Scanning electron microscopic study of normal and molar trophoblast

Scanning electron microscopic study of normal and molar trophoblast

GYNECOLOGIC ONCOLOGY 1, 95-110 (1972) Scanning Electron Microscopic Study Normal and Molar Trophoblast’ of ALEX FERENCZY AND RALPH M. RICHART In...

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GYNECOLOGIC

ONCOLOGY

1, 95-110

(1972)

Scanning Electron Microscopic Study Normal and Molar Trophoblast’

of

ALEX FERENCZY AND RALPH M. RICHART International Institute for the Study of Human Reproduction, the Departments of Pathology and Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University and the Obstetrical and Gynecological Service (The Sloane Hospital for Women) of the Presbyterian Hospital, New York, New York 10032 Received

October

6, 1972

The surface of molar syncytiotrophoblastic cells when examined by scanning electron microscopy had prominent, complexly branching microvillous processes. The degree of microvillous arborization was comparatively less abundant in the early normal placenta and considerably reduced in the mature placenta. These compamtive topographic features suggest a close surface similarity between the molar and immature trophoblast, reflecting increased proliferative and metabolic activity, which is exaggerated in the hydatidiform mole. The significance of these findings is discussed in relation to pathogenesis.

The trophoblast, a term first suggested by Hubrecht [l], is a metabolically complex cell system, which covers the placental villi. Structural variations in the microvilli of the cytoplasmic membrane occur as part of the normal maturation process and in response to abnormal metabolic conditions and neoplasia. Although several transmission electron microscopic investigations of normal placentas [2-41 and hydatidiform moles [5-71 have been published, studies focusing on alterations in the microvillous pattern of normal and abnormal trophoblastic tissue have not been performed. The purpose of the present study is to compare the surface ultrastructure of early (immature) and full-term (mature) trophoblastic tissue with that of hydatidiform moles by using the high-resolution, three-dimensional capability of the scanning electron microscope. MATERIALS

AND

METHODS

Immature placental tissue was obtained by suction curettage from 10 patients in the 8th and 12th week of gestation who underwent voluntary abortion. Other specimens were obtained from eight normal placentas at full-term. The patients all had normal, uncomplicated pregnancies. Molar vesicles were obtained by hysterotomy at the 5th to 6th months of gestation from three women aged 23, 26, and 38 years, respectively. The placental and molar tissues were washed in normal saline and immedi’ Paper presented at the Annual Meeting of the Canadian Association sity of Sherbrooke, Sherbrooke, Quebec, Canada, June 19, 1972. 95 Copyright @ 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.

of Pathologists,

Univer-

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FIG. 1. Immature placenta (8th week of gestation). Active trophoblastic growth the presence of numerous trophoblastic sprouts of various stages of development. tervillous spaces are large and circular (x200).

is suggested by Most of the in-

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FIG. 2. Higher magnification of Fig. 1 illustrating a relatively well-developed trophoblast tic sprout. The microvillous covering is partly agglutinated and appears smooth on the most expos ed surface (X600).

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FIG. 3. Immature placenta (12th week of gestation). Prominent chorionic villi give rise to a multitude of trophoblastic sprouts varying from minute (small arrow) to large cylindrical villous structures (large arrow). Note the large intervillous spaces and the short, noninterdigitating trophoblastic villosity (x200).

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FIG. 4. Micrograph illustrating the development of a terminal of blunt-ended, cone-shaped syncytial sprout (x560).

TROPHOBLAST

chorionic

villus

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by the formation

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FIG. 5. Full-term placenta. The terminal chorionic the immature placenta. Although a few trophoblastic others are cord-like (x600).

RICHART

villi are of uniform size but smaller than in sprouts are present, most are small, while

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FIG. 6. Detail of Fig. 5. Note the bulbous tion of the trophoblastic sprouts (x2000).

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MOLAR

(small arrow)

TROPHOBLAST

and cord-like

(larger arrow)

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configura-

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FIG. 7. The immature placenta (8th week of gestation) has an abundant syncytial surface pattern characterized by multibranching, bulbous promontories (X20,000).

microvillous

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FIG. 8. At full term the syncytium is covered by dense, hair-like, single microvilli. The licrovillous branching and the bulbous tips are inconspicuous in the mature placenta (~20,000).

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FIG. 9. Hydatidiform mole. The vesicular surface varying-sized, flat syncytial protrusions (X400).

RICHART

is slightly

undulant

and contains

several

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FIG. 10. Hydatidiform mole. Artificially envelope. Note the very fine “rhythmic” the lack of vessels (x800).

FIG. 11. Hydatidiform branching microvillous

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fractured molar vesicle covered by a thin trophoblastic pattern of the supporting connective tissue fibrils and

mole. The molar syncytial processes (X6000).

surface

contains

a profusion

of complexly

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FIG. 12. Detail of Fig. 11, illustrating the complicated microvillous arborization of the molar syncytiotrophoblast. Compare this photomicrograph with those of immature and mature placentas, taken at the same magnification (~20,000).

