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AMH Review


Anti-Müllerian Hormone in Female Reproduction

Introduction:
Anti-Müllerian hormone (AMH) is a member of the TGF-β superfamily and transduces its effects through a specific type II receptor called AMHR2 and type I receptors ALK2/ACVR1, ALK3/BMPR1A, or ALK6/BMPR1B and SMAD1, SMAD5, or SMAD8 proteins, which AMH shares with bone morphogenetic proteins (BMPs).
AMH is responsible for the regression of Müllerian ducts in male fetuses and is also expressed in females by the ovaries where it regulates folliculogenesis.
Serum AMH is a reliable marker of ovarian reserve and is notably increased in women with polycystic ovary syndrome (PCOS), the main cause of infertility in women.
AMH and AMHR2 have been detected in organs other than the ovaries, such as the hypothalamus, pituitary gland, breasts, uterus, placenta, lungs, motor neurons, and neurons, extending further the notion of extra-Müllerian effects of AMH.
The number of articles published on AMH in females, especially in women, has increased exponentially in the past 2 decades.
The review aims to focus on the main findings of the past 2 decades in respect to ovarian AMH regulation and its mechanism of action in mammals, the role played by AMH in female reproductive organs, and its clinical utility in gynecology.
AMH and AMH-specific Receptor (AMHR2)
Expression Profile in the Ovary
The AMH gene and cDNA were first cloned in 1986 in bovines and humans.
The human AMH gene is 2.8 kbp long and is carried by chromosome 19.
The human gene has a degenerated TATA box and several transcription initiation sites.
The AMH mRNA is 2.1-kb long and is the same in fetal testes and ovarian follicles.
The AMH protein is a 70-kDa monomer consisting of 560 amino acids in humans.
The N-terminal domain of AMH shows the least homology between species, while the C-terminal domain presents homology of roughly 30% with TGF-β family members.
In the ovary, AMH is produced by granulosa cells and its expression level depends on the follicle stages.
AMH synthesis is initiated in primary follicles, is highest in preantral and small antral follicles, and then subsequently decreases in large follicles with the exception of their cumulus cells.
AMH expression begins from the onset of folliculogenesis and continues until menopause.
Serum AMH levels reflect the growing pool of follicles and mainly originate from the ovary.
Serum AMH levels decrease with age, but do not vary significantly during menstrual cycles.
AMH has been shown to be expressed by reproductive organs other than the ovaries, such as the hypothalamus and the gonadotrope cells of the pituitary gland.
The physiological relevance of these facts requires further investigation to establish whether AMH effects are autocrine, paracrine, or endocrine.
AMHR2 gene, protein, and expression in
the ovary
The AMH-specific receptor, AMHR2, cDNA was first cloned in 1994 in the rat and in the rabbit.
The human AMHR2 gene is composed of 11 exons spread over approximately 8 kbp on the long arm of chromosome 12.
The human AMHR2 gene encodes a 2.05-kb mRNA that is translated into a 573-amino acid protein with a molecular weight of 82 kDa.
AMHR2 is highly specific and mutations in the AMHR2 gene induce in humans a persistent Müllerian duct syndrome, characterized by the persistence of Müllerian duct derivatives in normally virilized males.
AMHR2 is mainly expressed in GCs from growing follicles and in cumulus rather than in mural cells in large antral follicles.
AMHR2 is also expressed in the hypothalamus, pituitary gland, breasts, uterus, and placenta, supporting the relevance of the effects of AMH observed in these tissues.
Controversies and future directions
High-sensitivity technological tools have been developed since the AMH and AMHR2 genes were cloned.
Both genes are expressed in females in organs other than the ovaries.
Technical issues, such as selecting specific primers for PCR due to the high level of sequence similarity with other members of the TGF-β family, need to be addressed.
Validation studies of protein expression in these organs and responsiveness to AMH of AMHR2-expressing cells are required.
Further investigation is needed to understand the physiological relevance of AMHR2 mRNA variants.
Mechanism of Action of AMH
The AMH 140-kDa precursor needs to be cleaved to become active in humans.
The cleavage occurs at a consensus monobasic cleavage site between Arg427 and Ser428, resulting in a 110-kDa N-domain and a 25-kDa C-terminal domain that carries the AMH bioactive site.
The AMH precursor and noncovalent forms have similar half-lives in female serum, although measurements of AMH half-life vary depending on the method used.
The cleavage of the AMH precursor likely occurs in producing cells, and the AMH cleavage enzyme in the fetal testis could be the kex2/subtilisin-like endoprotease PC5.
