The role of AMH in anovulation associated with PCOS: a hypothesis

Human Reproduction, Vol.29, No.6 pp. 1117 1121, 2014 Advanced Access publication on April 25, 2014 doi:10.1093/humrep/deu076 OPINION The role of AMH in anovulation associated with PCOS: a hypothesis Roy Homburg and Giselle Crawford* Homerton Fertility Centre, Homerton University Hospital, London E96SR, UK *Correspondence address. E-mail: giselle.crawford@homerton.nhs.uk Submitted on January 15, 2014; resubmitted on March 17, 2014; accepted on March 20, 2014 abstract: Polycystic ovary syndrome (PCOS) is the most common cause of infertility due to anovulation. Despite its prevalence, the precise cause of the anovulation is yet to be clearly defined. There is an increased number of pre-antral and antral follicles in the polycystic ovary, many of which individually produce increased amounts of anti-müllerian hormone (AMH) compared with those in the normal ovary. In this article, it is hypothesized that the high AMH concentrations present in women with PCOS play an integral role in causing anovulation due to its inhibitory influence on the actions of follicle-stimulating hormone, which normally promotes follicular development from the small antral to the ovulatory stage. Key words: polycystic ovarian syndrome / anovulation / anti-müllerian hormone / pathophysiology Introduction Polycystic ovary syndrome (PCOS) is the most common cause of infertility due to anovulation and it is often diagnosed for the first time in the fertility clinic. Women with anovulatory or oligo-ovulatory PCOS will frequently exhibit other traits of the syndrome including hyperandrogenism, whether clinical and/or biochemical, and ultrasonic polycystic ovaries (Adams et al., 1986). The large disease burden of PCOS, affecting up to 10% of the female population, has led to much research into its aetiology, pathophysiology and management. In the beginning Stein and Leventhal first described PCOS in 1935 from their observations in seven patients with enlarged ovaries, amenorrhoea, infertility and hirsutism. They incorrectly hypothesized that the sclerocystic thickened cortex of the ovary was the primary aetiology of the condition and this appeared to be supported by the fact that ovulation was restored with bilateral wedge resection (Stein and Leventhal, 1935). It is now widely agreed that the basic lesion of PCOS lies in the ovaries themselves and that the hyperproduction of androgens by the ovaries is the basal endocrinological disturbance. Associated extra-ovarian factors such as insulin resistance and the consequent hyperinsulinaemia and elevated concentrations of luteinizing hormone(lh) play a part in exacerbating the problems and are all intertwined into an androgen circle (Homburg, 2009). Although PCOS is clearly familial in a large majority of cases, many years of searching for the culprit polymorphisms or combination of causative genes have not produced cogent results. This has led to the developmental theory of PCOS, based on the Barker hypothesis (Barker, 1990), that the offending genes are programmed by hyper-exposure to androgens in utero (Abbott et al., 2002). When Abbott et al. (2002) injected pregnant rhesus monkeys with testosterone, shown to reach the fetus, in adolescence the female offspring had a polycystic morphology of the ovaries, high-lh concentrations, hyperinsulinaemia and oligomenorrhoea compared with controls. Should the developmental theory of PCOS be verified in humans, the question remains as to the source of the excess androgens: are they coming from the mother or from the fetus? Numerous theories for the aetiology of the associated anovulation have been described, including an excess of small antral follicles, hyperandrogenaemia, hyperinsulinaemia and dysfunctional feedback mechanisms. While the severity of hyperandrogenaemia seems to correlate well with the severity of the ovulatory disturbance, there is a paucity of evidence for a direct effect of androgens in the inhibition of ovulation. Hyperinsulinaemia increases androgen production but again evidence for a direct inhibitory effect of insulin on the ovulatory process has not being forthcoming. While dysfunctional feedback mechanisms obviously play a part in the occurrence of anovulation, the changes in the feedback mechanisms are widely thought to be secondary to the primary pathophysiological changes in the ovary itself (Franks et al.,2006). Here, we present a hypothesis based upon recent research and introducing into the puzzle some new concepts, which may help to coordinate past theories. Follicle numbers in PCOS The density of pre-antral and small antral follicles in the polycystic ovary is six times that of the normal ovary (Webber et al., 2003). There are two possible reasons for this which are probably working in combination. & The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

