1Department of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, Departments of 2 Obstetrics and Gynecology and 4 Medicine, University of Washington, Seattle WA and 3 Reproductive Endocrine Unit, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA
5 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, 4225 Roosevelt Way NE, Suite 305, Seattle, WA 98105, USA. Email: nklein{at}u.washington.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: antral follicles/inhibin/ovarian ageing/ovarian reserve
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ageing in women is associated with depletion of the primordial follicle pool (Gougeon, 1979; Richardson et al., 1987
; Faddy et al., 1992
), disorganization of the oocyte meiotic spindle (Battaglia et al., 1996
, 1997
) and a marked decrease in fertility (Menken et al., 1986
; van Noord Zaadstra et al., 1991
). In spite of these early changes in the reproductive ageing process, ovarian secretory capacity remains equivalent to (or surpasses) that of younger reproductive aged women (Burger et al., 1998
; Lee et al., 1988
; Klein et al., 1996a
; Reame et al., 1998
; Welt et al., 1999
; van Zonneveld et al., 2003
). Furthermore, women in these early stages of reproductive ageing [equivalent to stage 3 to 2 according to the Stages of Reproductive Ageing Workshop (STRAW), Soules et al., 2001
] continue to be ovulatory, and can be indistinguishable from their younger counterparts in the absence of endocrinological testing. Women in these early stages of reproductive ageing were selected as subjects for this study, as these are the women who frequently experience age-related infertility after intentionally delaying childbearing, often for career and other social reasons.
One mechanism whereby ovarian secretion of hormones and growth factors may remain adequate despite age-related decreases in oocyte quality and number (Gougeon, 1979; Menken et al., 1986
; Richardson et al., 1987
; Faddy et al., 1992
; Battaglia et al., 1996
, 1997
) is through a compensatory effect of the early follicular phase FSH rise. In this model, early follicular phase FSH elevations observed in older reproductive aged women may not only be due to a decrease in the number of recruitable follicles (and the resulting decrease in negative feedback), but may also be due to a relative resistance of the follicles to gonadotrophins. To investigate the relative role of FSH elevations in ovarian follicular development and secretion, we administered equal doses of recombinant FSH to a group of older (4045, n=9) and younger (2025, n=10) reproductive aged subjects after suppression of endogenous FSH secretion. A relatively low (but effective) dose of FSH (150 IU) was chosen specifically to lessen the possibility that decreased ovarian reserve would be the limiting factor in the secretory response of the older subjects. Outcome measures included serum estradiol, inhibin A, inhibin B, and follicular development as determined by serial transvaginal ultrasound examinations. We also investigated the association between ovarian secretory capacity and basal parameters of ovarian reserve, including cycle day 3 FSH, estradiol, inhibin B and the antral follicle count (AFC).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protocol and procedures
Baseline blood samples and ultrasound examinations for AFC were obtained on day 3 of a spontaneous menstrual cycle. Seven days after detection of a urinary LH surge, subjects were suppressed with a GnRH agonist, leuprolide acetate (Lupron, Tap Pharmaceuticals) 0.5 mg s.c. daily. Beginning with the first day of the subsequent menses, daily venipuncture was performed and serum estradiol levels were determined. Once suppression was confirmed (estradiol <73 pmol/l), the subjects continued on Lupron for an additional 5 days prior to beginning FSH. Both groups were then treated with 150 IU of recombinant FSH (Gonal F, Serono) s.c. per day for 5 days. Blood samples were obtained on a daily basis throughout the treatment interval and for 4 days after FSH was discontinued. Daily Lupron injections were continued throughout the FSH treatment interval and for 2 days after treatment was completed. All blood samples were stored at 20°C for subsequent analysis of FSH, estradiol, and inhibin A and B.
Transvaginal ultrasound
Ultrasound examinations for baseline AFC and follicle growth during the treatment interval were performed by one of following investigators: M.R.S, N.A.K., A.C.T. or K.R.H. We recently have demonstrated that inter-observer differences in AFC are minimal (Hansen et al., 2003). Sonolucent structures visualized within the ovary and having regular contour were considered to be follicles. All follicles 210 mm in size were considered antral follicles. AFC refers to the total antral follicle count for both ovaries, and this number was used for calculations. Ultrasound examinations for AFC and determination of the number of follicles >10 mm bilaterally were performed following suppression with Lupron and daily beginning after 3 days of FSH treatment. Transvaginal ultrasound examinations were performed with an ATL Ultramark 400 C with a 6.5 MHz vaginal transducer.
