Sage Journals: Discover world-class research

Abstract

Polycystic ovary syndrome (PCOS) has been traditionally perceived as a reproductive disorder due to its most common presentation with menstrual dysfunction and infertility. However, it is now clear that women with PCOS are at increased risk of metabolic dysfunction, from impaired glucose tolerance and type 2 diabetes mellitus to nonalcoholic fatty liver disease and cardiovascular disease. PCOS is characterised by androgen excess, with cross-sectional data showing that hyperandrogenism is directly complicit in the development of metabolic complications. Recent studies have also shown that C11-oxy _C19 androgens are emerging to be clinically and biochemically significant in PCOS, thus emphasising the importance of understanding the impact of both classic and C11-oxy _C19 androgens on women’s health. Here we discuss androgen metabolism in the context of PCOS, and dissect the role played by androgens in the development of metabolic disease through their effects on metabolic target tissues in women.

Keywords

adipose tissue androgens C11-oxy C19 androgens diabetes metabolic disease obesity PCOS

Introduction

Polycystic ovary syndrome (PCOS) is one of the most common endocrine conditions, affecting approximately 10% of all women across the globe.¹ Women with PCOS commonly present with menstrual irregularities, infertility and signs and symptoms of androgen excess. PCOS has been traditionally perceived as a predominantly reproductive disorder, with minimal focus on long-term metabolic complications. However, recent studies have shown that PCOS is associated with significant health consequences in affected women,² representing a lifelong metabolic disorder (Figure 1).

Figure 1.

Iceberg phenomenon: PCOS has been traditionally perceived as a primarily reproductive disorder. On a superficial level, clinical consequences of this condition include subfertility, irregular menses and signs and symptoms of androgen excess. However, clinicians should be aware of the significantly increased risk of metabolic complications across the female lifespan in women with PCOS, underpinned by androgen excess and insulin resistance.

Although controversies persist with regard to diagnostic criteria for PCOS, androgen excess remains a fundamental biological and diagnostic feature of the disorder.^3,4 This is recognised in the 2003 Rotterdam Consensus criteria, with androgen excess featuring as a defining criterion alongside irregular periods and polycystic appearances of the ovaries at ultrasound. Following this, the Androgen Excess Society recommended hyperandrogenism as a mandatory criterion for the diagnosis of PCOS, further highlighting PCOS as a disorder of androgen excess.^4–6 In cross-sectional clinical phenotyping as well as population studies, androgen excess is a major driver of metabolic risk in PCOS, with the presence and severity of androgen excess closely correlating with surrogate parameters of metabolic risk, including insulin resistance.⁷ Women with PCOS have been shown to be at increased risk of metabolic disease including type 2 diabetes mellitus (T2DM), nonalcoholic fatty liver disease (NAFLD), and cardiovascular disease (CVD).⁸

Most studies on PCOS have focused on classic C₁₉ androgens, their precursors and/or their downstream urinary metabolites for biochemical assessment of androgen excess. However, the newly described, adrenal-derived C11-oxy C₁₉ androgen subclass is emerging to be clinically and biochemically significant in the context of PCOS-related androgen excess.⁹ In our recent study, we found that C11-oxy C₁₉ androgens are the majority of circulating serum androgens in women with PCOS¹⁰, thus emphasising the importance of improving our understanding of the impact of both classic and C11-oxy C₁₉ androgens on women’s health. Furthermore, circulating levels of the active C11-oxy C₁₉ androgen 11-ketotestosterone (11KT) have recently been shown to remain consistent throughout the female lifespan,¹¹ contrary to classic androgen concentrations, which decrease post-menopause. However, recent study by Swart et al.¹² analysing steroid hormones of adolescents and young women with PCOS and adrenocortical dysfunction using UPC2-MS/MS found C19 steroids [A4, testosterone and dehydroepiandrosterone (DHEA)] concentrations higher than the combined C11-oxy C₁₉ androgens. The combined C19 steroid levels were higher in PCOS group with the combined C11-oxy androgens being similar in patients with PCOS and healthy people. Therefore, more studies on C11-oxy C₁₉ and their downstream metabolites will be needed before a conclusion can be drawn about the roles of C11-oxy _C19 androgens compared with the classic androgens.

Conditions characterised by androgen excess are PCOS, premature adrenarche, congenital adrenal hyperplasia, ovarian hyperthecosis, androgen secreting tumours and, to a degree, also Cushing’s syndrome.^13,14 Many of these conditions lack the necessary prevalence and frequency of life events and metabolic dysfunction to understand the overall impact of androgen excess.¹⁵ PCOS is a lifelong metabolic condition likely to affect female health from childhood through to post-menopause. Recent work by Risal et al. showed that daughters of mothers with PCOS have a fivefold increased risk for PCOS.¹⁶ More importantly, they showed that prenatal androgen exposure and not obesity lead to transgenerational reproductive and metabolic dysfunction in rodent models.¹⁷ Another study by Gunning et al. showed subtle cardiometabolic dysfunction in early childhood in otherwise healthy weight children of women with PCOS.^18,19 Based on its high prevalence in the general background population, and the correlation between PCOS and other features of metabolic syndrome, PCOS is a good model for studying the impact of androgen excess on metabolic risk.The origins of androgens and their mechanistic role inducing metabolic dysfunction in PCOS are discussed in detail in the following.

Review methodology

We included both in vitro and in vivo human and animal studies published in English up until March 2020 providing evidence on PCOS and metabolic dysfunctions. Articles were searched from PubMed using terms “(PCOS) OR (PCOS MeSH terms)” AND (Androgens) OR “(Androgens MeSH terms)” AND terms on metabolic dysfunctions as mentioned previously such as “Obesity”, “Diabetes” and with their associated MeSH terms. We also added specific organs such as “Adrenal”, “Ovary”, “Muscle”, “Pancreas”, “Liver” in separate searches to review their impact on PCOS and metabolic dysfunctions. Additional articles were also obtained from previously published reviews for respective subjects. Articles were then screened based on titles and abstracts and full texts were subsequently obtained.

Origins of androgen excess in PCOS and pre-receptor androgen metabolism

Androgens are responsible for the development of male characteristics such as male pattern body hair, muscle bulk, and deep voice; androgens also impact on sexual function and reproductive capacity in both sexes. In women, androgens also play an important role in general wellbeing, libido, energy levels, muscle mass and strength and bone mass.^20–24 Apart from their role in reproduction and sexual health, androgens also play an important role in human metabolic health in both men and women.²⁵ The origins of androgen excess in PCOS have not been comprehensively delineated, but adrenals, ovaries and adipose tissue collectively contribute to the bulk of circulating androgen burden in the disorder.⁸ Androgens are synthesized in the ovaries and adrenal glands catalysed by a series of steroidogenic enzymes, especially the rate limiting cytochrome P450 (CYP) 17A1 enzyme, responsible for the synthesis of the classic androgen pathway precursor DHEA. DHEA and its downstream product androstenedione are released into circulation, with androgenic signals further amplified after uptake into target tissues, where androgen precursor steroids are converted into the active androgens testosterone and 5α-dihydrotestosterone (DHT), the most potent natural androgen.²⁶ Several studies have reported a systemic upregulation of 5α-reductase activity as a significant feature in women with PCOS,^27–31 with consequently increased activation of testosterone to 5α-DHT. More recently, another pathway of DHT synthesis, referred to as ‘backdoor pathway’ has been discussed in the context of PCOS and congenital adrenal hyperplasia.^32–34 This pathway involves the synthesis of DHT bypassing testosterone in the classic pathway. There are also speculations of androgens transferring via mother’s milk into children as an early source of androgens in neonatal and infant life. However, currently there is a lack of scientific evidence to support this theory. As PCOS is a heterogeneous condition associated with features of the metabolic syndrome, in vivo studies involving women with PCOS are often confounded by coexisting obesity and insulin resistance, which are also contributors to hyperandrogenism in women with and without PCOS.³⁵

Ovaries and androgen excess

The origin of androgen excess from the ovaries is complex and mainly attributed to tonic hypersecretion of Luteinizing Hormone (LH), likely due to a dysregulated hypothalamic–pituitary–gonadal axis.³⁶ A theory of insulin-potentiated accelerated gonadotropin-releasing hormone (GnRH) pulse frequency, resulting in increased LH pulse is supported by the works of Roland and Moenter.^37,38 Insulin has also been shown to stimulate androgen productions in the ovarian theca cells of women with PCOS via insulin receptor rather than insulin growth factor 1 (IGF-1) receptor.^39,40 However, the IGF-1 receptor may also play a role in conditions with extreme levels of insulin resistance such as heridatery insulin receptor mutations or lipodystrophy contributing to their hyperandrogenism and PCOS.^41,42 High levels of IGF-1 in acromegaly have also been previously associated with PCOS⁴³ with the observation of improvement in IGF-1 and PCOS phenotype following reduction in growth hormone levels. This was followed by the normalisation of menstrual cycle and numbers of polycystic ovary morphoplogy suggesting that IGF-1 could play a role in hyperandrogenism and PCOS.⁴⁴ Low concentrations of progesterone have been proposed to increase LH secretion by disrupting the negative feedback of GnRH.⁴⁵ However, the presence of increased LH and androgen excess in prepubertal girls fails to support low progesterone as a sole source of LH increase in PCOS.⁴⁶ The increase in ovarian androgens in PCOS is not only LH- and insulin-dependent as some in vitro studies have shown that it could be due to an abnormality in primary theca cell steroidogenesis.^47,48 Subsequent clinical studies in women with PCOS have shown that ovarian androgen production is hyperresponsive to both administration of GnRH agonists and Human Chorionic Gonadatropin (hCG) challenges.^48–51 The role of upregulation of backdoor androgen synthesis pathway contributing to androgen excess in women with PCOS has been discussed by Marti et al.⁵² In their study, they have shown that the ovaries express all the enzymes required for DHT synthesis through the backdoor pathway. Comparing ovarian tissues of women with PCOS to women without PCOS, immunohistochemistry revealed higher reactivity for 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2), retinol dehydrogenase (RoDH), 5α-reductase type 1 (SRD5A1) and aldo-keto reductase family 1 member C2 (AKR1C2) genes indicating a possible higher activities of their respective enzymes involved in the backdoor pathway. SRD5A1, the gatekeeper of the backdoor pathways converting A4 into androstanedione, showed the highest difference in PCOS compared with non-PCOS highlighting the relevance of this pathway in women with PCOS. Derangement in the Kiss-1 system resulting in increased LH release has also been postulated; however, evidence supporting this theory is lacking to date.³⁶ Recently, the role of Gamma Aminobutyric Acid (GABA) activity in the arcuate nucleus stimulating LH secretion resulting in PCOS-like reproductive dysfunction has been explored in mice by Silva et al.⁵³

Adrenal glands and androgen excess

Nearly 50% of women with PCOS have a predominantly adrenal hyperandrogenism phenotype as shown by elevated concentrations of Dehydroepiandrosterone Sulphate (DHEAS) and 11β-hydroxyandrostenedione, two androgens that are exclusively produced by the adrenal glands.^54–56 This is particularly prominent in young women with PCOS.¹³ This may be due to upregulation of CYP17A1 activity through tonic hyperstimulation by Adrenocorticotropic hormone (ACTH) of the adrenal gland. Lack of a significant increase in 17-hydroxyprogesterone (17OHP) after exogenous stimulation with ACTH supports this theory.^54,57 One of the other proposed mechanisms of adrenal hyperandrogenism is a relative deficiency of HSD3B2. In women with PCOS and high DHEAS, a normal DHEA response and slightly elevated 17OHP response to ACTH suggested that elevated DHEAS was due to tonic hyperstimulation of the adrenal gland rather than deficiency of the enzymes.⁵⁴ Downregulation of 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) activity will result in a decreased rate of cortisol activation and hence could lead to adrenal androgen stimulation via hypothalamic–pituitary–adrenal axis feedback; however, no convincing evidence for decreased HSD11B1 activity was found in two independent studies in women with PCOS.^58,59 The relevance of the adrenal backdoor pathway in women with PCOS was also discussed by Saito et al. showing correlations between the levels of DHEAS to androstanedione and androsterone indicating a potential adrenal gland contribution in these two androgens involved in this pathway.⁶⁰

Importantly, the C11-oxy C₁₉ androgens are primarily of adrenal origin as this pathway starts with the conversion of A4 to 11-hydroxyandrostenedione (11OHA4), which can only be catalysed by 11β-hydroxylase activity of the exclusively adrenally expressed enzyme CYP11B1.^55,61 As C11-oxy C₁₉ androgens are particularly prominent in the serum women with PCOS¹⁰ as well as in girls with premature adrenarche,⁶² it is safe to assume that the adrenal glands provide a major contribution to circulating androgen excess in PCOS (Figure 2).

