Follicular Depletion during the Menopausal Transition SANDRA J. RICHARDSON" AND JAMES F. NELSONh Department of Medicine, and the Department of Obstetrics und Gynecology and Centre for Studies of Reproduction McGill University Montreal, Quebec
INTRODUCTION Although the menopause, the final cessation of menses, is the most dramatic biomarker of the reproductive aging process in women, age-related alterations in the pattern of cyclicity, as well as in fertility, are ongoing from menarche to menopause. Population studies of menstrual patterns'.* document that for the first few years after menarche the menses tends to be irregular. Thereafter and throughout the middle reproductive years, cycles are regular, although they become progressively shorter with advancing age primarily owing to a shortening of the follicular phase. Finally, in the 2-8-year period preceding the menopause, cycles again become highly variable, ranging from very short to very long. This last period of irregular menses is called the perirnenopause. Fertility begins to decline many years prior to the onset of irregular menses, being maximum in the mid- to late twenties and decreasing steadily until menopause .3,4 Age-associated alterations in the ovary, the neuroendocrine system, and the uterus may all influence the changing pattern of menses as well as the declining f e r t i l i t ~This . ~ paper will focus on the role of ovarian changes in these reproductive aging processes, with particular attention to the declining ovarian follicular reserve. FOLLICLE NUMBER IN THE PERIMENOPAUSE AND MENOPAUSE The human ovary steadily loses follicles from mid-fetal life onward.5-XUntil recently, the number of follicles remaining in the ovary at the time of menopause was unknown. Previous studies of follicle counts in human ovaries had been restricted to children and to women who were still menstruating regularly. Block estimated follicle number in autopsy specimens from 43 children and women from 6 to 44 years of age who had died accidently.5 Gougeon studied surgical specimens from 35 women aged 20-52 with regular mensesx A straight-line projection from either of these studies predicted that between 2,000 and 5,000 primordial Isolated reports follicles would remain at 50 years, the mean age of of the presence of follicles in postmenopausal ovaries supported this hypothesis."" We have recently studied follicle number in ovaries obtained at surgery from 17 otherwise healthy women, aged 45-55 yrs.'? The purposes of the study were ( I ) to clarify the relationship between follicle number and the perimenopausal transi13
ANNALS NEW YORK ACADEMY OF SCIENCES
14
tion from regular to irregular menses and (2) to estimate follicle number at the time of menopause. The women were divided into three age-matched groups according to the pattern of their menses in the previous 12 months: (i) a regular menstrual pattern [i.e., regular menses between 3 and 6 weeks without hot flashes]; (ii) a perimenopausal pattern [i.e., irregular menses with intervals of less than 3 weeks or more than 6 weeks for at least a year, with or without hot flashes]; and (iii) a postmenopausal pattern [i.e., no menses for at least a year]. Each ovary was sectioned at 10 microns (850-3,000 sections per ovary). Estimates of total follicle count were made by counting all the follicles in 10 consecutive sections in I , the mean number of primordial follicles in every 100. As can be seen in FIGURE the ovaries of women who were still menstruating regularly was ten-fold greater than that in the ovaries of perimenopausal women of the same age; there was little overlap in the range of follicle counts between the two groups (1392 k 355 vs. 142 & 72). Follicles were virtually absent from the ovaries of the four postmenopausal
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FIGURE 1. The relationship between primordial follicle number and menstrual status in age-matched women, 45-55 years of age ( n = 17). The effect of menstrual status on follicle number was significant ( p < 0.001, by ANOVA: age was the covariate). Mean follicle counts in each group differed from those in the other groups ( p < 0.05, by the Student-NewmanKeuls test). (From Richardson ei al." Reproduced by permission.)
