SCANNING ELECTRON MICROSCOPY OF THE HUMAN ENDOMETRIAL SURF ACE EPITHELIUM*

FERTILITY AND STERILITY Copyright 1972 by The Williams & Wilkins Co. Vol. 23, No.8, August 1972 Printed in U.S.A. SCANNING ELECTRON MICROSCOPY OF THE HUMAN ENDOMETRIAL SURF ACE EPITHELIUM* ALEX FERENCZY, M.D., RALPH M. RICHART, M.D.,t FREDERIC J. AGATE, JR., PH.D., MABEL L. PURKERSON, M.D., AND EDWARD W. DEMPSEY, PH.D. International Institute for the Study of Human Reproduction, and the Departments of Pathology, Obstetrics and Gynecology,andAnatomy, Columbia University College of Physicians and Surgeons, New York, New York 10032 Although the cyclic morphologic changes occurring in the human endometrial glands have been extensively investigated by means of light and, more recently, by transmission electron microscopy, 1-5 surprisingly little attention has been paid to the uterine surface epithelium. The question of whether the lining epithelium has similar structural characteristics to the glandular epithelium during the menstrual cycle, has not been determined and there have been no comprehensive, high resolution studies focusing on the geographical distribution and significance of ciliogenesis and cells with secretory potential. In contrast to the extremely limited areas which can be examined by the conventional transmission electron microscope, scanning electron microscopy (SEM) offers a new approach to the study of surfaces, three-dimensional topographic study of large fields of cells. This paper reports preliminary results obtained from the study of the human uterine surface lining epithelium during the normal reproductive cycle. MATERIALS AND METHODS Endometrial biopsies were obtained from the uterine corpus in women ranging from 18-40 years of age. Specimens without histologic abnormalities were Received January 10, 1972. * This investigation was aided by grants from the National Institutes of Health (GM 15289 and AM 50950), the Association for the Aid of Crippled Children, and by The Ford Foundation Grant 660-0032. t Reprint requests should be mailed to Dr. Ralph M. Richart, 630 West 168th St., New York, N. Y. 10032. dated according to the histologic criteria previously described by Noyes, Hertig, and Rock. 6 The endometria examined in this study were dated as proliferative (follicular) phase; 16th, 17th, 19th, 21st, and 28th day of the secretory (luteal) phase, and midmenstrual endometrium, respectively. Specimens for scanning electron microscopy were prepared as previously described 7 and examined with a Cambridge Stereos can microscope at 20 kv. RESULTS The appearance of the endometrial surface in the follicular, luteal, and mid menstrual phases is depicted in Figs. 1-6. The lining is smooth, slightly convoluted, and contains haphazardly distributed deep invaginations corresponding to the endometrial gland openings. By the early proliferative phase (4-8 days postmenstrual), the surface has been completely re-epithelialized and is covered by closely placed, dome-shaped cells mixed with numerous ciliated cells in an approximate 30: 1 ratio. The dome-shaped cells contain a profuse array of short, occasionally branching, hair-like projections, which are often obscured by agglutinated, granular secretory material. The ciliated cells occur principally in randomly distributed groups, although occasionally isolated tufts associated with a single cell are seen. The cilia are well developed and are morphologically similar to those seen in the fallopian tube. 7 The midproliferati ve (8-12 days), (Figs. 1 and 2) late proliferative (12-14 days), and 16-day secretory (Fig. 3) endometrial 515

FIG. 1 (top). Midproliferative endometrium. The surface is covered by groups of ciliated cells intermingled with microvillous cells, many of them obscured by granular, mucous-like secretions. x 2450. FIG. 2. (bottom). Midproliferative endometrium. Higher resolution view of well-developed cilia and neighboring cells covered by a thick layer of granular secretory material. x 5600. 516

