Induction of follicular growth using recombinant human follicle-stimulating hormone in two volunteer women with hypogonadotropic hypogonadism

Induction of follicular growth using recombinant human follicle-stimulating hormone in two volunteer women with hypogonadotropic hypogonadism

FERTILITY AND STERILITYt VOL. 69, NO. 2 (SUPPL. 1), FEBRUARY 1998 Copyright ©1998 American Society for Reproductive Medicine Published by Elsevier Sci...

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FERTILITY AND STERILITYt VOL. 69, NO. 2 (SUPPL. 1), FEBRUARY 1998 Copyright ©1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.

Induction of follicular growth using recombinant human follicle-stimulating hormone in two volunteer women with hypogonadotropic hypogonadism* Zeev Shoham, M.D.,† Bernadette Mannaerts, M.Sc,† Vaclav Insler, M.D.,† and Herjan J. T. Coelingh Bennink, M.D., Ph.D.‡ Department of Obstetrics and Gynecology, Kaplan Hospital, (Affiliated with the Medical School of the Hebrew University and Hadassah, Jerusalem), Rehovot, Israel

Objective: To examine the safety, tolerance, pharmacokinetics, follicular growth, and steroidogenesis after the administration of recombinant human FSH (Org 32489; Organon International, Oss, the Netherlands) in women with isolated hypogonadotropic hypogonadism. Design: An open phase I multiple rising dose study with recombinant FSH in two hypogonadotropic but otherwise healthy women. The drug was administered intramuscularly one time per day for a maximum of 21 days, i.e., 75 IU for the first 7 days, 150 IU for the next 7 days, and 225 IU during the last 7 days. Treatment was discontinued if serum E2 was #1,100 pmol/L and/or one or more growing follicle .14 mm in diameter was observed. After the last recombinant FSH injection, subjects were monitored for another 3 weeks. Setting: Specialist Reproductive Endocrinology and Infertility Unit. Volunteers: Two women with isolated hypogonadotropic hypogonadism who did not want to get pregnant anymore. Main Outcome Measure(s): Serum FSH, androstendione (A), T, P, LH, follicular growth, and endometrial thickness. Safety parameters: blood pressure, heart rate, urinalysis, hematology, blood biochemistry, and antirecombinant FSH antibodies. Result(s): Treatment with recombinant FSH resulted in dose-related increases of serum FSH. Both women showed follicular growth (diameter, 17 mm), whereas serum A concentrations were very low, and serum E2 concentrations rose to only 76.7 and 139.5 pmol/L, respectively. No antirecombinant FSH antibody formation or changes of safety variables were noted.

Received September 17, 1992; revised and accepted December 9, 1992. Reprint requests: Zeev Shoham, M.D., Department of Obstetrics and Gynecology, Kaplan Hospital, Rehovot 76100 Israel. * Supported by Organon International, Oss, the Netherlands. † Department of Obstetrics and Gynecology, Kaplan Hospital. ‡ Scientific Developmental Group, Organon International B.V., Oss, the Netherlands. 0015-0282/98/$19.00 PII S0015-0282(97)00517-7

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Conclusion(s): This study in two women with hypogonadotropic hypogonadism is consistent with the two-cell theory that FSH alone can induce follicular growth. The low concentrations of A and E2 indicate the need for LH to induce appropriate steroidogenesis. It was also found that recombinant FSH is well absorbed, safe, and well tolerated after daily treatment for up to 21 days. (Fertil Sterilt 1993;59:738 – 42. ©1993 by American Society for Reproductive Medicine.) Key Words: Recombinant human FSH, hypogonadotropic hypogonadism, ovulation induction

Gonadotropin preparations have been used in clinical practice for .30 years, but paradoxically there is still a need for more information about the pharmacokinetics of exogenous FSH under different physiological and pathological conditions. FSH produced by the anterior pituitary gland plays a major role in female reproduction by stimulating gonadal differentiation and maturation (1). The mechanism of FSH action includes binding to specific ovarian membrane receptors and subsequent activation of the adenylate cyclase system (1, 2), causing the enhancement of the synthesis of cyto-

chrome P450 aromatase, resulting in increased conversion of androgens to estrogens (Es) (1, 3). It also controls morphological and cellular events such as acquisition of the antral cavity, induction of LH receptors on granulosa cells (GCs), and it causes the activation of enzymes involved in P biosynthesis (4). It was Fevold (5) in 1941 who was the first to demonstrate that highly purified FSH could increase ovarian growth and follicular development in immature hypophysectomized rats without stimulating the release of E. Later on, Eshkol and Lunenfeld (6) in rodents and

