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In the 1940s, Alfred Jost proposed that a testicular factor distinct from testosterone caused regression of the Müllerian duct the female internal genital precursor during male sexual differentiation. This factor later became known as Anti-Müllerian hormone (AMH) or Müllerian inhibiting substance (MIS). It was discovered to be exclusively produced by the sertoli cells of the testis in males (Josso, 1974). The AMH human and bovine gene was first isolated successfully in 1986 and AMH was classified as a homodimeric disulphide-linked glycoprotein of 560 amino acids and member of the TGF beta superfamily of growth factors. (1)
In females, AMH is produced by the granulosa cells of follicles in the ovary (8). It is thought to play a key role in folliculogenesis (8, 9, 10). In contrast to males, AMH levels at birth are largely undetectable in female cord blood. The levels then rise shortly after birth and continue to increase (4) to puberty. AMH concentrations then become stable from puberty onwards until the menopausal transition (5, 6, 7 ) . MIS declines to very low/ undetectable levels in women undergoing menopausal transition (5) and can therefore be implemented as a potential menopausal bio-marker. Since AMH is produced by growing follicles, it's correlation with the number of follicles can also be used as a measure of ovarian reserve in patient undergoing In-vitro fertilisation (IVF) treatment (11, 12). Raised levels of AMH have been reported in polycystic ovary syndrome (PCOS) (13-17) and certain ovarian cancers (18-20) and therefore measurement of this hormone may provide diagnostic and prognostic value. The roles of AMH are summarised in figure 1:
The physiological role of AMH during male sexual differentiation and its role in female folliculogenesis will be addressed. The clinical roles of AMH will then be discussed, particularly assessing its value in menopause, IVF, PCOS and finally ovarian cancer.
Role of AMH in male sexual differentiation
Approximately six weeks after conception, the embryo is bi-potential (has primordia for both male and female internal genitalia). The Wolffian duct and Müllerian duct form the male and female internal genitalia respectively. If the germ cell possesses XY sex chromosomes, the conceptus is destined to become male and MIS is secreted from the male gonads resulting in Müllerian duct regression and Wolffian duct formation under the influence of testosterone. Regression of the Müllerian duct prevents the development of the oviducts, uterus, cervix and superior vagina (10).
Sertoli cells of the testes are responsible for secreting AMH. SOX-9 and SF1 are the primary transcription factors that drives the production of AMH by increasing cAMP via the protein kinase A and P13k/PKB pathway (21). AMH achieves its action upon binding to the MIS receptor which is understood to be located on the mesenchymal cells surrounding the Müllerian duct (22).
There are two receptors for MIS, type 1 and 2. They are transmembrane-spanning serine/threonine kinase receptors. When MIS type 2 receptor binds to MIS, a heterotetrameric receptor complex is formed which subsequently recruits the type 1 receptor via phosphorylation. Activation of the type 1 receptor consequently phosphorylates downstream Smad proteins to form phosphorylated receptor-specific Smads, these migrates to the nucleus and regulate gene expression (8, 23).
Figure 2 demonstrates the intracellular mechanism of action of AMH:
The regression of the Müllerian duct follows a similar pattern to its formation in that it occurs in a cranial to caudal direction. Possible mechanisms involved in the regression of the Müllerian duct include migration of Müllerian epithelial cells, dissolution of the extracellular matrix and apoptosis (23). Failure of AMH production or defective AMH receptors results in a condition termed persistent Müllerian duct syndrome whereby genotypic males possess female internal genitalia (25).
Role of AMH in Folliculogenesis:
Folliculogenesis is the process of follicle growth and differentiation. The highly complex process is controlled by autocrine, paracrine and endocrine factors as well as cross talk between follicle cells and oocytes. The process can be simplified into two stages, initial recruitment of primordial follicles from the dormant primordial follicle pool, followed by cyclic recruitment whereby a few follicles are rescued under the influence of gonadotrophins in order to select a dominant follicle (26).