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ately fixed in 10% phosphate-buffered formalin for light microscopy and in 2.5% phosphate-buffered (pH 7.3) gluteraldehyde for scanning electron microscopy. Individual molar vesicles measuring up to 1 x 1 cm, and blocks of normal and neoplastic placental tissue, measuring 0.5 x 0.5 cm were freezedried, or critical point-dried, and coated with a 150- to SOO-A-thick film of gold-palladium. The specimens were examined in a JSM-US scanning electron microscope at 25 kV. RESULTS Stunning

Electron

Microscopy

Placentu. The chorionic villi in both the early and the term placentas had a complex branching and interlacing pattern which had the appearance of a series of labyrinthine channels (Figs. 1, 3, and 5). Several of these spaces in the immature placenta were large and circular, measuring up to 500 ,um in diameter, but in the mature trophoblastic tissue, they appeared smaller, often reduced to narrow intervillous slits. The terminal villi, which were especially prominent in the immature placentas, had the appearance of thick cylinders and contained irregular blunt-ended syncytial sprouts, of all stages of development, most often oriented perpendicularly to the long axis of the villus (Figs. 1, 2, 3, and 4). The trophoblastic buddings were considerably more numerous in the immature than in the mature placentas (Figs. 5 and 6). In the latter, the trophoblastic buds were generally smaller and infrequent but larger cone-shaped sprouts were occasionally noted, ending in a cord-like syncytial belt. This feature was most commonly seen in the term placentas and was interpreted as representing focal atrophic changes (Figs. 5 and 6). The syncytiotroblastic sprouts had a spongy appearance due to the abundant microvilli which were often agglutinated by the adherent intervillous plasma (Fig. 2). At a higher magnification the syncytial surface of the immature trophoblast contained a multitude of single or multibranching hairlike cytoplasmic projections measuring 2-4 pm in length with prominent bulbous tips (Fig. 7). In the term placentas, the intermicrovillous space was comparatively more distinct, the microvilli were single rather than branching, and the bulbous promontories were less pronounced (Fig. 8). Hydatidifoorm moles. The distended trophoblastic membrane of the molar vesicles often became wrinkled as a result of the preparative procedures, but the folding did not seem to affect the syncytial microvillous architecture. The pattern did vary over regions of the same vesicle and from one vesicle to another in all three cases examined, however. Some molar surface regions were smooth, with scant and dispersed microvilli, a feature interpreted as being due to early degenerative changes; others, however, appeared spongy and were covered by a tightly packed carpet of fine cytoplasmic projections which were partially obscured by a thick layer of granular material. The surface of most of the molar vesicles was covered by clusters of varying sized and shaped buds or sprouts (Fig. 9), thought to be composed of syncytio- and cytotrophoblast, and which generally appeared as

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flat overgrowths, in contrast to the usual cylindrical configuration of comparable structures in the normal, immature placenta. The surface microvilli were arranged in a profusion of complexly branching cytoplasmic processes (Fig. 11) and the number of microvilli and the degree of arborization exceeded that found in the immature and mature placenta (Fig. 12). After intentional fracturing of frozen molar vesicles the exposed supporting stroma consisted of a delicate network of collagen fibrils, devoid of vascular structures (Fig. 10). DISCUSSION All the chorionic villi examined in this study contained prominent trophoblastic sprouts which were more numerous in the immature than in the fullterm placenta. Both the immature placentas and the molar vesicles were covered by a large number of trophoblastic protrusions but the molar tissues lacked the morphologic evidence of gradual maturation which was seen in the immature placenta. These protrusions have been recognized as developing terminal chorionic villi [8,9] and their great number in the early placentas is probably a reflection of active trophoblastic growth. In the hydatidiform moles, however, these syncytial sprouts, despite their abundance, appeared immature and were more haphazardly arranged. The cell surfaces contained abundant microvilli with comparatively more branching and more bulbous apices in the immature than in the mature chorionic tissue. This observation provides topographic support for the previous transmission electron microscopic observation that the microvilli were generally longer in the first trimester than in the term placenta [2,8,10]. On the other hand, in examining large surface areas at high magnification, there were less dramatic differences in the configuration and distribution of the syncytial microvillous covering between young and old placentas than has been reported at the transmission ultrastructural level. With the scanning electron microscope, the most significant modification was the degree of microvillous arborization rather than the presence or absence of microvillous processes [2,8,11], their number, or height. Although correlative scanning electron microscopic studies of normal and abnormal trophoblastic tissues have not previously been published, an essentially similar microvillous architecture in immature 14,121 and older placentas 141 has been reported. The present findings, however, are not concordant with earlier stereoscopic investigations [ 13-161 in which a “poorly developed” syncytial microvillous surface at full term has been described. In view of the present observations and the previous [4,12] reports of abundant syncytial surface specialization, this discrepancy we believe is most likely due to different techniques in specimen preparation. The molar syncytial layer has a microvillous covering which is more abundant and multibranching than that of the normal trophoblast. The morphologic recognition and comparative evaluation of this special surface organ-

