REVIEWS Genetics of androgen metabolism in women with infertility and hypoandrogenism Aya Shohat-Tal, Aritro Sen, David H. Barad, Vitaly Kushnir and Norbert Gleicher Abstract | Hypoandrogenism in women with low functional ovarian reserve (LFOR, defined as an abnormally low number of small growing follicles) adversely affects fertility. The androgen precursor dehydroepiandrosterone (DHEA) is increasingly used to supplement treatment protocols in women with LFOR undergoing in vitro fertilization. Due to differences in androgen metabolism, however, responses to DHEA supplementation vary between patients. In addition to overall declines in steroidogenic capacity with advancing age, genetic factors, which result in altered expression or enzymatic function of key steroidogenic proteins or their upstream regulators, might further exacerbate variations in the conversion of DHEA to testosterone. In this Review, we discuss in vitro studies and animal models of polymorphisms and gene mutations that affect the conversion of DHEA to testosterone and attempt to elucidate how these variations affect female hormone profiles. We also discuss treatment options that modulate levels of testosterone by targeting the expression of steroidogenic genes. Common variants in genes encoding DHEA sulphotransferase, aromatase, steroid 5α-reductase, androgen receptor, sex-hormone binding globulin, fragile X mental retardation protein and breast cancer type 1 susceptibility protein have been implicated in androgen metabolism and, therefore, can affect levels of androgens in women. Short of screening for all potential genetic variants, hormonal assessments of patients with low testosterone levels after DHEA supplementation facilitate identification of underlying genetic defects. The genetic predisposition of patients can then be used to design individualized fertility treatments. Shohat-Tal, A. et al. Nat. Rev. Endocrinol. advance online publication 5 May 2015; doi:10.1038/nrendo.2015.64
Introduction
Androgens have an important role in regulating ovarian function by stimulating the early stages of follicular growth and promoting granulosa and theca cell proliferation.1,2 Whereas excess androgen production in women is frequently associated with polycystic ovary syndrome (PCOS), hypoandrogenism is associated with low functional ovarian reserve (LFOR, defined as an abnormally low number of small growing follicles).3 LFOR, and therefore hypoandrogenism, can be found in women, sometimes aged 0.04 IU/l in women 20 copies of CAG repeat in exon 1)148
Direct testosterone administration
Low testosterone levels and low DHEAS levels
SULT2A1 (Ala63Pro substitution) SULT2A1 (Lys227Glu substitution)67 oPOI (POA) or age-related impairment of adrenal zona reticularis59
Low testosterone levels, low DHEAS levels and elevated TNF‑α levels
Acute-phase inflammatory response71,72
TNF inhibition79,80
Low testosterone levels and high DHEAS levels
CYP19A1 (C/T substitution in exon 10 of 3'‑UTR)94 FMR1 (>26 copies of CGG repeat in 5'–UTR)59
Androgen supplementation with either DHEA or testosterone12,14,25
High estradiol:testosterone ratio
BRCA1 mutations96
Aromatase inhibition (for example, letrozole and NSAIDs)111,116,119
High 3α-androstandiol glucuronide levels High DHT:testosterone ratio Low free testosterone levels, high SHBG levels and/or high T4 levels
Hyperthyroidism132,166,167
Antithyroid drugs if hyperthyroidism is diagnosed132 Steroid 5α-reductase inhibitors (such as finasteride) if only levels of DHT and its metabolites are increased130–132
67
Abbreviations: DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulphate; DHT, dihydrotestosterone; oPOI, occult primary ovarian insufficiency; POA, premature ovarian ageing; SHBG, sex hormone-binding globulin; TNF, tumour necrosis factor; UTR, untranslated region.
(DHEA-ST, also known as bile salt sulphotransferase), which is encoded by the SULT2A1 gene. SULT2A1 is highly expressed in the zona reticularis of the adrenal cortex and in the liver and small intestines, where orally administered DHEA is first metabolized.52,53 DHEAS is bound by albumin and has a slower metabolic clearance rate and longer half-life than DHEA.54 Consequently, DHEAS is often considered to be the storage form of DHEA. In response to metabolic demand, circulating DHEAS is hydrolysed back to DHEA by steroid sulphatases, located in ovarian and peripheral steroidogenic tissues, for use as a precursor in androgen and estrogen synthesis.55 In addition to being the substrate for androgen and estrogen synthesis, DHEAS has a role in stimulating steroidogenesis: incubation of the human adrenocortical cell line, NCI‑H295R, with DHEAS significantly increased mRNA expression of the steroidogenic acute regulatory protein (also known as StAR), which is responsible for translocation of cholesterol from the cytosol to mitochondria—a rate-limiting step in steroid biosynthesis.56 Adrenal, rather than ovarian, deficiencies might cause failure to properly ‘prime’ DHEA metabolism via sulphonation, as sulphonation occurs almost exclusively in the adrenal cortex.57 This conclusion is supported by the observation that levels of DHEA-ST decline in adrenal cortexes of ageing men and women, which leads to proportional loss of DHEAS formation.58 Adrenal pathologies that affect sulphonation might also be observed in female patients 38 years, and women whose levels of DHEAS and/or testosterone fail to increase have significantly reduced IVF pregnancy rates.7,59 The duration of DHEA supplementation is important as the small follicles targeted are still weeks to months from becoming gonadotropin-sensitive in the later stages of folliculo genesis during the IVF cycle.14,25 The observed variability in hormonal responses to exogenous DHEA administration is probably due, at least in part, to genetic factors. SNPs that affect serum levels of androgens Approximately 40–60% of the variability in serum levels of DHEAS seems to be related to inheritance, with women displaying higher heritability than men.60–65 African American women have significantly lower levels of DHEAS than white women.66 Sequencing of DNA samples from 60 white American and 60 African American individuals identified two single-nucleotide polymorphisms (SNPs) in SULT2A1 that were only present in African American individuals: rs11569681 (resulting in an Ala63Pro mutation) and rs11569680 (resulting in a Lys227Glu mutation), at frequencies of 0.050 and 0.008, respectively.67 Transfection of constructs expressing these SNPs into COS‑1 cells reduced DHEAS production, DHEA-ST activity and levels of DHEA-ST protein. These results suggest ethnic-specific pharmacogenetic variations
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REVIEWS in DHEA-ST-catalysed sulphation of both endogenous and exogenous substrates.66,67 African American women with low testosterone levels and infertility might, therefore, be good candidates to be genotyped for these two SNPs. Presence of these two SNPs could, at least partially, account for the widely reported racial disparities in IVF cycle outcomes, with women of African descent almost uniformly demonstrating lower pregnancy rates than white or Asian women.68 Endocrine and immune infertility Even though POI (POF) is generally associated with low levels of DHEAS, androstenedione and testosterone,59,69 autoimmune-associated forms of oPOI (POA) and POI (POF) seem to be primarily associated with abnormally low levels of DHEAS.69 This observation suggests that SULT2A1 expression is regulated by immune system components, and that at least some cases of hypoandrogenic LFOR are the consequence of adrenal autoimmune processes. Animal studies support such a hypothesis.70–74 In a murine model, acute-phase inflammatory responses reduced SULT2A1 mRNA expression, DHEA-ST enzymatic activity and circulating levels of DHEAS in a timedependent and dose-dependent manner.70 Furthermore, proinflammatory cytokines, such as tumour necrosis factor α (TNF‑α) and IL‑1, lowered