Initial activation and recruitment of follicles in folliculogenesis are thought to be under the control of both positive and negative factors. Several growth factors are implicated in the recruitment and initiation of growth arrested primordial follicles (26). This is shown in more detail in figure 3:
The rationale behind AMH as a key regulator of follicle development perhaps came from early studies on AMH gene knockout mice. Durlinger et al studied a population of homozygous AMH knockout female mice and wild type mice. Comparisons between the two sets of mice revealed that AMH null mice had more growing pre-antal and small antral follicles than the wild type; furthermore the primordial follicles were depleted earlier in life. This study provided foundation for AMH as a vital regulator of folliculogenesis through the suppression of initial primordial follicle recruitment (27).
The role of AMH during follicular development in humans was studied by Weenen et al who measured the levels of AMH expressed in follicles at various stages of development. In this study, a monoclonal anti-body to AMH was formed and ovaries were collected in 12 subjects undergoing oophorectomy. Tissue samples were collected and immunostaining was performed after follicles were grouped according to their size (9). Results showed that the size of the follicle strongly correlated to the levels of AMH expressed (Figure 4):
This study supports the notion of AMH as a key regulator in both initial and cyclic recruitment of follicles as reported in animal models, since similar patterns were observed. It is possible that the pool of growing follicles produces AMH which subsequently acts neighbouring primordial follicles to inhibit their recruitment (28). The increasingly lower expression of AMH as the follicle size increases suggests that the expression is lost in follicles selected for dominance (cyclic recruitment). Despite this, it's very important to note that the study did not address follicles larger than 10mm in diameter and therefore did not definitively determine whether AMH expression is lost in the follicles selected for dominance (9).
The mechanism by which AMH exerts its inhibitory effects on cyclic recruitment has been linked to Follicle stimulating hormone (FSH). In Durlinger's study involving AMH null mice, FSH levels were expressed in low amounts (27). Interestingly, there was an increase in growing follicles indicating that in the absence of AMH, the sensitivity of follicles to FSH is increases. This concept has been confirmed by a further study which found that in the presence of exogenous AMH, FSH induced pre-antral follicle growth was inhibited and the results were time dependent (29). Depending on the developmental stage, each follicle has its own threshold for FSH concentrations which must be reached to ensure the process of selection (30). Since AMH appears to reduce the sensitivity of follicles to FSH, it plays a key role in the selection of a dominant follicle. It is likely that AMH modulates FSH sensitivity via the regulation of aromatase activity as Grossman et al reported that serum and follicular MIS inhibits FSH augmented cytochrome P450 aromatase activity (31).
A summary of the inhibitory actions of AMH on folliculogenesis is illustrated below (figure 5):
Value of measuring AMH in premature ovarian failure/ Menopause:
Menopause refers to the inevitable depletion of potentially functioning primordial follicles associated with the complete cessation of menses and reproductive life. When this occurs before the age of 40 it is termed premature ovarian failure (POF) (33). Since AMH is produced by growing follicles, the logic of using AMH as a predictor of menopause is based on the age-related decline in follicle numbers (5-7).
Rooij et al investigated this notion (7). AMH, AFC, FSH and inhibin B levels were significantly correlated with menopausal transition. According to logistic regression analyses, AMH, AFC and age had the highest predictive accuracy. After accounting for age, AMH and inhibin B were significantly correlated with cycle irregularity. Sensitivities of AMH, inhibin and FSH as potential biomarkers for menopausal transition show that AMH levels declined to very low or undetectable levels on average 5 years before the FMP. This was accompanied by rises in FSH levels and falls in inhibin levels, these were less sensitive however (7). The relationship between AMH to the age distribution of menopause has also been studied. AMH levels were obtained from healthy volunteers and data on age of menopausal onset was obtained from a very large cohort of the same female Caucasian society. A strong correlation between observed and predicted distributions of age at menopause and declining levels of AMH was reported using percentage quantiles. They also worked out a threshold diagnostic of menopause using combinations of data obtained on menopausal age and predictive distribution derived from a threshold of AMH level below which women experience menopause (5). These studies are summarised in more detail (figure 6):
One of the standout features of AMH is its limited menstrual cycle fluctuations (34) and so measurements at fixed intervals of the menstrual cycle are not required. A competitive bio-marker, inhibin, has been reported to be affected by body mass (35) and possibly ethnicity (36), adding complexity to the interpretation of results. Furthermore, AMH is a more sensitive bio-marker compared to inhibin B as undetectable levels are observed at an earlier stage of menstrual transition (6). Moreover, AMH is the only ovarian test that changes longitudinally in young women (7) and levels are almost always undetectable after menopause (5-7, 37). Lastly FSH only increases substantially when cycles become irregular, AMH starts to decline before this and is therefore more sensitive (7).
