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Polycystic ovary syndrome: Syndrome XX?
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Polycystic ovary syndrome: Syndrome XX? Susan Sam and Andrea Dunaif Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University, Chicago, IL 60611, USA
Polycystic ovary syndrome (PCOS) is now recognized as an important metabolic and reproductive disorder. It is associated with substantial defects in insulin action and secretion that confer a markedly increased risk for type 2 diabetes mellitus. Insulin resistance modifies reproductive function both by the direct actions of insulin on steroidogenesis and by disruption of insulin signaling pathways in the central nervous system. These insights have led to a new therapy for PCOS with insulin-sensitizing agents. Hyperandrogenemia and insulin resistance cluster in PCOS families, consistent with a genetic susceptibility to these abnormalities. There is evidence for both linkage and association of the hyperandrogenemia phenotype with an allele of a marker locus on chromosome 19, in the region of the gene encoding the insulin receptor. Polycystic ovary syndrome (PCOS) is among the most common endocrine disorders in premenopausal women, and is characterized by increased androgen production and disordered gonadotropin secretion [1]. It is now clear that PCOS is associated with profound insulin resistance and pancreatic b-cell dysfuntion, defects that confer a substantially increased risk for glucose intolerance [2– 5]. Indeed, PCOS is a leading risk factor for impaired glucose tolerance and type 2 diabetes mellitus (T2DM) in adolescent girls as it is in premenopausal women [6,7]. Women with PCOS might also have an increased risk for cardiovascular disease [8]. Investigation of these metabolic abnormalities has revealed that insulin resistance plays an important role in the pathogenesis of the reproductive disturbances of PCOS (Fig. 1). Familial clustering of PCOS is well documented and it is a complex genetic disease reflecting the interplay of genetic and environmental factors [9,10].
GnRH pulse frequency selectively increases LH release [11]. The raised LH levels enhance thecal androgen production, and these androgens are incompletely aromatized into estrogens by the granulosa cells, because of arrested follicular development as a consequence of lowlevel cyclic FSH release. The so-called vicious cycle of PCOS is created, in which disordered gonadotropin secretion begets increased ovarian androgen production, which in turn alters gonadal steroid feedback, perpetuating disordered gonadotropin release (Fig. 1) [1]. Whether the primary defect is in the regulation of gonadotropin secretion or of gonadal steroidogenesis has been debated for decades. Adrenal androgen production is also frequently increased in PCOS [12]. This finding might reflect a common defect in ovarian and adrenal androgen biosynthesis because adrenocorticotropin hormone (ACTH) release is not increased [13]. Increasing evidence suggests that there is a primary defect in ovarian steroidogenesis in PCOS. PCOS theca cells show increased activity of multiple steroidogenic enzymes, resulting in raised androgen production, both Insulin resistance
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Reproductive abnormalities in PCOS The cardinal reproductive features of PCOS are increased androgen production and disordered gonadotropin secretion. Both luteinizing hormone (LH) pulse frequency and amplitude are increased, whereas follicle-stimulating hormone (FSH) levels remain tonically in the midfollicular range [11]. The frequency of gonadotropin-releasing hormone (GnRH) release is increased secondary to decreased sensitivity of the GnRH pulse generator to the negative feedback effects of estradiol and progesterone. This increased Corresponding author: A. Dunaif ([email protected]).
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Fig. 1. The frequency of GnRH release is increased in polycystic ovary syndrome (PCOS), leading to a selective increase in LH relative to FSH secretion. PCOS theca cells have increased activity of multiple steroidogenic enzymes, resulting in raised androgen (A) production, both basally and in response to LH. There is decreased intraovarian aromatization of androgen to estradiol (E2) because follicular maturation is arrested as a result of relatively decreased FSH release. Insulin directly stimulates theca cell steroidogenesis in synergy with LH via its cognate receptor or, in cases of extreme insulin resistance, via the type 1 IGF receptor (IGFR). Abbreviations: FSH, follicle-stimulating hormone, GnRH, gonadotropin-releasing hormone, IGF, insulin-like growth factor, LH, luteinizing hormone. Reproduced with permission from Andrea Dunaif.
