Cyclic changes in ciliation, cell height, and mitotic activity in human tubal epithelium during reproductive life

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1 FERTILITY AND STERILITY Copyright" 1985 The American Fertility Society Vol. 43, No.4, April 1985 Printed in U.SA. Cyclic changes in ciliation, cell height, and mitotic activity in human tubal epithelium during reproductive life Jacques Donnez, M.D. * Fram;oise Casanas-Roux, B.S. Jacques Caprasse, M.D. Jacques Ferin, M.D. Karl Thomas, M.D. Physiology of Human Reproduction Research Unit, Department of Obstetrics and Gynecology, University of Louvain, Brussels, Belgium The percentage of ciliated cells, the height, and the mitotic index of human oviductal epithelium were studied during the menstrual cycle and under progestogens. A total of 141 fallopian tubes were examined. During the menstrual cycle, a process of ciliation and deciliation was observed. Maximal ciliation was attained around the time of ovulation, particularly in the fimbria where significant differences were noted. Deciliation was observed under progestogen therapy, but this progestogen effect was easily reversible. The decrease of epithelial height observed after ovulation was also found after progestogen therapy. Cyclic mitotic activity was noted during the menstrual cycle. Estrogens influence the mitotic activity of tubal epithelium, whereas endogenous progesterone and progestogens inhibit this estrogenic effect. Fertil Steril43:554; 1985 Both secretory and ciliated cells are consistently present in the normal epithelium of the oviducts. Although it is widely accepted that tubal epithelium undergoes morphologic changes during the ovarian cycle, there is disagreement concerning specific changes in the structure and dis~ tribution of the ciliated cells during the cycle. 1-4 Oberti and Gomez-Rogers 5 and Verhage et a1. 4 observed ciliogenic cells during the proliferative phase of the menstrual cycle and suggested that ciliation was influenced by 17J3-estradiol. Experimental studies 6, 7 have shown that administration of estrogens to castrated monkeys induces ciliogenesis. In a recent study, we proved that estrogens strongly influence the process of ciliation and deciliation of oviductal epithelium after the menopause. 8 The aim of this investigation was to study the variations in the percentage of ciliated cells, the height, and the mitotic index of human oviductal epithelium during the menstrual cycle and under progestogen therapy. Received September 14,1984; revised and accepted December 19, *Reprint requests: Jacques Donnez, M~D., Physiology ofhuman Reproduction Research Unit, Department of Obstetrics and Gynecology, Universi~y of Louvain, Avenue Em. Mounier, 53, 1200 Brussels, Belgium. 554 Donnez et ai. Cyclic changes in human tubal epithelium MATERIALS AND METHODS A total of 141 fallopian tubes were obtained from a sample of subjects comprising fertile women with regular ovulatory cycles (group I); women receiving daily progestogen therapy (lynestrenol, 5 mg/day, Organon, Oss, The Netherlands) for Fertility and Sterility

2 Table L Classification of Fallopian Tubes According to the Menstrual Phase or the Duration of Progestogen Therapy Group Menstrual phase or medication No; of tubes Menstrual cycle 98 Early follicular phase 8 (days 1-6) Late follicular phase 38 (days 7-13) Early luteal phase (days ). Late luteal phase (days ) II Progestogen therapy 31 1 to 3 months 17 > 1 year 14 III Progestogen therapy taken 12 for. 1 year and stopped 1 month before surgery uterine myomata (group 11); and women in whom progestogen therapy, taken for 1 year, was stopped 1 month before surgery (group III) (Table 1). Patients with a medical history of salpingitis or with endometriosis were excluded from the study. The indications for operation were sterilization, pedunculated serous myomata, in situ epithelioma of the cervix, and prolapse. At the time of surgery, endometrial biopsy and blood samples were collected for confirmation of the tubal biopsy dating and assessment of the luteal function. Women with luteal phase deficiency were excluded from the study. In women in whom progestogen therapy was stopped 1 month before surgery, only fallopian tubes removed in the proliferative phase were studied. Tubal biopsy specimens were taken from the fimbria, ampulla, and isthmus. Biopsies were performed on the side opposite the broad ligament, at the fimbrial edge, the midampulla, and the midisthmus. After fixation and embedding, 2-j.Lmthick sections. were stained with Gomori trichrome or toluidine blue. The percentage of ciliated cells. was determined on a "blind basis" by counting the number of ciliated cells per 1000 epithelial cells with a Wild M 501 microscope (Wild, Heerbrugg, Switzerland) with diascopic vision. Cell height was measured with an ocular micrameter. Fifty cells were selected in which the plane of section clearly passed through the cell nucleus, parallel to the longitudinal axis of the cell. The mitotic index was calculated by counting mitotic figures (prometaphase, metaphase, anaphase, telophase) for 10,000 epithelial cells. This is the only method available in women because administration of colchicine or tritiated thymidine is not ethical. Results were expressed as the mean ± standard deviation. The Wilcoxon test and median test were used for statistical analysis. RESULTS PERCENTAGE OF CILIATED CELLS In each group (Fig. 1), the highest percentage of ciliated cells was found in the fimbria. A significantly lower percentage (P < 0.001) was observed in the ampulla. The percentage in the isthmus was significantly lower (P < 0.001) than in either the fimbria or the ampulla. During the menstrual cycle, the highest percentage of ciliated cells was found around the FIMBRIA MeNSTRUAl.: CYCLE._.11IUAL CYCLE EO...a _.- AMPULLA COlI"""'".. OG 'MIRa'" IT""'! - -.' ;"t.., '7.3 '62 t8.3 til '710!l5.2 t6.8 "=8 "=]6 ":26 nz22 "=17 n:fs; "='2 Results are.xpr.ss.d as man. SO Prog. stogen, 'Iken during mor., than on. re... was stopped on, month before surger.. Figure 1 Percentage of ciliated cells (mean ± standard deviation) in human tubal epithelium during the menstrual cycle and under progestogen therapy. Vol. 43, No.4, April 1985 Donnezet at Cyclic changes in human tubal epithelium 555