Both the AMH precursor and noncovalent complex are present in the follicular fluid and serum of women, and further cleavage or degradation of the AMH precursor occurs in the serum.
The noncovalent complex binds to AMHR2, and the N-terminal fragment of AMH potentiates the biological activity of the C-terminal domain.
Following AMH binding to AMHR2, the N-terminal domain of the noncovalent complex is removed to allow AMH signaling.
AMH mainly transduces its signals through the canonical SMAD pathway, mobilizing the ALK2/ACVR1, ALK3/BMPR1A, or ALK6/BMPR1B type I receptors and inducing the phosphorylation of the SMAD1, SMAD5, or SMAD8 proteins.
The AMH-induced SMAD1, 5, and 8 signaling pathway is down-regulated by the inhibitory SMAD, SMAD6.
How AMH regulates the promoter of its target genes remains unknown.
Controversies and future directions
Uncertainty remains about the mechanisms of action of AMH.
It is unclear if ovarian AMH is cleaved in GCs or at the level of target cells.
The proteolytic enzymes responsible for cleaving AMH are not yet known.
It is unknown if AMH has binding proteins other than its N-terminal domain.
Investigating different type I receptors, SMAD proteins, and other signaling pathways could yield fruitful results.
Determining how these different pathways transduce different effects of AMH is an important area of research.
Main Regulators of AMH and AMHR2 Expression in the Ovary
Regulation by transcription factors/cofactors and epigenetic mechanisms
The human AMH promoter contains binding sites for transcription factors involved in sexual differentiation.
In males, SF1, SRY, and GATA4 promote the expression of SOX9, which initiates the differentiation of Sertoli cells and triggers AMH expression.
SF1 and GATA4 sustain the expression of AMH in males, whereas WT1 and DAX-1 modulate it.
The inhibition of AMH expression in the fetal ovary is induced by genetic cascades involving FOXL2 and the RSPO1/WNT pathway.
FOXL2 and WNT4 may trigger AMH expression at the beginning of folliculogenesis.
SF1, GATA4, and WT1 could play a role in ovarian AMH expression because they are up-regulated in the GCs of preantral follicles.
Epigenetic mechanisms may also regulate AMH and AMHR2 expression.
H19lncRNA and lncRNA-AMHR2 are required for the full and specific activation of AMH and AMHR2, respectively.
Regulation by gonadotropins
AMH, FSH, and LH are hormones involved in reproductive processes and are secreted by different glands in the body.
FSH promotes the growth and differentiation of follicles, while LH supports their final maturation and triggers ovulation.
GCs of all growing follicles express FSH receptors, while LH receptors are present in the theca cells of early preantral follicles, in the GCs of the preantral follicles, and in the luteal cells of the corpora lutea.
In vitro studies suggest that FSH has a stimulatory effect on AMH expression, while LH has no effect on AMH expression.
However, the addition of androgens, estrogens, serum, or growth factors to maintain the cells in an environment close to in vivo conditions interferes with the single effect of FSH on AMH secretion by GCs.
In vivo studies show that AMH and FSH levels are inversely correlated during controlled ovarian stimulation with FSH, as treatment with FSH stimulates estrogen production, which inhibits AMH expression.
In women with hypothalamic hypogonadism, AMH levels are higher than in control women due to an increased pool of small antral follicles.
The positive effect of FSH on AMH expression is likely counteracted by estrogens from the terminal growth of folliculogenesis in normal conditions.
Few studies have addressed the single effect of LH on AMH expression, as LH induces the luteinization of GCs, and high concentrations mimic a pathological condition such as PCOS.
The effects of FSH and LH on AMHR2 expression have received less attention, but in vitro studies suggest that LH down-regulates AMHR2 mRNA expression, while FSH has no effect on AMHR2 expression.
Regulation by steroids
Estrogens
Estrogens are produced by the aromatization of androgens secreted by theca cells under the action of FSH on GCs of growing follicles
Most studies suggest that estrogens, particularly estradiol (E2), have an inhibiting effect on AMH and AMHR2 expression during follicular growth
In vitro studies have shown that E2 reduces AMH and AMHR2 mRNA levels in human luteinized GCs cultured without adjuvant
In vivo, AMH secretion declines when estradiol levels increase during folliculogenesis
GCs sensitivity to E2 could depend on their differentiation stage
During controlled ovarian stimulation, most studies reported a negative correlation between AMH and estradiol levels both in the serum and in the follicular fluids until the day of hCG administration
E2 has a differential effect on a human AMH reporter gene depending on the relative levels of the estrogen nuclear receptors ESR1 and ESR2 in GCs
The decrease in AMH expression during folliculogenesis is likely due to the inhibitory effect of E2, which progressively overcomes the stimulatory effects of FSH
Androgens
Androgens are produced by the theca cells of follicles under the influence of tonic levels of LH.
