REPRODUCTIVE aging is associated with a decline in

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1 X/99/$03.00/0 Vol. 84, No. 1 Journal of Clinical Endocrinology and Metabolism Printed in U.S.A. Copyright 1999 by The Endocrine Society Female Reproductive Aging Is Marked by Decreased Secretion of Dimeric Inhibin* CORRINE K. WELT, DENNIS J. MCNICHOLL, ANN E. TAYLOR, AND JANET E. HALL Reproductive Endocrine Unit, Reproductive Endocrine Sciences Center and National Center for Infertility Research, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts ABSTRACT The increase in serum FSH that accompanies female reproductive aging occurs before changes in estradiol (E2). A decrease in negative feedback from inhibin A (a product of the dominant follicle and corpus luteum) and/or inhibin B (secreted by developing follicles) may explain the rise in FSH with age. To test the hypothesis that decreases in inhibin A or inhibin B occur at an age at which the first increase in follicular phase FSH is evident, daily blood samples were obtained across the menstrual cycle from younger ( 35 yr; n 23) and older (35 46 yr; n 21) cycling women. These cross-sectional studies were complemented by longitudinal data in 3 women studied at a 10-yr interval. In the early follicular phase, mean inhibin B was lower in older cycling women (88 7 vs pg/ml; P 0.05) and FSH was higher ( vs IU/L in older vs. younger, respectively; P 0.04). In the mid- and late follicular phases, inhibin B was also lower in the older women (117 9 vs and 85 8 vs pg/ml; P 0.04), whereas E2 was higher ( vs and vs pg/ml; P 0.02), and no differences in FSH were observed in the two groups at these times. In women REPRODUCTIVE aging is associated with a decline in fertility, which begins in a moderate and steady fashion starting in the third to fourth decade (1, 2) but accelerates rapidly after age 35 (1). A gradual diminution of the pool of ovarian follicles seems to underlie the decline in fertility (3 5). This age-related decrease in follicle number and fertility is marked by a rise in follicular-phase FSH (6 9), which also commences after age 35 (7) and is followed several years later by an increase in LH (6, 7, 9). Although changes in estradiol (E2) were initially sought to explain this differential increase in FSH, E2 has variably been reported to decrease (8, 10, 11), remain constant (7, 12), or even increase (9, 13 15) in the follicular phase of older women with ovulatory menstrual cycles. Inhibin, a dimeric glycoprotein composed of an -subunit and a A-subunit (inhibin A) or B-subunit (inhibin B), was initially identified based on its ability to suppress FSH (16, 17). Thus, changes in inhibin levels have been examined to explain the monotropic FSH rise that occurs with age. During Received April 17, Revision received August 11, Rerevision received September 15, Accepted September 24, Address all correspondence and requests for reprints to: Dr. Corrine K. Welt, Reproductive Endocrine Unit, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts * This work was supported by National Institutes of Health Grants U-54-HD-29164, M-01-RR-01066, and P-30-HD studied longitudinally, FSH and inhibin B varied inversely in the follicular phase. In the early luteal phase, mean inhibin B was lower in the older group (64 6 vs pg/ml; P 0.03), and FSH was higher ( vs IU/L; P 0.03). In the mid- and late luteal phases, inhibin B was also lower in older subjects (21 2 vs and 22 2 vs pg/ml; P 0.02). No difference in inhibin A, E2, or progesterone was observed across the luteal phase, between the two groups. However, in all subjects studied longitudinally, increased age was associated with a decrease in inhibin A, inhibin B, and progesterone in the absence of changes in E2. Our conclusions were: 1) reproductive aging is accompanied by decreases in both inhibin B and inhibin A; 2) the decrease in inhibin B precedes the decrease in inhibin A and occurs in concert with an increase in E2, suggesting that inhibin B negative feedback is the most important factor controlling the earliest increase in FSH with aging; 3) these studies suggest that the decrease in inhibin B is the earliest marker of the decline in follicle number across reproductive aging. (J Clin Endocrinol Metab 84: , 1999) the normal menstrual cycle, inhibin B levels are highest in the early to midfollicular phases (18) and decrease in the late follicular phase (19), suggesting secretion