SULT2A1 mRNA levels in human Hep3B hepatoma cells.70 In cultured human fetal adrenal cells, ACTH-mediated increases in SULT2A1 mRNA levels were ameliorated by TNF‑α and transforming growth factor β (TGF‑β),71 which also inhibited DHEAS production independent of ACTH.72–74 Autoimmunity and elevated levels of proinflammatory cytokines, including TNF‑α, have been associated with female infertility, recurrent implantation failure and pregnancy loss.75–78 The TNF‑α inhibitor adalimumab, in combination with intravenous administration of immuno globulin, improved live birth rates associated with IVF treatments and in women with autoimmune-associated recurrent spontaneous abortions.79,80 Additionally, autoimmunity to various steroidogenic enzymes is associated with POI (POF).81–84 All of these observations, which link the immune system to SULT2AI expression, suggest that investigations of inflammatory and autoimmune markers in infertile women with hypoandrogenism or hyperandrogenism, especially if characterized by abnormal DHEAS levels, are of interest. Confirmation of these associations would help substantiate a role for an inflammatory and/or immunologic system that regulates levels of androgens.3 Owing to the natural immunosuppressant properties of testos terone,85,86 such studies might also elucidate complex interactions between the immune system and the endocrine system, and their effects on female fertility.87 Pituitary, and even hypothalamic, pathologies of an autoimmune and/or inflammatory nature should also be considered.88
CYP19A1 CYP19AI encodes aromatase, which converts androgens (testosterone and androstenedione) to estrogens (estradiol and estrone).89
SNPs that affect serum levels of androgens SNPs that affect aromatase function are associated with either aromatase hypoactivity (low estradiol:testos terone ratio) or aromatase hyperactivity (high estradiol: testosterone ratio). The A allele of SNP rs173591 and the TC allele of SNP rs2470152 are associated with aroma tase hypoactivity, increased serum testosterone levels and exacerbation of the PCOS phenotype.90,91 The TT allele of SNP rs936306 is associated with aromatase hyperactivity and reduced testosterone levels, and is more prevalent in African American women than white women.92 This SNP might, therefore, be yet another contributing factor to the reported ethnic disparities in female fertility and IVF outcomes.68 A C/T substitution (rs10046) in the 3'-untranslated region of exon 10 of CYP19A1 is also significantly associated with increased levels of CYP19A1 mRNA, increased aromatase activity 93 and reduced levels of testosterone, androstenedione and DHEAS,94 and seems more prevalent in women with breast cancer than in healthy women.93 BRCA1-mediated regulation Albeit not undisputed,95 BRCA1 expression is increasingly associated with early declines in FOR.96–98 Two mechanisms have been proposed that might explain these declines. The first mechanism involves a role for BRCA1 in repair of DNA double-strand breaks (DSBs). Brca1–/– mice demonstrate impaired reproductive capacity compared with wild-type littermates. This reduced reproductive capacity is intimated by significantly reduced numbers of primordial follicle per ovary, reduced oocyte production in response to ovarian stimulation, reduced litter sizes after mating and considerably increased numbers of DSBs in oocytes.96 In addition, when Brca1 is downregulated in mice oocytes by use of small interfering RNAs (siRNAs), mean numbers of DSB foci are markedly increased and animal survival is reduced following in vitro exposure to genotoxic stress.96 Interestingly, Brca1–/– mice did not demonstrate similar increases in the number of DSBs in granulosa cells.96 In these cells, BRCA1 might function primarily as a transcription factor that regulates aromatase gene expression, as 85% of BRCA1 mutation carriers (characterized by an absence of functional BRCA1 protein) have increased levels of CYP19A1 mRNA in their ovaries and breast tissue.99 In the human granulosa cell line KGN, BRCA1 expression inversely correlates with levels of CYP19A1 mRNA and aromatase protein. 65,100 Murine Brca1 is highly expressed in the granulosa cells of developing (small growing) follicles, but is significantly decreased in granu losa cells of large antral and preovulatory follicles after aromatase expression peaks.101,102 At these late follicular developmental stages, BRCA1 expression is restricted to the cumulus granulosa cells, which contain much lower levels of aromatase protein than mural granulosa cells.101 In women with germline mutations in BRCA1, FOR (as determined by peripheral levels of AMH), has been reported to be prematurely decreased.96–98 In addition, carriers of BRCA1 mutations seem to have worse ovarian responses to stimulation with gonadotropins
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REVIEWS than would be expected for women with LFOR.103 This impairment in reproductive function is possibly the consequence of an increased number DSBs in oocytes of BRCA1 mutation carriers.96 The second possible explanation for LFOR is the overexpression of CYP19A1 in granulosa cells of preantral follicles, which is characterized by increased expression of androgen receptor protein 104–106 and increased AMH production.107 Haploinsufficiency in BRCA1 mutation carriers might primarily affect preantral follicles, as BRCA1 is highly expressed in these follicles.101 Low testosterone levels, typically observed in women with oPOI (POA) as a result of various causes,3 might, therefore, be the consequence of increased aromatase activity in other wise androgen-dominant stages of follicular development. Pharmacological interventions at various stages of follicular development, with the aim of modulating CYP19A1 expression, might improve understanding of the role of aromatase in follicular dynamics. Aromatase inhibitors Supraphysiologic levels of estrogen during ovarian stimu lation negatively affect developing oocytes, embryos, blastocyst hatching and the endometrium.108,109 Aromatase inhibitors with short half-lives, such as letrozole, (usually taken for only ~5 days during an ovarian stimulation cycle), are, therefore, used to temporarily block the conversion of androgens to estrogen. Two mechanisms have been proposed for the short-term blockade of this conversion. Firstly, by temporarily blocking the conversion of androgens to estrogen, negative feedback from estrogen is blocked, thereby increasing gonadotropin secretion in the early phase of the menstrual cycle.110 Secondly, aromatase inhibitors promote an androgen-rich intrafollicular environment, which increases the density of FSH receptors and, consequently, enhances the gonadotropin sensitivity of granulosa cells.111,112 Indeed, letrozole subtly increases levels of testosterone and androstenedione in patients undergoing IVF.113 Poor response to ovarian stimulation with gonado tropins (often a manifestation of LFOR114) seems to improve with letrozole treatment, as demonstrated by increased follicular fluid levels of testosterone and androstenedione, improved oocyte yields and increased embryo implantation rates in association with IVF.115 Levels of AMH also seem to increase concomitantly with increasing follicular fluid levels of testosterone,116 which suggests that increased expression and/or proliferation of granulosa cells and, therefore, increased granulosa cell mass rather than improved FOR, is an underlying mechanism for at least some of the improvements reported in IVF outcomes. Ageing of the immediate microenvironment, rather than the ageing of oocytes themselves, has been suggested to primarily characterize ovarian ageing.14,15,25 Within a therapeutic androgen range, more cumulus and/or granulosa cell mass might, therefore, reflect an improved nutritional supply for growing oocytes and increased oocyte quality, whereas improvements in FOR might be the consequence of the increased oocyte yields observed in these patients.