women. These observations indicate that the size of the follicular reserve is the major determinant of both the transition from regular menses to the perimenopause as well as to the menopause itself. Are there any follicles remaining in the human ovary at the time of menopause? This can be determined by extrapolating from the perimenopausal and postmenopausal follicle counts in this study and reviewing the anatomic and hormonal studies of others.s",13-'8 The paucity of follicles in many of the perimenopausal ovaries together with the absence of follicles in all of the postmenopausal ovaries suggest that, at least in some women, few, if any, follicles remain at menopause. This does not guarantee, however, that the ovaries of all women are completely depleted of follicles by the last cycle. A few hundred follicles were present in some of the perimenopausal ovaries. The virtually depleted postmenopausal ovaries were all from women more than three years past their last menses. There have been reports of follicles seen in postmenopausal ovaries. Saunrama observed an unspecified number of primordial and growing follicles, all
RICHARDSON & NELSON: FOLLICULAR DEPLETION
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showing evidence of degeneration, in the ovaries of two women, 3 and 5 years post m e n o p a ~ s eGuraya .~ also saw “some follicles” in the ovaries of six women, 45-52 years of age who were 1-5 years post menopause.I0 Costoff and Mahesh reported postmenopausal primordial follicles in all ovarian samples studied. I They did not, however, give the number of cases, their ages, the time from menopause, or the number of follicles seen. Most provocative are two reports of “fresh corpora lutea” in a 53-year-old woman 3 years post menopausei3and in a 63-year-old woman, 12 years post menopause.I4 Hormonal studies also indicate that follicles capable of secreting estrogen persist in some women in the early post menopause. In a longitudinal study, Metcalf studied weekly urinary hormone excretion in eight women from 5-15 weeks before to 22-30 weeks after their final menstruation.I5 She observed eight episodes of elevated estrogen excretion compatible with follicular development in five women during the postmenopausal period. Although ovulatory cycles occurred as late as the final menses in three women, there was no evidence of ovulation after menopause. Cross-sectional studies of blood hormone levels over the months after menopause suggest that from 20-40% of women have estrogen levels indicative of follicular function during the first 6- 12 months after the cessation of m e n ~ e s . ’ ~By , ’ ~12 to 24 months, the estrogen levels have fallen into the menopausal range in almost all subjects.’”’* JuddI9 and VermeuelenZ0found no evidence of estradiol secretion by the ovaries of women who were more than 3 years post menopause. Longcope” and Lucisano,22on the other hand, observed that the ovaries of some women, who were also at least 3 years past their last menses, secreted significant amounts of estradiol. This may be explained by the recent evidence that the amount of estradiol secreted by the ovaries of women who were at least 2 years post menopause correlated with the degree of ovarian stromal hyperplasia.” No mention was made of the presence of follicles in these ovaries. In summary, when the current hormonal and anatomic evidence is compiled, it would appear that, while the ovaries of some women are virtually depleted of follicles when menses ceases, others still have some follicles remaining for a year or more after menopause which are capable of secreting estrogen but, with rare exception, incapable of ovulating.
DECLINING FOLLICULAR RESERVES AND AGE-RELATED INFERTILITY Are the follicles that remain in the human ovary as the reserve nears exhaustion normal or defective? While the causes of the declining fertility rate24 and rising incidence of congenital anomalies25with advancing maternal age are not known, it is possible that age-related defects in the remaining follicles play a role. Such a hypothesis remains speculative, however, in view of the difficulty in distinguishing the intrinsic changes in the ova from such confounding factors as male fertility, frequency of intercourse and age-related changes in t h e hormonal milieu of the As long as menses remains regular, women over 40 continue to ovulate consistently, that is, in more than 95% of cycles.26Luteal phase progesterone profiles in women over 45 are identical to those of younger women.27The only aberration noted in these women is a slightly higher incidence of cycles with a shortened luteal phase.*SIn the perimenopause, however, the proportion of ovulatory cycles drops to 60%.26
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The technology of “assisted fertilization” provides the opportunity to study the effect of maternal age at successive steps of the fertilization process. Several studies have addressed the issue of fertility in women over 40 years of age.2s33 The technique of superovulation used for in uitro fertilization or gamete intrafallopian transfers (GIFT) produces either the same29or a slightly lower number of follicle^^^.^' in women over 40 compared to younger women. The ability of the ova from women over 40 to be fertilized also appears to be undiminished.*’,” Despite apparently normal ovulation and fertilization rates in older women, fecundity, or the ability to become pregnant, is reduced. Pregnancy rates in uitro fertilization and GIFT programs are diminished in women over 40 in most,30,32.33 but not reports. In these procedures, however, the effect of maternal age cannot be separated from that of paternal age as, in most cases, older women tend to have older partners. However, the CECOS report of their experience in artificial insemination by donor (AID) shows an age-associated decline in the reproductive capacity of the women independent of such variables as male fertility or ~ ’ reported the relationship between materfrequency of sexual i n t e r c o ~ r s e . They nal age and fecundity in 2,193 women undergoing artificial insemination with sperm from young fertile donors. Implantation of the embryo, as indicated by rising circulating hCG levels after 12 attempts, was successful in 73% of women under 31,62% of women 31-35, and only 54% of women over 35. The 16 women over 40 also had a pregnancy rate of 54% after twelve cycles. The subsequent abortion rate was not reported in this study. It is, however, consistently higher in women over 40 than in younger women (50-60%) in both in uitro fertili~ation?’,~~ and GIFT,)O as it is in natural p r e g n a n c i e ~ . ~ Despite the insights provided by the technology of assisted fertilization into the relationship between fertility and maternal age, the contribution of the aging follicle to the decreased fecundity, elevated abortion rate, and higher incidence of congenital anomalies observed in older women must remain speculative. While the rate of chromosome abnormalities in the ova of women over 35 is considerably higher than in those less than 35 (47% vs. 25%),34it is presently not possible to isolate the role of the ovum from that of other potential influences such as the sperm or the endometrium.
