients, men from the general populations, and sperm donors of known fertility. Materials and Methods: All study couples had normal comprehensive RPL evaluations including: hysterosalpinogram, thyroid stimulating hormone, lupus anticoagulant, anti-cardiolipin, factor V Leiden, mid-luteal progesterone, and parental chromosome karyotyping. A semen sample was collected following 2-5 days of sexual abstinence and analyzed using WHO techniques for sperm concentration, motility, morphology, and HOS reactivity. Additionally, decondensed sperm preparations were simultaneously analyzed for aneuploidy of chromosomes X, Y, 13, 18, 21 using commercially available probes, and techniques. Results: Percent of sperm with normal morphology was significantly (pcO.05) decreased in the RPL patients (43.0 ? 4.3) compared to the general population group (55.6 2 2.4) and sperm donors (68.1 + 1.3). The percentage of tapered sperm was increased (pcO.05) in the RPL group compared to those same controls groups (42.8 2 5.2 versus 27.3 2 2.4 and 21.9 2 1.7). The percentage of total sperm aneuploidy for the five probes used in the RPL group was increased (pcO.05) compared to the donor control group (2.38 + 0.33 and 1.47 2 0.24). In 5/18 RPL patients, the aneuploidy rates were substantially increased. Conclusions: To date, the basic evaluation for couples with RPL has concentrated on female factors. Semen analyses obtained during the evaluation of couples with otherwise unexplained infertility demonstrated an increased proportion of abnormal sperm morphology, specifically an increase in tapered spermatozoa compared to control groups of sperm donors and men from the general population. Furthermore, aneuploidy rates detected by fluorescence in situ hybridization for five chromosomes were higher among the sperm of the RPL group than the control group.

Tuesday,

September

2:30

28, 1999

P.M.

o-134 Aneuploidy in Sperm From Fifty-four Oligoasthenoteratozoospermic (OAT) Patients Undergoing Intracytoplasmic Sperm Injection (ICSI). M. G. Pang’, S. F. Hoegennat?, .I. Pfeffers, M. Stacey4, L. Lunsford4, C. Osgood4,5, G. Donce16, S. Oehninger6, A. A. Acosta6, W. G. Kearns4@,‘. ‘Biomedical Research Center, Korea Advanced Institute of Science and Technology, Taejon, Korea, *College of William and Mary, Williamsburg, VA., ‘DHUYS IVF Center, Zerah-Tsar-Pfeffer Laboratory, Bagnolet, France, 4Center for Pediatric Research, Eastern Virginia Medical School, Norfolk, VA., 50ld Dominion University, Norfolk, VA., 6Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, VA., ‘Johns Hopkins University School of Medicine, Baltimore, MD. Objective: We summarize data on aneuploidy in sperm from 54 OAT patients undergoing ICSI. Study Design: This study determined aneuploidy in sperm from OAT patients and tabulated fertilization and pregnancy rates following ICSI. Materials and Methods: Aneuploidy frequencies were determined in sperm from 54 OAT patients and 18 proven fertile donors using multiprobe, multi-color fluorescence in situ hybridization (FISH). Direct labelled DNA specific for chromosomes 1, 4, 6, 8, 9, 10, 11, 12, 13, 17, 18, 21, X and Y were used and over 200,000 sperm were scored. Where appropriate, Chi-square analysis or Fisher’s Exact Test were performed. Results: Per chromosome disomy frequencies for the gonosomes and autosomes ranged between 0 and 5.7% for patients and 0 to 0.3% for controls. The frequency of diploid sperm ranged from 0 to 9.6% in patients and 0 to 1.2% for controls. The OAT patient group was heterogeneous for the type and frequency of numerical chromosome abnormalities observed. Total aneuploidy in sperm from the 54 OAT patients was estimated, using formulae developed by Hoegerman et al., to range between 33 and 74%. In contrast, total aneuploidy in sperm from the 18 proven fertile donors was between 3.9 and 7.7%. Thirty-six ICSI cycles were performed. Sixty-nine percent (175/252) of oocytes fertilized. Following fresh embryo transfer, 2 preclinical abortion’s, 1 first trimester loss and 2 healthy term deliveries (2/26 = 7.7%) resulted. Conclusions: There were significant increases in the frequencies of dip-

s53

loidy, autosomal disomy, sex chromosome aneuploidy and total cytogenetic abnormalities in sperm from 54 OAT patients versus controls. The data suggest that meiotic errors occur at highly elevated frequencies in the germ cells of severely affected OAT patients. Males with severe OAT donating sperm for ICSI are at risk of transmitting genetic abnormalities to their offspring.

Tuesday, September 28, 1999 2145 P.M.

their most common mode of inheritance included: Trisomy 21 (aneuploidy-maternal origin), Turner syndrome (aneuploidy-paternal origin), cleft lip/palate (multifactorial), VATER sequence (Vertebral defects, imperforate anus, trachea-esophageal fistula, radial and renal dysplasia-sporadic inheritance), and Hurler syndrome (autosomal recessive). Possible responses included: A) remove donor from program, B) inform potential recipients of prior pregnancy outcomes and continue to use donor or C) further study donor to assess karyotype/mutations. Results: A total of 64 surveys were available for analysis, reproductive endocrinologists (27, 42%) and medical geneticists (37, 58%). Trisomy 21

o-135 Screening of the Differential Expressed Genes in Testes With Normal and Spermatogenic Defect Patients. ‘G. .I. Song, ‘H. S. Lee, ‘Y. S. Park, ‘J. W. Kim, sH. J. Lee, 2,4J. T. Seo, 2,4J. H. Kim, 2.4Y. S. Lee, 3.41. S. Kang. ‘Infertility Research Laboratory, *Department of Urology, 3Department of Obstetrics/Gynecology, Samsung Cheil Hospital and Women’s Healthcare Center, 4College of Medicine, Sungkyunkwan University, sDepartment of Physiology, College of Eulji Medicine, Seoul, Korea. Objectives: Spermatogenesis is controlled by many genes and spermatogenic failure may be induced by abnormal gene expression. Non-obstructive azoospermic patients showed spermatogenic defects in their testes. Therefore, we expect the differential expression pattern in patients with spermatogenic defects compared to normal spermatogenesis. Differential display-polymerase chain reaction (DD-PCR) technique is a powerful tool for identifying and cloning differentially expressed genes in patients. The aim of this study is to identify genes which are associated with spermatogenie defects in human testis. Design: DD-PCR was used to screen the differential expressed genes. Method: Testicular biopsies were taken from 4 patients, two with Sertoli cell only syndrome and two patients with normal spermatogenesis. Total RNA was extracted and mRNA DD-PCR technique was performed to identify the differentially expressed genes. The expressed gene products were cloned, sequenced and sequence-analyzed. Results: Seventeen differentially expressed amplification products were identified and isolated. Nine out of 17 differential bands were detected only in men with normal spermatogenesis (Norl-Nor9 clones), the other eight bands were detected only in SC0 patients (Scol-Sco8 clones). Norl, Nor2, Nor& Scol, Sco2 clones were sequenced and analyzed. Nor8, Scol and Sco2 clones showed no significant similarity with previously reported nucleotide sequences. On the other hand, Nor1 clone showed 70% homology to X-linked apoptotic inhibitory protein (XAIP) and Nor2 clone had 98% similarity to translation initiation factor eIF-2 gene. Conclusion: From these results, we could confirm that differentiation of sperrnatogenic cell was related with apoptosis and translational regulation. In addition, several novel genes and homologous genes, may be important factors in normal spermatogenesis, were obtained. Further studies will study the functions of these genes and their effects on spermatogenic defects.

Tuesday, September 28, 1999 3:00 P.M. O-136 Management of Anonymous Sperm Donors Whose “Offspring” Have a Chromosomal or Structural Abnormality. ‘K. D. Traynor, ‘W. R. Meyer, ‘J. A. Kuller, ‘K. F. Dorman. *Department of Obstetrics and Gynecology, University of North Carolina, Chapel Hill, NC. Objectives: To determine how clinicians would manage sperm donors whose “offspring” are found to have chromosomal or structural abnormalities. Design: A directed, multiple-choice survey was administered to reproductive endocrinologists and medical geneticists to assess their management of sperm donors whose “offspring” were found to have chromosomal or structural abnormalities. Materials and Methods: A survey questionnaire was completed by 64 reproductive endocrinologists and medical geneticists. The questionnaire addressed what action should be taken if a sperm donors’ “offspring” was found to have a chromosomal or structural abnormality. Abnormalities and

s54

Abstracts

A B C A B to C

12% 79% 9%

Turner Syndrome 33% 53% 14%

Cleft Lip/Palate 38% 60% 2%

VATER Sequence 33% 65% 2%

= Remove donor from program = Inform potential recipients of prior pregnancy outcomes use donor = Further study donor to assess karyotype/mutations

Hurler Syndrome 82% 12% 6% and continue

Conclusions: Well-established guidelines exist for screening of potential gamete donors. However, no such guidelines have been established to address the sperm donor whose “offspring” is found to have a chromosomal or structural abnormality. Our data indicate that mode of inheritance strongly influences the decision to remove these donors from sperm banks; however, a clear consensus does not exist. This indicates a critical need for further study and the development of appropriate guidelines for the management of sperm donors with untoward pregnancy outcomes.

Tuesday, September 28, 1999 3:45 P.M. o-137 Detection of Testicular Cancer Pasqualotto, H. Kobayashi, A. Advanced Research in Human Urology, The Cleveland Clinic

in Men Presenting With Infertility. F. F. Agarwal, A. J. Thomas, Jr. Center for Reproduction & Infertility, Department of Foundation, Cleveland, OH.

Objectives: The increase in the incidence of testis cancer appears to coincide with signs of general decline in male reproductive health. It has been suggested that the development of testis cancer is etiologically and pathogenetically linked to other forms of gonadal dysfunction. Epidemiologic reports suggest that testicular cancer and impaired spermatogenesis may be biologically related. Infertility is one of the less common presenting features of testicular tumors. We sought to evaluate the histological and biochemical findings, and pregnancy outcome in patients presenting with infertility who were found to have incidentally discovered testicular tumors. Design: Retrospective study. Materials and Methods: From 1983 to 1998, 7 patients with infertility were also diagnosed as having testicular cancer by a single examiner (AJT) at a tertiary care hospital. Of the seven patients presenting with primary or secondaty infertility, two had orchialgia. Age, physical exam, histological findings, hormonal status, tumor markers, and pregnancy outcome results were recorded from the patients medical chart. Results: The mean age was 30.7 years (range 25-34). All patients had testicular ultrasound evaluation, which demonstrated a hypoechoic or irregular intratesticular mass. The indications for the ultrasound were either testicular pain in 2 patients or a suspicious palpable mass in 5 patients. Two men had elevated serum FSH and LH levels, one of these had abnormally low semm testosterone level. Tumor markers were normal in all patients. In four patients the tumor was on the right side and in three, on the left. All tumors were considered pT1. The histological diagnoses were seminoma (n = 5), Leydig cell tumor (n = l), and carcinoma in situ (n = 1). Of the 7 patients, 5 underwent adjuvant radiation therapy. Two patients had sperm cryopresemed. Follow up on fertility status was available in 6 cases. One patient has subsequently established pregnancy, and 5 did not achieve pregnancy after treatment for their cancer. Conclusions: Patients presenting primarily with infertility may be at higher risk for testicular tumors. We suggest that men who present with

Vol. 72, No. 3, Suppl. 1, September 1999

infertility should be thoroughly investigated to rule out any other serious, concomitant disease along with their infertility.

Tuesday,

September

4:00

28, 1999

P.M.

Detected, Non-Palpable A. Zini, M. Buckspan.

Testicular University

Masses in of Toronto,

Objectives: There is evidence that testicular cancer and some types of male infertility are interrelated, possibly as part of a disorder of normal spermatogenesis. The links between testis cancer and infertility are based on 1) Increased testis cancer in infertile men. 2) Decreased fertility in men with testis cancer. 3) Increased rates of testis cancer and infertility in men with undescended testis. The ultrasound characteristics of testis tumors are well documented. Usually a testicular ultrasound is performed once the testicular mass is palpable. The potential to use ultrasounds for early diagnosis of testis cancer (i.e. prior to the tumor being palpable) has not been explored, but early identification of tumors is an important strategy in the management of all cancers. Materials and Methods: All men with infertility (excluding men with a previous vasectomy) presenting for investigation at the Mount Sinai Hospita1 andrology centre had a scrotal ultrasound. If a suspicious mass was identified, the ultrasound was repeated by a second ultrasonographer. Men with a suspicious mass had a standard inguinal exploration, ultrasound guided testis bionsv and radical orchiectomv if warranted. Results: A no&alpable, ultrasound de&able, testis mass was identified in 3/450 men in the past year. All of the masses were hyperechoic: 2/3 had well defined margins. The average size was 1.2 cm. All were felt to be suspicious for cancer. One of the men had a previous orchiopexy, while the other 2 had no previous surgery. At surgery the exposed testis was carefully palpated, and even at surgery no masses were felt. The ultrasound identified mais was removed for all 3 men. None of the specimens had any evidence of a malignancy. In 2/3 specimens, no abnormality was detected while in one man there was evidence of scar tissue. Conclusion: Ultrasound detected, non-palpable testicular masses are relatively common features in men with infertility. In our small series none of the masses were a malignancy. This remains a small series, and until these findings are confirmed by larger series, we continue to recommend that all men with an incidentally discovered testis mass with the ultrasound characteristics of cancer, be treated as thought they may potentially have a cancer of the testis. Tuesday,

September

4:15

28,

1999

P.M.

o-139 Testicular Function in HIV-Infected Males. S. Fiore, F. P. G. Leone, A. Bulfoni, V. Savasi, E. Garzia, M. Oneta, S. Giuntelli, and A. E. Semprini. Department of Obstetrics and Gynaecology, San Paolo Biomedical Institute, University of Milan, Medical School. Objective: Evaluate testicular function in HIV positive males according to their immunological and clinical status by investigating their hormonal profile and seminal analysis. Design: Retrospective study of HIV-positive men with seronegative partners enrolled in a program of assisted conception. Materials and Methods: Two hundred sixty eight HIV-positive males requiring reproductive assistance to reduce the risk of infecting their partner. All patients were asked a blood sample to be tested for FSH, LH, PRL and Testosterone by radioimmunoassay. Seminal analysis was conducted according to the WHO recommendations in the screening for infertility factors prior to the insemination procedure. Results: The Table shows mean values of seminal parameters and hormonal concentrations obtained in 240 (89%) CDC II-III and in 28 (11%) CDC IV patients.

FERTILITY

& STERILITY@

pramenters

Volume Motility

(mL) (%)

Sperm count (million/mL) Sperm count (specimen)

O-138 Significance of Ultrasound Infertile Males. K. Jarvi, Toronto, Canada.

ax Seminal

Motile count (million/mL) Motile count (specimen) Round cells (X 106) Normal sperm (%) Hormonal concentrations

FHS (mUI/mL) LH (mUJ/mL) Testosterone (~&IL) Prolactin (n&IL)

Level II + III

CDC Level

(n = 240)

(n = 28)

3.16 46.8 87.7 264.4 45.8 127.9 2.79 48

2.1 40.3 86.3 137 40.1 66 3.56 43.6

4.85 5.84 8.9 26

8.82 12.1 8.44 10.3

IV P <0.0001 ~0.06 co.9


Conclusions: Testicular function is affected by the progression of patient’s disease as males classified as in class IV of disease have a reduced seminal output volume. These men have more frequently a reduced total sperm count (P
THE SOCIETY

OF REPRODUCTIVE

Tuesday,

September

2:00 SRS Prize

28,

SURGEONS 1999

P.M.

Paper

O-140 Successful High Intensity Focused Ultrasound (HIFU) Treatment of Uterine Fibroid Tumors in a Nude Mouse Model. ‘V. Y. Fujimoto, *C. Walker, 3G. Keilman, 4*6R. Martin, ‘L. A. Crum, ‘?S. Vaezy. ‘Department of Obstetrics and Gynecology, 4Department of Anesthesiology, and 6Department of Bioengineering, University of Washington School of Medicine, Seattle WA. ‘Applied Physics Laboratory, University of Washington, Seattle WA. *Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithsville, TX, 3Sonic Concepts, Inc (Woodinville, WA). Objectives: Uterine leiomyomata represent a common gynecologic surgical indication in the United States. We are developing a method of treatment for uterine fibroids (leiomyomata) using High Intensity Focused Ultrasound (HIFU). HIFU provides a modality for delivering large amounts of thermal energy using ultrasonic waves to induce tissue necrosis, without damaging intervening tissues. In contrast to diagnostic ultrasound where intensity levels are below 0.1 Watts/cm’, HIFU can deliver l,OOO-10,000 Watts/cm* at the focal spot. The HIFU transducer emits ultrasonic waves that converge to create a focal spot size of 9 X 1 mm. To establish efficacy of uterine fibroid treatment, we utilized a nude mouse model with subcutaneous tumors derived from ELT-3, a uterine fibroid cell line. Design: Athymic nude mice were injected with ELT-3 cells and the resulting fibroid tumors were treated and observed. Materials and Methods: Thirty-nine female athymic nude mice were inoculated with 5 X lo6 cells of the ELT-3 cell line injected subcutaneously into the suprascapular region. Fibroid tumors became visible within 2-3 weeks after injection. Tumor volume was calculated using transcutaneous caliper measurements of the long and short dimensions of the tumors. Animals were designated to one of two groups: (1) sham-treated group (n=ll) or (2) HIFU-treated group (n=28). All animals underwent an incision in the skin overlying the tumor to expose the tumor. HIFU was administered using a 3.5 MHz transducer with an intensity of 2000 Watts/ cm’. Histological analyses of tumors was conducted. Unpaired t-testing was used to compare tumor volumes between the two groups.

s55

Results: We observed a maximal percent tumor volume reduction (mean 2 SEM) in the HIFU-treated group of 87.0 -C 4.7% (d. 4-28 after treatment). Sixteen of the 28 animals in the HIFU-treated group (57%) had complete elimination of their tumors after a single treatment of HIFU. Of the 12 animals without complete tumor shrinkage after the initial HIFU treatment, five animals underwent a second treatment of HIFU after d.28 of initial treatment that successfully resulted in complete reduction of the tumors. The remaining five animals were sacrificed due to scab loss or restrictions on tumor bulk without further treatment. In contrast, the tumors in the sham-treated animals continued to grow with a percent tumor volume increase (mean 2 SEM) of 177.3 2 44.8% observed d. II-14 after treatment (vs. -65.4 2 10.3% in the HIFUtreated group, p
Tuesday, September 28, 1999 2:15 P.M. o-141 Robotically Assisted Human Pilot Study. tion, Cleveland, OH.

Laparoscopic T. Falcone,

Microsurgical Tubal Anastomosis: .I. Goldberg. Cleveland Clinic Founda-

Objective: Microsurgical tubal anastomosis requires extremely fine, precise suturing technique, which is difficult to achieve by conventional laparoscopy. The recent development of robotic technology has demonstrated that robots can outperform humans in several tasks that require a precise repetitive motion. The primary objective of this study was to evaluate a robotic system in performing laparoscopic tubal anastomosis in the human. This is the first human trial to use a robot to perform a surgical procedure. Design: Prospective human pilot study. Materials and Methods: IRB approval was obtained prior to the study. The FDA approved a 10 patient pilot study. Patients with a reversible tubal ligation were recruited for the study. All male partners had a normal semen analysis. All of the patients had ovulatory cycles and a hysterosalpingram (HSG) that demonstrated at least 1 cm of proximal tube. The anastomosis was performed in 2 layers with interrupted 8-O Vicryl suture. Four sutures were placed through the mucosa and muscularis and 4 sutures were used for the serosal layer. The ZEUS system (Computer Motion Inc) was used to perform the anastomosis. The system incorporates three robotic arms, allowing a single surgeon to remotely manipulate the laparoscopic camera and two laparoscopic surgical instruments. The surgeon sits at a console away from the operating room table. Although this console could be placed in a different room, in this study it was placed in the room where the surgery was performed. The console consists of a monitor, two handles and the computer interface. The surgeon directs the position of the camera by voice activation. The two handles control the movement of the two robotic arms that hold the needle holder and tissue grasper. The computer interface filters, scales, and then translates the surgeon’s movements to the robotic arms and instrumentation. Results: The mean age of the patients was 3123.6 years and mean body mass index was 25 kg/M’. Tubal anastomosis was accomplished by robotic laparoscopic microsurgery on all tubes that were accessible (19 tubes/IO patients). All tubes demonstrated patency with chromotubation at the end of the procedure. It took 6?2 minutes to set up the robotic arms and 159?33 minutes to perform the tubal anastomosis. There were no complications and 800 patients were discharged home the same day. A six week postop HSG demonstrated patency in 17119 (89%) tubes anastomosed. All patients had at least one tube open. The trial occurred from June to Sept 1998. Three ongoing pregnancies have been reported so far. Conclusion: Robotic technology can be used safely to perform a laparoscopic tubal anastomosis. Robotic technology has the potential to make laporascopic microsuturing easier. This technology also has the potential to make telesurgery a reality.

S56

Abstracts

This work

was supported

in part by Computer

Tuesday,

September

2:30

Motion

Inc. Goleta,

CA.

28, 1999

P.M.

O-142 Uterine Balloon Therapy vs. Rollerball Endometrial Ablation in the Treatment of Excessive Uterine Bleeding. ‘D. A. Grainger, ‘B. W. Walsh, 3W. R. Meyer, 4L. M. Peacock, ‘F. D. Loffer, ‘J. F. Steege. ‘Center for Reproductive Medicine, Wesley Medical Center, Wichita, KS, *Department of Obstetrics and Gynecology, Brigham and Women’s Hospital, Chestnut Hill, MA, 3Department of Obstetrics and Gynecology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 4Department of Obstetrics and Gynecology, Emory University School of Medicine, Atlanta, GA, ‘Maricopa Medical Center, Phoenix, AZ. Objectives: To assess the safety and efficacy of the THERMACHOICE* Uterine Balloon Therapy System (Gynecare/Ethicon, Inc.) compared to rollerball ablation in the treatment of excessive uterine bleeding, and to identify late complications and adverse effects of uterine balloon therapy (UBT). Design: A randomized, prospective, controlled multicenter clinical investigation at 14 investigational sites in the United States and Canada. Materials and Methods: 275 women with menorrhagia, as established by menstrual flow diary, were randomly assigned to treatment with UBT or rollerball therapy. 50% were assigned to each group. All investigators were experienced rollerball users and were trained in the use of THERMACHOICE* Uterine Balloon Therapy System. Uterine bleeding and quality of life indicators were documented pretreatment, monthly through the first year, and then at year 1 and 2. All complications and adverse events were documented. Results: 255 patients underwent treatment. Two-year results are reported herein. With success defined as absence of menorrhagia, the success rate for UBT was 89.1%, and for rollerball 90.4%. Amenorrhea was reported in 13.3% of UBT patients and 22.1% of rollerball patients (p=O.28). 86.1% of UBT patients and 86.7% of rollerball patients reported a high level of patient satisfaction. Adverse events at 2 years were confined to 15 hysterectomies (4 UBT, 11 rollerball) with 6 hysterectomies performed for menorrhagia (2 UBT, 4 rollerball). One pregnancy was reported in a rollerball patient 2.5 years post-ablation. Conclusions: THERMACHOICE uterine balloon therapy is as safe and effective as rollerball therapy in the treatment of excessive uterine bleeding. (Sponsored by Gynecare/Ethicon, Inc.)

Tuesday,

September

2145

28, 1999

P.M.

o-143 Hypoxia Regulates Activin A Expression in Fibroblasts. ‘W. W. Zhang, ‘,‘F. D. Yelian, ‘K. A. Wei, ‘G. M. Saed, 3D. B. Seifer, ‘M. P. Diamond. ‘Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI; ‘Department of Obstetrics and Gynecology, Michigan State University College of Human Medicine, Saginaw, MI; 3Department of Obstetrics and Gynecology, UMDNJ, New Brunswick, NJ. Objectives: Previous studies have shown that activin modulates cell proliferation in fibroblasts and vascular smooth muscle cells suggesting that activin may play an important role in wound healing and adhesion formation. Our recent studies have demonstrated that hypoxia directly alters cellular adhesion to various extracellular matrix (ECM) proteins. In this study, we are investigating the effects of hypoxia on the mRNA and protein levels of activin A in fibroblast cells. Design: NIH3T3 cells, a fibroblast cell line, were incubated in normal or hypoxic condition for 48 h. The mRNA and protein levels of activin A were analyzed using RT-PCR and ELISA assays. Materials and Methods: NIH3T3 cells, mouse activin A and P-actin primers, and an activin A ELISA kit were purchased from ATCC, GIBCO BRL, and Serotec Inc., respectively. 3T3 cells were incubated in normal (20% oxygen) or hypoxic condition (5% oxygen) for 48 h. Total RNA from these cells was isolated and reverse transcription polymerase chain reaction

Vol. 72, No. 3, Suppl. 1, September 1999

(RT-PCR) was performed. Following densitometry, mRNA levels were standardized by p-actin. Meanwhile the supematants from these cell cultures were collected, and activin A protein levels were measured using an ELISA assay. Results: Activin A mRNA was detected in these fibroblast cells. Interestingly, using a densitometry, we observed a significant increase in mRNA expression in cells treated with hypoxia. We also detected a high protein level of activin A in these culture supematants. The levels of activin A in both normal and hypoxia-treated samples were 5.70 and 8.25 @ml, respectively. It indicates that hypoxia increases the activin A expression by 44% in these fibroblast cells. Conclusion: Our study showed that mRNA and protein of activin A were detected from fibroblasts. We also demonstrated for the first time that hypoxia modulate the activin A expression in these cells. Since hypoxia results in an increased activin A expression and cellular adhesion to ECM proteins, it suggests that activin A may have an important role in cellular adhesion. The significance of activin A in wound healing and adhesion development remains to be explored. (Supported in part by Genzyme Corporation).

Tuesday,

September 3:00 P.M.

28, 1999

o-144 Clinical Evaluation of INTERGEL Adhesion Prevention Solution For the Reduction of Adhesions Following Peritoneal Cavity Surgery. D. Alan Johns. Texas Healthcare, Fort Worth, Texas, and the INTERGEL International Adhesion Study Group. Objectives: To assess the safety and efficacy of INTERGEL Adhesion Prevention Solution (Ethicon, Inc.) in reducing adhesions in patients undergoing peritoneal cavity surgery by laparotomy with a planned second-look laparoscopy. Design: Randomized parallel group, third party blinded, placebo controlled (lactated Ringer’s solution) study at 16 centers in the U.S. and Europe. Materials and Methods: Primary indications for surgery include infertility, pain, and/or irregular vaginal bleeding in patients desirous of retaining their fertility. INTERGEL Adhesion Prevention Solution, a hyaluronic acid gel, manufactured by Lifecore Biomedical Inc., Chaska, MN and lactated Ringer’s solution were provided in sealed cartons. A total of 213 female patients who met all preoperative and intraopcrative criteria received 300 mL of either the treatment or control solution as an intraperitoneal instillate at the completion of a laparotomy procedure as determined by the randomization schedule. At second-look laparoscopy 6-12 weeks after the laparotomy, the incidence, extent and severity of adhesions was assessed at 24 anatomical sites throughout the peritoneal cavity. The principle surgical procedures performed at the initial laparotomy included myomectomy, adhesiolysis, ovarian surgery, tubal surgery, and surgical treatment of endometriosis. Hematology, serum chemistries, concomitant medications, and adverse events were monitored throughout the study interval. Results: Patients treated with INTERGEL Solution had significantly reduced adhesions compared to the patients who received lactated Ringer’s solution, including both de nova (26%. p=O.OlO) and reformed adhesions (33%, p
FERTILITY

& STERILITY@

IMAGING

IN REPRODUCTIVE

Wednesday,

September 2:00 P.M.

MEDICINE 29, 1999

o-145 Basal Antral Follicle Number and Ovarian Volume Predict Cycle Cancellation and Ovarian Responsiveness in In Vitro Fertilization. ‘J. L. Frattarelli, ‘D. F. Lauria-Costa, ‘B. T. Miller, ‘R. T. Scott Jr, ‘Combined Federal Program of Reproductive Endocrinology at WRAMC, NNMC, USUHS, and NIH, Bethesda, MD., and ‘The Institute for Reproductive Medicine and Science of Saint Barnabas Medical Center, Livingston, NJ. Objectives: Ovarian responsiveness is a clinical variable that is difficult to predict. The functional ovarian reserve as measured by basal antral follicle count and ovarian volume has recently been suggested to predict follicular response in IVF. The objective of this study was to determine the predictive value of pretreatment transvaginal ultrasound measurements of estimated ovarian volume and basal antral follicle count in patients undergoing IVF treatment. Design: An analysis of 278 infertility patients at a tertiary care center undergoing ovarian measurements prior to IVF stimulation. Materials and Methods: All women had basal ovarian measurements performed on cycle day 3 prior to starting gonadotropins. The main outcome measure was the number of recovered oocytes. All ovarian follicles were counted and the total number of follicles per patient was used for calculations. Ovarian volume was estimated by a simplified two diameter measurement, which was then averaged for each patient. To assess ovarian responsiveness, the pretreatment ovarian ultrasound measurements were compared with respect to number of follicles retrieved, age, basal serum estradiol, day 3 FSH level, day 3 FSH:LH ratio, peak estradiol level, ampules of gonadotropins, days of stimulation, pregnancy rate, and cancellation rate. A linear regression model was used to identify any correlation between two continuous variables. Additional statistical analysis was performed using a one-way Analysis of Variance test, Kruskal-Wallis ANOVA, Wilcoxon-Mann-Whitney test for nonparametric data, t-test for parametric data, Fisher’s Exact test for percentages, and a Chi-square test for trends. An alpha error of 0.05 was considered significant for all calculations. Results: The mean age of the patients was 34.8 ? 3.8. The clinical pregnancy rate per retrieval was 60.8% (155/255); of these, 15.6% (24055) experienced a first trimester loss. The cancellation rate per cycle start was 8.3% (23/278). There was a direct linear correlation between average ovarian diameter and basal follicle number (r = 0.30, p < 0.001). The mean antral follicle number was 10.1 t 8.57 (range 0 to 66). Basal antral follicle number demonstrated a positive linear correlation with recovered oocytes (r = 0.260, p < O.OOl), basal estradiol (r = 0.186, p = 0.03), and peak estradiol (r = 0.214, p < 0.001). Basal antral follicle number demonstrated a negative linear correlation with ampules of gonadotropins (r = 0.458, p < O.OOl), days of stimulation (r = -0.330, p < O.OOl), age (r = -0.32, p < O.OOl), and day 3 FSH (r = -0.244, p < 0.001). Mean ovarian size was 22.3 2 3.65 mm (range 11.25 to 34.3). Ovarian size correlated directly with recovered oocytes (r = 0.234, p < O.oOl), and basal estradiol (r = 0.194, p = 0.002). Ovarian size correlated inversely with day 3 FSH (r = 0.208, p = O.OOl), ampules of gonadotropins (r = 0.326, p < O.OOl), and days of stimulation (r = -0.231, p < 0.01). Using a Chi-square for trends, an antral follicle count less than < 11 (p = 0.007, OR 4.7 1,95% CI 1.28 to 20.47) and ovarian size <20 mm (p = 0.009, OR 3.08, 95% CI 1.2 to 7.9) was associated with an increased risk in cycle cancellation. Likewise, cycle cancellation was significantly effected by total ovarian size (p = 0.001) FSH:LH ratio (p = O.Ol), and age (p < 0.001). Both antral follicle count and ovarian size demonstrated an inverse relationship to pregnancy outcome but statistical significance was not reached. Conclusions: Ovarian size and antral follicle number can help identify patients who are at risk to have a poor response to IVF stimulation. Both ovarian parameters correlated directly with recovered oocytes and basal estradiol levels and correlated inversely with ampules of gonadotropins, days of stimulation, and day 3 FSH levels. An antral follicle count (11 and ovarian size <20 mm were associated with an significant increase in cycle cancellation. Both basal follicle number and ovarian size demonstrated an inverse relationship with pregnancy outcome; however, a larger sample size may be needed to have sufficient power to reach significance. Nevertheless, transvaginal ultrasound ovarian measurements can help predict poor re-

sponse; thereby, allowing physicians to counsel patients that they are at an increased risk for cancellation and subsequently to optimize stimulation protocols and resources.

Wednesday,

September 2:1.5 P.M.