Berger and Taymor (7) in patients with hypogonadotropic amenorrhea demonstrated that treatment with pituitary FSH alone did not result in follicular E2 secretion and concluded that LH was essential for final follicular maturation and ovulation induction. On the basis of these and other observations, the two-cell, two-gonadotropin theory of follicular steroidogenesis was raised. According to this hypothesis the ovarian theca cells stimulated by LH secrete androgens that transverse through the basal membrane into the GCs where the FSH-augmented excessive enzyme system turns them into Es (8, 9). Thus the synergistic action of both gonadotropins is presumed to be necessary for follicular maturation and steroidogenesis. However, not everyone agreed with this notion. It was Kenigsberg and his colleagues (10) who showed that in monkeys rendered hypogonadotropic by the administration of GnRH antagonists, injecting increased doses of purified urinary FSH caused multiple follicular development, and ovulation could apparently be induced by the administration of hCG. This theory received some support from studies of ovulation induction and IVF programs because the fertilization rate after oocyte retrieval was similar with urinary FSH or urinary hMG preparations, the latter containing considerable amounts of LH activity. Thus, remaining endogenous LH during GnRH suppression seems to be sufficient for follicular maturation (11). Recombinant FSH has been produced by a Chinese hamster ovary cell line transfected by the genes encoding the subunit genes (12). The isolated recombinant hormone has been shown to contain carbohydrate chains that in structure and charge heterogeneity bear a close resemblance to the natural FSH glycans (13, 14). This offers the opportunity to examine the role of this gonadotropin in the induction of ovarian steroidogenesis. We chose women with isolated hypogonadotropic hypogonadism as the best model to test this theory. The results in this report are part of a multicenter study that was conducted to examine pharmacokinetics, pharmacodynamics, safety, and tolerance of a recombinant human FSH preparation devoid of LH activity, i.e., recombinant human FSH (Org 32489; Organon International, Oss, The Netherlands). The results give additional support to the two-cell, two-gonadotropin hypothesis by showing that FSH-induced increases of E2 are subnormal in the presence of minute amounts of LH and androstenedione (A). Nevertheless, recombinant FSH was able to induce normal follicular growth.

MATERIALS AND METHODS Patients Two women volunteered to participate in an open phase I clinical trial with recombinant FSH to assess its tolerance, safety, and pharmacokinetics. Their age was 38 and 39 years, respectively, and they both had a body mass index (BMI [weight/height2]) of 23. Both women suffered from isolated FERTILITY & STERILITYt

hypogonadotropic hypogonadism, with delayed puberty, amenorrhea, and low serum gonadotropin concentrations (FSH and LH ,1.5 IU/L). Ovarian function was proven to be normal in both women during previous gonadotropin treatment. Both women did not wish pregnancy, and both of them refrained from E therapy at least 30 days before entering the study. Serum thyroid-stimulating hormone (TSH), PRL, and cortisol concentrations were within normal limits. Both women were in good health without chronic illness. They gave their written informed consent to participate in the study, according to the Tokyo Amendment to the Declaration of Helsinki. The study protocol was approved by the Kaplan Hospital and the Israeli Health Authority Ethical Committee.

Study Protocol Before entering the study, the women underwent a pretreatment evaluation that consisted of a general medical history, physical examination, and laboratory evaluation, including liver and kidney function, hematology tests, and hormonal measurements. Autoimmunity was excluded by antinuclear and specific antirecombinant FSH antibody assays. A baseline ultrasound (US) scan of the ovaries was performed to determine ovarian morphology. Recombinant human FSH (Org 32489, batch number CP 90073, specific activity 15.064 [14.000 to 16.170] IU/mg protein; Organon International) was supplied as a lyophilized powder in ampules containing 75 IU of FSH standardized according to the Steelman/Pohley in vivo FSH bioassay (15). For injection, one Org 32489 ampule was reconstituted with 0.5 mL sterile water and administered intramuscularly in the upper lateral quadrant of the buttocks. The women were scheduled to receive one ampule for 7 days followed by two ampules for another 7 days and three ampules per day for another 7 days. Treatment was to continue according to this protocol up to 21 days or maximum dose of 3.150 IU, as long as serum E2 concentrations were ,1,100 pmol/L or follicles .14 mm were not observed. Because the women were not wishing procreation and this study was a phase I clinical trial, no hCG for induction of the final stage of ovulation was administered. Before each injection, one 10-mL blood sample was collected on days 1, 3, 5, 8, 10, 12, 15, 17, and 19 during the treatment period and on days 22, 24, 26, 29, 31, 33, and 36 during the follow-up period. Monitoring was done by serial US scans that were done on alternate days before the administration of the drug. Ultrasound machine (RT 3000; General Electrics, Tokyo, Japan) with two probes for abdominal scanning (3.5 and 5 mHz) and a 5-mHz vaginal probe was used. Safety analysis included clinical observations, i.e., blood pressure and heart rate as well as laboratory assessments such as routine urinalysis (pH, protein, acetone, glucose, hemoglobin), blood biochemistry (sodium, potassium, phosphorus, calcium, glucose, urea, creatinine, uric acid, alkaline phosphatase, alanine and aspartate aminotransfer11S