THE RATE OF FOLLICULAR DEPLETION IN THE HUMAN OVARY FIGURE 2 shows the data from the three quantitative studies of primordial follicles in human ovaries. Note that the follicle counts from the ovaries of women with regular cycles from the three studies fall within the same range. There is also a striking acceleration in the rate of follicle loss in the last decade before menopause. This accelerated loss can be seen even in the older women still having regular cycles. This observation has been made previously in separate analyses of Block’s and can also be seen in Gougeon’s data.x The mechanism responsible for this apparent increase in the rate of follicular depletion is unknown. It is noteworthy that the age at which the rate of follicular depletion begins to accelerate coincides with the age at which there is a selective increase in circulating FSH levels in women who are still menstruating regularly.374) We have hypothesized that this increased FSH stimulates a greater proportion of primordial follicles to enter the growing pool and subsequently become atretic. IZ Evidence to support such a hypothesis is drawn from animal data. Hypophysectomy, food restriction, and chronic administration of an analogue of LHRH, all of which have been shown to lower gonadotropin levels, retard the rate of follicular depletion in
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FIGURE 2. The relationship between age and primordial follicle number is compared using data from four studies. Follicle depletion appears to accelerate in the decade preceding menopause. * = Block’s studies of stillborns6and of girls and women with regular menses, aged 6-44 yrs’; x = extrapolation from Figure 8 in Gougeon’s study of women aged 20-52, all with regular menses8; and in the study of Richardson et d.”of women aged 45-55 years, + = women with regular menses, 0 = perimenopausal women, and 0 = postmenopausal women.
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FSH has been shown to increase recruitment of primordial rats and follicles in u i t r but ~ ~ not ~ in u ~ u o . ~ ~ Evidence that FSH affects the rate of folticular depletion in humans is circumstantial. Increased parity has been shown to be associated with a relative delay in the age of m e n o p a ~ s e . ~ If ~ .the ~ ’ exhaustion of follicular reserves is the major determinant of the onset of menopause, one could hypothesize that the cumulative time that FSH is suppressed during successive pregnancies slows the rate of follicular loss, which, in turn, delays menopause. Circulating FSH levels are also suppressed during oral contraceptive use. While there is no evidence that the use of oral contraceptives is associated with a later menopause, this may be due to the fact that the cohort of women using “the pill” in their twenties are only now reaching the age of menopause. Alternatively, one might expect the greatest effect of such use after the age of 40 when circulating FSH levels rise. Most women have stopped using oral contraceptives in their forties, however.
SUMMARY From mid-fetal life, the human ovary steadily loses follicles. While age-related alterations at the level of the hypothalamus-pituitary appear to determine the timing of menarche, the number of follicles remaining in the mature ovary is the major determinant of the timing of both the perimenopause and the menopause. As the residual follicle reserve nears exhaustion, some of the remaining follicles appear to be defective, as evidenced by the higher incidence of chromosomal abnormalities in the ova of older women. It is presently not possible, however, to distinguish the specific contribution of the ovum to the age-related decrease in fecundity and higher abortion rate because of such confounding variables as aging sperm or changes in the endometrium. When follicle counts from peri- and postmenopausal ovaries are placed with those from previous studies of younger women and children there appears to be an acceleration in the rate of follicle loss in the decade preceding menopause. We hypothesize that the elevated FSH levels observed in normal women in the decade preceding the menopause may be responsible for this apparent acceleration in the rate of follicle loss. REFERENCES 1.
2. 3. 4.
5. 6. 7. 8.
TRELOAR, A. E., R. E. BOYNTON, B. G. BEHN& B. W. BROWN.1967. Variation of the human menstrual cycle through reproductive life. Int. J . Fertil. 12: 77-127. VOLLMAN, R. F. 1977. Major Problems in Obstetrics and Gynecology. Vol. 7. Philadelphia, PA. W.B. Sdunders. FRANCIS. W. J. A. 1970. Reproduction at menarche and menopause in women. J . Reprod. Fertil. Suppl 12: 89-98. NELSON,J. F. & L. S. FELICIO.1985. Reproductive aging in the female: An etiological perspective. Rev. Biol. Res. Aging 2: 251-314. BLOCK,E. 1952. Quantative morphological investigations of the follicular system in women. Acta Anat. 14 108-123. BLOCK,E. 1953. A quantative morphological investigation of the follicular system in newborn female infants. Acta Anat. 17: 201-206. BAKER,T. G. 1963. A quantative and cytological study of germ cells in human ovaries. Proc. R. SOC.Lond. (Biol.) 158: 417-433. GOUGEON, A. 1984. Cardctkre qualitative et quantdtif de la population folliculaire dans I’ovaire humain adulte. Contracep. Fertil. Sexual. 12: 527-535.