Figure 2.

Adrenal, ovarian and peripheral androgen metabolism in PCOS. Androgenic precursors are secreted predominantly by the adrenal glands, and activated to potent androgens in the ovaries and peripheral tissues. Expression of key androgen-activating enzymes in peripheral tissues such as adipose tissue highlight an important role for extra-adrenal and -ovarian androgen generation.

Peripheral tissues and androgen excess

After the secretion by the ovaries and/or adrenals into the circulation, androgenic precursors are further activated in peripheral target tissues into more potent androgens that bind to the cytosolic androgen receptor (AR), which subsequently translocates to the nucleus where it functions as a transcription factor after binding to DNA, resulting in increased transcription of genes encoding for factors important for androgenic biologic activity. The concentration of androgens in peripheral target tissues of sex steroid action are determined by (1) the total concentration of the androgenic steroids in the circulation (2) whether they are bound to sex hormones binding globulin (SHBG) or albumin (3) important regulators of bioavailability, and the availability of mechanisms for cellular influx and efflux. Circulating androgens must cross the plasma membrane of the target cells to be metabolized by intracellular enzymes and/or to activate AR. While unconjugated steroids can freely diffuse across the plasma membrane, conjugated steroids are hydrophilic and hence need to be actively transported inside the cell. Once in the cell, these steroids will need to be deconjugated before they can be metabolized and/or interact with the androgen receptors.^25,63

Combining human-based in vivo and ex vivo study approaches, our group has shown that the androgen-activating enzyme aldoketoreductase type 1 C3 (AKR1C3), which converts androstenedione to testosterone as well as 11OHA4 to 11KT, is a major driver of adipocyte-specific androgen excess and that its activity in adipose tissue is a major source of PCOS-related androgen excess.⁶⁴ This will be discussed in detail in the adipose tissue section in the following.

MicroRNA and androgen excess

MicroRNAs (miRNAs) are present widely in the body playing a role in the regulation of gene expression by inhibiting post-transcriptional or post-translational expression of messenger RNA (mRNA).⁶⁵ Alteration in levels of miRNA have been implicated in conditions such as cervical cancer, ovarian cancer, endometriosis and CVDs.^66–68 Studies have shown differential expression of miRNA in women with PCOS compared with controls, suggesting a potential role of miRNA in PCOS.^69,70 Chen et al. have extensively discussed the role of miRNA in PCOS.⁷¹ Muri et al. have previously shown that levels of miR-21, miR-103 and miR-155 were positively associated with levels of free testosterone in women with PCOS⁷² and several miRNAs have been shown to be negatively correlated with levels of testosterone and A4 in these women.^73,74 Moreover, comparing miR-130b-3p in normal and theca cells of women with PCOS have shown a decreased expression in PCOS which was correlated with increased expression of CYP17A1 and DHEA synthesis, which may contribute to the androgen excess in PCOS.⁷⁵

In isolated porcine ovaries, overexpression of miR-378 was shown to inhibit the expression of aromatase enzymes in the ovaries, which contributed to hyperandrogenaemia.⁷⁶ Furthermore, miRNA181a also downregulates oestrogen synthesis via CYP19A1 expression in mouse granulosa cells.⁷⁷ However, there is no evidence of miRNA overexpression in women with PCOS, hence its role in increasing levels of androgens in this population. miR-193a-5p and miR-199a-3p have been positively associated with oestrogen and SHBG and negatively associated with free testosterone in women with PCOS with predictions of target genes, indicating the role of these miRNA in regulating some enzymes in the steroidogenesis pathways.⁷⁸ These evidences taken together suggested that more detailed studies will be needed to investigate the role of miRNA in contributing to androgen excess in women with PCOS. The role of miRNA in propagating metabolic diseases will be discussed in the relevant section in the following.

Androgens in metabolic target tissues

Critical metabolic target organs such as adipose tissue, muscle and pancreatic beta cells are also targets of androgen action (Figure 3). Here, we summarise the potential mechanisms and metabolic dysregulation due to androgen action in each of these organs.

Figure 3.

Impact of androgen excess on metabolic target tissues- (A) Increased fat accumulation in the hepatocytes in response to androgen excess, eventually resulting in NAFLD, (B) Androgen excess is proportionally related to beta-cell dysfunction, (C) Ectopic lipid accumulation in skeletal muscle with androgen excess influences glucose-regulating pathways resulting in insulin resistance, (D) under the influence of androgens, women with PCOS have been shown to have more central obesity phenotypes.

Adipose tissue, obesity, fat mass distribution and androgen excess

Adipose tissue forms the largest store of conserved energy, in the form of triglycerides, in the human body. The process of adipocyte differentiation from stem cells is complex and involves a variety of signals for gene regulation, histone modification, and protein modification by ubiquitin.⁷⁹ There are two distinct types of adipose tissue, brown adipose tissue and white adipose tissue. Several studies have established the role of adipose tissue beyond an energy storehouse to regulating several critical physiological processes for example, through adipokine signals such as leptin and adiponectin. The various roles of adipokines is discussed in detail by Ouchi et al. and Dimitriadis et al.^80,81

Women with PCOS have been shown to have more central obesity phenotypes compared with weight and body mass index (BMI)-matched controls,^82–85 which is associated with various metabolic conditions in PCOS.^86–88 However, these data have been inconsistent.^89,90 Some studies have also shown that although women with PCOS have android fat mass distribution, these might not be accompanied by increased visceral fat.^91,92 On the other hand, gluteo-femoral fat or gynecoid adiposity, low in PCOS, has been shown to be independently associated with protective lipid and glucose profile along with decreased cardiovascular and metabolic risk.⁹³ Visceral adiposity index (VAI) has similar utility to the gold-standard computed tomography scan in the evaluation of visceral adiposity.⁹⁴ VAI is a sex-specific empirical mathematical model based on BMI, waist circumference, triglycerides and high-density cholesterol in healthy normal and overweight populations.⁹⁵ Lim et al. reported a similar VAI in women with PCOS and healthy controls in few studies. However, subgroup analysis comparing three groups (PCOS and obesity, obesity alone, and PCOS alone) revealed that women with PCOS and obesity have higher VAI compared with the other two groups.⁹⁶ In a study on women with PCOS by Amato et al., VAI was only associated with compensatory hyperinsulinemia and there was no independent association between VAI and insulin sensitivity index (by HOMA-IR and ISI Matsuda), androgen profiles and PCOS.⁹⁷ Visceral adiposity has also been previously, but not consistently, associated with elevated FAI⁸² and total testerosterone.⁸⁵ However, the number of studies available is too low for a systematic review to examine whether there is an association between PCOS, central obesity and androgens.

Adipose tissue is a key target of androgen action. Androgens are known to inhibit adipocyte differentiation in both animal and human cell lines.^98–101 The effects of androgen on adipose tissue can be noted from birth. Roland et al. noted higher fasting glucose and impaired glucose tolerance in prenatally androgenised female mice which closely mimics PCOS model.³⁷ Prior to this, Nilsson et al. showed that exposure to testosterone in the neonatal period resulted in insulin resistance, change in adipose tissue distribution and lean mass without significant changes in circulating androgen concentrations.¹⁰² Further, their groups showed this early exposure to androgen resulted in long-lasting effects on insulin sensitivity, adipose tissue and lipid profile.¹⁰³ We have previously published a detailed review of the abnormalities in adipose tissue function, fat distribution and lipid metabolism in the context of androgen excess.¹⁰⁴

We have demonstrated that expression of the androgen-activating enzyme, AKR1C3, which converts the weak androgen precursor androstenedione into potent testosterone, is increased in subcutaneous (SC) adipose tissue in women with PCOS. Locally generated androgens enhance de novo lipogenesis within SC female adipocytes, potentially predisposing to fatty acid overspill into the systemic circulation, with consequent lipotoxicity driving an adverse metabolic phenotype.⁶⁴ We demonstrated that associated insulin resistance and hyperinsulinemia then drive AKR1C3 expression and activity within the adipocyte, raising the possibility of a vicious circle of intra-adipose androgen activation, lipid accumulation and hyperinsulinaemia.

AKR1C3 also converts the C11-oxy C₁₉ androgen precursor 11-ketoandrostenedione (11KA4) into 11-ketotestosterone (11KT), which binds and activates the AR with similar potency to testerosterone.¹⁰⁵ Recent data from Storbeck’s group also suggest that 11KA4 is the preferred substrate for AKR1C3, which more efficiently converts it to active 11KT than its counterpart reaction in the classic pathway with K_M values of 0.11 μM and 0.06 μM for 11KA4 and A4 respectively.¹⁰⁶ Our findings of increased AKR1C3 expression in SAT of women with PCOS were corroborated by a recent study describing similar results.¹⁰⁷ Amer et al. have shown an upregulated AKR1C3 and CYP17A1 mRNA expression in SAT of women with PCOS as well as a higher levels of T. But, it is important to note that the authors did not assess if the higher levels of testerosterone in the adipose tissue was a result of increased AKR1C3 or CYP17A1 expression. This was previously shown by our group showing a reduction of testerosterone levels following inhibition of AKR1C3 by 3-4 trifluoromethyl-phenylamino-benzoic acid in healthy female adipose tissue. Studies on women with PCOS will be needed to confirm if the increased levels of testerosterone in SAT are in fact related to overexpression of AKR1C3 per se or related to both CYP17A1 and AKR1C3 activities. These findings define a novel intra-adipose mechanism that could contribute to androgen excess and metabolic dysfunction in PCOS.

These data strongly implicate adipose tissue as a major peripheral site of activation and metabolism of C11-oxy C₁₉ androgens in PCOS and suggest that locally generated C11-oxy C₁₉ androgens play a significant role in adipose tissue lipotoxicity. To date, however, this hypothesis has not been tested in adipose tissue of human participants using in vivo physiology approaches.

The association between adiposity, androgen excess and PCOS has been further complicated by hyperinsulinaemia and insulin resistance, which exacerbate the metabolic, reproductive and steroidogenic abnormalities observed in the disorder.⁸ A systematic review by Lim et al.⁹⁶ found women with obesity and PCOS have a higher total and free testerosterone and lower SHBG compared with those with normal BMI. Furthermore, women with both PCOS and obesity also have a more severe clinical evidence of hyperandrogenism (such as hirsutism, menstrual abnormalities and anovulation) compared with women with PCOS and normal BMI. This effect tends to be more pronounced in the abdominal/visceral obesity phenotypes^96,108–110 and weight loss in women with PCOS has been shown to improve clinical, metabolic and reproductive outcomes.^110–112

Insulin resistance and compensatory hyperinsulinemia in obesity inhibits hepatic production of SHBG, with a consequent increased bioavailability of androgens. Further, insulin modulates hypothalamic-gonadal and steroid enzymatic machinery at various levels as described previously.