Inhibitors of cyclooxygenase (COX, also known as prostaglandin G/H synthase), such as aspirin and NSAIDs, have also been used to suppress CYP19A1 expression in estrogen-dependent pathologies.117,118 The action of COX‑2 produces prostaglandin E2, which activates the CYP19A1 promoter and downregulates expression of BRCA1.119,120 Investigations of this family of drugs are, therefore, of interest for the treatment of infertile women with low testosterone levels.
SRD5A2 In some cell types that express androgen receptors, testos terone undergoes local conversion to the more potent androgen, DHT, by the action of steroid 5α-reductase 1 (also known as 3‑oxo‑5α-steroid 4‑dehydrogenase 1; encoded by SRD5A1) and steroid 5α-reductase 2 (also known as 3‑oxo‑5α-steroid 4‑dehydrogenase 2; encoded by SRD5A2). In theca and granulosa cells, the expression of SRD5A2 is ~threefold higher than that of SRD5A1, which makes steroid 5α-reductase 2 the predominant 5α-reductase in ovarian tissue.121,122 Given that very little DHT diffuses into the extracellular compartment and general circulation,123 soluble 3α-androstanediol glucuronide (commonly known as 3α-diolG) is commonly used as marker for 5α-reductase activity. 3α-diolG is a DHT metabolite produced by reduction of DHT to 5α-androstane-3α,17β-diol by 3α-hydroxysteroid dehydrogenase, followed by inactivating glucuronidation (Figure 1).124 5α-reductase inhibition Owing to its twofold–threefold higher affinity for the androgen receptor, DHT is considered a more potent androgen than testosterone.41,125,126 In addition, studies have suggested that androgen receptor signalling in response to DHT and testosterone differs qualitatively rather than quantitatively, and is promoter and cell-type specific. These conclusions are supported by crystallographic studies, which revealed different androgen receptor conformations upon binding to testosterone and DHT that resulted in distinct androgen receptor–DNA and androgen receptor–coactivator interactions.126 In vivo, testosterone, but not DHT, upregulates CYP19A1 expression in nonluteinized bovine granulosa cells,127 a stimulation shown to be directly mediated by testost erone and not by the conversion of testosterone to DHT or estradiol.128 In the absence of gonadot ropins, expression of nuclear receptor sub family 5 group A member 2 (LHR‑1, a transcription factor known to regulate expression of CYP19A1) in rat granulosa cells is exclusively stimulated by testosterone in an androgen receptor-dependent manner; 128 this effect is more pronounced in the presence of inhibitors of steroid 5α-reductase.128 Finasteride, a selective steroid 5α-reductase inhibitor, significantly reduces serum levels of DHT and 3α-diolG, and either maintains or increases total levels of testosterone without altering serum levels of DHEAS or androstenedione.129–130 Given that the aberrant conversion of testosterone to DHT might potentially contribute to low testosterone levels, measuring levels
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REVIEWS of 3α-diolG could be useful in assessing this possibility in infertile women. If increased conversion of testos terone to DHT is confirmed, treatments involving steroid 5α-reductase inhibitors might prove therapeutically useful for infertile women with low levels of testosterone. 5α-reductase function and infertility The Val89Leu substitution in steroid 5α-reductase 2 (encoded by the SNP rs523349) reduces 5α-reductase activity by 10–40% in vitro.131,132 This SNP is found in the general population with a frequency of 0.292 and seems to be associated with a decreased prevalence of PCOS. Com pared with Val/Val homozygotes, Val/Leu heterozygotes and Leu/Leu homozygotes are 24% and 61%, respectively, less likely to develop PCOS.131,133 Furthermore, the relative ratio of testosterone:DHT correlated with presence of the metabolic syndrome in women with PCOS.134 Hyperthyroidism was shown to be associated with changes in the DHT:testosterone ratio and levels of 5α-reduced androgens are increased in association with hyperthyroidism. Women with Graves disease present with increased levels of DHT and 3α-androstane‑3α, 17β-diol, and a lower than normal DHT:testosterone ratio, which normalizes with successful treatment of the hyperthyroidism.135 Investigations of SNP-affected androgenic hormone profiles in women with hypoandrogenic LFOR might also be of interest. Given that low levels of testosterone are, at least partially, the consequence of the abnormal conversion of testosterone to DHT, measurement of levels of 5α-androstanediol glucuronide (5α-diolG) might prove diagnostically useful. Directed treatments with steroid 5α-reductase inhibitors could then prove therapeutically useful in women with hypoandrogenic LFOR.
Genes beyond the steroidogenic pathways AR The androgen receptor (encoded by AR) is closely involved in ovarian folliculogenesis 104 and is highly expressed in granulosa cells of small growing follicles,15 peaking at preantral and early antral stages,15 when the trophic effects of androgens are maximal and synergistic with follicle-stimulating hormone.104 Expression of the androgen receptor declines as follicular maturation approaches the preovulatory stage.15,25,104–106 The most extensively studied variation in AR in humans is the polymorphic CAG trinucleotide repeat in exon 1. This repeat varies by ethnicity and encodes an uninterrupted polyglutamine tract in the N‑terminal transacti vation domain,136 which has been shown to be involved in the interaction between the androgen receptor and tissue-specific coactivators.137 CAG repeat—infertility and serum testosterone An inverse association exists between the CAG repeat number and transactivation capacity of the androgen receptor, with 19 CAG repeats.149 Combined, these observations suggest that the length of the CAG repeat tract affects androgen receptor-dependent transcriptional activity at the promoters of genes (such as LHR1), which are involved in the metabolism of DHEA to testosterone, thereby affecting the relative ratios of DHEA conversion products.