by developing follicles, whereas inhibin A is highest in the late follicular and luteal phases as a product of the preovulatory follicle and, subsequently, the corpus luteum (20, 21). Therefore, decreased secretion of either inhibin B in the follicular phase or inhibin A in the luteal phase could account for the early follicular-phase increase in FSH in older cycling women. In women over age 45, higher follicular-phase levels of FSH were associated with a decrease in total inhibin in both follicular and luteal phases (11), as measured by an assay which uses an antibody directed against the -subunit and detects all forms of inhibin (22 24). Recent studies that demonstrated lower inhibin B in the follicular phase of 40- to 45-yr-old cycling women with elevated FSH levels provide indirect evidence that inhibin B may also act as an important regulator of follicular-phase FSH in the human (25, 26). The first detectable rise in follicular-phase FSH levels occurs much earlier than age 40 (7), however, indicating the importance of examining the relationship between inhibin A and inhibin B and FSH in younger subjects and in subjects whose FSH levels are not yet outside the normal range. Daily blood samples were obtained, across an entire menstrual cycle, in a group of 44 women, ranging in age from yr, with regular menstrual cycles and normal early- 105

2 106 WELT ET AL. JCE&M 1999 Vol 84 No 1 follicular-phase FSH levels. Results in women 35 yr old or older were then compared with those in women less than 35 yr, based on the age at which the first marked decline in fertility occurs (1) and in which the first rise in follicularphase FSH is detectable (7). To determine whether differences seen in cross-sectional studies were representative of changes in individual women over time, 3 women were studied on 2 occasions at least 10 yr apart. Subjects Materials and Methods Two studies were undertaken to evaluate inhibin A and B levels across reproductive aging. The first was a cross-sectional study in which 23 younger cycling women (20 34 yr old) and 21 older cycling women (35 46 yr old) were studied. All subjects had normal PRL and TSH levels, were on no medication, had normal body weight (with BMI of kg/m 2 ), had no history of excessive exercise, and showed no evidence of androgen excess on physical examination, as previously described (27, 28). All subjects had a history of regular (25 35 days) menstrual cycles with evidence of ovulation in the preceding cycle, as indicated by a serum progesterone (P4) level of more than 6 ng/ml. Blood samples were drawn daily for the duration of 1 menstrual cycle in all subjects. In a subset of subjects ( 35 yr, n 14; and 35 yr, n 13), transvaginal ultrasounds (Toshiba SAL 77B, 5 MHz convex array transducer, Toshiba Corp., Japan) were performed, starting in the midfollicular phase, to assess follicular development. Subsequent ultrasounds were performed every 2 5 days until ovulation was documented by collapse of the dominant follicle, with or without internal echoes. Three younger subjects and 1 older subject had only 1 ultrasound in the midfollicular phase. The number and maximum diameter of all follicles of more than 6 mm was recorded. Three additional women, now aged 37, 42, and 47 yr, had daily blood samples drawn across a menstrual cycle approximately 10 yr previously, and repeated daily blood sampling for the present study, to provide longitudinal data. The 47-yr-old subject obtained blood samples across two cycles at each age. All blood samples were measured for LH, FSH, E2, P4, inhibin A, and inhibin B. The study was approved by the Subcommittee on Human Studies of the Massachusetts General Hospital. Informed consent was obtained from each subject before participation. Assays Serum LH, FSH, E2, and P4 were measured by RIA, as previously described (29, 30). The 95% confidence limits for early-follicular-phase FSH levels (days 1 5, starting at menses) in this assay were IU/L, based on data from 109 normal cycling women, yr old. All samples for LH, FSH, E2, and P4 were analyzed in duplicate, and all samples from an individual were analyzed in the same assay. The interand intraassay coefficients of variation were similar to those previously described (31). Gonadotropin levels are expressed in IU/L, as equivalents of the Second International Reference Preparation 71/223 of human menopausal gonadotropins. Inhibin A was measured in duplicate by enzyme-linked