Pancreas, type 2 diabetes and androgen excess

Studies have shown that there is a clear sex difference in the way the foetal pancreas reacts to androgen excess. Insulin secreted from the pancreatic beta cells is critical for glucose utilisation in the peripheral tissues. Defects in insulin secretion or action predispose to insulin resistance and T2DM. Beta cells expand rapidly during late gestation and any alteration to its expansion during this period results in long-lasting deleterious effects. Navarro et al. demonstrated potentiating effects of DHT on male mice beta cell function via the glucagon-like peptide-1 (GLP-1) receptor.¹¹³ Further, they showed male mice lacking AR had decreased glucose-stimulated insulin secretion, leading to glucose intolerance. Castration of male rats resulted in approximately 30% reduction in beta cell mass.¹¹⁴ Xu et al. studied the transcriptome of AR-deficient islet beta cells of male mice and found altered expression of genes involved in inflammation and insulin secretion.¹¹⁵

Studies in women with PCOS have also shown that androgen excess is proportionally related to beta cell dysfunction. This has been discussed in detailed by Mauvaus-Jarvis in a review of sex steroid effects on pancreatic beta cell function.¹¹⁶ This hypothesis was further tested in female mice by exposing them to chronic androgen excess, which resulted hyperinsulinemia and insulin resistance. However, this was not observed in female βARKO mice lacking AR expression in pancreatic beta cells.¹¹⁷ Testosterone prevents beta cell apoptosis and increases insulin mRNA levels both in vitro and in vivo in a series of studies conducted by Morimoto et al.^118,119

Harada et al. demonstrated an in vivo effect of androgens on beta cell mass and expansion in a sex-specific manner. They found reduced beta cell mass and proliferation in male rats following administration of flutamide to pregnant dams. Although beta cell mass was restored after feeding the mice a high fat diet, they had persisting glucose intolerance suggesting decreased insulin secretion.¹²⁰

Androgen exposure during intrauterine life results in several phenotypic characteristics of PCOS in nonhuman primates and sheep. When rats were exposed to testosterone during late gestation, their female offspring exhibited significant weight gain, increased adipose tissue and other traits of metabolic dysfunction.¹²¹ Exposing rhesus monkey dams to testosterone pre-conception resulted in hyperinsulinaemia and a reduction in glucose clearance following accelerated weight gain during testosterone treatment. This transient hyperglycaemic and relative hyperinsulinaemic episodes were sufficient to induce differential programming of insulin action and secretion in their female offspring.¹²² Intrauterine exposure to testosterone resulted in increased beta cell numbers in prenatally androgenised female foetuses with no effect on alpha cells.¹²³ The same group reported upregulation of genes involved in beta cell development and function.¹²⁴ Neither studies found any such changes in male offspring, even when they were exposed to androgens before the male programming window. Roland et al. reported fasting hyperglycaemia and impaired glucose tolerance independently of age and changes in body composition or peripheral insulin sensitivity in prenatally androgenised female mice. Once again, no such changes were noted in male counterparts.³⁷

Liu et al.¹²⁵ demonstrated improved differentiation of pluripotent stem cells into insulin-producing cells when testosterone was added to routine differentiation medium, resulting in an upregulation of NGN3, NEUROD1 and INS genes. Zhang et al. investigated the association between free-androgen index, a marker of androgen excess, with glucose tolerance in PCOS. They found a positive correlation suggestive of beta cell dysfunction in women with PCOS.¹²⁶

Glucose-stimulated insulin secretion significantly decreases in islets treated with DHT in mice studies. This is associated with significant reductions in expression of several key genes involved in islet cell mitochondrial biogenesis and mitochondrial ATP production.¹²⁷ Chronic exposure to androgens resulted in increased oxidative stress in islet cells resulting in secondary beta cell failure in female mice fed on a western diet.^117,128 The study also reported direct androgen receptor-dependant impairment of beta cell function by inducing mitochondrial dysfunction in vitro.¹²⁸ This is important in connection to skeletal muscle insulin resistance and failing insulin secretion would predispose women with PCOS to develop T2DM.

There are limitations that prevent direct translation findings from in vitro and animal studies into human physiology. Some of these limitations may be due to variability in animals for study, methods of randomization, choice of comparison therapy, blinding investigators to interventions and analysis, and variable duration of follow up.^129,130 Nevertheless, these studies provide plausible pathophysiology and mechanisms linking pancreas and androgen excess, which have been explored in a few human studies described in the following.

PCOS is strongly linked with insulin resistance and T2DM in population and other large-scale studies. A meta-analysis of 35 studies of women with PCOS by Moran et al. reported increased odds for insulin resistance (OR 2.54, CI 1.44–4.47) and T2DM (OR 4.00, CI 1.97–8.10) compared with BMI-matched studies.¹³¹ Legro et al. conducted one of the earliest prospective studies to assess the prevalence of T2DM in PCOS. They found the prevalence of T2DM in women with PCOS to be 7.5%; it was 1.5% in lean women with PCOS, further supporting the synergistic harmful effect of obesity on insulin sensitivity in PCOS. Interestingly, 31.1% of their cohort had impaired glucose tolerance speculating whether standards of diagnosing T2DM may not identify all index cases in PCOS cohort.¹³² Another study by Glintborg et al. later found no differences in the prevalence of Impaired Glucose Tolerance (IGT) and T2DM in healthy weight women with PCOS compared with healthy controls.¹³³ Boudreaux et al. reported an incidence of 13.4% and 5.8% in women with PCOS and healthy controls respectively, with a relative risk of 2.3-times after prospective follow up over 8 years. Further, obesity increased the risk by fivefold in their cohort.¹³⁴ One of the largest population-based survey of T2DM in PCOS was reported from Australia by Joham et al. They surveyed 9145 women who self-reported the diagnosis of PCOS, T2DM and gestational diabetes mellitus. The prevalence of T2DM was 5.1%. After adjusting to sociodemographic and other known risk factors, the odds for T2DM were significantly increased in PCOS [odds ratio 8.8, 95% confidence interval (CI) 3.9–20.1, p = 0.001]. BMI was independently associated with T2DM in PCOS with every BMI increment increasing the risk of T2DM by 10%.¹³⁵ A Danish national register study by Rubin et al. studying about 18,000 women with PCOS and 54,000 controls has also shown a higher event rate of T2DM in PCOS compared with well-matched controls, and diabetes was diagnosed on average 4 years earlier than in the background population.¹³⁶ The prevalence of glucose intolerance and T2DM in a cohort of 122 women with PCOS studied by Ehrmann and colleagues was 35% and 10% respectively.¹³⁷ They further followed up a subset of the glucose-intolerant women with PCOS and found them to have higher post-prandial glucose compared with their baseline, suggesting a progressively worsening insulin resistance in the absence of any intervention. The prevalence of diabetes in the Dutch population was much less (2.3%) compared with the American cohorts described previously. However, the risk was 2.3-times higher compared with the generally healthy cohorts. And this risk increased from 1.3-times in 25–34 years to 9.4-times in 45–54 years.¹³⁸ The study by Kauffman and colleagues suggests that ethnicity plays an additive effect on insulin resistance in PCOS. Mexican American women had significantly higher insulin resistance compared with white American women, thus challenging a single population-wide screening tool.¹³⁹

First reported in 1980, the association of hyperandrogenism and hyperinsulinemia is firmly established.^64,140–142 Our group have also shown in a retrospective cohort study in a UK primary care database that women with increased serum testosterone levels have an increased risk of incident T2DM.¹⁴³ This was recently highlighted further by Ruth et al. in their study of 455,097 UK Biobank samples, which showed that the risk of T2DM in women was increased by 37% for every increased standard deviation of free testosterone from baseline. A higher fasting insulin was also reported in these women.¹⁴⁴ However it is important to note that these are associations rather than causation and hence more prospective studies will be needed to investigate the impact of androgen excess on the development of T2DM.

Dunaif et al. were amongst the first to prove that women with PCOS have significant insulin resistance independent of obesity. However, obesity seemed to have a synergistic deleterious effect on glucose tolerance.¹⁴² Further, they proved in their study that insulin resistance was not due to decreased insulin clearance but of a different pathology.¹⁴⁵ Since then, several researchers have studied the exact mechanisms of insulin resistance and its consequent deleterious effect on metabolism. Hypotheses to explain intrinsic insulin resistance in PCOS include mitochondrial dysfunction and lipid accumulation affecting the insulin-signalling pathway. Hansen et al.¹⁴⁶ found that lean women with PCOS have 25% lower insulin sensitivity and 40% lower plasma adiponectin levels compared with age- and BMI-matched controls. The finding of low levels of adiponectin has been shown to predict women with PCOS who are at high risk for developing T2DM.¹⁴⁷ They also reported an increased accumulation of triacylglycerol, diacylglycerol and ceramide in skeletal muscles of PCOS women supporting the lipid accumulation theory.

Muscle

Androgen excess may play a role impacting skeletal muscles in women with PCOS by altering their insulin sensitivity. Muscle is one of the key organs responsible for disposing 70–80% of glucose load. Insulin stimulates a canonical signalling cascade composed of insulin receptor substrate (IRS)-1, phosphoinositide 3-kinase (PI3K), protein kinases PDK1, Akt and Rab. The overall cascade ends with translocation of glucose transporter (GLUT) 4 to the cell membrane which permits the intake of glucose from blood into the cell.¹⁴⁸ Several groups have tried to tease out the role of androgen excess in metabolic dysfunction using hyperglycaemic and euglycaemic-hyperinsulinaemic clamps. Most of these studies concluded that high levels of testosterone resulted in reduction in whole-body glucose uptake in healthy women.^149–152 Furthermore, they reported that these testosterone-induced insulin resistance were not attributed to hepatic insulin resistance supporting the role of muscles in androgen-mediated insulin resistance.¹⁵³ Insulin resistance is a common finding in PCOS. Stepto et al. found that 75% of lean women with PCOS and 95% of overweight women with PCOS are intrinsically insulin resistant.¹⁵⁴ Various scientists have attempted to explain insulin resistance in PCOS by one of three mechanisms: (1) dysregulation and/or dysfunction at several steps in the insulin-signalling cascade, (2) lipid accumulation and (3) mitochondrial dysfunction.^155,156

Most of the theories about a dysfunctional insulin-signalling pathway come from studies on diabetes. The earliest proposed mechanism of insulin resistance in skeletal muscle of women with PCOS was increased phosphorylation of serine residue on IRS-1 limiting the signal cascade.¹⁵⁷ This was also shown in a later study by Corbould et al.¹⁵⁸ However, these findings could not be replicated by other groups. Instead, defects distal to IRS-1/IRS-2 involving Akt substrate 160 kDa^159,160 and other pathways were found. This theory is supported by a study on the impact of exercise on hyperandrogenized mice, showing improvement in insulin sensitivity via PI3K-Akt pathway with associated reduction in 5αR1 expression in skeletal muscle of the exercise group versus stationary group.¹⁶¹ A cross-sectional study looking at impact of habitual physical activity on women with PCOS showed an association of having more than 7500 steps per day (active group) with reduction of BMI, waist circumference, lipid accumulation product, androgen levels and fasting and 120-min insulin levels. In their study, HOMA-IR and 2000 daily steps increment were also found to be an independent predictor of free-androgen index.¹⁶² A randomized trial on women with PCOS have also shown improvement in some androgen levels (total testosterone and SHBG) as well as insulin resistance shown by the decrease in HOMA-IR following 12 weeks of high-intensity interval training, further strengthening the link between androgens excess and insulin resistance in skeletal muscle.¹⁶³

Nilsson et al. found aberrant gene expression and DNA methylation in skeletal muscles of women with PCOS, mainly DYRK1A, which encodes an inhibitor of glycogen synthase kinase-3, SCP2, which is involved in lipid metabolism, SYNPO2, which encodes synaptopodin protein involved in oxidation in the muscles, KLF10, which is a transcriptional repressor regulating circadian expression of various genes involved in lipid and glucose metabolism, and NAMPT, which encodes visfatin and promotes glucose uptake into skeletal muscle.¹⁶⁴ However, many of these changes had contrasting results in the presence of androgens in vitro and hence remain inconclusive.