The nuclear androgen receptor (AR) is found in all 3 components of the ovarian follicle, but it is most abundant in GCs.
Studies on the regulation of AMH expression by androgens have yielded positive, negative, or no effects due to the effects of androgens on basal follicle growth and the aromatization of androgens into estrogens that inhibit AMH expression.
Most studies in normo-ovulatory women did not find a correlation between AMH and androgen levels in the plasma or follicular fluid.
Chronic exposure of prepubertal mice to non-aromatic androgen 5α-DHT did not affect serum AMH levels or the pattern of AMH expression in the ovaries when the treatment did not induce an increased number of growing follicles.
In vitro, in human luteinized GCs from control women cultured without any additives, 5α-DHT did not affect AMH and AMHR2 mRNA levels. Similarly, treatment with 5α-DHT did not change AMHR2 immunostaining in rat preantral follicles.
Regulation by BMPs
Intraovarian factors, specifically members of the TGF-β family (BMPs and AMH), regulate early follicular growth and the threshold/window of follicle sensitivity to FSH.
BMPs are expressed in a cell-specific manner in the ovary and their pattern of expression depends on the stage of follicular development.
BMPs signal through the canonical BMP signaling pathway, resulting in the activation of SMAD1, 5, or 8 proteins.
BMPs, particularly BMP15, stimulate AMH and AMHR2 expression in both in vitro and in vivo studies.
BMP15 stimulates AMH expression and GDF9 increases its effect on AMH expression through the formation of cumulin, which is more potent than homodimers.
BMP2, BMP4, and BMP6 have also been found to stimulate ovarian AMH expression in various species in vitro.
BMPs might be involved in the onset of both AMH and AMHR2 expression by GCs, explaining their up-regulation in the first stages of folliculogenesis as well as their high expression in cumulus cells of antral follicles.
Regulation by metabolic and inflammatory factors
Metabolic and inflammatory factors can regulate female reproductive function, so it is likely that AMH expression is also regulated by these factors.
Insulin can up-regulate AMH mRNA levels in a dose-dependent manner in human luteinized GCs cultured without additives.
Leptin can stimulate or inhibit AMH expression in the same model of GCs.
Vascular endothelial growth factor and TNF-α can repress AMH mRNA levels in human luteinized GCs and GCs from small bovine follicles, respectively.
Numerous in vivo studies suggest that AMH expression is regulated by metabolic and inflammatory factors, but contradictory results have been obtained.
AMHR2 expression is also regulated by metabolic and inflammatory factors such as IGF and vascular endothelial growth factor, which up-regulate it, and leptin, which inhibits it.
Regulation by other factors
AMH and AMHR2 expression may be regulated by environmental factors such as vitamin D, tobacco, and advanced glycation end products, as well as endocrine disruptors.
Results regarding the effect of these factors are often contradictory and require further investigation.
Negative emotions such as depression and anxiety are associated with a decrease in fertility, and abnormal AMH levels have been found to be positively associated with chronic abdominal pain and urinary cortisol.
In female rats presenting chronic unpredictable mild stress, serum levels of AMH were significantly decreased.
Controversies and future directions
Many studies have been conducted to identify factors that influence AMH and AMHR2 secretion by GCs.
BMPs, FSH, LH, and estrogens have been found to influence AMH and AMHR2 expression under normal conditions.
The extent to which other important regulators of folliculogenesis, such as inhibin B, IGF-1, metabolic, inflammatory, societal, and environmental factors, could also regulate their expression is still unclear.
Most in vivo investigations have been correlation studies, and further in vitro studies are required to demonstrate the action of individual factors and decipher the molecular mechanisms underlying the regulation of AMH and AMHR2 expression by GCs.
AMH levels are also influenced by the large vs small follicles ratio, which varies in response to different hormonal stimuli.
Effects of AMH on the Female Reproductive Organs
Effects of AMH on the ovaries
Effects of AMH on folliculogenesis
AMH regulates folliculogenesis at an early stage.
In rats, it inhibits the assembly of primordial follicles and recruitment from the pool of primordial follicles.
AMH down-regulates extracellular growth and regulatory factors, particularly members of the TGF-β signaling pathway.
In mice, AMH attenuates FSH-stimulated preantral follicle growth, but in rats, it increases the growth of preantral follicles.