1118 Homburg and Crawford First, in the normal ovary, there is a constant dynamic progression of development from primordial follicles to primary, secondary, pre-antral and small antral follicles (Webber et al., 2003). This process seems to be accelerated in the polycystic ovary. In vitro experiments suggest that this acceleration is due to androgens (Hillier and Tetsuka, 1997; Vendola et al., 1999). Secondly, the fact that these small follicles do not develop into ovulatory follicles and also have a reduced rate of apoptosis compared with those in a normal ovary (Beloosesky et al., 2004) gives rise to the typical morphology of a polycystic ovary, a so-called stockpiling effect. Anti-Müllerian hormone As the name suggests, anti-müllerian hormone (AMH) has an integral role in the intrauterine development and sex differentiation of the male fetus. It is secreted from the Sertoli cells of the developing testes inhibiting ipsilateral mullerian duct development and thereby allowing the Wolffian duct system to prevail (Behringer et al., 1994). However, the role of AMH across the female reproductive life-span has only more recently come to light. It is a member of the transforming growth factor-beta (TGF-beta) protein super family and is produced exclusively by the gonads (Pepinsky et al., 1988). Along with other members of the TGF-beta superfamily, AMH plays an important role in folliculogenesis. In females, AMH is secreted by the granulosa cells of the early primordial follicles, thereafter its secretion increases and reaches peak concentration in small antral follicles before its level subsides in the granulosa cells of pre-ovulatory follicles (Durlinger et al., 2002; Andersen and Byskov, 2006). Owing to its strong correlation with the number of antral follicles, the AMH serum level is now well utilized as a marker for ovarian reserve and also as a prognostic marker for fertility potential (Fanchin et al., 2003; Muttukrishna et al., 2005; van Rooij et al., 2005). As women with PCOS have an increased number of small follicles in the pre-antral and antral stage, it is also not surprising that their AMH serum concentrations are higher than in women with normal ovaries. Since, Fallat et al. first reported this in 1997, several clinical studies have confirmed that serum AMH levels are two to three times higher in PCOS compared with levels in women with normal ovaries (Cook et al., 2002; Laven et al., 2004; Pigny et al., 2006). Laven et al. (2004) showed significantly elevated AMH levels in WHO II patients (that is normogonadotrophic normoestrogenic anovulatory patients; Rowe et al., 2000) compared with controls. Further, AMH levels were found to positively correlate with individual features of PCOS, including LH concentrations, testosterone, mean ovarian volume and the number of ovarian follicles (Laven et al., 2004). The overexpression of AMH and its receptors in oligo/anovulatory PCOS women could be due to increased LH levels and/or inhibition of its repressive action. This dysregulation is observed in oligo/anovulatory, but not in normo-ovulatory, PCOS women and this implicates LH in the follicular arrest of PCOS (Pierre et al., 2013). However, the positive correlation of AMH with testosterone concentrations does not necessarily imply a causal relationship. In fact, after androgen suppression AMH concentration was shown to be unchanged (Carlsen et al., 2009). A recent study supports the finding that the level of AMH can help to delineate between women with PCOS compared with women with polycystic ovarian morphology alone compared with controls (Homburg et al., 2013). Thus, not only is AMH elevated in women with PCOS but it also correlates with the severity of PCOS. However, it is not just the mere increased numbers of these follicles that produce raised AMH levels, as individual follicles from a polycystic ovary produce more AMH than their size-matched counterparts from a normal ovary. The AMH production from the granulosa cells of a patient with ovulatory PCOS is three times higher compared with granulosa cells from normal ovaries while, incredibly, the AMH production from sized matched granulosa cells of a patient with anovulatory PCOS is up to 75 times higher (Pellatt et al., 2007). Very recently, we have confirmed clinically that the individual granulosa cell produces more AMH than its counterpart in the normal ovary by calculating the ratio of AMH to antral follicle count. Interestingly, this ratio for women with polycystic ovaries but who are ovulatory falls between that of women who have normal ovaries and women with anovulatory PCOS (Bhide et al., submitted for publication). The role of AMH in the normal ovary While the exact function of AMH is yet to be completely clarified, there is evidence that it counteracts the actions of follicle-stimulating hormone (FSH) on aromatase activity and in the development of an ovulatory follicle (Pellatt et al., 2010, 2011). A number of animal and human studies collectively demonstrate an inhibitory role of AMH in follicular fluid. In murine granulosa cells, the FSH- and camp-stimulated aromatase activity was significantly reduced after treatment with AMH (Di Clemente et al., 1994). The same study revealed reduced mrna expression in campstimulated cells and decreased LH receptor numbers in porcine granulosa cells with AMH treatment (Di Clemente et al., 1994). Pellatt et al. (2011) revealed that AMH inhibits factors that promote follicle progression and growth in human granulosa cells. Grynberg et al. (2012) demonstrated in humans that AMH expression can be differentially regulated by estradiol depending on the estradiol receptors suggesting that its decrease in the granulosa cells of growing follicles, which mainly express estradiol receptor beta (ERb), is due to the effect of estradiol. Collectively, these effects of AMH would be expected to inhibit folliculogenesis or premature maturation in the normal ovary when the follicles remain at the small antral or antral stage. AMH production becomes almost undetectable once follicles reach.10 mm in size (Pellatt et al., 2007), allowing these follicles to become responsive to FSH and thereby allowing follicle selection to occur. Anovulation in PCOS It was first suggested by Dewailly et al. (2007) that the size of the 2 5 mm follicle pool is an independent and important contributor to the follicular arrest in PCOS. They had previously demonstrated that AMH serum concentrations were significantly higher in PCOS women who had amenorrhoea compared with those with oligomenorrhoea who, in turn, had higher levels than those who had normal cycles (Pigny et al., 2003). We have also demonstrated that oligo/anovulatory women with PCOS have significantly higher serum concentrations of AMH compared with women who have the typical morphology of a polycystic ovary but who are ovulatory (Homburg et al., 2013). Serum concentrations of FSH, although within normal limits, are generally lower in women with PCOS compared with women who have normal ovaries (Laven et al., 2004). This is not, however, enough to explain the cause of anovulation. Rather, there is an endogenous