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SAURAMA, H. 1952. Histology, histopathology, and function of the senile ovary. Ann. Chir. Gynaecol. Fenn. (Suppl. 1) 41: 1-62. GURAYA, S. S. 1976. Histochemical observation on the corpus luteum atreticum of the human postmenopausal ovary with reference to steroid hormone synthesis. Arch. Ital. Anat. Embriol. 81: 434-455. COSTOFF,A. & V. B. MAHESH.1975. Primordial follicles with normal oocytes in the ovaries of postmenopausal women. J . Am. Geriat. Soc. 23: 193-196. RICHARDSON, S. J., V. SENIKAS & J. F. NELSON.1987. Follicular depletion during the menopausal transition: evidence for accelerated loss and ultimate exhaustion. J. Clin. Endocrinol. Metab. 65: 1231-1237. NOVAK,E. R. 1970. Ovulation after fifty. Obstet. Gynecol. 3 6 903-910. DAWOOD,M. Y., M. STROGIN, E . E. KRAMER & R WIECHE.1980. Recent ovulation in a postmenopausal woman. Int. J . Gynaecol. Obstet. 18: 192-194. METCALF,M. G., R. A. DONALD& J. H. LIVESEY.1982. Pituitary-ovarian function before, during and after menopause: A longitudinal study. Clin. Endocrinol. (Oxford) 17: 489-494. RANNEVIK, G., K. CARLSTROM, S. JEPPSSON,B. BJERRE& L. SVANBERG. 1986. A prospective long-term study in women from pre-menopause to post-menopause: Changing profiles of gonadotropins, oestrogens and androgens. Maturitas 8: 297307. TREVOUX, R., J. DE BRUX,M. CASTANIER, K. NAHOUL,J.-P. SOUII & R. SCHOLLER. 1986. Endometrium and plasma hormone profile in the peri-menopause and postmenopause. Maturitas 8: 309-326. LONGCOPE, C., C. FRANZ,C. MORELLO, R. BAKER& C. C. JOHNSON.1986. Steroid and gonadotropin levels in women during the peri-menopausal years. Maturitas 8: 189- 196. JUDD,H. J., G. E. JUDD,W. E . LUCAS,& S. C. YEN. 1974. Endocrine function of the postmenopausal ovary: Concentrations of androgens and estrogens in ovarian and peripheral vein blood. J. Clin. Endocrinol. Metab. 39: 1020-1024. VERMEULEN, A. 1976. The hormonal activity of the postmenopausal ovary. J. Clin. Endocrinol. Metab. 42: 247-253. LONGCOPE,C., K. HUNTER& C. FRANZ.1980. Steroid secretion by the postmenopausal ovary. Am. J . Obstet. Gynecol. 138: 564-568. LUCISANO, A., M. G. ACAMPORA, N. Russo, E. MANICCA, A. MONTEMURRO & S. DELL’ACQUA.1984. Ovarian and peripheral plasma levels of progestogens, androgens and oestrogens in postmenopausal women. Maturitas 6 45-53. LUCISANO,A., N. Russo, M. G. ACAMPORA, A. FABIANO. M. FATTIRENE,E. PARLATI,E . MANICCIA & S. DELL’ACQUA.1986. Ovarian and peripheral androgen and oestrogen levels in postmenopausal women: correlations with ovarian histology. Maturitas 8: 57-65. BIGGERS, J. D. 1988. Fecundibility. Ann. N. Y. Acad. Sci. 541: 706-714. SIMPSON,J. L. 1980. Antenatal diagnosis of chromosomal disorders. Clin. Obstet. Gynaecol. 7: 13-26. METCALF,M. G. 1983. Incidence of ovulation from the menarche to the menopause: observations of 622 New Zealand women. N.Z. Med. J. 9 6 645-648. LEE,S. J., E. A. LENTON,L. SEXTON & I. D. COOKE.1988. The effect of age on the cyclic patterns of plasma LH, FSH, oestradiol and progesterone in women with regular menstrual cycles. Hum. Reprod. 3: 851-855. LENTON,E. A., B. M. LANDCREN & L . SEXTON.1984. Normal variation in the length of the luteal phase of the menstrual cycle: Identification of the short luteal phase. Br. J. Obstet. Gynaecol. 91: 685-689. ROMEU,A., S. J. MUASHER, A. A. ACOSTA,L . L . VEECK,J. DIAZ, G. S. JONES,H. W. JONES& Z. ROSENWAKS. 1987. Results of in vitro fertilization attempts in women over 40 years of age and older: The Norfolk Experience. Fertil. Steril. 47: 130-136. CRAFT, I., T. AL-SHAWAF,P. LEWIS,P. SERHAL,E. SIMONS,M. AH-MOYE,W. FIAMANYA, D. ROBERTSON, P., SHRIVASTAV & P. BRINSDEN. 19XX. Analysis of 1071 GIFT procedures-the case for a flexible approach to treatment. Lancet i: 10941098.
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31. 32. 33. 34. 35. 36. 37. 38.
39. 40. 41. 42. 43. 44. 45. 46. 47.