29,

1999

O-146 Comparison of Subendometrial Blood Flow On Day 2-3 of Cycle and Day of bCG Administration. C. B. Coulam, C. Goodman, J. S. Rinehart. The Center for Human Reproduction-Illinois, Chicago, IL, Objective: At the 1998 ASRM Meeting we reported that a subendometrial peak systolic velocity (PSV) of 7.0 cm/set at the time of hCG administration provides an index of uterine receptivity for implantation after IVFIET. Earlier identification of decreased uterine receptivity would allow more time for correction of the problem. Design: To assess differences in subendomettial blood flow, PSV were measured on both cycle day 2-3 and day of hCG administration and compared. Methods: 74 women undergoing IVF treatment for infertility had subendometrial PSV measurements taken on both cycle day 2-3 (baseline) and the day of hCG administration. Mean PSV, standard deviations, standard error or the mean, minimum and maximum values were compared between day 2-3 and day hCG using two-way ANOVA. Results: Cycle

Day

2-3 hCG

N

Mean

SD

SEM

MIN

74 74

8.6 8.9

2.8 2.9

0.5 0.5

4.0 4.2

MAX 19.9 16.6

No difference was observed when PSV determined on cycle day 2-3 was compared with PSV on day hCG (F = 0.4, P > 0.5). Conclusion: No significant difference in subendometrial cycle day 2-3 or day hCG exists allowing prediction endometria earlier in the cycle.

Wednesday,

September 2:30 P.M.

PSV determined of nonreceptive

29, 1999

o-147 Magnetic Resonance Image Attributes of Ovarian Follicles At Specific Phases of Development and Regression: II. The Follicle Wall. J. L. Hilton, G. E. Sarty, G. P. Adams, R. A. Pierson. WHIRL, ObstetricsGynecology, University of Saskatchewan, Saskatoon, SK, Canada. Objective: To determine whether MR image attributes of the follicular wall would reflect the physiologic status of ovarian follicles. Design: Linear analysis of numerical pixel intensities (NPV), area under the curve, length, and standard deviation (heterogeneity, PH), slope, intercept and correlation coefficient of dominant and largest subordinate follicles in T,, T, and diffusion in vitro MR images at defined stages of the bovine ovarian cycle. Materials and Methods: The ovaries of 36 cows were removed on day 3 of wave 1 (D3W ‘l,n= lO),dav6ofwavel(D6Wl.n=9).davlofwave 2 (Dl W2, n = 9) or on at least hay 17 (D 2 17, n = 8; preovulatbry. Images were acquired using a 1.5 Tesla SP Magnetom MR imager with sequences weighted by proton density (PD) and T, (TR/TE = 480/15), 16 echo T,(TmE = 2000/50) and diffusion sequences. Images were analyzed by assessing NPV and heterogeneity along a line traversing the dominant and largest subordinate follicle wall from the annum to the stroma. NPV at the fluid follicle interface were used to calculate a regression line from which the intercept, slope, area and correlation coefficient (r’) were determined. Statistical analyses used ANOVA and protected LSD. Results: In T, images, NPV across the wall of the dominant follicles were less likely to reflect a linear relationship than at any other phase (p < 0.003). The subordinate follicle in the presence of a preovulatory dominant follicle (D 2 17) had lower NPV and intercepts than the subordinate follicle of the anovulatory wave (55.7 t 3.8 vs 69.3 t- 2.9 and 66.4 t 5.8 NPV

S58

Abstracts

respectively; p < 0.02 and 51.1 2 3.5 vs 63.5 + 2.8 and 63.3 2 5.5 respectively; p < 0.02). In T, images, the area under the curve of the dominant anovulatory follicle was higher at Dl W2 than at D3W1, D6Wl and D 2 17 (930 -t 75 vs 6802 30, 719 5 30 and 760 2 80 respectively; p < 0.02). Similarly, NPV and the intercept of the anovulatory dominant follicle wall were higher at DlW2 than at D3Wl and D6Wl (134.9 -C 7.2 vs 103.0 2 5.5 and 109.1 2 7.9 NPV respectively; p < 0.03 and 181.6 -t 8.3 vs 141.5 ? 4.1 and 145.7 2 7.9 respectively; p < 0.01). Area, NPV and intercepts of anovulatory dominant follicle walls (DlW2) were higher than preovulatory dominant follicle walls (D 2 17; p < 0.04). The wall of the anovulatory follicle was more heterogeneous and had an increasingly negative slope as the follicle regressed (DlW2 and D 2 17; p < 0.08). The subordinate follicle in the presence of a preovulatory dominant follicle (D 2 17) had lower NPV than the subordinate follicle from the anovulatory wave (p < 0.02). Subordinate follicles exhibited higher Tz wall area, NPV, PH and intercepts than dominant follicles at D3Wl and D6W 1 (p < 0.04). Conclusion: MR imaging of the ovarian follicular wall reflected the physiologic status of dominant and first subordinate follicles. Follicles with acute linear transformations at the fluid-follicle interface were atretic. Follicles that showed gradual transformations and less linear relationships were viable. Supported by the Medical Research Council of Canada. Wednesday,

September 2145 P.M.

29, 1999

O-148 Simultaneous Multigated Spectral Doppler Imaging - A Novel Independent Tool To Explore Uterine Receptivity. I. Bar-Hava, R. Yoeli, J. Ashkenazi, R. Orvieto, I. Meizner, J. Shalev, I. Sherizli, T. Petri, Z. Ben-Rafael. Department of Obstetrics and Gynecology, Rabin Medical Center, Golda Campus, Petah Tiqva, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Introduction: Simultaneous multigated spectral Doppler imaging provides simultaneous multiple measurements of blood flow velocity waveforms in the endometrium. The purposes of this study were: 1. To establish the role of simultaneous multigated spectral Doppler imaging in the evaluation of endometrial blood flow patterns; 2. To evaluate the correlation between these patterns and the endometrial thickness, E, level and patient age. Design: Prospective cohort study. Materials and Methods: The study group was comprised of women enrolled in our IVF unit who were being evaluated during the induction of ovulation stage. Blood flow velocity waveforms of the endometrium were recorded with multigated simultaneous spectral Doppler (Diasonic Synergy ultrasound scanner), which enables the acquisition and analysis of blood flow in multiple sites over a large endometrial area. Time average maximal velocity (tamax) was calculated automatically for the selected area. Endometrial thickness and serum E, level were recorded. Results: In all, 113 Doppler scans were performed. No significant correlation was demonstrated between tamax and the endometrial thickness, E, level and patient age. Conclusion: Endometrial blood flow is apparently an independent factor, unrelated to endometrial thickness or E, level. Its measurement with simultaneous spectral Doppler imaging may provide a novel tool to predict endometrial receptivity.

Wednesday,

September 3:00 P.M.

29, 1999

o-149 3 Dimensional Measurement of Gestational and Yolk Sac Volumes as Predictors of Pregnancy Outcome in the First Trimester. A. B. Copperman, A. Babinszki, T. Nyari, S. Jordan, A. Nasseri, T. Mukherjee. Division of Reproductive Endocrinology, Department of Obstetrics, Gynecology and Reproductive Science, Mount Sinai School of Medicine, New York, USA and Department of Medical lnformatics, Albert Szent-Gyorgyi Medical University, Szeged, Hungary*. Objective: CRL has been found to be the most accurate method to determine gestational age in the first trimester, and it is used to predict

Vol.

72, No.

3, Suppl.

1, September

1999

abnormal outcome as well. In addition, the size (diameter) of embryonic structures like gestational sac (GS) and yolk sac (YS) has also been found to have prognostic value for embryonic abnormalities. We proposed that first trimester volume calculations of these structures may have value as predictors of adverse reproductive outcome. Design: A prospective study of 49 consecutive patients who achieved singleton pregnancies in an infertility practice. Materials and Methods: Forty-nine patients (treated for infertility) with singleton pregnancies were included in this prospective study. Ninety-eight examinations were performed in normal pregnancies, and 14 with abnormal outcome. Each calculation of GS and YS volume was repeated 3 times using 3 different planes, and the mean of the 3 measurements was calculated. The volumes were plotted against gestational age (GA) (16-6.5 days post ovulation) to create nomograms for normal outcome. Measurements of abnormal pregnancies were compared with these nomograms. In addition, a calculation of the ratio of yolk sac volume to gestational sac volume was performed. Results: Regression analysis revealed a power correlation between GS volumes and GA, while logarithmic relationship was observed when YS volumes were plotted against GA (correlation coefficient: r = 0.904). The normal range for volumes was established within the limits of the fifth percentile. Using the nomograms, it was found that the difference between pregnancies with normal and abnormal outcome in terms of GS volumes is statistically significant (p < 0.05). However, no statistically significant difference was found when YS volumes of normal and abnormal pregnancies were compared (p = 0.2). The mean YS/GS ratio for patients with a good obstetrical outcome (ongoing pregnancy) was 0.02, while the ratio for patients who went on to have a first trimester spontaneous abortion was 0.05 (p < 0.05). Conclusion: Volume determination of YS and GS can be quickly and simply performed applying 3 dimensional sonography. This technique provides the possibility of evaluating the structures of early pregnancy with tomographic precision. Volumetry of GS proved to be predictive for pregnancy outcome, and the growth of GS volume was found to have a power correlation with GA. YS volumes increased logarithmically with GA, and when used in conjunction with overall GS volume was predictive of adverse pregnancy outcome. Ongoing studies using 3D technology will allow us to create reliable nomograms and may enable us to predict pregnancy outcome earlier and more accurately.

CLINICAL

FEMALE

INFERTILITY

Wednesday,

September 2:00 P.M.

AND

GYNECOLOGY

29, 1999

and 56.1% at 48 months, reproductive technology

as shown in Figure

excluding 6 patients who conceived (Figure 1). Post-operative ovulation

2.

6

Wednesday,

Objective: To determine cumulative ovulation and pregnancy rates following laparoscopic ovarian cautery (LOC) for clomiphene-resistant anovulatory infertility in women with polycystic ovarian syndrome (PCOS). Design: A retrospective review of 59 patients with PCOS undergoing LOC for clomiphene-resistant anovulatory infertility between January 1990 and August 1998. Materials and Methods: Through chart review and personal follow-up, response to LOC was determined. All but 9 patients had demonstrated resistance to clomiphene (i.e. did not ovulate) in doses up to 150-200 mg, and all had ~6 months of post-operative follow-up. Response to LOC was defined as spontaneous conception, or ovulation for ~6 months postoperatively; ovulation <6 months post-operatively was defined as a non-response. Data was collected with respect to post-operative ovulation rates, duration of ovulatory cycles, pregnancy rates and outcomes, including time to conception. Duration of patient follow-up ranged from 6 to 76 months. Results: Overall, 69.5% (41/59) responded to LOC. Pregnancy rate was 73.2% in responders, versus 44.4% in non-responders, with a “take home baby” rate of 68.3% and 27.8%, respectively. Cumulative spontaneous pregnancy rate for all patients was 47.4% at 12 months, 52.6% at 18 months,

FERTILITY

& STERILITY@

12 24 33 Number of cycles

46 60 from 0.R

72

Conclusions: Response to LOC results improves pregnancy rates. Our post-operative ovulation and pregnancy rates are somewhat lower than other published series. We attribute this to our selection criteria for surgery, as it was generally offered to patients with all of the following inclusion criteria: oligomenorrhea, PCO-like ovaries on ultrasound, a demonstrated resistance to ovulation induction with clomiphene, and clinical or biochemical evidence of hyperandrogenism. The lower ovulation rates with time likely reflect a trend that “successful” patients (i.e. those who ovulated and conceived) tended to be lost to follow-up earlier than unsuccessful patients.

O-150 Longterm Follow-Up After Laparoscopic Ovarian Cautery for Clomiphene-Resistant Anovulatory Infertility in Polycystic Ovarian Syndrome. A. M. Case, E. M. Greenblatt. Reproductive Biology Unit, Toronto Hospital, University of Toronto, Toronto, Ontario, Canada.

with advanced rates declined

September 2:15 P.M.

29, 1999

o-151 Women with Unexplained Recurrent Pregnancy Loss Have Elevated Day 3 Serum FSH Levels. ‘S. W. Trout, *C. W. Benito, *E. T. Vostrovsky, ‘D. B. Seifer. ‘Department of Obstetrics/Gynecology/Reproductive Sciences, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ and ‘Division of Maternal Fetal Medicine, St. Peters University Hospital, New Brunswick, NJ. Objective: 70% of recurrent pregnancy loss has no identifiable explanation despite extensive testing. Diminished ovarian reserve, as reflected by elevated day 3 serum FSH, has been associated with an increased rate of spontaneous abortion. We designed this study to determine the potential contribution that diminished ovarian reserve may have in women with unexplained recurrent pregnancy loss. Design: A retrospective comparative analysis of day 3 serum FSH and/or estradiol values of women with an identifiable cause for recurrent pregnancy loss versus those with unexplained recurrent pregnancy loss. Materials and Methods: 41 women who presented to the Pregnancy Loss Evaluation Service underwent an evaluation for anatomical, genetic, hormonal, infectious, and immunological causes for recurrent pregnancy loss and were assigned to one of two groups. Group A (n = 15) were women with a known or explained cause of recurrent miscarriage. Group B (n = 26) were women with unexplained recurrent pregnancy loss. Mean age, parity, day 3 serum FSH and estradiol levels,

s59

and presence or absence between groups. Results:

of a history

D 3 FSH 2 10 mIU/ml* D 3 estradiol 2 70 pg/ml Elevated FSH or estradiol**

of infertility

were

compared

Group A (explained)

Group B (unexplained)

1115 (7%) 111.5 (7%) 2/15 (13%)

lo/26 (38%) 2/26 (8%)

Age and presence or absence of infertility did not differ between the two groups. Conclusions: Women with unexplained recurrent miscarriage have elevated day 3 serum FSH values when compared to women with a known cause of recurrent pregnancy loss. Thus, diminished ovarian reserve may contribute to recurrent pregnancy loss in women with no identifiable cause.

September

2:30

29, 1999

P.M.

o-152 Meta-Analysis Comparing The Efficacy of Single Dose vs. Multidose Systemic Methotrexate Therapy For The Treatment Of Ectopic Pregnancy. ‘R. K. Ashby, ‘G. G. Gosman, ‘.‘K. T. Barnhart. ‘Center for Reproductive Medicine and Surgery, and the ‘Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania Medical Center, Philadelphia, PA. Objectives: The management of ectopic pregnancy (EP) with methotrexate (MTX) has been proposed using a “single dose” or a “multidose” protocol. The most appropriate dosing protocol has not yet been determined. The purpose of this study was to review and evaluate the literature describing the use of both dosing protocols to determine differences in efficacy and side effect profiles. Design: A Medline search was performed using the key words: methotrexate, ectopic pregnancy, and tubal pregnancy. Twenty-six articles were identified that described medical management of EP with intramuscular MTX administered using the single dose or the multidose protocol. The comparable results of these studies were combined and evaluated using logistic regression. Materials and Methods: The articles were stratified based on comparability of dosage and protocols, inclusion and exclusion criteria, diagnostic methods of EP, number of patients, overlap of previously published results, and quality of data reporting. Analysis was conducted on all articles identified, and also after restricting data to only those studies considered of high quality. Results: 1067 patients were treated with the single dose protocol for a combined success rate of 88.1% (95% CI 86.0-90.0%) and 260 patients were treated with the multidose protocol for a combined success rate of 92.7% (88.8-98.5%). Overall the chance of failure using single dose protocol was 71% higher compared to the multidose protocol (Odds ratio (OR) 1.71 (95% CI 1.04-2.82) P = 0.03). Restricting analysis to high quality studies demonstrated a success rate of 88.0% (85.8-89.9%) and 93.5% (88.9-96.6%) for single dose (n = 1022) and multidose (n = 184) respectively. The OR of failure using the single dose protocol was 1.96 (1.073.60) P = 0.029. Controlling for hCG level before treatment increased the OR of failure of single dose to 2.34 (1.05-5.23) P = 0.038. The presence of cardiac activity was independently associated with failure using either protocol (OR = 9.09 (3.76-21.95) P = 0.002). A total of 14.5% of patients treated with the “single dose” protocol received >l dose of MTX. These patients tended towards a lower failure rate compared to those who actually received one dose (OR = 0.64 (0.031-1.30)). Of patients receiving the multidose protocol, 6.8% received > 1 course (>4 doses). These patients were almost 5 times more likely to fail compared to those who received one course (OR = 4.9 (1.49-16.56) P = 0.002). The incidence side effects was lower in subjects using single dose (29% vs 48%, OR 0.44 (0.31-0.63), P < 0.0001) while the incidence of abdominal pain was similar (23.3% vs

20.2%).

S60

Abstracts

Wednesday,

12/26(46%)

* (Group A vs. B, p < 0.04 by Fisher’s exact test). ** (Group A vs. B, p < 0.03 by Fisher’s exact test).

Wednesday,

Conclusions: The overall success rate of systemic MTX of EP is excellent. The actual number of doses administered even within the two standard protocols. Analysis of 1320 demonstrates that the single dose-protocol has almost twice of the multidose protocol. The true optimal number of doses up to four.

September

in the treatment varies widely subjects treated the failure rate may be > 1 and

29, 1999

2145 P.M. o-153 The Effect of Follicular Fluid Reactive Oxygen Species on the Outcome of in Vitro Fertilization. ‘,‘R. K. Sharma, ‘M. Attaran, ‘,*E. B. PasqualOtto, ‘.I. M. Goldberg, ‘K. F. Miller, *A. Agarwal, ‘T. Falcone. Center for Advanced Research in Human Reproduction and Infertility, Departments of ‘Gynecology and Obstetrics, and *Urology. The Cleveland Clinic Foundation, Cleveland, OH. Objectives: To quantify follicular fluid reactive oxygen species (ROS) and total antioxidant capacity (TAC) in patients undergoing in vitro fertilization (IVF) and compare these with the pregnancy outcome. Design: Prospective, blinded, pilot study. Materials and Methods: Fifty-three women undergoing ART procedure at the IVF center were enrolled in the study. Following ovarian stimulation protocol, oocytes were retrieved, and clear follicular fluid (FF) from each follicle was obtained. Conventional IVF was performed in women with female factor only (n = 30), and ICSI in male-factor cases (n = 23). We examined the number of oocytes recovered, number of oocytes fertilized, and the presence of white blood cells in FF. The clear FF specimens were analyzed for the presence of reactive oxygen species (ROS) measured by the chemiluminescence method using luminol as the probe. Total antioxidant capacity (TAC) was determined from aliquots of frozen seminal plasma by the enhanced chemiluminescence method. Patients were categorized according to their diagnosis (endometriosis, tubal infertility, and male factor). Levels of ROS and TAC were compared for an association between patients who achieved pregnancy to those who did not. Results: ROS levels were significantly lower (0.69 2 0.08) in patients who failed to establish a pregnancy compared to those who did (1.01 + 0.14; p = 0.03). When overall comparisons were made, the groups did not differ in age, number of oocytes recovered, percentage of oocytes fertilized, or TAC levels. However, when grouped according to clinical diagnosis, women with endometriosis, who became pregnant had significantly depressed level of ROS (0.60 + 0.17) compared to those who established pregnancies (1.31 C 0.19; and P < 0.01). Similarly, in women whose partners were severely oligospennic, low ROS levels were seen in those women who did not get pregnant (0.67 2 0.17) compared to those who had successful pregnancies (1.31 * 0.19; P < 0.01). Conclusion: Follicular fluid ROS may be an important indicator of oocyte quality and may be utilized as a potential marker in predicting success in IVF patients. This work was supported by a research grant from The Cleveland Clinic Foundation.

Wednesday,

September

3:00

29, 1999

P.M.

o-1.54 Chlamydial Serology Screening in Infertility. M. D. Keltz, M. Moustakis. Division of Reproductive Endocrinology, St. Lukes-Roosevelt Hospital Center, Columbia University, New York, NY. Objectives: Upper genital tract infections and adhesions are responsible for at least 20% of infertility which is often caused by asymptomatic chlamydial infection. While there has been considerable literature confirming the correlation between elevated chlamydial serologies and tubal infertility, this test has not been widely used for screening. Design: Starting in July 1995, we prospectively screened all patients referred for an infertility evaluation with a Chlamydia Trachoma IgG titre at a commercial laboratory. Material and Methods: 188 patients diagnosed with infertility, who had a chlamydia serology, a subsequent HSG and/or laparoscopy, if indicated,

Vol.

72, No.

3, Suppl.

1, September

1999

were included. The serologic results were categorized as negative or positive and the positives were further divided into low positive (less than a two fold increase over a negative titre) or high positive. Results: While only 7088 (3.7%) patients reported a prior chlamydial infection or PID, 79/188 (42.0%) were seropositive. When compared to laparoscopy, a chlamydial serology was 93.2% sensitive and 78.9% specific in detecting tubal disease, and a high positive serology was 97.8% sensitive. HSG was 81.3% sensitive and 81.0% specific for tubal factor, but when combined with chlamydial serology the sensitivity increased to 98.3% and specificity to 97.8%. HSG documented tubal disease in 22/100 (22.0%) seronegative subjects, 11/26 (42.3%) subjects with low serologies, and 40/46 (87.0%) subjects with high serologies, (p < 0.001). Laparoscopy documented tubal disease in 12/57 (21.0%) seronegative subjects, 1408 (77.9%) in low positive serologies, and 44/45 (97.8%) in high positive serologies (p < 0.001)~ Clinical pregnancies during follow-up care were documented in 47/98 (48.0%) seronegative subjects, lo/23 (43.7%) low positive subjects, and only 7/49 (14.3%) high positive subjects (p < 0.001). Pregnancy loss without subsequent live birth occured in 6/17 (35.3%) seropositive and 8/47 (17.0%) seronegative subjects. Conclusions: Chlamydia serology is a highly sensitive and specific screening test for infectious tubal infertility, a problem infrequently detected by history alone. When used in conjunction with HSG, it enhances patient selection for laparoscopic tubal assessment. In addition to detecting tubal disease, a high positive chlamydia serology appears to be a significant negative predictive factor for conception and may be associated with increased pregnancy wastage.

Wednesday,

September

3:45

29, 1999

P.M.

o-155 Definition of High-Risk Patients for High-Order Multiple Pregnancies With Ovarian Stimulation by Gonadotropins. ‘,*N. Gleicher, ‘G. Kaberlein, ‘,‘D. Pratt, ‘J. Rinehart, r,‘R. Morris, ‘,‘R, Rao, ‘,‘V. Karande. ‘The Center for Human Reproduction-Illinois and ‘The University of Illinois at Chicago, Chicago, IL, USA. Objective: It is estimated that controlled ovarian stimulation with gonadotropins (COS) contributes over 2/3 of the increase in multiple conceptions which has been observed as a consequence of infertility therapy. It has been also reported that the contribution of COS to the occurrence of high-order multiples (~3) is even higher since IVF and other ART procedures (the principal other contributor to the observed increase in multiple births) allow through a limitation of the number of embryos transferred a better control of high-order multiples. We have reported elsewhere that the prevention of high-order multiples with COS using standard monitoring criteria seem basically impossible. This study addresses the question whether a high-risk group of patients can be identified during COS which, if either cancelled or converted to ART, would reduce the previously reported 1.2% risk of high order multiples with COS. Design: Analysis of 4,035 consecutive COS cycles between l/l/97 and 1 l/30/98 at a medical school-affiliated infertility program. Materials and Methods: Estradiol (Ea) levels, number of follicles of ~16 mm diameter (F-16) and total number of follicles (F-T) on day of hCG administration during COS were compared for singletons, low-order and high-order multiples. Results: Mean E, levels (2 1 SD), F-16 and F-T are reported in the table. n Singletons Twins Triplets Quadruplets Quintuplets Sextuplets

314 88 22 10 5 2

E, ? (1 SD) 1,015 1,328 1,534 1,506 1,168 1,573

? 2 k 2 ? +

677 830 358 874 457 652

F-16 + (1 SD) 2.99 3.68 4.09 3.70 3.00 2.50

+ ‘2 ? k lr

1.94 2.06 2.02 1.68 1.10 1.50

F-T ? (1 SD) 17.51 20.22 24.23 24.80 29.40 30.00

2 Ifi 2 k !I 2

9.85 9.54 9.76 14.46 8.36 10.00

Neither E,, F-16 nor F-T levels differed statistically between singletons and any of the low-order or high-order multiples. (Student-Newman-Keuls Multiple Comparisons Test). While F-T demonstrated a slight upward trend with high order multiples, neither E, nor F-16 (the current standard criteria

FERTILITY

& STERILITY@

used to reach a decision on whether to trigger a cycle or not) did even demonstrate such a trend. Conclusions: In addition to confirming that high-order multiples are unpreventable with COS, this study also demonstrates that it seems impossible to screen out a high-risk group of patients for high-order multiples during a COS cycle. This observation further confirms that efforts should be undertaken to redirect treatment algorithms from COS to IVF for young women who are principally at risk for high-order multiples.

Wednesday,

September

4:00

29, 1999

P.M.

O-156 Ultrasonographic Measurement of Cervical OS Diameter in Non-Pregnant Women Was Diagnostic of Cervical Incompetence. A. Nagji, S. Daya, J. Gunby. McMaster University, Hamilton, ON, Canada. Objectives: Cervical incompetence (CT) is frequently observed in women with recurrent spontaneous abortion (RSA, 2 or more pregnancy losses). The ability to identify CI prior to pregnancy would allow early monitoring, treatment, and possibly prevention of pregnancy loss or premature birth due to CI. The purpose of this study was to determine which test or combination of tests would provide the most accurate diagnosis of CI in non-pregnant women. Design: Prospective cohort study. Materials and Methods: Vaginal ultrasonography was carried out on a series of 164 non-pregnant women with RSA, measuring cervical length, width, and OS diameter, both longitudinally and in cross section (2 dimensions). Assessment of cervical status by the Hegar test and by hysteroscope was also performed. The women were judged to have clinical evidence of CI if they had experienced a pregnancy loss at 2 18 wks gestation or if they had delivered at 532 wks. Test results were compared between women with CI and those who had delivered at >32 wks. Test were compared using receiver operating characteristics (ROC) curves. Results: In 64 women, ultrasound measurements were obtained on 2 different days. The 2 sets of measurements were significantly correlated, with r values ranging from 0.31 to 0.67. Pregnancy outcome beyond 18 wks was known for only 66/164 women and 16 of these were judged to have Cl Of the ultrasonographic measurements, only the cross-sectional diameters were predictive of CI (area under the ROC curve 0.79). The median value (range) of the longitudinal diameter for women with CI was 3 (2-6) mm and for those without CI was 2 (l-5) mm (p < 0.05). The probability of CI was 14% in women with a value <3 mm and 50% in those with a value 23 mm (p < 0.002). The median value (range) of the transverse diameter for women with CI was 7.5 (4-12) mm and for those without CI was 4 (3-9) mm (p < 0.05). The probability of CI was 9% in women with a value <5 mm and 43% in those with a value ~5 mm (p < 0.002). The two diameters were used to calculate the OS area, and the median value (range) of this parameter was 16.5 (6.2-47) mm2 for women with CI and 6.3 (2.4-31) mm2 for those without CI. The probability of CI was 8% when the OS area was <7 mm’, 33% when it was 7-10.9 mm*, and 50% when it was ~11 mm2 (p < 0.002). The Hegar test and hysteroscopic assessment were also significant predictors of CI, but inclusion of the results of these tests did not increase the accuracy of CI diagnosis over that of ultrasonographic measurement (area of cervical OS) alone (area under the ROC curve 0.80 vs 0.81, respectively). Conclusion: Ultrasonographic measurement of the cross-sectional diameters of the cervical OS was the most accurate test for the diagnosis of cervical incompetence in non-pregnant women.

Wednesday,

September

4:15

29, 1999

P.M.

o-157 A Randomized Controlled Trial of a “Stage-of-Change” Smoking Cessation Intervention for Subfertile and Pregnant Patients. E. G. Hughes, M. L. Beecroft, D. Lamont, S. Rice, ‘D. Wilson, M. Freebury, J. Gunby, ‘H. Frey, B. Brennan, A. Pinto. Departments of Obstetrics and Gynecology, and ‘Family Medicine, McMaster University Faculty of Health Sciences, Hamilton, ON, Canada.

S61

Objective: The burden of illness caused by smoking is enormous. In addition, a significant correlation has been demonstrated between smoking and impaired reproductive outcome at various levels. However, smoking cessation strategies remain relatively ineffective. The objective of this study is to assess the effectiveness of a five minute scripted intervention, tailored to the “stage-of-change” of each individual in the smoking cessation continuum. These stages may be defined as: 1) not contemplating quitting, 2) planning to quit within 6 months, 3) planning to quit within one month, 4) actively quitting and 5) maintained cessation. Design: Randomized controlled trial, conducted at three university hospitals in Hamilton, ON. Materials and Methods: 91 subfertile and 112 pregnant women were enrolled. Randomization was stratified by patient type: subfertility and first trimester pregnancy. Women were randomized to receive either a scripted structured intervention, based on the subject’s stage-of-change, or to receive standard of care i.e. information currently given by clinicians. Intervention subjects also received a booklet targeted to their stage-of-change and were offered a follow-up appointment in the smoking cessation clinic. Women were followed for a period of 12 months with reassessment of stage-of-change. Carbon monoxide monitoring was done at all visits in the intervention group. Pre- and post-study questionnaires were administered, to assess the subjects satisfaction with the study. The primary end point was delta stage-of-change. Wilcoxen’s rank sum test was used to assess this. Results: Preliminary analysis suggests that the scripted intervention is superior to control at 12 months, only for subfertile women (p = 0.013). Active or sustained cessation was achieved by 22% of intervention compared with 16% of control subjects. Among pregnant women, neither intervention nor control significantly altered the course of progress. In fact, pregnant women tended to revert to smoking after childbirth at 12 month follow-up. At enrollment, 50% of control and 41% of intervention women who were pregnant had achieved cessation, but by 12 months these numbers had fallen to 10% and 15% respectively. Conclusion: Subfertile and pregnant women differ significantly in their stage-of-change at presentation and respond differently to cessation interventions. A scripted stage-of-change oriented intervention appears to be more effective than standard of care among subfertile women. It was ineffective in pregnant women, with a significant return to active smoking after delivery. The pre- and post-trial questionnaires will be explored and presented, in order to more fully understand the implications of these findings. This trial was supported by grants from the Father Sean O’Sullivan Foundation and MUMC Foundation.

Wednesday,

September

4:30

29, 1999

P.M.

O-158 An Immunohistochemical Analysis of Fibroid Vasculature. B. J. Vollenhoven, R. Casey, P. A. Rogers. Department of Obstetrics and Gynaecology, Monash University, Melbourne, Victoria, Australia. Objective: Uterine fibroids or leiomyomas affect at least 50% of women over 30 years. They are a major public health issue with 22% of hysterectomies in Australia being due to fibroids. Despite their frequency, little is known of fibroid etiology and in particular their vasculature. This study aimed to compare different vascular parameters between fibroid and adjacent myometrium. Design: Quantitative analysis of vascular density, structure and arrangement of fibroid and myometrium using immunohistochemistry. Materials and Methods: From 10 uteri the following specimens were taken: a small fibroid (CO.5 cm), inner and outer parts of a large fibroid (~3 cm), and myometrium. Antibodies to the endothelial cell (EC) markers CD31, CD34, factor VIII related antigen (FVIII) and to the lectin Ulex europaeus were used in routine immunohistochemistry protocols and the information digested for computer aided image analysis. Parameters calculated were vascular area (VA: proportion of image immunostained), microvascular density (MD: counts of stained microvessels), vascular luminal diameter (VLD) and vascular variability (VV: a measure of the regularity of the distribution of blood vessels). Statistical analysis used paired T-tests. Data is presented as mean ? standard deviation, Results: For VA for CD31 the mean proportional area stained (%) in

S62

Abstracts

myometrium (2.0 2 1.1) was significantly greater than small fibroids (0.5 ? 0.3) (p = 0.046) and inner regions of large fibroids (1.2 5 0.8) (p = 0.015). For CD34 there were no significant differences between regions studied. For FVIII related antigen myometrium (3.6 t 1.8) was significantly greater than for small fibroids (2.0 t 1.1) (p = 0.007) and for both inner (2.0 ? 1.0) (p = 0.05) and outer regions of large fibroids (1.5 t 1.2) (p = 0.01). For Ulex myometrium (4.1 -C 2.1) was significantly greater than for small fibroids (1.4 2 1.2) (p = 0.05) and for outer regions of large fibroids (1.6 2 1.0) (p = 0.028). For MD for CD31 the mean microvascular density (vessels/mm*) in myometrium (317.5 2 137.5) was significantly greater than for small fibroids (191.3 2 86.2) (p = 0.008) and for outer regions of large fibroids (212.1 5 89.7) (p = 0.012). There were no differences for CD34. For FVIII related antigen myometrium (395.6 2 157.7) was significantly greater than for small fibroids (252.9 i 84.1) (p = 0.013) and for outer (210.4 + 91.7) (p = 0.003) and inner regions of large fibroids (255.6 2 97.6) (p = 0.038). For Ulex myometrium (394.6 5 SD 135.5) was significantly greater than for small fibroids (197.1 + 84.5) (p = 0.02) and for the outer (232.5 2 142.1) (p = 0.024) and inner regions of large fibroids (245.4 ? 78.7) (p = 0.001). For VLD for CD31, CD34 and Ulex there were no differences between groups. For FVIII related antigen the mean vascular luminal diameter (mm) for myometrium (0.2 2 0.1) was significantly greater man for small fibroids (0.1 ? 0.0) (p = 0.032). For VV the vessels in outer areas of large fibroids were generally more clumped than that for inner regions for all stains. Conclusion: Differences in vascular density as measured by VA and MD between myometrium and fibroids may represent a difference in angiogenesis and vascular remodelling in these vascular beds.

