Histological Evidence of Alternating Ovulation in Women A

Home , Follicular phase, Menstrual cycle, Ovulation

Histological evidence of alternating ovulation in women A. Gougeon and Brigitte Lef\l=e`\vre Physiologie et psychologie de la reproduction humaine, INSERM U-187, 32 rue des Carnets, 92140 Clamart, France

Summary. Detailed histological analysis of ovaries obtained from 25 women by ovariectomy for medical reasons demonstrated that the degeneration of the cyclic corpus luteum is a long process, taking several cycles. The ages of the various regressing corpora lutea were identified to permit classification of the side of ovulation during the preceding 4 or 5 cycles. In the 25 women studied, with regular menstrual cycles of 28 \m=+-\3 days (48 cycles), ovulation occurred most often (87\m=.\6%)in an alternating manner.

Introduction

It is not known whether the single follicle that ovulâtes in each cycle of primates does so by chance in either of the two ovaries or is influenced by the side of ovulation of the preceding cycle. The follicle destined to ovulate and/or the cyclic corpus luteum could influence follicular development in such a way that the selection of the dominant follicle is not by chance. Riihl (1925) suggested that ovulation in women was alternate, but his results, obtained from post-mortem ovaries, were not quantitative or illustrated. The question is also unresolved in non-human primates. Wallach, Virutamasen & Wright (1973) and Clark, Dierschke & Wolf (1978) reported that ovulation occurred by chance in either ovary, uninfluenced by the side of ovulation in the previous cycle in the rhesus monkey. However, Dukelow (1977) and Hodgen (1982) consider that ovulation in the cynomolgus monkey and rhesus monkey, respectively, occurs alternately as long as the cycles are not perturbed. The present study was undertaken to determine whether human ovulation is alternate and was based upon histological examination of pairs of ovaries. The studies of Corner, Bartelmez & Hartman (1936) and Corner (1942) have shown that the ages of involuting corpora lutea in rhesus monkeys can be determined. In the normal human ovary, this assessment is facilitated by the absence of the corpora aberrantia that occur in the rhesus monkey ovary (Corner, 1942) and the identification of corpora lutea of different ages permits deduction of the side of ovulation during the preceding cycles.

Materials and Methods Sampling Pairs of ovaries were obtained during gynaecological surgery (ovariectomy for carcinoma of the breasts or cervix, hysterectomy for fibroids). They were fixed in Bouin's fluid or a mixture of alcohol, formaldehyde and acetic acid, processed by routine histological methods and then serially sectioned at 10 pm.

Downloaded from Bioscientifica.com at 10/04/2021 04:33:08AM via free access Ovarian function was considered to be normal after verification of (i) the absence of morphological pathology of the ovary; (ii) regular cycles, i.e. 28 + 3 days, determined over the 2 previous cycles, and (iii) the occurrence of ovulation in each of the preceding 3 cycles, corroborated by the presence of corpora lutea at various stages of degeneration (see below). After verification of these criteria 25 pairs of ovaries were retained for study. The women were aged 19-52 years (mean + s.d. = 40-0 ± 8-5 years).

Calculations

Corpus luteum dimensions were measured using an eyepiece micrometer. The diameter was taken as the mean of three measurements: the first two being made at right angles to each other in the section showing maximal corpus luteum area and the third was the number of sections in which the corpus luteum was present, multiplied by 0-01 mm (the thickness of each section). Cellular and nuclear diameters were measured using an eyepiece micrometer and an oil immersion 100 objective.

Methodology Histological examination of the 50 ovaries revealed the presence of large structures (2-10 mm in diameter) at various stages of involution. Comparison of the histological appearance of the functional corpus luteum of the cycle and the least involuted of these structures revealed similar histological characteristics, indicating that these involuting structures are corpora lutea at different stages of regression. All the ovaries taken during the same half of the cycle (i.e. follicular or luteal) showed an identical number of regressing corpora lutea (5 and 4 respectively), the difference indicating that the corpora lutea in samples taken during the follicular phase were, on average, half a cycle younger than those in luteal phase samples (Text-fig. 1). The oldest recognizable corpus luteum in the follicular phase therefore represents the last regression stage that can be dated reliably ; the ages of older corpora lutea cannot be estimated due to their transformation into corpora albicantia.

Follicular 0 5 1-5 phase 2 5 3-5 4-5

ma _ _ _ [ _ F CL 2a CL 3a CL 4a CL 5a CL 6a CL1 CL 2b CL 3b_CL 4b CL 5b "ET w

luteal phase Text-fig. 1. Age of the involuting corpus luteum observed in the samples obtained during the follicular or luteal phases of the menstrual cycle, m = menstruation.