Hormone assays
Serum FSH levels were determined using a solid-phase two-site monoclonal enzyme-linked immunosorbent assay (ELISA; DELFIA, Wallace Inc., Gathersburg, MD). The intra- and inter-assay coefficients of variation (CVs) were 1.9 and 8.9%, respectively.
Serum estradiol measurements were performed with a radioimmunoassay using reagents supplied by ICN Biomedicals, Inc. (Costa Mesa, CA). The intra- and inter-assay CVs were 9 and 18%, respectively.
Inhibin B assays were performed with a solid-phase sandwich ELISA (Serotec, Oxford, UK) based on the use of plates coated with a monoclonal antibody specific for the inhibin B -subunit with a second monoclonal antibody specific for the
-subunit for detection. The sensitivity of the assay was 15.6 pg/ml, and inhibin A had a 0.5% cross-reaction in the inhibin B ELISA. The assay was controlled in triplicate using samples with mean concentrations of 155.3, 316.3 and 919.3 pg/ml, with inter-assay CVs of 11.6, 7.6 and 9.7%, respectively.
A solid-phase sandwich ELISA (DSL, Webster, TX) was also used to measure inhibin A. The first monoclonal antibody is specific for the -subunit of inhibin A, with a second monoclonal antibody specific for the
-subunit and labelled with horseradish peroxidase for detection. The assay sensitivity was 0.1 IU/ml. The assay standard provided by the manufacturer was calibrated using the World Health Organization's First International Standard for Inhibin (recombinant human inhibin, Lot 91/624). The assay was controlled in duplicate using aliquots of specimens containing 2.00 or 9.56 IU/ml, with inter-assay CVs of 8.7 and 3.3%, respectively.
Data analysis
Power analysis
This report represents an interim analysis of what was intended to be a larger study (30 older subjects and 30 younger controls) designed to investigate the relative role of FSH elevations in ovarian follicular development and function. Based upon earlier work, we anticipated a peak estradiol of 1100 pmol/l in the younger group and 880 pmol/l in the older group with an equivalent SD of 290 pmol/l. With this sample size, we would have had an 80% power to detect a difference of 200 pmol/l between groups. The interim analysis demonstrated significantly smaller differences in peak estradiol levels between the groups (812 pmol/l, younger, and 749 pmol/l, older) and greater SD (540 pmol/l) than anticipated. These recent calculations would indicate the need for a study population that would amount to >200 subjects in order to demonstrate a statistical difference in estradiol levels between groups. These large subject numbers would be impractical considering the intensity of this study. As a result, the decision was made to discontinue enrolment.
Statistics
Differences between groups for hormone levels and follicle numbers were evaluated with a two-factor repeated measures ANOVA. Single point measurements were compared with the MannWhitney (rank sum) test. Spearman correlation coefficients were used to determine correlations. Statistical evaluations were performed with StatView software, version 5.0.1 (Abacus Concepts, Inc., Berkeley, CA). Statistical significance was defined as P<0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Numerous investigations have determined that one of the earliest detectable changes in reproductive ageing is the early follicular phase rise in serum FSH (Sherman and Korenman, 1975; Reyes et al., 1977
; Lee et al., 1988
; Rannevik et al., 1995
; Klein et al., 1996a
; Burger et al., 1998
, 1999
; van Zonneveld et al., 2003
). Although the exact cause of this FSH rise remains to be fully elucidated, a key component of this mechanism appears to be a decrease in negative feedback as a result of lower early follicular phase inhibin B levels as observed in older reproductive aged women (Burger et al., 1998
, 1999
; Roseff et al., 1989
; Hall et al., 1992
; Reame et al., 1998
; Santoro et al., 1999
; Klein et al., 2004
). Lower serum inhibin B levels, in turn, are likely to be due to the smaller pool of pre-antral and early antral follicles remaining in the ovaries of older reproductive aged women (Faddy et al., 1992
; Scheffer et al., 1999
).