The other commonly proposed mechanisms explaining insulin resistance in PCOS include lipid accumulation and mitochondrial dysfunction. Lipid accumulation in skeletal muscle influencing glucose-regulating pathways is hypothesised to cause insulin resistance in diabetes.^165,166 Intramuscular lipid levels (triacylglycerol, sn-1.3 diacylglycerol and ceramide) were higher in women with PCOS compared with healthy controls in a study done by Hanssen et al. In this study, they found women with PCOS had 25% higher whole-body insulin resistance compared with healthy controls. They also found lower AMP-activated protein kinase and Thr172 phosphorylation in association with lower plasma adiponectin levels suggesting a role of the latter in insulin resistance.¹⁴⁶ Studies on the theory of mitochondrial dysfunction causing insulin resistance has resulted in contrasting evidence over the last decades.^167,168 A nontargeted metabolomics analysis of skeletal muscle of mice which was treated with DHEA revealed 32 metabolites and five metabolic pathways that are significantly different compared with controls.¹⁶⁹ Among these, the reduced NAD+/NADH ratio affecting ATP generation was a key finding, which may influence downstream activation of the insulin-signalling pathway, supporting the mitochondrial dysfunction theory. Skov et al. showed that impaired insulin-stimulated glucose disposal in women with PCOS was associated with downregulation of mitochondrial oxidative phosphorylation genes using global genetic pathway analysis.¹⁷⁰ Interestingly, Hutchinson et al. could not explain the difference in insulin resistance between women with and without PCOS with either lipid accumulation or mitochondrial dysfunction.¹⁷¹ The ability of high-intensity interval training to improve insulin sensitivity and androgen excess may also be related to increase in mitochondrial density and capacity during the training as well as increased in fat oxidation reducing lipid accumulation.^163,172

Taken together, the essence of these studies suggests there is perhaps a distinct mechanism of insulin resistance in PCOS. Future studies on the specific influence of androgens on insulin resistance in skeletal muscle may answer this question.

Liver

Several cross-sectional studies have shown an increased prevalence of NAFLD in women with PCOS; NAFLD is now on its way to become the most frequent cause of liver transplantation, driven by the global obesity pandemic, and is a forerunner of CVD.¹⁷³ A study by Petta et al. concluded that PCOS is an independent risk factor for hepatic steatosis and possibly progression to fibrosis and cirrhosis, with hyperandrogenism and insulin resistance as main determinants.¹⁷⁴ These associations have been observed in other studies.^175–177 Our group has carried out a cohort study utilising a large primary care database in the United Kingdom, including 63,000 women with PCOS and 121,000 matched controls, revealing that the risk of NAFLD was significantly increased in women with PCOS, even in women with a normal BMI (hazard ratio = 2.23, 95% CI 1.86–2.66, p < 0.001).¹⁷⁸ Androgen excess (high testosterone, low SHBG) was found to be a contributing risk factor for the development of NAFLD in PCOS in this study.¹⁷⁸ These associations have been recently supported by a meta-analysis showing a 2.3-fold increased rate of NAFLD in women with PCOS, especially those who have hyperandrogenism.¹⁷⁹

There are numerous proposed mechanisms linking this hepatic manifestation of metabolic syndrome with PCOS.^179,180 Androgen excess in PCOS suppressed low-density lipoprotein receptor RNA expression both in the adipocytes and the liver.They speculate the suppressed receptor expression might prolong plasma half-life of Very low density lipoprotein (VLDL) and low density lipoprotein (LDL), potentially leading to lipid accumulation both in the adipocytes and liver.¹⁸¹ Tumour necrosis factor (TNF)-α levels related to hyperandrogenism have also been implicated in the development of NAFLD.¹⁸² DHEA-induced hyperandrogenism in healthy women of reproductive age resulted in increased fasting AR mRNA content and TNF-α levels, which were both potentiated by glucose ingestion.¹⁸³ TNF-α is one of the proinflammatory cytokines involved in many inflammatory disorders, including metabolic syndrome and has also been shown to induce insulin resistance via promotion of IRS-1.¹⁸⁴ TNF-α is also involved in inducing enzymes involved in lipid metabolism, proinflammatory cytokines and fibrosis-associated protein in the liver, thus playing a pivotal role in development of NAFLD. A study by Shimomura et al. showed that the adipocyte-specific nuclear form of sterol regulatory element-binding protein 1c (nSREBP-1c) transgenic mice developed hepatic changes similar to the ones seen in NAFLD, with increased TNF-α.¹⁸⁵ Follow-up studies by Kakino et al. using the same mouse model showed that TNF-α is responsible for the development of NAFLD.¹⁸² In this study, TNF knockout nSREBP-1c mice showed an improved glucose tolerance and there was a significantly reduced prevalence of hepatic steatosis compared with the original nSREBP-1c model. This finding was also supported by culturing primary hepatocytes in the presence of TNF-α.¹⁸² These observations support that increased TNF-α, possibly mediated by hyperandrogenism, is an important mechanism in the development and progression of NAFLD in women with PCOS.

Our group has demonstrated androgen-mediated suppression of lipolysis and increased de novo lipogenesis in vivo and in vitro.¹⁸⁶ This would result in net positive fat accumulation beyond the adipocyte storing capacities causing fatty acid overspill, systemic lipotoxicity, insulin resistance and fat accumulation in the liver, thus leading to the development of NAFLD in women with PCOS. In the same study, serum metabolomics showed increased concentration of glycerophospholipids and lysoglycerophospholipids in women with PCOS with androgen excess, but not in controls with normal androgen concentrations, at baseline. Acute androgen exposure yielded a further increased in these metabolites whereas a decrease was observed in the BMI-matched healthy controls.⁶⁴ Both glycerophospholipids and lysoglycerophospholipids were previously observed to be increased in people with NAFLD and have been identified as potential markers of risk and progression of the condition.¹⁸⁷ These data shows that women with PCOS have a distinct metabolic response to androgens which might contribute to the development of NAFLD and other related conditions.

The role of microRNA

Interestingly, miRNA differential expression was previously associated with the increased risk in the development of T2DM.^188,189 These expressions have been investigated in women with PCOS in the context of insulin resistance (IR).⁷¹ miR-222 has been shown to be positively associated with hyperinsulinaemia in women with PCOS, suggesting its role in IR in PCOS.⁶⁹ Treating an IR adipose tissue cell line with high levels of glucose and insulin increased levels of miR-320 significantly which in turns reversed the IR via increasing the expression of GLUT4. Moreover, miR-320 have been found in the follicular fluid of women with PCOS which may present a future therapeutic target for women with PCOS and IR.¹⁹⁰ Jiang et al. added to the literature of miRNA and IR in PCOS later showing the upregulation of miR-122, miR-193b and miR-194 in women with IGT and PCOS compared with those with normal glucose tolerance. miR-33b-5p expression in ovarian tissues has also been found to play a role in propagating IR in PCOS.¹⁹¹ This miRNA has been found to be increased in rats with PCOS and IR with levels negatively correlated with the expression GLUT4, high mobility group A2 (HMGA2) and sterol regulatory element-binding protein 1 (SREBF1). The overexpression was also shown in IR adipose tissue in vivo with a similar reduced expression of GLUT4, HMGA2 and SREBF1. Furthermore, expression of GLUT4, HMGA2 and SREBF1 was increased following the inhibition of miR-33b-5p, further strengthening the role of this miRNA in promoting IR in PCOS via this pathway.

The role of miRNA, obesity and dyslipidaemia have also been discussed by Chen et al. in their review.⁷¹ Several studies have also shown differential expression of miRNA with their expressions correlating to markers of adiposity such as BMI and waist-to-hip ratio.^72,78,192 Arancio et al. have also shown association of miRNA levels with LDL cholesterol levels in women with PCOS and hyperandrogenism, adding into the evidence on androgen excess and miRNA in propogating metabolic diseases in women with PCOS.⁷³

In summary, miRNAs have been shown to be play a role in the metabolic derrangements in women with PCOS with evidences linking them to androgen excess. More studies will be needed to thoroughly investigate these associations to enable for successful therapeutic targets specifically for reducing the metabolic risk in women with PCOS.

Androgen excess and cardiovascular disease

Earlier studies investigating the association between PCOS and CVD reported no increased prevalence. Pierpoint et al. reviewed the case notes of 786 women who were diagnosed with PCOS between 1930 and 1979 and were followed up for an average 30 years. They did not find any increased deaths due to CVDs in this cohort compared with national rates.¹⁹³ Interestingly, the same group reported higher prevalence of cardiovascular risk factors and nonfatal cerebrovascular disease in this cohort.¹⁹⁴ While they concluded that PCOS may have protective effects on CVD, most of these patients did not have a hormonal profile, challenging the diagnosis and the conclusion of the study. Dahlgren et al. reported a 7.4-times higher risk for myocardial infarction in women with PCOS compared with those without, using an early risk factor model.¹⁹⁵ However, the number of participants was relatively small. Birdsall et al. reported a more extensive coronary artery disease in postmenopausal women with polycystic ovaries on ultrasound.¹⁹⁶ Although the the diagnosis of PCOS in this cohort could not be ascertained from their report, the study reported associations between the polycystic ovaries with hirsutism and high levels of free testosterone. Christian et al. found that women with PCOS had higher coronary artery calcification compared with their age-matched controls, independently of obesity.¹⁹⁷ The incidence of coronary artery disease was four-times higher in Czech women with a history of PCOS compared with the general population.¹⁹⁸ Although these women also had higher prevalence of T2DM, the remaining cardiovascular risk factors were comparable with the rest of the population, questioning whether PCOS is an independent risk factor for coronary artery disease.¹⁹⁸ Based on these findings, the Androgen Excess and PCOS Society acknowledged that women with PCOS have a moderate-to-high risk for CVD, depending on the presence of other risk factors.¹⁹⁹

A meta-analysis evaluating the effect of androgen excess in PCOS on metabolic parameters has shown that androgen excess is associated with higher levels of total cholesterol and lower high-density cholesterol levels.²⁰⁰ A study by Luque-Ramírez et al. comparing hyperandrogenic with nonhyperandrogenic PCOS showed an increased mean carotid intima-media thickness independently of BMI, with the main determinant being the concentration of serum total testosterone and A4.²⁰¹ Furthermore, testosterone has also been shown to inhibit bradykinin-induced intracellular calcium kinetics resulting in endothelial dysfunction.²⁰² Androgens are also known to impact the renal system through the upregulation of sodium channels in the proximal tubules increasing the rate of fluid reabsorption, hence increasing extracellular volume and blood pressure.^203,204 Orio et al.²⁰⁵ clinically confirmed endothelial dysfunction in brachial arteries and increased cardiac intima-media thickness directly proportional to androgen excess and independently of obesity in PCOS compared with age- and BMI-matched controls. Kravariti et al. and Luque-Ramírez et al. independently confirmed both these findings in PCOS women of differing ethnicities.^201,206 Levels of serum highly sensitive C-reactive protein (hsCRP) has been previously shown to be higher in women with PCOS, which may play a role in increased risk of CVD in this population.^207,208 Möhlig et al. later found that increased levels of hsCRP were due to obesity rather than PCOS alone²⁰⁹, which was confirmed by another study showing increased hsCRP in women with PCOS that diminishes after adjustment for BMI. The study also reported no association between levels of androgens and that of hsCRP.²¹⁰ Hyperhomocysteinaemia and oxidative stress are widely known as risk factors for the development of CVD.^211,212 Several studies have previously shown elevated levels of homocysteine and oxidative stress in women with PCOS which may contribute to their risk of developing CVD.^213–216 A study by Yilmaz et al.²¹⁷ also showed a positive correlation between levels of oxidative stress and free testosterone levels in women with PCOS, suggesting the potential role of androgen excess in increasing risk of CVD via this mechnanism. Although there is no evidence on the relationship between androgen excess and homocysteine levels, many studies using anti-androgenic oral contraceptives containing 35 μg ehtynylestroadiol and 2 mg cyproterone acetate have demonstrated rapid reductions of homocysteine levels in women with PCOS.^218–220 However, this was not the case for anti-androgen-containing drosperinone.²²¹ Recently, the role of androgens in attenuating endothelin-1-induced vasodilation and endothelin B receptor-mediated nitric oxide production resulting in endothelial dysfunction was described by Usselman et al. in women with PCOS.²²² These and other studies support that androgen excess is implicated either directly or indirectly in almost all proposed mechanisms to explain the increased risk for CVD in PCOS.

Conclusion

Androgen excess plays a pivotal role in increasing risk of metabolic dysfunction in women with PCOS. Currently, there are no disease-specific therapeutic options available to modify metabolic risk in women with PCOS, and treatments are limited to the use of insulin-sensitising agents, anti-hypertensives and lipid lowering agents. Consensus guidelines² have consistently acknowledged the need to identify novel biomarkers and therapies for metabolic risk in women with PCOS, and targeting androgen excess is likely to represent the most promising future therapeutic avenue. With the emerging role of the less-well-characterised C11-oxy C₁₉ androgen subclass, it is important to establish the origins and roles of specific androgens in metabolic pathophysiology in order to identify potential therapeutic targets. There is now a strong rationale for therapies targeting androgen synthesis or action for the amelioration of metabolic risk in women with PCOS.