ANNALS NEW YORK ACADEMY OF SCIENCES SPIRA,A. 1988. The decline of fecundity with age. Maturitas Suppl. 1: 15-22. HUGHES,E . G . , C. KING& E. C. WOOD. 1989. A prospective study of prognostic factors in in vitro fertilization and embryo transfer. Fertil. Steril. 51: 838-844. PADILLA,S. L., & J. E. GARCIA.1989. Effect of maternal age and number of in vitro fertilization procedures on pregnancy outcome. Fertil. Steril. 52: 270-273. PLANCHOT, M., J. DE GROUCHY, A. M. JUNCA,J. MANDELBAUM, J. SALAT-BAROUX & J. COHEN.1988. Chromosome analysis of human oocytes and embryos in an in vitro fertilization program. Ann. N. Y. Acad. Sci. 541: 384-397. ASCHHEIM, P. 1983. Le vieillissement ovarien. In Les Menopauses et le Vieillissement, Colloque d e Nice. M. Lagrange, C. Nahmanivici & P. Pras, Eds. :24-32. Duphar et Cie, Lyons. GOSDEN,R. G . 1985. Biology of Menopause: The Causes and Consequences of Ovarian Aging. Academic Press. London. SHERMAN, B. M., J. H . WEST& S. G. KORENMAN. 1976. The menopausal transition: Analysis of LH, FSH, estradiol, and progesterone concentrations during menstrual cycles of older women. J . Clin. Endocrinol. Metab. 4 2 629-636. REYES,F. I., J. S. D. WINTERS& C. FAIMAN. 1977. Pituitary-ovarian relationships preceding menopause. 1. A cross-sectional study of serum follicle-stimulating hormone, luteinizing hormone, prolactin, estradiol and progesterone levels. Am. J. Obstet. Gynecol. 129: 557-564. METCALF,M. G. & J . H. LIVESEY.1985. Gonadotropin excretion in fertile women: Effect of age and the onset of the menopausal transition. J. Endocrinol. 105: 357362. LENTON,E. A , , L . SEXTON, S. LEE& I. D. COOKE.1988. Progressive changes in L H and FSH and L H : FSH ratio in women through reproductive life. Maturitas 1 0 35-43. JONES,E. C. & P. L. KROHN.1961. The effect of hypophysectomy on age changes in the ovaries of mice. J. Endocrinol. 21: 497-505. MERIDITH, S.. D. KIRKPATRICK-KELLER & R. L. BUTCHER.1986. The effects of food restriction and hypophysectomy on numbers of primordial follicles and concentrations of hormones in rats. Biol. Reprod. 35: 68-73. ATAYA,K. M., J. A. MCKANNA, A. M. WEINTRAUB, M. R. CLARK& W. J. LEMAIRE. 1985. A luteinizing hormone-releasing hormone agonist for the prevention of chemotherapy-induced follicular loss in rats. Cancer Res. 45: 3651-3656. RYLE,M. 1972. The growth in vitro of mouse ovarian follicles of different sizes in response to purified gonadotropins. J . Reprod. Fertil. 30: 395-405. PETERS,H., A. G. BYSKOV,S. LINTERN-MOORE, M. FABER & M. ANDERSON. 1973. The effect of gonadotropin on follicle growth initiation in the neonatal mouse ovary. J. Reprod. Fertil. 35: 139-141. JEUNE,B. 1986. Parity and age at menopause in a Danish sample. Maturitas 8 359365. STANFORD, J . L., P. HARTGE,L. A. BRINTON,R. N. HOOVER& R. BROOKMEYER. 1987. Factors influencing the age at natural menopause. J. Chron. Dis. 40: 995-1002.
INTRODUCTION Although the menopause, the final cessation of menses, is the most dramatic biomarker of the reproductive aging process in women, age-related alterations in the pattern of cyclicity, as well as in fertility, are ongoing from menarche to menopause. Population studies of menstrual patterns'.* document that for the first few years after menarche the menses tends to be irregular. Thereafter and throughout the middle reproductive years, cycles are regular, although they become progressively shorter with advancing age primarily owing to a shortening of the follicular phase. Finally, in the 2-8-year period preceding the menopause, cycles again become highly variable, ranging from very short to very long. This last period of irregular menses is called the perirnenopause. Fertility begins to decline many years prior to the onset of irregular menses, being maximum in the mid- to late twenties and decreasing steadily until menopause .3,4 Age-associated alterations in the ovary, the neuroendocrine system, and the uterus may all influence the changing pattern of menses as well as the declining f e r t i l i t ~This . ~ paper will focus on the role of ovarian changes in these reproductive aging processes, with particular attention to the declining ovarian follicular reserve. FOLLICLE NUMBER IN THE PERIMENOPAUSE AND MENOPAUSE The human ovary steadily loses follicles from mid-fetal life onward.5-XUntil recently, the number of follicles remaining in the ovary at the time of menopause was unknown. Previous studies of follicle counts in human ovaries had been restricted to children and to women who were still menstruating regularly. Block estimated follicle number in autopsy specimens from 43 children and women from 6 to 44 years of age who had died accidently.5 Gougeon studied surgical specimens from 35 women aged 20-52 with regular mensesx A straight-line projection from either of these studies predicted that between 2,000 and 5,000 primordial Isolated reports follicles would remain at 50 years, the mean age of of the presence of follicles in postmenopausal ovaries supported this hypothesis."" We have recently studied follicle number in ovaries obtained at surgery from 17 otherwise healthy women, aged 45-55 yrs.'? The purposes of the study were ( I ) to clarify the relationship between follicle number and the perimenopausal transi13
ANNALS NEW YORK ACADEMY OF SCIENCES
14
tion from regular to irregular menses and (2) to estimate follicle number at the time of menopause. The women were divided into three age-matched groups according to the pattern of their menses in the previous 12 months: (i) a regular menstrual pattern [i.e., regular menses between 3 and 6 weeks without hot flashes]; (ii) a perimenopausal pattern [i.e., irregular menses with intervals of less than 3 weeks or more than 6 weeks for at least a year, with or without hot flashes]; and (iii) a postmenopausal pattern [i.e., no menses for at least a year]. Each ovary was sectioned at 10 microns (850-3,000 sections per ovary). Estimates of total follicle count were made by counting all the follicles in 10 consecutive sections in I , the mean number of primordial follicles in every 100. As can be seen in FIGURE the ovaries of women who were still menstruating regularly was ten-fold greater than that in the ovaries of perimenopausal women of the same age; there was little overlap in the range of follicle counts between the two groups (1392 k 355 vs. 142 & 72). Follicles were virtually absent from the ovaries of the four postmenopausal
1000
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FIGURE 1. The relationship between primordial follicle number and menstrual status in age-matched women, 45-55 years of age ( n = 17). The effect of menstrual status on follicle number was significant ( p < 0.001, by ANOVA: age was the covariate). Mean follicle counts in each group differed from those in the other groups ( p < 0.05, by the Student-NewmanKeuls test). (From Richardson ei al." Reproduced by permission.)