SHBG Approximately 10–15% of intracellular androgens diffuse from intracellular compartments to the extracellular space and general circulation, where they are bound to the glycoprotein sex hormone-binding globulin (SHBG) and, to a lesser extent, to serum albumin. SHBG binds androgens with high affinity and estrogens with low affinity, and regulates access of both hormones to target tissues.150 Hormone fractions not bound by SHBG are considered bioavailable and can bind to and activate androgen receptors or estrogen receptors in target cells.151,152 Revisiting bioavailable androgens Even though synthesized primarily in the liver, SHBG expression has been detected in various tissues of the female reproductive tract, which suggests a role for this protein in regulating the intracellular availability of sex steroids in reproductive organs.150–155 Some reproductive tissues, indeed, express high-affinity SHBG receptors on cell membranes that interact with steroid-bound SHBG. Steroid–SHBG complexes might undergo endocytic internalization with subsequent release of steroids. Alternatively, these complexes might transduce signals from the cell surface that, via G‑proteins, trig ger transcriptional activity of classic intracellular steroid hormone receptors.156.157 In women with infertility, it might, therefore, be benefi cial to consider both free testosterone and total testosterone as markers of peripheral androgen status, as the contribution of the SHBG-bound androgen fraction to the bioactive hormonal milieu might be considerable. By studying conversion rates of DHEA to testosterone, the relevance of both testosterone measurements for assessing pregnancy potential in women with oPOI (POA) was demonstrated,59 with measurements of total testosterone being slightly more reliable in a predictive model than measurements of free testosterone.7
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REVIEWS Box 2 | Potential future studies on DHEA supplementation ■■ Investigation of the prevalence of Ala63Pro and Lys227Glu substitutions in dehydroepiandrosterone sulphotransferase in African American women, correlation of these mutations with androgen status, and comparison of African American women with white and Asian controls; increased prevalence of these mutations might at least partially explain the widely reported reduced IVF pregnancy rates in African American women ■■ Investigation of the TT allele of SNP rs936306 in infertile African American women, as this allele is associated with hypoandrogenaemia (low testosterone levels due to aromatase hyperactivity), which is potentially treatable with aromatase inhibitors; increased prevalence of this allele might be a contributing factor in the lower IVF pregnancy rates in African American women than in white women ■■ Determination of whether African women would benefit more from supplementation with testosterone than from DHEA, as these women have at least three distinct polymorphisms that are associated with decreased conversion of DHEA to testosterone ■■ Investigation of the Val89Leu substitution in steroid 5α-reductase 2 in women with DOR and low testosterone levels; similar substitutions have been shown to be associated with PCOS, which is considered to be the extreme opposite of DOR ■■ Investigation of levels of 5α-androstanediol glucuronide in infertile women with low testosterone levels to identify abnormal conversion of testosterone to dihydrotestosterone; if confirmed, inhibition of steroid 5α-reductase might improve abnormally low testosterone levels ■■ Investigation of levels of inflammatory markers in women with hypoandrogenaemia or hyperandrogenaemia, oPOI (POA), and PCOS, respectively, to clarify the relationship between endocrine and immune systems and support the hypothesis of an immune system component controlling adrenal androgen production ■■ Investigation of COX inhibitors (which suppress CYP19A1 expression) as a potential infertility treatment for women with a high estradiol:testosterone ratio ■■ Investigation of whether CAG repeats in AR in infertile women supplemented with DHEA affect the conversion of DHEA to testosterone and how these repeats relate to IVF cycle outcomes ■■ Investigation of the effects of DHEA or testosterone supplementation on levels of SHBG in infertile women with low testosterone levels Abbreviations: COX, cyclooxygenase; DHEA, dehydroepiandrosterone; DOR, diminished ovarian reserve; IVF, in vitro fertilization; oPOI; occult primary ovarian insufficiency; PCOS, polycystic ovary syndrome; POA, premature ovarian ageing; SHBG, sex hormone-binding globulin; SNP, single nucleotide polymorphism.
Regulated expression Serum levels of SHBG vary significantly among individuals and are affected by endocrine conditions and lifestyle.158–160 For instance: SHBG is induced by estrogen but suppressed by androgens, insulin and prolactin;161–164 BMI is inversely related to circulating levels of SHBG;165 hypothyroidism in elderly women is strongly associated with decreased levels of SHBG, whereas, in young women hyperthyroidism significantly correlates with increased SHBG levels;166,167 and treatment with antithyroid drugs gradually decreases SHBG levels in association with the hyperthyroidism.132 Hypoandrogenic infertility Among the various polymorphisms that affect levels of SHBG and androgens, the pentanucleotide TAAAA repeat polymorphism has been vigorously investigated.168–170 This repeat is located in the promoter region of SHBG and is uniquely distributed across populations.148 Extended TAAAA repeat polymorphisms have mostly been studied in the context of hyperandrogenism and PCOS;170–172 polymorphisms leading to hypoandrogenism have not, so far, been described.
Levels of SHBG are significantly higher in women with familial POI (POF) than in those with sporadic POI (POF), which suggests a hereditary component to SHBG levels.173 A multiethnic study of premenopausal and perimenopausal women demonstrated that African American women had higher levels of SHBG and a lower free androgen index than white women.174 By contrast, Chinese women had lower levels of SHBG and a higher free androgen index than white women.174 Studies of SHBG variants in hypoandrogenic women with infer tility, therefore, seem warranted. Such studies might be of particular interest in relation to studies of the fragile X mental retardation protein I gene (FMR1), in which a strikingly similar association exists, with so-called ‘low’ alleles (34 CGG repeats) in Chinese women.181,182 Given that DHEA supplementation significantly reduces levels of SHBG in infertile women with LFOR25 and in those with adrenal insufficiency,175 the efficacy of DHEA supplementation in lowering SHBG levels in women with POI (POF) warrants further investigation.