Wednesday,

September

4:45

29, 1999

P.M.

o-159 /.S3 Integrin and MAG Mucin in Luteal Phase Glandular Endometrium: A Comparative Marker Study. ‘H. J. Kliman, *P. G. Van Deerlin, ‘B. A. Lessey, ‘,4R. F. Feinberg. ‘Department of Obstetrics and Gynecology, Yale University, New Haven, CT, ‘South Jersey Fertility Center, Marlton, NJ, 3Department of Obstetrics and Gynecology, University of North Carolina, Chapel Hill, NC, 4Reproduction Associates, Wilmington, DE. Objectives: Temporal expression of specific endometrial markers, such as the /33 integrin and the MAG mucin, have been independently linked to disorders of endometrial receptivity. The purpose of this study was to investigate the immunoreactivity of both p3 (normal expression days 2024) and MAG (normal expression days 5-19) within the same biopsies, and to correlate the normal or aberrant expression of these markers in luteal phase glands. Design: In a double-blinded fashion, tissue sections from the same luteal phase endometrial biopsies were immunohistochemically stained for p3 integrin and MAG mucin, scored for staining intensity, and the results tabulated and compared by an independent investigator. Materials and Methods: 58 natural cycle luteal phase endometrial biopsies from infertility patients were immunohistochemically stained for p3 (JCI, 90:188-95, 1992), MAG mucin (AJP, 146:166-181, 1995). ABO blood group antigens, the sialomucin MUCl (positive control), and NMA (negative control). Biopsies were tabulated as negative marker staining with a p3 HSCORE c1.2, and/or glandular MAG glandular reactivity
MAG

-

5 (16%) 10 (31%)

P3 03 +

Vol.

72, No.

+

13 (41%) 4 (13%)

3, Suppl.

1, September

1999

Conclusions: In this series, p3 integrin and MAG mucin immunoreactivity are correlated in a reciprocal fashion in 72% of the biopsies, consistent with the expression patterns previously described for these markers. Biopsies that were concomitantly p3 negative and MAG positive (41%) have a pattern suggestive of a significant endometrial receptivity defect; 31% were appropriately p3 positive and MAG negative, consistent with normal luteal phase glands. In the remaining 28%, however, the concomitant presence or absence of p3 and MAG may denote a subset of abnormal biopsies not readily detected by a single marker. We conclude that evaluation and detection of endometrial differentiation abnormalities may be enhanced by the concomitant use of p3 and MAG mucin immunohistochemical assays. (Supported by the Donaghue Foundation to HJK, NIH 35041 to BAL and NIH 29729 to RFF).

PEDIATRIC

AND ADOLESCENT Wednesday,

September 290 P.M.

two rudimentary uterine horns, right salpingo-oophorectomy, resection of a right endometrioma, lysis of extensive adhesions, and treatment of endometriosis. Results: The unicomuate uterus was situated in the left hemipelvis and communicated with the left fallopian tube. The two rudimentary horns were situated in the right hemipelvis with the first situated superior to yet sharing serosa with the second. The two horns were cavitated and did not communicate with each other. The inferior horn contained endometrial polyps. Each horn contained a functional endometrium and “chocolate” fluid. Conclusions: Laparoscopy is a viable alternative to laparotomy for management of congenital mullerian anomalies. For the case presented, the presence of two cavitated, noncommunicating rudimentary horns situated one on top of the other suggests that one mullerian duct, after failure of lateral fusion underwent incomplete canalization initiated from two distinct sites offering some elucidation of mullerian duct embryology.

GYNECOLOGY 29, 1999

O-160

Wednesday,

September 2:30 P.M.

29, 1999

O-162

Clitoral Inclusion Cyst: Another Reason To cision. ‘A. A. Rouzi, ‘Othman Sindi, ‘Bandar Departments of Obstetrics and Gynecology at Hospital, ‘King Fahd Armed Forces Hospital, Guard Hospital.

Abandon Female CircumRadhan, 3Hassan Ba’aqeel. ‘King Abdulaziz University and 3King Khalid National

Objectives: Around the world there are between 100 and 132 million girls and women who have been subjected to female circumcision. The procedure and its consequences are not familiar to Western health care professionals. The development of clitoral inclusion cyst after female circumcision proper is rare. Its surgical treatment is not very well described in the literature. Design: Case-series. Materials and Methods: Between August 1991 and August 1998, 19 girls and women presented with clitoral inclusion cyst after female circumcision proper. The age (mean ? SD, range) was 17.4 ? 7.6, 5-29 years. The duration of symptoms was 10.5 2 5, 2-20 years. The size was 4 t 1.9, 2-7 X 3.1 -C 1.3, 1.5-5 cm. They were treated by surgical excision of the cyst with particular attention to preserve the clitoris. The technique involves making a vertical incision in the skin, dissection and excision of the cyst, removal of the excessive skin, and reapproximation of the skin edges. Results: The procedure was done in all patients without intraoperative complications. All patients except one were sent home on the second postoperative day. One patient developed postoperative fever, required therapeutic antibiotics, and was discharged home on the fourth postoperative day. Final pathology showed epidermal inclusion cysts in all patients. Follow-up of the patients showed satisfaction and no recurrence of symptoms. Conclusions: The surgical technique of excision of clitoral inclusion cyst is simple and safe. However, efforts should be directed towards abandoning female circumcision altogether.

Wednesday,

September 2:15 P.M.

29, 1999

Intraoperative Use of Ultrasonography to Facilitate Repair of a High Transverse Vaginal Septum. S. C. Murray, B. Hurst. University of Kentucky Medical Center, Lexington, KY. Objectives: Surgical repair of a high transverse vaginal septum may be technically challenging. When the surgical plane is not obvious vaginally, a combined abdominal-vaginal approach is the traditional approach. The need for laparotomy increases the patient’s potential morbidity and her recovery time. We present a case of a high transverse vaginal septum repair in which concurrent transabdominal ultrasonography allowed for the procedure to be performed completely by the vaginal approach. Materials and Methods: Case report. Results: The patient presented at age 12 with severe lower abdominal pain, and primary amenorrhea. CT scan at an outside facility had revealed a large abdominal-pelvic mass. Physical examination was consistent with a transverse vaginal septum, with a small vaginal dimple. MRI scan revealed the presence of a cervix, and an extensive transverse vaginal septum. She was found to have a single pelvic kidney. Surgical repair was undertaken by the vaginal approach. Despite dissection and attempts at needle localization transvaginally, the upper vagina could not be identified. The urinary bladder was then filled, and transabdominal ultrasonography was performed. The uterus and upper vagina were easily localized sonographically. The image was consistent with an approximate 4 cm hematocolpos. Under ultrasound guidance a 14 gauge angiocatheter was introduced directly into the upper vagina, which was then further distended with 60 cc of saline. The needle tract was then progressively dilated, allowing exposure to the upper vagina. The septum was then excised. The cervix could be visualized, and was found to be unremarkable. After undermining the upper and lower vaginal mucosa, primary reanastomosis was feasible. The patient tolerated the procedure well, and has no postoperative complications. Conclusions: Concurrent transabdominal ultrasonography is a helpful modality during surgical repair of congenital reproductive anomalies. Accurate visualization of the reproductive anatomy by ultrasound simplifies the surgical correction of a high transverse vaginal septum.

O-161 Laparoscopic Management of a Unicornuate Uterus with Two Cavitated, Noncommunicating Rudimentary Horns. ‘,*.?. R. Nezhat, 1.3K. S. Smith. ‘Department of Gynecology and Obstetrics and ‘Department of Surgery, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA, ?Stanford University Endoscopy Center for Training and Technology, MSOB, Ste X340, Stanford, CA. Objectives: An 18 year old nulligravid patient presented with severe dysmenorrhea secondary to rAFS stage IV endometriosis, right hydrosalpinx, right endometrioma, unicomuate uterus and two cavitated, noncommunicating rudimentary uterine horns. We report the laparoscopic management of this previously unreported variant of congenital mullerian anomaly and examine its significance with respect to mullerian duct embryology. Design: Case report and review of the world literature. Materials and Methods: Following three months of GnRH agonist therapy, our patient underwent operative videolaparoscopy with resection of

FERTILITY

& STERILITY@

Wednesday,

September 2:45 P.M.

29, 1999

O-163 Characterization of Adolescents Presenting with Amenorrhea. M. Attaran, M. McDaniel, G. P. Gidwani. Department of Gynecology and Obstetrics, The Cleveland Clinic Foundation, Cleveland, OH. Objective: The objective of this study was to characterize adolescent patients presenting with amenorrhea regarding their etiology and outcome. Design: Retrospective chart review of adolescents presenting with amenorrhea from 1980 to 1998. Materials and Methods: Ninety-five patients with age up to 21 were identified with primary complaint of amenorrhea. Data was collected in regards to age of onset, etiology, laboratory evaluation, and treatment.

S63

Descriptive data are presented as means. Fishers exact test was performed when appropriate. P < .05 was considered statistically significant. Results: Thirty patients were diagnosed with primary amenorrhea and 65 patients were diagnosed with secondary amenorrhea. Mean age of primary amenorrhea versus secondary amenorrhea was 15 2 1.8 and 16.6 ? 2.3 respectively. Specific causes of primary infertility included: ovarian failure (30%), mullerian agenesis (26.7%), androgen insensitivity (lo%), and eating disorder (6.7%). The most common etiology of secondary amenorrhea was anovulation due to hyperandrogenism and weight gain (33.8%) followed by eating disorder (27.7%). A history of weight change was elicited in 57% of secondary amenorrhea patients as opposed to only 6.7% of primary amenorrhea patients. Forty percent of primary amenorrhea and 75% of secondary amenorrhea patients responded to therapy (P = ,006). Conclusions: Familiarity with the more common causes of amenorrhea may assist the clinician in expediting the evaluation of these patients.

Wednesday,

September

3:00

29, 1999

P.M.

o-164 The Levels of FSH, LH, Insulin and Androgen Hormones in Adolescent Girls Suffering from Dysfunctional Uterine Bleeding. ‘Ubavka Radivojevic, ‘Gordana Lazovic, ‘Tatjana Starovic-Medan. ‘Centre for family planning, Institute for mother and child health care, New Belgrade “Institute of gynecology and obstetrics, Clinical Centre of Serbia, Belgrade, Yugoslavia. Objectives: Dysfunctional uterine bleeding (DUB) is a common complaint in adolescent girls, often caused by anovulatory cycles. Overproduction of adrenal and ovarian androgens, along with hyperinsulinemia appears to be one of the features of DUB. Some evidence suggest that hyperinsulinemia augments the levels of androgens by influencing pituitary release of gonadotrophins. The aim of this study was to establish the levels of FSH, LH, testosterone, androstenendione, DHEAS and insulin in 14 adolescent girls in whom DUB was diagnosed. Design: The levels of FSH, LH, testosterone, androstenendiole, DHEAS and insulin were measured in 14 patients. Body mass index (BMI) was calculated for every girl. Materials and Methods: 14 adolescent girls suffering from DUB were investigated. Serum samples were obtained, and the quantitative measurements were performed by the use of radioimmunoassay procedures. BMI of these girls was determined. Results: The mean levels of measured hormones in serum of 14 adolescent girls were: FSH = 3,951 IUlL t 0.781 IU/L; LH = 10,021 IU/L & 10,521 III/L; insulin = 294,14 pmol/l? 171,33 pmol/L; testosterone = 3,73 nmol/L 2 0,85 nmol/L; androstenendione = 8,13 nmol/ L 2 3,99 nmol/L; DHEAS = 07,95 pmol/L ? 4,66 pmol. In 8 girls hyperinsulinemia was detected and elevated one, two or all androgen hormones. In 10 girls hyperandrogenemia was seen. Three girls were normoinsulinemic and with normal ranges of androgens. The levels of LH (14,86 IU/L t 11,32 III/L) were higher in hyperinsulinemic group than in normoinsulinemic girls (3,75 III/L 2 1,35 IU/L). In the hyperandrogenemic girls LH was also higher (11,92 IU/L 2 lo,76 IU/L) than in girls with normal androgens (LH = 3,43 IU/L t 0,81 IU/L). The levels of FSH were similar in all groups. Mean BMI level was 20,92 2 3,04 (one girl was obese and 2 were underweight). The positive correlation between insulin levels and levels of LH was found to be significant. The correlation between FSH and insulin and between BMI and insulin was not significant. Conclusion: In this study higher levels of LH were found in hyperinsulinemic group and lower levels in normoinsulinemic group. FSH levels did not differ in these groups. While the positive correlation between the levels of insulin and the levels of LH was noticed, the significant correlation between BMI and insulin and FSH and insulin were not noticed, These findings indicate that hyperinsulinemia might influence pituitary release of LH and thus augments androgen production, all of which contributes to the mechanism of anovulation.

S64

Abstracts

MENOPAUSE

SPECIAL

Wednesday,

INTEREST

September

3:45

GROUP

29, 1999

P.M.

O-165 Functional Neurochemistry Using MRSI in Menopausal Women. ‘P. A. Keenan, ‘G. J. Moore, ‘K. A. Ginsburg. Departments of ‘Psychiatry and Behavioral Neurosciences and *Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI. Background: Cognitive capacity, particularly memory, has become one of the variables considered in the risk/benefit ratio of postmenopausal hormone replacement. Ovariectomy-induced morphologic changes in the hippocampi of rats stimulated the search for clinical correlates. Despite reports of an association between impaired memory and hypoestrogenic states in women, there have been no studies of brain structure or functional neurochemistry analogous to this animal work. Study Design: ‘H Magnetic Resonance Spectroscopy Imaging (MRSI) was used to measure regional levels of N-acetyl-aspartate (NAA), creatine compounds and choline compounds which are putative measures of neuronal viability, energy metabolism, and cell membrane function respectively, in the left or right hippocampus or prefrontal cortex in postmenopausal women. The hippocampus is critical to the formation of new memories while working memory, attention, and cognitive flexibility among others are dependent upon the integrity of the prefrontal cortex. Methods: Five women on hormone replacement therapy (HRT) and five non-replaced women closely matched for years of menopause, estimated IQ, and education underwent MRSI and a small battery of neuropsychological tests. Radioimmunoassay of serum estrogen concentrations confirmed that the hormonal status of the women on HRT differed significantly from that of the non-replaced women (p < .05 for all assays). Results: There were no differences between groups in measures of hippocampal NAA, creatine, choline, or any component of memory as tested by list learning and paragraph recall. In contrast, there were significant group differences in the ratios of creatine, NAA and choline in the left dorsolateral prefrontal cortex (p < .Ol). Comparison of these ratios suggests a higher level of creatine and hence greater energy metabolism in this area in the women on HRT. In addition, we found a trend toward a significant group difference in scores on Trails B, a test of frontal lobe function. The replaced women were more adept at this task. Conclusions: These preliminary data suggest greater energy metabolism in the left prefrontal cortex in postmenopausal women on HRT which warrants more extensive investigation. Furthermore, these results indicate a possibility of a primary attentional deficit underlying the reported loss of memory function in non-replaced postmenopausal women.

Wednesday,

September

4:00

29, 1999

P.M.

O-166 Protein-l The Effect of Tamoxifen on Monocyte Chemoattractant (MCP-1) Production in Human Coronary Artery Endothelial Cells. T. Pehlivan, B. Selam, N. Mulayim, E. Seli, A. Arici. Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology. Yale University School of Medicine, New Haven, CT. Objective: Recruitment of macrophages to the arterial wall is one of the initial events in the formation of the atheroma plaque. MCP-1 is expressed in atherosclerotic arteries, and MCP-1 is the factor that recruits macrophages to the arterial wall. We have previously shown that estradiol decreases MCP-1 levels in human coronary artery endothelial cells in culture. Tamoxifen is a selective estrogen receptor modulator with known antiestrogenic effects in mammary tissue and estrogen-like effects on bone density and blood cholesterol levels in postmenopausal patients. Tamoxifen is shown to decrease coronary heart disease in breast cancer patients receiving long term tamoxifen therapy. We hypothesized that tamoxifen might play a protective effect on atherosclerosis through inhibition of MCP-1 levels, and we studied the effect of tamoxifen on MCP-1 production by human coronary artery endothelial cells in culture. Design: MCP- 1 production in primary cultures of human coronary artery endothelial cells were quantified by ELISA.

Vol.

72, No.

3, Suppl.

1, September

1999

Materials and Methods: Human coronary artery endothelial cells were obtained from Clonetics (San Diego, CA), were replicated to confluence in endothelial cell growth medium containing epidermal growth factor, bovine brain extract, and 5% fetal bovine serum. Prior to each experiment, cells were incubated for 24 hours with phenol red-free medium containing 5% charcoal-stripped fetal bovine serum, and then they were treated with media alone (as control), and with tamoxifen (lo-‘) in the same medium. After 24 hours of incubation, supematants were collected and MCP-I protein was quantified using an ELISA. Statistical analysis was performed using ANOVA with a post hoc Tukey test. Results: Human coronary artery endothelial cells in culture secreted the MCP-1 protein and tamoxifen decreased the levels of MCP-I protein by more than 50% below control (p < 0.05). Conclusion: One of the protective effects of tamoxifen against coronary heart disease might be through decreasing MCP- 1 levels in human coronary artery endothelial cells. Lower MCP-1 levels would decrease atheroma formation. This data along with the clinical data on tamoxifen’s cardioprotective effects in breast cancer patients shows that tamoxifen has a possible estrogen-like effect on the cardiovascular system. Wednesday,

September

4:15

29, 1999

P.M.

O-167 Effect of Hormone Replacement Therapy on Left Ventricular Functions, Late Potential and Heart Rate Variability. ‘A. Yildinir, ‘F. Aybar, ‘H. Yarah, ‘Cl. Kabakci, ‘0. Btikiilmez, ‘E. Akgiil, ‘A. Oto, ‘L. TokgSzoglu, ‘T. Glirgan. Hacettepe University, Faculty of Medicine, Departments of ‘Cardiology and ‘Obstetrics and Gynecology, Ankara, Turkey. Objective: Hormone replacement therapy (HRT) is associated with decreased cardiovascular disease in postmenopausal women but the underlying mechanism(s) are not fully understood. There are a paucity of data on the direct effect(s) of HRT on various cardiac indices. The objective of this study is to evaluate the impact of HRT on LV sysytolic and diastolic functions, late potential (LP) and heart rate variability (HRV). Design: The effect of HRT on LV systolic and diastolic functions, LP and HRV parameters were determined before and 3 months after HRT use. Materials and Methods: Fifteen consecutive postmenopausal women with a mean age of 47 -C 5 y (range 40-56 y) with no cardiac symptoms and negative treadmill exercise testing were included. Oral continuous 0.625 mg/day conjugated equine estrogen plus 2.5 mg/day medroxyprogesterone acetate was used for HRT. LV ejection fraction and fractional shortening were calculated with M-mode echocardiography as parameters of systolic function. The examined diastolic functions included peak early diastolic velocity (E peak), peak late diastolic velocity (A peak), early-to-late peak velocity ratio (E/A) and isovolumic relaxation time (IVRT). Recordings for LP and HRV analyses were obtained with Kardiosis am-LP high resolution ECG analysis system. Filtered QRS duration, root mean square and low amplitude signals were used as LP parameters. Powers of low and high frequency components (LF and HF) and the ratio of LF/HF were analysed as HRV parameters. Paired sample t-test was used for statistical analyses. Results: LV systolic functions were not changed with HRT (p > 0.05); whereas significant improvement was noted in some of the diastolic functions (Table 1). LP positivity was noted in one woman at the baseline examination but normalized after HRT. HRV analysis showed significantly increased HF component (p < 0.05) and decreased LF/HF ratio (p < 0.05) with HRT. Before

HRT

After

0.10 0.16 0.14 7.6

0.68 0.63 1.09 100

HRT

fibrosis. Our preliminary data suggest that a 3-month HRT use is associated with significantly improved left ventricular diastolic functions and increased parasympathetic (HF) component of HRV.

Wednesday,

September

4:30

29, 1999

P.M.

O-168 Elevated Estradiol Causes Luteinizing in Perimenopausal Women: Preliminary Division of Reproductive Endocrinology, icine, Bronx, NY 10461.

Hormone (LH) Surge Failure Results. N. Santoro, T. Adel. Albert Einstein College of Med-

Objective: Women in their later reproductive years sometimes fail to ovulate despite the production of supraphysiologic midcycle estradiol. This preliminary study was performed to test the hypothesis that exposure to high concentrations of circulating estradiol can result in LH surge failure in perimenopausal women. Design: We challenged two perimenopausal women with estradiol in a step-up fashion designed to duplicate the peak concentrations seen in perimenopausal anovulatory cycles. LH was measured daily throughout an untreated control cycle and an exogenous estrogen replacement cycle. Materials and Methods: Estradiol was administered by transdermal patch (Estraderm; CIBA) and serum levels of estradiol were checked periodically to assure attainment of expected circulating concentrations of estradiol. All exogenous hormones were started on Day 2 after a menstrual period and ultrasound monitoring was carried out every 2-4 days to assure that there was no follicle growth. Patches were adjusted to achieve the desired concentration of estradiol. In supraphysiologic cycles, we attained a peak estradiol of 800-1000 pg/ml over 7 days, or 3-4 time normal midreproductive midcycle estradiol (Santoro, JCEM 81: 1495, 1996). In one volunteer, estradiol was also administered exogenously to duplicate a normal midcycle peak of approximately 250-300 pg/ml as an additional control for the method. LH was measured using a sensitive and specific immunofluorometric assay (DELFIA; Pharmacia) and normalized for urinary creatinine. Results: The first two women have been analyzed. In control cycles, both women had evidence of clear-cut LH surges (peak LH 73 and 54 BJ/L, respectively), lasting for 3 and 6 days. The summated areas under the curve were 149 and 202 IU/surge, respectively. When supraphysiologic estradiol was administered, both women had evidence of impairment of their LH surge (peak LH 10 and 12 IU/L, respectively). Duration of the attenuated LH response was also reduced from 3 days to 1 day in one woman and from 6 days to 4 days in the other, when the normal cycle and the supraphysiologic estradiol cycle were compared. Summated areas under the curve were 10 and 48 IU, respectively, representing 93% and 76% inhibition of the natural cycle LH surge. Conclusion: The phenomenon of perimenopausal anovulation in the face of high circulating estradiol may be due to hypothalamic-pituitary impairment of the LH surge mechanism on the basis of excessive estradiol exposure. These findings may have importance in the understanding of perimenopausal dysfunctional uterine bleeding. (Supported by NIH AG12222 and CIBA)

Wednesday,

September

4:45

29,

1999

P.M.

P value O-169

E peak ‘4 peak WA IVRT

0.65 0.68 0.99 107

5 2 2 2

2 k t ?

0.09 0.12 0.20 6.2

NS co.05 co.05 co.01

?able 1: Alteration of LV diastolic functions before and after 3-mounts HRT. Data are expressed as mean 2 SD. NS: not significant

Proliferation and Status of Estrogen and Progesterone Receptors in the Endometrium of Aging and Old Rats. L. L. Staneva-Dobrovski. Department of Neuroanatomy, H. Heine University of Duesseldorf, Duesseldorf, Germany. of

Conclusion: The mechanisms of favourable effects of HRT on heart are complex and may include improved myocardial relaxation patterns, altered autonomic nervous system innervation with decreased sympathetic and increased parasympathetic activity and possible regression of myocardial

FERTILITY

& STERILITY@

Objectives: Dysregulation of the balance between cell proliferation and apoptosis by hormonal, genetic or other factors during female reproductive aging may play a crucial role in endomettial carcinogenesis. In the course of studies on aging alterations of endometrium homeostasis, the present study simultaneously examines the proliferative activity and the status of estrogen and progesterone receptors in rat endometrial luminal and glandu-

S65

lar epithelial cells and stromal fibroblasts in relation to age and vaginal cytology. Design: Immunohistochemical survey of proliferating cell nuclear antigen (PCNA), estrogen and progesterone receptors (ER and PR) on formalinfixed uterine sections of Wistar rats. The animals were longitudinally monitored for at least 180 days by daily vaginal smears and used at 18, 24, 28 and 32 months of age (no = 24; six rats per age group). Materials and Methods: Immunohistochemical expression of PCNA, ER and PR was demonstrated applying monoclonal antibodies (clones PClO; ER88 and PR88) and Bio-Strep&AlPase Immunodetection System (BioGenex, CA, USA) on deparaffinized uterine sections (4 em thick) after microwave antigen retrieval. To calculate the percentage of immunopositive cells (nuclear staining for all three markers under study) at X400 magnification by light microscopy, labeled nuclei were counted in 300 randomly selected surface and glandular epithelial cells and stroma fibroblasts from two sections per animal uterus. Results: The vaginal cycles of Wistar rats at 18, 24, 28 and 32 months of age were extremely irregular with a predominance of either persistent estrus (PE) or persistent die&us (PD) over many days, weeks and even months. In the aging rats (18 and 24 month of age) persistent estrus predominated (in 5 out of 6 rats), whereas in the old rats (28 and 32 months of age) a nearly equal number of animals was found to be in PE and in PD (4 PE at 28 months of age; 3 PE at 32 months of age). In all age groups ER immunoexpression was very high in glandular (75-98%), surface epithelial cells (62-95%) and central stroma fibroblasts (55-88%) in the endometria of persistent estrus rats and coincided with a high PR labeling (71-95% in glandular, 55-96% in surface epithelia and 68-956 in central stroma fibroblasts). This receptor status was paralleled by a variable PCNA expression in surface (12-93%) and glandular epithelia (34-82%), as well as in stroma cells (32-65%). The generally low immunolabeling for estrogen and progesterone receptors in endometria of ageing and old rats in persistent diestrus was associated with a low (5-30%) PCNA expression in central stroma fibroblasts and an often surprisingly high one in glandular (52-83%) and surface (56-92%) epithelial cells. Conclusions: Our findings show, for the first time, that under continuous, long-acting estrogens, as in PE rats, ER and PR expression can be upregulated and associated with a variable, often very high proliferative activity in the endometrial epithelial cells and stroma fibroblasts of even old rats, at an age of 28 and 32 months. Particularly interesting is the immunohistochemically detected proliferative activity in endometrial epithelial cells of aging and old rats, killed at persistent dies&us, irrespectively of their low ER and PR status. A combined immunohistochemical and electromncroscopical trial on apoptosis rate and expression of apoptosis-related proteins in the endometrium of aging and old rats is underway at the Department of Neuroanatomy, Duesseldorf.

REPRODUCTIVE Wednesday,

ENDOCRINOLOGY September 2:00 P.M.

29, 1999

O-170 A Metoclopramide Induced Midcycle Prolactin Surge Does Not Effect LH Pulsatility or Luteal Phase Length. ‘R. .I. Karne, ?I. M. Goldberg, ‘A. E. Mehta. Departments of ‘Endocrinology and ‘Gynecology and Obstetrics, The Cleveland Clinic Foundation, Cleveland, Ohio. Objective: Luteal phase defects or a short luteal phase may contribute to infertility and/or recurrent pregnancy loss. While the etiology of these luteal phase abnormalities are unclear hyperprolactinemia appears to play a role. It has also been suggested that an abnormal midcycle prolactin surge may be cause in some normoprolactinemic patients. We sought to investigate the effect of inducing an acute rise in prolactin during the midcycle LH surge on LH pulsatility and subsequent luteal phase length. Design: Midcycle pulse evaluation of LH and prolactin were compared in de novo cycles and those with metoclopramide (MCP) induced prolactin surge. Materials and Methods: Twelve women were admitted to the CRC in anticipation of their midcycle LH surge. Blood was drawn every I minutes for 24 hours and assayed for LH and prolactin using standard RIA techniques. Seven women were studied de novo and five women were given MCP 10 mg four hours after the blood drawing was initiated. Four women

S66

Abstracts

in the de novo and three given MCP were of the cycle with daily blood progesterone using the Veldhius Cluster program for detection. Full IRB approval and patient tained. Results:

Group

n

No. of LH Peaks

de novo MCP

7 5

13 (12-18) 11 (8-13)

Group

LH Peak Interval (min.)

* results expressed

LH Peak % over basal ht

103 (73-l 13) 98 (76-101)

No. of Prolactin peaks

de novo MCP

followed through the luteal phase levels. The results were evaluated pulse amplitude and frequency informed consent had been ob-

125 (120-130) 132 (118-183)

Prolactin peak int. (min.)

11 (8-13) 6 (5-10)

Prolactin peak % over basal ht

123 (88-174) 145 (98-200)

as median

135 (124-200) 121(113-140)

(range)

Thus there were no difference in the LH pulse frequency, peak interval or pulse amplitude between the two groups in spite of a four hour period of sustained prolactin rise which obliterated prolactin pulses for about 3-4 hours after MCP administration and therefore decreased the media number of prolactin pulses over the 24 hours. In those patients followed through the cycle, the results showed that the luteal lengths were no different [median 14( 1 l-15) days de nova vs. 13(13-l 5) days MCP group] and the integrated progesterone for the luteal phase was also no different [ 118 5 20 ng/mI. de novo vs. 104 ? 32 ng/mL MCP group (mean and SD)]. Conclusion: An abnormal prolactin surge in the midcycle ovulatory surge is unable to disrupt LH pulse frequency or amplitude and is unable to disrupt the luteal phase durations or progesterone production.

Wednesday,

September 2:15 P.M.

29, 1999

o-171 The Effect of Ovarian Cyst Presence on the 7th Day of Gonadotropin Releasing Hormone Agonist (GnRH-a) Administration in IVF Treatment. M. M. Biljan, L. Lapansee, F. Bissonnette, N. G. Mahutte, R. Hemmings, S. L. Tan. McGill Reproductive Center, Royal Victoria Hospital, McGill University, Montreal, Canada. Objective: The impact of the presence of an ovarian cyst detected during pituitary suppression has been controversial. In this study, our objective was to observe the differences in the dynamics of hormonal changes and subsequent treatment outcome in patients who developed a cyst with those who did not. Design: Prospective observational study. For a study to have a 80% power to detect a reduction, from 65% to 20%, in a proportion of patients who achieved pituitary suppression following 7 days of GnRH-a administration (cu = 0.05), at least 22 patients were required in each group. Materials and Methods: Patients undergoing in vitro fertilization (IVF) treatment. Pituitary suppression with GnRH-a, s/c 500 pg/day was initiated on day two of the menstrual cycle. Follow-up visits, including transvaginal ultrasound and blood tests to estimate E2, FSH, LH and P levels were performed on days 1, 7, 11 and 14 after commencement of GnRH-a. If after 14 days of GnRH-a administration criteria for pituitary suppression, defined as serum level of estrogen < 150 pmol/ml, were not met, then the GnRH-a was increased to 500 pg twice per day, and an injection of progesterone was given. If a week later, the criteria for pituitary suppression were still not met, ovarian cysts were aspirated, and gonadotropin stimulation was started a minimum of 3 days later. Results: Forty-eight patients underwent 51 cycles of treatment. In twentyseven cycles (52.9%) a cyst was detected 7 days following initiation of GnRH-a administration. There was no difference in age (p = 0.07) or baseline FSH levels (p = 0.72) between patients who developed and those who did not develop a cyst. The length of time required to achieve pituitary suppression was significantly shorter in patients who did not develop a cyst (7 vs 21 days p = 0.0057). Additionally, patients who developed cysts had lower FSH at the time of initiation of gonadotropin therapy (1.8 IU/mL vs

Vol.

72, No.

3, Suppl.

1, September

1999

4.4 IU/mL p = 0.02). Furthermore, patients who developed a cyst required more gonadotropin to achieve ovarian stimulation (45 vs 41 amps. p = 0.05), and developed less follicles (13 vs. 17.5 p = 0.04). Interestingly, cumulative embryo scores (CES) were higher in patients who developed cysts (36 vs. 28 p = 0.05). However, similar implantation (23.5% vs 17.2%) and pregnancy rates (37.2% vs 29.2%) were observed in both groups of patients. Conclusions: Formation of a cyst prolongs the period required to achieve pituitary suppression, increases gonadotropin requirements, and decreases follicular recruitment. It, however, has no negative effect on embryo quality, implantation and pregnancy rates. Therefore, the presence of a cyst at an initial ultrasound scan should not deter the continuation of an IVF treatment.