In each of the 25 pairs of ovaries, the 4 or 5 involuting corpora lutea could be distinguished from each other by their morphological characteristics, although fragmentation of the oldest corpora lutea, caused by follicular development, may have made estimation of their age more difficult, particularly from the stage 4a (Table 1). Each corpus luteum may therefore be considered as representative of a stage of luteal regression. These various stages of regression, showing the same morphological characteristics, were seen in all the samples taken in the same halfof the cycle (PI. 1, Figs 1-6; PI. 2, Figs 7-12). Downloaded from Bioscientifica.com at 10/04/2021 04:33:08AM via free access =^ ei·? c>? =>? e£> m en — t -S —e <3\ — o CN OV o o ° 6 ó s C c +IS +IS +,: +1 « te +18 +i» +1 ?"ef-C 9 c -r c •T- c " ? c eò en 3 en 3 en 3 "* 2 S

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Downloaded from Bioscientifica.com at 10/04/2021 04:33:08AM via free access The findings indicate the existence of an ovulation during each of the 4-5 cycles preceding ovariectomy. If this had not been the case then at least one involuting corpus luteum would have been absent, reflecting the existence of an anovulatory cycle. Such anovulatory cycles do exist in women. In several other ovaries that we have studied one or more involuting corpora lutea were missing, but since the aim of the present study was the investigation of normal folliculogenesis, which implies an ovulation in each cycle, such samples were excluded from the study material. After determination of the ages of the involuting corpora lutea it was necessary to identify the ovulating ovary during the cycle of ovariectomy. This ovary showed the following characteristics : (i) during the first half of the follicular phase it was the ovary that contained the largest healthy follicle (Gougeon & Lefèvre, 1983); (ii) during the second half of the follicular phase it was the ovary that contained a large healthy follicle which in turn showed the histological characteristics of follicles destined to ovulate (Bomsel-Helmreich et al, 1979); and (iii) during the luteal phase it was the ovary that contained the most recent corpus luteum. Together, the data relating to the side of ovulation during the cycle of ovariectomy and the ages and localization of the involuting corpora lutea permitted easy determination of the side of ovulation during the preceding 4 or 5 cycles.

Results

The side of ovulation was analysed for 5 consecutive cycles in 13 women operated on during the follicular phase, and for 4 consecutive cycles in 12 women operated on during the luteal phase (Table 2). The mean duration of the cycles in the whole group of 25 patients was 27-8 + 31 days (mean + s.d., 48 cycles).

Table 2. Distribution of contralateral and ipsilateral ovulations during 4 or 5 consecutive cycles in 25 women

Corpus luteum stage CL2 CL3 CL4 CL5 CL6

No. of contralateral ovulations* 20 22 22 23 12 No. of ipsilateral ovulations* 5 3 3 2 1

* Compared with corpus luteum or the dominant follicle.

PLATE 1

Figs 1 & 2. CL 1 : a cyclic corpus luteum (Day 20-24 of a 28-day cycle). The central cavity is filled with a fibrin network (f.n.), and the luteal cell nuclei are spheroidal with many visible nucleoli. Unlike the theca lutein cells (th.l.c), the granulosa lutein cells (gr.l.c.) are hypertrophied. Figs 3 & 4. CL 2a : the fibroblasts (fb) have replaced the fibrin network in the central cavity and the luteal cells show spherical nuclei and very vacuolated cytoplasm. While the size of the granulosa lutein cells (gr.l.c.) is greatly reduced, they still remain easily distinguishable from the theca lutein cells (th.l.c). Figs 5 & 6. CL 3a : the central cavity is partly obscured by fibrohyalin substance (f.h.s.). Theca lutein cells (th.l.c.) conserve their spherical nuclei, unlike the granulosa lutein cells (gr.l.c.) whose nuclei are shrunken and dark after condensation of the chromatin. Downloaded from Bioscientifica.com at 10/04/2021 04:33:08AM via free access PLATE 1

(Facing p. 10)

Downloaded from Bioscientifica.com at 10/04/2021 04:33:08AM via free access PLATE 2

Downloaded from Bioscientifica.com at 10/04/2021 04:33:08AM via free access From a total of 113 cycles analysed there were 14 ipsilateral and 99 (87-6%) contralateral ovulations. Therefore, alternate ovulation in the human female showing regular menstrual cycles is the general rule (P < 0-001, 2 test). Among the 25 women studied, 14 always ovulated in an alternate manner, 8 showed one ipsilateral ovulation, and 3 showed two ipsilateral ovulations.