FMR1 The fragile X mental retardation protein 1 (FMRP, which is encoded by FMR1) is associated with cognitive development and female reproductive function.176,177 Classical expansion of the CGG repeat (to 55–199 copies) in the 5'-untranslated region of FMR1 leads to partial promoter heterochromatinization (premutation), whereas >200 copies of the CCG repeat (full mutation) leads to complete promoter heterochromatinization.176,177 Silencing of transcription, which is associated with >200 copies of the CCG repeat, results in fragile X syndrome, which is characterized by mental retardation, autism, developmental delays and other neuropsychiatric deficits.178,179 In women, the premutation range is associated with a significantly increased risk of POI (POF).178,179 CGG repeat polymorphisms and androgen levels New FMR1 haplotypes, distinct from the classification of normal, premutation and full mutation genotypes have been described, further subdividing what has, so far, been considered the normal CGG repeat range for this gene.180,181 This advance has enabled new definitions of FMR1 repeat classes, which are associated with specific ovarian phenotypes. In this classification, ~25% of infertile women carry a low allele, defined as
Introduction
Androgens have an important role in regulating ovarian function by stimulating the early stages of follicular growth and promoting granulosa and theca cell proliferation.1,2 Whereas excess androgen production in women is frequently associated with polycystic ovary syndrome (PCOS), hypoandrogenism is associated with low functional ovarian reserve (LFOR, defined as an abnormally low number of small growing follicles).3 LFOR, and therefore hypoandrogenism, can be found in women, sometimes aged 0.04 IU/l in women 20 copies of CAG repeat in exon 1)148
Direct testosterone administration
Low testosterone levels and low DHEAS levels
SULT2A1 (Ala63Pro substitution) SULT2A1 (Lys227Glu substitution)67 oPOI (POA) or age-related impairment of adrenal zona reticularis59
Low testosterone levels, low DHEAS levels and elevated TNF‑α levels
Acute-phase inflammatory response71,72
TNF inhibition79,80
Low testosterone levels and high DHEAS levels
CYP19A1 (C/T substitution in exon 10 of 3'‑UTR)94 FMR1 (>26 copies of CGG repeat in 5'–UTR)59
Androgen supplementation with either DHEA or testosterone12,14,25
High estradiol:testosterone ratio
BRCA1 mutations96
Aromatase inhibition (for example, letrozole and NSAIDs)111,116,119
High 3α-androstandiol glucuronide levels High DHT:testosterone ratio Low free testosterone levels, high SHBG levels and/or high T4 levels
Hyperthyroidism132,166,167
Antithyroid drugs if hyperthyroidism is diagnosed132 Steroid 5α-reductase inhibitors (such as finasteride) if only levels of DHT and its metabolites are increased130–132
67
Abbreviations: DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulphate; DHT, dihydrotestosterone; oPOI, occult primary ovarian insufficiency; POA, premature ovarian ageing; SHBG, sex hormone-binding globulin; TNF, tumour necrosis factor; UTR, untranslated region.
(DHEA-ST, also known as bile salt sulphotransferase), which is encoded by the SULT2A1 gene. SULT2A1 is highly expressed in the zona reticularis of the adrenal cortex and in the liver and small intestines, where orally administered DHEA is first metabolized.52,53 DHEAS is bound by albumin and has a slower metabolic clearance rate and longer half-life than DHEA.54 Consequently, DHEAS is often considered to be the storage form of DHEA. In response to metabolic demand, circulating DHEAS is hydrolysed back to DHEA by steroid sulphatases, located in ovarian and peripheral steroidogenic tissues, for use as a precursor in androgen and estrogen synthesis.55 In addition to being the substrate for androgen and estrogen synthesis, DHEAS has a role in stimulating steroidogenesis: incubation of the human adrenocortical cell line, NCI‑H295R, with DHEAS significantly increased mRNA expression of the steroidogenic acute regulatory protein (also known as StAR), which is responsible for translocation of cholesterol from the cytosol to mitochondria—a rate-limiting step in steroid biosynthesis.56 Adrenal, rather than ovarian, deficiencies might cause failure to properly ‘prime’ DHEA metabolism via sulphonation, as sulphonation occurs almost exclusively in the adrenal cortex.57 This conclusion is supported by the observation that levels of DHEA-ST decline in adrenal cortexes of ageing men and women, which leads to proportional loss of DHEAS formation.58 Adrenal pathologies that affect sulphonation might also be observed in female patients 38 years, and women whose levels of DHEAS and/or testosterone fail to increase have significantly reduced IVF pregnancy rates.7,59 The duration of DHEA supplementation is important as the small follicles targeted are still weeks to months from becoming gonadotropin-sensitive in the later stages of folliculo genesis during the IVF cycle.14,25 The observed variability in hormonal responses to exogenous DHEA administration is probably due, at least in part, to genetic factors. SNPs that affect serum levels of androgens Approximately 40–60% of the variability in serum levels of DHEAS seems to be related to inheritance, with women displaying higher heritability than men.60–65 African American women have significantly lower levels of DHEAS than white women.66 Sequencing of DNA samples from 60 white American and 60 African American individuals identified two single-nucleotide polymorphisms (SNPs) in SULT2A1 that were only present in African American individuals: rs11569681 (resulting in an Ala63Pro mutation) and rs11569680 (resulting in a Lys227Glu mutation), at frequencies of 0.050 and 0.008, respectively.67 Transfection of constructs expressing these SNPs into COS‑1 cells reduced DHEAS production, DHEA-ST activity and levels of DHEA-ST protein. These results suggest ethnic-specific pharmacogenetic variations
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REVIEWS in DHEA-ST-catalysed sulphation of both endogenous and exogenous substrates.66,67 African American women with low testosterone levels and infertility might, therefore, be good candidates to be genotyped for these two SNPs. Presence of these two SNPs could, at least partially, account for the widely reported racial disparities in IVF cycle outcomes, with women of African descent almost uniformly demonstrating lower pregnancy rates than white or Asian women.68 Endocrine and immune infertility Even though POI (POF) is generally associated with low levels of DHEAS, androstenedione and testosterone,59,69 autoimmune-associated forms of oPOI (POA) and POI (POF) seem to be primarily associated with abnormally low levels of DHEAS.69 This observation suggests that SULT2A1 expression is regulated by immune system components, and that at least some cases of hypoandrogenic LFOR are the consequence of adrenal autoimmune processes. Animal studies support such a hypothesis.70–74 In a murine model, acute-phase inflammatory responses reduced SULT2A1 mRNA expression, DHEA-ST enzymatic activity and circulating levels of DHEAS in a timedependent and dose-dependent manner.70 Furthermore, proinflammatory cytokines, such as tumour necrosis factor α (TNF‑α) and IL‑1, lowered SULT2A1 mRNA levels in human Hep3B hepatoma cells.70 In cultured human fetal adrenal cells, ACTH-mediated increases in SULT2A1 mRNA levels were ameliorated by TNF‑α and transforming growth factor β (TGF‑β),71 which also inhibited DHEAS production independent of ACTH.72–74 Autoimmunity and elevated levels of proinflammatory cytokines, including TNF‑α, have been