Anti-Müllerian hormone as a biochemical marker of gonadal development and fertility status

Anti-Müllerian hormone (AMH) plays a critical role in sex differentiation in fetal development and goes on to be important in the regulation of folliculogenesis in women. This article discusses the role of AMH in reproductive physiology and the many different situations where assessment of AMH levels is useful, as well as touching on methods of AMH analysis and reference ranges.

Synthesis/processing of anti-Müllerian hormone

Anti-Müllerian hormone (AMH), also known as ‘Müllerianinhibiting hormone’ and ‘Müllerian-inhibiting factor’ is a glycoprotein that belongs to the transforming growth factor-beta (TGF-β) superfamily. It is synthesized as a large precursor with a short signal sequence followed by the pro-prohormone that forms homodimers. Prior to its secretion, the mature hormone undergoes glycosylation and dimerization to produce a 144-kDa dimer composed of identical disulphide-linked 72-kDa monomer subunits [1]. Each monomer contains a large N-terminal ‘pro’ region and a much smaller C-terminal ‘mature’ region, in contrast to other TGF-β family members. The hormone requires the N-terminal domain to potentiate the activity of the C-terminal domain for full bioactivity [1,2]. During cytoplasmic transit, part of AMH is cleaved at specific region to produce the biologically active 110-kDa N-terminal and 25-kDa C-terminal homodimers, which remain together in a non-covalent complex [1]. This hormone typically binds to a specific AMH type II receptor (AMH II) on the cell membrane of its target organs (including mesenchymal cells of the Müllerian ducts, granulosa cells of the ovary, and Sertoli and Leydig cells of the testes) [1,2]. The human gene coding for AMH is located on the short arm of chromosome 19p13.3; this has been sequenced and isolated [3].

Physiology of AMH

AMH is produced by Sertoli cells of the testes in males and by ovarian granulosa cells in females. Expression during male fetal development prevents the Müllerian ducts from developing into the uterus, resulting in male sexual differentiation and gonadal development. Hence, AMH is essential for the involution of the Müllerian ducts (the anlagen of the internal female genitalia in the male fetus) which would otherwise develop into the uterus and fallopian tubes [4]. Sex differentiation in the male fetus is completely dependent on the normal development of testes that will produce two hormones: testosterone by Leydig cells and AMH by Sertoli cells [5]. The AMH-induced regression of the Müllerian duct allows Wolffian ducts to develop into the male reproductive tract under the influence of testosterone; this process occurs as early as 8 weeks of gestation [6]. AMH is continuously produced by the testicles until puberty, and then levels decrease slowly to post-puberty residual values for the rest of life [7]. In the absence of AMH, the embryo develops into a female, allowing the Müllerian duct to differentiate into the upper vagina, uterus and oviduct. AMH is not present in ovarian tissues until the 36th week of gestation and is mainly produced by granulosa cells of prenatal and early antral follicles. The circulating levels reflect the number of remaining primordial follicles (termed ‘ovarian reserve’).

AMH has an essential role as an autocrine and paracrine regulator of ovarian folliculogenesis and maturation in females [8]. Follicle development in the ovaries consists of two stages: initial recruitment during which the primordial follicles start to mature under the control of follicle-stimulating hormone (FSH), which leads to the growth of a group of small antral follicles among which the dominant follicle, which is destined to ovulate, is subsequently selected. The expression of AMH in granulosa cells starts in primary follicles and typically reaches its maximum in granulosa cells of preantral and small antral follicles up to approximately 6 mm in diameter. At this stage, the follicle growth becomes FSH dependent. The hormone expression then diminishes and becomes undetectable. AMH involvement in its inhibitory role occurs in two distinct stages. First, AMH inhibits the transition of follicles from the primordial maturation stages; thus, it contributes in regulating the number of follicles remaining in the primordial pool. Second, AMH inhibits the follicular sensitivity to FSH and therefore has a role in the process of follicular selection [9].

Clinical utility of AMH in health and disease

The role of AMH in reproductive physiology has attracted the scientific interest in the use of AMH in many fields in medicine, as discussed below.

