Role of anti-Müllerian hormone and testosterone in follicular growth: a cross-sectional study

Gonadal hormone anti-Müllerian hormone (AMH), a member of transforming growth factor-β (TGF-β) superfamily, is a glycoprotein synthesized by granulosa cells [1]. It is now considered the best serum biomarker of ovarian reserve, reflecting the number of primordial follicles and its response to exogenous gonadotropins [2]. It is generally agreed that intra-individual variation of serum AMH is less than true biological variability [3, 4], and AMH level is not disturbed by menstrual cycles and exogenous sex steroids [5], so the test of AMH can be performed on any day of menstrual cycle, which is much more convenient than basal follicle-stimulating hormone (FSH) test. Nowadays AMH is widely used in menopause prediction, predicting pregnancy chances in infertility couples, management in IVF, and management of women with cancer [1].

Some studies investigated the relationship between AMH and gonadotropins. It was found that after gonadotropin-releasing hormone agonist (GnRHa) treatment AMH levels decreased significantly together with increase of FSH and luteinizing hormone (LH), and then all hormone levels reversed, in which there was a moderate negative correlation between AMH and FSH [6]. However, others reported that after GnRHa administration AMH level changed significantly, and changes of AMH levels did not correlate with changes of gonadotropins, estradiol, or progesterone [7]. Evenmore, it was found that AMH receptor type 2 (AMHR2) was expressed in pituitary gonadotrope cells and AMH directly stimulated the secretion of FSH and LH [8‐10]. In all, the relationship between AMH and sex hormones is complicated and inconsistent, further investigation is required to address the correlation.

AMH inhibits follicle development in two critical points: the recruitment of primordial follicles into the pool of growing follicles and the responsiveness of growing follicles to FSH [11‐13]. An assessment of the correlation of AMH and individual sex hormone would be valuable for understanding the regulatory function of AMH in follicle development. Diminished ovarian reserve (DOR) and polycystic ovarian syndrome (PCOS) are representative of two extremes of ovarian reserve [14, 15], to explore the predictive factors of DOR and PCOS helps to understand the regulation mechanism of follicle development.

The aim of the present study was to determine the association between serum AMH level and serum basal sex hormone concentrations. Firstly, correlation analysis was performed to explore the individual correlation between AMH and basal sex hormones, and further multiple regression analysis was carried out to select predictive determinants of serum AMH level. Secondly, sex hormone levels were compared in DOR and PCOS populations, and logistic regression analysis was used to further determine the predictive factors of DOR and PCOS.

In this study we demonstrated that age was moderately negatively correlated with AMH concentration. Risk of DOR increased and risk of PCOS decreased significantly with age. Since it is widely accepted that oocyte number and quality decline with age [16, 18], it is reasonable that age is a dominant factor of DOR. However, PCOS is considered a lifelong disease. What is the underlying reason for the decreased risks in aged population? In PCOS patients, due to the increased number of antral follicles, there is higher AMH concentration and lower basal FSH concentration compared to women with normal cycle [19], which leads to rare or no ovulation in PCOS. Aging leads to decreased follicle number, with decreased AMH concentration and increased basal FSH level, which is in favor of follicular growth and regular ovulation [20]. Therefore, it is possible that the diagnosis of PCOS cannot be made any more if it is not diagnosed in earlier age, so the incidence of PCOS decreased as age increased. In younger women with PCOS the main manifestations are hyperandrogenism and chronic anovulation, whereas in older women with PCOS, the main manifestations are obesity, insulin resistance, and metabolic disturbances [21]. A study comparing postmenopausal women with and without PCOS found that PCOS women had similar menopausal age, body weight, BMI, LH, total testosterone, and estradiol, except lower FSH [22]. In a word, the decreased risk of PCOS in aged population may be due to the difficulty of diagnosis.

In the present study, basal FSH was mildly correlated with AMH negatively. This result is in line with the previous studies. It is reported that there is a negative correlation between AMH concentration and serum FSH concentration in PCOS, and AMH may reduce the concentration of FSH and decrease follicle sensitivity to FSH by inhibition of aromatase [23, 24]. This study demonstrates that elevated FSH level is associated with increased risk of DOR, and this is generally accepted worldwide [16].