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basally and in response to LH [14,15]. This phenotype persists in theca cells propagated in longterm culture, which is suggestive of a genetically determined trait [14,15]. Consistent with this hypothesis, family studies indicate that hyperandrogenemia is the major reproductive phenotype in brothers and sisters of women with PCOS [16,17]. Recent human and animal studies provide several mechanisms by which a primary defect resulting in hyperandrogenemia could cause PCOS. First, the decreased sensitivity of the GnRH pulse generator to gonadal steroid negative feedback appears to be a consequence of raised circulating androgen levels, because androgen receptor blockade with flutamide abolishes this defect in PCOS [18]. Second, studies of rhesus monkeys suggest that prenatal androgen exposure produces many of the features characteristic of the PCOS phenotype in adult females, such as increased LH secretion, ovarian hyperandrogenism, central obesity and defective insulin secretion [19]. Third, permanent alterations in LH secretion are demonstrable in women who were exposed to excess androgens during in utero development, such as women with congenital adrenal hyperplasia or with neonatal androgensecreting neoplasms [12]. Metabolic abnormalities in PCOS Insulin resistance is a prominent feature of PCOS, independent of obesity [1 –5]. Many but not all women with PCOS are insulin resistant. Obesity and PCOS have an additive deleterious effect on insulin sensitivity [2]. The molecular mechanisms of this defect differ from those in other common insulin resistant conditions, such as T2 DM and obesity, suggesting that PCOS-related insulin resistance has a distinct genetic etiology [1]. Studies of PCOS adipocytes suggest that there is a post-binding defect in insulin receptor-mediated signal transduction, and this observation has recently been confirmed in skeletal muscle, the major site of insulin-mediated glucose uptake (Fig. 2) [1,20]. Studies of insulin receptors isolated from PCOScultured skin fibroblasts suggested that the signaling defect results from a decrease in insulin receptor tyrosine kinase activity, secondary to a constitutive increase in receptor serine phosphorylation [1]. Mixing and immunopurification studies suggest that a factor extrinsic to the insulin receptor is responsible for the serine phosphorylation [1]. Recent studies have suggested that a serine kinase accounts for this finding by demonstrating that a non-specific serine kinase inhibitor normalizes insulin receptor tyrosine autophosphorylation in cultured skin fibroblasts from PCOS patients [21]. The signaling defect produces selective insulin resistance, affecting metabolic but not mitogenic actions of insulin [22]. Insulin acts through its cognate receptor, in synergy with LH, to stimulate theca cell steroidogenesis in PCOS (Fig. 1) [23]. Hence, it is possible that the selective insulin resistance of PCOS accounts for the continued actions of insulin on steroidogenesis, in spite of defects in insulin-mediated glucose metabolism. Insulin also contributes to increased adrenal androgen secretion in PCOS, in part by enhancing adrenal sensitivity to ACTH [24]. Ek and colleagues have identified fat depot-specific abnormalities in the regulation of lipolysis in PCOS [25]. http://tem.trends.com
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Fig. 2. Insulin resistance in some polycystic ovary syndrome (PCOS) tissues (e.g. skin fibroblasts) results from the constitutive serine phosphorylation of the insulin receptor secondary to an as yet unidentified serine kinase. The insulin resistance is selective, affecting the metabolic but not mitogenic actions of insulin. Serine phosphorylation of P450c17 (CYP 17), the rate-limiting enzyme for androgen biosynthesis, increases its activity. It is possible that the same serine kinase phosphorylates the insulin receptor, producing insulin resistance, and P450c17, producing hyperandrogenemia. Cytokines and free fatty acids (FFA) can activate intracellular serine kinases and might play a role in the pathogenesis of PCOS. It is possible that the serine kinase phosphorylates downstream signaling molecules, such as insulin receptor substrates (IRS) 1 or 2, further compromising insulin signaling. Minus symbols (2) denote serine phosphorylation, which inhibits signal transduction. Reproduced with permission from Andrea Dunaif.
