1. Introduction
Anti-Mullerian hormone (AMH), a hormone originally known to inhibit the development of Mullerian ducts in male embryos, has been shown to be an excellent marker of ovarian reserve in women.1 AMH is secreted by the granulosa cells of the preantral and small antral follicles and inhibits the initial and cyclic recruitment of follicles.2 Serum AMH declines as age increases in women over 25 years of age,3 and has been shown to be as effective of a predictor of the ovarian response during controlled ovarian hyperstimulation as antral follicle counts (AFCs).4 Moreover, serum AMH exhibits less intra- and intercycle variation than AFCs, suggesting that AMH may be a more reliable means of assessing ovarian reserve.5 However, age-related changes in serum AMH levels in Taiwanese women have not been established.
The expression of AMH can first be detected in human fetal ovaries at 36 weeks of gestation.6 AMH shows a rise in the weeks following birth and reaches its highest level after puberty.2 In adult women, serum AMH levels decline with age and become undetectable after menopause.4 This decline in AMH levels reflects the decrease in the number of follicles and the resulting reduction in reproductive capacity.7 Several studies have suggested that a single AMH measurement may be a good predictor of the onset of menopause in aging women.8–10
Polycystic ovary syndrome (PCOS) is characterized by chronic oligoanovulation, excessive androgen, and enlarged polycystic ovaries, and women with PCOS are at greater risk for the development of diabetes, cardiovascular disease, and ovarian hyperstimulation syndrome during ovarian stimulation.4,11 Because women with PCOS show an increased number of antral follicles, circulating AMH levels in these women are two to three times higher than levels in healthy controls.12 Measuring AMH has been found to be a useful diagnostic marker for PCOS.13 However, the AMH-based diagnostic criteria for PCOS in Taiwanese women remain to be determined.
The purpose of this study was to establish the age-related changes in AMH levels that occur in Taiwanese women of reproductive age and to determine if AMH measurement is a highly sensitive and specific tool for diagnosing PCOS in Taiwanese women. Our results may provide a basis for the application of AMH measurement in Taiwanese women in the field of reproductive medicine.
2. Methods
2.1. Subjects
In order to determine age-related changes in AMH levels that occur in healthy, fertile Taiwanese women and in women with premature ovarian failure or menopause and the sensitivity and specificity of AMH for diagnosing PCOS, we recruited three types of patients. This study was approved by the ethics committee of Taipei Veterans Hospital, and informed consent was obtained from all participating patients.
A group of 59 healthy, fertile, regularly cycling women aged 26–49 years was enrolled from the community, with all subjects meeting the following criteria: 1) having at least one natural pregnancy carried to term; 2) regular menstrual cycle with an interval of 21–35 days; 3) no medical history of hirsutism or severe acne; 4) no evidence of endocrine disease; 5) no history of ovarian abnormalities; 6) no history of ovarian or uterine surgery; and 7) no history of taking medicines that contained hormones within the previous 2 months.
A second group of seven patients, aged 31–55 years with premature ovarian failure or menopause, were recruited. All patients met the following criteria: 1) absence of spontaneous menstrual bleeding for more than 12 months; 2) serum estradiol level <25 pg/mL; and 3) serum follicle-stimulating hormone level >20 mIU/mL.
A third group of 45 PCOS patients aged 25–40 years was also recruited. These patients met the Rotterdam criteria, i.e., they demonstrated an association between at least two of the three following features: 1) oligo- or anovulation; 2) clinical and/or biochemical signs of hyperandrogenism; or 3) polycystic ovaries on transvaginal ultrasound; in addition to the exclusion of related diseases such as hyperprolactinemia, hyperthyroidism, congenital adrenal hyperplasia, etc.
2.2. Hormonal immunoassays
Blood samples were obtained during the early follicular phase (days 3–5) from the healthy fertile controls and PCOS patients. In the case of amenorrhoeic women with PCOS, intramuscular progesterone (100 mg) or oral medroxyprogesterone acetate (10 mg/day for 7 days) was given to induce withdrawal bleeding. Patients with premature ovarian failure or menopause were required to give blood samples on random days. Serum AMH concentrations were assayed using the ACTIVE MIS/AMH ELISA kit (reference DSL-10-14400; Diagnostic Systems Laboratories, Inc., Webster, TX, USA) according to the manufacturer's instructions. The lowest detectable level was 0.006 ng/mL. Intra- and interassay coefficients of variation were less than 4.6 and 8.0%, respectively. Serum concentrations of luteinizing hormone (LH), testosterone and dehydroepiandrosterone sulfate (DHEA-S) were measured using a radioimmunoassay (RIA) (Beckman Coulter, Inc., Brea, CA, USA).