women. These observations indicate that the size of the follicular reserve is the major determinant of both the transition from regular menses to the perimenopause as well as to the menopause itself. Are there any follicles remaining in the human ovary at the time of menopause? This can be determined by extrapolating from the perimenopausal and postmenopausal follicle counts in this study and reviewing the anatomic and hormonal studies of others.s",13-'8 The paucity of follicles in many of the perimenopausal ovaries together with the absence of follicles in all of the postmenopausal ovaries suggest that, at least in some women, few, if any, follicles remain at menopause. This does not guarantee, however, that the ovaries of all women are completely depleted of follicles by the last cycle. A few hundred follicles were present in some of the perimenopausal ovaries. The virtually depleted postmenopausal ovaries were all from women more than three years past their last menses. There have been reports of follicles seen in postmenopausal ovaries. Saunrama observed an unspecified number of primordial and growing follicles, all
RICHARDSON & NELSON: FOLLICULAR DEPLETION
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showing evidence of degeneration, in the ovaries of two women, 3 and 5 years post m e n o p a ~ s eGuraya .~ also saw “some follicles” in the ovaries of six women, 45-52 years of age who were 1-5 years post menopause.I0 Costoff and Mahesh reported postmenopausal primordial follicles in all ovarian samples studied. I They did not, however, give the number of cases, their ages, the time from menopause, or the number of follicles seen. Most provocative are two reports of “fresh corpora lutea” in a 53-year-old woman 3 years post menopausei3and in a 63-year-old woman, 12 years post menopause.I4 Hormonal studies also indicate that follicles capable of secreting estrogen persist in some women in the early post menopause. In a longitudinal study, Metcalf studied weekly urinary hormone excretion in eight women from 5-15 weeks before to 22-30 weeks after their final menstruation.I5 She observed eight episodes of elevated estrogen excretion compatible with follicular development in five women during the postmenopausal period. Although ovulatory cycles occurred as late as the final menses in three women, there was no evidence of ovulation after menopause. Cross-sectional studies of blood hormone levels over the months after menopause suggest that from 20-40% of women have estrogen levels indicative of follicular function during the first 6- 12 months after the cessation of m e n ~ e s . ’ ~By , ’ ~12 to 24 months, the estrogen levels have fallen into the menopausal range in almost all subjects.’”’* JuddI9 and VermeuelenZ0found no evidence of estradiol secretion by the ovaries of women who were more than 3 years post menopause. Longcope” and Lucisano,22on the other hand, observed that the ovaries of some women, who were also at least 3 years past their last menses, secreted significant amounts of estradiol. This may be explained by the recent evidence that the amount of estradiol secreted by the ovaries of women who were at least 2 years post menopause correlated with the degree of ovarian stromal hyperplasia.” No mention was made of the presence of follicles in these ovaries. In summary, when the current hormonal and anatomic evidence is compiled, it would appear that, while the ovaries of some women are virtually depleted of follicles when menses ceases, others still have some follicles remaining for a year or more after menopause which are capable of secreting estrogen but, with rare exception, incapable of ovulating.
