REPRODUCTIVE ENDOCRINOLOGY

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1 FERTILITY AND STERILITY VOL. 77, NO. 2, FEBRUARY 2002 Copyright 2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. REPRODUCTIVE ENDOCRINOLOGY Antimüllerian hormone serum levels: a putative marker for ovarian aging Annemarie de Vet, M.D., a Joop S. E. Laven, Ph.D., a Frank H. de Jong, Ph.D., b Axel P. N. Themmen, Ph.D., b and Bart C. J. M. Fauser, Ph.D. a Erasmus University Medical Center, Rotterdam, The Netherlands Received May 1, 2001; revised and accepted October 10, Supported by Stichting Voortplantingsgeneeskunde Rotterdam and by the European Commission (QLRT ). Reprint requests: Bart C. J. M. Fauser, Ph.D., Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Erasmus University Medical Center Rotterdam, Dr Molewaterplein 40, 3015 GD, Rotterdam, The Netherlands (FAX: ; a Division of Reproductive Medicine, Department of Obstetrics and Gynecology. b Department of Internal Medicine /02/$22.00 PII S (01) Objective: To investigate whether serum concentrations of antimüllerian hormone may be used as a marker for ovarian aging. Design: Longitudinal observational study. Setting: Academic research center. Patient(s): Forty-one normo-ovulatory premenopausal women and 13 healthy postmenopausal women. Main Outcome Measure(s): Concentrations of serum antimüllerian hormone (assessed on two occasions years apart), FSH, inhibin B, and estradiol and number of ovarian follicles on ultrasonography. Result(s): Concentrations of antimüllerian hormone decreased significantly over time (median value, 2.1 g/l [range, g/l] at visit 1 vs. 1.3 g/l [range, g/l] at visit 2), whereas the number of antral follicles and levels of FSH and inhibin B did not change. During visits 1 and 2, concentrations of antimüllerian hormone correlated with age (r.40, P.01 and r.57, P.001, respectively); number of antral follicles (r.66, P.001 and r.71, P.001); and, to a lesser extent, with FSH level (r.29, P.07 and r.37, P.05) but not with inhibin B levels. Conclusion(s): Serum concentrations of antimüllerian hormone decreased over time in young normoovulatory women, whereas other markers associated with ovarian aging did not change. Concentrations of antimüllerian hormone correlate with the number of antral follicles and age and less strongly with FSH level. Concentrations of antimüllerian hormone may be a novel marker for ovarian aging. (Fertil Steril 2002;77: by American Society for Reproductive Medicine.) Key Words: Ovarian aging, antimüllerian hormone, follicle number, FSH, inhibin B Several factors involved in the regulation of the early stages of follicle development have been identified, such as growth and differentiation factors 9 and 9b (1, 2), antimüllerian hormone (3), Wilms tumor protein (4), Bax (5), and Bcl-2 (6). Transgenic animal models (7 9) established the involvement of most of these factors in follicle development and primordial follicle pool depletion (5). Antimüllerian hormone, a member of the transforming growth factor- family produced by granulosa cells of early developing follicles (3), seems to be the only factor expressed exclusively in the gonads (10, 11). Recent transgenic studies indicate that antimüllerian hormone knock-out mice are born with a normal number of follicles. However, depletion of the stock is accelerated in the absence of antimüllerian hormone (8), which may compromise reproductive life span. This observation strongly suggests that antimüllerian hormone has a direct or indirect inhibitory effect on follicle pool depletion. Throughout life, the stock of primordial follicles decreases steadily until it is depleted around menopause (12). Morphometric studies suggest a correlation between the size of the stock of primordial follicles and the number of small growing follicles in the primate and human ovary (13). The role of antimüllerian hormone in the regulation of human folliculogenesis remains speculative; however, production of antimüllerian hormone by a reduced number of growing follicles may decrease over time. Antimüllerian hormone is detectable in the peripheral circulation of healthy women (14, 15). Only minor fluctuations in serum concentrations have been observed during the normal menstrual cycle, consistent with continuous noncyclic growth of small follicles (16). 357