ase, lactic dehydrogenase, bilirubin, protein, albumin), hematology (hemoglobin, hematocrit, erythrocytes, differentiated leukocytes, thrombocytes).

Hormone Assays Immunoreactive FSH and LH were measured by an immunofluorometric assay, time-resolved fluoroimmunoassay technique, using reagent kits 1244-017 for human FSH and 1244-31 for human LH (Delfia; Pharmacia, Woerden, The Netherlands). These sandwich assays employ a b-directed capturing monoclonal antibody (mAb) and an a-directed europium labeled detection mAb. The assays were performed as described by the manufacturer using the Delfia instrumentation system and MultiCalc software (Pharmacia). FSH and LH immunoreactivity was expressed in terms of the Second International Reference Preparation of pituitary FSH (code no. 78/549) and the Second International Standard for pituitary LH (code no. 80/552). The sensitivity of immunofluorometric assay was 0.05 IU/L for both gonadotropins, and the intra-assay and interassay coefficients of variation (CVs) were below 4.8% and 4.3%, respectively, for FSH and 4.7% and 7.5%, respectively, for LH. The cross-reactivity of the FSH kit with LH was ,0.08%, and the LH kit with FSH ,0.01%. Serum E2 was measured by a double antibody E2 RIA (reagent kit KE2D1 DPC; Diagnostic Products Corporation, Los Angeles, CA). The intra-assay and interassay CVs were ,4% and 5%, respectively. Serum A concentrations were measured, after extraction with diethylether, using the antiserum described by Frolich et al. (16). The intra-assay CV was ,7%. Serum samples were analyzed for the presence of antirecombinant FSH antibodies using a sensitive radioimmunoprecipitation assay and 125I-recombinant FSH as a tracer. When testing a mixture of two mouse mAbs raised against recombinant FSH and recognizing an aand b-specific epitope, the sensitivity of the assay was 0.5 pmol/L, and the intra-assay and interassay CVs ranged from 4.3% to 9.6% and from 0.8% to 2.7%, respectively. The induction of antirecombinant FSH antibodies after recombinant FSH treatment was judged by comparing pretreatment and post-treatment samples according to criteria, allowing a probability of false-positive results of ,0.1%. All serum samples were tested in duplicate, and the mouse mAb mixture was used as a positive control in all experiments. Antinuclear antibodies were measured using immunofluorescence technique in a Hep-2 cell line (Bio-Lab, Amsterdam, the Netherlands). Serum T concentrations were measured by RIA using a coat-a-count T RIA (reagent kit TKTT1 DPC, Diagnostic Products Corporation) with a detection limit of 0.27 nmol/L. The intra-assay and interassay CVs were ,9% and 13%, respectively. All blood samples were taken just before administration of recombinant FSH, i.e., 24 hours after the previous injection. The blood samples were then centrifuged, and the serum was removed and stored at 220°C until analyzed. Serum samples of one subject were examined in one run. 12S