AMH represses the differentiation of growing follicles and inhibits their responsiveness to FSH.
FOXL2 up-regulation in human luteinized GCs is involved in the repression of CYP19A1 by AMH.
Effects of AMH on follicular atresia
AMH has been suspected to protect follicles from atresia based on indirect evidence
Indirect evidence includes absence of AMH expression in atretic follicles and increased number of atretic follicles in Amh null mice
Recent studies have confirmed this hypothesis, showing that AMH treatment reduces number of atretic follicles in mice and prepubertal human ovarian cortex transplanted in mice
Knocking down AMH expression negatively affected follicle survival in macaque preantral follicles
Research has shown that AMH has antiapoptotic effects on ovarian cell apoptosis
However, recent findings in cancer GC lines and primary cultures of mouse and human GCs did not concur with this
Integrative biology analyses of AMH target genes identified in the AT29C GC line highlighted 307 AMH target genes potentially involved in reducing cell death, providing mechanistic evidence that AMH protects follicles from atresia
Effects on meiosis
Meiosis in mammalian oocyte is arrested immediately after birth and resumed before ovulation.
It is unclear whether AMH inhibits meiosis in mammals as it has only been shown to do so in transgenic mice overexpressing AMH.
The expression of AMHR2 on the oocyte is controversial.
In vitro, an AMH preparation from calf testes prevented the spontaneous resumption of meiosis of rat oocytes, but this effect was not confirmed when AMH was purified to homogeneity, except when a detergent was added to the medium.
Controversies and future directions
AMH has been shown to regulate various stages of folliculogenesis and has multiple target genes.
However, its role in primordial follicle recruitment and follicle growth is still debated, and further research is needed.
The protective effect of AMH on follicular atresia may influence some results and needs to be taken into consideration.
The signaling pathways used by AMH to mediate its effects are still unknown and require further investigation.
The role of AMH on other ovarian cells such as theca cells and oocytes needs to be studied because their interaction is important for proper follicle development.
Effects of AMH on the hypothalamopituitary axis
Recent research has revealed new aspects of the role of AMH on the hypothalamopituitary axis.
AMH can promote the migration of GnRH neurons towards the brain, possibly through an autocrine mechanism.
AMHR2 is expressed in migratory GnRH neurons and along olfactory axons in both mice and human fetuses, supporting these findings.
Electrophysiological investigations have shown that AMH can activate 50-70% of GnRH neurons, leading to an increase in GnRH-dependent LH pulsatility and secretion in female mice.
AMH was also shown in vivo in rats to act on the pituitary gland to regulate FSH but not LH secretion, specifically in females and before puberty.
AMH was shown to potentiate activin signaling and activin-dependent Fshb expression in vitro, and GnRH interferes with AMH in vitro in LβT2 cells.
AMH expression was detected in both the pituitary gland of the rat and in LβT2 cells, suggesting that AMH could act as an autocrine/paracrine regulator in this organ.
The physiological relevance of AMH expression in the pituitary gland remains to be confirmed.
The issue of whether AMH directly regulates FSH and LH secretions in adulthood remains to be investigated further.
Effects of AMH in the uterus
AMHR2 is expressed in oviducts and uterus from birth to adulthood
In rats, AMHR2 expression decreases during postnatal development coinciding with endometrial stroma expansion and AMH secretion from the ovaries
In humans, AMHR2-positive cells recede from fetal uterus around 24 weeks gestation coinciding with the start of AMH production in the ovary
Some AMHR2-positive endometrial cells may be necessary for proper uterine development
AMH can prevent the decrease of uterine AMHR2 expression and inhibit endometrial stroma expansion in rats
AMH inhibits growth of endometrial stromal cells and endometrial cancer cell lines
AMHR2 is also expressed in adult myometrium and cervical tissues
AMH may play additional roles in the adult uterus, but only cervical cancer cell growth has been reported to be repressed by AMH so far
Effects of AMH on the placenta
Maternal circulating AMH levels decrease during pregnancy.
AMH and AMHR2 are expressed by placenta and fetal membranes in humans and mice.
AMH could be playing a physiological role in the placenta as indicated by the lower expression of Cyp19a1 and Hsd3b1 and higher expression of LHR in the placenta of AMH-treated mice.
Effects of AMH on the breast
MHR2 mRNA and protein are detected in human and rat normal breast epithelial cells.
AMH expression is inversely proportional to the state of proliferation of the breast in rats.
AMH inhibits the proliferation and induces the apoptosis of breast cancer cell lines.