DECLINING FOLLICULAR RESERVES AND AGE-RELATED INFERTILITY Are the follicles that remain in the human ovary as the reserve nears exhaustion normal or defective? While the causes of the declining fertility rate24 and rising incidence of congenital anomalies25with advancing maternal age are not known, it is possible that age-related defects in the remaining follicles play a role. Such a hypothesis remains speculative, however, in view of the difficulty in distinguishing the intrinsic changes in the ova from such confounding factors as male fertility, frequency of intercourse and age-related changes in t h e hormonal milieu of the As long as menses remains regular, women over 40 continue to ovulate consistently, that is, in more than 95% of cycles.26Luteal phase progesterone profiles in women over 45 are identical to those of younger women.27The only aberration noted in these women is a slightly higher incidence of cycles with a shortened luteal phase.*SIn the perimenopause, however, the proportion of ovulatory cycles drops to 60%.26
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The technology of “assisted fertilization” provides the opportunity to study the effect of maternal age at successive steps of the fertilization process. Several studies have addressed the issue of fertility in women over 40 years of age.2s33 The technique of superovulation used for in uitro fertilization or gamete intrafallopian transfers (GIFT) produces either the same29or a slightly lower number of follicle^^^.^' in women over 40 compared to younger women. The ability of the ova from women over 40 to be fertilized also appears to be undiminished.*’,” Despite apparently normal ovulation and fertilization rates in older women, fecundity, or the ability to become pregnant, is reduced. Pregnancy rates in uitro fertilization and GIFT programs are diminished in women over 40 in most,30,32.33 but not reports. In these procedures, however, the effect of maternal age cannot be separated from that of paternal age as, in most cases, older women tend to have older partners. However, the CECOS report of their experience in artificial insemination by donor (AID) shows an age-associated decline in the reproductive capacity of the women independent of such variables as male fertility or ~ ’ reported the relationship between materfrequency of sexual i n t e r c o ~ r s e . They nal age and fecundity in 2,193 women undergoing artificial insemination with sperm from young fertile donors. Implantation of the embryo, as indicated by rising circulating hCG levels after 12 attempts, was successful in 73% of women under 31,62% of women 31-35, and only 54% of women over 35. The 16 women over 40 also had a pregnancy rate of 54% after twelve cycles. The subsequent abortion rate was not reported in this study. It is, however, consistently higher in women over 40 than in younger women (50-60%) in both in uitro fertili~ation?’,~~ and GIFT,)O as it is in natural p r e g n a n c i e ~ . ~ Despite the insights provided by the technology of assisted fertilization into the relationship between fertility and maternal age, the contribution of the aging follicle to the decreased fecundity, elevated abortion rate, and higher incidence of congenital anomalies observed in older women must remain speculative. While the rate of chromosome abnormalities in the ova of women over 35 is considerably higher than in those less than 35 (47% vs. 25%),34it is presently not possible to isolate the role of the ovum from that of other potential influences such as the sperm or the endometrium.
THE RATE OF FOLLICULAR DEPLETION IN THE HUMAN OVARY FIGURE 2 shows the data from the three quantitative studies of primordial follicles in human ovaries. Note that the follicle counts from the ovaries of women with regular cycles from the three studies fall within the same range. There is also a striking acceleration in the rate of follicle loss in the last decade before menopause. This accelerated loss can be seen even in the older women still having regular cycles. This observation has been made previously in separate analyses of Block’s and can also be seen in Gougeon’s data.x The mechanism responsible for this apparent increase in the rate of follicular depletion is unknown. It is noteworthy that the age at which the rate of follicular depletion begins to accelerate coincides with the age at which there is a selective increase in circulating FSH levels in women who are still menstruating regularly.374) We have hypothesized that this increased FSH stimulates a greater proportion of primordial follicles to enter the growing pool and subsequently become atretic. IZ Evidence to support such a hypothesis is drawn from animal data. Hypophysectomy, food restriction, and chronic administration of an analogue of LHRH, all of which have been shown to lower gonadotropin levels, retard the rate of follicular depletion in
0
10
I
*
*
20
I
Age (years)
30
I
40
I
0
-
. I .
50
A
n
”
60
I
FIGURE 2. The relationship between age and primordial follicle number is compared using data from four studies. Follicle depletion appears to accelerate in the decade preceding menopause. * = Block’s studies of stillborns6and of girls and women with regular menses, aged 6-44 yrs’; x = extrapolation from Figure 8 in Gougeon’s study of women aged 20-52, all with regular menses8; and in the study of Richardson et d.”of women aged 45-55 years, + = women with regular menses, 0 = perimenopausal women, and 0 = postmenopausal women.
I
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ANNALS NEW YORK ACADEMY OF SCIENCES
FSH has been shown to increase recruitment of primordial rats and follicles in u i t r but ~ ~ not ~ in u ~ u o . ~ ~ Evidence that FSH affects the rate of folticular depletion in humans is circumstantial. Increased parity has been shown to be associated with a relative delay in the age of m e n o p a ~ s e . ~ If ~ .the ~ ’ exhaustion of follicular reserves is the major determinant of the onset of menopause, one could hypothesize that the cumulative time that FSH is suppressed during successive pregnancies slows the rate of follicular loss, which, in turn, delays menopause. Circulating FSH levels are also suppressed during oral contraceptive use. While there is no evidence that the use of oral contraceptives is associated with a later menopause, this may be due to the fact that the cohort of women using “the pill” in their twenties are only now reaching the age of menopause. Alternatively, one might expect the greatest effect of such use after the age of 40 when circulating FSH levels rise. Most women have stopped using oral contraceptives in their forties, however.
SUMMARY From mid-fetal life, the human ovary steadily loses follicles. While age-related alterations at the level of the hypothalamus-pituitary appear to determine the timing of menarche, the number of follicles remaining in the mature ovary is the major determinant of the timing of both the perimenopause and the menopause. As the residual follicle reserve nears exhaustion, some of the remaining follicles appear to be defective, as evidenced by the higher incidence of chromosomal abnormalities in the ova of older women. It is presently not possible, however, to distinguish the specific contribution of the ovum to the age-related decrease in fecundity and higher abortion rate because of such confounding variables as aging sperm or changes in the endometrium. When follicle counts from peri- and postmenopausal ovaries are placed with those from previous studies of younger women and children there appears to be an acceleration in the rate of follicle loss in the decade preceding menopause. We hypothesize that the elevated FSH levels observed in normal women in the decade preceding the menopause may be responsible for this apparent acceleration in the rate of follicle loss. REFERENCES 1.