Anovulation in PCOS 1119 inhibition of the action of FSH in promoting follicular development in women with PCOS. This endogenous inhibition may be overcome by administering exogenous FSH or, indeed, by promoting an FSH surge with clomiphene citrate. As AMH has been demonstrated to counteract the actions of FSH (Pellatt et al., 2010, 2011), it is suggested to be implicated in the cause of the anovulation in these women. This suggestion is reinforced by the fact that the higher the concentrations of AMH, the greater the severity of the ovulatory disturbance (Pigny et al., 2003; Laven et al., 2004). A further contribution of AMH to the mechanism of anovulation may be its positive correlation with LH concentrations (Laven et al., 2004; Piouka et al., 2009). LH is the principal promoter of androgen production by the ovary and more androgens are thought to encourage an acceleration of the progress of primordial to pre-antral and small antral follicles. These, in turn, produce AMH, thus creating a kind of vicious circle. The hypothesis of a positive feedback on the pituitary/hypothalamus is still poorly documented and we have cast further doubt on this with our finding of normal AMH values in 7 of 14 women with hypogonadotrophic hypogonadism (presently unpublished data). However, an additional factor could be that the lack of circulating progesterone in anovulatory women promotes the release of LH. This hypothesis for the aetiology of the anovulation associated with PCOS is summarized in Fig. 1. Indirect evidence to strengthen this hypothesis is provided by the fact that a reduction in the number of follicles is capable of restoring regular ovulation in oligo/anovulatory women with PCOS. When ovarian tissue is removed or destroyed, as in wedge resection of the ovaries or laparoscopic ovarian drilling, ovulation is frequently reinstated. Further, women with PCOS, over the age of 40, not infrequently resume normal regular cycles (Elting et al., 2003). These women, as is the case in normal women, have an accelerated loss of follicles at this age (and a decrease in AMH concentrations) and this, it is assumed, is the reason for the regulation of the cycle. Further indirect evidence to strengthen this hypothesis can be gathered from the practice of ovulation induction. Those with very high concentrations of circulating AMH are less likely to respond with ovulation to treatment with weight loss (Moran et al., 2007), clomiphene citrate or laparoscopic ovarian drilling (Amer et al., 2009). A possible reason that the metformin is less effective and certainly slower than clomiphene in inducing ovulation is the fact that AMH levels only decrease very slowly during this treatment (Fleming et al., 2005; Fabreques et al., 2011). Finally, AMH concentrations decrease during successful low-dose FSH therapy (Catteau-Jonard et al., 2007). Together, this clinical evidence accentuates the fact that the greater the number of follicles, the higher the AMH concentrations, the more severe the symptoms of PCOS. Other factors may influence AMH action. Activin may counter AMH action by inhibiting thecal androgen production, and follistatin, an activinbinding protein, neutralizes activin bioactivity (Duleba et al., 2001). Notably, in PCOS activin-a concentrations are decreased and follistatin concentrations are increased (Eldar-Geva et al., 2001; Norman et al., 2001). A decrease in concentration or functional activity of activin, as well as an increase in follistatin, might therefore also contribute to the pathophysiology of PCOS. Conclusions The greater the number of pre-antral and small antral follicles in the polycystic ovary, the greater the ovulatory disturbance. The number of these follicles is reflected by AMH concentrations in the serum. Both in vitro and clinical evidence has been presented here to support the hypothesis that the high-amh concentrations present in women with PCOS play an integral role in causing anovulation due to its inhibitory influence on the actions of FSH that normally promotes follicular development from the small antral stage to ovulation. Authors roles R.H. was responsible for the conception of the article. G.C. constructed the format of the manuscript and both authors were responsible for the writing and approval of the final version. Funding No funding was used and neither author had any conflict of interest. Conflict of interest None declared. Figure 1 A schematic representation of the proposed mechanism of anovulation in women with PCOS. AMH, anti-mullerian hormone; FSH, follicle stimulating hormone; LH, lutenizing hormone. References Abbott DH, Dumesic DA, Franks S. Developmental origin of polycystic ovary syndrome a hypothesis. J Endocrinol 2002;174:1 5. Adams J, Polson DW, Franks S. Prevalence of polycystic ovaries in women with anovulation and idiopathic hirsutism. Br Med J (Clin Res Ed) 1986; 293:355 359.

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