Although numerous studies support the role AMH, there are potential drawbacks. Most studies are based on small Caucasian populations and generalisations cannot be drawn about other populations. Additionally, key pieces of information gathered e.g. menstrual history are based on information recall making them susceptible to recall bias. Although many advantages of AMH have been highlighted, using AMH may not be required since existing measures such as chronological age provide a reliable means of predicting menopausal transition (7) and requires no measurement. The practicality of using AMH as a potential biomarker of menopause is also questionable since it not readily assayed in most laboratories (38).
The table below (figure 7) summarises the advantages and disadvantages of AMH in detecting menopause compared with other bio-markers:
The American society of reproductive medicine (ASRM) devised the stages of reproductive aging workshop (STRAW) to define menopausal transition (39). They defined menopausal transition according to variations in menstrual cycle length in conjunction with a monotrophic rise in FSH (39). Based on the evidence provided, this definition can be refined by using combinations of AMH and inhibin B as they possess advantages over FSH (7, 34, 40).
Value of measuring AMH in IVF treatment
The assessment of ovarian response in those receiving IVF is essential for the purpose of identifying individuals who are at high risk of poor ovarian response (POR) in order for the patient and clinician to decide whether it would be worth taking part in the program to begin with, moreover to aid the clinician in choosing an optimum stimulation program and to provide a valuable tool for patient counselling.
According to the ASRM, no uniform definition of decreased ovarian reserve (DOR) exists but the term may reflect oocyte quality, quantity and reproductive potential (41). For years, chronological age and FSH have been used as markers for determining ovarian reserve in those receiving assisted reproduction (42, 43). Recently, AFC and ovarian volume are utilised more as they provide the better prognostic information of poor ovarian response to IVF (44).
A prospective study investigated the significance of using MIS as a serum marker of ovarian response in individuals receiving GnRH IVF treatment and found that AMH levels were significantly correlated with AFC in addition to oocytes retrieved after hyperstimulation of the ovary. AFC was very good at predicting POR which was almost identical to AMH. When high response was used as the primary outcome of measure, AFC and AMH once again performed the best (12). These results were reproduced by Jayaprakasan et al more recently who found that AFC and AMH were the most significant predictors of POR (45). The significant correlation between AMH levels and the outcome of IVF treatment further supports the role AMH in determining ovarian reserve.
Muttukrishna et al investigated the use of several biomarkers (FSH, AMH and inhibin B) for predicting IVF response. Serum assays were measured in patients before receiving gonadotrophin stimulation. 17 patients had to cancel due to a poor response. Mean AMH levels were significantly lower in the cancelled group. Surprisingly however, 6 of the patients who had completed IVF cycle treatment had undetectable levels of AMH. Sensitivities and specificities of all 3 bio-markers at specific cut-off points are shown in the table below (figure 8) describes the results:C:\Users\mohsin\Desktop\Untitled.png
AMH displayed the best sensitivity and specificity compared with FSH and inhibin B by a significant margin and suggests AMH is a superior bio-marker over others in detecting ovarian reserve (11). These studies including one more are summarized below (figure 9):C:\Users\faiz\Desktop\Untitled.jpgC:\Users\mohsin\Desktop\Untitled.png
From cumulative evidence it is clear that AMH is a promising predictor of ovarian response to IVF treatment and thus its measurement should be strongly considered as part of the IVF programme. AMH has supremacies over other bio-markers, it is the best predictor of ovarian response when using regression analysis (12), sensitivity and specificity measurements (11). AFC has provided equivalent or better determination of ovarian reserve than AMH (12), though its subjective nature, invasiveness, the need for additional ultrasound and high intra- and inter-cycle variability is questionable (47).