3 time of ovulation in all tubal portions. A significant (P < 0.01) increase in the percentage of ciliated cells was observed in the fimbria during the late follicular phase. In both the ampulla and the isthmus, maximal ciliation was observed during the late follicular and early secretory phases. In the three tubal portions, some deciliation had occurred by the end of the menstrual cycle. For women who took progestogens for 1 to 3 months, the percentages of ciliated cells were lower than those observed during the menstrual cycle but similar to those observed during the first phase of the menstrual cycle. Significant deciliation (P < 0.001) occurred in the three tubal portions of women who took progestogens for > 1 year when compared with the menstrual cycle. Similar significant (P < 0.001) differences were noted for the secretory phase of the menstrual cycle. A significant decrease (P < 0.01) was also observed in both the fimbria and the ampulla when the comparison was made between women taking progestogens for 1 to 3 months and women taking progestogens for > 1 year. Significant reciliation (P < 0.01) was observed in both the fimbria and ampulla when progestogens, taken for> 1 year, were stopped 1 month before surgery. EPITHELIAL HEIGHT During the menstrual cycle (Fig. 2), significant variations were noted. The epithelial height increased significantly (P < 0.005) during the late follicular phase and decreased significantly (P < 0.005) after ovulation. The cyclic variation observed was the same in the three tubal portions. During the different phases, the isthmus appeared slightly higher than either the ampulla or the fimbria. For women who took progestogens for 1 to 3 months, the epithelial height was significantly different (P < 0.001) from that observed during the late follicular phase but similar to that found during the early follicular phase. When compared with the early and late secretory phases, significant differences (P < 0.05) were noted only in the ampulla. For women who took progestogens for> 1 year, the epithelial height decreased significantly (P < 0.001) when compared with values observed during the menstrual cycle. Significant (P < 0.01) differences were also noted in comparison with women receiving progestogens for 1 to 3 months. For women in whom progestogens, taken for > 1 year, were stopped 1 month before surgery, a significant (P < 0.001) increase of epithelial height was observed when they were compared with women taking this drug for> 1 year. MITOTIC INDEX Mitosis was quite evident in the tubal epithelium (Fig. 3). Figure 4 shows the mitotic index mean and median values in the three tubal portions. During the menstrual cycle, significant differences were noted in both the fimbria and the ampulla. Indeed, the mitotic index is significantly (P < 0.01) higher during the follicular phase than during the luteal phase. In the isthmus, differences were also noted but not significant. Progestogen, taken during more than 1 yell', was stopped on. month before SUfg'fY Results a', I. pressed as mean t SO Figure 2 Epithelial height (mean ± standard deviation) of human tubal epithelium during the menstrual cycle and under progestogen therapy. 556 Donnez et ai. Cyclic changes in human tubal epithelium Fertility and Sterility

4 DISCUSSION Figure 3~ Jiuman tubal epithelium (day 13). Mitosis was observed in two ciliated cells (arrows). For women who took progestogens for 2 to 3 months or> 1 year, the mitotic index was similar to that observed during the luteal phase of the menstrual cycle but significantly lower (P < 0.01) than that found during the follicular phase. For women in whom lynestrenol was stopped 1 month before surgery, the mitotic index in both the ampulla and the fimbria was similar to that observed during the follicular phase and significantly different (P < 0.01) from that observed in women who took progestogens for 2 to 3 months or> 1 year. Results.re pr... d as median I I :m n..un Vol. 43, No.4, April 1985 During the menstrual cycle, the oviductal epithelium attained maximum ciliation around the time of ovulation, although the difference between maximum and minimum ciliation was slight. The process of ciliation and deciliation observed in this study, particularly in the fimbria, where significant differences were noted, confirms the earlier observations of Verhage et a1. 4 but is in disagreement with other reports. 2, 9-11 An explanation of some of the discrepancies could be that, in some of the previous studies, only two phases of the menstrual cycle were compared: the follicular and the luteal phases. The cyclic process of ciliation and deciliation has been observed in other species. In dogs and cats,12, 13 total deciliation occurred by parturition, whereas reciliation was noted during proestrus and estrus. In rhesus monkeys6 and pigtailed macaques,14 the fimbria undergoes almost complete deciliation followed by reciliation with each cycle. The administration of estrogens to postpartum women increased the number of ciliated cells in the oviducts and progestogens inhibited this estrogenic effect. 15 Recently, we proved that significant deciliation occurred in late menopause but was prevented by estrogen substitution. 8 Moreover, the administration of estrogens to postmenopausal women increased the number of ciliated cells. The estrogens seem to influence strongly the process of ciliation of the oviductal epithelium. In the present study, the data found during the late luteal phase of the menstrual cycle and in Figure 4 Mitotic index mean and median values (%00) of human tubal epithelium during the menstrual cycle and under progestogen therapy. Donnez et ai. Cyclic changes in human tubal epithelium 557