Wednesday,

September

2:30

29, 1999

P.M.

O-172 What Are the True Cumulative Pregnancy Rates a Standard Treatment Algorithm for Infertility? lam, ‘,*V Karande , ‘,*N. Gleicher. ‘The Center for Illinois and ‘The University of Illinois at Chicago,

of Patients Entering i.aD. Pratt, ‘,*C. CouHuman ReproductionIL, USA.

Objectives: Cumulative pregnancy rates for sequential cycles of individual treatment options such as ovarian stimulation with clomiphene citrate (CC), cumulative cycles of gonadotropin stimulation (GS) and sequential 1VF cycles have been reported in the literature independently. A cumulative pregnancy rate for patients who undergo a standard treatment algorithm of 3 CC cycles, followed by 3 gonadotropin cycles and, finally, IVF has, however, never been reported. Patient selection biases obviously vary depending on whether patients enter any of these treatment steps “fresh” or as a consequence of treatment failure with a prior treatment option. Cumulative pregnancy rates of individual treatment steps can, therefore, not be simply added in an attempt to calculate a true cumulative pregnancy rate for sequential treatments. Patients need to be followed longitudinally through such a treatment algorithm in order to achieve such a goal. This was done in the here presented study. Design: Retroactive longitudinal case study of 359 patient couples without prior infertility treatment, conducted at a medical school-affiliated infertility center. Materials and Methods: This study involved 359 consecutive patients who enrolled between July 1, 1996 and December 1, 1996 as previously untreated infertility patients at CHR-Illinois. A routine fertility work-up confirmed that they were eligible for CHR’s standard treatment algorithm of 3 CC, 3 GS followed by IVF. This meant that the principal cause of the couple’s infertility was either an ovulatory dysfunction in the female, unexplained infertility or a mild male factor infertility. Ovarian stimulation cycles were routinely accompanied by 2 intrauterine inseminations (IUI) per cycle unless couples objected. Over 90% of cycles involved IUIs. Cumulative pregnancy rates were calculated for all patients who stayed in treatment through at least 2 IVF cycles unless they conceived earlier. Patients who dropped out from treatment or patients who conceived spontaneously between treatment cycles were not considered in the study. Results: Cumulative pregnancy rates were 19% after 3 CC cycles, 38% after 3 CC and 3 GS cycles and 80% after 2 IVF cycles. In a subgroup of patients these results were further evaluated for effects of female age and cycle number. CC cycles achieved similar pregnancy rates through all 3 consecutive cycles, though patients ~31 years lost efficacy of treatment after cycle 2. GS cycles progressively lost efficacy from cycle 1 to 3, with only patients between 30-35 years demonstrating any efficacy in a third GS cycle. IVF cycles also lost efficacy progressively between cycles l-3, with basically no pregnancies occurring after IVF cycle 3 and only in younger patients did pregnancies occur after cycle 2. Conclusions: These data demonstrate 1) that the literature underestimates pregnancy success with infertility treatment; 2) in fact, if intercycle pregnancies were to be included, over 90% of infertile couples who stay in treatment can expect to conceive; 3) the efficacy of CC, GS and IVF in sequential cycles varies from published reports suggesting that, especially in older patients, new treatment algorithms, involving fewer cycles within each treatment option should be considered.

FERTILITY

& STERILITY@

Wednesday,

September

2:45

29, 1999

P.M.

o-173 Long-Term Female Fertility Status Following Treatment for Hodgkin’s Disease. ‘L. Lemer-Geva, ‘0. Shpilberg, *I. Ben-Bassat, ‘A. Chetrit, ‘A. De-Medonsa, 3J. Rabinovici. ‘Department of Clinical Epidemiology, ‘Institute of Hematology and 3Department of Obstetrics and Gynecology, Chaim Sheba Medical Center, Tel Hashomer, Israel. Objectives: This study was undertaken to evaluate fertility among females treated for Hodgkins disease (HD) and to assess the role of oral contraceptive use in preserving fertility. Design: Retrospective analysis. Material and Methods: The study group comprised of 74 women aged 25 Z 6.0 years (range 15-42) who were at least 1 year in remission (mean follow-up 87 2 69 months). All women were interviewed using a preconstructed questionnaire regarding their marital status, menstrual cycles, pregnancies, fertility treatment and the use of oral contraceptives (OC) before, during and after treatment for HD. Clinical data related to stage of disease, chemotherapy and/or radiotherapy protocols and treatment outcome was abstracted from the patients medical records. Results: More than half of the patients (59%) were diagnosed at Stages I and II and treated with 2 cycles of ABVD followed by localized irradiation. Regular menstrual cycles following treatment for HD were observed in 76% (48/63) of the patients who reported regular cycles prior to treatment. Among patients who used OC, the rate of regular menstrual cycles was higher as compared to patients who did not use OC during treatment, although not statistically significant (87% vs. 70%, respectively). Among the married patients (5 l/74,68.9%), 34 (66.7%) became pregnant following the treatment for HD. Patients younger than 30 at time of diagnosis, had higher probability of being pregnant (OR = 4.98; 95% CI 1.25-19.8) as compared to patients over 30 after adjustment for disease stage, use of OC, and parity prior to treatment. Patients older than 30 who used OC during treatment, had higher pregnancy rates compared to those who did not use OC (80% vs. 12.5%; p = 0.06). Conclusions: Menstrual irregularities and possible infertility is evident in almost 25% of patients treated for HD. Use of OC may improve menstrual cycle preservation in all patients, and pregnancy rate in patients older than 30 years.

Wednesday,

September

3:00

29, 1999

P.M.

o-174 Outcome of Pregnancy in Mothers Given Cabergoline, An Ergot Derivative. ‘T. Motta, ‘C. I. Ferrari, 3K. Annoni, ‘S. N. Severino, ‘E. Fontana, ‘A. D’Alberton. ‘Department of Obstetrics and Gynecology, University of Milan, Milan, Italy, *Department of Medicine, S. Pio X Hospital, Milan, Italy, 3R & D Pharmacia & Upjohn, Milan, Italy. Objectives: The demonstration that exposure to cabergoline in early pregnancy is safe for the mother and the fetus is of great importance for the evaluation of this new ergot derivative. Cabergoline crosses the placenta in animal models but such studies have failed to demonstrate any teratogenic effects of the drug. It is not known whether cabergoline crosses the placenta in humans. The purpose of this study is to review available data on cabergoline-induced pregnancies to assess the reproductive safety of this inhibitor of prolactin secretion. Design: Preenancies were ascertained between 1987 and 1998 in 37 endocrinology and gynecology clinics in 6 European and 2 American countries. Materials and Methods: A total of 245 cabergoline-associated pregnancies have been documented in 217 women. Results: Of these pregnancies, 28 were terminated by elective abortion, 6 were lost to follow-up and data are not yet available on 17 others (pregnancy in progress). Of the remaining 194, 25 (12.9%) ended in spontaneous abortion (a rate that corresponds with that observed in the general population and in bromocriptine-associated pregnancies), 1 (0.5%) in tubal pregnancy and 1 (0.5%) in hydatidiform mole. One hundred and sixty-four pregnancies ended in childbirth: all were single births. No stillbirths were ”

-

S67

observed. The birthweights of these 164 livebom infants ranged from 1600 to 4350 g. There were no maternal complications during pregnancy. Congenital abnormalities have been reported in 11 of 222 (5%) evaluable cabergoline-associated pregnancies: 5 were classed as minor malformations (atrial septal defect with spontaneous closure, inguinal and umbilical hernias in a pre-term infant, talipes with associated hip dysplasia, slight labiognatopalatoschisis, mild chordee of the penis), the remaining 6 being major (craniosynotosis, left megaureter, Edwards’s and Down’s syndrome, hydrocephalus with cerebral atrophy and facial dysmorphia, “limb-body wall defect”). In the last 3 cases therapeutic abortions were performed. All other infants were normal at birth and no disturbances have been observed in the physical, psychomotor, and intellectual development in a 25-yearfollow-up, although limited to 123 babies. Conclusions: In view of our limited data, we may conclude that the use of cabergoline to restore fertility in hyperprolactinemic women is not associated with an increased risk of abortion, multiple pregnancy or the occurrence of malformations in infants.

Wednesday, September 29, 1999 3145

P.M.

o-175 Extracellular Matrix and Activin-A Interaction: A Novel Concept in Initiation of Follicle Growth. i,*K. Oktay, ‘,aG. Karlikaya, ‘0. Akman, *G. Ojakian, 3M. Hrzenjak Oktay. ‘Departments of Obstetrics and Gynecology Cornell University Weill Medical College, NYMH, NY; *Department Anatomy and Cell Biology, SUNY HSC, Brooklyn; 3Memorial Sloan Kettering Cancer Center, NY. What initiates primordial follicle growth is still unknown. Extracellular matrix (ECM) exerts stringent control on cell proliferation by either inducing specific signals via integrins, or by modifying cell responsiveness to growth factors and hormones. We hypothesized that ECM also plays a role in initiation of primordial follicle growth. Here we provide evidence that initiation of primordial growth depends on specific cell-ECM interactions and is modulated by recombinant human Activin-A (rhAA). Materials and Methods: Ovaries were obtained from 5-day-old B6D2Fl mice. Four ovaries were fixed and sectioned for baseline counts. The remaining ovaries were cultured in groups of six on culture plate filterinserts. Duplicate inserts were coated with either poly-L-lysine (PL, negative control), collagen type IV (C) or laminin (L). Ovarian explants were cultured for 10 days in Waymouth media supplemented with 20% FBS, 10 @ml EGF and FGF. rhAA 100 t&ml was added to one of the two C, L or PL coated wells. Half of the media was refreshed every day. After 10 days in culture, the tissues were embedded in LR-White resin and serially sectioned. Every tenth section was histologically evaluated for follicle density, and the ratios of primary/primordial follicle (PY/PD, growth initiation index), primary/total # follicles (PY/TF) and # multilayer follicles/TF (ML/-IF). Results: When compared to PL, both C and L showed significantly higher PY/PD and PY/TF ratio (fig 1, *p < 0.0001, logistic regression: odds ratio

la

Wdhoul

Activin]

FGURE 1

3.3 and 2.5 respectively) and higher follicle densities (p < 0.01). rhAA treatment did not influence these ratios. When ML/TF was considered, &AA treatment negatively influenced follicle growth in C (fig 2, *p < 0.01) while it enhanced the ratio in L (fig 2 +p = 0.004, OR 10.3 in L compared to C). rhAA treatment did not influence the ML/TF in PL. Comparing baseline to all treatment groups, the ratio of growing follicles was signifi-

S68

Abstracts

cantly higher and follicle densities were significantly lower in treatment groups (p < 0.001). Conclusions: These findings suggest that transition from primordial to primary stage is dependent on both collagen and laminin but not rhAA. Transition from primary to multilayer stage is enhanced by an interaction between laminin and rhAA, and suppressed by an interaction between collagen and rhAA. These novel concepts provide new insights into the enigmatic process of follicle growth initiation. (Supported by the ASRM-Mead Johnson and ASRM-Serono Research Grants) (&AA provided by NIDDK’s NHPP)

Wednesday, September 29, 1999 490

P.M.

O-176 Gonadotropin Receptor Expression on Human Granulosa Cells Assessed by Flow Cytometry. ‘P. Thiruppathi, *J. Dias, ‘J. McKenna, ‘E. Radwanska, ‘J. Luborsky. ‘Department of Obstetrics and Gynecology, Rush Medical College, Chicago, IL, and ‘Wadsworth Center, NYSDOH, Albany, NY. Multiple potential mechanisms may affect hormone responsiveness such as altered local blood flow, receptor expression, genetic defects in receptor structure, and post receptor aberrations of second messengers and steroidogenesis. Expression of gonadotropin receptors is essential for normal gonadal responsiveness to gonadotropins. This study focuses on FSH receptors (hFSHR) on human granulosa cells (GC) assessed by flow cytometry. Labeling of hFSHR with rabbit antiserum to a peptide domain (R265-S296) of the human receptor (Liu et al., Endocrinol, 1994, 135:682) and mouse antirabbit-FITC was optimized with CHO cells transfected with hFSHR (Ares Advanced Technologies). Controls included normal rabbit serum and antiserum adsorbed with antigen. Binding and antiserum dilution (1:50-l: 500) were linearly related. Binding saturation was achieved at 100,000 cells/O.2 ml antiserum (1: 100). The fluorescent signal was converted to number of bound molecules with beads containing a known amount of antimouse immunoglobulin (FCS Corp). The estimated receptors/CHOhFSHR cell was 15,423 or about IO-fold less than estimates by radiolabeled hormone binding. A similar procedure was used to assess FSHR on GCs obtained at the time of oocyte retrieval (36 hours post-hCG stimulation) during IVF with IRB approval. In the 12 subjects studied to date (mean age 31 2 6.5), peak estradiol (E,) response to human menopausal gonadotropin (hMG) averaged 1332.8 2 599 pg/ml (range 604-2340 pg/ml). In addition to peak E,, the ratio of peak E$# ampules of hMG and the pattern of sequential E, responses were explored as additional measures of response. The ratio of peak E,/# ampules averaged 31.4 (range 6.9-65.0). The E, response increased logarithmically with sequential hMG (average RZ = 0.96) and the slopes ranged from 0.12-.26 (mean 19.3). In order to assess hFSHR on GC, lymphocytes were identified and gated out with antiCD45PE. FSHR were identified on all cells. The number of FSHR/GC ranged from 1,806-6,113 (3,612 ? 1,874). In separate aliquots of GC, bound FSH and hCG were also detected using rabbit antibody to FSH or hCG, correlating with the presence of both FSHR and LHR. Thus, hFSHR could be detected and receptor number estimated by flow cytometry on transfected CHO cells and GC. A clear relation between responsiveness and receptor number was not seen in this small sample. However, the results show FSHR expression on human GC at the transition into the early luteal phase. It also provides a basis for further study of FSHR in order to determine if gonadotropin resistance in some women with compromised fertility can be ex-

Vol. 72, No. 3, Suppl.

1, September 1999

plained by altered reserve. Supported

receptor expression rather in part by HD18407.

than by diminished

Wednesday,

29, 1999

September

4:15

ovarian

P.M.

o-177 The Synergistic Effect of VIP and FSH In Vitro On the Expression of FSH Receptor Gene in Human Fetal Ovary. ‘,‘K. R. Kim, ‘C. J. Lee, ‘B. R. Do, ‘H. S. Yoon, ‘S. I. Roh, ‘Y. M. Choi, ‘5. Y. Lee. ‘Infertility Research Center, Jeil Women’s Hospital, *Department of Obstetrics and Gynecology, Seoul National University, Seoul, Korea. Objectives: Though it has been reported that the earlier stage of folliculogenesis is not dependent on follicle stimulating hormone (FSH), the level of pituitary gonadotropins is high at about 20 to 23 weeks of gestation in human. At this stage of gestation, folliculogenesis which initiated at 12 to 16 weeks was mostly abundant. Vasoactive intestinal peptide (VIP) is known to occur in ovarian nerves and immunoreactive VIP was detected at cortical region around follicles, interstitial cells, and perivascular region in ovary. It was reported that VIP also acted on the formation of primordial follicles. This suggests that VIP have an action on folliculogenesis in the milieu of high concentration of FSH during fetal ovarian development. Therefore, on the basis of hypothesis that FSH in fetal ovary acted through its receptor, the present study was carried out to evaluate VIP action on the expression of FSHR gene with or without FSH in vitro. Design: The expression and locality of FSHR gene were measured by semiquantitative reverse transcription polymerase reaction (RT-PCR) and PCR in situ hybridization respectively after the cultivation of 22 week human fetal ovarian tissue supplemented with VIP and FSH. Materials and Methods: Twenty-two weeks old human fetal ovaries were used. After freezing and thawing, the ovaries were sliced and placed in TCM 199 culture medium supplemented with 0.6% bovine serum albumin (BSA only). Experimental groups were separately further supplemented with 10 pg/ml or 100 wg/ml of VIP, or 10 IU/ml recombinant human FSH (rhFSH), or 10 IU/ml rhFSH + 10 pg/ml VIP, or 10 IU/ml rhFSH + 100 Fg/ml VIP and then cultured for 48 hours at 37°C in 5% CO, condition. To know the expression and locality of FSHR gene, RT-PCR and PCR in situ hybridization techniques were applied, respectively. In addition, immunohistochemical studies for proliferating cell nuclear antigen (PCNA) were carried out to detect the change of the cell cycle protein synthesis. Results: PCNA immunohistochemical staining was strong in the VIP and FSH co-treatment group than in the FSH or VIP alone. Transcripts of FSHR gene, which detected by RT-PCR, were identified in the frozen-thawed and BSA only ovarian tissues. In case of VIP treated group, FSHR gene was not expressed or weak. The ratio of FSHR transcripts to beta-actin in cotreatment group (rhFSH 10 IU/ml + VIP 10 pg/ml) was as high as in the positive control of the immature granulosa cells used as positive control. But the ratio was decreased in condition with FSH-alone and 10 IU/ml rhFSH + 100 pg/ml VIP. The localization of the expressed FSHR gene identified by PCR in situ hybridization was shown in the granulosa cells of primordial follicles and the surrounding interstitial cells. Conclusion: The present results clearly show the maintenance effect of FSH on the expression of FSHR only in the presence of VIP during culture of 22 week human fetal ovarian tissues. It suggests that VIP and FSH act synergistically on the ovarian development at fetal life. Also, it can be thought that FSH, in the presence of VIP, has its action on the regulation of the folliculogenesis at mid-term gestation of human fetus.

Wednesday,

September

4:30

29, 1999

P.M.

O-178 Regulation of the Progesterone Receptor (PR) in Human Granulosa Cells (hGC): An In Vitro Model. K. Pagidas. Division of Reproductive Medicine and Infertility, Women and Infants Hospital and Brown University School of Medicine, Providence, Rhode Island. Objectives: The autocrine role of intraovarian progestins in the primate ovary in follicular development is speculated to be via a receptor-mediated

FERTILITY

& STERILITY@

pathway. We devoted our efforts in developing an in vitro model of human granulosa cells (hGC) to assess the role of intraovarian progestins and estrogens, as a paracrine/autocrine regulator of human follicular development. Design: Human granulosa cells were obtained from preovulatory-ovulatory follicles of women undergoing in vitro fertilization-embryo transfer. The modulation of the expression of the hGC PR by agonists and antagonists was assessed by immunocytochemical techniques. Materials and Methods: The hGC obtained were isolated and purified from the follicular aspirates using Ficoll density gradients and gravitational sedimentation techniques, and then plated on tissue culture chamber slides. The granulosa cells were kept in culture with frequent media change until 7-10 days post harvest. The modulation of the expression of the hGC PR by agonists such as FSH, LH, and by protein kinase C (PKC) agonist and antagonists, and CAMP agonists, estrogen and antiestrogens was assessed. Results: The PR was identified on hGC since the time of the harvest. The hGC cultured for 9 days no longer expressed the PR. After seven days of culture, in the absence of FSH, the cells were cultured with LH (500 rig/ml); this resulted in a marked increase in the expression of the PR. No induction of the PR was seen with an LH concentration of 50 nglml, providing a dose-dependent role of the gonadotropin surge in PR induction. A further enhancement of PR expression occurred in hGC preincubated with FSH (50 nglml) 24 hours and then LH added. Culture of the hGC with phorbol myristate acetate (PMA), 0.2 yM, for 24 or 48 hours did not lead to an induction of PR. The addition of LH at 500 ng/mI with or 24 hours after the addition of the PMA led to complete inhibition of the LH induction of the PR. Human GC incubated with PMA and isomethyl xanthine (IMX), a phosphodiesterase inhibitor, with the LH, IMX was able to reverse the inhibitory effect of PMA on the induction of PR by LH. If staurosporine (S) was added 2 or 24 hours prior to LH (500 rig/ml), there was approximately a 70% decrease in the number of PR positive nuclei in the hGC, but if S is added 24 hours after hGC were incubated with LH, there was no inhibition. When H-7, a more specific PKC inhibitor was used at 1 FM, with or without LH present, there was no effect on the number of PR positive cells. CAMP caused an increasing number of PR positive cells in these hGC by 80% over control, and this increase can be inhibited by the preincubation with S. The addition of estradiol (E2) at 10-s M and/or ICI 164,384 (10e6 M), a pure antagonist of estrogen action at 72 and 24 hours before LH in cells primed with FSH when ICI 164,384 was added alone there was a complete obliteration of the hGC. Addition of E, or ethinyl estradiol (EE) with the ICI completely reversed the ICI effect, and the number of PR positive cells was similar to that observed with LH alone or LH with either E, or EE. Conclusions: PKC is a modulator of PR in human granulosa lutein cells, it is a “turn-off’ of LH induction of the PR expression. The presence of PR in the hGC-lutein cell is a balance between the endogenous PKC (inhibition) and protein kinase A (activation) pathways.

Wednesday,

September

4:45

29, 1999

P.M.

o-179 Corticotrophin Releasing Hormone, Vasopressin, and Pro-Opiomelanocortin mRNA Levels in the Non-Human Primate Hypothalamus Following Hypoglycemia-Induced Activation of the HypothalamicPituitary-Adrenal Axis. r.‘D. A. Van Vugt, ‘B. N. Roy, ‘S. Shridhar, ‘R. L. Reid. Department of Obstetrics and Gynecology and Physiology, Queen’s University, Kingston, Ontario. Objectives: Ovarian steroids influence the activity of the hypothalamicpituitary-adrenal (HPA) axis and sensitize the hypothalamic-pituitary-ovarian (HPO) axis to stress-induced inhibition. HPA axis activity is higher in females compared to males. This gender difference is dependent on estrogen. Furthermore, inhibition of luteinizing hormone in response to either acute food deprivation or insulin-induced hypoglycemia is more pronounced in animals primed with estrogen. The objective of the present study was to determine in the non-human primate, the impact of insulin-induced hypoglycemia on messenger RNA (mRNA) of three hypothalamic peptides that respond to stress and control the HPA and HP0 axes. Design: Hypothalamic mRNA levels of corticotrophin releasing hormone

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(CRH), arginine vasopressin (AVP), and pro-opiomelanocortin (POMC) in response to an insulin challenge were compared in estrogen primed ovariectomized monkeys and ovariectomized monkeys not primed with estrogen. Materials and Methods: Ovariectomized cynomolgus and rhesus monkeys were primed with ovarian steroids to mimic the menstrual cycle. Hypoglycemia was induced in unprimed monkeys and in estrogen primed monkeys on day 10 or 11 of the second menstrual cycle by administering insulin (1 U/kg). Controls in both primed and unprimed groups received saline. Two hours after injection, animals were deeply anesthetized with Saffan and ketamine, and brains were perfused with paraformaldehyde via the carotid arteries. Coronal sections through the supraoptic nucleus (SON), paraventricular nucleus (PVN), and arcuate nucleus (ARC) were subjected to in situ hybridization histochemistry for AVP, AVP and CRH, and POMC mRNA respectively. Results: Estrogen priming increased both CRH and AVP mRNA levels in the rostra1 PVN. Hypoglycemia also increased AVP but not CRH mRNA levels in the rostra1 PVN. This effect of hypoglycemia was not dependent on estrogen priming. Neither estrogen priming nor hypoglycemia, alone or in combination, affected AVP or POMC mRNA levels in the SON and ARC respectively. Conclusions: These data suggest that the mechanism whereby estrogen increases HPA axis activity in females involves an effect of estrogen on CRH and AVP transcription. Furthermore, since hypoglycemia increased AVP but not CRH mRNA levels, we speculate that hypoglycemia activates the HPA axis and inhibits the HP0 axis in the non-human primate by a mechanism involving AVP rather than CRH. The current data do not support the hypothe& that estrogen priming sensitizes the HP0 axis to stress through any of the three neuropeptides studied (this work was supoorted bv the MRC of Canada).

SURGICAL Wednesday,

PICKS AND PEARLS September 3145 P.M.

29, 1999

Wednesday,

September 4:00 P.M.

29, 1999

O-181 Transumbilical Direct Trocar Entry for Operative Videolaparoscopy. i.‘,?. R. Nezhat, 1.3K. S. Smith, 4M. Helmy. Department of Gynecology and Obstetrics and ‘Department of Surgery, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA, 3Stanford University Endoscopy Center for Training and Technology, MSOB, Ste X340, Stanford, CA, 4Menoufiya Faculty of Medicine, Shebin, El Kom, Egypt. Objectives: Complications associated with initial abdominal entry are a prime concern for laparoscopic surgeons. We describe our current technique of transumbilical direct trocar entry and evaluate the effectiveness and safety of this approach to primary trocar entry based on five years’ experience. Design: Complications were evaluated by retrospective review of patient charts. Methods: Between September 1993 and November 1998 nine hundred and eighty-three patients underwent 2159 operative laparoscopic procedures at Stanford University Medical Center, a tertiary care facility. The mean age of the patients was 39.4 years (13-90). Eight hundred and seventy-eight patients underwent transumbilical direct trocar entry. One hundred and five patients considered to be at high risk for intra-abdominal adhesions underwent initial pneumoperitoneum using the Veress needle prior to initial trocar placement. Results: Complication rates associated with primary trocar placement were evaluated. No major complications were observed. Two of the 878 patients who underwent transumbilical direct trocar placement experienced the minor complication of umbilical infection. Conclusions: Transumbilical direct trocar placement is a safe and effective method of primary trocar entry capitalizing on the anatomy of the anterior abdominal wall at the level of the umbilicus in combination with proven advantages of direct trocar entry.

O-180 Wednesday, Efficacy of Sclerotherapy for Management of Recurrent Benign Adnexal Cysts. K. H. Chang, C. H. Lee, M. R. Kim, K. J. Hwang, H. C. Kwon, K. S. Oh, Department of Obstetrics and Gynecology, Ajou University School of Medicine, Suwon, Korea. Objective: To evaluate the applicability and efficacy of the sclerotherapy for management of recurrent benign ovarian cysts. Design: A prospective study was performed from 35 patients who underwent sclerotherapy at the Ajou University Hospital. Material and Method: From February 1997 to October 1998, thirty-five patients with suspected benign recurrent adnexal cyst (31 cases of endometrioma: 4 cases of serous cystadenoma) who had undergone previous pelvic surgery were included. All patients underwent pelvic color Doppler sonography (CDS), and their blood sampled for the tumor makers ol-FP, CEA, S-hCG, and CA-125. The patients without suspicion of malignancy were scheduled for sclerotherapy. Under intravenous analgesia, the cysts were first irrigated with 213 volume dehydrated alcohol and then with 10% volume of 5% doxycycline was injected after complete cyst aspiration under transvaginal ultrasonography. The aspirated contents were sent for cytologic examination. All the patients were followed up monthly with serum FSH, CDS for over 4 months. At 4 months follow-up in 7 cases, IVF-ET showed that there was no adverse effect on the number and quality of oocytes retrieved, or on the embryo development. Also, three other cases later became pregnant, demonstrating preservation of reproductive functions. Results: All cysts revealed to be benign upon cytologic examination, were 4cm-8cm in size, and the aspirated volume was 25cc-120~~. In four patients with serous cystadenoma confirmed by cytology, pelviscopic surgery were performed. In thirty-one patients with recurrent benign adnexal cyst, there was excellent ovarian parenchymal preservation and no recurrences with this procedure after follow-up for 4-20 months. Conclusion: Sclerotherapy is an easy, safe and highly effective procedure and considered as valid alternative method for management of recurrent benign adnexal cyst. However, considering the limitations of ultrasonography and tumor markers in determining tumor malignancy, this procedure has to be confined to patients with the recurrent benign adnexal cyst confirmed by previously operative procedures.

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Abstracts

September 4:15 P.M.

29, 1999

O-182 Ovarian Remnant Syndrome After Laparoscopic Oophorectomy. ‘C. H. Nezhat, ‘.*F. R. Nezhat, ‘S. A. Mirmalek, 3D. S. Seidman, ‘C. R. Nezhat. ‘Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, CA, *Department of Obstetrics and Gynecology Mount Sinai Medical Center, New York, NY, 3Department of Obstetrics and Gynecology, Sheba Medical Center, Israel. Objective: To review the preoperative history, clinical characteristics and surgical technique used in patients with ovarian remnant syndrome after laparoscopic oophorectomy. Design: An observational study. Materials and Methods: Fifteen patients diagnosed with ovarian remnant syndrome after laparoscopic oophorectomy were evaluated for prior surgical history, type and technique of previous laparoscopic oophorectomy, associated pelvic pathology, time interval after recent oophorectomy, and clinical findings. Results: The average age was 40 years old, with a range of 25 to 48 years. The patient underwent an average of 4.5 previous surgical procedures (range 2 to 7). Ten patients had undergone bilateral oophorectomy, and the other five a unilateral procedure. The infundibulopelvic ligament had been secured with bipolar desiccation in 8 patients, pre-tied surgical loops in four, a linear stapler in 2, and two 0 vicryl sutures in one. In all but 1 patient, prior oophorectomy had been performed for pelvic endometriosis. The other had severe post surgical adhesions. Cystic ovarian remnants were identified by pelvic sonography in 9 women, and by CT scan in one. One patient had both CT scan and pelvic sonography identifying an abdominal mass. Benign ovarian cysts or tissue were found in all women. Pelvic pain was completely resolved in eight women and partially resolved in four. Three women underwent recent treatment and remain pain free to date. Conclusions: Although in severe cases of adhesions and endometriosis chances of ovarian remnant exists in both laparoscopy and laparotomy, the laparoscopic approach may risk incomplete removal of the ovary by mis-

Vol.

72, No.

3, Suppl.

1, September

1999

application or improper use of suture, linear stapler, or bipolar electrodesiccation on the infundibulopelvic ligament. Ovarian morcellation and failure to remove all fragments may lead to transplantation on the peritoneal surface. Meticulous attention to proper surgical technique and complete removal of the entire ovary from the peritoneal cavity can minimize the risk of this syndrome.

Wednesday,

September

4:30

29, 1999

P.M.

O-183 “Twisted Tubes” At Laparoscopy Are Associated with Internal Isthmic Adhesions on Transcervical Falloposcopy. S. Palter. Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT, USA. Objectives: Traditionally, the fallopian tube is assessed indirectly via HSG, SHG, or laparoscopy. These techniques do not address the functional lumen of the tube. Falloposcopy (FS) is a new technique claimed to better assess tubal function. Others have suggested Chlamydial serology is an equally accurate method of determining risk for tubal disease. Design: A prospective surgical study. 21 Women with suggestions of tubal disease on HSG (suggestions of narrowing, obstruction or loculated spill) underwent transcervical falloposcopic evaluation of their tubes, laparoscopy and Chlamydial serology. The protocol was approved by the institutional review board and informed consent was obtained. Materials and Methods: All procedures were performed in the ambulatory surgical center of a tertiary-care University affiliated hospital. Healthy infertile patients underwent laparoscopy and concomitant transcervical falloposcopy. Subjects also had laparoscopic scoring of pelvic adhesions and pathology. Transcervical falloposcopy was performed using a linear everting catheter and a modified prototype 0.5 mm fiberoptic falloposcope under direct visualization (“Proboscus” scope-designed to improve ease of tubal catheterization and visualization). Intramural, isthmic, ampullary, and fimbrial segments of the tube were separately scored according to standardized falloposcopic scoring systems. Chlamydial serology was obtained in all subjects. Tubes were scored in real-time during the procedures. Results: A new observation of “twisted tubes” (TT) was observed in 6/21 (29%) of subjects. TT were seen when there was a rotational twist along the long axis of the tube and no other associated tubal adhesive disease. Often this was associated with a contraction band along the axis of the tube when it was placed on stretch at laparoscopy. Internally on falloposcopy isthmic adhesions were observed 100% of the time when TT were seen with the adhesion occurring at the site of the twist. These tubes were otherwise normal laparoscopically. The adhesions noted were able to be lysed using the falloposcope balloon. Chlamydial serology did not predict the presence of twisted tubes. Conclusions: Transcervical falloposcopy has allowed the identification of a new variant of tubal adhesive pathology-the “Twisted Tube”. Clinicians should be familiar with its appearance at laparoscopy since these tubes may otherwise look normal. The adhesions associated with TT’s can be lysed using the falloposcope. Ongoing trials will assess the impact of this lysis of intratubal adhesions on fertility.

Wednesday,

September

4:45

29, 1999

P.M.