Discussion

The present study has been based on histological observations of human ovaries. The presence of an identical number of involuting corpora lutea (5 when ovariectomy was performed in the follicular phase, and 4 when it was during the luteal phase) showing the same morphological characteristics is clear proof of the formation of a corpus luteum during each of the cycles before ovariectomy. However, it was not possible to ascertain whether true ovulation, i.e. follicular rupture with the release of a mature oocyte, actually occurred. The 'luteinized unruptured follicle' syndrome has been proposed to explain some cases of sterility (Marik & Hulka, 1978). This defect cannot be detected from basal body temperature charts or from serial peripheral hormone analyses since the secretion of progesterone and oestradiol-17ß by a luteinized follicle cannot be distinguished from that by a cyclic corpus luteum (Marik & Hulka, 1978). Only serial ultrasono- graphic examinations during the periovulatory period (Coulam, Hill & Breckle, 1982) and the measurement of progesterone and oestradiol-17ß in peritoneal fluid (Koninckx, de Moor & Brosens, 1980) would permit confirmation offollicular rupture. Unfortunately, for clinical reasons, it is difficult to perform these two techniques in several consecutive cycles. Consequently, histo¬ logical observations represent the only feasible source of information. Our present results show that in women with tegular menstrual cycles of about 28 days, ovulation would usually alternate between the two ovaries. Di Zerega & Hodgen (1982) have shown that after extirpation of the corpus luteum in the rhesus monkey the subsequent ovulation occurs in the ovary secreting the least progesterone. At the end of the luteal phase of a normal unperturbed cycle, the ovary containing the cyclic corpus luteum probably has a greater intra-ovarian concentration of progesterone than does the contralateral ovary. Since progesterone has the ability to slow follicular growth in an ovary that is synthesizing it, or in an ovary containing an implant (rhesus monkey: Resko, Norman, Niswender & Spies, 1974; Clark et al, 1981 ; human: Hoffman, Kayser & Bergk, 1970), follicles present in the ovary bearing the corpus luteum may therefore show retarded growth with respect to the follicles present in the contralateral ovary. Koering, Baehler, Goodman & Hodgen (1982) provided some support for this hypothesis, having shown that 4 days after removal of the corpus luteum in the rhesus monkey the contralateral ovary contained significantly more medium-sized healthy follicles than did the ovary that had contained the corpus luteum. However, Nilsson, Wikland & Hamberger (1982) showed that removal of corpus luteum from women in the early luteal phase was associated with the next ovulation in either ovary. Two hypotheses may be envisaged to explain how ovulation can occur consecutively in the same ovary.

PLATE 2

Figs 7 & 8. CL 4a : the central cavity has been filled with fibrohyalin substance (f.h.s.) which has also begun to invade the intercellular spaces. Distinction between granulosa and theca lutein cells is no longer possible, and degenerate luteal cells (d.l.c) are filled with many small droplets of lipidie material (l.d.). The edges of these cells remain visible. Figs 9 & 10. CL 5a : cellular boundaries are no longer visible, only numerous condensed nuclei (en.) remain and the luteal tissue has given way to a mass of fibro-hyalin substance (f.h.s.). Figs 11 & 12. Cl 6a: some condensed nuclei (c.n.) and cellular debris scattered among a fibro¬ hyalin mass (f.h.s.) occupy the place of the degenerate corpus luteum. Downloaded from Bioscientifica.com at 10/04/2021 04:33:08AM via free access Firstly, at the time of the spontaneous demise of the cyclic corpus luteum some ovarian tissues (stroma, interstitial gland tissue, regressing corpora lutea) of the contralateral ovary may be synthesizing an amount of progesterone equivalent to that synthesized by the ovulatory ovary. This would result in the populations of follicles in the two ovaries being subjected to similar endocrinological conditions. Secondly, at the start of the cycle the selected follicle, although situated in the contralateral ovary, might cease growing for some unknown reason and be replaced by a follicle situated in either ovary. Ovulation might thereby occur in the ovary bearing the corpus luteum of the previous cycle. The consequences and significance of the alternate nature of primary ovulation are unclear. In rhesus monkeys ovulation occurs in either ovary, with no relationship to the side of ovulation in the preceding cycle (Wallach et al., 1973). However, the duration of the cycle was related to the side of ovulation: when ovulation occurred twice in succession from the same ovary, the mean + s.d. duration of the cycle (46-0 + 10-0 days) was significantly greater (P < 0-01) than when ovulation occurred alternately (33-5 + 4-0 days). In the present sample of women showing a high level of alternate ovulations the mean cycle length of 27-8 days was very close to the theoretical 28-days duration of the human menstrual cycle. In conclusion, alternate ovulation, which may have its origin in a progesterone gradient favouring those follicles situated in the ovary not containing the cyclic corpus luteum, would enable maintenance of a regular cyclicity close to the 28 days characteristic of the primate ovarian cycle. Kinetic analysis of follicular growth in the two ovaries during the late luteal phase, comparison of consecutive cycle lengths as a function of the side of ovulation, and also serial ultrasonography, should be performed in women to test the hypothesis originating from experimental studies carried out in non-human primates.

This study was supported by grants from the University Paris Sud U.E.R. médicale Kremlin- Bicêtre (Grant no. 797) and from the Institut National de la Santé et de la Recherche Médicale (INSERM). We thank Mlle M. C. Levasseur and Dr J. de Mouzon for their advice, Dr D. Mortimer for translating the manuscript, and Dr E. Martin, Dr H. Mazabraud, Dr E. Papiernik and Dr M. Prade for the clinical material.

References

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