The evidence available on AMH however is limited by small samples sizes, heterogeneity in study design and analysis as well as the lack of validated outcome measures (41). Furthermore Muttakrishna found that 35% of patients that completed the IVF cycle had undetectable levels of AMH (11). This overlap makes the use of AMH less favourable as a single marker of predicting cancelled IVF treatment. Taking everything into account, the combination of AFC and AMH would probably provide the best means of predicting ovarian response to IVF.
Comparisons between 3 potential bio-markers (AMH, FSH, inhibin B) of ovarian reserve are summarised in figure 10.
Value of measuring AMH in PCOS:
PCOS is a condition characterised by ovulatory dysfunction, hyperandrogenism (HA) and insulin resistance (51). PCOS patients have increased amounts of growing follicles and lack of dominant follicle selection (10). Based on increased follicle numbers, several studies have demonstrated that serum AMH is significantly higher in PCOS patients compared with healthy individuals (figure 11) (52, 17, 16 ).C:\Users\mohsin\Desktop\Untitled.png
The production of AMH by different follicle cell sizes were compared between healthy and PCOS subjects undergoing bilateral oophorectomy (Pellatt et al). Cells were isolated, cultured and AMH follicular fluids were compared. AMH levels were up to sixteen times greater in ovaries of ovulatory PCOS women and 75 times greater in anovulatory PCOS patients compared to subjects with normal ovaries (13). This study provided new evidence that elevated AMH observed in PCOS patients not only is a result of increased growing follicles but also because of increased secretion per granulosa cell.
The pathogenesis of PCOS is unclear and the increased number of developing follicles and lack of dominant follicle selection may perhaps be associated with the elevated production of AMH as illustrated in figure 12:C:\Users\mohsin\Desktop\Untitled.png
The diagnosis of PCOS is one of the most challenging issues in endocrinology and higher levels of AMH observed in PCOS may provide help. A study explored whether AMH can be used in place of AFC in the Rotterdam criteria for diagnosing PCOS (see figure 13). AMH was strongly correlated to AFC and androgen levels. It was therefore suggested that AMH levels in suspected PCOS women with HA can be used for diagnosis when reliable ultrasound scan is unavailable. The sensitivity of picking up PCOS using AMH was fairly poor however (67%), yet the specificity was very good (92%). With a sensitivity and specificity of 75% and 100% respectively using a 12 follicle count cut off value, AFC proved to be a better diagnostic tool (16).
Very recently, the replacement of polycystic ovarian morphology based on ultrasound with AMH measurement was examined. PCOS was defined according to two well-known criteria, The Rotterdam criteria (PCOS-R) and Androgen Excess PCOS society (PCOS-AES). AMH at a cut off value of 20 pmol gave a sensitivity of 94.6% and a specificity of 97.1% according to the PCOS-R . PCOS-AES criteria produced sensitivities and specificities of 97.2 and 95.5% respectively (56). This study provided strong evidence that AMH can be used in place of AMH, though AMH should be set at lower cut-off values than previously suggested.
AMH provides objective measure, displays very little menstrual cycle fluctuation (34) and is not significantly affected by treatments including weight loss, OCP and metformin (57-59) indicating that AMH appears not have added benefits of not being influenced by hormonal suppression in PCOS. Interestingly, it has been suggested that smaller follicles not readily detected by ultrasound, contribute significantly to AMH levels and therefore is a more sensitive marker of ovarian dysfunction than ultrasound (57). Serum AMH levels are also significantly correlated with other indications of ovarian dysfunction for instance raised LH and testosterone concentrations and therefore provides prognostic value to diagnosis (52).
Conversely, AMH has missed out up to a third of patients with PCOS (16). It is also very important to note that though AMH serum levels were highest in anovulatory PCOS patients, anovulatory women without PCOS also expressed elevated levels of AMH and there is the possibility that AMH may only be related with anovulatory cycles and not PCOS (17). Further studies are required to investigate this notion. Overall, Ultrasound will probably remain central to diagnosis since it has high sensitivity and specificity (16) and can determine the number of follicles as well as the ovarian volume, which is equally as important in defining polycystic ovaries (55).
Value of measuring AMH in Granulosa cell tumours (GCT):
Ovarian cancer is principally divided into three major types of tumours; epithelial, germ cell and sex cord- stromal. GCTs fall into the sex cord- stromal category (60).