Although women at these stages of reproductive ageing have decreased natural fecundity (Menken et al., 1986; van Noord Zaadstra et al., 1991
) and experience earlier recruitment and ovulation of the dominant follicle (Klein et al., 2002
; van Zonneveld et al., 2003
), serum levels of inhibin A and estradiol remain at levels similar to or greater than those observed in younger women (Burger et al., 1998
; Lee et al., 1988
; Klein et al., 1996a
; Reame et al., 1998
; Welt et al., 1999
; van Zonneveld et al., 2003
). Furthermore, follicular fluid levels of steroids and inhibins have also been determined to be equivalent to those seen in younger reproductive aged women (Klein et al., 1996c
, 2000
). These observations suggest that FSH elevations may serve a compensatory mechanism to maintain dominant follicle development and secretion in the presence of a diminishing primordial follicle pool. If true, then at equivalent FSH levels, the secretory capacity of the older ovary would be less than that of the younger ovary. To test this hypothesis, we treated a group of older (4045 years) and younger (2025 years) women with equivalent doses of FSH following pituitary suppression. To limit the influence of differences in ovarian reserve impacting the ovarian secretory response, we specifically chose a relatively low dose of exogenous FSH (150 IU).
At equivalent induced levels of FSH, we were unable to detect a difference in serum levels of the major secretory products of large follicles (estradiol and inhibin A), although both tended to be greater in the younger than in the older group. Similarly, when evaluated on a per large (>10 mm) follicle basis, secretion of estradiol and inhibin A was not significantly different between the groups. These findings would suggest that at equivalent serum FSH levels, the secretory capacity of the older and younger follicle is roughly equivalent. Furthermore, it would suggest that FSH elevations seen in the early follicular phase of older reproductive aged women are a reflection of the diminishing size of the primordial follicle pool and are not necessary to maintain adequate follicular secretion. The higher levels of estradiol and inhibin A observed in some natural cycles of older reproductive aged women may result from increased FSH levels in the presence of normally functioning (but fewer) follicles.
Although we detected no statistically significant differences in the secretion of estradiol and inhibin A between the groups, we did detect significant differences in the number of large pre-ovulatory size follicles. Additionally, inhibin B levels were also greater in the younger than in the older subjects, although this difference did not quite reach statistical significance (P=0.08). Both of these parameters are indicative of ovarian reserve (Eldar-Geva et al., 2000) and would be anticipated to be greater in the younger than in the older subjects.
The complexity and expense of recruiting and treating large numbers of women with this study design precluded a large number of subjects. With small prospective studies such as ours, the possibility of a type II error exists. In particular this seems likely in the case of inhibin B differences between the groups. However, even with this small sample size, we were able to detect a difference in follicular recruitment between the groups. Although a larger sample size might have detected statistical differences in estradiol and inhibin A levels between groups, in absolute terms, these differences would probably be small and of uncertain clinical significance.
It is also important to note that elevated serum FSH levels and decreased AFC were not part of the entry criteria for our older subjects. Although the study and control groups were statistically different in terms of these basal parameters, several of the older subjects had basal FSH levels and AFC that overlapped those of the younger control group. It is possible that an investigation of the ovarian secretory capacity of a more highly selected group of older reproductive aged subjects (those clearly more advanced in the reproductive ageing process) would have given different results.
Consistent with previous investigations, we determined that the AFC and basal FSH (Scott and Hofmann, 1995; Tomás et al., 1997
; Bancsi et al., 2002
; Yong et al., 2003
) were significantly correlated with parameters reflecting ovarian reserve (the number of large follicles developing and inhibin B levels obtained during stimulation). Nevertheless, neither of these basal parameters accurately reflected ovarian secretory capacity. Given the lack of statistical differences in secretory capacity between the two age groups, these findings are not surprising. We also detected no significant correlations between basal inhibin B and ovarian stimulatory or secretory capacity. These findings are consistent with recent investigations suggesting that both basal FSH and AFC are better markers of ovarian reserve than is basal inhibin B (Scott and Hofmann, 1995
; Bancsi et al., 2002
; Yong et al., 2003
).
In summary, we detected no significant differences in ovarian secretion of estradiol and inhibin A in older reproductive aged women compared with younger controls at equivalent serum FSH levels. Per large follicle, estradiol and inhibin A secretion also tended to be similar between the two groups. These observations suggest that in older reproductive aged women, higher FSH levels are not necessary to maintain adequate follicular secretion, but more probably are due to the diminished size of the primordial follicle pool and the resulting decreased negative feedback. While the AFC and basal FSH measurements reflect ovarian reserve, they are not significantly correlated with ovarian secretory capacity.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Battaglia DE, Goodwin P, Klein NA and Soules MR (1996) Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum Reprod 10, 22172222.