O-184 Decreasing the Rate of Persistent Ectopic Pregnancy After Laparoscopic Salpingostomy by Proximal Extension of the Tubal Incision. G. C. Hsieh, .I. J. Klutke, D. R. Mishell Jr. Department of Obstetrics and Gynecology, University of Southern California School of Medicine, Los Angeles, CA, USA. Objectives: The major surgical treatment of unruptured ectopic pregnancy is laparoscopic salpingostomy. Persistent ectopic pregnancy (PEP) is defined as continued growth of viable trophoblastic tissue after conservative treatment of unruptured ectopic pregnancy. This entity is manifested by P-HCG levels that fail to decline to undetectable levels within a few weeks postoperatively. Following laparoscopic salpingostomy, the incidence of

FERTILITY

& STERILITY”

PEP has been reported to range from 3% to 20%. In a recent report from this institution in which unruptured ectopic pregnancy was treated by laparoscopic salpingostomy without prophylactic administration of methotrexate, the PEP rate was 14.5%. Stock reported that in five women with PEP treated by laparoscopic salpingostomy, the residual trophoblastic tissue was always located medial to the original incision. The purpose of this study was to determine if an extension of the tubal incision proximal to the visible ectopic gestation would decrease the PEP rate. Design: One hundred-one consecutive unruptured ectopic pregnancies were treated by laparoscopic salpingostomy with proximal extension of the incision for one centimeter beyond the visible enlargement. /3-HCG levels were measured weekly until they declined to less than 15 mIU/ml. Materials and Methods: All subjects that underwent a laparoscopic salpingostomy for ectopic pregnancy at Los Angeles County-University of Southern California Medical Center from July 1997 to February 1999 were followed postoperatively until the P-HCG level declined to less than 15 mIU/mL, or a diagnosis of PEP was made by leveling or rising P-HCG levels. An exact one-sample test for binomial proportions was used to compare the PEP rate in this cohort to the historical PEP rate at this institution of 14.5%. Results: Following this surgical procedure, 8 of 110 subjects in which follow up was obtained were diagnosed with PEP. Nine subjects were lost to follow up and not included in the analysis. The PEP rate of 7.92% was significantly lower than the historical rate of 14.5% (p-value < 0.025) at this institution. Conclusions: When performing a laparoscopic salpingostomy, extension of the incision one centimeter proximal to the visible ectopic gestation appears to significantly decrease the incidence of PEP.

REPRODUCTIVELABORATORY Wednesday,

September

2:00

29, 1999

P.M.

O-185 Potential of Expanding Hypo-osmotic Swelling (HOS) Test from Andrology to Embryology: A Novel Indication. ‘A. Hossain, *S. Batik, ‘B. Rizk, ‘I. H. Thomeycroft. Departments of ‘Obstetrics/Gynecology, and ‘Biochemistry & Molecular Biology, University of South Alabama, Mobile, AL. Objectives: The HOS test, originally designed for evaluating the functional integrity of sperm membrane, is now approved by the World Health Organization as one of the sperm function tests. To this date, the application of this test has remained confined to sperm quality assessment, In this study, we have examined the hypo-osmotic responses of oocyte, embryo and blastocyst to explore the possibility of expanding the HOS test to evaluate the quality of the female gamete and its postfertilization stages. Design: Chronological and morphological changes that occur in spermatozoa, oocyte, embryo, and blastocyst exposed to hypo-osmotic solution were documented and compared to assess the similarities and differences of their HOS response patterns. Materials and Methods: Human sperm, mouse oocyte (n=7), and mouse embryo, representing 2-cells (n=39), 4-6 cells (n=36), morula (n=7) and blastocyst (n=46), were incubated in hypo-osmotic solution, consisting of 150 mOsmo1 sodium citrate and fructose, at 37’C. An appropriate control (iso-osmotic culture) for each experimental group was maintained. Origin and development of the morphological changes occurring due to the hypoosmotic exposure were monitored under microscope continuously for the first 10 min and subsequently at 20 min intervals up to 3 hrs. The lo-min exposed embryos were cultured to blastocyst development. Viability was determined by the trypan blue exclusion technique. Results: The time required by sperm, oocyte, and embryo to exhibit visible response was lO min to exhibit response. The HOS responses of spermatozoa were always irreversible while the responses of the oocyte and its postfertilization stages were reversible when the exposure duration was 510 min. The HOS reactions of oocyte, embryo and blastocyst consisted of initial defensive response followed by a gradual collapse of the defense. The HOS responses that we observed were: development of space between zona and oolemma, swelling of the celhblastomere, rupture of the membrane, compaction and fragmentation of the cell mass. About 38% 2-cell embryos and 57% mul-

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ticell embryos (4-6 cells) of the 10 min exposure were able to reach blastocyst. Furthermore, in 10 min exposed groups, there were identifiable differences between the ones that were able to produce blast and the ones that did not. In 35% of the 2-cell embryos, the two blastomere exhibited dissimilar HOS responses possibly indicating some differences between the blastomere. The embryos and blastocyst were viable following the HOS treatment, and became trypan positive only upon longer exposure (> 1 hr). Conclusions: To our knowledge this is the first study that illustrated the HOS responses of the female gamete and its postfertilization stages. Since the HOS-exposed embryo and blastocyst expressed varied growth and development we postulate that the HOS test could be utilized in developing an embryo scoring system. Currently we are working on such a scoring system utilizing different hypo-osmotic solutions for various lengths of exposure.

Wednesday,

September 2:15 P.M.

29, 1999

O-186 The Significance of Early Human Embryo Fragmentation in Development Post Genomic Activation. ‘M. Alikani, ‘G. Calderon, ‘G. J. Garrisi, ‘J. Cohen. ‘The Institute for Reproductive Medicine and Science of Saint Barnabas, West Orange, NJ USA. Objectives: Early fragmentation in human embryos is an aberration in development and leads to decreased potential for implantation. This study is an effort toward elucidating the manner in which fragments interfere with development, taking advantage of the availability of a reiiable cell-free culture system for extended culture of embryos (Gardner et al, Hum Reprod, 13: 3434; 1998). Design: Day-3 human embryos were placed in extended culture either following a day-3 transfer or for a day 5 transfer. Day-3 fragmentation was correlated to potential for development to day-6. Materials and Methods: Day-3 embryos (n=2416) were cultured to day 6. Second phase culture media used were G2.2 or S2. Embryos were evaluated daily. Fragmentation rates were determined on day 3. Results: On day-4, the ability of the embryo to compact was significantly reduced when fragmentation reached or exceeded 20% of the embryonic volume (p<.OOl) (see Table I). On day-5, formation of a single cavity was severely compromised when fragmentation was 30% or more. Also on day-5, the embryo was virtually unable to form an inner cell mass, once fragmentation reached or exceeded 30% (p<.OOl) (see Table 1). Conclusions: 1) Three major developmental events post genomic activation, namely, compaction, single cavity, and inner cell mass formation were significantly compromised with increasing fragmentation. 2) These findings indicate that a majority of fragmented embryos, despite their chromosomal integrity, do not reach their full potential because of the presence of fragments. 3) These data have important implications in selection of embryos for transfer. Table 1. Development of day 3 embryos to day 5 according to the degree of fragmentation on day-3 D3 Frag (%)

D4 Comp

o-5 l&-15 2g-25 30-35 >35

(%)

57.8” 53 43.6” 25.6 17.1

D5 Cav (%) 43.6b 43.1 39.5 28b 18.7

D5 ICM

September 2:30 P.M.

29,

14.9’ 14.5 14.9 4.9” 5.3

1999

O-187 Cumulus Cell-embryo nancy Rates. .I. Ding, Center, Oak Brook, IL.

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Abstracts

Coculture

Female Age

Cumulus None

35.0 2 5.0 33.1 2 5.2

Coculture

# of Embryos Transferred

Cumulus None

3.4 2 1.4 3.2 k 1.0

Coculture Improves Implantation and PregN. Rana, W. P. Dmowski. Oak Brook Fertility

# Retrievals

# Transfers

39 70

37 68

Clinic Pregnancy Rate/Transfer

Implantation Rate

54.1% 33.8%

29.2% 17.9%

Conclusion: Cumulus cell-embryo coculture may improve implantation and pregnancy rate. Autologous cumulus cells are readily available for coculture and autologous cumulus cell coculture offers advantages over coculture with animal cell lines and with non-autologous human cells.

Wednesday,

(%)

a p<.OOl. b p<.OOl. ‘p<.OOl.

Wednesday,

Objective: It has been demonstrated previously that coculture of human IVF embryos with somatic cells such as oviductal epithelial cells, vero cells and endometrial cells improves embryo quality and increases pregnancy rates. The objective of the present study was to evaluate the effect of autologous cumulus cell coculture on pregnancy and implantation rates. Design: A prospective study. Materials and Methods: Between Feb. 1996 and Dec. 1998, IVF with cumulus cell coculture was performed during 39 cycles, while there was no coculture in 70 cycles. The coculture cycles were selected at random. In cumulus coculture cycles, good-looking expanded cumuli were selected and cut off from retrieved oocytes with 25G BD syringe needles. Cumuli were washed twice in fertilization/embryo culture medium (IVF-50, Scandinavian IVF Sci. AB, Sweden) and then transferred to 401.~1 embryo culture drops (IVF-50), one cumulus per drop, covered with oil (Ovoil-150, Scandinavian IVF Sci. AB, Sweden). Oocytes immediately after KS1 or fertilized oocytes after conventional IVF were transferred to embryo culture drops (2 to 3 per drop) containing expanded cumulus. Embryos were cultured to Day-3 without medium change. If extended embryo culture were used, Hatch-50 or S2 medium (Scandinavian IVF Sci. AB, Sweden) was used to culture embryos up to Day-6. Embryo transfer was made on Day-3 or Day-5. On Day-3, embryo morphology was graded 1 to 4 (4 best, 1 worst), fragmentation scores (0 best, 4 worst) were assigned, and cell numbers were recorded. Embryo quality scores and IVF outcomes were collected and compared with non-coculture group during the same period. Results: IVF outcomes were shown in Table 1. Mean cell number on Day-3 in cumulus coculture group was 6.65? 1.18, significantly higher than in non-coculture group (5.77+ 1.32, P=O.O016). There were no significant differences between cumulus coculture and non-coculture groups in grades and fragmentation scores. In cumulus coculture group, embryo implantation rate was significantly higher than in non-coculture group (29.2% vs 17.9%, P=O.O26, 2 test). Clinical pregnancy rate was marginally higher in cumulus coculture group than in non-coculture group (54.1% vs 33.3%hransfer, P=O.O7, J test).

September 2~45 P.M.

29, 1999

O-188 Human Frozen Blastocyst Transfer: Pre and Post-thaw Morphology and Patient Parameters Associated with Positive Outcomes. N. Desai, V. Gindlesperger, A. Loeb, J. Goldfarb. Department Reproductive Biology, Case Western Reserve University, Cleveland, OH 44106. Objective: Laboratories considering a shift to Day 5 transfer of blastocysts will need to establish a successful freezing program to store “spare” untransferred blastocysts. The purpose of this investigation was to: (1) analyze pre and post-thaw morphological parameters of human blastocysts and their relationship to pregnancy outcome, and (2) to consider other patient and cycle parameters that might impact on outcomes. Design: A retrospective analysis of 94 frozen transfer cycles carried out over a two year interval at University MacDonald Women’s Hospital. Materials and Methods: Human blastocysts were frozen using Menezo’s two-step glycerol protocol. Blastocyst thaw was performed using a seven step rehydration protocol. All freeze thaw solutions were prepared in either B2 or 01 MEM with 10% SSS. Blastocysts were graded pre and post thaw

Vol.

72, No. 3, Suppl.

1, September

1999

according to the following parameters: maturity, inner cell mass development, trophectoderm and presence of necrotic regions. Maturity was graded as follows: A- cavity just starting, B- cavity less than % volume, Cexpanded with cavity greater than %, D- fully expanded. The inner cell mass was graded as 0- not visible, 1- scanty/disorganized and 2- well developed. The trophectoderm was scored as: l- poorly developed; 2adequate cell number; 3- extremely well developed with large cell number. Necrotic areas (if present) were identified as 1- minor, isolated sites; or 2severe, throughout the embryo. The day of culture that the blastocyst was frozen was also incorporated into the scoring system. Other parameters examined were patient age, method of fertilization, day 3 embryo grade, hours in culture prior to transfer, degree of post-thaw re-expansion and day of frozen transfer. Clinical pregnancy (fetal heart) and implantation rates were compared with transfer of different grades of blastocysts. Results: Eighty-six frozen embryo transfers were performed with an average of 2.2 embryos being transferred. Thiiy two percent of transfers resulted in a positive pregnancy test. The clinical pregnancy rate per transfer was 26% and the implantation rate was 14%. Eight thaw cycles resulted in no embryo survival. Blastocyst thaw survival rate was 75% (204/273). Approximately 81% of thawed embryos showed some degree of re-expansion within 4.5 hours of thawing. Blastocyst maturity and re-expansion after thaw correlated strongly with pregnancy outcome. Transfers with at least one re-expanded blastocyst of maturity grade C or D resulted in clinical pregnancy rates of 30% (6/20) and 40% (14/35), respectively. Conversely, transfer cycles with only non-expanded blastocysts, or else early blastocysts (A or B), had poor pregnancy outcomes (O/7, O/9, and 2/14 respectively). ICM and trophectoderm were well differentiated in blastocysts resulting in clinical pregnancies. No pregnancies were established with transfer of Day 7 blastocysts. Conclusion: Critical assessment of pre and post thaw blastocyst morphologies can yield valuable insight into factors predicating success. Selection criteria for blastocyst cryopreservation may need to be adjusted.

Wednesday,

September

3:00

29, 1999

P.M.

O-189 Isolation of Developmental Competence Markers in Cattle. ‘M. A. Sirard, ‘D. Gag&, ‘F. L. Barnes. ‘Laval University, Quebec, Canada and ‘Pacific Fertility Medical Centre, San Francisco, Ca. Objectives: Human oocytes are not equally competent to induce embryonic development and/or pregnancy after in vitro fertilisation. Results obtained in cattle indicate that oocyte developmental competence improves as follicle size increases. The bovine model may therefore be used to increase our knowledge regarding the acquisition of oocyte developmental competence in women. Since follicle granulosa cells (GC) play a key role in assisting the oocyte to acquire developmental competence, differences in gene expression in GC from small versus large follicles should lead to the identification of competence markers or products. Design: Prospective study evaluating the differences in mRNA expression from bovine granulosa cells derived from different follicle size categories. Materials and Methods: Bovine GC from follicles of different size categories (large>8 mm and small <4 mm) were subject to 4 hours of culture in TCM and subsequent RNA extraction and analysis. Samples were standardised using RT-PCR for actin and LHr. Comparisons between and within follicle size groups were performed using Differential Display (DD)technology. Results: Fifteen bands were cloned and sequenced. Band 1 correspond to the expression of a ribosomal L27a protein in GC from F<4 mm. Band 11 correspond to expression of 3’UTR of a Human mRNA (DRAKI), from both follicle categories but unique to treatments where LH was present. Northern blot analysis indicates that the major form of bovine DRAKl mRNA (1.9 kb) appears to be ubiquitous showing different splicing patterns of the human variant. Conclusion: The use of differential display is useful in isolating differences in gene products from cultured bovine granulosa cells. The differences observed may be related to the acquisition of oocyte developmental competence and relevant to human ART. The study is ongoing.

FERTILITY

& STERILITY@

Wednesday,

September

3:45

29, 1999

P.M.

O-190 Mutation Patients man, ‘M. ‘Section Obstetrics ‘Genetics

Analysis of the Follicle Stimulating Hormone-beta Gene in with Idiopathic Hypogonadotropic Hypogonadism. ‘L. C. LayR. Gray, ‘K. L. Layman, ‘J. Xie, ‘D. P. Bick, *R. J. Sherins. of Reproductive Endocrinology & Infertility, Department of & Gynecology, The University of Chicago, Chicago, IL, and & IVF Institute, Fairfax, VA.

Objective: Mutations in the gene for the beta subunit of follicle stimulating hormone (FSHP) have been described in several men and women with isolated FSH deficiency. Isolated FSH deficiency causes delayed puberty and infertility in women, and azoospermia with or without normal pubertal development in men. Although LH levels have been elevated in the reports to date, it is possible that a “normal” or low LH could be present in some patients depending upon the amount of sex steroid produced and the sensitivity of the pituitary gonadotropes. The purpose of the present study was to determine the prevalence of FSHP gene mutations in patients with IHH. Design: DNA analysis of IHH patients and controls in a university medical center. Methods: DNA extracted from 120 IHH patients and 30 controls was subjected to polymerase chain reaction (PCR) for the exons of the FSHP gene encoding the protein. GC-clamped PCR products were electrophoresed on agarose gels to document the presence of the fragment and then on denaturing gradient gels to screen for DNA sequence differences. Results: All PCR products from exons 2 and 3 were demonstrated by agarose gel electrophoresis in study subjects and controls. Exon 2 fragments did not reveal any variation by denaturing gradient gel electrophoresis (DGGE). In contrast, exon 3 fragments demonstrated a two allele polymorphism in both patients and controls, but an additional variant fragment was identified in one IHH patient, but not in any control. Conclusion: If DNA sequencing and in vitro analysis of this FSHE exon 3 DNA sequence difference demonstrate that it is a true mutation, this will confirm FSHS as another candidate gene for mutations in IHH patients. However, the prevalence of FSH@ gene mutations is low in IHH patients, suggesting that other genes are involved in the pathogenesis of IHH. This work was supported by USPHS-NICHD HD33004 (L.C.L.)

Wednesday,

September

490

29,

1999

P.M.

o-191 Multiplication of Female Pronuclei in the Hamster Oocyte as a Model for Enhancement of Fertility Potential in Women with Impaired Oocyte Quality and Number. ‘Y. L. Feng, *M. Millett-Johnston, ‘D. Engel, ‘J. L. Hall. ‘Center for Reproductive Research and Testing, Inc., Rockville, MD and ‘Aut Even Hospital, Kilkenny, Ireland. Objectives: In a previous study, we were able to retrieve second polar bodies (PBII) from white mice and transfer them by electrofusion to embryos derived from black mice devoid of a female pronucleus. Birth of white mice proved the term developmental potential of the polar body genome (Feng and Hill, 1997). We established that the PBII must be retrieved and used within two to three hours of extrusion or, consequently, degenerative changes lessen the likelihood of fusion and embryo development. Since the lifetime of the polar body can be short, we sought to extend the time in which the genetic material of the PBII is useful and to protect it from degeneration by suppressing PBII extrusion with cytochalasin B. Using this approach, we evaluated the success at which the second polar body would transform into a viable pronucleus within the ooplasm in order to multiply the genetic material usable for nuclear transfer. Design: Metaphase II (MB) oocytes were derived by superovulation. The first polar body (PSI) was removed and the oocyte activated by exposure to ethanol. PBII extrusion was suppressed by cytochalasin B. Oocytes were cultured until artificial activation was assessed. Materials and Methods: MB oocytes from superovulated golden hamsters were denuded with 1% hyaluronidase and placed into 7% ethanol in isotonic PBS for 5 minutes. MI1 oocytes were made zone free and devoid of PBI

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with 0.1% trypsin followed by micropipetting. MI1 oocytes without PBI were cultured in cytochalasin B (10 uglml) for 2.5 hours to suppress PBII extrusion and to retain the genome as a pronucleus in the ooplasm. Oocytes with PBI removed and treated with ethanol, but not exposed to cytochalasin B, served as a control. Results: After 2.5-3 hours, 56 of 60 control oocytes (93%) that were exposed to ethanol only were observed to have a single pronucleus and extruded PBII. Of 64 experimental oocytes treated with both ethanol and cytochalasin B, 32 (50%) exhibited two pronuclei by two hours of treatment and by 2.5 hours 57 (89%) had two approximately equal-sized haploid pronuclei in the cytoplasm. Clearly, PBII extrusion was suppressed which resulted in a second female pronucleus identical to the first. Conclusions: The second polar body has the same developmental capacity as its identical maternal haploid genome retained inside the ooplasm during meiosis. The short life of the PBII and poor cryo-survival, however, has limited its therapeutic application in IVF. Effectively protecting the PBII from degeneration by preventing extrusion and cryopreservation as a pronucleus will enhance utility of the PBII nuclear material in cases of poor oocyte quality and reduced oocyte number. Nuclear transfer, necessary in this procedure, will be a commonplace assisted reproductive technique in the future, if proven safe and effective. Our results with this animal model suggest that it may be feasible to improve female fertility potential by doubling the usual number of pronuclei available for transfer to enucleated healthy donor oocytes. This could be of considerable benefit to women with a limited ovarian reserve, especially because of the purported long-term health risks they would be exposed to if multiple cycles of controlled ovarian hyperstimulation were otherwise required.

Wednesday,

September 4:15 P.M.

29,

1999

o-192 Effects of Cytochalasins B and D on Murine Fertilization: Implications for Blocks to Polyspermy. ‘C. J. Williams and ‘5. P. Evans. ‘Center for Research on Reproduction and Women’s Health, University of Pennsylvania School of Medicine, Philadelphia, PA, and *Division of Reproductive Biology, Department of Biochemistry, Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD. Objective: Mouse eggs establish blocks to polyspermy at the level of the egg’s extracellular matrix (the zona block) and at the level of the plasma membrane (the membrane block). In this study, we examined the possibility that the actin cytoskeleton of the egg mediates the establishment of the membrane block and the zona block to polyspermy. Design: To examine the role of the actin cytoskeleton in the membrane and the zona blocks to polyspermy, mouse eggs were treated with varying concentrations of two drugs that perturb actin microfilaments, cytochalasin B and cytochalasin D, and then inseminated. The effects of these drugs on different endpoints of fertilization were assessed. Materials and Methods: Mouse eggs were inseminated in the presence of cytochalasin B (5-20 pg/ml) or cytochalasin D (2.5-10 pg/ml), or 1% DMSO (control). Sperm-egg binding and fusion to zona-free eggs, oscillations in intracellular calcium, and zona conversion were assessed as previousiy described (Biol. Reprod. 59: 145, Dev. Biol. 198:116). Results: Treatment with cytochalasin B resulted in decreases in spermegg binding and fusion (maximal effect with 20 Fg/ml; p
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Abstracts

a different effect was observed with cytochalasin D, which does not inhibit glucose transport. This work was supported by the American Society for Reproductive Medicine, Serono, Inc., Organon, Inc., the Burroughs-Wellcome Fund, and the National Institutes of Health (HD07903, HD37696, HD22732).

Wednesday,

September 4:30 P.M.

29, 1999

o-193 Integrin Adhesion Molecules Expression During Development of Preimplantation Human Embryos. A. K. Dubey, J. R. Cruz, B. Hartog, P. R. Gindoff. Department of Obstetrics and Gynecology, The George Washington University Medical Center, Washington, DC. Objectives: Multiple functions have been attributed to integrins in reproductive physiology such as sperm-oocyte binding and embryo implantation. Several studies have investigated integrin expression in mouse embryos and endometrium and in human endometrium, but limited information is available about integrins and human embryo development. The purpose of this study was to investigate the developmental expression of (YV integrin as well as the presence of 012, ru4, and /32 integrins in human oocytes and embryos. Design: The following is an observational descriptive study using immunofluorescence microscopy and computerized image analysis to evaluate integrin expression in human embryos and unfertilized oocytes. Materials and Methods: IRB approval was obtained for the use of donated human embryos and unfertilized oocytes from patients in our IVF program. Morphological normal (bi-pronucleate) fresh embryos at different developmental stages were fixed in 2% formaldehyde in PBS. The unfertilized oocytes were fixed two days after insemination or ICSI. After washing with PBS-BSA, samples were incubated with mouse monoclonal antibodies against human integrin subunits OIV, o2, a4, or p2 for 60 minutes. Samples were then washed again and incubated with goat anti-mouse IgG-FITC labeled (human serum adsorbed) for 60 minutes. Specimens were washed again and mounted using mounting media to minimize quenching. Immunofluorescence microscopy and computerized image analysis using IP Lab Scientific Image Software (Scanalytics, Inc., Fairfax, VA, USA) to evaluate both qualitative and quantitative expression of integrins in these human embryos and unfertilized oocytes. Results: a2, a4, and 02 integrin subunits were found to be constantly expressed in human embryos throughout their development (from 2.cell stage up to blastocyst). The (YV integrin subunit expression gradually increases throughout embryo development as measured quantitatively by image analysis. Conclusions: Based on this data it is observed that multiple integrin subunits ((Yv, 012, cx4, and (~2) are constantly expressed during preimplantation development. Our study also shows that OIV expression gradually increases throughout preimplantation embryo development. This might be due to an increase in surface area associated with increase in the number of cells as the embryo develops or due to an increase in the expression of cyv integrin receptors per cell. These observations might be useful in understanding the mechanisms responsible for implantation of human embryos.

Wednesday,

September 4:45 P.M.

29, 1999

o-194 Increased Oxidative Stress in the Peritoneal Fluids of Women With Endometriosis. ‘C. R. Tzeng, ‘K. Y. Wu, *S. H. Lee, ‘H. K. Au, ‘Y. Y. Chien, ‘C. T. Cehn. ‘Department of Obstetrics & Gynecology, Taipei Medical College Hospital, Taiwan, Taiwan and ‘Department of Biochemistry, Taipei Medical College, Taipei, Taiwan, Objective: To evaluate the activity of antioxidant enzyme such as catalase (CAT) and glutathione reductase (GRx), iron (Fe) and selenium (Se) concentrations in the peritoneal fluid (PF) from infertile patients with or without endometriosis.

Vol.

72, No.

3, Suppl.

1, September

1999

Design: The activity of antioxidant enzyme and the free iron which is responsible for Fenton reaction and enhances oxidative stress in PF were determined. The ratio of Fe/CAT indicating the generation activity of hydroxyl radical was also measured. Materials and Methods: Between Feb. and May 1998, peritoneal fluid was sampled from 36 cases during laparoscopic examination. According to the revised American Fertility Society Classification, there are 10 normal infertile cases in group 1 (control), 17 cases with minimal to mild endometriosis in group 2 and 9 cases with moderate to severe endometriosis in group 3. By using the spectrophotometric method from measuring the breakdown of hydrogen peroxide by CAT, we could evaluate the activity of CAT in the PF. By using the method from Bergmeyer, the activity of GRx could be measured from the changes of optical absorbance in spectrophotometry caused by decreased amount of NADPH. The activity of each enzyme was measured repeatedly for three times. Fe and Se concentrations was determined by atomic absorption spectrophotometer, respectively. Results: The activity of CAT (units/mg protein) in the PF (mean?SE) was 7.58kO.55, 8.49kO.57 and 12.61+1.85 (p
REPRODUCTIVE IMMUNOLOGY INTEREST GROUP Wednesday,

September

2:00

SPECIAL

29, 1999

P.M.

o-195 Estradiol and Progesterone Up-regulate Fas Ligand Expression in Endometrial Glandular Cells: A Novel Mechanism in Implantation. Belgin Selam, Junhui Zhang, Naciye Mulayim, Aydin Arici. Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT. Objective: Glandular apoptosis is suggested to be a requisite, physiologic phenomenon for successful implantation. Apoptosis in the glandular cells of the human endometrium occurs in the mid-luteal phase, which coincides with the implantation window. Fas ligand (FasL) is a specific mediator of apoptosis. Cross-linking of Fas/FasL induces apoptosis of Fas-bearing cells. We hypothesized that apoptosis of glandular cells in the human endometrium is hormonally regulated by way of timely expression of FasL in these cells. Design: Regulation of FasL mRNA expression and protein production by estradiol and progesterone were analyzed in human endometrial glandular cells in culture. Material and Methods: Endometrial tissue samples were obtained from human uteri after hysterectomy for benign diseases. Endometrial glandular cells were prepared by standard enzyme digestion and filtration and were plated on six-well plates previously coated with growth factor reduced matrigel, a basement membrane for the attachment of cells. Glandular cells were replicated to confluence in Dulbecco’s minimum essential media containing D-valine (substituted for L-valine to inhibit the stromal cell growth) and were incubated in serum-free, phenol red-free medium for 24

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& STERILITY@

hours prior to each experiment. Cells in primary cultures were treated with various concentrations of estradiol (lo-” to 10m7 M) and a fixed concentration of estradiol (5 X lo-* M) with different concentrations of progesterone (10-i’ to 10m7 M). After 2 hours of treatment, total RNA was extracted and the level of FasL expression was evaluated by RT-PCR. Twenty-four hours after treatment, FasL protein production was analyzed by Western blot following SDS-polyacrylamide gel electrophoresis. Results: We detected FasL mRNA expression and protein production in endometrial glandular cells using RT-PCR and Western blot. Estradiol alone up-regulated FasL expression in endometrial glandular cells in a concentration dependent manner. Progesterone had an additive effect in the expression of FasL. Conclusion: FasL expression in the glandular cells of human endometrium is regulated by estradiol and progesterone. This up-regulation of FasL may have a crucial role in the timely apoptosis of glandular cells in mid-luteal phase of the cycle.

Wednesday,

September

2:1.5

29, 1999

P.M.

O-196 Expression of the Interleukin-1 (IL-l) System in Human Fallopian Tubes With Ectopic Pregnancy: Possible Implication For “Abnormal” Embryo Implantation. ‘H. Y. Huang, ‘H. S. Wang, *M. L. Polan, ‘Y. K. Soong. ‘Department of Obstetrics and Gynecology, Lin-Kou Medical Center, Chang Gung Memorial Hospital, Taipei, Taiwan and ‘Department of Gynecology and Obstetrics, Stanford University Medical Center and School of Medicine, Stanford, CA. Objectives: The IL-l system is a major regulator in local cellular interactions during embryonic implantation. The complete IL-l system including IL-l/$ IL-1 receptor antagonist (IL-lra) and IL-l receptor has been demonstrated in human endometrium during embryo implantation. Human fallopian tube normally provides the environment for early embryonic development. For most ectopic pregnancies, abnormal implantation occurs in the fallopian tubes. Little information is available regarding the regulation and synthesis of these cytokines in the pathogenesis of fallopian tube ectopic implantation. The aim of this study was to investigate IL-l mRNA and protein expression in fallopian tubes during ectopic pregnancy. Design: The mRNA levels in fallopian tube were determined by RT-PCR and protein levels were determined with immunohistochemistry. Materials and Methods: Segments of human fallopian tubes with ectopic pregnancy, side portion close to ectopic pregnancy (n= 18) were collected from women undergoing laparoscopic salpingectomy after informed consent. Segments of fallopian tubes from women undergoing tubal ligation (n=5) and tubal ligation with elective termination of early pregnancy (n=2) were used as control groups. Total extracted RNA was reverse transcribed into cDNA and amplified by PCR using specific primers for p-actin (838bp), IL-lp (549 bp), IL-lra (422 bp) and IL-1R t1 (284 bp) target cDNA. To determine the presence of IL-l system proteins in fallopian tubes, tissues were fixed and processed for immunohistochemical staining using the avidin-biotin alkaline phosphatase method. Human luteal phase endometrium was used as a positive control. Deletion of the primary antibody was used as a negative control. Results: B-actin and IL-1R t1 mRNA was detected in all samples of fallopian tubes both with ectopic pregnancies and normal controls. IL-l/3 (9 of 18) and IL-lra (2 of 18) mRNA were detected only in fallopian tubes from ectopic pregnancies. Immunoreactive IL-lfl and IL-lra at the protein levels was present in tubes from ectopic pregnancies. Immunoreactive IL-1R t1 at the protein level was present in tubes both with ectopic pregnancies and normal controls. Conclusion: These results suggest that fallopian tube IL-l expression may play a crucial role in embryo-maternal interaction during the process of early embryonic implantation. This expression of agonist and receptor in fallopian tubes may indicate an earlier “dialogue” between the early developing embryo and fallopian tubal epithelium prior to uterine implantation.