associated with female infertility, recurrent implantation failure and pregnancy loss.75–78 The TNF‑α inhibitor adalimumab, in combination with intravenous administration of immuno globulin, improved live birth rates associated with IVF treatments and in women with autoimmune-associated recurrent spontaneous abortions.79,80 Additionally, autoimmunity to various steroidogenic enzymes is associated with POI (POF).81–84 All of these observations, which link the immune system to SULT2AI expression, suggest that investigations of inflammatory and autoimmune markers in infertile women with hypoandrogenism or hyperandrogenism, especially if characterized by abnormal DHEAS levels, are of interest. Confirmation of these associations would help substantiate a role for an inflammatory and/or immunologic system that regulates levels of androgens.3 Owing to the natural immunosuppressant properties of testos terone,85,86 such studies might also elucidate complex interactions between the immune system and the endocrine system, and their effects on female fertility.87 Pituitary, and even hypothalamic, pathologies of an autoimmune and/or inflammatory nature should also be considered.88
CYP19A1 CYP19AI encodes aromatase, which converts androgens (testosterone and androstenedione) to estrogens (estradiol and estrone).89
SNPs that affect serum levels of androgens SNPs that affect aromatase function are associated with either aromatase hypoactivity (low estradiol:testos terone ratio) or aromatase hyperactivity (high estradiol: testosterone ratio). The A allele of SNP rs173591 and the TC allele of SNP rs2470152 are associated with aroma tase hypoactivity, increased serum testosterone levels and exacerbation of the PCOS phenotype.90,91 The TT allele of SNP rs936306 is associated with aromatase hyperactivity and reduced testosterone levels, and is more prevalent in African American women than white women.92 This SNP might, therefore, be yet another contributing factor to the reported ethnic disparities in female fertility and IVF outcomes.68 A C/T substitution (rs10046) in the 3'-untranslated region of exon 10 of CYP19A1 is also significantly associated with increased levels of CYP19A1 mRNA, increased aromatase activity 93 and reduced levels of testosterone, androstenedione and DHEAS,94 and seems more prevalent in women with breast cancer than in healthy women.93 BRCA1-mediated regulation Albeit not undisputed,95 BRCA1 expression is increasingly associated with early declines in FOR.96–98 Two mechanisms have been proposed that might explain these declines. The first mechanism involves a role for BRCA1 in repair of DNA double-strand breaks (DSBs). Brca1–/– mice demonstrate impaired reproductive capacity compared with wild-type littermates. This reduced reproductive capacity is intimated by significantly reduced numbers of primordial follicle per ovary, reduced oocyte production in response to ovarian stimulation, reduced litter sizes after mating and considerably increased numbers of DSBs in oocytes.96 In addition, when Brca1 is downregulated in mice oocytes by use of small interfering RNAs (siRNAs), mean numbers of DSB foci are markedly increased and animal survival is reduced following in vitro exposure to genotoxic stress.96 Interestingly, Brca1–/– mice did not demonstrate similar increases in the number of DSBs in granulosa cells.96 In these cells, BRCA1 might function primarily as a transcription factor that regulates aromatase gene expression, as 85% of BRCA1 mutation carriers (characterized by an absence of functional BRCA1 protein) have increased levels of CYP19A1 mRNA in their ovaries and breast tissue.99 In the human granulosa cell line KGN, BRCA1 expression inversely correlates with levels of CYP19A1 mRNA and aromatase protein. 65,100 Murine Brca1 is highly expressed in the granulosa cells of developing (small growing) follicles, but is significantly decreased in granu losa cells of large antral and preovulatory follicles after aromatase expression peaks.101,102 At these late follicular developmental stages, BRCA1 expression is restricted to the cumulus granulosa cells, which contain much lower levels of aromatase protein than mural granulosa cells.101 In women with germline mutations in BRCA1, FOR (as determined by peripheral levels of AMH), has been reported to be prematurely decreased.96–98 In addition, carriers of BRCA1 mutations seem to have worse ovarian responses to stimulation with gonadotropins
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REVIEWS than would be expected for women with LFOR.103 This impairment in reproductive function is possibly the consequence of an increased number DSBs in oocytes of BRCA1 mutation carriers.96 The second possible explanation for LFOR is the overexpression of CYP19A1 in granulosa cells of preantral follicles, which is characterized by increased expression of androgen receptor protein 104–106 and increased AMH production.107 Haploinsufficiency in BRCA1 mutation carriers might primarily affect preantral follicles, as BRCA1 is highly expressed in these follicles.101 Low testosterone levels, typically observed in women with oPOI (POA) as a result of various causes,3 might, therefore, be the consequence of increased aromatase activity in other wise androgen-dominant stages of follicular development. Pharmacological interventions at various stages of follicular development, with the aim of modulating CYP19A1 expression, might improve understanding of the role of aromatase in follicular dynamics. Aromatase inhibitors Supraphysiologic levels of estrogen during ovarian stimu lation negatively affect developing oocytes, embryos, blastocyst hatching and the endometrium.108,109 Aromatase inhibitors with short half-lives, such as letrozole, (usually taken for only ~5 days during an ovarian stimulation cycle), are, therefore, used to temporarily block the conversion of androgens to estrogen. Two mechanisms have been proposed for the short-term blockade of this conversion. Firstly, by temporarily blocking the conversion of androgens to estrogen, negative feedback from estrogen is blocked, thereby increasing gonadotropin secretion in the early phase of the menstrual cycle.110 Secondly, aromatase inhibitors promote an androgen-rich intrafollicular environment, which increases the density of FSH receptors and, consequently, enhances the gonadotropin sensitivity of granulosa cells.111,112 Indeed, letrozole subtly increases levels of testosterone and androstenedione in patients undergoing IVF.113 Poor response to ovarian stimulation with gonado tropins (often a manifestation of LFOR114) seems to improve with letrozole treatment, as demonstrated by increased follicular fluid levels of testosterone and androstenedione, improved oocyte yields and increased embryo implantation rates in association with IVF.115 Levels of AMH also seem to increase concomitantly with increasing follicular fluid levels of testosterone,116 which suggests that increased expression and/or proliferation of granulosa cells and, therefore, increased granulosa cell mass rather than improved FOR, is an underlying mechanism for at least some of the improvements reported in IVF outcomes. Ageing of the immediate microenvironment, rather than the ageing of oocytes themselves, has been suggested to primarily characterize ovarian ageing.14,15,25 Within a therapeutic androgen range, more cumulus and/or granulosa cell mass might, therefore, reflect an improved nutritional supply for growing oocytes and increased oocyte quality, whereas improvements in FOR might be the consequence of the increased oocyte yields observed in these patients.