Battaglia DE, Klein NA and Soules MR (1997) Changes in centrosomal domains during meiotic maturation in the human oocyte. Mol Hum Reprod 2, 845851.
Burger HG, Cahir N, Robertson DM, Groome NP, Dudley E, Green A and Dennerstein L (1998) Serum inhibins A and B fall differentially as FSH rises in perimenopausal women. Clin Endocrinol 48, 809813.[CrossRef][ISI][Medline]
Burger HG, Dudley EC, Hopper JL, Groome N, Guthrie JR, Green A and Dennerstein L (1999) Prospectively measured levels of serum follicle-stimulating hormone, estradiol, and dimeric inhibins during the menopausal transition in a population-based cohort of women. J Clin Endocrinol Metab 84, 40254030.
Danforth DR, Arbogast LK, Mroueh J, Kim MH, Kennard EA, Seifer DB and Friedman CI (1998) Dimeric inhibin: a direct marker of ovarian aging. Fertil Steril 70, 119123.[CrossRef][ISI][Medline]
Eldar-Geva T, Robertson DM, Cahir N, Groome N, Gabbe MP, MacLachlan V and Healy DL (2000) Relationship between serum inhibin A and B and ovarian follicle development after a daily fixed dose administration of recombinant follicle-stimulating hormone. J Clin Endocrinol Metab 85, 607613.
Faddy M, Godsen R, Gougeon A, Richardson S and Nelson J (1992) Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause. Hum Reprod 7, 13421346.[Abstract]
Gougeon A (1979) Qualitative changes in medium and large antral follicles in the human ovary during the menstrual cycle. Ann Biol Anim Biochem Biophys 19, 14611468.[ISI]
Hall JE, Schoenfeld DA, Martin KA and Crowley WF, Jr (1992) Hypothalamic gonadotropin-releasing hormone secretion and follicle-stimulating hormone dynamics during the luteal-follicular transition. J Clin Endocrinol Metab 74, 600607.[Abstract]
Hansen KR, Morris JL, Thyer AC and Soules MR (2003) Reproductive aging and variability in the ovarian antral follicle count: application in the clinical setting. Fertil Steril 80, 577583.[CrossRef][ISI][Medline]
Klein NA, Battaglia DE, Fujimoto VY, Davis GS, Bremner WJ and Soules MR (1996a) Reproductive aging: accelerated ovarian follicular development associated with a monotropic follicle-stimulating hormone rise in normal older women. J Clin Endocrinol Metab 81, 10381045.[Abstract]
Klein NA, Illingworth PJ, Groome NP, McNeilly AS, Battaglia DE and Soules MR (1996b) Decreased inhibin B secretion is associated with the monotropic FSH rise in older, ovulatory women: a study of serum and follicular fluid levels of dimeric inhibin A and B in spontaneous menstrual cycles. J Clin Endocrinol Metab 81, 27422745.[Abstract]
Klein NA, Battaglia DE, Miller PB, Branigan EF, Giudice LC and Soules MR (1996c) Ovarian follicular development and the follicular fluid hormones and growth factors in normal women of advanced reproductive age. J Clin Endocrinol Metab 81, 19461951.[Abstract]
Klein NA, Battaglia DE, Woodruff TK, Padmanabhan V, Giudice LC, Bremner WJ and Soules MR (2000) Ovarian follicular concentrations of activin, follistatin, inhibin, insulin-like growth factor I (IGF-I), IGF-II, IGF-binding protein-2 (IGFBP-2), IGFBP-3, and vascular endothelial growth factor in spontaneous menstrual cycles of normal women of advanced reproductive age. J Clin Endocrinol Metabol 85, 45204525.
Klein NA, Harper AJ, Houmard BS, Sluss PM and Soules MR (2002) Is the short follicular phase in older women secondary to advanced or accelerated dominant follicle development? J Clin Endocrinol Metab 87, 57465750.
Klein NA, Houmard BS, Hansen KR, Woodruff TK, Sluss PM, Bremner WJ and Soules MR (2004) Age-related analysis of inhibin A, inhibin B, and activin A relative to the intercycle monotropic follicle-stimulating hormone rise in normal ovulatory women. J Clin Endocrinol Metab 89, 29772981.