August 1972 SCANNING ELECTRON MICROSCOPY 517 surfaces had the same general topographic pattern as described for the early proliferative phase, except for an increased number of ciliated cells, with a ratio of approximately 15 nonciliated to 1 ciliated cell. The latter occur in greater concentration in the plateaus surrounding the glandular ostia (Fig. 3). Cilia were also noted in the gland mouths and deeper glandular segments, but they were infrequent. A thick layer of mucus-like material appeared to cover large fields of the surface cells and secretory activity appeared to be manifest by protuberont cytoplasmic protrusions (macroapocrine secretion). Although striking surface changes were not noted during the mid or late luteal phases, a number of subtle alterations occurred. Gland orifices underwent gradual widening and had round, rather than narrow ellipsoidal, contour as was seen in the follicular and early secretory phases. Ciliated cells remained abundant, especially in the immediate vicinity of the gland openings. From about the 21st day, (Fig. 4) the number of ciliated cells decreased slightly with a ratio of 40: 50 nonciliated to 1 ciliated cell. In addition, the length and the number of cilia became somewhat reduced at Day 20-21, but thereafter remained unchanged through Day 28 (Fig. 5). Nonciliated cells retained their cobblestone appearance with a microvillous cell surface, and macroapocrine secretory activity was pronounced between the 20th and 24th day of the cycle (Fig. 4). When partial surface cell loss occurred (27-28th day) the cell outlined had a hexagonal pattern with tight intercellular junctions, which was similar to that seen in the oviductal epithelium. 7 Specimens taken during the midmenstrual phase (2-3rd day) contained leukocytic, stromal, and gland cellular debris enmeshed in masses of fibrillar material and red blood cells. Occasional endometrial gland necks with rolled edges were noted to have epithelial extensions onto the surrounding tissue, suggesting an early stage of re-epithelialization (Fig. 6). DISCUSSION The most striking feature of the endometrial surface was the large number of ciliated cells which were present throughout the menstrual cycle. This was a surprising finding in view of the light and electron microscopic observations of the uterine mucosa in which a peak occurrence of ciliated epithelium was reported during midcycle,8-10 and the cells were said to be more numerous in the glands than on the surface. 11 Our observation that abundant ciliated cells are present in the secretory phase is in contrast to the previous reports, in which a decrease,4, 5. 11 or even an absence8-1o of ciliated epithelium was described during the late postovulatory period. This discrepancy may have arisen because most previous studies were principally concerned with the endometrial glands4. 5, 8-11 rather than surface epithelium. The morphogenesis of cilia in the endometrial epithelium has been studied by means of light and phase contrast microscopy, 10. 12 indicating increased cilia formation in the proliferative phase. There have been no systematic studies of human endometrial ciliogenesis. The structure of the cilium, including basal body and ciliary bud formation, is similar to that of cilia found in the mammalian oviduct. 13. 14 The turnover of endometrial cilia must be considerably greater than that of the tubal epithelial cells with a peak in ciliogenesis immediately following the menstrual period, since a well developed and abundant ciliated cell population was observed in the early follicular phase. Detailed SEM observations coupled with transmission electron microscopic studies during the early postmenstrual period would be useful in evaluating the precise mechanisms of endometrial regeneration and ciliogenesis.

FIG. 3 (top). Sixteenth day endometrium. Note the narrow gland opening, characteristically surrounded by a "collarette" of ciliated cells. x 770. FIG. 4 (bottom). Twenty-first day secretory endometrium. Ciliated cells appear to contain slightly shorter and fewer cilia. Increased secretory activity is suggested by numerous mucous droplets and macroapocrine cytoplasmic buddings. Note the abundant hair-like microvilli covering the secretory cells. x 3550. 518

FIG. 5 (top). Premenstrual (28th day) endometrium. Numerous ciliated cells are still visible and the surrounding nonciliated cells preserve their general dome-shaped appearance. x 3400. FIG. 6 (bottom). Midmenstrual (2-3rd day) endometrium. A gland neck with rolled margin contains tongues of epithelium extending onto the neighboring stroma, suggesting early re-epithelialization. x 510. 519