Inhibitors of cyclooxygenase (COX, also known as prostaglandin G/H synthase), such as aspirin and NSAIDs, have also been used to suppress CYP19A1 expression in estrogen-dependent pathologies.117,118 The action of COX‑2 produces prostaglandin E2, which activates the CYP19A1 promoter and downregulates expression of BRCA1.119,120 Investigations of this family of drugs are, therefore, of interest for the treatment of infertile women with low testosterone levels.
SRD5A2 In some cell types that express androgen receptors, testos terone undergoes local conversion to the more potent androgen, DHT, by the action of steroid 5α-reductase 1 (also known as 3‑oxo‑5α-steroid 4‑dehydrogenase 1; encoded by SRD5A1) and steroid 5α-reductase 2 (also known as 3‑oxo‑5α-steroid 4‑dehydrogenase 2; encoded by SRD5A2). In theca and granulosa cells, the expression of SRD5A2 is ~threefold higher than that of SRD5A1, which makes steroid 5α-reductase 2 the predominant 5α-reductase in ovarian tissue.121,122 Given that very little DHT diffuses into the extracellular compartment and general circulation,123 soluble 3α-androstanediol glucuronide (commonly known as 3α-diolG) is commonly used as marker for 5α-reductase activity. 3α-diolG is a DHT metabolite produced by reduction of DHT to 5α-androstane-3α,17β-diol by 3α-hydroxysteroid dehydrogenase, followed by inactivating glucuronidation (Figure 1).124 5α-reductase inhibition Owing to its twofold–threefold higher affinity for the androgen receptor, DHT is considered a more potent androgen than testosterone.41,125,126 In addition, studies have suggested that androgen receptor signalling in response to DHT and testosterone differs qualitatively rather than quantitatively, and is promoter and cell-type specific. These conclusions are supported by crystallographic studies, which revealed different androgen receptor conformations upon binding to testosterone and DHT that resulted in distinct androgen receptor–DNA and androgen receptor–coactivator interactions.126 In vivo, testosterone, but not DHT, upregulates CYP19A1 expression in nonluteinized bovine granulosa cells,127 a stimulation shown to be directly mediated by testost erone and not by the conversion of testosterone to DHT or estradiol.128 In the absence of gonadot ropins, expression of nuclear receptor sub family 5 group A member 2 (LHR‑1, a transcription factor known to regulate expression of CYP19A1) in rat granulosa cells is exclusively stimulated by testosterone in an androgen receptor-dependent manner; 128 this effect is more pronounced in the presence of inhibitors of steroid 5α-reductase.128 Finasteride, a selective steroid 5α-reductase inhibitor, significantly reduces serum levels of DHT and 3α-diolG, and either maintains or increases total levels of testosterone without altering serum levels of DHEAS or androstenedione.129–130 Given that the aberrant conversion of testosterone to DHT might potentially contribute to low testosterone levels, measuring levels
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REVIEWS of 3α-diolG could be useful in assessing this possibility in infertile women. If increased conversion of testos terone to DHT is confirmed, treatments involving steroid 5α-reductase inhibitors might prove therapeutically useful for infertile women with low levels of testosterone. 5α-reductase function and infertility The Val89Leu substitution in steroid 5α-reductase 2 (encoded by the SNP rs523349) reduces 5α-reductase activity by 10–40% in vitro.131,132 This SNP is found in the general population with a frequency of 0.292 and seems to be associated with a decreased prevalence of PCOS. Com pared with Val/Val homozygotes, Val/Leu heterozygotes and Leu/Leu homozygotes are 24% and 61%, respectively, less likely to develop PCOS.131,133 Furthermore, the relative ratio of testosterone:DHT correlated with presence of the metabolic syndrome in women with PCOS.134 Hyperthyroidism was shown to be associated with changes in the DHT:testosterone ratio and levels of 5α-reduced androgens are increased in association with hyperthyroidism. Women with Graves disease present with increased levels of DHT and 3α-androstane‑3α, 17β-diol, and a lower than normal DHT:testosterone ratio, which normalizes with successful treatment of the hyperthyroidism.135 Investigations of SNP-affected androgenic hormone profiles in women with hypoandrogenic LFOR might also be of interest. Given that low levels of testosterone are, at least partially, the consequence of the abnormal conversion of testosterone to DHT, measurement of levels of 5α-androstanediol glucuronide (5α-diolG) might prove diagnostically useful. Directed treatments with steroid 5α-reductase inhibitors could then prove therapeutically useful in women with hypoandrogenic LFOR.
Genes beyond the steroidogenic pathways AR The androgen receptor (encoded by AR) is closely involved in ovarian folliculogenesis 104 and is highly expressed in granulosa cells of small growing follicles,15 peaking at preantral and early antral stages,15 when the trophic effects of androgens are maximal and synergistic with follicle-stimulating hormone.104 Expression of the androgen receptor declines as follicular maturation approaches the preovulatory stage.15,25,104–106 The most extensively studied variation in AR in humans is the polymorphic CAG trinucleotide repeat in exon 1. This repeat varies by ethnicity and encodes an uninterrupted polyglutamine tract in the N‑terminal transacti vation domain,136 which has been shown to be involved in the interaction between the androgen receptor and tissue-specific coactivators.137 CAG repeat—infertility and serum testosterone An inverse association exists between the CAG repeat number and transactivation capacity of the androgen receptor, with 19 CAG repeats.149 Combined, these observations suggest that the length of the CAG repeat tract affects androgen receptor-dependent transcriptional activity at the promoters of genes (such as LHR1), which are involved in the metabolism of DHEA to testosterone, thereby affecting the relative ratios of DHEA conversion products.