immunosorbent assay (Serotec, Oxford, England), as previously described (20). The assay uses a lyophilized human follicular fluid calibrator standardized as equivalents of the World Health Organization recombinant human inhibin A preparation 91/624, and values are reported as IU/mL. The limit of detection of the assay was 0.6 IU/mL. The intraassay coefficient of variation for the dimeric inhibin A assay was 3.9% at the ED 20 dose, and the interassay coefficient of variation was 6.8% at the ED 30 dose. All samples for a given individual were run in the same assay. Inhibin B was measured as single samples by enzyme-linked immunosorbent assay (Serotec), as previously described (32). The limit of detection of the inhibin B assay (mean 2 sd of multiple zero standard measurements) was 15.6 pg/ml. The intraassay coefficient of variation was 4 6%, and the interassay coefficient of variation was 15 18% for serum spiked with 121, 250, and 723 pg/ml inhibin B. All samples with levels in excess of 500 pg/ml were appropriately diluted. All samples for a given individual were run in the same assay. Data analysis and statistics Data were centered to ovulation for comparison of hormonal dynamics across the follicular and luteal phases of the menstrual cycle using three of four of the following criteria: 1) day of LH peak; 2) day of the midcycle FSH peak; 3) day of or after the midcycle E2 peak; and 4) day that the P4 doubled from baseline or reached 0.6 ng/ml (27). Mean values for each hormone were calculated across the follicular phase from menses to the day before ovulation and the luteal phase from the day after ovulation to the day before menses. In addition, menstrual cycles were standardized to a 28-day cycle length with the day of ovulation centered to day 0, and mean hormone levels were determined in the early (days 13 to 9), mid- (days 8 to 5), and late (days 4 to 1) follicular phase and early (days 1 4), mid- (days 5 9) and late (days 10 14) luteal phase, as previously described (33). The mean value approximates the area under the curve for hormonal values at each cycle stage, based on the trapezoidal rule. Mean values were compared between older and younger cycling women studied cross-sectionally using two-tailed, unpaired t tests, whereas comparisons between older and younger cycles in women studied longitudinally were performed using paired t tests. Pearson correlations were used to examine associations between follicle size and inhibin A, inhibin B, and E2 levels obtained on the day of the ultrasound measurement. The ratio of inhibin A and E2 to the maximum diameter of the dominant follicle was then determined, and resulting values were compared between older and younger cycling subjects by t test. Results are expressed as mean sem unless otherwise indicated. A P value of less than 0.05 was considered significant. Results Cross-sectional studies Inhibin B and inhibin A were differentially secreted across the menstrual cycle in older and younger cycling women (Fig. 1), following a pattern that is qualitatively similar to results in previous smaller series (18 21). Follicular phase Follicular-phase length did not differ between older and younger cycling women ( and days, respectively). Although mean FSH across the entire follicular phase was not different between older and younger women selected for regular menstrual cycles, the peak FSH level from the day of menses to the day before ovulation occurred earlier in the older women (day vs ; P 0.001), and FSH was higher in the early follicular phase (Table 1, Fig. 2). Inhibin B was lower in the older group in the early follicular phase, whereas E2 was not different between the two groups (Table 1, Fig. 2). In the mid- and late follicular phase, inhibin B was lower and E2 was higher in older subjects, compared with younger subjects, perhaps accounting for the absence of differences in FSH levels at these cycle stages (Table 1, Fig. 2). Inhibin A was not different between the older and younger groups in the follicular phase, and there were no differences in LH or P4. Age correlated inversely with inhibin B (r 0.4; P 0.03) and positively with FSH (r 0.3; P 0.03) across the menstrual cycle. Age correlated with E2 (r 0.3; P 0.05) in the follicular phase only. Mean follicular-phase FSH correlated inversely with E2 (r 0.402, P 0.03) and showed a trend toward an inverse correlation with inhibin B (r 0.334, P 0.06) when adjusted for age.