2.3. Analysis
AMH levels are expressed as the means ± standard deviation (SD). Statistical significance in experiments with three or more groups was determined by one-way analysis of variance using the general linear model, followed by Fisher's post-hoc least significant difference test. Statistical significance in experiments with two groups was determined using the Student t-test. The sensitivity of AMH for diagnosing PCOS was defined as the proportion of PCOS patients that were correctly identified using AMH measurements. The specificity of AMH for diagnosing PCOS was defined as the proportion of patients without PCOS that were correctly identified by AMH testing. However, since there are clinical, biochemical, and ultrasonic criteria for diagnosing PCOS, our aim was to increase specificity by using AMH and to keep the sensitivity as close as possible to the specificity. Correlations were assessed by calculating the Spearman coefficient.
3. Results
3.1. Age-related decline in AMH levels
A total of 59 healthy fertile women were recruited. As shown in Fig. 1, AMH levels in healthy fertile women with regular menstrual cycles demonstrated an age-related decline. Analysis showed that there was a rapid drop in AMH levels between 30–40 years of age, followed by a slow decrease after 40 years (Fig. 2; p < 0.05 between the ages of 25–30 and 31–35 years; p < 0.05 between the ages of 31–35 and 36–40 years; p > 0.05 between the ages of 36–40 and 41–45 years). All patients with premature ovarian failure and menopause had undetectable AMH levels (Fig. 1).
;)
Fig. 1: Serum anti-Mullerian hormone (AMH) levels in normal fertile controls and patients with ovarian failure (n = 59 for normal fertile control group; n = 7 for ovarian failure group).
Fig. 2: Serum AMH concentrations in normal fertile controls of different ages. The means of groups without the same letter are significantly different (n = 7 for patients aged 25–30 years; n = 12 for patients aged 31–35 years; n = 20 for patients aged 36–40 years; n = 16 for patients aged 41–45 years; and n = 4 for patients aged 46–49 years).
3.2. Significantly higher AMH levels in PCOS patients
Because women with PCOS have an increased number of antral follicles, serum AMH levels are expected to be higher in these women. Patients aged 25–40 years who met the Rotterdam criteria for diagnosing PCOS were recruited. AMH levels in PCOS patients were found to be higher than those in healthy fertile controls (Fig. 3). Moreover, the rate of age-related decline in AMH levels was lower in PCOS patients compared with that of healthy fertile patients (Fig. 3). Statistical analysis showed that AMH levels in all age-related subgroups were significantly higher in PCOS patients than healthy fertile controls (Fig. 4).
Fig. 3: Comparison of serum AMH levels in PCOS versus normal fertile controls (n = 45 for PCOS group; n = 39 for normal fertile controls).
Fig. 4: Statistical analysis of serum AMH concentrations in PCOS versus normal fertile control patients in different age subgroups (n = 24 for PCOS patients aged 25–30 years; n = 7 for normal fertile controls aged 25–30 years; n = 14 for PCOS patients aged 31–35 years; n = 12 for normal fertile controls aged 31–35 years; n = 7 for PCOS patients aged 36–40 years; and n = 20 for normal fertile controls aged 36–40 years). * p < 0.05.
In patients with PCOS, AMH levels were positively correlated with LH (r = 0.297, p < 0.001), testosterone (r = 9.365, p = 0.001), and DHEA-S (r = 0.421, p = 0.016), but the correlation between AMH levels and body mass index (BMI) was not statistically significant (r = -0.242, p = 0.064).
To calculate the sensitivity and specificity of AMH for diagnosing PCOS, the age factor was controlled by narrowing the age range to 29–38 years (the mean ages of the control and PCOS groups were 33.25 ± 3.04 and 32.32 ± 2.93 years, respectively; p = 0.257; n = 24 for the control group and n = 31 for the PCOS group). The sensitivity and specificity of AMH for diagnosing PCOS in patients aged 29–38 years were calculated to be 74% and 79%, respectively, using an AMH cut-off value of 3.5 ng/mL.