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ization of the syncytial cells is important since qualitative and quantitative modifications are thought to mirror changes in the function of the trophoblastic membrane. It has generally been accepted that the microvilli of the syncytiotrophoblast increase the cell surface and, therefore, its absorptive area [4,8,10,12]. McKay et al. [171 have demonstrated dissimilar osmotic pressure and amino acid concentrations between the molar vesicular core and the maternal plasma, indicating imbibition and accumulation of fluid within the vesicles. The conspicuous microvillous surface of the immature placenta and the highly complex microvillous processes in molar syncytial cells may be regarded as a reflection of this specialized cellular activity and of the demonstrated correlation between surface specialization and active fluid and ion transport, in both normal [3,4,8-111 and abnormal [6] trophoblast. Although scanning electron microscopy alone cannot resolve the question of the pathogenesis of hydatidiform mole, it does provide additional support for the original concept championed by Hertig and Edmonds [18] that the initial step in the formation of a mole is related to metabolic disturbances of the trophoblastic tissue secondary to abnormal pregnancy [18]. Whether the abnormal molar tissue is due to a purely hyperplastic, rather than a neoplastic process, is still a matter for discussion, although several investigators have considered a neoplastic continuum to exist as evidenced by the gradual change in chromosome number and nuclear DNA content of the molar trophoblast [19]. REFERENCES 1. HUBRECHT, A. A. W. Studies in mammalian embryology. 1. The placentation of ~~~~~~~~~ europneus, with remarks on the phylogeny of the placenta. Quart. J. Microsc. Sci. 30, 283-404 (1889). 2. WISLOCKI, G. B., ANU DEMPSEY, E. W. Electron microscopy of the placenta of the rat. Awt. Rec. 123, 33-63 (1955). 3. LISTER, U. M. Ultrastructure of the early human placenta. J. Olxtet. Gywecol. Brit. Enlp. 71, 21-32 (1964). 4. DEMPSEY, E. W., AND LUSE, S. A. Regional specializations in the syncytial trophoblast of early human placentas. J. Anclt. 108, 545-561 (1971). 5. RIGANO, A., AND SERMANK, R. Preliminary observations on the ultrastructure of the chorionic villus in vesiculo-molar degeneration. Riw. O&et. Ginecol. 18, 12-36 (1963). 6. WYNN, R. M., AND DAVIES, J. Ultrastructure of hydatidiform mole: correlative electron microscopic and functional aspects. Amer. /. Ohstet. Gynecol. 90,293-307 (1964). 7. WYNN, R. M., AND HARRIS, J. A. Ultrastructure of trophoblast and endometrium in invasive hydatidiform mole (chorioadenoma destruens). Amer. /. Ohstet. Gynecol. 99, 1125-1135 (1967). 8. BOYD, J. D., AND HAMILTON, W. J. Development and structure of the human placenta from the end of the 3rd month of gestation. /. Obstet. Gynaecol. Brit. Emp. 74, 161-226 (1967). 9. ALADJEM, S. Morphopathology of the human placental villi and the fetal outcome (A study by phase-contrast microscopy). J. O&et. Gynuecol. Brit. Emp. 75, 1237-1244 (1968). 10. TIGHE, J. R., GARROD, P. R., AND CURRAN, R. C. The trophoblast of the human chorionic villus. J. Puthol. Bacterial. 93, 559-567 (1967). 11. SCHIEBLER, T. H., AND KAUFMANN, P. Uber die Gliederung der menschlichen Plazenta. Z. Zellforsch. 102, 242-265 (1969).

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12. BERGSTR~~M, S. Surface ultrastructure of human amnion and chorion in early pregnancy: A scanning and electron microscope study. Obstet. Gynecol. 38, 513-524 (1971). 13. MULTIER, A. M., HERBST, R., AND BLASCHKE, R. Face maternelle du placenta et villosites choriales observees au “Scanning Microscope.” Bull. Fed. Sot. Gyn&col. Obstet. Lang. FT. 20,411-414 (1968). 14. LUDWIG, H., JUNKERMANN,H., AND KLINGELE, H. Oberflachenstrukturen der menschlichen plazenta im rasterelektronenmikroskop. Arch. Gynuekol. 210, l-20 (1971). 15. LUDWIG, H. Surface structure of the human term placenta and of the uterine wall post partum in the screen scan electron microscope. Amer. J. Obstet. Gyn&ol. 111, 328-344 (1971). 16. KAUFMANN, P. The demonstration of cytoplasmic polyps from the human trophoblast by scanning electron microscopy. Arch. Gynuekol. 211, 523-526 (1971). 17. MCKAY, D. G., RICHARDSON,M. V., AND HERTIG, A. T. Studies ofthe function of early human trophoblast. III. A study of the protein structure of mole fluid, chorionic and amniotic fluids by paper electrophoresis. Amer. /. Obstet. Gynecol. 75, 699-707 (1958). 18. HERTIC, A. T., AND EDMONDS, H. W. Genesis of hydatidiform mole. Arch. Puthol. 30, 260-291 (1940). 19. GOLDFARB, S., RICHART, R. M., AND OKAGAKI, T. A cytophotometric study of nuclear DNA content of cyto- and syncytiotrophoblast in trophoblastic disease. Cancer 27,83-92 (1971).