3 DIMERIC INHIBIN IN REPRODUCTIVE AGING 107 FIG. 1. Mean inhibin B, FSH, E2, inhibin A, LH, and P4 levels in younger (E; yr) and older (F; yr) cycling women, centered to the day of ovulation. Notice that FSH is higher and inhibin B is lower in the early follicular phase of older subjects. In contrast, FSH is not different between older and younger subjects during the remainder of the follicular phase, when E2 is higher and inhibin B is lower in older subjects. FSH is also higher in the early luteal phase of older subjects when inhibin B is lower. Inhibin A is lower in older subjects only on the day after the LH peak. (**, P 0.02; *, P 0.04). TABLE 1. Mean follicular phase FSH, inhibin B, inhibin A, and estradiol levels Follicular phase (FP) Early FP Mid FP Late FP FSH (IU/L) 35 yr a yr b c Inhibin B (pg/ml) 35 yr yr 96 6 d 88 7 c c 85 8 c Inhibin A (IU/mL) 35 yr yr Estradiol (pg/ml) 35 yr yr d d e a n 23. n 21. c P 0.05 between subjects 35 yr and 35 yr. d P e P Twenty-seven of the subjects studied cross-sectionally underwent ultrasound evaluation of follicular development. In this subset, older and younger subjects developed a single preovulatory follicle of similar size ( vs mm in older and younger subjects, respectively). Three older subjects and two younger subjects had 1 2 additional follicles of 11 mm, whereas one younger subject had an additional follicle of 20 mm, and one older subject had an additional follicle of 14 mm, neither of which seemed to ovulate, as determined by ultrasound. Maximum follicle diameter correlated positively with inhibin A and E2 levels drawn on the same day but not with inhibin B (Fig. 3). When expressed in relation to size of the dominant follicle, older women had similar levels of serum inhibin A ( vs IU/mL mm) but higher levels of E2 ( vs pg/ml mm; P 0.05), compared with younger women. Luteal phase Luteal phase length did not differ between the two groups ( vs days for older and younger women, respectively). FSH levels were higher in the early luteal phase in older, compared with younger, cycling women (Table 2; Fig. 1). Inhibin B was lower both in the early luteal phase and across the entire luteal phase in older women (Table 2; Fig. 1). Inhibin A was also lower on the day after the LH peak in the older group ( vs IU/mL; P 0.04; Fig. 1). However, there were no further differences in inhibin A across the luteal phase, nor were differences observed in LH, E2, or P4. Longitudinal cycles In all three subjects studied longitudinally, FSH was higher across the menstrual cycle after 10 yr (Fig. 4), although these differences did not reach statistical significance, because of small numbers and interindividual variation. At the time of the earlier study, the average inhibin B levels in these subjects had already reached the levels seen in the older subjects studied cross-sectionally. Despite these low baseline levels, the average inhibin B in the early phase (87 15 vs pg/ml; P 0.09) and midfollicular phase (111 10

4 108 WELT ET AL. JCE&M 1999 Vol 84 No 1 FIG. 2. Mean FSH, inhibin B (Inh B), and E2 in younger (open bars; yr) and older (closed bars; yr) women in the early (EFP), mid- (MFP), and late follicular phase (LFP) of the menstrual cycle (**, P 0.05). vs pg/ml; P 0.09) showed a trend toward a further decrease after 10 yr. In the oldest woman, 47 yr old, cycles had become less regular by the second study. FSH was elevated above the 95% confidence limit of normal women and demonstrated remarkable cycle-to-cycle variability. The cycle in which FSH was highest in the follicular phase also showed the lowest inhibin B levels (Fig. 5). These data complement the similar inverse relationship between inhibin B and FSH observed in the cross-sectional data. Inhibin A levels were lower in the late follicular phase in all three subjects after 10 yr, where E2 was not different (Figs. 4 and 5). Interestingly, increased age was associated with a decrease in luteal phase secretion of inhibin A and P4 in all three subjects after 10 yr, in the absence of changes in E2 (Fig. 4). This was true, even for the 37-yr-old subject, whose cycles remained regular and in whom FSH was not elevated. FIG. 3. Correlation between maximum follicle diameter on ultrasound and inhibin A, E2, and inhibin B levels on the same day. Correlation coefficients and P values are indicated. Discussion Lower inhibin levels in the follicular and luteal phases of older cycling women have been demonstrated in some (11), but not all (10, 13), previous studies, using an assay directed against the inhibin -subunit and measuring a combination of inhibin A, inhibin B, and the nonbiologically active -subunit (22 24). Using assays