5 women taking progestogens show that progesterone and progestogens provoke a decrease in ciliation of the oviductal epithelium. The deciliation observed after 2 to 3 months of progestogen therapy increased to attain significantly lower values after 1 year of treatment. This deciliation effect is easily reversible. Indeed, 1 month after cessation of treatment, a significant increase in the percentage of ciliated cells was noted. Our observations of a ciliation and deciliation process during the menstrual cycle and of a deciliation effect by progestogens support the view that estrogens are the key promoters of the ciliogenesis in the oviduct, whereas progestogens antagonized this estrogenic effect in spite of the continued estradiol in the system, as has been reported in subhuman primates7 and cats.13 Cyclic variations in the epithelial height were reported for the first time in and later confirmed by many investigators. The maximum height observed before ovulation coincided with maximum secretory activity.4 After ovulation, there was a significant decrease corresponding to the loss of secretory activity. Maximum height was seen in the isthmus during each phase of the menstrual cycle. This is in agreement with the data reported by Jansen,16 who found tenacious luminal isthmic mucus in the fallopian tubes of women in the late follicular phase and immediately after ovulation. Significant decrease in cell height was also observed in women taking progestogens, as noted in women on continuous low-dose oral progestin contraception. 17, 18 Variations of cell height found in our study support the hypothesis of the antiestrogen effect of progestogens in the human tubal epithelium. The striking absence of mitosis in the human tubal epithelium has been mentioned by many investigators; and so signs of mitotic activity in the oviduct were one of the criteria on which malignancy was based. 19 In a recent work,20 we reported an increase of mitotic activity in the tubal isthmus after sterilization, when compared with normal isthmi. For evident ethical problems, neither the colchicine technique nor the in vivo method of tritiated thymidine were applicable to humans. By counting the number of mitotic figures on 10,000 epithelial cells, we were able to find a cyclic variation of mitotic activity in the human tubal epithelium. In both the fimbria and the ampulla, the mitotic index was significantly higher during the follicu- 558 Donnez et al. Cyclic changes in human tubal epithelium lar phase than during the luteal phase. This is the first report of a relationship between tubal epithelium renewal and the endogenous hormones. Estrogens seem to influence the mitotic activity of tubal epithelium, whereas progesterone partially inhibits this effect. In women taking progestogens, the mitotic index was very low, and this observation could explain the Significant deciliation observed in this group. When the administration of progestogens was stopped 1 month before surgery, a significant increase of the mitotic activity was found, suggesting that progestogens, when present, were able to block the mitotic activity controlled by estrogens in spite of the continued presence of estrogens in the system. However, this antiestrogenic effect of progestogen therapy is easily reversible, as confirmed by data found in women in whom therapy was stopped 1 month before surgery. In conclusion, ciliation, increase of epithelial height, and increased mitotic activity occurred during the follicular phase when serum progesterone was essentially undetectable; whereas deciliation, decrease of epithelial height, and loss of mitotic activity coincided with elevated levels of serum progesterone. Continuous progestogen therapy exerts the same antiestrogenic effect as observed during the secretory phase of the menstrual cycle. Therefore, it seems reasonable to conclude that the induction and maintenance of a mature tubal epithelium in women are strictly controlled by estrogens, whereas this effect is partially antagonized by progestogens. REFERENCES 1. Novak E, Everett HS: Cyclical and other variations in the tubal epithelium. Am J Obstet Gynecol 16:499, Patek E, Nilsson L, Johanisson E: Scanning electron microscopic study of the human fallopian tube. Report I. The proliferative and secretory stages. Fertil Steril 23:459, Seki K, Rawson J, Eddy CA, Smith NK, Pauerstein CJ: Deciliation in the puerperal fallopian tube. Fertil Steril 29:75, Verhage HG, Bareither ML, Jaffe RC, Akbar M: Cyclic changes in ciliation, secretion and cell height of the oviductal epithelium in women. Am J Anat 156:505, Oberti C, Gomez-Rogers C: "De novo" ciliogenesis in the human oviduct during the menstrual cycle. In Biology of Reproduction: Basis and Clinical Studies, Edited by JT Velardo, BA Kraspow. Presented at the Symposium on Biology of Reproduction, m Pan American Congress of Anatomy, 1972, New Orleans Fertility and Sterility

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