These effects are mediated through the NF-κB signaling pathway, which is abrogated by the inhibition of NF-κB induction.
These effects are amplified by interferon γ.
IEX-1S, interferon regulatory factor-1, and Gro- β are AMH target genes involved in these effects.
The expression of these genes depends on the activation of the NF-κB pathway by AMH and both NF-κB and SMAD1 pathways for Gro-β.
AMH plays a physiological role in the growth regulation of the mammary epithelium, which undergoes proliferation, differentiation, and extensive remodeling during puberty and pregnancy.
Controversies and future directions
AMH plays a role in female reproductive tissues other than the ovaries
Its effects on GnRH neurons are well-documented, but it is unclear whether these are of an autocrine or endocrine nature
Further investigation is needed to understand the physiological effects of AMH on the uterus, placenta, and breast.
AMH in Gynecology
AMH and the main pathological conditions of
women’s reproductive organs
PCOS
PCOS is a common cause of female infertility affecting 5% to 10% of women of reproductive age worldwide.
The Rotterdam criteria for the diagnosis of PCOS are based on at least 2 of the following features: irregular ovulatory function, evidence of hyperandrogenism, and presence of a polycystic ovarian morphology.
Women with PCOS also present increased GnRH/LH pulsatility and elevated serum LH levels and/or LH/FSH ratio, as well as several metabolic disturbances such as obesity, insulin resistance, dyslipidemia, and type 2 diabetes.
AMH concentrations are 2- to 4-fold higher in both the serum and the follicular fluid of women with PCOS and in daughters of women with PCOS.
The high serum AMH levels observed in women with PCOS might be due to both the increased number of small antral follicles that express AMH the most and an overexpression of AMH and AMHR2 by their GCs.
Hyperandrogenism induces an overexpression of AMH by GCs from women with PCOS, and LH levels play a part in the overexpression of AMH in PCOS GCs.
The increased ESR1/ESR2 ratio in PCOS GCs might prevent the inhibitory effect of estrogens on AMH expression observed in GCs from normo-ovulatory women and thus might also contribute to the overexpression of AMH in PCOS GCs.
Ovarian tumors
Granulosa cell tumors (GCTs) account for 1-2% of all ovarian tumors but 6-10% of malignant ovarian tumors.
The malignant potential of GCTs is low but they frequently recur and have a poor prognosis.
Serum AMH levels rise in GCTs until tumor removal and are an early marker of recurrence.
In situ, AMH-secreting cells of GCTs produce smaller quantities of AMH than normal cells.
Elevated serum AMH levels in patients with progressive tumors are due to increased number of GCs secreting AMH.
AMH expression decreases in large GCTs, with 53% of tumors more than 10 cm being AMH negative.
AMHR2 is highly expressed in GCs from GCTs and its downstream receptors and SMAD effectors are active, suggesting AMH could be involved in the pathogenesis of this cancer.
The effect of the AMH/AMHR2 system on GCT growth is unknown, but only antiproliferative and pro-apoptotic effects of AMH on cancer GCs have been described.
AMH and AMHR2 have been detected in other ovarian tumors but their expression there is lower than in sex cord stromal tumors.
In epithelial ovarian cell lines, AMH inhibits cell proliferation and promotes apoptosis, but whether AMH is involved in epithelial ovarian tumors remains unknown.
Primary ovarian insufficiency
Primary or premature ovarian insufficiency (POI) is when ovarian functions cease before the age of 40, resulting in amenorrhea, low estrogen levels, and elevated FSH levels.
POI affects about 1% of women and can be iatrogenic (caused by medical treatment) or spontaneous (with autoimmune, genetic, or idiopathic causes).
Iatrogenic POI is becoming more common due to the gonadotoxic effects of cancer treatments.
AMH levels are undetectable in most cancer survivors who have received pelvis irradiation or chemotherapy with alkylating agents, but this is likely due to the cessation of ovarian activity.
Spontaneous POI cases are mostly autoimmune, genetic, or idiopathic, and AMH levels are reduced but not undetectable in some cases.
Genetic POI cases, such as those with FRM1 premutation or Turner syndrome, are associated with low serum AMH levels.
Several sequence and missense variants of AMH and AMHR2 with reduced activity have been identified in idiopathic POI cohorts, suggesting that AMH could contribute to POI in these cases.
FOXL2 mutations have been found in induced POI, and missense mutations of WT1 have been shown to repress AMH expression in some cases of POI.
However, in most POI cases, the decrease in AMH levels mainly reflects the ovarian reserve and is a consequence and not a cause of POI.
Endometriosis
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