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Wednesday, September 29, 1999 2:30 P.M. o-197 Hydrosalpinx Fluid Inhibits Tropboblast Cell Proliferation In Vitro Culture System: Implication For Early Implantation Failure In Women With Hydrosalpinx Fluid (HF). ‘B. C. Choi, ‘M. K. Koong, ‘J. A. Lee, ‘H. K. Byun, ‘J. Y. Han, ‘I. P. Son, ‘5. A. Hill. Recurrent Miscarriage Clinic, Division of Reproductive Endocrinology and Infertility, Department of Obstetrics/Gynecology, ‘Samsung Cheil Hospital and Women’s Healthcare Center, College of Medicine, Sungkyunkwan University, Seoul, Korea; *Brigham and Women’s Hospital, Harvard Medical School, MA., USA. Objective: It has been recently suggested that the presence of hydrosalpinges has a negative impact on successful pregnancy; however, the pathological basis for this mechanism is poorly understood. Since cytokines has been associated with inflammatory processes as well as with embryotoxic characteristics, we hypothesized that HF plays a role in early implantation failure. We used the Jeg-3 choriocarcinoma cell line as a source of trophoblast cell and exposed them to varying concentrations of hydrosalpinx fluid to evaluate if this fluid affects trophoblast cell proliferation in vitro. Design: 3-day proliferation assay of trophoblast cell in response to hydrosalpingeal fluid from 10 patients undergoing laparoscopy was performed. We also checked the levels of several cytokines (INF--y, TNF-ol, IL-IO, IL-6) in this fluid. Materials and Methods: Hydrosalpinx fluid was aspirated during laparoscopy. All samples were centrifuged at 10,000 RPM for 10 minutes to remove cellular debris and frozen at 20°C until analysis. Trophoblast cell (Jeg-3 choriocarcinoma cell line; ATTC, Bethesda, MD) proliferation in vitro was determined by a calorimetric immunoassay (Boehringer Mannheim), based on the measurement of BrdU incorporation during DNA synthesis, using a kit. The optical absorbance of the samples was measured in an ELISA reader at 450 nm. Cytokines were assayed by a two step sandwich enzyme immunoassay technique (Biosource, CA; lower limit of sensitivity, 4 pg/mL for IFN-y, 1 pg/mL for TNF-ol, 2 pg/mL for IL-6, 5 pg/mL for IL-IO). Data are presented as mean?SEM. Statistical analysis was performed by regression analysis. Results: Samples from 7 out of 10 patients significantly suppressed trophoblast proliferation in a dose dependent manner (r=-0.0673, p < 0.05). Trophoblast cell proliferation was assayed in the presence of six different concentrations of hydrosalpinx fluid (0%, 25%, 50%, 75%, 90%, 100%). IFN-7 and IL-6 were present in 2 out of 10 HF samples with a mean concentration of 10.45pg/ml ? 0.95 pg/ml and 0.814 pg/mL?O.59 pg/mL. IL-10 was present in 4 out of 10 samples with concentration of 2.83 pg/mLt 1.27pg/mL. TNF-a was checked in all samples, with a mean concentration of 13.12pg/mL ? l.lOpg/mL. The mean concentration of TNF-(U was greater in the hydrosalpinx fluid from suppressor group than nonsuppressor group (14.74pg/mL * 1.05 pg/mL versus 9.33 pg/mL 2 0.55 pg/mL, respectively). Conclusion: In our study, fallopian tube fluid from the majority of women with hydrosalpinx significantly inhibited trophoblast proliferation in vitro model system. Fallopian tube fluid may play a similar role in vivo. Given the possible role of cytokines in the regulation of pregnancy, and maintenance of a proper hormonal milieu, we postulate that high levels of the Thl cytokine, TNF-cu, may represent a potential mechanism for early implantation failure in women with hydrosalpinx.

Wednesday, September 29, 1999 2:4.5 P.M. RISIG Group

Prize Paper

O-198 Expression of Telomerase Activity in Chorionic Villi From Missed Abortion and Undergoing Surgical Termination of First Trimester Pregnancy. ‘B. C. Choi, ‘S. C. Kim, ‘H. S. Lee, 3K. H. Baek, 4D. J. Anderson, 4J. A. Hill, *K. C. Kimm. Division of Infertility and Reproductive Endocrinology, Department of Obstetrics/Gynecology, ‘Samsung Cheil Hospital and Women’s Healthcare Center, College of Medicine, Sungkyunkwan University, ‘Division of Oncology, NIH, Seoul, Korea;

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Abstracts

jDFC1, Harvard

4Fearing Medical

Research Laboratory, School, MA, USA.

Brigham

and Women’s

Hospital,

Objectives: Telomerase activation is required for cellular immortalization and is found in most malignant tumors. Normal somatic cells are generally telomerase-negative, except for stem cells in renewing tissues. During pregnancy, human trophoblast continues to proliferate and acts as a source of proliferating stem cells for the development of chorion and the formation of the placenta. Trophoblast cells infiltrate into the endometrium and the maternal blood vessels under strict control but, unlike malignant cells, never metastasize. We hypotheze that maintenance of trophoblast proliferation during the first trimester of pregnancy may be regulated by telomerase activity. Design: A total (n=44) of chorionic villi from first trimester gestations were examined using the telomeric repeated amplication protocol (TRAP) assay. Materials and Methods: We assessed hTRT (human telomerase catalytic subunit) expression pattern in chorionic villi from missed abortions (n=36) and therapeutic abortions (n=8) by RT-PCR using primers from a region of the transcript upstream of the reverse transcriptase domain. The expression of human telomerase RNA component (hTR) and hTRT are believed to be the core components of telomerase enzyme, so we examined whether one or both of these subunits were limiting factors for the expression of telomerase activity. Results: PCR with the 2164/2620 primer set revealed alternate splicing of the hTRT gene in the 4 controls, yielding two PCR bands. These two bands represent the full-length hTRT transcriptase and a deletion transcript (457bp/421bp), and the a-P deletion and p deletion transcripts (275bp/ 239bp). Only the full-length hTRT transcript would be expected to code for an active reverse transcriptase. We assessed telomerase activity in chorionic villi obtained from 8 therapeutic abortions, and 36 missed abortions. Telomerase activity was detected in 4/8 (50%) chorionic villi samples from therapeutic abortion. In contrast, no telomerase activity was exhibited in the chorionic villi from any of the 36 missed abortion (p = 0.0005, Fishers exact test). Conclusions: Our data suggest that the placenta inevitably deteriorates following embryonic demise, and that the degree of placental histological change is related to the cell proliferation and telomerase activity. These findings suggest that telomerase activity in chorion is closely correlated with the survival of embryo, and is associated with hTRT expression. These studies also appear to support the emerging concept that normal somatic cells with stem cell-like characteristics can express telomerase activity.

Wednesday, September 29, 1999 3:00 P.M. o-199 Homocysteine (H) Induces the Chorionic Hormone man Trophoblast, but Does Not Alter the Levels Inducible Nitric Oxide Synthase (eNOS and iNOS) Vitro. ‘,‘E. Hambartsoumian, ‘R. K. Srivastava, ‘M. Centre for Reproductive Medicine, Faulkner Hospital; School of Medicine, Department of Obstetrics and MA.

Production in Huof Endothelial and Gene Expression In M. Seibel. ‘Faulkner ‘Boston University Gynecology, Boston,

Objectives: Elevated levels of H are implicated in several pregnancy pathologies and endothelial disorders. This suggests that hyperhomocysteinemia may affect placental function and the production of NO during pregnancy. To investigate this possibility, the effect of different doses of H on the production of hCG and eNOS and iNOS by human trophoblast were investigated. Design: Purified human cytotrophoblast (CTB) cells were cultured in vitro until they differentiated into syncytiotrophoblast (STB) cells. Both CTB and STB were treated with H and the levels of hCG (mUI/ml) and eNOS and iNOS mRNA were examined. Materials and Methods: Full IRB approval was obtained. Two protocols, short term treatment of CTB and STB cells with H (24 h) and long term treatment (72 h) were used. The production of hCG by trophoblast cells was examined in cultured media by immunofluorochemical method. The expression of eNOS and iNOS mRNA by trophoblast cells was examined using polymerase chain reaction (PCR) using specific primers for eNOS and iNOS. Data was analysed using Student’s paired f test.

Vol. 72, No. 3, Suppl. 1, September 1999

Results: Treatment of CTB and STB by H in a dose 10 mM during 24 b significantly stimulate the hCG production (mean ? SEM, 38.7 2 14.2 vs. 26.3 2 11.5 and 1291.7 2 334.2 vs. 804.3 ? 221.5 respectively; p
Wednesday,

September 3:45 P.M.

29, 1999

O-200 Unexplained Recurrent Abortions Associated With Up-regulation of a Novel Prothrombinase on Trophoblast and In Decidua, are Inhibited By Maternal y6 T Cells That Produce Th2 and Th3 Cytokines in Response to The Novel Tolerance-promoting Molecule, 0X-2, Expressed at These Sites. ‘D. A. Clark, 2J. W. Ding, ‘K. Yu, *G. A. Levy, ‘R. M. Gorczynski. Department Medicine, Molecular Medicine & Pathology, & Obstetrics & Gynecology, McMaster University, Hamilton, Ontario, Canada & MRC Group on Organ Injury, Toronto Hospital, University of Toronto, Toronto, Ontario, Canada. Objectives: Recurrent miscarriages are associated with increased classical NK cells in endometrium and production of proinflammatory Thl cytokines (TNF-ru, IFN-y) inadequately counterbalanced by Th2/3-type cytokines (IL-lo, TGF-P). In the CBA X DBA/2 mouse recurrent abortion model, and in recurrent first trimester human abortion material, Thl cytokine-upregulate the novel prothrombinase fg12 in decidua and on trophoblast cells. Trophoblast activates certain y8 T/NK cells to produce IL-10 & TGPP2 that antagonize Thl cytokines, clotting, and polymorphonuclear cell (PMN) activation. Iniecting allogeneic cells into the portal vein activates similar allograft-tolerance y6 T cells via OX-2 molecules on hepatic antigenpresenting cells. Does OX-2 act similarly in the uterus? Design: CBA/J female mice were mated to DBA/2J (high abortion rate matings) or to BALB/c (low abortion rate matings). In situ hybridization to detect fg12 mRNA and OX-2 mRNA was canied out. The effect of antiOX-2 monoclonal antibody on abortion rates measured on day 14.5 of pregnancy (equivalent to human first trimester) was evaluated. Materials and Methods: Using CCAC-approved methods, timed-pregnant mice were injected with control medium or TNF-a + IFN-7 on day 7.5 of gestation. Some animals were sacrificed on day 8.5, 1 day prior to the onset of abortions (resorptions), the uteri were fixed in 4% paraformaldehyde, serial sections were cut and processed for in situ hybridization using digoxigenin-labeled riboprobes for fg12 (650bp fragment) and OX-2 (586bp fragment). Fibrin was detected by immunohistochemistry. Rat monoclonal anti-OX-2 was generated from hybridoma supematant and injected at a dose of 100 @g at various times during pregnancy. Results: The proportion of embryos showing evidence of intense fg12 upregulation on trophoblast and in decidua was significantly higher than the spontaneous abortion rate, but similar with cytokine treatment on day 7.5. OX-2 was expressed on trophoblast and at similar sites as fg12 in decidua, but there was a reciprocal relationship with fg12, and cytokine injection reduced OX-2 expression. In uninjected mice, some implants expressed both OX-2 and fg12, and when anti-OX-2 antibody was injected, the abortion rate increased to the value predicted by % fgl2++ implants. Anti-OX-2 was only effective if given day 8.5 or thereafter, the time when IL-lo+ TGF-P2+ y6 T cells are activated, and when the PMN-dependent abortions occur. Conclusion: Maternal tolerance of her semiallogeneic fetus appears to be dependent on a Thl+Th2/3 cytokine shift mediated via the tolerancepromoting molecule 0X-2.

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Wednesday,

September 490 P.M.

29, 1999

O-201 Role of Protein Kinase C in Reactive Oxygen Species (ROS) Generation by Human Spermatozoa. S. C. Sikka, J. S. Armstrong, W. J. G. Hellstrom. Department of Urology, Tulane University School of Medicine, New Orleans, LA, USA. Objectives: ROS production by human spermatozoa has recently been implicated in the physiological control of sperm capacitation and the acrosome reaction, but the mechanism(s) involved is unknown. ROS production by phagocytic leukocytes is regulated by protein kinase C (PKC), but it is not known whether PKC plays a similar role in the human spermatozoon. The purpose of our study was to determine the involvement of PKC in the production of ROS by human spermatozoa. Design: Washed sperm exposed to xanthone-xanthine oxidase and used for measuring protein kinase C activity. Materials and Methods: Percoll purified donor sperm and purified human leukocytes were used for these studies. We employed: (1) chemiluminescence (CL) using the superoxide radical (O,-) specific, membrane impermeant probe MCLA after PKC activation with phorbol myristate acetate (PMA) and inhibition by GF-109203X; (2) nitroblue tetrazolium (NBT) dye reduction; and (3) electron paramagnetic resonance (EPR) spectroscopy with “spin trapping” using specific probe DEPMPO. Luciferase assay was employed to quantitate ATP levels. Results: The CL response (RLLVmin) of PMA-stimulated spermatozoa was not altered by 200 u/ml superoxide dismutase (SOD), PMA-stimulated PKC, or by PKC inhibitor. However, there was about 40-fold inhibition of peak CL response in leukocytes by SOD or PKC inhibitor. NBT dye (OD units) was not reduced by PKC activated spermatozoa compared to a 2- to 4-fold reduction by activated leukocytes or PKC inhibitor. EPR spectra of control leukocytes in the presence of spin trap DEPMPO produced the characteristic signals for Oi-; however,.this spectra was no; observed in PKC-activated spermatozoa. Leukocyte ATP levels decreased by 40% compared to non-PMA stimulated or treated with PKC inhibitor, while spermatozoa ATP levels were not altered under these conditions. Conclusions: We conclude that PKC is not involved in the production of ROS by human spermatozoa unlike in leukocytes and that R&S generation by the human spermatozoon is probably regulated by a yet unknown mechanism.

Wednesday,

September 4:15 P.M.

29, 1999

O-202 Regulation of Interleukin-1 Receptor Antagonist (IL-lra) Production by Sertoli Cells. ‘.4D. Zeyse, ls3E. Lunenfeld, 31. Prinsloo, 1,2.3M. Huleihel. ‘Department of Obstetrics and Gynecology, Soroka University Medical Center and *Department of Microbiology and Immunology, 3Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel. 4Christian-Albrechts-University of Kiel Medical School, Kiel, Germany. Objectives: Interleukin-1 has been suggested to be involved in the cellcell cross talk within the testis. It is produced by Leydig cells, Sertoli cells, germ cells and testicular macrophages. It has been shown to interfere with steroidogenesis, transferrin production and meiotic DNA synthesis. A unique feature of the IL-l system is the naturally occurring IL-l receptor antagonist. To identify the testicular cell source and factors involved in the regulation of IL-lra production, mouse Sertoli-cells were isolated, purified, cultured and examined for IL-lra levels. Design: IL-lra levels were determined in the supematant and the lysates of non-stimulated and stimulated primary mouse Sertoli cell cultures. Immunohistochemical staining for IL-lra was performed on Sertoli cell-germ cell cocultures with or without stimulation. Materials and Methods: Sertoli cells from the testes of 15-day old Balb/c mice were isolated by subsequential enzymatic digestion. On day 3 Sertoli cells were purified by hypotonic shock treatment and incubated on day 4 for 24 h in the presence of 0,2% BSA (control) or fetal calf serum (FCS; 5%), and FSH (0.1; 1 IU), FCS + phorbol-myristate-acetate (PMA; 0.1; 1; lOng/ml) or lipopolysaccharides (LPS; 10 pg/ml). Supematant and lysates

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of Sertoli cell cultures were examined for IL-lra by ELISA. Sertoli cellgerm cell cocultures were established on Permanox 4-chamber culture slides and incubated with or without FSH, PMA or LPS. Slides were immunohistochemically stained for IL- 1ra. Results: 1) Immunoreactive IL-lra could be detected by immunohistochemical staining in unstimulated and stimulated Sertoli cells and germ cells, as well as in the lysates of unstimulated Sertoli cells by ELISA. IL-lra levels in the supematants were below detection levels of the ELISA kit. 2) The addition of FCS to Sertoli cell cultures did not alter their capacity to produce IL-lra. 3) Addition of FSH or PMA enhanced intracellular IL-lra production by Sertoli cells in a dose-dependent fashion. 4) No additive effect was observed between FSH and PMA concerning the IL-lra production. 5) LPS did not affect IL-lra production in purified Sertoli cell cultures, but induced intracellular IL-fra in Sertoli cell-germ cell cocultures as examined by immunohistochemical staining. 6) Differences between the levels of cellular IL-lra in unstimulated and stimulated immature germ cells were not observed. Conclusions: Sertoli cells and immature germ cells constitutively express IL-lra, and both FSH and PMA can induce this expression. The observation that LPS has a potent effect on IL-lra production by Sertoli cell-germ cell cocultures and not on purified Sertoli cell cultures may indicate its effect on germ cells to produce IL-lra and/or other factors that may induce Sertoli cells to produce IL- lra. The fact that Sertoli cells produce IL- 1ra and do not secrete it may indicate its physiological involvement in Sertoli cell function as an autocrine factor. Our results indicate the involvement of IL-lra in the regulation of physiological functions of the testis, playing a role in the reproductive system and male fertility.

Wednesday,

September

4:30

29,

1999

P.M.

O-203 Effect of Interleukin Concentration in Follicular Fluid on Intracytoplasmic Sperminjection (ICSI) Outcome. M. E. Hammadeh, S. Baltes, B. Bremert, P. Rosenbaum, W. Schmidt. Department of Obstetrics & Gynecology, University of Saarland, Germany. Objective: The aim of this study was to determine the presence and concentration of IL-6, IL-8 and granulocyte-macrophage-colony stimulating factor (GMCSF) in preovulatory ovarian follicular fluid (FF) of patients undergoing ovarian hyperstimulation for ICSI therapy on the one hand, and to find out whether these cytokine concentrations have any influence on ICSI outcome. Design: The levels of IL&, IL-8 and GMCSF were measured from women that underwent from women underwent ICSI therapy and the results were compared between the patients who become pregnant after ICSI and those who didn’t. Material and Method: Follicular fluid was obtained from 85 women participating in our ICSI program. The male factor was the main criteria for including the women in these study. These patients underwent ovarian hyperstimulation either with FSH (n= 19), HMG (n=38) or both FSH and HMG (n=28) after they had been treated with Gonadotropin-Releasing hormone agonist (GnRHa) for pituitary down regulation. FF was collected by transvaginal aspiration 36 hours after HCG ovulation induction. The concentration of IL-6, IL-8 and GMCSF was measured by the highly sensitive ELISA method. 27 women became pregnant (GI) and the other 56 (G II) did not. The statistical analysis was made by non-parametric test (Kruskal-Wallis-test) as the group was not normally distributed. In addition, the possible relationship between the IL-6, IL-8 and GMCSF levels and oocyte retrieval, fertilization and pregnancy rate was analysed with spearman rank correlation test. Results: The main concentrations of IL-6, IL-8 and GMCSF in follicular fluids of patients who became pregnant were (5.4 ? 2.9, median 4.8 pg/ml; 207.9 2 118.3, median 165.8 pg/ml and 1.01 + 0.8, median 1.37 pg/ml) respectively and the corresponding values in patients who did not become pregnant were (7.0 t- 5.1, median 5.4 pg/ml; 230.8 + 183, median 189.5 pg/ml and 1.34 2 0.9, median 1.00 pg/ml). No significant difference was found between the two groups with regard to the IL-6 (p=O.21), IL-8 (~~0.93) an GMCSF (~~0.12). Furthermore, no significant difference was found between the two groups concerning the mean age of the patients (32.5 2 4.8 vs 32.4 2 4.6, p = 0.72), so the mean number of retrieved oocytes (11.3 ? 6.9 vs 9.8 t 5.6, p = 0.55). mean number of M II oocyte

S78

Abstracts

(injected) (10.3 r+_ 6.8 vs 8.8 ? 4.1, p = 0.52), the mean number of fertilized oocyte (5.8 2 4.0 vs 4.7 ? 3.3, p = 0.255) and the fertilization rate (59.0 -C 23.0 vs 47.0 i 21.0, p = 0.098). Conclusions: I.) Interleukin IL-6, IL-8 and GMCSF were found in all follicular fluid investigated either from patients who became pregnant after ICSI treatment or from those who did not. II.) There were no significant differences between the IL-6, IL-8 and GMCSF concentrations in patients who became pregnant or who did not. III.) The mean oocyte retrieval, fertilization and cleavage rates were not significant between the two groups. IV.) No correlation between the IL-6, IL-8 and GMCSF concentration in FF can not be used as a reliable prognostic index for ICSI outcome.

Wednesday,

September

29, 1999

4:45 P.M. O-204 Does Thyroid Autoimmunity kian. Pacific Fertility Medical

Affect Center,

IVF Pregnancy Rates? Los Angeles, CA.

V. Saha-

Objective: Some studies have suggested an association between increased spontaneous abortion rates and thyroid autoimmunity (Fertil Steril 1995;63: 303-7). The purpose of this analysis was to evaluate the pregnancy rates in patients with serum thyroid antibodies during their first IVF attempt. Design: A retrospective analysis in a private practice setting. Materials and Methods: Between July 1997 and October 1998, all patients undergoing IVF in our institution were tested for thyroid peroxidase and thyroglobulin antibodies prior to the initiation of an IVF cycle. Patients who had elevated levels of either one of these antibodies were considered antithyroid antibody (ATA) positive. All patients who met the following criteria were included in this analysis: (1) Age < 40 years, (2) No previous IVF cycles (3) No previous spontaneous abortions, (4) Absence of phospholipid antibodies and (5) Cycle day 3 FSH of less than 10. Group 1 included those patients who were ATA positive and Group 2 those patients who tested negative for thyroid antibodies. All patients were treated with a luteal phase GnRH agonist (long) protocol and embryo transfer was done 3 days after egg retrieval. Patients who were past 12 weeks of gestation were considered pregnant. Student-t and Chi-square was used for data analysis. Results: In Group 1, 25 patients met the inclusion criteria versus 106 patients in Group 2. Outcome data is summarized in the table below. There was no significant difference in the two groups in age, day 3 FSH, number of oocytes retrieved and number of embryos transferred. In Group 1,4 of 25 (16%) have delivered or have an ongoing pregnancy past 12 weeks versus 45 of 106 (43%) in Group 2 (p = 0.014).

Group Group

1 (ATA 2 (ATA

positive) negative)

N

Age

Day 3 FSH

Pregnancy Rate

25 106

32.7 2 3.6 32.4 ? 3.4

6.6 2 1.9 6.7 & 1.7

16% 43%

Conclusion: This analysis suggests an association between the presence of thyroid autoimmunity and lower IVF success rates. Further prospective studies are needed.

ENDOMETRIOSIS Wednesday,

September

2:00

29, 1999

P.M.

O-205 The Shed Endometrial Tissues in Menstrual Fluid Can Adhere to Intact Amniotic Epithelium: Observation of Ultrastructure. ‘M. K. Koong, ‘J. H. Jun, ‘K. N. Ko, 3E. S. Kim, ‘I. S. Kang. ‘Division of Reproductive Endocrinology and Infertility, Department of Obstetrics & Gynecology, ‘Infertility Research Laboratory, College of Medicine, Sungkyunkwan University, Samsung Cheil Hospital & Women’s Healthcare Center and “Department of Biology, College of Science, Konkuk University, Seoul, Korea. Objectives: The nature of adhesion, implantation and invasion of the endometrial cells into peritoneum still remains to be elucidated, although Sampson’s transplantation theory is the most widely accepted for patho-

Vol.

72, No.

3, Suppl.

1, September

1999

genesis of endometriosis. The adhesion of the shed endometrial tissues in menstrual fluid (MF) has been demonstrated on epithelial layer of amniotic membrane (AM) after 5 days of in-vitro culture in our previous study. Therefore. the purposes of this study were to observe ultrastructure of the adhesion site and to evaluate the adhesion status between endometrial tissues and amniotic epithelium. Design: Ultrastructural observation of the adhesion sites after in-vitro culture of the shed endometrial cells using AM. Materials and methods: The MFs from five women with regular menstruation were collected with Wallace catheter by aspiration from the uterine cavity on the second or third day of the menstrual period. The fresh term placenta was obtained from term delivery without any complication and the AM was separated out from the chorionic membrane. The cells in each MF were placed on epithelial layer of two dissected AM pieces and cultured for 5 days. The adhesion sites on AM were observed under a stereomicroscope and prepared for scanning and transmission electron microscopy (SEM & TEM). Results: After 5 days of culture, the adhesion sites were observed in all cultured samples. The cultured amniotic epithelial cells still had well preserved intra- and inter-cellular structures, compared with fresh amniotic epithelial cells. In scanning electron micrographs, the endometrial fragment consisted of two kinds of cells, one was larger and located in periphery and the other was smaller and located inside. The endometrial tissues attached to amniotic epithelial layer. In the observation of the adhesion site with TEM, the large cells at periphery were epithelial cells and the small cells at inside were stromal cells. The endometrial epithelial cells directly attached amniotic epithelial cells and the stromal cells contacted with the basement membrane of amniotic epithelium. The amniotic epithelial cells in contact with endometrial epithelial cells lost their own intercellular digits and had several junctional complexes in intercellular space between these two cells, which were the characteristics of cell-to-cell adhesion. Conclusion: As a result of our study, we can conclude that the shed endometrial tissues adhere to intact amniotic epithelium. These observations indicate that the shed endometrial tissues in MF have adhesive potentials and intact epithelial layer itself does not protect the cell adhesion. Our culture system using MF and AM could be a suitable model for studying pathogenesis of endometriosis.

Wednesday, September 29, 1999 2:15 P.M. O-206 Apoptosis in Endometrial Glandular and Stromal Cells in Women with and Without Endometriosis. ‘J. Ding, r?l. Shen, ‘I’D. P. Braun, ‘,‘N. Rana, 3B. B. Femandez, r,*W P Dmowski. ‘Institute for the Study and Treatment of Endometriosis, Chicago, IL, ‘Rush Medical College, Chicago, IL, 3Department of Pathology, Elmhurst Hospital, Elmhurst, IL. Objectives: We have previously reported that spontaneous apoptosis as determined with the Cell Death Detection ELISA kit is significantly reduced in the uterine endometrium from women with endometriosis (Gebel et al, Fertil Steril 1998;69: 1042-7). The objectives of the present study were: 1) to confirm previous findings using a different cell apoptosis assay and 2) to analyze the location of apoptotic cells in the endometrial glands and stroma in relation to different phases of the menstrual cycle. Design: Uterine endometrial tissues from women with and without endometriosis were evaluated for spontaneous apoptosis in the epithelial and stromal cells. Materials and Methods: Sixty-one paraffin blocks of uterine tissues from patients who underwent laparoscopy and endometrial curretage between 1995-1998 were retrieved from the pathology laboratory. Of these, 48 patients were laparoscopically positive and 13 were negative for endometriosis. Tissue sections were made and apoptotic cells were identified by TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assay. Apoptotic cells were counted with a cytometer in an area of IO-50 mm’ depending on the size of the samples and presented statistically as the number of apoptotic cells per 10 mm2 area (mean+SD). Endometrial phases were classified according to the histological criteria. Results: The mean number of total apoptotic cells per 10 mm2 area of the uterine endometrium, including both glands and stroma, was 121.7 2 I 19.1, n = 13, in women without endometriosis, significantly greater than the

FERTILITY

& STERILITY@

corresponding number for women with endometriosis (55.1 5 33.8 n = 48; P = 0.001). When the concentration of apoptotic cells was determined separately for the glandular epithelium and stroma the same relationship remained. In women without endometriosis there were 63.1 t 77.1 apoptotic cells per 10 mm’ in the glandular epithelium as compared to 24.0 2 21.1 in women with endometriosis (P = 0.002). In the endometrial stromal cells the concentration of apoptotic cells was 58.6 -C 58.2 in women without endometriosis as compared to 31 .l f 20.48 in women with endometriosis (P = 0.007). In women without endometriosis spontaneous apoptosis in both endometrial glandular and stromal cells was the highest during early proliferative and late secretory phases representing an approximately 3 to 4 fold increase as compared to women with endometriosis. No significant differences in apoptotic cells between different stages of endometriosis were detected in women with endometriosis. Conclusions: The results of this study demonstrate that spontaneous apoptosis in both glandular and stromal endometrial cells is significantly decreased in women with endometriosis, as compared to normal controls. The decrease in spontaneous apoptosis, especially during the menstrual phase, may result in the increased viability of shed endometrial cells, allowing their survival and implantation in ectopic locations.

Wednesday, September 29, 1999 2:30

P.M.

O-207 A Clinical Trial of Aiendronate for Prevention of Bone Loss Associated with Gonadotropin-Releasing Hormone Analogues. ‘B. A. Ripps, ‘K. Vangilder, *MI. Welford, ?Z. Mamish. ‘Department of Obstetrics/Gynecology, University of Florida-Pensacola, ‘Sacred Heart Hospital Pharmacy and ‘Department of Medical Endocrinology at Sacred Heart Hospital, Pensacola FL. Objective: To investigate the impact of alendronate on bone metabolism and the lipid profile in young women treated with gonadotropin releasing hormone agonist (GnRHa). Design: Prospective, randomized, doubly-blinded, clinical trial. Materials and Methods: After full IRB approval, twelve premenopausal women (mean age 29.7, range 21-38) planning to initiate 6 months of leuprolide acetate for medical indications were referred and screened for participation. Subjects were excluded if they smoked, had prior GnRHa use in the preceeding year, had past or present steroid use or history of hypothyroidism or autoimmune disease. Compliance with the random assignment of alendronate 10 mg or identical placebo was monitored by the pharmacy and initiated with the first GnRHa injection. Changes in bone mineral density (BMD) at the total lumbar spine and femur neck were evaluated by dual energy x-ray absorptiometry (DEXA) baseline and at the end of the sixth month of intervention. Bone turnover was evaluated by longitudinal urinary N-telopeptide. Total cholesterol, HDL and triglyceride were measured before and after GnRHa therapy. Data were analyzed by paired and unpaired t-test where appropriate. Results: Eleven women completed the study; one enrollee withdrew prior to starting treatment due to a relocation out-of-state. Alendronate (5) and placebo (6) groups did not differ in age, lumbar, femur BMD. body mass index or lipid parameters at baseline. Following 6 months of GnRHa, both treated and control groups had significant reductions in femur BMD (p=O.O2 and p=O.Ol, respectively). Femur BMD loss was greater in the placebo group (-3.4%) than in alendronate recipients (- 1.2%) (p=O.O4). Lumbar BMD showed a mean loss of -4.1% with placebo compared to a mean gain of + 1.3% with alendronate, p=O.O02. While on GnRHa, bone turnover increased above baseline in both alendronate and placebo group (p=O.O03 and 0.05, respectively). There were no significant changes or differences between groups in total cholesterol, HDL or triglyceride levels. Conclusions: In this limited clinical trial, alendronate appears to impede loss of BMD compared to placebo in a young population using GnRHa for six months, Although well tolerated and with insignificant impact on pelvic symptoms, hot flashes and lipid changes, alendronate in this young population did not induce the gain in BMD usually seen in postmenopausal women.

s79

Wednesday,

September 2:45 P.M.

29, 1999

O-208 Prolonged GnRH Agonist and Add-Back Therapy for Symptomatic Endometriosis Patients: Long Term Follow-up of a 12 Month Clinical Trial. ‘E. Surrey, *M. Homstein, Add-back Study Group. ‘Reproductive Medicine and Surgery Associates, UCLA School of Medicine, Los Angeles, CA and ‘Harvard University School of Medicine, Boston, MA. Objectives: We have recently demonstrated that symptomatic endometriosis patients can be safely and effectively treated for up to 12 months with GnRHa when combined with norethindrone acetate (NEtAc) alone or in combination with conjugated equine estrogens (CEE) (Obstet Gynecol 91:16, 1998). The results of post-treatment follow-up (F/U) are presented. Design: Prolonged F/U of patients enrolled in a multicenter prospective randomized placebo controlled trial. Materials and Methods: Patients with laparoscopically confirmed symptomatic endometriosis were enrolled in the initial 12 month trial (N=201). All patients received a depot preparation of the GnRHa leuprolide acetate (TAP Holdings, Deerfield, IL) 3.75 mg IM X 13 and were randomized to 1 of 4 add-back groups: I - Placebo; II - NEtAc 5 mg daily; III - NEtAc 5 mg + CEE 0.625 mg daily; IV - NEtAc 5 mg + CEE 1.25 mg daily. 150 patients entered F/U. The F/u analysis of dysmenorrhea and pain scores included only patients who successfully completed treatment (118 and 97, respectively). All received calcium carbonate 1000 mg daily only. F/U bone mineral density (BMD) was determined by serial DEXA scans of Ll-4. Return of symptoms and physical findings were quantified by 4 point Biberoglu and Behrman scales (I =none, 2=mild, 3=moderate, 4=severe). Results:

Return

to menses (median

days)

Gr.1

Gr.11

71

63

F/U Month 12: Mean 2 SE dysmenorrhea score Mean t SE pain score % without return to baseline pain BMD (% change from baseline)

2.6 2 0.2t

2.5 k 0.27

2.2 ? 0.2$ 42 -2.5 t- 0.6**

2.4 5 0.2$ 33 -0.7 '-+ 0.3

Gr.111 Return

to menses (median

~

49*

2.5 5 0.2 2.2 k 0.2$

2.8 z 0.2 2.1 + 0.2$ 43 0.5 2 0.5

Mean t SE dysmenorrhea score Mean t SE pain score % without return to baseline pain BMD (% change from baseline) * PcO.05 vs. Gr.1 and II. t PcO.05 decrease from baseline. ** P
39

0.8 5 0.2 decrease

from

baseline.