4. Discussion
AMH was first discovered as a secretion from the testis of a male fetus that induced regression of the Mullerian ducts.14 Later, AMH was found to be produced by the granulosa cells of preantral and small antral follicles in females.15 The AMH level is low in females at birth, rises rapidly during 0–3 months and 4–8 years of age, and remains constant during 8–25 years of age, and is followed by a gradual decline after 25 years of age.3 After menopause, AMH levels become undetectable. Although the age-related decline in AMH levels has been established in several studies, it has not been determined in Taiwanese women. In the present study, we recruited 59 healthy, fertile, regularly cycling Taiwanese women aged 26–49 years and established age-related changes in AMH levels. Using these reference data as the control, we found that AMH levels in PCOS patients were significantly higher. The sensitivity and specificity of AMH for detecting PCOS in patients aged 29–38 years were calculated to be 74% and 79%, respectively, using an AMH cut-off value of 3.5 ng/mL.
AMH is a 140-kilodalton glycoprotein homodimer that belongs to the transforming growth factor family.4 In females, AMH inhibits the initial and cyclic recruitment of follicles.2 Circulating AMH is solely of ovarian origin in women, and AMH levels become undetectable 3–5 days after oophorectomy.16 Serum AMH levels are stable throughout the menstrual cycle and are unmodified by pregnancy, gonadotropin-releasing hormone treatment, and the administration of short-term oral contraceptives, making AMH an ideal marker of ovarian reserve. In fact, AMH and antral follicle counts have been accepted by most reproductive endocrinologists as the best methods for testing ovarian reserve.1 AMH measurement may be superior to AFCs because it is less operator-dependent and can be performed on any day of the menstrual cycle. In this study, we found that there was a quick decline in AMH levels between 30–40 years of age that was followed by a gradual decrease after 40 years of age. The age-related decline in AMH levels for Taiwanese women found in this study can be utilized by future studies that involve AMH measurement in Taiwanese women.
The serum AMH level, when measured at a certain age, has been used to predict the age of menopause.8–10 The AMH-based prediction of age at menopause can be utilized for reproductive planning. Moreover, it may be useful for choosing treatment strategies for gynecological diseases. In women with severe endometriosis who do not desire further pregnancies, for example, the selection of treatments that require a definitive hysterectomy versus other medical therapies may depend on when the patient will go through menopause, which could be predicted by AMH measurement. More normal fertile women and menopausal women need to be enrolled in additional studies in order to predict the age of menopause onset in Taiwanese women.
In this study, AMH levels in PCOS women were found to be approximately three-fold higher that those of healthy fertile control. Although the increase in AMH levels in PCOS women was thought to be due to the increase in small antral follicles, a recent study showed that AMH production is 75 times higher per granulosa cell in PCOS patients than in granulosa cells in normal ovaries.17 Furthermore, AMH concentrations in the follicular fluid were five times higher in the follicles of women with anovulatory PCOS compared with women who were ovulatory.18 In this study, the rate of age-related decline in AMH concentrations was found to be smaller in PCOS patients compared with healthy fertile patients. Whether or not the aforementioned finding is due to a slower depletion of the follicular pool in PCOS patients requires further research. The sensitivity and specificity of AMH-based detection of PCOS in Taiwanese women aged 29–38 years were calculated to be 74% and 79%, respectively, using an AMH cut-off value of 3.5 ng/mL. Whether it is helpful or not to use AMH as a screening tool for PCOS requires future investigations.
Racial difference in the serum levels of AMH have been reported in the literature.19 Compared with white women, AMH concentrations are lower among black (25.2% lower) and Hispanic (24.6% lower) women, following adjustment for other variables. Our data showed that AMH values in Taiwanese women were similar to or slightly higher than those of white women that have been reported in previously published articles, but variations in different AMH measurement techniques used by differ laboratories could result in different results, and other confounding variables will have to be controlled in future studies. AMH levels were also reported to be significantly higher in PCOS patients in studies conducted in France, Finland, and other countries, indicating that higher AMH values in women with PCOS may be a universal finding among different races.11–13
In conclusion, we have provided data on Taiwanese women that demonstrate an age-related decline in AMH levels and establish an AMH-based method for diagnosing PCOS, which may be used as reference data for future AMH studies on Taiwanese women. Future studies are required to evaluate the value of a single AMH measurement for the prediction of the age of menopause onset.
Acknowledgments
This work was supported, in part, by the National Science Council, Taiwan (NSC 98-2314-B-075-032-MY3), Taipei Veterans General Hospital, Taiwan (V99E1-009), and HealthBanks Biotech Company Limited, Taipei, Taiwan (R92-001-9).
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