Footnotes

Author contribution(s)

Punith Kempegowda: Data curation; Formal analysis; Investigation; Methodology; Project administration; Resources; Supervision; Validation; Visualization; Writing-original draft; Writing-review & editing.

Eka Melson: Data curation; Formal analysis; Investigation; Methodology; Project administration; Resources; Supervision; Validation; Visualization; Writing-original draft; Writing-review & editing.

Konstantinos Manolopoulos: Formal analysis; Methodology; Supervision; Validation; Visualization; Writing-original draft; Writing-review & editing.

Weibke Arlt: Conceptualization; Formal analysis; Methodology; Project administration; Resources; Supervision; Validation; Visualization; Writing-original draft; Writing-review & editing.

Michael O’Reilly: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Resources; Supervision; Validation; Visualization; Writing-original draft; Writing-review & editing.

Conflict of interest statement

The authors declare that there is no conflict of interest.

Funding

This review paper is supported by WA’s Wellcome Trust Investigator award grant and Emerging Clinician Scientist Award (HRB ECSA-2020-001) to MOR.

ORCID iD

Eka Melson

References

Skiba

Islam

Bell

, et al. Understanding variation in prevalence estimates of polycystic ovary syndrome: a systematic review and meta-analysis. Hum Reprod Update 2018; 24: 694–709.

Teede

Misso

Costello

, et al. Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome. Hum Reprod 2018; 33: 1602–1618.

Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril 2004; 81: 19–25.

Azziz

Carmina

Dewailly

, et al. The androgen excess and PCOS society criteria for the polycystic ovary syndrome: the complete task force report. Fertil Steril 2009; 91: 456–488.

Azziz

Carmina

Dewailly

, et al. Criteria for defining polycystic ovary syndrome as a predominantly hyperandrogenic syndrome: an androgen excess society guideline. J Clin Endocrinol Metab 2006; 91: 4237–4245.

Azziz

Carmina

Dewailly

, et al. Positions statement: criteria for defining polycystic ovary syndrome as a predominantly hyperandrogenic syndrome: an androgen excess society guideline. J Clin Endocrinol Metab 2006; 91: 4237–4245.

O’Reilly

Taylor

Crabtree

, et al. Hyperandrogenemia predicts metabolic phenotype in polycystic ovary syndrome: the utility of serum androstenedione. J Clin Endocrinol Metab 2014; 99: 1027–1036.

Bozdag

Mumusoglu

Zengin

, et al. The prevalence and phenotypic features of polycystic ovary syndrome: a systematic review and meta-analysis. Hum Reprod 2016; 31: 2841–2855.

Pretorius

Arlt

Storbeck

KH.

A new dawn for androgens: novel lessons from 11-oxygenated C19 steroids. Mol Cell Endocrinol 2017; 441: 76–85.

10.

O’Reilly

Kempegowda

Jenkinson

, et al. 11-Oxygenated C19 steroids are the predominant androgens in polycystic ovary syndrome. J Clin Endocrinol Metab 2017; 102: 840–848.

11.

Nanba

Rege

Ren

, et al. 11-Oxygenated C19 steroids do not decline with age in women. J Clin Endocrinol Metab 2019; 104: 2615–2622.

12.

Swart

du Toit

Gourgari

, et al. Steroid hormone analysis of adolescents and young women with polycystic ovarian syndrome and adrenocortical dysfunction using UPC2-MS/MS. Pediatr Res 2020: 1–9.

13.

Elhassan

Idkowiak

Smith

, et al. Causes, patterns, and severity of androgen excess in 1205 consecutively recruited women. J Clin Endocrinol Metab 2018; 103: 1214–1223.

14.

Idkowiak

Elhassan

Mannion

, et al. Causes, patterns and severity of androgen excess in 487 consecutively recruited pre- and post-pubertal children. Eur J Endocrinol 2019; 180: 213–221.

15.

Schiffer

Kempegowda

Arlt

, et al. Mechanisms in endocrinology: the sexually dimorphic role of androgens in human metabolic disease. Eur J Endocrinol 2017; 177: R125–R143.

16.

Risal

Pei

, et al. Prenatal androgen exposure and transgenerational susceptibility to polycystic ovary syndrome. Nat Med 2019; 25: 1894–1904.

17.

Yan

Dai

Wang

, et al. Prenatal androgen excess programs metabolic derangements in pubertal female rats. J Endocrinol 2013; 217: 119–129.

18.

Wilde

MAD

Eising

Gunning

, et al. Cardiovascular and metabolic health of 74 children from women previously diagnosed with polycystic ovary syndrome in comparison with a population-based reference cohort. Reprod Sci 2018; 25: 1492–1500.

19.

Gunning

Sir Petermann

Crisosto

, et al. Cardiometabolic health in offspring of women with PCOS compared to healthy controls: a systematic review and individual participant data meta-analysis. Hum Reprod Update 2020; 26: 103–117.

20.

Arlt

Callies

Van Vlijmen

, et al. Dehydroepiandrosterone replacement in women with adrenal insufficiency. N Engl J Med 1999; 341: 1013–1020.

21.

Raisz

Wiita

Artis

, et al. Comparison of the effects of estrogen alone and estrogen plus androgen on biochemical markers of bone formation and resorption in postmenopausal women. J Clin Endocrinol Metab 1996; 81: 37–43.

22.

Shifren

Braunstein

Simon

, et al. Transdermal testosterone treatment in women with impaired sexual function after oophorectomy. N Engl J Med 2000; 343: 682–688.

23.

Miller

Sesmilo

Schiller

, et al. Androgen deficiency in women with hypopituitarism. J Clin Endocrinol Metab 2001; 86: 561–567.

24.

Snyder

PJ.

Editorial: the role of androgens in women. J Clin Endocrinol Metab 2001; 86: 1006–1007.

25.

Schiffer

Arlt

Storbeck

K-H.

Intracrine androgen biosynthesis, metabolism and action revisited. Mol Cell Endocrinol 2018; 465: 4–26.

26.

Pretorius

Africander

Vlok

, et al. 11-Ketotestosterone and 11-ketodihydrotestosterone in castration resistant prostate cancer: potent androgens which can no longer be ignored. PLoS One 2016; 11: e0159867.

27.

Stewart

Edwards

CRW

Shackleton

CHL

, et al. 5 α-Reductase activity in polycystic ovary syndrome. Lancet 1990; 335: 431–433.

28.

Chin

Shackleton

Prasad

, et al. Increased 5α-reductase and normal 11β-hydroxysteroid dehydrogenase metabolism of C19 and C21 steroids in a young population with polycystic ovarian syndrome. J Pediatr Endocrinol Metab 2000; 13: 253–259.

29.

Fassnacht

Schlenz

Schneider

, et al. Beyond adrenal and ovarian androgen generation: increased peripheral 5α-reductase activity in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2003; 88: 2760–2766.

30.

Vassiliadi

Barber

Hughes

, et al. Increased 5α-reductase activity and adrenocortical drive in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2009; 94: 3558–3566.

31.

Torchen

Idkowiak

Fogel

, et al. Evidence for increased 5α-reductase activity during early childhood in daughters of women with polycystic ovary syndrome. J Clin Endocrinol Metab 2016; 101: 2069–2075.

32.

Auchus

RJ.

The backdoor pathway to dihydrotestosterone. Trends Endocrinol Metab 2004; 15: 432–438.

33.

Miller

Auchus

RJ.

The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocr Rev 2011; 32: 81–151.

34.

Kamrath

Hochberg

Hartmann

, et al. Increased activation of the alternative “backdoor” pathway in patients with 21-hydroxylase deficiency: evidence from urinary steroid hormone analysis. J Clin Endocrinol Metab 2012; 97: E367–E375.

35.

Barber

McCarthy

Wass

JAH

, et al. Obesity and polycystic ovary syndrome. Clin Endocrinol 2006; 65: 137–145.

36.

Baskind

Balen

AH.

Hypothalamic–pituitary, ovarian and adrenal contributions to polycystic ovary syndrome. Best Pract Res Clin Obstet Gynaecol 2016; 37: 80–97.

37.

Roland

Nunemaker

Keller

, et al. Prenatal androgen exposure programs metabolic dysfunction in female mice. J Endocrinol 2010; 207: 213–223.

38.

Roland

Moenter

SM.

Prenatal androgenization of female mice programs an increase in firing activity of gonadotropin-releasing hormone (GnRH) neurons that is reversed by metformin treatment in adulthood. Endocrinology 2011; 152: 618–628.

39.

Nestler

Jakubowicz

Falcon

Vargas

, et al. Insulin stimulates testosterone biosynthesis by human thecal cells from women with polycystic ovary syndrome by activating its own receptor and using inositolglycan mediators as the signal transduction system. J Clin Endocrinol Metab 1998; 83: 2001–2005.

40.

Divall

Nwaopara

, et al. Obesity-induced infertility and hyperandrogenism are corrected by deletion of the insulin receptor in the ovarian theca cell. Diabetes 2014; 63: 1270–1282.

41.

Rosenfield

Ehrmann

DA.

The pathogenesis of polycystic ovary syndrome (PCOS): the hypothesis of PCOS as functional ovarian hyperandrogenism revisited. Endocr Rev 2016; 37: 467–520.

42.

Ehrman

Barnes

Rosenfield

RL.

Polycystic ovary syndrome as a form of functional ovarian hyperandrogenism due to dysregulation of androgen secretion. Endocr Rev 1995; 16: 322–353.

43.

Kaltsas

Androulakis

Tziveriotis

, et al. Polycystic ovaries and the polycystic ovary syndrome phenotype in women with active acromegaly. Clin Endocrinol 2007; 67: 917–922.

44.

Hashimoto

Yatabe

Midorikawa

, et al. Inhibition of growth hormone excess reduces insulin resistance and ovarian dysfunction in a lean case of polycystic ovary syndrome with a growth-hormone-producing pituitary adenoma. Horm Res Paediatr 2003; 59: 149–155.

45.

Soules

Steiner

Clifton

, et al. Progesterone modulation of pulsatile luteinizing hormone secretion in normal women. J Clin Endocrinol Metab 1984; 58: 378–383.

46.

Apter

Bützow

Laughlin

, et al. Accelerated 24-hour luteinizing hormone pulsatile activity in adolescent girls with ovarian hyperandrogenism: relevance to the developmental phase of polycystic ovarian syndrome. J Clin Endocrinol Metab 1994; 79: 119–125.

47.

Gilling-Smith

Story

Rogers

, et al. Evidence for a primary abnormality of thecal cell steroidogenesis in the polycystic ovary syndrome. Clin Endocrinol 1997; 47: 93–99.

48.

Gilling-Smith

Willis

Beard

, et al. Hypersecretion of androstenedione by isolated thecal cells from polycystic ovaries. J Clin Endocrinol Metab 1994; 79: 1158–1165.

49.

Chang

Cook-Andersen

Disordered follicle development. Mol Cell Endocrinol 2013; 373: 51–60.

50.

Levrant

Barnes

Rosenfield

RL.

A pilot study of the human chorionic gonadotrophin test for ovarian hyperandrogenism. Hum Reprod 1997; 12: 1416–1420.

51.

Ibañez

Hall

Potau

, et al. Ovarian 17-hydroxyprogesterone hyperresponsiveness to gonadotropin-releasing hormone (GnRH) agonist challenge in women with polycystic ovary syndrome is not mediated by luteinizing hormone hypersecretion: evidence from GnRH agonist and human chorionic gonadotropin stimulation testing. J Clin Endocrinol Metab 1996; 81: 4103–4107.

52.

Marti

Galván

Pandey

, et al. Genes and proteins of the alternative steroid backdoor pathway for dihydrotestosterone synthesis are expressed in the human ovary and seem enhanced in the polycystic ovary syndrome. Mol Cell Endocrinol 2017; 441: 116–123.

53.

Silva

MSB

Desroziers

Hessler

, et al. Activation of arcuate nucleus GABA neurons promotes luteinizing hormone secretion and reproductive dysfunction: implications for polycystic ovary syndrome. EBioMedicine 2019; 44: 582–596.

54.