Over a decade ago, alterations in AMH secretion were reported in patients suffering from GCTs. One patient had levels of AMH drop substantially after oophorectomy resections which became undetectable after subsequent resections. AHM was raised in two other women with GCTs which returned to normal post-operatively. Six women that had undergone resections of GCTs had normal levels of serum MIS. There was no significant correlation between MIS and other types of ovarian cancers (18). A few years later Rey et al found that eight of nine patients with a progressive GCT had raised serum AMH levels and serum AMH concentrations were below the detection limit in 10 of 11 patients who were in clinical remission after successful treatment (61). Lane et al retrospectively analysed MIS data from subjects prior to and following initial tumour resection and used this to predict remissions in patients who have undergone surgery. Prior to tumour resection MIS was elevated in 75% and 78% of subjects with juvenile GCT and adult GCT respectively. In the post-operative group, 71% had MIS determinations that were normal or undetectable; the remaining 29% had either elevated values just after surgery which persisted or elevated for a period of time before decreasing to normal levels (19). An ultrasensitive ELISA assay was developed some years ago to increase the sensitivity of detecting GCTs. In remission subjects, fifteen had undetectable levels of serum AMH after bilateral oophorectomy; apart from one which remained high despite lack of clinical features of occurrence (20).
Early studies investigating the use of AMH as marker for GCT are summarised in figure 14:
These studies have provided remarkable sensitivities and specificities for diagnosing GCTs. MIS levels can also be measured postoperatively as a marker for efficacy of surgery and tumour recurrence (19, 20). Compared to other candidate bio-markers currently used (Inhibin and oestrogen), AMH is exclusively produced by granulosa cells and is therefore more specific. Oestrogen shows significant fluctuations due to disease or response to treatment, lacks sensitivity and its secretion is dependent on testosterone produced by adjacent theca cells which are not always hormonally active (62).
On the other hand the ability to correlate the clinical courses of the disease with MIS values is limited by the retrospective nature of studies and very small study populations. Larger prospective studies are required to address temporal sequence between serum MIS concentrations and the presence of tumour and staging. Furthermore, evidence that inhibin B has a greater sensitivity and specificity exists (61, 63). Major concerns over the inverse relationship between MIS and advanced GCTs have also been reported by Antonnen et al who found that MIS gene expression was significantly reduced in 87% of tumour sizes greater than 10cm (64). Further limitations of AMH in elderly women have also been reported very recently in elderly women with GCTs where serum levels of AMH were undetectable (65).
MIS has very important roles both physiologically as well as clinically. AMH is responsible for the regression of the Müllerian duct during male sexual differentiation, but it also plays a key role in females after birth.
AMH is produced exclusively by the graunola cells of the primary and pre-antral follicle is thought to directly inhibit initial recruitment of primordial follicles. It is very likely that as follicles produce increasing AMH as they progress through the pre-antral stage of folliculogenesis, they prevent the recruitment of other primordial follicles. AMH also plays a key inhibitory role in the cyclic recruitment stage of folliculogenesis by reducing the sensitivity of follicles to FSH.
As the expression of AMH strongly declines or becomes undetectable during the menopausal transition it can be used to predict the onset of menopause and more importantly assess the ovarian reserve of females undergoing IVF treatment. AMH correlates very well with AFC which is readily used to detect ovarian reserve and can be used as a replacement or as an aid.
AMH is highly elevated in patients with PCOS because of increased growing follicles and increased production per granulosa cell. AMH can be a very useful diagnostic tool for PCOS since it has little cycle variability, very good specificity, lacks hormonal suppression in treatment and provides prognostic information to diagnosis. AFC on the other hand is more sensitive and there are concerns that AMH levels correlate with anovulatory cycles and not necessarily PCOS. Taking everything into account AMH as an additional diagnostic tool would be more reasonable rather than a replacement.
Early studies have suggested that AMH hormone provides a very sensitive and specific predictive value for initial diagnosis and predicting remissions of GCTs. Most of these studies are retrospective and small-scaled however. Larger and longer follow ups are required before it can be potentially implemented in clinical practise.