2. 3. 4.
5. 6. 7. 8.
TRELOAR, A. E., R. E. BOYNTON, B. G. BEHN& B. W. BROWN.1967. Variation of the human menstrual cycle through reproductive life. Int. J . Fertil. 12: 77-127. VOLLMAN, R. F. 1977. Major Problems in Obstetrics and Gynecology. Vol. 7. Philadelphia, PA. W.B. Sdunders. FRANCIS. W. J. A. 1970. Reproduction at menarche and menopause in women. J . Reprod. Fertil. Suppl 12: 89-98. NELSON,J. F. & L. S. FELICIO.1985. Reproductive aging in the female: An etiological perspective. Rev. Biol. Res. Aging 2: 251-314. BLOCK,E. 1952. Quantative morphological investigations of the follicular system in women. Acta Anat. 14 108-123. BLOCK,E. 1953. A quantative morphological investigation of the follicular system in newborn female infants. Acta Anat. 17: 201-206. BAKER,T. G. 1963. A quantative and cytological study of germ cells in human ovaries. Proc. R. SOC.Lond. (Biol.) 158: 417-433. GOUGEON, A. 1984. Cardctkre qualitative et quantdtif de la population folliculaire dans I’ovaire humain adulte. Contracep. Fertil. Sexual. 12: 527-535.
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ANNALS NEW YORK ACADEMY OF SCIENCES SPIRA,A. 1988. The decline of fecundity with age. Maturitas Suppl. 1: 15-22. HUGHES,E . G . , C. KING& E. C. WOOD. 1989. A prospective study of prognostic factors in in vitro fertilization and embryo transfer. Fertil. Steril. 51: 838-844. PADILLA,S. L., & J. E. GARCIA.1989. Effect of maternal age and number of in vitro fertilization procedures on pregnancy outcome. Fertil. Steril. 52: 270-273. PLANCHOT, M., J. DE GROUCHY, A. M. JUNCA,J. MANDELBAUM, J. SALAT-BAROUX & J. COHEN.1988. Chromosome analysis of human oocytes and embryos in an in vitro fertilization program. Ann. N. Y. Acad. Sci. 541: 384-397. ASCHHEIM, P. 1983. Le vieillissement ovarien. In Les Menopauses et le Vieillissement, Colloque d e Nice. M. Lagrange, C. Nahmanivici & P. Pras, Eds. :24-32. Duphar et Cie, Lyons. GOSDEN,R. G . 1985. Biology of Menopause: The Causes and Consequences of Ovarian Aging. Academic Press. London. SHERMAN, B. M., J. H . WEST& S. G. KORENMAN. 1976. The menopausal transition: Analysis of LH, FSH, estradiol, and progesterone concentrations during menstrual cycles of older women. J . Clin. Endocrinol. Metab. 4 2 629-636. REYES,F. I., J. S. D. WINTERS& C. FAIMAN. 1977. Pituitary-ovarian relationships preceding menopause. 1. A cross-sectional study of serum follicle-stimulating hormone, luteinizing hormone, prolactin, estradiol and progesterone levels. Am. J. Obstet. Gynecol. 129: 557-564. METCALF,M. G. & J . H. LIVESEY.1985. Gonadotropin excretion in fertile women: Effect of age and the onset of the menopausal transition. J. Endocrinol. 105: 357362. LENTON,E. A , , L . SEXTON, S. LEE& I. D. COOKE.1988. Progressive changes in L H and FSH and L H : FSH ratio in women through reproductive life. Maturitas 1 0 35-43. JONES,E. C. & P. L. KROHN.1961. The effect of hypophysectomy on age changes in the ovaries of mice. J. Endocrinol. 21: 497-505. MERIDITH, S.. D. KIRKPATRICK-KELLER & R. L. BUTCHER.1986. The effects of food restriction and hypophysectomy on numbers of primordial follicles and concentrations of hormones in rats. Biol. Reprod. 35: 68-73. ATAYA,K. M., J. A. MCKANNA, A. M. WEINTRAUB, M. R. CLARK& W. J. LEMAIRE. 1985. A luteinizing hormone-releasing hormone agonist for the prevention of chemotherapy-induced follicular loss in rats. Cancer Res. 45: 3651-3656. RYLE,M. 1972. The growth in vitro of mouse ovarian follicles of different sizes in response to purified gonadotropins. J . Reprod. Fertil. 30: 395-405. PETERS,H., A. G. BYSKOV,S. LINTERN-MOORE, M. FABER & M. ANDERSON. 1973. The effect of gonadotropin on follicle growth initiation in the neonatal mouse ovary. J. Reprod. Fertil. 35: 139-141. JEUNE,B. 1986. Parity and age at menopause in a Danish sample. Maturitas 8 359365. STANFORD, J . L., P. HARTGE,L. A. BRINTON,R. N. HOOVER& R. BROOKMEYER. 1987. Factors influencing the age at natural menopause. J. Chron. Dis. 40: 995-1002.