2 Concentrations of antimüllerian hormone may therefore be used as a marker for ovarian aging. Serum levels of FSH and inhibin B are endocrine markers used to assess ovarian aging. Inhibin B is produced by healthy growing follicles after cyclic recruitment during the early follicular phase of the menstrual cycle (17, 18). Levels of FSH start to increase over time because of reduced inhibin B and E 2 production by the diminished cohort of growing follicles (19, 20). The increase in FSH level occurs late in the sequence of events associated with ovarian aging (21). If fertility is considered the end point this increase may be of limited clinical use as a marker (22). Although inhibin B levels change earlier than do FSH levels, clinical studies have had conflicting results concerning the value of cycle day 3 levels of inhibin B in predicting the chance of pregnancy (23 25). Recent data suggest that a decreased number of ovarian follicles on ultrasonography is a sensitive marker of advanced aging of the ovaries (26), providing improved prediction of the outcome of fertility therapy (27). Decreased fertility and absolute infertility seems to precede menopause by a fixed interval (28), indicating that the natural decrease in fertility starts earlier in women who are destined to undergo menopause at a relatively young age. The proper assessment of ovarian aging at an early stage seems crucial for counseling patients about their chances for pregnancy, either spontaneously or during fertility therapy (29). We investigated whether serum antimüllerian hormone levels decrease over time in young normo-ovulatory women and whether antimüllerian hormone concentrations correlate with other factors associated with ovarian aging. MATERIALS AND METHODS Participants We studied 41 normo-ovulatory women who participated in previous studies (30 32) between 1993 and Inclusion criteria were age years, regular menstrual cycle (cycle length, days), body mass index of kg/m 2, absence of endocrine disorders or any other relevant disease, and no receipt of medical or hormonal treatment for at least 3 months before the study. Women were requested to prevent pregnancy during the study by using an intrauterine device, tubal ligation, or condoms. Serum antimüllerian hormone was also measured in 13 healthy women at least 2 years after menopause (mean age [ SD], 56 2 years). The study was approved by the institutional review board of Erasmus University Medical Center. All subjects were recruited by advertisement in the local news media and paid for their participation. Written informed consent was obtained from each participant. Outcome Variables All women visited our unit for another early follicular phase assessment during Blood was withdrawn on day 3 in all cycles. Before the second observation cycle, participants answered a questionnaire about cycle, reproductive, and medical history during the interval between visits. All cycles were proven to be ovulatory by measuring the midcycle LH surge and mid-luteal progesterone levels. Transvaginal ultrasonography was performed on day 3 as described elsewhere (33). All visible antral follicles present in the ovaries were counted. Blood samples were centrifuged within 2 hours after withdrawal and stored at 20 C until assay. Serum FSH was assessed by using a chemiluminescent immunoassay (Immulite; Diagnostic Products Corp., Los Angeles, CA). Intraassay and interassay coefficients of variation were 8% and 7%, respectively. Inhibin B was measured by using an immunoenzymometric assay (Serotec, Oxford, United Kingdom), as described elsewhere (34). Intraassay and interassay coefficients of variation were 9% and 15%, respectively. Estradiol was measured by using radioimmunoassay kits (Diagnostic Products Corp.); intraassay and interassay coefficients of variation were 15% and 18%. Serum antimüllerian hormone levels was measured by using an ultrasensitive enzyme-linked immunosorbent assay (Immunotech-Coulter, Marseilles, France), as described elsewhere (35). The limit of detection (defined as negative control 3 SD of the negative control) was 0.05 g/l. For positive quality control, high and low serum samples from separate assays were used. For negative quality control, samples from postmenopausal women were used. Intraassay and inter-assay coefficients of variation were 5% and 8%, respectively. Statistical Analysis Statistical analysis was performed by using SPSS software (SPSS, Inc., Chicago, IL). Data were analyzed for normal distribution. Data are presented as means ( SD) or medians and ranges. To determine whether variables changed over time, a one-sample t-test was used. P.05 was considered statistically significant. Correlations between different parameters were determined by using bivariate correlation statistics and are expressed as Spearman correlation coefficients. Linear regression of age on the log-transformed antimüllerian hormone values was performed, and 95% prediction limits were calculated. For graphical display, results of the linear regression were back-transformed. RESULTS The mean age of the normo-ovulatory women was 29 4 years at visit 1 and 32 4 years at visit 2. Mean body mass index was 22 3 kg/m 2 at visit 1 and 23 4 kg/m 2 at visit 2. The median of the average cycle length during both visits was 28 days (range, days). The interval between the two visits ranged from 1.1 years to 7.3 years (mean, years). 358 de Vet et al. Antimüllerian hormone and ovarian aging Vol. 77, No. 2, February 2002