1. Marker of ovarian reserve in female reproduction

AMH appears to be a reliable hormonal marker with respect to ovarian reserve and fertility assessment in females. A normal AMH level (adjusted for age) usually has a positive reflection with fertility. Also, there are different disorders that may affect women which are associated with ovarian insufficiency or diminished ovarian reserve, i.e. women who were born small for gestational age; type 1 diabetics; autoimmune diseases; and women having undergone ovarian or uterine surgery. All may be at risk of various degrees of reduced ovarian reserve, and ovarian reserve testing by AMH level is significantly important in these groups to assess their overall ovarian reserve [8,9].

2. Measurement of ovarian response and reserve in assisted reproduction in the management of women with infertility, including in vitro fertilization (IVF) and prediction of pregnancy

Measurement of serum AMH is important in assessing the ovarian reserve, particularly in assisted reproductive medicine. It is a good prognostic marker of the ovarian response to controlled ovarian stimulation during IVF cycles, and reflects both the number of oocytes retrieved during follicular aspiration and the number of arrested cycles. It may predict poor ovarian response to ovarian hyperstimulation in IVF cycles and women who are prone to ovarian hyperstimulation syndrome (OHSS) [10]. Women who have high AMH levels prior to induction with human gonadotrophins exhibit high rates of live births. This may be attributed to the high level of AMH that corresponds to a greater number of oocytes retrieved from women when using AMH level as a predictor for pregnancy outcome [11]. However, in a cohort of fecund women with a history of one or two prior losses, higher or lower AMH values were not associated withthe fecund ability in unassisted conception [12].

3. Menopause prediction and diagnosis

Menopause, which is defined as the final menstrual period, marks the end of the female reproductive life span. This is marked by the decline in the number of ovarian follicles (ovarian reserve) with increasing age. When follicle numbers fall below a critical threshold of a few thousand, the menstrual cycle pattern becomes irregular. At menopause, less than 1000 follicles remain. For human fertility, optimal conditions are present until an average age of 31 years, followed by a gradual decline until natural sterility. It has been postulated that these events follow a time sequence with a more or less fixed interval, with the end of natural fertility occurring some 10 years before menopause. AMH is a stronger predictor of late menopausal transition compared with age, AFC or ovarian volume. AMH might be a better surrogate measure of reproductive age than chronological age alone [13].

Additionally, menopause is associated with some general health issues, such as osteoporosis, breast cancer, cardiovascular disease, Alzheimer’s disease, and stroke. The ability to predict menopause in premenopausal women by assessing AMH at a relatively young age may help in establishing screening and prevention programmes according to the risk factors for each individual woman [14]. An inverse relationship between AMH and subsequent atherosclerosis risk has been reported that can be applied to humans [15].

4. Diagnosis and management of women with polycystic ovarian syndrome

Ovarian dysfunction in women with polycystic ovarian syndrome (PCOS) is characterized by the arrest of follicle maturation and disturbed dominant follicle selection with involvement of AMH in its pathogenesis. As a result, increased serum AMH concentrations (2–3-fold) occur in PCOS, reflecting the increased number of early antral follicles. Evidence suggests that assessment of AMH levels may replace polycystic ovarian morphology (PCOM) assessment and features of hyperandrogenism or anovulation as diagnostic criteria for PCOS [16]. Additionally, AMH level in PCOS is associated with the extent of disease, and improved reproductive performance in relation to weight loss, improved ovulatory function with age and ovarian response to infertility treatment by laparoscopic ovarian diathermy can be predicted by initial AMH levels. The observation of elevated AMH levels in prepubertal and adolescent girls with PCOS and daughters of mothers with PCOS allows the use of AMH measurements in early detection of subclinical disease in siblings of PCOS women [16].

5. AMH and cancer in women

AMH could play a role in many aspects of cancer management in women. Its measurement may help identify which chemotherapeutic agents are toxic to the ovaries [17]. In addition; AMH can identify reduced ovarian reserve when ovulatory cycles are restored following cessation of cancer treatment. AMH levels are typically reduced during chemotherapy with some recovery 3–6 months thereafter [18]; AMH is also a good marker of ovarian function both before and after radiotherapy treatment. In women with breast cancer, the pre-treatment AMH levels are a useful predictor of the long-term post-chemotherapy loss of ovarian function. Pre-treatment AMH measurements may aid in determining treatment options and the need for applying fertility-preservation procedures [19]. A mosaic chart has been instructed for classification of ongoing menses or chemotherapy-related amenorrhea in women with early breast cancer using pre-chemotherapy serum AMH and chronological age [20]. Also, in patients with hormone-sensitive breast cancer, it is important to know when the ovarian reserve is depleted given that this information helps in making decision regarding optimal adjuvant hormonal treatment [21]. There is no significant association between age-specific AMH levels and risk of breast cancer [22,23]; however, a modulatory genetic effect has been investigated [24]. In addition, serum AMH is a tumour marker for the diagnosis and follow-up of ovarian granulosa cell tumours. Postoperatively, serum AMH levels may be used as a marker for the efficacy of surgery and for disease recurrence [13].