520 FERENCZY ET AL. Vol. 23 The precise significance of cilia in the endometrium, especially on the surface is not understood. Ciliary motion in the endometrium is in the form of uniform stroke and counter-stroke movements, 10 unlike the alternating strong beat and slow recovery of the tracheal mucosa. Most probably it promotes fluid circulation or sperm and egg transport. In this regard, concentration of ciliated cells around gland openings may facilitate the distribution of glandular secretions. Observations from the present study that the surface epithelium does not respond to hormonal stimuli as do the endometrial glands, support a previously suggested possibility.4, 5 Ciliogenesis in the uterine glands is thought to be influenced chiefly by endogenous estrogen as evidenced by the apparent abundance of cilia in proliferative and hyperplastic endometria 8, 9, 15 and their scarcity in secretory and atrophic endometria. The uterine lining epithelium does not undergo major morphologic surface alterations in response to hormonal stimuli and, in this respect, it bears a close resemblance to the tubal mucosa, in which ciliogenesis and secretory activity undergo minimal cyclical changes. 7, 14 The development and maintenance of ciliated cells in both types of epithelia, however, seem to be under estrogenic control since an absence of estrogen 7-10, 14 results in deciliation and cessation in secretory activity. Additional evidence indicating a commonality of response in the tubal and the endometrial lining epithelia is provided by similar findings in transmission electron microscopy 1, 4, 5, 14 and histochemical studies. 16 Indeed, the similarity between tubal and endometrial ciliated epithelium has led several authors to consider ciliated cells in the endometrium as a "tubal metaplastic" phenomenon. 8, 9, 15 The large number of ciliated cells in the endometrium and their regular distribution as shown in the present SEM study, however, preclude the supposition that they are the result of a metaplastic reaction_ These cells appear to be normal cellular constituents of the endometrial surface rather than a reflection of metaplastic alterations. SUMMARY Stereoscopic morphologic characteristics of the human endometrial surface include a surprisingly large number of ciliated cells and relatively scant secretory activity throughout the menstrual cycle. These characteristics bear close resemblance to changes previously noted in oviductal epithelium, perhaps a reflection of similar endocrine behavior. REFERENCES 1. CAVAZOS, F., GREEN, J. A., HALL, D. G., AND Lu CAS, F. V. Ultrastructure of the human endometrial glandular cell during the menstrual cycle. Amer J Obstet Gynec 99:833, 1967. 2. NILSSON, O. Electron microscopy of the glandular epithelium in the human uterus. 1. Follicular phase. Ultrastruct Res 6:413, 1962. 3. NILSSON, O. Electron microscopy of the glandular epithelium in the human uterus. II. Early and late luteal phase. Ultrastruct Res 6:422, 1962. 4. WYNN, R. M., AND HARRIS, J. A. Ultrastructural cyclic changes in the human endometrium. I. Normal preovulatory phase. Fertil Steril 18:632, 1967. 5. WYNN, R. M., AND WOOLEY, R. S. Ultrastructural cyclic changes in the human endometrium. II. Normal postovulatory phase. Fertil SteriI18:721, 1967. 6. NOYES, R. W., HERTIG, A. T., AND ROCK, J. Dating the endometrial biopsy. Fertil Steril 1:3, 1950. 7. FERENCZY, A., RICHART, R. M., AGATE, F., JR., PURKERSON, M. L., AND DEMPSEY, E. W. Scanning electron microscopy of the human fallopian tube. Science 175:783, 1972. 8. FRUIN, A. H., AND TIGHE, J. R. Tubal metaplasia of the endometrium. J Obstet Gynaec Brit Comm 74:93, 1967. 9. MADDI, F. V., AND PAPANICOLAOU, G. H. Diagnostic significance of ciliated cells in human endometrial tissue cultures. Amer J Obstet Gynec 82:99, 1961. 10. SCHUELLER, E. F. Ciliated epithelia of the human uterine mucosa. Obstet Gynec 31:215, 1968.

August 1972 SCANNING ELECTRON MICROSCOPY 521 11. FLEMING, S., TWEEDDALE, D. N., AND RODDICK, J. W., JR. Ciliated endometrial cells. Amer J Obstet Gynec 102:186, 1968. 12. SCHUELLER, E. F. Epithelien und stromazellen des menschlichen endometrium. Arch Gynaek 196:49, 1961. 13. ANDERSON, R. G. W., AND BRENNER, R. M. The formation of basal bodies (centrioles) in the rhesus monkey oviduct. J Cell Bioi 50:10, 1971. 14. BRENNER, R. M., "The Biology of Oviductal Cilia." In The Mammalian Oviduct, Hafez, E. S. E., and Blandau, R. J., Eds. Univ. Chicago Press, Chicago, 1969. 15. NOVAK, E., AND RUTLEDGE, F. Atypical endometrial hyperplasia simulating adenocarcinoma. Amer J Obstet Gynec 55:43, 1948. 16. SORVARI, T. E., AND LAAKSO, L. Histochemical investigation of epithelial mucosubstances in the uterine isthmus. Obstet Gynec 36:76, 1970.