specific for dimeric inhibin (19 21), we have now shown in cross-sectional and longitudinal studies that reproductive aging is associated with changes in both dimeric inhibin A and inhibin B. Inhibin B is decreased in women 35 yr old or older, the age at which an increase in follicular-phase FSH is first detectable (7). Though less apparent in cross-sectional studies, changes in inhibin A are clearly seen in patients studied longitudinally. These studies suggest that inhibin B plays an important role in the early follicular-phase FSH rise that occurs with reproductive aging, but a role for inhibin A cannot be excluded. The decrease in inhibin B, demonstrated across the menstrual cycle in older cycling women in this study, confirms and extends the work of Klein et al., which indicated that inhibin B levels were lower in the 5 days surrounding the FSH peak in the follicular phase of women who already had modestly elevated FSH levels (25). In the current study, FSH is higher and inhibin B lower in the early follicular phase in subjects studied both cross-sectionally and longitudinally, and inhibin B varies inversely with FSH when controlled for age, thus providing further evidence for a negative feedback role for inhibin B. The increased FSH in the early follicular phase is not sustained through the mid- and late follicular

5 DIMERIC INHIBIN IN REPRODUCTIVE AGING 109 TABLE 2. Mean luteal phase inhibin B, inhibin A, estradiol and progesterone levels Luteal phase (LP) Early LP Mid LP Late LP FSH (IU/L) 35 yr a yr b * Inhibin B (pg/ml) 35 yr yr 37 3 c 64 6 c 21 2 d 22 2 d Inhibin A (IU/mL) 35 yr yr Estradiol (pg/ml) 35 yr yr Progesterone (ng/ml) 35 yr yr a n 23. b n 21. c P 0.05 between subjects 35 yr and 35 yr. d P FIG. 4. Mean FSH, Inh B, inhibin A (Inh A), P4, and E2 in the follicular and luteal phase of all three subjects studied longitudinally. Subjects were 28, 32, and 36 yr old in the initial study (open bars) and 37, 42, and 47 yr old, respectively, in the second study (closed bars). ELP, Early; MLP, mid-; and LLP, late luteal phase (**, P 0.05). phase, when E2 levels are clearly higher in older women, however, alluding to the important balance between E2 and inhibin B in the regulation of FSH. In vitro studies in nonhuman primates (34) and in the human (35) indicate that inhibin B is a product of small antral follicles. The rise in inhibin B across the luteal-follicular transition (18) when the pool of small follicles is recruited and the decrease in the late follicular phase in this study and others (19) is consistent with these findings. Further, no relationship was demonstrated between inhibin B and follicle size, in contrast to inhibin A and E2 which increase with increasing follicle size, suggesting that in normal cycles inhibin B is derived from follicles other than the dominant follicle. The decrease in inhibin B secretion in women 35 yr in this study occurs concurrent with the first marked decline in fertility with age (1) suggesting that it reflects a decrease in the number of follicles available for recruitment. The decrease in inhibin B is demonstrated even in the luteal phase, when levels are close to the lower limit of assay sensitivity and the coefficient of variation is high (32). When FSH is elevated and cycles have become slightly irregular, as seen in the woman studied longitudinally, a more marked decrease in inhibin B is observed in the follicular phase, paralleling the accelerated decrease in follicle number at the time of the perimenopausal transition (4). Thus, lower inhibin B levels in older cycling women may be the earliest marker of the decreased number of available follicles in reproductive aging. The changes in inhibin A secretion with age are more subtle. A decrease in inhibin A was not demonstrated with aging in the cross-sectional studies, but it was seen in individual subjects studied longitudinally over a 10-yr period. These changes were seen even in the youngest subject, who was 37 yr old at the time of the second study. Therefore, the changes in inhibin A may be detectable only when the large interindividual variability is eliminated. Alternatively, decreases in inhibin A may occur later than decreases in inhibin B, as suggested by recent data in women studied across the perimenopause (36). A decrease in P4 accompanied the decreased inhibin A in the luteal phase of subjects studied longitudinally in this study, whereas E2 was relatively preserved, as previously described (9, 15). Taken together, the results suggest that luteinized granulosa cell synthetic function is relatively preserved until late in aging but is disrupted before changes in aromatase function are manifested.