$ P
Conclusions: 1) GnRH a + NEtAc alone or with low dose CEE may be safely and effectively administered to symptomatic endometriosis patients for 12 months. 2) Pain relief and BMD preservation are maintained for prolonged periods after therapy completion. 3) Prolonged GnRHa therapy should include add-back to preserve bone density. This work was sponsored in part by a grant from TAP Holdings.

Wednesday,

September

3:00

29,

1999

P.M.

O-209 Novel Conservative Medical Therapy for Uterine Adenomyosis: Danazol-Loaded IUD Therapy. ‘*2,3M. Igarashi, ‘M. Fukuda, ‘Y. Nogami, ‘A. Ando, ‘M. Miyasaka, *Y. Taguchi, 3M. Yoshida. ‘Gunma Central General Hospital, Maebashi, ‘Ohmiya Red Cross Hospital, Ohmiya and 3Fujioka Genera1 Hospital, Fujioka, Japan. Objectives: The effects of pharmaceutical therapies osis are very limited and temporary, and hysterectomy

Abstracts

September

3:45

29, 1999

P.M.

O-210

F/U Month 12:

sso

Wednesday,

Gr.IV ~

48*

days)

infertile women. Thus, the development of a new effective pharmaceutical therapy has been eagerly anticipated. Recent in vitro researches have demonstrated a direct inhibitory action of danazol on endometriotic cells. Therefore, the effects of danazol-loaded IUD on adenomyosis were investigated. Design: A danazol-loaded IUD consisting of a core composed of contraceptive IUD (FD-l), danazol and medical silicone rubber, was prepared. The direct effects of danazol on adenomyosis were investigated by using this danazol-IUD. Materials and Methods: The danazol (approx. 200 mg)-loaded IUD was inserted into the uterine cavity of the adenomyotic uterus of 9 patients resistant to previous oral danazol therapy or nasal LH-RH analog therapy. The effects of the danazol-IUD on adenomyosis were evaluated by transvaginal ultrasonography, MRI, serum CA125, and with uterine sound, and on the basis of symptoms, such as dysmenorrhea, hypetmenorrhea, etc. The IUD was removed after confirming improvement in symptoms and signs 4-7 months later. Serum danazol levels were assayed by HPLC during both danazol IUD therapy and oral (daily 400mg) danazol therapy. Results: Reduction of the thickness of the myometrium and shortening of the uterine sound length were observed within 5 weeks in the 5 patients and within 12 weeks in the other 3 of the 9 patients. The serum CA125 levels decreased within 4 weeks in 3 patients and within 7 weeks in the another patient. In the other 5 patients, its levels remained within normal range since the pretreatment. Dysmenorrhea and hypermenorrhea resolved within 6 weeks in all 9 patients, Two of the 4 infertile women whose danazol-IUD had been removed, conceived 6.2 and 9 weeks after removal of IUD. Systemic side effects, often observed during oral danazol therapy, were never observed during danazol-IUD therapy, because serum danazol levels were undetectable in the danazol-IUD group, whereas they were always over 100 rig/ml during oral danazol therapy. Conclusions: I) Intrautetine insertion of the danazol-loaded IUD was very effective conservative therapy for uterine adenomyosis resistant to oral danazol therapy or nasal LH-RH analog therapy. 2) It cured dysmenorrhea and hypermenorrhea within 6 weeks. 3) It reduced the thickness of the myometrium, the length of the uterine sound within 12 weeks and semm CA125 levels within 7 weeks. 4) Conception could occur after removal of danazol-IUD. 5) Serum danazol levels were undetectable during insertion of danazol IUD, so systemic side-effects were never observed during danazolIUD therapy.

on uterine adenomyis contraindicated in

Stimulation of Eutopic and Ectopic Endometrial Cell Proliferation by Autologous Peritoneal Fluids in Women with Endometriosis is Due to Tumor Necrosis Factor-Alpha (TNFa).D. P. Braun, W. P. Dmowski. Institute for the Study and Treatment of Endometriosis and Rush Medical College, Chicago, IL. Objectives: Endometriosis depends on the capacity of endometrial cells to proliferate in ectopic sites that contain activated macrophages and the products of activated macrophages. The current study compared the effects of autologous peritoneal fluid on proliferation of endometrial cells from fertile controls and women with endometriosis. Design: Single cell suspensions of endometrial cells were cultured in the presence and absence of cell-free autologous peritoneal fluids to assess the effects on in vitro proliferation. Materials and Methods: Endometrial cells were prepared from biopsies of eutopic endometrium from fertile controls (n=5) and eutopic (n= 15) and ectopic (n=5) endometrium from women with endometriosis. Endometrial biopsies were dissociated with a mixture of collagenaselDNAse to obtain single cell suspensions of glands and stroma. Endometrial cells were cultured in the presence and absence of autologous peritoneal fluids for 72 hours prior to incubation with ‘H-Thymidine for 24 hours to assess DNA incorporation, Results: Autologous peritoneal fluids significantly stimulated proliferation of eutopic and ectopic endometrial cells in endometriosis (mean 3HThymidine incorporation in the absence and presence of peritoneal fluids was 6982 counts per minute (cpm) vs 9703 cpm respectively for eutopic endometrium; p=O.O03, and 4347 cpm vs 6018 cpm respectively for ectopic endometrium; p=O.O5). In contrast, autologous peritoneal fluids from fertile

Vol.

72, No.

3, Suppl.

1, September

1999

controls significantly suppressed proliferation of eutopic endometrial cells (mean ‘H-Thymidine incorporation in the absence and presence of peritoneal fluids was 3088 cpm vs 2192 cpm respectively; p=O.O3). The capacity of peritoneal fluids to stimulate endometrial cell proliferation in endometriosis was completely abrogated by treatment with TNFa blocking agents. Conclusions: The capacity of peritoneal fluids to stimulate endometrial cell proliferation in endometriosis appears to be due to TNFa, a cytokine which normally mediates apoptosis. Endometriosis may reflect a selection of endometrial cells that are resistant to apoptosis and that have acquired the capacity to utilize TNFa to stimulate growth in ectopic sites. Wednesday,

September

4:00

29, 1999

P.M.

o-211 Molecular DNA Evidence of Chromosome 17 Alterations in Endometriosis. ‘F. Z. Bischoff, ‘D. D. Nguyen, ‘D. A. Marquqz-Do, 3S. Elias, ‘R. L. Malinak, 4D. Mitchell-Leef, ‘,‘J. L. Simpson. Departments of ‘Obstetrics and Gynecology, and *Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 3Department of Obstetrics and Gynecology, University of Illinois, Chicago; ‘!Southeastem Fertility Institute, Atlanta, GA. Objectives: Genetic predisposition to endometriosis is established: however, the gene(s) and mechanisms involved remain unknown. Although considered a benign disease, endometriosis actually resembles the beginning of the multi-step process toward malignant transformation. Molecular studies of cancer have provided evidence of acquired somatic DNA alterations playing a role in the loss of cell cycle control and DNA repair. These studies have revealed DNA alterations and microsatellite instability, namely loss of heterozygosity in tumor suppressor genes and mutations in mutator genes. Because chromosome 17, 18 and X alterations are frequently observed in premalignant and malignant tissues (i.e., endometrial and ovarian carcinomas), we wanted to determine if similar somatic changes occur in late stage endometriosis. Design: To examine matched endometriotic and normal DNA for loss of heterozygosity and microsatellite instability involving chromosomes 17, 18 and X. Materials and Methods: Under IRB approval, we obtained surgical tissue specimens and a peripheral blood sample from 15 women undergoing extirpation of advanced stage disease. A total of 13 markers, three specific for chromosome 17 (HGH, D17S250, CHRNBl), two specific for chromosome 18 (D18S53 and D18S51) and eight specific for chromosome X (DXS8051, XHPRT, DXS1047, DXS986, DXS1060, DXS1214, DXS1073 and DXS991) were used in PCR amplification. Based on the primer set, PCR was carried out using either conventional, P3’-labeled or fluorescent methods. Loss of heterozygosity was determined by comparing the presence of one or two alleles in endometriotic DNA to matched informative normal cases. Positive evidence of microsatellite instability was based on the addition of one or more novel size alleles in endometriotic DNA as compared to matched normal DNA. Results: Overall, loss of heterozygosity involving chromosome 17 was observed in 4 of 15 (27%) cases. In three cases, loss of heterozygosity with marker HGH was detected. In one case, loss on D17S250 was observed. We observed no evidence of loss of heterozygosity for chromosomes 18 or X nor did we find evidence of microsatellite instability with the 13 markers tested. Conclusion: Our results provide evidence that alterations involving chromosome 17 occur in endometriosis, possibly representing early events toward a transformed phenotype. That microsatellite instability was not observed suggests that mutations in certain DNA repair genes (i.e. PTEN) are either absent or occur late in a multi-step process. Overall, these findings support a multi-step genetic pathway for the development and progression of endometriosis. Supported in part by a research grant from The Women’s Fund for HER (FZB). Wednesday,

September

4:1.5

29, 1999

P.M.

o-212 Expression of Tenascin, Integrins Eutopic and Ectopic Endometrium

FERTILITY

& STERILITY@

(cwp5 & cuvp6) and Catenins in of Women With Endometriosis.

L. A. Puy, C. L. Librach. Department of Obstetrics en’s College Hospital and The Toronto Hospital, Toronto, Ontario, Canada.

and Gynecology, WomUniversity of Toronto,

Objective: Evidences indicate that extracellular matrix (ECM) proteins and integrins are involved in the process of ectopic endometrial cell implantation in endometriosis. The objective of this study was to assess the expression of the ECM protein, tenascin, the cuvp5 & (~vp6 integrins and the cell-cell adhesion molecules, o( and p catenins in this disease. Design: We carried out an immunohistochemical study on the expression of these molecules in eutopic and ectopic endometrium of women with endometriosis, and in normal endometrium of control women, not affected by endometriosis. Material and Methods: We examined proliferative (n = 22) and secretory stage (n = 11) eutopic and ectopic endometrial samples from 33 patients with endometriosis who underwent operative laparoscopy and eutopic endometrium from 10 normal women as controls. Streptavidin-biotin complex immunoperoxidase assay was performed using monoclonal antibodies against tenascin, avfl5 and (~vp6 and the catenins. Immunotaining intensity was assessed and assigned a score 0 (negative), 1 (weak), 2 (moderate) or 3 (strong). The staining intensity score and the percentage of positive cells were evaluated using Student’s t-Test and ANOVA analysis. Results: The immunoreactivity for tenascin, (~v/35 & ruvfl6 and catenins was similar in eutopic endometrium from women with or without endometriosis. Major differences were observed in endometrium from patients with endometriosis. In eutopic endometrium weak tenascin immunoreactivity was detected around the glands during proliferative phase and no staining in the secretory phase. In contrast, diffuse and strong tenascin immunoreactive bands were detected in ectopic endometrium during both proliferative and secretory phases. Immunostaining for (~v/35 & avp6 and catenins was weak in eutopic endometrium during proliferative phase, but moderate during secretory phase. Ectopic endometrium during both proliferative and secretory phases, showed strong immunoreactivity for these molecules. The t-Test and ANOVA revealed significant differences (p
Wednesday,

September

4:30

29, 1999

P.M.

O-213 Urokinase Plasminogen Activator (UPA) and Urokinase Plasminogen Activator Receptor (uPAR) mRNA Expression in Ectopic and Eutopic Endometrium. ‘,‘H. W. Chung, ‘Y. Wen, ‘K. Smith, ‘H. Li, *H. K. Yu, ‘M. L. Polan. Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, California, Department of Obstetrics and Gynecology, Ewha Womans University Mokdong Hospital, Seoul, Korea. Objectives: The pathogenesis of endometriosis is controversial, but the theory of retrograde menstruation is widely accepted. Such retrograde menstruation is physiologic, but menstrual debris does not result in endometriosis in all women. In endometriosis patients, refluxed menstrual debris is more prone to implant and invade peritoneum or ovary possibly through the action of extracellular proteolysis. This proteolytic action may involve plasminogen activators, which play a critical role in the breakdown of extracellular matrix components and basement membrane in the processes of ovulation, implantation, tumor invasion, and metastasis. PAS bind to a specific receptor, uPAR, and are inhibited by Plasminogen activator inhibitor (PAI-1). We have previously reported that endometrium from endometriosis patients showed decreased expression of PAIcompared to endometrium from normal women. The higher expression of uPA and uPAR in ectopic and eutopic endometrium from women with endometriosis might favor increased proteolytic enzyme activity with subsequent endometriotic invasion. Design: uPA and uPAR mRNA expression in ectopic and eutopic endometrium from women with endometriosis and eutopic endometrium from control patients without endometriosis were determined throughout the menstrual cycle. Methods: Endometrial tissue was obtained from 14 patients with severe

S81

endometriosis (ectopic endometrium was also obtained in 6 of these women) and 13 patients without endometriosis undergoing hysterectomy or endometrial biopsy. Stage of endometrial cycle and a diagnosis of endometriosis were confirmed histologically. Total RNA was extracted and reverse transcribed into c-DNA. Quantitative, competitive PCR was performed to evaluate uPA and uPAR expression. Results were analysed by ANOVA. Results: uPA and uPAR were expressed throughout the menstrual cycle in both groups. Endometrium from endometriosis patients showed increased expression of uPA compared to endometrium from normal women in the luteal phase (p < 0.05). uPAR mRNA expression was not statistically different in ectopic and eutopic endometrium from women with endometriosis and normal endometrium. Conclusion: Our results suggest that uterine endometrium from women with endometriosis expresses higher levels of uPA than endometrium from normal women during luteal phase. uPAR expression is similar in both groups and may not be a marker of invasiveness. Endometrium from endometriosis patients may, thus, be more invasive and prone to peritoneal implantation than that from women without endometriosis because of greater uPA enzymatic activity and result in the development of endometriosis. (Supported by NIH Grant HD-31575)

Wednesday,

September

4:45

29, 1999

P.M.

O-214 Modulation of T Lymphocyte Subsets and CD14+, CD36+ or Aminopeptidase (CD13)+ Macrophages Coexpressing HLADR or CD44 Molecules in Peripheral Blood of Patients with Endometriosis. D. Gosselin, D. Gag+, P. Miron, P. Hugo. Division of Research and Development, PROCREA Biosciences, Montreal, Quebec, Canada. Objectives: A number of evidence suggests that several leucocyte populations are altered in the peritoneal cavity of patients with endomettiosis. The present study was aimed at investigating whether the immune abnormalities associated with endometriosis influence the leucocyte subsets found in other sites such as the peripheral blood. Design: The proportion of several subpopulations of T lymphocytes, B lymphocytes, NK cells and macrophages were compared in peripheral blood of patients with endometriosis and normal controls. Materials and Methods: Mononuclear cells were isolated from peripheral blood by centrifugation on Ficoll-Paque and stained with monoclonal antibodies directed against surface markers for T lymphocytes (CD3, CD4, CD8). B lymphocytes (CD20), macrophage subsets (CD14, CD36), NK cells (CD56, CD57) activation markers (CD5, CD16, CD44, CD45RA, CD45R0, CD69, CD 122, HLADR) or cell-surface aminopeptidase (CD 13). The proportion of CD45+ leucocytes expressing these antigens were compared by means of flow cytometric analysis, in patients with surgicallyconfirmed endomehiosis (stage I-II and/or III-IV) and in subjects who underwent surgery for tubal ligation and had no clinical evidence of endometriosis (controls). Results: The proportion of CD3+ and CD8+. but not CD4+ T lymphocytes was found to be significantly impaired in peripheral blood of patients with endometriosis compared to normal controls (P
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Abstracts

MALE

REPRODUCTION Wednesday,

AND UROLOGY

September

2:00

29, 1999

P.M.

o-215 Correlation Between Fine Needle Aspiration Cytology and Testis Biopsy Histology in Infertile Men. ‘M. V. Meng, ‘1. Cha, ‘P. .I. Turek. Departments of ‘Urology and *Pathology, University of California San Francisco, San Francisco, CA. Objectives: Open testicular biopsy remains the standard method for histopathological assessment of spermatogenesis. The need for testis biopsy has been questioned with the increased success of minimally invasive techniques such as fine needle aspiration (FNA) mapping. This study examines the ability of FNA aspiration to assess testicular histologic patterns by correlating “histology” as determined by FNA cytology with formal testis biopsy histology. Design: Retrospective study of infertile, azoospennic men. Materials and Methods: All men who had undergone both diagnostic FNA mapping and open testis biopsy in the evaluation of infertility were identified. Biopsies were assessed by recognized histologic patterns of normal, hypospermatogenesis, early and late maturation arrest, and Sertoli cell-only. FNA cytologic specimens were examined for adequacy and similarly classified. Mixed patterns were also identified that included at least two of the above groups. Results: A total of 77 men had both FNA cytology and testis biopsy findings. The correlation between the two methods in assessing histology and spermatogenesis is found below: FNA Cytology Biopsy

Histology

# Pts.

Normal Hypospermatogenesis Sertoli cell-only Early maturation arrest Late maturation arrest Total

12 13 28 13 11 77

# Agree

% Agree

11 9 21 12 9 68

92 69 96 92 82 88

Discrepancies between FNA and biopsy histology were primarily due to inadequate sampling and evidence of mixed patterns on FNA mapping. Conclusion: FNA aspiration cytology is a minimally invasive method of obtaining testicular tissue for diagnostic purposes. This data demonstrate that FNA cytology can accurately evaluate all histologic groups as classically defined by testis biopsy, and may have potential to replace testis biopsy in the assessment of spermatogenesis.

Wednesday,

September

2:15

29, 1999

P.M.

O-216 A Novel Diagnostic Model for Predicting the Probability of Testicular Sperm Extraction in Men with Nonobstructive Azoospermia. ‘U. I. 0. Ezeh, ‘,aH. D. M. Moore, and ‘I. D. Cooke. ‘.2University Departments of OBGYN and Molecular Biology and 2Biotechnology, Jessop Hospital For Women, Sheffield, UK. Objective: Testicular biopsies for sperm extraction may be associated with potential complications while testicular sperm extraction is successful in approximately 50% of men with nonobstructive azoospermia. Limiting testicular sperm extraction to azoosperrnic men with a high prospect of yielding testicular sperm would therefore be beneficial. A diagnostic testicular biopsy is less invasive than therapeutic biopsy required for testicular sperm extraction and visualisation of testicular spermatids or spermatozoa on testicular histology sections have been shown to be of predictive value. The objective of this study was to assess the value of a diagnostic model based on histological criteria in predicting testicular sperm extraction in men with nonobstructive azoospermia. Design: Logistic regression analysis was applied to a set of variables such

Vol.

72, No.

3, Suppl.

1, September

1999

as biophysical (patients age, maternal and paternal age at birth BMI, testicular volume), endocrine (plasma LH, testosterone and FSH) and morphological (Johnsen score and visualisation of testicular spermatids) profiles in order to derive a diagnostic model based on independent predicting variables. The accuracy of the diagnostic model in retrieving at least one testicular spermatozoon was compared with that of testicular spermatids or Johnsen score alone. Materials and Methods: Forty consecutive patients with nonobstructive azoospermia underwent open testicular biopsy performed general anaesthesia or outpatient analgesia. Sperm extraction was performed using both mincing and in vitro culture of testicular tissues. Biopsy samples for histology were preserved in Bouin’s solution, spermatogenesis was quantified using the Johnsen score method, and whether or not testicular spermatids were visualised on testicular histology were noted. Data on patients’ biophysical, endocrine and morphological profiles were collected prospectively. Results: Model: (i) Predicted score (x) = -5.14 ? [I.01 x J] + [II2 X S] whereby “J” = Johnsen score and “S” = testicular spermatid (ii) Probability of successful sperm extraction (P) = ex divided by 1 + eX whereby ex = exponential function applied to x. The accuracy of prediction of successful testicular sperm extraction was 87% for the diagnostic model compared with 77% for visualisation of testicular spermatids alone and 72% for Johnsen score alone. Conclusion: A diagnostic model based on the independent predictors of testicular sperm extraction represented by Johnsen score and testicular spermatids is more accurate than either of the parameters individually in predicting the likelihood of testicular sperm extraction in men with nonobstructive azoospennia.

Wednesday,

September

2:30

29, 1999

P.M.

O-217 Microsurgical Testicular Sperm Extraction (TESE) in Non-obstructive Azoospermia. ‘S. J. Silber. ‘Infertility Center of St. Louis, St. Luke’s Hospital, St. Louis, MO, USA. Objectives: Men with non-obstructive azoospermia caused by germinal failure can now be treated successfully in many cases using testicular sperm extraction (TESE - a term which we coined in 1993) and ICSI. We have already determined that a prior diagnostic testicle biopsy analyzed quantitatively can predict success or failure of TESE-ICSI 85% of the time. We have also shown that there is a threshold of quantitative sperm production in the deficient testis, below which no sperm will reach the ejaculate (azoospetmia). This threshold phenomenon of spermatogenesis is the reason that in approximately 50% of cases of non-obstructive azoospermia, sperm can be extracted from testicular tissue and be used successfully for ICSI. Design: We now wished to examine the quantitative presence of spermatogenesis in different regions of the testis in azoospermic men, in order to develop a rational microsurgical strategy for the TESE procedure. Materials and Methods: A prospective, quantitative histologic study of testicular tissue in azoospermic men undergoing sperm retrieval for ICSI, with microsurgical removal of large contiguous areas of testicular tissue, and careful analysis of tubular fullness compared to quantitative histology was performed. 45 patients with non-obstructive azoospermia caused by testicular failure underwent diagnostic testicle biopsy prior to a subsequent TESE-ICSI procedure. The diagnostic testicle biopsy was analyzed quantitatively and correlated with the results of subsequent attempts at ICSI. 15 azoospermic men underwent multiple testis biopsy sampling from different regions of the testis in a testicular mapping effort during TESE. An additional 11 men with non-obstructive azoospermia underwent removal of large contiguous strips of testicular tissue with microsurgical dissection and evaluation of tubular dilation. Results: The 71 men with non-obstructive azoospermia caused by germinal failure had a mean of 0 to 3 (3 cases over 3, but never greater than 6) mature spermatids per seminiferous tubule seen on a diagnostic testicle biopsy. This compared to 17 to 35 mature spermatids per tubule in men with normal spermatogenesis and obstructive azoospermia. This suggested that a certain threshold of quantitative spermatogenesis had to be exceeded (about 3 to 6 mature spermatids per tubule) in order for some sperm to “spill over” into the ejaculate. In greater than half of cases, there is some sperm in the testis despite none in the ejaculate. Testicular “mapping” by multiple biopsy

FERTILITY

& STERILITY@

revealed a basically diffuse quantitative distribution of spermatogenesis, in 13 out of 15 non-contiguous cases. In 2 cases, however, there was just a rare tubule with sperm in only 1 or 2 out of many biopsies. In the 11 such cases that underwent removal of contiguous strips of testicular tissue, the distribution of spermatogenesis, however sparse, was always diffuse. Conclusion: Incomplete testicular failure appears to involve a sparse multi-focal distribution of spermatogenesis throughout the entire testicle, rather than a patchy, or local distribution in just a few areas. This can often be observed under the operating microscope. Therefore, in most cases only one modest size biopsy is necessary. However, in a small number of cases where sperm are present, the distribution is so sparsely diffused (13%) that a microsurgical approach is absolutely critical. Wednesday,

September

2:45

29, 1999

P.M.

O-218 Identification of Meiotic and Post-meiotic Gene Expression in Testicular Tissue of Patients Histologically Classified as Sertoli Cell Only. ‘.‘M. S. Ricci, l,‘N. B. Hecht, 3J. E. Tomaszewski, i,‘P. Patrizio. ‘Center for Research on Reproduction and Women’s Health; ‘Department of Obstetrics and Gynecology, School of Medicine; 3Department of Pathology, Hospital University of Pennsylvania. Objective: Intracytoplasmic sperm injection (ICSI) has helped men with a variety of spermatogenic disorders to father their own children. Current assisted reproductive technologies cannot help men with histological diagnosis of Sertoli cell only (SCO) or pre-meiotic maturation arrest. This study used a molecular biology approach to determine whether meiotic and post-meiotic germ cell gene products could be detected in biopsies from SC0 patients. The germ cell specific gene, lactate dehydrogenase C4, was used as a marker for meiosis, and the nuclear condensation proteins, transition protein-l and protamines-l and -2, were used as markers for post-meiotic gene expression. Design: Correlation between results of open testicular biopsies divided among the IVF lab for sperm extraction, the pathology lab for histological analysis and the molecular lab for genetic marker probing. Materials and Methods: A total of 6 testicular biopsies were examined. Five biopsies were from patients diagnosed with non-obstructive azoospermia, 2 had a histological diagnosis of severe hypospermatogenesis and 3 were diagnosed as SCO. One biopsy, from a patient with obstructive azoospermia, served as a control. At the time of the biopsy, the specimens were divided into three parts, one for the IVF lab, one for histology and one for molecular analysis. A minimum of 30 seminiferous tubules were analyzed for each biopsy. Germ cell specific mRNAs were detected by extracting total RNA from each biopsy for use in either northern blotting or RT-PCR procedures. Results: In the IVF lab, spermatozoa were identified in 2 of the 6 biopsies (control and severe hypospermatogenesis); in the histology lab, one additional biopsy showed spermatogenesis with maturation arrest at the primary spermatocyte stage (Johnsen score 2.4 2 0.7) and 3 were diagnosed as SC0 (Johnsen score 2). Molecular analysis showed 5 out of 6 biopsies contained mRNA from meiotic and post-meiotically expressed genes, two of which were labeled as SC0 by the histology lab and in one which was labeled as pre-meiotic maturation arrest. Conclusions: This is the first study to document the presence of differentiating germ cells in biopsies of men diagnosed SC0 by testing for meiotic and post-meiotoically expressed genes. These molecular markers provide a powerful screening tool to identify spermatogenesis in human testicular tissue. The absence of these molecular markers strengthens histological diagnosis to better help the physician in counseling the infertile couple about their reproductive choices. Improved quantitation of these markers will provide good estimates of spermatid number present in biopsies from men previously considered irreversibly sterile. M.S.R. was supported by NIH training grant T32HD07305. Wednesday,

September

3:00

29,

1999

P.M.

O-219 Transplantation of Human Spermatogonia into the Seminiferous Tubules (STs) of Animal Testicles Results in the Completion of the Human

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Meiosis and the Generation of Human Motile Spermatozoa. ‘N. Sofikitis, ‘Y. Mio, ‘Y. Yamamoto, ‘I. Miyagawa. ‘Department of Urology, Tottori University School of Medicine and ‘Mio Fertility Clinic, Yonago, Japan. Objectives: Nonobstructed azoospermic (NOA) men negative for spermatozoa in the therapeutic testicular biopsy (TTB) material do not have a hope to father their own children. We evaluated the potential of spermatogonia (SRG) of the latter men to differentiate to round spermatids (RSs) or spermatozoa into the STs of recipient animals. Design: Animal testicles were evaluated for the presence of human spermatozoa 5 months post-transplantation (P-T) of human SRG and primary spermatocytes (PSs) into these animal testicles. Materials and Methods: TTB material of 18 NOA men with spermatogenie arrest at the SRG or PS stage was minced and filtered. SRG and PSs were recovered and transplanted microsurgically into the STs of immature immunosuppressed animals (one animal/man; 10 nude rats and 8 SCID mice). Ten STs per recipient testis were microinjected with human SRG/ PSs. Five months P-T, testicles, epididymes, and vas deferens of each recipient animal were evaluated for the presence of human spermatozoa. Fluorescent in situ hybridization (FISH) techniques using specific probes against human X-, 18-, and Y-chromosomes were also performed in some of the round germ cells found in the testicles of the recipient animals. The probes used could not react with/label rat or mouse chromosomes. Results: Human spermatozoa were found in the testicles, epididymes, and vas deferens of 3 nude rats and 2 SCID mice. Within the group of the latter five animals the % human motile spermatozoa ranged from 0% to 19% in testicular samples, 0% to 83% in the epididymal cauda samples, and 0% to 88% in the vas deferens samples. FISH techniques in round germ cells demonstrated human PSs (4n-DNA), human secondary spermatocytes (2nDNA-haploid cells), and human RSs (n-DNA-haploid cells). Transmission electron microscopy demonstrated human RSs (mitochondria are scattered in the cytoplasm) in contact with rat RSs (mitochondria are located peripherally only). Conclusions: Human spermatogonia transplanted into animal STs can complete meiosis and differentiate up to the spermatozoon stage. Human spermatozoa generated within animal STs have capacity to expose forward motility after epididymal passage. Rat or mouse Sertoli cells can regulate human spermatogenesis. Rat or mouse epididymis can induce human sperm maturation process. Transplantation of human spermatogonia into recipientSTs combined with assisted reproduction programs may serve as a unique mode of treatment for NOA men with premeiotic block in spermatogenesis.

Wednesday, September 29, 1999 3:45

P.M.

O-220 Pre-operative Semen Analysis as a Predictor of Seminal Improvement and Pregnancy Rates Following Varicocelectomy. ‘T. G. Matkov, ‘L. A. Levine, ‘M. Zenni, ‘J. I. Sandlow. ‘Department of Urology, Rush-Presbyterian-St. Luke’s Medical Center, Chicago, IL and 2Department of Urology, University of Iowa Hospitals and Clinics, Iowa City, Iowa. Objectives: Improvement in semen quality and pregnancy rate after varicocelectomy (Vx) in patients with clinical varicoceles has been well documented. The recent advent of procedures such as intrauterine insemination (IUI) and intra-cytoplasmic sperm injection (ICSI) may, however, alter the indications for Vx. Given these new techniques, stratification of patients based on potential benefit of Vx would be desirable. Design: This study examines the pre-operative total motile sperm count (TM) as a potential predictor of who would benefit most from Vx. Materials and Methods: Eighty-four consecutive subfertile patients with clinical varicoceles were stratified according to their preoperative TM (grade A, B, or C). Grade A (normal or near-normal) was defined as greater than 20 million, Grade B was 5 to 20 million, and grade C was less than 5 million total motile sperm. Following Vx, semen analyses were obtained, the TM was calculated, and pregnancy results were recorded. Results: Patients who began with TM grade A (n=2.5) showed a mean absolute increase in TM of 52.6 million with a mean relative change of 97%. Patients who began with TM grade B (n=28) had a mean absolute improvement of 39.9 million with a mean relative improvement of 440%. Patients who began with TM grade C (n=31) showed a mean absolute improvement

S84

Abstracts

of 10.5 million with a mean relative improvement of 470%. Statistical analysis of this data reveals that both TM groups A and B had significantly greater absolute improvement than grade C (p
3 were with IUI (8%), and 18 were with IVFKSI

(50%). Twelve of the

fifteen pregnancies with natural conception (80%) were in post-op grade A patients. Conclusions: The urologist’s goal is to improve the quality of sperm sufficiently to allow the couple to conceive with the least invasive and most economical method. Our data reveals the likelihood of patients with moderate asthenospermia (grade B), to be improved to normal or near normal (grade A) after Vx. This would theoretically allow them to conceive on their own, or with less invasive techniques. The overall pregnancy rates, however, do not reflect this trend. This is probably secondary to the early use of invasive techniques for reproduction in our patients. The twelve natural pregnancies in post-op group A patients is impressive, but nine pregnancies were obtained using IVF/ICSI. With the ubiquitous use of IVF/ICSI in infertility clinics, the true incidence of potential improvement of each of the groups is unknown, with IVFlICSI acting as the equalizer.