A recombinant FSH

RESULTS Serum concentrations of FSH after intramuscular administration of the recombinant FSH, the ovarian response as measured by serum E2 concentrations, and follicular development are illustrated in Figures 1 and 2. One of the women received treatment for 21 days (total dose of 3,150 IU), and the other was treated for 17 days (total dose of 2,250 IU). Serum concentration of FSH in both women was increased with the FSH dose given, indicating that the pharmacokinetics of the recombinant FSH were linear in the dose range tested. For each recombinant FSH dose applied, steady state levels of serum FSH concentrations were reached in 3 to 5 days. During the 3rd treatment week at day 17, plateau serum FSH concentrations of 11.8 and 10.1 IU/L, respectively, were reached after the administration of a total of 2,250 IU recombinant FSH. The increments were similar in magnitude and in time course in both women. After increasing the dose of recombinant FSH from 150 to 225 IU, steady state levels were reached at day 17 as illustrated in one woman who showed thereafter no further increases of FSH, although she received one time per day 225 IU recombinant FSH for 4 additional days (treatment days 18 to 21, total 890 IU). Serum FSH concentrations decreased to baseline levels after 10 and 12 days in each of the women, respectively, after the cessation of treatment. Treatment was stopped after the observation on US scan of at least one follicle .14 mm, which occurred on treatment day 17 and 21, respectively. Serum E2 concentrations showed a gradual increase to a maximum of 76.7 and 139.5 pmol/L, respectively, at the time of maximum follicular growth. Mean concentrations of serum LH, A, and T at the time of maximal follicular growth (0.11 IU/L, 2.2 nmol/L, and 0.48 nmol/L, respectively) were comparable with those measured at baseline (0.15 IU/L, 2.7 nmol, and 0.56 nmol/L, respectively). Serum P was undetectable during the whole study period. No change in endometrial thickness (4 and 5 mm, respectively), as measured by US scan, was observed during the treatment. After a multiple administration of the recombinant FSH, up to a total dose of 3,150 IU, the drug was found to be well tolerated, and no drug-related adverse experiences were noted. Neither pain nor skin redness was observed at the site of injections. Safety variables such as blood biochemistry, hematology, urinalysis, blood pressure, and heart rate were assessed once before treatment, twice during treatment before increasing the recombinant FSH dose, and once after the last recombinant FSH injection. Evaluation of these data did not reveal any change of clinical significance. No antirecombinant FSH antibody formation was observed.

DISCUSSION This study provides novel data regarding dose-related increases of serum FSH after multiple administrations of recomVol. 69, No. 2 (Suppl. 1), February 1998

FIGURE 1 Serum FSH and E2 concentrations and follicular development during the administration of the recombinant FSH (recFSH), 21 days with a total dose of 3,150 IU, are presented.

binant FSH in hypogonadotropic hypogonadism women. Analysis of serum FSH concentrations in hypogonadotropic hypogonadism women and men after the intramuscular administration of one single dose of 300 IU of the recombinant FSH revealed comparable elimination half-lives of 44 6 14 (mean 6 SD) and 32 6 12 hours, respectively (17).

The present study showing that serum concentrations of FSH are successively rising after the injections up till 17 days indicates that the absorption rate of the hormone from the injection site exceeds that of its redistribution in extravascular compartments, gonadal uptake, metabolism, and excretion. The absence of adverse effects and antirecombi-

FIGURE 2 Serum FSH and E2 concentrations and follicular development during the administration of the recombinant FSH (recFSH), 17 days with a total dose of 2,250 IU, are presented.

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nant FSH antibody formation gives further assurance about the safety of this drug. The present study provides more definite evidence in favor of the two-cell theory, confirming that FSH alone can stimulate follicular growth without increase of E2 secretion. The first data about his hypothesis came from studies in rats done by Fevold (5) and later by Short (8) and Ryan et al. (9). In a study done by Couzinet et al. (18), it was clearly shown that in women with hypogonadotropic hypogonadism who were treated with a fixed dose of purified urinary FSH, follicular development was noted, whereas serum A and E2 concentrations were inappropriately low. This study was confirmed by Shoham et al. (19) who treated hypogonadotropic hypogonadism patients for induction of ovulation, using urinary hMG versus purified urinary FSH. In the cycles in which purified FSH was administered, serum E2 concentrations were significantly lower (P , 0.002), and these levels were not enough even to cause an increase in endometrial thickness (P , 0.02). In the present study we found that recombinant FSH, applied to hypogonadotropic hypogonadism women, is sufficient for inducing follicular growth but ineffective to increase the synthesis of E2. This result is probably due to the fact that in the two women, circulating endogenous serum LH concentrations were extremely low or undetectable as recently described in a case report (20) along with low serum A concentrations. These findings further support the notion that normal follicular development is dependent predominantly on FSH and that steroidogenesis is mainly supported by the synergistic action of FSH and LH. References 1. Hsueh AJ, Adashi EY, Jones PB, Welsh TH Jr. Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocr Rev 1984;5:76 –127. 2. Sharp RM. Intratesticular control of steroidogenesis. Clin Endocrinol (Oxf) 1990;33:787– 807. 3. Dorrington JH, Armstrong DT. Effects of FSH on gonadal functions. Recent Prog Horm Res 1979;35:301– 42. 4. Zeleznik AJ, Hillier SG. The role of gonadotropins in the selection of the preovulatory follicle. Clin Obstet Gynecol 1984;27:927– 40. 5. Fevold HL. Synergism of follicle stimulating and luteinizing hormones in producing estrogen secretion. Endocrinology 1941;28:33– 6. 6. Eshkol A, Lunenfeld B. Purification and separation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from human menopausal gonadotropin (HMG). 3. Effects of a biologically appar-