6 110 WELT ET AL. JCE&M 1999 Vol 84 No 1 FIG. 5. Inhibin B, FSH, E2, inhibin A, LH, and P4 in a representative subject studied across two menstrual cycles at age 36 (E, ) and again at age 47 (F, f), when cycles had become slightly irregular (21 28 days) and FSH levels were elevated. Note the lower levels of inhibin B across the cycle, the decrease in inhibin A and P4 in the luteal phase, and the relative preservation of E2 with age and increasing FSH and LH. Data are centered to the day of ovulation. The current data are consistent with studies that uniformly demonstrate an increase in late follicular-phase E2 in older reproductive-age women (7, 10, 25), whereas a decrease in E2 across the follicular phase is eventually observed in women more than 45 yr old (8, 11). The results of the present study further demonstrate an increased E2 level, in relation to follicle size and in the absence of multifollicular development. Thus, the increased E2 indicates changes in aromatase activity, perhaps caused by increased FSH or substrate availability at the level of the dominant follicle. The higher late follicular-phase E2 levels may also indicate a decrease in pituitary sensitivity with age, resulting in the need for higher E2 levels to elicit the LH surge. The control of FSH is dependent not only on inhibin and E2 but also on the activin/follistatin system (16, 17). Some (26, 37), but not all (38), recent studies have demonstrated an increase in activin A levels across reproductive aging, suggesting that activin A may play an endocrine role in stimulating FSH secretion. However, an increase in follistatin (37) or similar levels of free follistatin (26), the major activin binding protein, accompanied the increase in activin A in these studies, suggesting that circulating activin A was not biologically active. Further, no correlation was demonstrated between FSH and activin A in the largest study (38). Previous work from our group demonstrated no difference in total activin A or total follistatin among premenopausal, postmenopausal (38 40), and castrate women (40). Therefore, it does not seem that the ovary is the major source of circulating activin A or follistatin in women or that activin A plays a major endocrine role in the control of FSH in aging. In summary, in a large population of regularly cycling women with normal day 3 FSH levels, we have demonstrated that reproductive aging is accompanied by changes in both inhibin B and inhibin A. Decreases in inhibin B precede those of inhibin A and occur in concert with increases in E2, suggesting that inhibin B negative feedback is the most important factor controlling the rise in FSH across aging. Our data also suggest that a decrease in inhibin B across the cycle may be the earliest marker of the decline in follicle number with reproductive aging. Acknowledgments We would like to thank Geralyn Lambert-Messerlian, Ph.D.; Patrick Sluss, Ph.D.; Rita Khoury, M.D.; and Patty Smith, B.S. for inhibin A and inhibin B assay development. We also thank the members of the P30 core laboratory for their technical assistance. We acknowledge Judy Adams, D.M.U., for her expertise in performing and analyzing the ultrasound data. We would also like to thank William F. Crowley Jr., M.D., for his careful review of the manuscript; Brooke Wexler, B.A., for her assistance in patient recruitment; and Douglas Hayden, Ph.D., and David Schoenfeld, Ph.D., for assistance with statistical analysis. References 1. Schwartz D, Mayaux MJ Female fecundity as a function of age: results of artificial insemination in 2193 nulliparous women with azoospermic husbands. New Engl J Med. 306: Menken J, Trussell J, Larsen U Age and infertility. Science. 233: Block E Quantitative morphological investigations of the follicular system in women. 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