Carmina

Rosato

Jannì

Increased DHEAs levels in PCO syndrome: evidence for the existence of two subgroups of patients. J Endocrinol Invest 1986; 9: 5–9.

55.

Swart

Schloms

Storbeck

, et al. 11β-Hydroxyandrostenedione, the product of androstenedione metabolism in the adrenal, is metabolized in LNCaP cells by 5α-reductase yielding 11β-hydroxy-5α-androstanedione. J Steroid Biochem Mol Biol 2013; 138: 132–142.

56.

Rege

Nakamura

Satoh

, et al. Liquid chromatography-tandem mass spectrometry analysis of human adrenal vein 19-carbon steroids before and after ACTH stimulation. J Clin Endocrinol Metab 2013; 98: 1182–1188.

57.

Ayers

JW.

Differential response to adrenocorticotropin hormone stimulation in polycystic ovarian disease with high and low dehydroepiandrosterone sulfate levels. Fertil Steril 1982; 37: 645–649.

58.

Walker

Rodin

Taylor

, et al. Endogenous inhibitors of 11β-hydroxysteroid dehydrogenase type 1 do not explain abnormal cortisol metabolism in polycystic ovary syndrome. Clin Endocrinol 2000; 52: 77–80.

59.

Tsilchorozidou

Honour

Conway

GS.

Altered cortisol metabolism in polycystic ovary syndrome: insulin enhances 5α-reduction but not the elevated adrenal steroid production rates. J Clin Endocrinol Metab 2003; 88: 5907–5913.

60.

Saito

Matsuzaki

Iwasa

, et al. Steroidogenic pathways involved in androgen biosynthesis in eumenorrheic women and patients with polycystic ovary syndrome. J Steroid Biochem Mol Biol 2016; 158: 31–37.

61.

van Rooyen

Gent

Barnard

, et al. The in vitro metabolism of 11β-hydroxyprogesterone and 11-ketoprogesterone to 11-ketodihydrotestosterone in the backdoor pathway. J Steroid Biochem Mol Biol 2018; 178: 203–212.

62.

Rege

Turcu

Kasa-Vubu

, et al. 11-Ketotestosterone is the dominant circulating bioactive androgen during normal and premature adrenarche. J Clin Endocrinol Metab 2018; 103: 4589–4598.

63.

Giorgi

Stein

WD.

The transport of steroids into animal cells in culture. Endocrinology 1981; 108: 688–697.

64.

O’Reilly

Kempegowda

Walsh

, et al. AKR1C3-mediated adipose androgen generation drives lipotoxicity in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2017; 102: 3327–3339.

65.

Yates

Norbury

Gilbert

RJC

. The long and short of microRNA. Cell 2013; 153: 516–519.

66.

Zhang

Tian

, et al. Value of diffusion-weighted magnetic resonance imaging combined with miR-18a level in predicting radiosensitivity of cervical cancer. Med Sci Monit 2018; 24: 7271–7278.

67.

Marí-Alexandre

Carcelén

Agababyan

, et al. Interplay between microRNAs and oxidative stress in ovarian conditions with a focus on ovarian cancer and endometriosis. Int J Mol Sci 2019; 20: 5322.

68.

Romakina

Zhirov

Nasonova

, et al. MicroRNAs as biomarkers of cardiovascular diseases. Kardiologiya 2018; 58: 66–71.

69.

Long

Zhao

, et al. Characterization of serum microRNAs profile of PCOS and identification of novel non-invasive biomarkers. Cell Physiol Biochem 2014; 33: 1304–1315.

70.

Ding

Chen

Zhu

, et al. Circulating microRNAs in patients with polycystic ovary syndrome. Hum Fertil 2015; 18: 22–29.

71.

Chen

, et al. Role of microRNA in the pathogenesis of polycystic ovary syndrome. DNA Cell Biol 2019; 38: 754–762.

72.

Murri

Insenser

Fernández-Durán

, et al. Effects of polycystic ovary syndrome (PCOS), sex hormones, and obesity on circulating miRNA-21, miRNA-27b, miRNA-103, and miRNA-155 expression. J Clin Endocrinol Metab 2013; 98: E1835–E1844.

73.

Arancio

Calogero Amato

Magliozzo

, et al. Serum miRNAs in women affected by hyperandrogenic polycystic ovary syndrome: the potential role of miR-155 as a biomarker for monitoring the estroprogestinic treatment. Gynecol Endocrinol 2018; 34: 704–708.

74.

Sørensen

Wissing

Salö

, et al. MicroRNAs related to polycystic ovary syndrome (PCOS). Genes 2014; 5: 684–708.

75.

Mcallister

Han

Modi

, et al. MiRNA profiling reveals miRNA-130b-3p mediates DENND1A variant 2 expression and androgen biosynthesis. Endocrinology 2019; 160: 1964–1981.

76.

Linher-Melville

Yang

, et al. Micro-RNA378 (miR-378) regulates ovarian estradiol production by targeting aromatase. Endocrinology 2011; 152: 3941–3951.

77.

Zhang

Sun

Jiang

, et al. MicroRNA-181a suppresses mouse granulosa cell proliferation by targeting activin receptor IIA. PLoS One 2013; 8: e59667.

78.

Murri

Insenser

Fernández-Durán

, et al. Non-targeted profiling of circulating microRNAs in women with polycystic ovary syndrome (PCOS): effects of obesity and sex hormones. Metabolism 2018; 86: 49–60.

79.

Lowe

O’Rahilly

Rochford

JJ.

Adipogenesis at a glance. J Cell Sci 2011; 124: 2681–2686.

80.

Ouchi

Parker

Lugus

, et al. Adipokines in inflammation and metabolic disease. Nat Rev Immunol 2011; 11: 85–97.

81.

Dimitriadis

Kyrou

Randeva

Polycystic ovary syndrome as a proinflammatory state: the role of adipokines. Curr Pharm Des 2016; 22: 5535–5546.

82.

Cosar

Üçok

Akgün

, et al. Body fat composition and distribution in women with polycystic ovary syndrome. Gynecol Endocrinol 2008; 24: 428–432.

83.

Carmina

Bucchieri

Mansueto

, et al. Circulating levels of adipose products and differences in fat distribution in the ovulatory and anovulatory phenotypes of polycystic ovary syndrome. Fertil Steril 2009; 91: 1332–1335.

84.

Strowitzki

Halser

Demant

Body fat distribution, insulin sensitivity, ovarian dysfunction and serum lipoproteins in patients with polycystic ovary syndrome. Gynecol Endocrinol 2002; 16: 45–51.

85.

Svendsen

Nilas

Norgaard

, et al. Obesity, body composition and metabolic disturbances in polycystic ovary syndrome. Hum Reprod 2008; 23: 2113–2121.

86.

Zheng

S-H

X-L.

Visceral adiposity index as a predictor of clinical severity and therapeutic outcome of PCOS. Gynecol Endocrinol 2016; 32: 177–183.

87.

Techatraisak

Wongmeerit

Dangrat

, et al. Measures of body adiposity and visceral adiposity index as predictors of metabolic syndrome among Thai women with PCOS. Gynecol Endocrinol 2016; 32: 276–280.

88.

Durmus

Duran

Ecirli

Visceral adiposity index levels in overweight and/or obese, and non-obese patients with polycystic ovary syndrome and its relationship with metabolic and inflammatory parameters. J Endocrinol Invest 2017; 40: 487–497.

89.

Barber

Golding

Alvey

, et al. Global adiposity rather than abnormal regional fat distribution characterizes women with polycystic ovary syndrome. J Clin Endocrinol Metab 2008; 93: 999–1004.

90.

Faloia

Canibus

Gatti

, et al. Body composition, fat distribution and metabolic characteristics in lean and obese women with polycystic ovary syndrome. J Endocrinol Invest 2004; 27: 424–429.

91.

Boumosleh

Grundy

Phan

, et al. Metabolic concomitants of obese and nonobese women with features of polycystic ovarian syndrome. J Endocr Soc 2017; 1: 1417–1427.

92.

Ribeiro

Kogure

Lopes

, et al. Association of measures of central fat accumulation indices with body fat distribution and metabolic, hormonal, and inflammatory parameters in women with polycystic ovary syndrome. Arch Endocrinol Metab 2019; 63: 417–426.

93.

Manolopoulos

Karpe

Frayn

KN.

Gluteofemoral body fat as a determinant of metabolic health. Int J Obes 2010; 34: 949–959.

94.

Brończyk-Puzoń

Jagielski

Kulik-Kupka

, et al. Usefulness of a new anthropometric indicator-VAI (visceral adiposity index) in the evaluation of metabolic and hormonal disorders in women with polycystic ovary syndrome. Adv Clin Exp Med 2017; 26: 825–828.

95.

Amato

Giordano

Galia

, et al. Visceral adiposity index: a reliable indicator of visceral fat function associated with cardiometabolic risk. Diabetes Care 2010; 33: 920–922.

96.

Lim

Norman

Davies

, et al. The effect of obesity on polycystic ovary syndrome: a systematic review and meta-analysis. Obes Rev 2013; 14: 95–109.

97.

Amato

Verghi

Galluzzo

, et al. The oligomenorrhoic phenotypes of polycystic ovary syndrome are characterized by a high visceral adiposity index: a likely condition of cardiometabolic risk. Hum Reprod 2011; 26: 1486–1494.

98.

Dieudonne

Pecquery

Leneveu

, et al. Opposite effects of androgens and estrogens on adipogenesis in rat preadipocytes: evidence for sex and site-related specificities and possible involvement of insulin-like growth factor 1 receptor and peroxisome proliferator-activated receptorγ 2. Endocrinology 2000; 141: 649–656.

99.

Gupta

Bhasin

Guo

, et al. Effects of dihydrotestosterone on differentiation and proliferation of human mesenchymal stem cells and preadipocytes. Mol Cell Endocrinol 2008; 296: 32–40.

100.

Chazenbalk

Singh

Irge

, et al. Androgens inhibit adipogenesis during human adipose stem cell commitment to preadipocyte formation. Steroids 2013; 78: 920–926.

101.

Blouin

Nadeau

Perreault

, et al. Effects of androgens on adipocyte differentiation and adipose tissue explant metabolism in men and women. Clin Endocrinol 2010; 72: 176–188.

102.

Nilsson

Niklasson

Eriksson

, et al. Imprinting of female offspring with testosterone results in insulin resistance and changes in body fat distribution at adult age in rats. J Clin Invest 1998; 101: 74–78.

103.

Alexanderson

Eriksson

Stener-Victorin

, et al. Postnatal testosterone exposure results in insulin resistance, enlarged mesenteric adipocytes, and an atherogenic lipid profile in adult female rats: comparisons with estradiol and dihydrotestosterone. Endocrinology 2007; 148: 5369–5376.

104.

O’Reilly

House

Tomlinson

JW.

Understanding androgen action in adipose tissue. J Steroid Biochem Mol Biol 2014; 143: 277–284.

105.

Storbeck

K-H

Bloem

Africander

, et al. 11β-Hydroxydihydrotestosterone and 11-ketodihydrotestosterone, novel C19 steroids with androgenic activity: a putative role in castration resistant prostate cancer? Mol Cell Endocrinol 2013; 377: 135–146.

106.

Barnard

Quanson

Mostaghel

, et al. 11-Oxygenated androgen precursors are the preferred substrates for aldo-keto reductase 1C3 (AKR1C3): implications for castration resistant prostate cancer. J Steroid Biochem Mol Biol 2018; 183: 192–201.

107.

Amer

Alzanati

Warren

, et al. Excess androgen production in subcutaneous adipose tissue of women with polycystic ovarian syndrome is not related to insulin or LH. J Endocrinol 2019; 241: 99–109.

108.

Plymate

Matej

Jones

, et al. Inhibition of sex hormone-binding globulin production in the human hepatoma (Hep G2) cell line by insulin and prolactin. J Clin Endocrinol Metab 1988; 67: 460–464.

109.

Pasquali

Gambineri

Pagotto

Review article: the impact of obesity on reproduction in women with polycystic ovary syndrome. BJOG An Int J Obstet Gynaecol 2006; 113: 1148–1159.

110.