In this study basal LH was mildly correlated with AMH positively, and elevated LH level was associated with increased risk of PCOS. This result is consistent with the previous studies [25‐27], indicating an interaction between AMH and LH secretion, which may contribute to the pathogenesis of PCOS [28]. On one hand, AMH activates the GnRH neuron through AMH receptors and increases GnRH-dependent LH pulsatility and secretion [10]. On the other hand, LH may increase AMH production in granulosa cells [29]. Therefore, increased AMH and LH stimulate each other mutually and play a role together in follicular arrest and pathogenesis of PCOS.

We found that T was mildly correlated with AMH positively. Moreover, elevated T level was associated with increased risk of PCOS and decreased risk of DOR. The positive correlation between serum AMH and total testosterone has been reported previously [24‐26, 30]. It is demonstrated that androgens stimulate follicular FSHR expression, amplify FSH effect, promote follicular growth, which may lead to increased AMH production [31, 32]. Meanwhile, elevated AMH increases LH secretion in GnRH-neurone through AMH receptors [10], and elevated AMH inhibites aromatase expression in granulosa cells disturbing the transformation of androgen to estrogen [13, 33], which leads to the elevated androgen level. The two hormones may mutually reinforce each other, which may be the reason for the positive correlation of them. Elevated T level is associated with increased risk of PCOS. In PCOS with high androgen, serum AMH levels increase significantly compared with PCOS with normal androgen [27]. Others suggest that serum AMH level is related to the severity of PCOS [34, 35]. Such an interaction may contribute to the pathogenesis of PCOS [28].

As shown in Fig. 4, in PCOS high androgen leads to increased AMH level, and high AMH inhibits follicular development, leading to accumulation of small follicles without development of dominant follicles. High AMH decreases expression of FSHR and inhibit the response to exogenous gonadotropin, which leads to the poor response of ovarian stimulation. On one hand AMH impedes the conversion of androgen to estrogen, and on the other hand accumulated androgen leads to the increase of AMH, which thus initiates a vicious cycle. In DOR women, reduced androgen concentration accompanies with decreased AMH level. Insufficient androgen level cannot supply sufficient substrates for ovarian steroids synthesis. Meanwhile, decreased AMH level leads to accelerated follicular consumption and reduced ovarian reserve.

Either excess or deficiency of androgen may do harm to follicle development and deteriorate fertility [36]. In PCOS, it is demonstrated that suppression of androgen can improve the results of ovulation induction in PCOS [37, 38]. In DOR, androgen supplement may lead to a PCOS-like phenotype that increases oocyte production. Some randomized controlled trials (RCTs) have shown that DHEA and testosterone supplement can improve fertility in women with DOR [39, 40]. It is estimated that about a quarter of women with DOR are treated with DHEA supplements in all IVF centers [41]. Both literatures and our study point out that appropriate androgen level is required for normal follicular growth and is important in controlled ovarian hyperstimulation (COH) process.

The present study has some limitations. Firstly, in this study the diagnosis of DOR was made based on solely AMH. Until now, there is no uniformly accepted definition of DOR, as stated in the committee opinion provided by American Society for Reproductive Medicine [16]. There is mounting evidence to support the use of AMH as a screening test for poor ovarian response, but the data are insufficient yet [16]. In the Bologna criteria abnormal ovarian reserve test included AFC less than 5–7 follicles or AMH below 0.5–1.1 ng/ml [42]. The definition of DOR might be more solid with more criteria such as AFC. Secondly, since this is a retrospective observational study, we cannot distinguish other factors participating in follicular growth. Further RCTs and fundamental studies are required to confirm the roles of T in follicular growth and explore the underlying mechanisms. Furthermore, in infertile population with IVF we can develop an algorithm using AMH and T to predict ovarian response, which can help in daily work.

In the present study, we found that age and FSH were main negative correlation factors, and LH and T were main positive factors of AMH. In DOR group, age, FSH and E₂ increased and T decreased, while in PCOS group, LH and T increased and FSH decreased. Binary logistic regression found that age, weight, FSH, E₂, and T were the significant factors which independently predicted the likelihood of DOR, and that age, BMI, AMH, LH, and T predicted the likelihood of PCOS. Since DOR and PCOS are manifested with insufficient and excessive AMH respectively, our study demonstrated that age and T were factors that most closely correlated with AMH level, and involved in DOR and PCOS. It is suggested that total testosterone plays an important role in follicular growth and more attention should be given to total testosterone level during COH process.