3 FIGURE 1 Box-and-whiskers plots of antimüllerian hormone levels in 41 normo-ovulatory women at two visits (mean [ SD] interval between visits, years) (left) and by age (right). All postmenopausal women presented with undetectable serum levels of antimüllerian hormone (data not shown). Figure 1 shows levels of antimüllerian hormone at visits 1 and 2 and the distribution of antimüllerian hormone levels by age group per visit. At both visits, levels of antimüllerian hormone were negatively correlated with age (r.40, P.01 at visit 1 and r.57, P.001 at visit 2). Table 1 shows levels of antimüllerian hormone, FSH, inhibin B, and E 2 and the number of antral follicles at visits 1 and 2. Serum levels of FSH, inhibin B, and E 2 and the number of follicles did not change significantly over time within participants, whereas levels of antimüllerian hormone significantly decreased over time (P.001). TABLE 1 Serum variables and follicle number assessed on two occasions in 41 normo-ovulatory women. Screening variable Visit 1 Visit 2 P value a Antimüllerian hormone 2.1 ( ) 1.3 ( ).001 level ( g/l) FSH level (IU/L) 6.0 ( ) 5.8 ( ).29 Inhibin B level (pg/l) 112 (12 213) 110 (4 206).92 E 2 level (pmol/l) 151 (64 404) 161 (70 620).52 No. of antral follicles 14 (6 28) 14 (2 24).27 Note: Values are medians (ranges). a One-sample t-test. Figure 2 shows serum levels of antimüllerian hormone and FSH and follicle counts in relation to age in individual women. In some women, both antimüllerian hormone levels and follicle counts increased over time rather than decreased. Individual data for inhibin B and E 2 are not presented. Levels of antimüllerian hormone correlated most strongly with follicle count (r.66, P.001 at visit 1 and r.71, P.001 at visit 2) and less strongly with FSH levels (r.29, P.07 at visit 1 and r.37, P.05 at visit 2) and age (r.40, P.01 at visit 1 and r.57, P.001 at visit 2) (Fig. 3). Levels of antimüllerian hormone correlated with inhibin B levels only at visit 2 (r.36, P.02) and did not correlate with E 2 levels at either visit (P.01, P.97 at visit and r.07, P.68) (data not shown). To exclude bias in analysis of serum antimüllerian hormone due to long-term storage of blood samples, we performed statistical analysis on the serum levels of antimüllerian hormone from the 15 participants in whom serum levels at both visits were assessed within 2 years after of blood storage (15). A one-sample t-test of these participants already showed a significant decrease in antimüllerian hormone levels. The slopes of the regression lines of follicle number compared with antimüllerian hormone level were not significantly influenced by storage time of serum (data not shown). Total follicle count did not correlate significantly with FERTILITY & STERILITY 359

4 FIGURE 2 Serum levels of antimüllerian hormone, FSH and follicle number in relation to age in 41 normo-ovulatory women. Serum levels at visit 1 and visit 2 in each woman are interconnected. Solid lines indicate the 95th (upper line), 50th (middle line), and 5th (lower line) percentiles. FIGURE 3 Correlation of age, number of antral follicles, and FSH level with antimüllerian hormone level at visit 1 (closed circles, solid line) and visit 2 (open circles, dotted line) in 41 normoovulatory women. inhibin B levels at either visit (r.31, P 0.07 at visit 1 and r.19, P.35 at visit 2) or with age during visit 1 (r.30, P.06), but they did correlate with age during visit 2 (r.41, P.03) (data not shown). Finally, serum levels of FSH, inhibin B, and E 2 did not correlate with age at either visit (data not shown). DISCUSSION Morphometric studies of the human ovary suggest a correlation between the diminished stock of resting primordial follicles and the decrease in number of small growing follicles with advancing age (13). Because antimüllerian hormone is produced exclusively by these small growing folli- 360 de Vet et al. Antimüllerian hormone and ovarian aging Vol. 77, No. 2, February 2002