Gambineri

Pelusi

Vicennati

, et al. Obesity and the polycystic ovary syndrome. Int J Obes 2002; 26: 883–896.

111.

Panidis

Farmakiotis

Rousso

, et al. Obesity, weight loss, and the polycystic ovary syndrome: effect of treatment with diet and orlistat for 24 weeks on insulin resistance and androgen levels. Fertil Steril 2008; 89: 899–906.

112.

Escobar-Morreale

Botella-Carretero

Álvarez-Blasco

, et al. The polycystic ovary syndrome associated with morbid obesity may resolve after weight loss induced by bariatric surgery. J Clin Endocrinol Metab 2005; 90: 6364–6369.

113.

Navarro

Jacobson

, et al. Extranuclear actions of the androgen receptor enhance glucose-stimulated insulin secretion in the male. Cell Metab 2016; 23: 837–851.

114.

Harada

Yoda

Yotsumoto

, et al. Androgen signaling expands β-cell mass in male rats and β-cell androgen receptor is degraded under high-glucose conditions. Am J Physiol Metab 2018; 314: E274–E286.

115.

Niu

, et al. Androgen receptor-deficient islet β-cells exhibit alteration in genetic markers of insulin secretion and inflammation. A transcriptome analysis in the male mouse. J Diabetes Complications 2017; 31: 787–795.

116.

Mauvais-Jarvis

Role of sex steroids in β cell function, growth, and survival. Trends Endocrinol Metab 2016; 27: 844–855.

117.

Navarro

Allard

Morford

, et al. Androgen excess in pancreatic β cells and neurons predisposes female mice to type 2 diabetes. JCI Insight 2018; 3: 6364–6369.

118.

Morimoto

Fernandez-Mejia

Romero-Navarro

, et al. Testosterone effect on insulin content, messenger ribonucleic acid levels, promoter activity, and secretion in the rat. Endocrinology 2001; 142: 1442–1447.

119.

Morimoto

Mendoza-Rodríguez

Hiriart

, et al. Protective effect of testosterone on early apoptotic damage induced by streptozotocin in rat pancreas. J Endocrinol 2005; 187: 217–224.

120.

Harada

Yotsumoto

Katsuki

, et al. Fetal androgen signaling defects affect β-cell mass and function, leading to glucose intolerance in high-fat diet-fed male rats. Am J Physiol Metab 2019; 317: E731–E741.

121.

Demissie

Lazic

Foecking

, et al. Transient prenatal androgen exposure produces metabolic syndrome in adult female rats. Am J Physiol Metab 2008; 295: E262–E268.

122.

Abbott

Bruns

Barnett

, et al. Experimentally induced gestational androgen excess disrupts glucoregulation in rhesus monkey dams and their female offspring. Am J Physiol Metab 2010; 299: E741–E751.

123.

Ramaswamy

Grace

Mattei

, et al. Developmental programming of polycystic ovary syndrome (PCOS): prenatal androgens establish pancreatic islet α/β cell ratio and subsequent insulin secretion. Sci Rep 2016; 6: 27408.

124.

Rae

Grace

Hogg

, et al. The pancreas is altered by in utero androgen exposure: implications for clinical conditions such as polycystic ovary syndrome (PCOS). PLoS One 2013; 8: e56263.

125.

Liu

Guo

Ruzi

, et al. Testosterone improves the differentiation efficiency of insulin-producing cells from human induced pluripotent stem cells. PLoS One 2017; 12: e0179353.

126.

Zhang

, et al. Analyses of risk factors for polycystic ovary syndrome complicated with non-alcoholic fatty liver disease. Exp Ther Med 2018; 15: 4259–4264.

127.

Wang

Zhu

, et al. Increased androgen levels in rats impair glucose-stimulated insulin secretion through disruption of pancreatic beta cell mitochondrial function. J Steroid Biochem Mol Biol 2015; 154: 254–266.

128.

Liu

Navarro

Mauvais-Jarvis

Androgen excess produces systemic oxidative stress and predisposes to β-cell failure in female mice. PLoS One 2010; 5: e11302.

129.

Pound

Ebrahim

Sandercock

, et al. Where is the evidence that animal research benefits humans? Br Med J 2004; 328: 514–517.

130.

Bracken

MB.

Why animal studies are often poor predictors of human reactions to exposure. J R Soc Med 2009; 102: 120–122.

131.

Moran

Misso

Wild

, et al. Impaired glucose tolerance, type 2 diabetes and metabolic syndrome in polycystic ovary syndrome: a systematic review and meta-analysis. Hum Reprod Update 2010; 16: 347–363.

132.

Legro

Kunselman

Dodson

, et al. Prevalence and predictors of risk for type 2 diabetes mellitus and impaired glucose tolerance in polycystic ovary syndrome: a prospective, controlled study in 254 affected women. J Clin Endocrinol Metab 1999; 84: 165–169.

133.

Glintborg

Henriksen

Andersen

, et al. Prevalence of endocrine diseases and abnormal glucose tolerance tests in 340 Caucasian premenopausal women with hirsutism as the referral diagnosis. Fertil Steril 2004; 82: 1570–1579.

134.

Boudreaux

Talbott

Kip

, et al. Risk of T2DM and impaired fasting glucose among PCOS subjects: results of an 8-year follow-up. Curr Diab Rep 2006; 6: 77–83.

135.

Joham

Ranasinha

Zoungas

, et al. Gestational diabetes and type 2 diabetes in reproductive-aged women with polycystic ovary syndrome. J Clin Endocrinol Metab 2014; 99: E447–E452.

136.

Rubin

Glintborg

Nybo

, et al. Development and risk factors of type 2 diabetes in a nationwide population of women with polycystic ovary syndrome. J Clin Endocrinol Metab 2017; 102: 3848–3857.

137.

Ehrmann

Barnes

Rosenfield

, et al. Prevalence of impaired glucose tolerance and diabetes in women with polycystic ovary syndrome. Diabetes Care 1999; 22: 141–146.

138.

Elting

Korsen

TJM

Bezemer

, et al. Prevalence of diabetes mellitus, hypertension and cardiac complaints in a follow-up study of a Dutch PCOS population. Hum Reprod 2001; 16: 556–560.

139.

Kauffman

Baker

DiMarino

, et al. Polycystic ovarian syndrome and insulin resistance in white and Mexican American women: a comparison of two distinct populations. Am J Obstet Gynecol 2002; 187: 1362–1369.

140.

Burghen

Givens

Kitabchi

AE.

Correlation of hyperandrogenism with hyperinsulinism in polycystic ovarian disease. J Clin Endocrinol Metab 1980; 50: 113–116.

141.

Chang

Nakamura

Judd

, et al. Insulin resistance in nonobese patients with polycystic ovarian disease. J Clin Endocrinol Metab 1983; 57: 356–359.

142.

Dunaif

Segal

Futterweit

, et al. Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. Diabetes 1989; 38: 1165–1174.

143.

O’Reilly

Glisic

Kumarendran

, et al. Serum testosterone, sex hormone-binding globulin and sex-specific risk of incident type 2 diabetes in a retrospective primary care cohort. Clin Endocrinol 2019; 90: 145–154.

144.

Ruth

Day

Tyrrell

, et al. Using human genetics to understand the disease impacts of testosterone in men and women. Nat Med 2020; 26: 1–7.

145.

Dunaif

Segal

Shelley

, et al. Evidence for distinctive and intrinsic defects in insulin action in polycystic ovary syndrome. Diabetes 1992; 41: 1257–1266.

146.

Hansen

Svendsen

Jeppesen

, et al. Molecular mechanisms in skeletal muscle underlying insulin resistance in women who are lean with polycystic ovary syndrome. J Clin Endocrinol Metab 2019; 104: 1841–1854.

147.

Sepilian

Nagamani

Adiponectin levels in women with polycystic ovary syndrome and severe insulin resistance. J Soc Gynecol Invest 2005; 12: 129–134.

148.

Satoh

Molecular mechanisms for the regulation of insulin-stimulated glucose uptake by small guanosine triphosphatases in skeletal muscle and adipocytes. Int J Mol Sci 2014; 15: 18677–18692.

149.

Barrett-Connor

Wedick

, et al. Endogenous sex hormones and the development of type 2 diabetes in older men and women: the Rancho Bernardo study. Diabetes Care 2002; 25: 55–60.

150.

Diamond

Grainger

Diamond

, et al. Effects of methyltestosterone on insulin secretion and sensitivity in women. J Clin Endocrinol Metab 1998; 83: 4420–4425.

151.

Shamma

Rossi

HajHassan

, et al. The effect of Norplant on glucose metabolism under hyperglycemic hyperinsulinemic conditions. Fertil Steril 1995; 63: 767–772.

152.

Polderman

Gooren

Asscheman

, et al. Induction of insulin resistance by androgens and estrogens. J Clin Endocrinol Metab 1994; 79: 265–271.

153.

Navarro

Allard

, et al. The role of androgens in metabolism, obesity, and diabetes in males and females. Obesity 2015; 20: 713–719.

154.

Stepto

Cassar

Joham

, et al. Women with polycystic ovary syndrome have intrinsic insulin resistance on euglycaemic-hyperinsulaemic clamp. Hum Reprod 2013; 28: 777–784.

155.

Carnagarin

Dharmarajan

Dass

CR.

Molecular aspects of glucose homeostasis in skeletal muscle: a focus on the molecular mechanisms of insulin resistance. Mol Cell Endocrinol 2015; 417: 52–62.

156.

Stepto

Moreno-Asso

McIlvenna

, et al. Molecular mechanisms of insulin resistance in polycystic ovary syndrome: unraveling the conundrum in skeletal muscle? J Clin Endocrinol Metab 2019; 104: 5372–5381.

157.

Dunaif

Xia

Book

, et al. Excessive insulin receptor serine phosphorylation in cultured fibroblasts and in skeletal muscle: a potential mechanism for insulin resistance in the polycystic ovary syndrome. J Clin Invest 1995; 96: 801–810.

158.

Corbould

Kim

Y-B

Youngren

, et al. Insulin resistance in the skeletal muscle of women with PCOS involves intrinsic and acquired defects in insulin signaling. Am J Physiol Metab 2005; 288: E1047–E1054.

159.

Hojlund

Glintborg

Andersen

, et al. Impaired insulin-stimulated phosphorylation of Akt and AS160 in skeletal muscle of women with polycystic ovary syndrome is reversed by pioglitazone treatment. Diabetes 2008; 57: 357–366.

160.

Martens

JWM

Geller

Arlt

, et al. Enzymatic activities of P450c17 stably expressed in fibroblasts from patients with the polycystic ovary syndrome. J Clin Endocrinol Metab 2000; 85: 4338–4346.

161.

Jiang

Wei

, et al. Exercise activates the PI3K-AKT signal pathway by decreasing the expression of 5α-reductase type 1 in PCOS rats. Sci Rep 2018; 8: 1–10.

162.

Mario

Graff

Spritzer

PM.

Habitual physical activity is associated with improved anthropometric and androgenic profile in PCOS: a cross-sectional study. J Endocrinol Invest 2017; 40: 377–384.

163.

Samadi

Bambaeichi

Valiani

, et al. Evaluation of changes in levels of hyperandrogenism, hirsutism and menstrual regulation after a period of aquatic high-intensity interval training in women with polycystic ovary syndrome. Int J Prev Med 2019; 10: 187.

164.

Nilsson

Benrick

Kokosar

, et al. Transcriptional and epigenetic changes influencing skeletal muscle metabolism in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2018; 103: 4465–4477.

165.

Samuel

Petersen

Shulman

GI.

Lipid-induced insulin resistance: unravelling the mechanism. Lancet 2010; 375: 2267–2277.

166.

Borén

Taskinen

M-R

Olofsson

S-O

, et al. Ectopic lipid storage and insulin resistance: a harmful relationship. J Intern Med 2013; 274: 25–40.

167.

Yamada

Ida

Yamaoka

, et al. Two distinct patterns of glucose intolerance in icteric rats and rabbits. Relationship to impaired liver mitochondria function. J Lab Clin Med 1975; 86: 38–45.

168.

Montgomery

Turner

Mitochondrial dysfunction and insulin resistance: an update. Endocr Connect 2015; 4: R1–R15.

169.