SHBG Approximately 10–15% of intracellular androgens diffuse from intracellular compartments to the extracellular space and general circulation, where they are bound to the glycoprotein sex hormone-binding globulin (SHBG) and, to a lesser extent, to serum albumin. SHBG binds androgens with high affinity and estrogens with low affinity, and regulates access of both hormones to target tissues.150 Hormone fractions not bound by SHBG are considered bioavailable and can bind to and activate androgen receptors or estrogen receptors in target cells.151,152 Revisiting bioavailable androgens Even though synthesized primarily in the liver, SHBG expression has been detected in various tissues of the female reproductive tract, which suggests a role for this protein in regulating the intracellular availability of sex steroids in reproductive organs.150–155 Some reproductive tissues, indeed, express high-affinity SHBG receptors on cell membranes that interact with steroid-bound SHBG. Steroid–SHBG complexes might undergo endocytic internalization with subsequent release of steroids. Alternatively, these complexes might transduce signals from the cell surface that, via G‑proteins, trig ger transcriptional activity of classic intracellular steroid hormone receptors.156.157 In women with infertility, it might, therefore, be benefi cial to consider both free testosterone and total testosterone as markers of peripheral androgen status, as the contribution of the SHBG-bound androgen fraction to the bioactive hormonal milieu might be considerable. By studying conversion rates of DHEA to testosterone, the relevance of both testosterone measurements for assessing pregnancy potential in women with oPOI (POA) was demonstrated,59 with measurements of total testosterone being slightly more reliable in a predictive model than measurements of free testosterone.7
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REVIEWS Box 2 | Potential future studies on DHEA supplementation ■■ Investigation of the prevalence of Ala63Pro and Lys227Glu substitutions in dehydroepiandrosterone sulphotransferase in African American women, correlation of these mutations with androgen status, and comparison of African American women with white and Asian controls; increased prevalence of these mutations might at least partially explain the widely reported reduced IVF pregnancy rates in African American women ■■ Investigation of the TT allele of SNP rs936306 in infertile African American women, as this allele is associated with hypoandrogenaemia (low testosterone levels due to aromatase hyperactivity), which is potentially treatable with aromatase inhibitors; increased prevalence of this allele might be a contributing factor in the lower IVF pregnancy rates in African American women than in white women ■■ Determination of whether African women would benefit more from supplementation with testosterone than from DHEA, as these women have at least three distinct polymorphisms that are associated with decreased conversion of DHEA to testosterone ■■ Investigation of the Val89Leu substitution in steroid 5α-reductase 2 in women with DOR and low testosterone levels; similar substitutions have been shown to be associated with PCOS, which is considered to be the extreme opposite of DOR ■■ Investigation of levels of 5α-androstanediol glucuronide in infertile women with low testosterone levels to identify abnormal conversion of testosterone to dihydrotestosterone; if confirmed, inhibition of steroid 5α-reductase might improve abnormally low testosterone levels ■■ Investigation of levels of inflammatory markers in women with hypoandrogenaemia or hyperandrogenaemia, oPOI (POA), and PCOS, respectively, to clarify the relationship between endocrine and immune systems and support the hypothesis of an immune system component controlling adrenal androgen production ■■ Investigation of COX inhibitors (which suppress CYP19A1 expression) as a potential infertility treatment for women with a high estradiol:testosterone ratio ■■ Investigation of whether CAG repeats in AR in infertile women supplemented with DHEA affect the conversion of DHEA to testosterone and how these repeats relate to IVF cycle outcomes ■■ Investigation of the effects of DHEA or testosterone supplementation on levels of SHBG in infertile women with low testosterone levels Abbreviations: COX, cyclooxygenase; DHEA, dehydroepiandrosterone; DOR, diminished ovarian reserve; IVF, in vitro fertilization; oPOI; occult primary ovarian insufficiency; PCOS, polycystic ovary syndrome; POA, premature ovarian ageing; SHBG, sex hormone-binding globulin; SNP, single nucleotide polymorphism.
Regulated expression Serum levels of SHBG vary significantly among individuals and are affected by endocrine conditions and lifestyle.158–160 For instance: SHBG is induced by estrogen but suppressed by androgens, insulin and prolactin;161–164 BMI is inversely related to circulating levels of SHBG;165 hypothyroidism in elderly women is strongly associated with decreased levels of SHBG, whereas, in young women hyperthyroidism significantly correlates with increased SHBG levels;166,167 and treatment with antithyroid drugs gradually decreases SHBG levels in association with the hyperthyroidism.132 Hypoandrogenic infertility Among the various polymorphisms that affect levels of SHBG and androgens, the pentanucleotide TAAAA repeat polymorphism has been vigorously investigated.168–170 This repeat is located in the promoter region of SHBG and is uniquely distributed across populations.148 Extended TAAAA repeat polymorphisms have mostly been studied in the context of hyperandrogenism and PCOS;170–172 polymorphisms leading to hypoandrogenism have not, so far, been described.
Levels of SHBG are significantly higher in women with familial POI (POF) than in those with sporadic POI (POF), which suggests a hereditary component to SHBG levels.173 A multiethnic study of premenopausal and perimenopausal women demonstrated that African American women had higher levels of SHBG and a lower free androgen index than white women.174 By contrast, Chinese women had lower levels of SHBG and a higher free androgen index than white women.174 Studies of SHBG variants in hypoandrogenic women with infer tility, therefore, seem warranted. Such studies might be of particular interest in relation to studies of the fragile X mental retardation protein I gene (FMR1), in which a strikingly similar association exists, with so-called ‘low’ alleles (34 CGG repeats) in Chinese women.181,182 Given that DHEA supplementation significantly reduces levels of SHBG in infertile women with LFOR25 and in those with adrenal insufficiency,175 the efficacy of DHEA supplementation in lowering SHBG levels in women with POI (POF) warrants further investigation.
FMR1 The fragile X mental retardation protein 1 (FMRP, which is encoded by FMR1) is associated with cognitive development and female reproductive function.176,177 Classical expansion of the CGG repeat (to 55–199 copies) in the 5'-untranslated region of FMR1 leads to partial promoter heterochromatinization (premutation), whereas >200 copies of the CCG repeat (full mutation) leads to complete promoter heterochromatinization.176,177 Silencing of transcription, which is associated with >200 copies of the CCG repeat, results in fragile X syndrome, which is characterized by mental retardation, autism, developmental delays and other neuropsychiatric deficits.178,179 In women, the premutation range is associated with a significantly increased risk of POI (POF).178,179 CGG repeat polymorphisms and androgen levels New FMR1 haplotypes, distinct from the classification of normal, premutation and full mutation genotypes have been described, further subdividing what has, so far, been considered the normal CGG repeat range for this gene.180,181 This advance has enabled new definitions of FMR1 repeat classes, which are associated with specific ovarian phenotypes. In this classification, ~25% of infertile women carry a low allele, defined as