5 cles and is secreted into the circulation, levels of the hormone may decrease with advancing age. We found that serum levels of antimüllerian hormone significantly decreased over time in young normo-ovulatory women, whereas other markers associated with ovarian aging, such as serum levels of FSH and inhibin B and the number of antral follicles, did not change during the same interval. In addition, levels of antimüllerian hormone were correlated with age. Markers that directly represent the size of the primordial follicle stock have not been discovered. Because resting follicles have extremely low metabolic activity, it seems unlikely that such a marker would be detectable in the peripheral blood. Current markers associated with ovarian aging are all distantly related to initial development of primordial follicles. At least 3 months after follicles have left the resting stage (initial recruitment), they reach a diameter of about 2 mm (17). Follicles as small as 2 mm in diameter can be seen on ultrasonography. These antral follicles express inhibin B and E 2 after cyclic recruitment elicited by the intercycle increase in FSH level (17, 18). The increase in FSH level at the end of the reproductive life span, which is due to diminished feedback by reduced inhibin B and E 2 output, seems to be even more distantly related to the size of the primordial follicle stock. In the rat, expression of antimüllerian hormone by granulosa cells of the growing follicle starts directly after growth initiation of primordial follicles and disappears just after the small antral follicle stage has been reached (3). Because many factors are involved in the regulation of follicle growth and atresia during the life span of the follicle (20), substances related to early follicle development, such as antimüllerian hormone, may correlate better with the size of the primordial follicle pool. Furthermore, in contrast to other markers associated with early follicle development, serum levels of antimüllerian hormone appear to reflect ovarian activity only, since expression of antimüllerian hormone in females of various species is seen exclusively in granulosa cells (10, 11). The undetectable levels of antimüllerian hormone that we observed in our postmenopausal participants support this idea. We found no change in other markers associated with ovarian aging, such as levels of FSH and inhibin B and the number of antral follicles, during the study period, whereas levels of antimüllerian hormone levels decreased. In the rat, no differences were observed in immunostaining of follicles from similar stages but obtained from animals of different ages (A. P. N. Themmen. Unpublished observations). Therefore, less efficient production of antimüllerian hormone with advancing age is not a likely explanation for the observed decrease in antimüllerian hormone serum levels with age. These observations are in line with the hypothesis that the best marker for size of the stock of primordial follicles is a substance produced in the early stages of follicle development, such as antimüllerian hormone. We observed a strong correlation between antimüllerian hormone and age and between antimüllerian hormone and follicle number. The latter finding suggests that the antral follicle count reflects the amount of small growing follicles. Recently, the age-related decrease in ovarian follicle number on ultrasonography was shown to correspond with the agerelated decrease in primordial follicles (26), suggesting that antral follicle counts not only reflect the primordial follicle pool but also the number of early growing follicles producing antimüllerian hormone. However, we could not establish a decrease in follicle number or change in other agingrelated variables. Studies with larger sample or longer study period would be expected to observe these changes over time. In conclusion, our data establish that serum levels of antimüllerian hormone in normo-ovulatory women decrease over time and decrease with advancing age before changes occur in currently known aging-related variables. These observations, in combination with the strong correlation of antimüllerian hormone with the number of antral follicles, indicate that serum levels of antimüllerian hormone may be a novel marker for ovarian aging. Acknowledgment: The authors thank M. J. C. Eijkemans for statistical advice. References 1. Aaltonen J, Laitinen MP, Vuojolainen K, Jaatinen R, Horelli-Kuitunen N, Seppa L, et al. Human growth differentiation factor 9 (GDF-9) and its novel homolog GDF- 9B are expressed in oocytes during early folliculogenesis. J Clin Endocrinol Metab 1999;84: McGrath SA, Esquela AF, Lee SJ. Oocyte-specific expression of growth/differentiation factor-9. Mol Endocrinol 1995;9: Baarends WM, Uilenbroek JT, Kramer P, Hoogerbrugge JW, van Leeuwen EC, Themmen AP, et al. 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