Shen

, et al. Nontargeted metabolomic analysis of skeletal muscle in a dehydroepiandrosterone-induced mouse model of polycystic ovary syndrome. Mol Reprod Dev 2019; 86: 370–378.

170.

Skov

Glintborg

Knudsen

, et al. Reduced expression of nuclear-encoded genes involved in mitochondrial oxidative metabolism in skeletal muscle of insulin-resistant women with polycystic ovary syndrome. Diabetes 2007; 56: 2349–2355.

171.

Hutchison

Teede

Rachoń

, et al. Effect of exercise training on insulin sensitivity, mitochondria and computed tomography muscle attenuation in overweight women with and without polycystic ovary syndrome. Diabetologia 2012; 55: 1424–1434.

172.

Cassidy

Thoma

Houghton

, et al. High-intensity interval training: a review of its impact on glucose control and cardiometabolic health. Diabetologia 2017; 60: 7–23.

173.

Pais

Barritt

Calmus

, et al. NAFLD and liver transplantation: current burden and expected challenges. J Hepatol 2016; 65: 1245–1257.

174.

Petta

Ciresi

Bianco

, et al. Insulin resistance and hyperandrogenism drive steatosis and fibrosis risk in young females with PCOS. PLoS One 2017; 12: e0186136.

175.

Cai

Zhang

, et al. High-free androgen index is associated with increased risk of non-alcoholic fatty liver disease in women with polycystic ovary syndrome, independent of obesity and insulin resistance. Int J Obes 2017; 41: 1341–1347.

176.

Kim

Yim

, et al. Polycystic ovary syndrome with hyperandrogenism as a risk factor for non-obese non-alcoholic fatty liver disease. Aliment Pharmacol Ther 2017; 45: 1403–1412.

177.

Macut

Tziomalos

Božić-Antić

, et al. Non-alcoholic fatty liver disease is associated with insulin resistance and lipid accumulation product in women with polycystic ovary syndrome. Hum Reprod 2016; 31: 1347–1353.

178.

Kumarendran

O’Reilly

Manolopoulos

, et al. Polycystic ovary syndrome, androgen excess, and the risk of nonalcoholic fatty liver disease in women: a longitudinal study based on a United Kingdom primary care database. PLoS Med 2018; 15: e1002542.

179.

Yao

X-Y

Shi

R-X

, et al. A potential link between polycystic ovary syndrome and non-alcoholic fatty liver disease: an update meta-analysis. Reprod Health 2018; 15: 77.

180.

Macut

Božić-Antić

Bjekić-Macut

, et al. Management of endocrine disease: polycystic ovary syndrome and nonalcoholic fatty liver disease. Eur J Endocrinol 2017; 177: R145–R158.

181.

Baranova

Tran

Afendy

, et al. Molecular signature of adipose tissue in patients with both non-alcoholic fatty liver disease (NAFLD) and polycystic ovarian syndrome (PCOS). J Transl Med 2013; 11: 133.

182.

Kakino

Ohki

Nakayama

, et al. Pivotal role of TNF-α in the development and progression of nonalcoholic fatty liver disease in a murine model. Horm Metab Res 2018; 50: 80–87.

183.

González

Sia

Bearson

, et al. Hyperandrogenism induces a proinflammatory TNFα response to glucose ingestion in a receptor-dependent fashion. J Clin Endocrinol Metab 2014; 99: E848–E854.

184.

Hotamisligil

Peraldi

Budavari

, et al. IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-α- and obesity-induced insulin resistance. Science 1996; 271: 665–670.

185.

Shimomura

Hammer

Richardson

, et al. Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy. Genes Dev 1998; 12: 3182–3194.

186.

O’Reilly

Gathercole

Capper

, et al. Effect of insulin on AKR1C3 expression in female adipose tissue: in-vivo and in-vitro study of adipose androgen generation in polycystic ovary syndrome. Lancet 2015; 385(Suppl.): S16.

187.

Anjani

Lhomme

Sokolovska

, et al. Circulating phospholipid profiling identifies portal contribution to NASH signature in obesity. J Hepatol 2015; 62: 905–912.

188.

Shi

Zhao

Guo

, et al. Differential expression of microRNAs in omental adipose tissue from gestational diabetes mellitus subjects reveals miR-222 as a regulator of ERα expression in estrogen-induced insulin resistance. Endocrinology 2014; 155: 1982–1990.

189.

Ortega

Mercader

Moreno-Navarrete

, et al. Profiling of circulating microRNAs reveals common microRNAs linked to type 2 diabetes that change with insulin sensitization. Diabetes Care 2014; 37: 1375–1383.

190.

Sang

Yao

Wang

, et al. Identification of microRNAs in human follicular fluid: characterization of microRNAs that govern steroidogenesis in vitro and are associated with polycystic ovary syndrome in vivo. J Clin Endocrinol Metab 2013; 98: 3068–3079.

191.

Yang

Jiang

Xiao

, et al. MicroRNA-33b-5p is overexpressed and inhibits GLUT4 by targeting HMGA2 in polycystic ovarian syndrome: an in vivo and in vitro study. Oncol Rep 2018; 39: 3073–3085.

192.

Xiong

Lin

, et al. Circulatory microRNA 23a and microRNA 23b and polycystic ovary syndrome (PCOS): the effects of body mass index and sex hormones in an Eastern Han Chinese population. J Ovarian Res 2017; 10: 10.

193.

Pierpoint

McKeigue

Isaacs

, et al. Mortality of women with polycystic ovary syndrome at long-term follow-up. J Clin Epidemiol 1998; 51: 581–586.

194.

Wild

Pierpoint

McKeigue

, et al. Cardiovascular disease in women with polycystic ovary syndrome at long-term follow-up: a retrospective cohort study. Clin Endocrinol 2000; 52: 595–600.

195.

Dahlgren

Janson

Johansson

, et al. Polycystic ovary syndrome and risk for myocardial infarction: evaluated from a risk factor model based on a prospective population study of women. Acta Obstet Gynecol Scand 1992; 71: 599–604.

196.

Birdsall

Farquhar

White

HD.

Association between polycystic ovaries and extent of coronary artery disease in women having cardiac catheterization. Ann Intern Med 1997; 126: 32.

197.

Christian

Dumesic

Behrenbeck

, et al. Prevalence and predictors of coronary artery calcification in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2003; 88: 2562–2568.

198.

Cibula

Cífková

Fanta

, et al. Increased risk of non-insulin-dependent diabetes mellitus, arterial hypertension and coronary artery disease in perimenopausal women with a history of the polycystic ovary syndrome. Hum Reprod 2000; 15: 785–789.

199.

Wild

Carmina

Diamanti-Kandarakis

, et al. Assessment of cardiovascular risk and prevention of cardiovascular disease in women with the polycystic ovary syndrome: a consensus statement by the Androgen Excess and Polycystic Ovary Syndrome (AE-PCOS) Society. J Clin Endocrinol Metab 2010; 95: 2038–2049.

200.

Yang

, et al. Effects of hyperandrogenism on metabolic abnormalities in patients with polycystic ovary syndrome: a meta-analysis. Reprod Biol Endocrinol 2016; 14: 67.

201.

Luque-Ramírez

Mendieta-Azcona

Álvarez-Blasco

, et al. Androgen excess is associated with the increased carotid intima-media thickness observed in young women with polycystic ovary syndrome. Hum Reprod 2007; 22: 3197–3203.

202.

Rubio-Gayosso

Garcia-Ramirez

Gutierrez-Serdan

, et al. Testosterone inhibits bradykinin-induced intracellular calcium kinetics in rat aortic endothelial cells in culture. Steroids 2002; 67: 393–397.

203.

Quan

Chakravarty

Chen

J-K

, et al. Androgens augment proximal tubule transport. Am J Physiol Ren Physiol 2004; 287: F452–F459.

204.

Quinkler

Bujalska

Kaur

, et al. Androgen receptor-mediated regulation of the α-subunit of the epithelial sodium channel in human kidney. Hypertension 2005; 46: 787–798.

205.

Orio

Palomba

Cascella

, et al. Early impairment of endothelial structure and function in young normal-weight women with polycystic ovary syndrome. J Clin Endocrinol Metab 2004; 89: 4588–4593.

206.

Kravariti

Naka

Kalantaridou

, et al. Predictors of endothelial dysfunction in young women with polycystic ovary syndrome. J Clin Endocrinol Metab 2005; 90: 5088–5095.

207.

Kelly

CCJ

Lyall

Petrie

, et al. Low grade chronic inflammation in women with polycystic ovarian syndrome. J Clin Endocrinol Metab 2001; 86: 2453–2455.

208.

Boulman

Levy

Leiba

, et al. Increased C-reactive protein levels in the polycystic. J Clin Endocrinol Metab 2004; 89: 2160–2165.

209.

Möhlig

Spranger

Osterhoff

, et al. The polycystic ovary syndrome per se is not associated with increased chronic inflammation. Eur J Endocrinol 2004; 150: 525–532.

210.

Lee

, et al. Serum C-reactive protein levels in normal-weight polycystic ovary syndrome. Korean J Intern Med 2009; 24: 350–355.

211.

Clarke

Robinson

Graham

, et al. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 1991; 324: 1149–1155.

212.

Garibaldi

Valentini

Aragno

, et al. Plasma protein oxidation and antioxidant defense during aging. Int J Vitam Nutr Res 2001; 71: 332–338.

213.

Yarali

Yildirir

Aybar

, et al. Diastolic dysfunction and increased serum homocysteine concentrations may contribute to increased cardiovascular risk in patients with polycystic ovary syndrome. Fertil Steril 2001; 76: 511–516.

214.

Schachter

Raziel

Friedler

, et al. Insulin resistance in patients with polycystic ovary syndrome is associated with elevated plasma homocysteine. Hum Reprod 2003; 18: 721–727.

215.

Loverro

Lorusso

Mei

, et al. The plasma homocysteine levels are increased in polycystic ovary syndrome. Gynecol Obstet Invest 2002; 53: 157–162.

216.

Sabuncu

Vural

Harma

, et al. Oxidative stress in polycystic ovary syndrome and its contribution to the risk of cardiovascular disease. Clin Biochem 2001; 34: 407–413.

217.

Yilmaz

Bukan

Ayvaz

, et al. The effects of rosiglitazone and metformin on oxidative stress and homocysteine levels in lean patients with polycystic ovary syndrome. Hum Reprod 2005; 20: 3333–3340.

218.

Cagnacci

Tirelli

Renzi

, et al. Effects of two different oral contraceptives on homocysteine metabolism in women with polycystic ovary syndrome. Contraception 2006; 73: 348–351.

219.

Gul

Somunkiran

Yucel

, et al. The effect of ethinyl estradiol-cyproterone acetate treatment on homocysteine levels in women with polycystic ovary syndrome. Arch Gynecol Obstet 2008; 277: 25–30.

220.

Luque-Ramírez

Mendieta-Azcona

del Rey Sánchez

, et al. Effects of an antiandrogenic oral contraceptive pill compared with metformin on blood coagulation tests and endothelial function in women with the polycystic ovary syndrome: influence of obesity and smoking. Eur J Endocrinol 2009; 160: 469–480.

221.

Mancini

Cianciosi

Persico

, et al. Drospirenone and cardiovascular risk in lean and obese polycystic ovary syndrome patients: a pilot study. Am J Obstet Gynecol 2010; 202: 169.

222.

Usselman

Yarovinsky

Steele

, et al. Androgens drive microvascular endothelial dysfunction in women with polycystic ovary syndrome: role of the endothelin B receptor. J Physiol 2019; 597: 2853–2865.

Implicating androgen excess in propagating metabolic disease in polycystic ovary syndrome

Abstract

Keywords

Introduction

Review methodology

Origins of androgen excess in PCOS and pre-receptor androgen metabolism

Ovaries and androgen excess

Adrenal glands and androgen excess

Peripheral tissues and androgen excess

MicroRNA and androgen excess

Androgens in metabolic target tissues

Adipose tissue, obesity, fat mass distribution and androgen excess

Pancreas, type 2 diabetes and androgen excess

Muscle

Liver

The role of microRNA

Androgen excess and cardiovascular disease

Conclusion

Footnotes

Author contribution(s)

Conflict of interest statement

Funding

ORCID iD

References