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ently pure FSH preparation on ovaries and uteri of intact immature mice. Acta Endocrinol (Copenh) 1967;54:91–5. Berger MJ, Taymor ML. The role of luteinizing hormone in human follicular maturation and function. Am J Obstet Gynecol 1971;111: 708 –10. Short RV. Steroids in the follicular fluid and the corpus luteum of the mare: a ‘‘two-cell type’’ theory of ovarian steroid synthesis. J Endocrinol 1962;24:59 – 63. Ryan KJ, Petro Z, Kaiser J. Steroid formation by isolated and recombinant ovarian granulosa and theca cells. J Clin Endocrinol Metab 1968;28:355– 8. Kenigsberg D, Littman BA, Williams RF, Hodgen GD. Medical hypophysectomy. II. Variability of ovarian response to gonadotropin therapy. Fertil Steril 1984;42:116 –26. Bentick B, Shaw RW, Iffland CA, Burford G, Bernard A. A randomized comparative study of purified follicle stimulating hormone and human menopausal gonadotropin after pituitary desensitization with buserelin for superovulation and in vitro fertilization. Fertil Steril 1988;50:79 – 84. Van Wezenbeek P, Draaier J, Van Meel F, Olijve W. Recombinant follicle stimulating hormone. I. Construction, selection and characterization of a cell line. In: Crommelin DJA, Schellekens H, editors. From clone to clinic, developments in biotherapy. Dordrecht, Kluwer: Academic Publications, 1990:245–51. Hard K, Mekking A, Damm JBL, Kamerling JP, de Boer W, Wijnands RA, et al. Isolation and structure determination of the intact sialyated N-linked carbohydrate chains of recombinant human follitropin (hFSH) express in Chinese hamster ovary cells. Eur J Biochem 1990;193:263– 71. De Boer W, Mannaerts B. Recombinant follicle stimulating hormone. II. Biochemical and biological characteristics. In: Crommelin DJA, Schellekens H, editors. From clone to clinic, developments in biotherapy. Dordrecht, Kluwer: Academic Publications, 1990:253–9. Mannaerts B, de Leeuw R, Geelen J, Van Ravestein A, Wezenbeek PV, Schuurs A, et al. Comparative in vitro and in vivo studies on the biological characteristics of recombinant human follicle stimulating hormone. Endocrinology 1991;129:2623–30. Frolich M, Brand EC, van Hall EV. Serum levels of unconjugated aeticholanolone, androstendione, testosterone, dehydroepiandrosterone, aldosterone, progesterone, and oestrogens during the normal menstrual cycle. Acta Endocrinol (Copenh) 1976;81:548 – 62. Mannaerts B, Shoham Z, Schoot D, Bouchard P, Harlin J, Fauser B, et al. Single dose pharmacokinetics and pharmacodynamics of recombinant human follicle-stimulating hormone (Org 32489) in gonadotropindeficient volunteers. Fertil Steril 1993;59:108 –14. Couzinet B, Lestart N, Brailly S, Forest M, Schaison G. Stimulation of ovarian follicle maturation with pure follicle-stimulating hormone in women with gonadotropin deficiency. J Clin Endocrinol Metab 1988; 66:552– 6. Shoham Z, Balen A, Patel A, Jacobs HS. Results of ovulation induction using human menopausal gonadotropin or purified follicle-stimulating hormone in hypogonadotropic hypogonadism patients. Fertil Steril 1991;56:1048 –53. Schoot DC, Coelingh-Bennink HGT, Mannaerts BMJL, Lamberts SWJ, Bouchard P, Fauser BCJM. Human recombinant follicle-stimulating hormone induces growth of preovulatory follicles without concomitant increase in androgen and estrogen biosynthesis in a woman with isolated gonadotropin deficiency. J Clin Endocrinol Metab 1992;74: 1471–3.

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