Lee SJ, Lenton EA, Sexton L and Cooke ID (1988) The effect of age on the cyclical patterns of plasma LH, FSH, oestradiol and progesterone in women with regular menstrual cycles. Hum Reprod 3, 851855.[Abstract]
Menken J, Trussell J and Larsen U (1986) Age and infertility. Science 233, 13891394.[ISI][Medline]
Muttukrishna S, Child T, Lockwood GM, Groome NP, Barlow DH and Ledger WL (2000) Serum concentrations of dimeric inhibins, activin A, gonadotropins and ovarian steroids during the menstrual cycle in older women. Hum Reprod 15, 549556.
Rannevik G, Jeppsson S, Johnell O, Bjerre B, Laurell-Borulf Y and Svanberg L (1995) A longitudinal study of the perimenopausal transition: altered profiles of steroid and pituitary hormones, SHBG and bone mineral density. Maturitas 21, 103113.[CrossRef][ISI][Medline]
Reame NE, Wyman TL, Phillips DJ, de Kretser DM and Padmanabhan V (1998) Net increase in stimulatory input resulting from a decrease in inhibin B and an increase in activin A may contribute in part to the rise in follicular phase follicle-stimulating hormone of aging cycling women. J Clin Endocrinol Metab 83, 33023307.
Reyes FL, Winter JS and Faiman C (1977) Pituitary-ovarian relationships preceding the menopause. I. A cross-sectional study of serum follicle-stimulating hormone, luteinizing hormone, prolactin, estradiol, and progesterone levels. Am J Obstet Gynecol 129, 557564.[ISI][Medline]
Richardson S, Senikas V and Nelson J (1987) Follicular depletion during the menopausal transition: evidence for accelerated loss and ultimate exhaustion. J Clin Endocrinol Metab 65, 12311237.[Abstract]
Roseff SJ, Bangah ML, Kettel LM, Vale W, Rivier J, Burger HG and Yen SS (1989) Dynamic changes in circulating inhibin levels during the luteo-follicular transition of the human menstrual cycle. J Clin Endocrinol Metab 69, 10331039.[Abstract]
Santoro N, Adel T and Skurnick JH (1999) Decreased inhibin tone and increased activin A secretion characterize reproductive aging in women. Fertil Steril 71, 658662.[CrossRef][ISI][Medline]
Scheffer GJ, Broekmans FJ, Dorland M, Habbema JD, Looman CW and te Velde ER (1999) Antral follicle counts by transvaginal ultrasonography are related to age in women with proven natural fertility. Fertil Steril 72, 845851.[CrossRef][ISI][Medline]
Scott RT Jr and Hofmann GE (1995) Prognostic assessment of ovarian reserve. Fertil Steril 63, 111.[ISI][Medline]
Sherman B and Korenman S (1975) Hormonal characteristics of the human menstrual cycle throughout reproductive life. J Clin Invest 55, 699706.[ISI][Medline]
Soules MR, Sherman S, Parrott E, Rebar R, Santoro N, Utian W and Woods N (2001) Executive summary: stages of reproductive aging workshop (STRAW). Fertil Steril 76, 874878.[CrossRef][Medline]
Tomás C, Nuojua-Huttunen S and Martikainen H (1997) Pretreatment transvaginal ultrasound examination predicts ovarian responsiveness to gonadotrophins in in-vitro fertilization. Hum Reprod 12, 220223.[Abstract]
van Noord Zaadstra BM, Looman CW, Alsbach H, Habbema JD, te Velde ER and Karabaat J (1991) Delaying childbearing: effect of age on fecundity and outcome of pregnancy. Br Med J 302, 13161365.
van Zonneveld P, Scheffer GJ, Broekmans FJM, Blankenstein MA, de Jong FH, Looman CWN, Habbema JDF and te Velde ER (2003) Do cycle disturbances explain the age-related decline of female fertility? Cycle characteristics of women aged over 40 years compared with a reference population of young women. Hum Reprod 18, 495501.
Welt CK, McNicholl DJ, Taylor AE and Hall JE (1999) Female reproductive aging is marked by decreased secretion of dimeric inhibin. J Clin Endocrinol Metab 84, 105111.
Yong PYK, Baird DT, Thong KJ, McNeilly AS and Anderson RA (2003) Prospective analysis of the relationships between the ovarian follicle cohort and basal FSH concentration, the inhibin response to exogenous FSH and ovarian follicle number at different stages of the normal menstrual cycle and after pituitary down-regulation. Hum Reprod 18, 3544.
Submitted on June 21, 2004; accepted on September 10, 2004.