Wednesday, September 29, 1999 4:00

P.M.

o-221 Role of Oxidative Stress and Interleukind in Varicocele Patients R. K. Sharma, F. F. Pasqualotto, H. Kobayashi, J. A. Daitch, B. H. Hendin. ‘D. R. Nelson, A. I. Thomas, Jr., A. Agarwal. Center for Advanced Research in Human Reproduction & Infertility, Departments of Urology and ‘Biostatistics & Epidemiology, The Cleveland Clinic Foundation, Cleveland, OH. Objectives: Varicocele affects sperm quality thereby affecting male fertility. The exact mechanism of fertility disturbance induced by a varicocele is unknown. Both varicoceles and cytokines are involved in the production of reactive oxygen species (ROS). Cytokines released by various immunocompetent cells in the male urogenital tract are capable of influencing sperm function and fertility. It is known that IL-6 levels are elevated in the seminal plasma of infertile men. We measured the levels of ROS, nonenzymatic total antioxidant capacity (TAC), a composite ROS-TAC score, and interleukin-6 (IL-6) levels in infertile patients with varicocele comparing them to normal healthy men. Design: Prospective study. Material and Methods: Semen specimens from 39 infertile patients with varicocele were compared for semen characteristics, levels of ROS, IL-6 and ROS-TAC score. Sixteen normal healthy men were included as controls. ROS production in the semen specimens was measured by the chemiluminescence assay and the results were expressed as Log (ROS + 1) X IO6 counted photons/minute/20 X lo6 sperm. Total antioxidant capacity was measured in the seminal plasma and expressed as molar Trolox equivalent. A composite ROS-TAC was calculated. A score lower than 45 was considered as abnormal. IL-6 levels were measured in the seminal plasma by an enzyme-linked immunosorbant assay (ELISA) and results were expressed as pg/mL of seminal plasma. Results: Infertile varicocele patients had higher levels of ROS (1.96 2 0.16) compared to controls (1.29 ? 0.27; p = 0.04). The TAC levels and ROS-TAC scores were lower in the varicocele group (1112.48 t 69.81 Vs 1494.99, p = 0.09; 34.90 t 2.2, and 48.08 2 2.2; p = 0.004) respectively. High levels of IL-6 were seen in-patients with varicocele compared to controls [median and 25%, 75% interquartile range; patients: 131.61 (43.87, 267.61) versus controls: 4.39 (0, 87.74; p
Vol. 72, No. 3, Suppl. 1, September 1999

This work Foundation.

was supported

by a research

grant

from

the Cleveland

SOCDETY FOR ASSISTED REPRODUCTIVE TECHNOLOGY

Clinic

Wednesday, September 29, 1999 2:00 P.M.

Wednesday, September 29, 1999 4:1.5

P.M.

SART Prize o-222

O-223

Measurement of the Oxidation Metabolite S-Epiprostaglandin F,, in Media Incubated with Sperm. R. W. Ke, C. Sommerfelt, R. Ahokas. Department of Obstetrics and Gynecology, University of Tennessee, Memphis, Memphis, TN. Introduction: The generation of spermicidal cytotoxic end products by oxygen-free radicals is believed to be relevant in human male infertility. Excessive production of these reactive oxygen species may cause peroxidative damage to the cell lipid membranes of spermatozoa and must be minimized during sperm preparation for assisted reproductive techniques. While the presence of reactive oxygen species in semen has been well documented, direct measurement of cell membrane damage is difficult and nonspecific. Therefore we set out to measure the accumulation of free 8-epiprostaglandin FZa (8-epi-PGF,,), the end product of plasma membrane bound arachadonic acid oxidation, in a in vitro fertilization (IVF) sperm preparation. This represents a novel, specific determination of possible sperm oxidative damage in artificial culture environments.

New Look at Endometrial Echogenicity (EC): Objective Computerassisted Measurements Predict Endometrial Receptivity in IVF-ET. R. Fanchin, C. Righini, 0. Sefrioui, N. Frydman, C. Torrisi, N. MdCe, F. Olivennes, R. Frydman. Department of Obstetrics Gynecology and REI, Hapita A B&l&e, Clamart, France. Objective: Numerous investigators have attempted to correlate endometrial EC with receptivity to embryo implantation in IVF-ET. Divergent findings, poor precision, and operator-dependent variability, however, have prevented the finding of a consensus. Further, endometial EC has been shown to evolve from the base to the surface of the endometrium and to reflect its histological changes (Grunfeld et al. Obstet Gynecol, 1991;78: ZOO). To clarify this issue, we analyzed objectively the possible predictive value of end follicular phase endometrial EC on IVF-ET outcome.

-60 e 8

200

- 30 B is B LO

6-S@PGFZa (PW)

M

31-40

4150

endometial

3

Hours

16

Design: In vitro measurement of culture media levels of free I-epi-PGF,, over time. Materials and Methods: Complete ejaculate specimens from five infertile men were prepared through a 3-hour swim-up preparation into human tubal fluid with 0.3% human serum albumin (HTF, Irvine, Santa Ana, CA, St. Louis, MO) media. The swim-up specimen was washed twice and resuspended to a concentration of 1.5 million cells/cc. This sample was then divided into two equal aliquots. After 3 hours of incubation at room temperature, the first aliquot was centrifuged and 1 cc of the supernatant removed for 8-epi-PGF,, determination. The second aliquot was incubated for 16 hours and then assayed for 8-epi-PGF,,. Concentration of free 8-epi-PGF,, was measured by enzyme linked immune assay (Cayman Chemical Co., Ann Arbor, MI). Results: Over time, four of five men demonstrated increased levels of 8-epi-PGF,, between 3 hours and 16 hours (see figure). The fifth patient exhibited no change in lipid peroxidation metabolite. Conclusion: In this preliminary study, it appears that metabolites of lipid peroxidation accumulate in sperm culture over time. This would suggest an accumulation of oxidative damage within the artificial culture environment and may prove useful in determining optimal parameters for sperm performance at time of in vitro fertilization.

FERTILITY

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& STERILITY@

51-60

61-70

EC (%)

Design: Prospective study on computerized estimation, at hCG administration, of endometrial EC and IVF-ET outcome. Materials and Methods: 221 women undergoing 228 GnRH-a and FSW hCG cycles for IVF-ET were studied. To limit the possibility of confounding factors in the analysis of our results, we selected only women aged <38 years (range, 25-37), whose uteri were morphologically normal and who had 22 grade A or B embryos transferred. On the day of hCG administration, 2-minute sagital uterine scans were obtained by US with a 7.2 MHz vaginal probe (Siemens Elegra, France) and digitized with an image analysis system (IBDP, Paris, France). Endometrial EC was calculated semiautomatically as the ratio of the extent of the submyometrial hyperechogenie transformation over the whole endometrial surface. Cycles were arbitrarily sorted into 6 groups according to endometrial EC measurements: <30% (n=34), 31-40% (n=37), 41-50% (n=37), 51-60% (n=55), 6170% (n=37), or >70% (n=28). Results: Patients included in all groups were similar in regard to population characteristics, ovarian response to COH, and embryology data. Overall clinical pregnancy and implantation rates were 33% and 15%, respectively. As illustrated, pregnancy rates (59%. 57%. 35%, 20%. 16%, and 1 l%, respectively, closed bars) and implantation rates (35%, 23%, 17%, 6%, 7%. and 3%, respectively, open bars) fell progressively from the low EC group to the high EC group. Conclusions: 1. As this investigation was strictly controlled for confounding variables and EC was assessed objectively by a computerized system, its results confirm and extend prior observations that high endometrial EC values are associated with poor IVF-ET outcome. Therefore, we suspect that reports that failed to find such association were probably flawed by the poor quality of measurements. 2. It is possible that hyperechogenic endometrial patterns reflect altered endometrial receptivity due to hastened secretory transformations of the endometrium (Fanchin et al, Fertil Steril, 1999;71: 174).

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Wednesday, September 29, 1999

M. C. Schiewe. California Fertility search, Santa Monica, CA 90404.

2: 15 P.M. O-224 A Successful Human Oocyte Cryopreservation Regime: Survival, Implantation and Pregnancy Rates are Comparable to That of Cryopreserved Embryos Generated From Sibling Oocytes. D. S. Yang, P. L. Blohm, L. Cramer, K. Nguyen, Y. L. Zhao, K. L. Winslow. Florida Institute for Reproductive Medicine, Baptist Medical Center, Jacksonville, FL. Objectives: We reported last year (Yang et al., Fertility & Sterility Vol. 70 suppl. 1. 1998, P S86) that our specially developed oocyte cryopresetvation regime significantly improved the cryosutvival rate of human fresh and unfertilized MB oocytes compared to oocyte cryopreservation utilizing traditional embryo cryopreservation protocol. To further assess the efficiency of the protocol, this study was designed to examine the survival, fertilization, implantation, and pregnancy rates of cryopreserved oocytes based on cumulated clinical cases, and to compare these rates to those of sibling fresh oocytes and cryopreserved embryos obtained from the same cycles. Design: Prospective descriptive clinical study. Oocytes from donors and patients were used for the study. Materials and Methods: One hundred and twenty oocytes (fifty from donors out of 10 fresh cycles and seventy from patients out of 7 fresh cycles) collected after controlled ovarian stimulation went through the oocyte cryopreservation process. The oocytes were preequilibrated in 1.5M PROH while cooling from 37°C to 32°C over 5 minutes, and then frozen in 1.5M PROH with 0.2M sucrose. The oocytes were then stored in liquid nitrogen for 1 to 12 months. After thawing, the cryoprotectants were removed by stepwise dilution and the survived oocytes were cultured for 1 to 2 hours before ICSI. Fertilization (2PN) was confirmed 16 to 18 hours after ICSI and embryo transfer was carried out next day. The protocol for preparation of the endometrium was identical to that used for cryopreserved embryo transfer. Positive pregnancy tests were confirmed by ultrasound examination four weeks after embryo transfer. Results: The outcome of cryopreserved oocytes is illustrated on the table. Oocyte origin

Thawing cycles (n)

MI1 thawed 00

Survival (%)

Donor Patient Total

9 8 17

50 70 120

64.0% 52.9% 57.5%

Oocyte origin Donor Patient Total

ET cycle 0-d 9 8 17

# Embryos trans. 24 27 51

ICSIed (n) 31 37 68 Clinical preg. (%) 5 (55.6%) 2 (25.0%) 7 (41.2%)

# of 2PN (%) 26 (83.9%) 28 (75.7%) 54 (79.4%) Implantation (%) 8 (33.3%) 3(11.1%) 11 (21.6%)

Two sets of healthy twins have been born, and 4 additional ongoing pregnancies have been established. The fertilization rate of cryopreserved oocytes (79.4%) is similar to that of fresh sibling oocytes obtained from the same cycle (85.2%, 150/176, P=O.823). The survival, implantation and pregnancy rates of cryopreserved oocytes are comparable to those of frozen embryos (60.5%, 57.1% and 18.5%, respectively, P>O. 1) generated from the same cycles. Conclusions: In this study, cryopreserved oocytes achieved fertilization, survival, embryo development and pregnancy rates comparable to their fresh sibling oocytes and cryopreserved embryos. It is thought that the increased equilibration temperature and sucrose concentration in freezing solution is responsible for the successful oocyte cryopreservation. These data suggest that the human oocyte cryopreservation regime reported here is as efficient as our current embryo cryopreservation program.

Wednesday, September 29, 1999 2:30 P.M. o-225 Improved Thawed

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Pregnancy Rates Using Assisted Hatching on Day 3 FrozenEmbryos. G. E. Ringler, R. P. Marts, A. L. Stein, J. M. Varygas,

Abstracts

Associates/Institute

for Fertility

Re-

Objective: Cryopreserved embryos undergo physiochemical changes that could inhibit blastocyst hatching by altering zona pellucida properties and/or the embryonic production of a zona lysin. Since frozen embryo transfer (FET) pregnancy rates are typically low (>20%), this study was conducted to determine if FET success could be enhanced by applying assisted hatching (AH). Design: In 1998, a prospective study was conducted on 122 frozen embryo transfer cycles. Patients were arbitrarily assigned to AH (n=66) or no AH treatment (n=56) prior to transfer of cleavage stage embryos (Day 3) derived from patient (n=47 and 38, respectively) or donor (n= 19 and 18, respectively) egg sources. Treatment selection was not based on any specific criteria (e.g., patient history, age, zona thickness or embryo quality). Materials and Methods: Good to excellent quality pronucleate and 2- to 8-cell embryos were cryopreserved by a 3-step dilution into 1.5M propylene glycol + 0.2M sucrose (20 min interval; mHTF + 2O%SSS stock solution), placed in 0.5ml straws, loaded into a Planara Kryo 10 cryogenic unit at 20°C and cryopreserved (-2.0”C/min to -5°C seeded, -0.3”C/min to -30°C plunged into LN,). Straws containing embryos were thawed at room temperature (20°C) for 30 set and then submersed into a 35°C water bath for 30 set before expelling contents into 3ml of 0.2M sucrose-mHTF/ lO%SSS solution at 20°C for 5 min. Embryos were then diluted into O.lM sucrose solution for 5 min, washed (2X, 5 min each) in mHTF + lO%SSS and then placed into 0.1 ml microdrops of D3 medium (Conception Tech., San Diego, CA) + 10% SSS under oil in a 5% CO> humidified atmosphere at 37°C. Embryos were cultured 24-48hr up to the 8-cell stage where control transfers were performed or AH, using acidic Tyrodes solution, was done before transfer. All FET cycles involved hormone replacement therapy, with the FET performed on cycle Day 19. Results: Our overaIl results indicate that assisted hatching of frozenthawed embryos significantly enhanced clinical pregnancy rates (fetal sac with cardiac activity) from 16.1% in FET-controls to 40.9% in the FET-AH group. Ongoing pregnancy rates were more than doubled when AH was applied to cycles using patient (34%) or donor egg (36.8%) sources compared to no AH treatment (13.2% or 16.7%, respectively). 549 embryos were thawed (423-2PN, 126-cleaved) and 430 survived post-thaw (78.3%). Of the 419 embryos transfened 37 implanted (8.8%). While the overall implantation rate was higher (P
Wednesday, September 29, 1999 2:45

P.M.

O-226 Improved Pregnancy and Implantation Rates Following Transfer of Day 5 Embryos Compared with Day 3 Embryos in Non-Selected InVitro Fertilization Patients. ‘D. Marek, ‘M. Langley, ‘D. K. Gardner, ‘N. Confer, ‘L. Cram, ‘L. Underwood, ‘K. M. Doody, ‘K. J. Doody. ‘Center for Assisted Reproduction, Bedford, TX and *Colorado Center for Reproductive Medicine, Englewood, CO. Objective: Previous investigation has demonstrated that extended embryo culture to blastocyst (BL) can be applied to selected “good prognosis” patients with resulting high implantation and pregnancy rates (PR). The purpose of this study is to evaluate the non-selective application of extended embryo culture on in-vitro fertilization (IVF) outcome. Design: A retrospective analysis of embryo transfer results from January 1, 1997 through December 31, 1998.

Vol. 72, No. 3, Suppl. 1, September 1999

Materials and Methods: In 1997 and early 1998, embryo transfers (ET) were performed on Day 3. Beginning early 1998, extended embryo culture to Day 5 in sequential media (Sl, S2, IVF Science) was performed in all patients undergoing IVF (good and poor prognosis). For ET on Day 3, multi-cell (MC) embryos were cultured in Human Tubal Fluid (Irvine) + 12% Synthetic Serum Substitute (Irvine). For Day 5 ET, embryos were cultured for 48 hours in Sl media followed by 48 hour culture in S2 media. Results: Age <3.5 Day 3 Day 5

Transfer Day: Mean Patient Age: Retrievals/Transfers: Total Oocytes/Z Pronuclear: Pregnancies (+hCG)/Clinical: Biochemical/Miscarriages: PlUClinical Rate Per TVR (%): PRfClinical Rate per ET (%): Total Embryos/Mean ET: Total BL/Morulah4C for ET: Patients/No. Embryos for cryo: No. Clinical Sacs (l/2/3/4): Implantation Rate (%):

31.4 2421235 3354/1740 134/109 12/13

30.9 1641155 229311331 107/82 11114

36.9 1651158 1765/890 70152 6112

37.1 124/l 14 1392f786 65148 918

55.4145.0 57.0146.4 692/2.9** 0181684

65.U50.0 69.0152.9 375/2.4** 304/53/18

42.4131.5 44.3132.9 492/3.1** 0141488

52.4138.7 57.0142.1 294/2.6** 193/85/16

911505 51/49/15/1 29.5**

791346 50/36/8/38.9**

37/200 36/16/10/l 20.7*

40/137 3111 l/8/28.2+

Age >39 Day3 Day 5

Transfer Day: Mean Patient Age: Retrievals/Transfers: Total Oocytesl2 Pronuclear: Pregnancies (+hCG)/Clinical: Biochemical/Miscarriages: PR/Clinical Rate per TVR (lo): PR/Clinical Rate per ET (%): Total Embryos/Mean ET: Total BL/Momla/MC for ET: Patients/No. Embryos for cryo: No. Clinical Sacs (l/2/3/4): Implantation Rate (%):

Age 35-39 Day 3 Day 5

Total Day 3

Day 5

41.7 25123 2221143 817 110

34.9 4771463 565812906 2201171 22J27

34.2 3 131292 390712260 180/137 21122

22.9114.3 32.0/28.0 22.9114.3 34.8130.4 214/3.1 6913.0 o/2/2 12 3812615

46.1135.9* 47.5/36.9** 1398/3.0** 01411394

57.5/43.8* 61.6/46.9** 738/2.5** 5351164139

131/715 100/68/27/2 23.3**

121/500 91/50/16/32.4**

42.3 70170 5391276 1600 412

3/10 7/3/2/o 8.9

2117 4/3/o/14.5

Significant difference using Fisher’s Exact or “t” tests for Day 3 compared to Day 5: *P < .05; **P < .Ol. Conclusion: These results demonstrate the feasibility of applying extended embryo culture to IVF couples in a non-selective manner without affecting cancellation rates. The data show that overall, extended embryo culture was associated with a significant increase in pregnancy rates, a significant increase in implantation rates and a significant decrease in number of embryos transferred. The multiple implantation rates associated with patients <35 years of age warrants consideration of single blastocyst transfers for this group.

Wednesday, September 29, 1999 3:00

P.M.

O-227 PCR Detection of Chlamydia Trachomatis in Semen Samples from a Semenbank. ‘S. Repping, ‘Y. Pannekoek, ‘J. W. A. de Vries, ‘L. Spanjaard, ‘5. Dankert, ‘F. van der Veen. ‘Center for Reproductive Medicine, Department of Gynecology and Obstetrics, Academic Medical Center Amsterdam, The Netherlands. *Department of Medical Microbiology, Academic Medical Center Amsterdam, The Netherlands. Objective: Chlamydia trachomatis infections are now recognized as the most prevalent sexually transmitted disease in the United States and Europe. ‘To reduce the risk of spreading this infection through donor insemination,

FERTILITY

& STERILITY@

the ASRM recommends urethral swab or urine testing of semen donors, repeated at six-months intervals or more frequently if clinically indicated. To our knowledge it is unknown whether negative test outcomes of these specimens are representative for semen. We therefore developed a controlled polymerase chain reaction (PCR) assay to detect C. trachomatis in donor semen samples. Since C. truchomatis infections in men are mostly asymptomatic we used our test on every semen sample in our bank instead of applying the recommended six months interval. Design: Ejaculates, cryopreserved between 1989 and 1999 from 80 regular semen donors from our institution were analyzed as follows: Two separate samples from the same straw were processed for the assay. The first sample was directly subjected to DNA extraction. The second sample was, prior to DNA extraction, spiked with bacteria and served as a control. DNA was extracted using a silica-based purification procedure and eluted in water. PCR was carried out targeting the C. trachomatis plasmid. PCR product was analyzed by agarose gel electrophoresis, blotted and detected by hybridization using an internal probe. The sensitivity of the PCR assay was determined using repeatedly negative tested semen pools and single semen samples to which a dilution line of bacteria was added. Results: Using a dilution line of bacteria in semen, the detection limit of our PCR assay corresponded to 5 bacteria per ~1 semen. Among 259 ejaculates derived from 80 donors tested, 11 ejaculates of 4 donors were positive. Donors were not contiguously positive or negative for C. trachomatis. Data on the accuracy of urine LCR compared with semen PCR will also be presented. Conclusion: We recommend testing of every semen sample used for AID instead of urethral and urine testing of semen donors.

Wednesday, September 29, 1999 3:45

P.M.

O-228 Nuclear-Cytoplasmic Interactions Determine Apoptosis in Preimplantation Mouse Embryos. ‘.‘D. L. Keefe, ‘,‘L. Liu, *J. Trimarchi. ‘Laboratory for Reproductive Medicine, Department of Obstetrics/Gynecology, Women & Infants Hospital and Brown University, Providence, RI and the ‘Marine Biological Laboratory, Woods Hole, MA. Objectives: Abnormal human preimplantation embryos undergo apoptosis in culture. Although apoptosis classically is defined by nuclear condensation and fragmentation, mitochondria mediate the commitment to apoptosis in many tissues. We sought to test the hypothesis that mitochondrial dysfunction is involved early in apoptosis by attempting to rescue apoptotic mouse embryos by nuclear transfer. Apoptosis was induced by treatment with the uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP). Materials and Methods: Mouse zygotes were collected in Hepes-buffered KSOM (HKSOM) from oviductal ampule at 20-21 h after hCG injection and mating, removed of cumulus cells, washed and incubated in KSOM at 37°C in 7% CO2 and humidified air. Shortly after incubation, zygotes were treated with 750 nM FCCP. Treated zygotes were assessed for mitochondrial function with JC-I and confocal or fluorescence microscopy. Micromanipulation was performed in HKSOM with cytochalasin B and nocodazole. Pronuclear exchanges between normal zygotes served as nuclear transfer controls. Pronuclei from FCCP treated zygotes (FCCP PN) were transferred to untreated zygotic cytoplasts. Alternatively, normal zygotic pronuclei were transferred to cytoplasts derived from FCCP treated zygotes (FCCP cyto). Reconstituted zygotes were obtained by electrofusion of the nuclei-cytoplast couplets at l-l.5 h after the end of FCCP treatment. Reconstituted zygotes were cultured in vitro for 3 days. They were checked for cleavage at day 2, development to morula at day 3, and morula and blastocyst at day 4. Apoptotic cells and cell number of blastocysts were assessed by TUNEL assay, with In Situ Cell Death Detection Kit (Boehringer) and counterstaining with PI. Results: FCCP inhibited cleavage in 64% of zygotes, reduced development to morula and blastocysts (to 36%), depleted mitochondrial membrane potential and led to blebby degeneration by 24 h after treatment. 100% of nuclear transfer control zygotes cleaved by day 2 and 95% developed to morula or blastocyst by day 4. Transfer of treated PN into normal cytoplasts (FCCP PN) improved cleavage at day 2 (62%) and development to morula and blastocyst (60%) of reconstituted zygotes (P
ss7

observed at day 3, compared to other groups. Surprisingly, after normal PN were transferred into FCCP cytoplasm (FCCP cyto), reconstituted zygotes showed significantly (PcO.01) higher rates of cleavage (75%) and momla and blastocyst formation (72%), compared to FCCP treated zygotes, although it was lower than the untreated, nuclear transfer controls. The number of apoptotic cells in blastocysts derived from either FCCP cyto or FCCP PN groups was lower than from FCCP treated zygotes (P
September 4:00

29,

1999

P.M.

O-229 Chromosome Analysis of Testicular Spermatozoa Retrieved from Patients with Klinefelter Syndrome. ‘Jacob Levron, 3Ayala Aviram, *Igal Madgar, ‘Gil Raviv, 3Gad Barkai, 3Boleslav Goldman, ‘Shlomo Mashiach, ‘Jehoshua Dor. ‘IVF Unit, Department of Obstetrics and Gynecology, ‘The Unit of Andrology and 3The Center for Human Genetics, The Sheba Medical Center, Tel Hashomer, Israel. Objectives: Klinefelter syndrome is one of the most common chromosomal abnormalities found in newborns. These patients who were considered eternally infertile until recently, can reproduce now using testicular sperm extraction (TESE) and in vitro fertilization (IVF). There is, however, a major concern about the risk of transmitting sex chromosome abnormalities to the next generation during assisted reproduction in these patients. Surprisingly, the few pregnancies that have been reported so far worldwide, including in our center, indicated birth of normal babies only. In the light of these observation we have studied the chromosome constitution of testicular spermatozoa from patients with Klinefelter syndrome and the probable risk of aneuploidy after their use in IVF. Design: Couples with Klinefelter syndrome that underwent TESE and IVF in our center and became pregnant gave us the permission to analyze part of their frozen testicular samples for sperm chromosomes. Materials and Methods: Over 100 metaphases in the peripheral blood were analyzed to confirm the state of non-mosaic 47:XXY karyotype. The spermatozoa were collected from the testicular tissue with an ICSI pipette, fixed on a slide using acetomethanol solution and analyzed by Fluorescent In-Situ Hybridization (FISH) for chromosomes X, Y and 18. Results: 92.5% of the sperm analyzed were found to be normal with a sex ratio of 0.45:0.55 Xl8 to Yl8 respectively. The few abnormal cells that were found represented errors in meiosis-I and -11 (XY 18 and XX1 8NY 18 respectively). Due to religious restrictions the patients did not agree for genetic testing of the babies during pregnancy. Two normal healthy males and one normal healthy female were born and another pregnancy is still ongoing. Conclusions: Our findings may indicate that the spermatogonia populating the testis of males with non-mosaic Klinefelter syndrome are of normal 46XY karyotype and therefore produces normal sperm population. The presence of few chromosome abnormalities among the cells tested here is probably the consequence of meiotic errors of normal spermatocytes, as seen in other patients with testicular failure. It appears therefore, that the risk of transmitting chromosome abnormalities during assisted reproduction in patients with Klinefelter syndrome do not exceed the risk found in the general patients population with severe male factor infertility. We speculate that during multiplication of the embryonic primordial germ cells there are mitotic “correcting errors” that produce normal germ lines that survive in the testis to produce normal mature sperm during adulthood. Wednesday,

September 4:15

29,

1999

P.M.

O-230

Mosaicism in the Human Reimplantation Embryo. M. M. Bielanska, S. L. Tan, A. Ao. McGill University, Royal Victoria Hospital, Department of Obstetrics and Gynecology, Montreal, Quebec, Canada.

St38

Abstracts

Objective: In order to determine the effect of chromosomal mosaicism on fetal development, it is important to investigate the origin and impact of mosaicism during early embryogenesis. The aim of this study is to determine the incidence of mosaicism and the allocation of the chromosomally abnormal cells throughout the progressive stages of preimplantation development. Study Design: A total of 74 spare day-2 to day-6 embryos were analyzed for the presence of mosaicism using multi-color fluorescence in situ hybridization (FISH) with probes specific to chromosomes X, Y, 13,16,18,21 and 22. Materials and Methods: Seventy four normally fertilized (2PN) preimplantation embryos were donated to research by 28 patients undergoing IVF treatment at the McGill Reproductive Centre. A total of 744 blastomeres from two 2-cell, nineteen 4-cell, thirty nine &cell, eleven morula, and three blastocyst-stage embryos were spread by the HCLTween 20 method. Nuclei were treated with pepsin and dehydrated. Nuclei were hybridized with fluorescently labeled DNA probes specific to chromosomes X, Y, 18 and/or chromosomes 13, 16, 21 and 22. Results: Of all embryos analyzed twenty (27%) were normal for chromosomes tested, and fifty four (73%) had detectable chromosomal aberrations. Out of the fifty four abnormal embryos four (7.4%) were aneuploid, and nineteen (35%) were chaotic with blastomeres of variable chromosome patterns. Thirty one (57%) of embryos with detectable aberrations were mosaic, consisting of both chromosomally normal and abnormal blastomeres. The frequency of mosaicism at the 4.cell, g-cell, morula, and blastocyst-stage was 16%, 46%. 55%. and 100% respectively. The incidence of mosaicism was not higher in women of advanced maternal age. Conclusions: Multi-color FISH is a useful method for the detection of chromosomal mosaicism at various stages of preimplantation development. So far our data suggests that mosaicism may originate before the 4-cell stage. The frequency of mosaicism increases with successive cleavage divisions, and mosaicism persists until the blastocyst stage. Analysis of a larger number of embryos at various stages of cleavage division, particularly investigation of the distribution of the abnormal cells into the inner cell mass and trophectoderm at the blastocyst stage, will provide further insight into the mechanism of post zygotic cell selection and allocation.

Wednesday,

September 4:30

29, 1999

P.M.

O-231 Hyaluronic Acid-A Physiological Replacement for Polyvinylpyrrolydon (PVP) in Intracytoplasmic Sperm Injection (ICSI) Procedure. ‘Y. Barak, *Y. Menezo, 3A. Veiga, ‘A. Kogosowski, 42. Nevo. ‘IVF unit, Herzliya Medical Center, Herzliya-on-Sea, Israel, ‘Laboratoire Marcel Merieux, Bron, France, sIVF Laboratory, Department of Obstetrics and Gynecology, Institute Dexeus, Barcelona, Spain, 4Department of Clinical Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel. Objectives: PVP is being routinely used during the procedure of ICSI in order to slow down the motile spermatozoa and to facilitate the injection of the sperm into the ooplasm. It is known as one of the two substances composing the molecule of the Percoll, which was lately withdrawn from usage in human in vitro fertilization (IVF) due to problems of toxicity. Thus, the usage of PVP for injecting sperm into human oocytes has become doubtful. Hyaluronic acid (HA), the chief component of the extracellular matrix which appears in large amounts between the cumulus cells of the matured oocyte-cumulus complex, is the natural material through which the sperm penetrates on its way to fertilize the oocyte. The aim of the current study was, therefore, to examine the efficiency of HA as a _. nhvsiologicalreulacement for PVP in _ the ICSI procedure. Design: Prospective, randomized controlled study. Materials and Methods: In 123 ICSI cycles involved in the study, collected oocytes were denuded from their cumulus cells in droplets of Hepes-buffered medium containing final concentration of 40 iu/ml of hyaluronidase, under oil. MI1 oocytes were injected as follows; Group A: In 58 ICSI cycles, 1~1 of suspension which contained motile spermatozoa was introduced into the center of 5~1 viscous suspension of HA.

Vol.

72, No.

3, Suppl.

1, September

1999

Motility of the sperm cells was slowed down. After breaking the sperm tail with the injection pipette, the spermatozoa were injected into the oocytes. Group B: In 65 ICSI cycles, MI1 oocytes were injected with sperm cells which were immobilized and aspirated for injection in 10% PVP solution. Embryo transfer (ET) was performed on day 2 or day 3. Fertilization, on going pregnancy and implantation rates were compared in both groups. Results: In group A (Ha group), 2PN were observed in 372 out of the 525 MI1 injected oocytes [fertilization rate (FR) - 70.9%]; 484 out of 649 MI1 “PVP injected” oocytes were fertilized (FR - 74.6%; NS). No difference was found in pregnancy rate between both groups-29 pregnancies (50%) in group A vs. 25 (38.5%) in group B, nor in implantation rate-41 embryonic sacs resulted from 221 transferred embryos (18.6%) in group A vs. 35 observed sacs resulted from 250 transferred embryos (14%) in group B. In addition we would like to add that to date 8 healthy babies-3 girls and 5 boys, resulted from the “HA injected spermatozoa” were born. Conclusions: HA was found to be as efficient as PVP for the ICSI procedure. As it is the natural agent which is introduced to the sperm on its “journey” towards the oocyte and as PVP is an artificial agent which might be toxic, as well as it might interfere with the physiological events-e.g.: timing of calcium oscillations, we highly recommend HA as the physiological replacement for PVP.

Wednesday,

September 4:45 P.M.

29,

1999

O-232 Low Mean LH Concentrations in the Late Follicular Phase of IVF Cycles Down Regulated with Low Dose Luteal GnRHa and Stimulated with Recombinant FSH (Gonal-F) Affect Fertilization Rates. P. Patrizio, M. Esposito, K. Barnhart, L. Blasco, R. Tureck, L. Mastroianni, Jr., S. Pfeifer, S. Sondheimer, C. Coutifaris. Center for Reproductive Medicine

FERTILITY

& STERILITY@

and Surgery, University tem, Philadelphia, PA.

of Pennsylvania

Medical

Center

and Health

Sys-

Objective: Refinements in ovulation induction protocols for IVF have suggested that recombinant human FSH (r-hFSH), is the agent of choice. Given the observation that after down regulation with GnRH analogs, patients receiving r-hFSH as the sole agent for follicular growth, have varying levels of endogenous LH, we examined the correlation between varying LH concentrations on fertilization and pregnancy rates. Design: Retrospective analysis of the association between mean LH concentrations on fertilization and pregnancy rates. Material and Methods: A total of 94 IVF cycles (patient age: 35 ? 3.9; range: 27-42) using r-FSH (Gonal-F) were reviewed for this study. Downregulation was achieved with leuprolide acetate (Lupron) at a daily dose of 0.5 mg starting in the luteal phase and decreased to 0.25 mg when ovarian stimulation was commenced (day 1). 10,000 U of hCG (Profasi) were administered between days 11-13. For each patient the mean of three LH serum concentrations from stimulation days 6-7, 8-9 and 10-l 1, were computed for statistical analysis using STATA software and means were evaluated by the Student t-test. Results: After data stratification analysis, an arbitrary cut-off of 3 mIU/mL as mean LH concentrations was used. At this cut-off, 68 (72%) patients had late follicular mean LH values of 53 mIU/mL and 26 (28%) LH values ~3 mIU/mL. The fertilization rate was significantly lower in patients with LH 53 mIU/mL (51% vs. 65%, P