6. Assessment of intersexuality, cryptorchidism, anorchia and puberty disorders

The concentrations of AMH are sexually dimorphic in children: the measurement of AMH can assist in evaluating those with gonadal disorders. In boys with cryptorchidism, serum AMH is proportional to the testicular tissue. An undetectable value is highly suggestive of anorchia, whereas a measurable is associated with undescended testes. AMH can also help in the differential diagnosis of children with intersex states, with a value above the normal range for females being predictive of the presence of testicular tissue, whereas an undetectable result is suggestive of the absence of testicular tissue. Additionally, AMH can help in the diagnosis of delayed puberty and its differentiation from hypogonadotrophic hypogonadism [5].

Methods for measuring AMH

AMH assays have evolved in the last two decades, starting as enzymelinked immunosorbent assays (ELISA). In 1999, the first kit was introduced by Immunotech (IOT, France), then in 2003 a second AMH kit was manufactured by Diagnostic Systems Laboratories (DSL, USA). In 2010 Beckman Coulter developed a second generation single two-step; sandwich-type (Gen II) kit, based on analytical collaboration from the aforementioned two firms [25]. This new kit gained increased analytical sensitivity and specificity compared with the other previous assays.

Since then, more automated assays were added to the market, including kits Ultrasensitive AMH and picoAMH assay (Ansh Labs), UniCel DxI 800, Cobas E601, iFlash 3000 [26–28]. Currently, Cobas E601, UniCel DxI 800 and iFlash3000 are the most widely used immunoassay platforms to measure AMH levels. Comparison studies were conducted with numerous observations and conclusions were made in relation to the analytical performance in the clinical setting [25–29]. The analytical progress and advancement has expanded the utilization of the different assays in clinical practice. Also, studies have revealed that some interchangeability between AMH assays (Roche Elecsys or Beckman Coulter Access) is associated with modest differences in AMH values and seems to have little effect for personalizing FSH dose in IVF [30].

Reference range of serum AMH in healthy males and females

At birth, cord blood samples from newborn males typically exhibit high AMH levels, whereas AMH levels are typically undetectable or very low in cord sera from female newborns. At approximately 3 months of age, the levels are typically increased dramatically to its highest levels in both sexes. However, the concentrations in females are greatly reduced compared with male infants. AMH levels typically start to decline at 12 months of age and remain relatively stable during childhood (1–9 years of age) in both sexes. During this time frame, boys typically exhibit levels that are increased approximately 35-fold compared with girls. In males, after adolescence and during puberty, AMH levels are significantly reduced with the onset of testosterone synthesis, reaching low levels thereafter [31]. Serum levels of AMH also show a variability between individuals depending on the number of antral follicles among women with different ages, as well as ethnicity: African-American and Hispanic women tend to have lower serum AMH levels compared with Caucasians [32]. The AMH level is also affected by body mass index (where a negative relationship with AMH has been noted), and possibly smoking (which may reduce AMH level) [33,34]. Also, conditions associated with gonadotrophin suppression, particularly hormonal oral contraception use and pregnancy (particularly in the second and third trimesters), usually lower serum AMH levels. The levels usually return to normal after stopping the contraception and following delivery. Serum AMH levels usually do not change significantly and is relatively stable throughout the menstrual cycle making it an attractive determinant of ovarian activity [35]. However, the derivation of age-specific reference values of AMH is worth considering for improved interpretation of the results of AMH tests in practice.

The authors
Elham Said AlRisi1* MD, OMSB, FRCPath and
Prof. Jamal Sharef Mula- Abed2 MBChB, PhD
1 Dept. Pathology and Blood Bank, Sohar Hospital, Sohar, Sultanate of Oman
2 Faculty of Pharmacy, Philadelphia University, Amman, Jordan
*Corresponding author

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