Isolated subcutaneous abdominal adipocytes are resistant to catecholamine-induced lipolysis, whereas the opposite phenomenon, markedly enhanced sensitivity, is observed in visceral adipocytes from women with PCOS [26]. These abnormalities are independent of obesity. Analogous to the defects in glucose metabolism, the molecular mechanisms of the lipolytic abnormalities in PCOS are unique. In contrast to adrenoceptor-mediated alterations in lipolysis in the metabolic syndrome, in PCOS there is a postreceptor increase in protein kinase A activity [25]. The increase in visceral fat lipolysis could lead to an increase in free fatty acid (FFA) release directly into the portal circulation. Portal FFA levels are major positive modulators of hepatic glucose production [27]. Accordingly, enhanced visceral fat lipolysis could be one mechanism for the increased risk for glucose intolerance in PCOS [28]. Prenatal androgen administration can increase visceral adiposity and alter lipolysis in animals [29]. The possible role of androgens in the pathogenesis of the lipolytic defects in PCOS merits further investigation. Increased serine kinase activity: a unifying hypothesis for PCOS? Serine phosphorylation of the rate-limiting enzyme for androgen biosynthesis, P450c17 (CYP17), increases its activity (Fig. 2) [1]. This observation led to the parsimonious hypothesis that the same kinase was responsible for serine phosphorylation of the insulin receptor, producing insulin resistance, and P450c17-a, producing hyperandrogenism in PCOS [1]. The one study that has tested this hypothesis by transfecting P450c17 into skin fibroblasts
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failed to detect increased serine phosphorylation of P450c17 in PCOS fibroblasts with increased serine kinase activity [30]. Nevertheless, this study only fails to support rather than disprove the serine kinase hypothesis, because fibroblasts are not steroidogenic cells and might have lacked essential cofactors for phosphorylation of P450c17. Further investigation of this hypothesis is clearly warranted, and genes encoding serine kinases are highpriority candidate genes for PCOS. Phenotypes in PCOS families: both men and women are affected Familial clustering of PCOS is well documented, suggesting that there is a genetic susceptibility to the disorder [10,16,17,31]. It was found that , 40% of premenopausal sisters of women with PCOS have elevations of total or biologically available testosterone levels [16]. There are two phenotypes in the affected sisters: (1) raised testosterone levels with irregular menses (classic PCOS); and (2) raised testosterone levels with regular menstrual cycles [16]. Although ovulation was not documented in the sisters with hyperandrogenemia and regular menses, their fecundity was significantly greater than that in sisters with the PCOS phenotype and similar to that seen in unaffected sisters, suggesting that the hyperandrogenemic sisters had ovulatory cycles [16]. Dehydroepiandrosterone sulfate (DHEAS) levels were also raised in affected sisters, suggesting that there was an adrenal component to hyperandrogenemia [16]. A possible candidate gene to account for this finding would be one that regulated both ovarian and adrenal steroidogenesis. Androgen levels were similarly raised in sisters with classic PCOS and those with hyperandrogenemia and regular menses [16]. Sisters with classic PCOS and those with hyperandrogenemia and regular menses both had similarly raised markers of insulin resistance, whereas sisters who had normal circulating androgen levels and menstrual cycles (i.e. unaffected sisters) had no evidence of insulin resistance [31]. This study also demonstrates that hyperandrogenemia and markers of insulin resistance track together in affected sisters, suggesting that these findings might have a common pathogenesis or might be the result of closely linked genes [31]. Furthermore, these findings suggest that using the hyperandrogenemia phenotype in linkage studies will also identify genes associated with insulin resistance. There have been limited studies of male relatives in PCOS families and few of these have used concurrently studied control subjects. Several studies suggested that premature male pattern balding was a male phenotype, but more comprehensive studies of male relatives have questioned this [9]. A large group of brothers of women with PCOS were found to have significantly raised DHEAS levels compared with control men [17]. Furthermore, DHEAS levels were significantly correlated between the brothers and their proband sisters with PCOS, consistent with a heritable trait. Testosterone levels were not raised in brothers, as they were in the proband sisters with PCOS; however, testosterone secretion in men is tightly regulated by hypothalamic – pituitary feedback of androgens [17]. It is also possible that subtle differences in http://tem.trends.com
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testosterone levels escaped detection. The elevation in adrenal androgen DHEAS suggests that affected brothers might have the same putative defect in steroidogenesis as affected sisters do [16]. There was no increased prevalence of balding in the brothers of women with PCOS [17]. The endocrine syndrome and polycystic ovarian morphology The significance of polycystic ovarian morphology has been debated for decades. Polycystic ovaries (PCO) also cluster in families, consistent with a heritable trait, and anovulatory women with PCO can have ovulatory sisters with PCO [9,16]. The probands and their sisters with PCO have similarly raised androgen concentrations (Rush, K. et al. (2002) Serum androgen and insulin concentrations in probands and affected sisters with polycystic ovaries. Presented at the 84th Annual Meeting of the Endocrine Society, San Francisco, California, USA) analogous to the finding of sisters with hyperandrogenemia and regular menses. The presence of these ovulatory (hyperandrogenemia and/or PCO alone) and anovulatory (PCOS) reproductive phenotypes within one family suggests that such phenotypes reflect variable expression of the same genetic trait. Most women with PCO have hyperandrogenemia and raised LH levels [1]. Functional ovarian hyperandrogenism can be unmasked with long-acting GnRH analogs in women with PCO who have normal basal androgen levels [32]. Theca cells from women with PCO consistently secrete increased androgens, independent of ovulatory function or circulating androgen levels [14]. Taken together, these findings suggest that PCO are intrinsically abnormal. The family studies suggest that PCO and hyperandrogenemia are heritable traits. We propose that hyperandrogenemia is the biochemical correlate of PCO. Genetic analyses of PCOS There have been no genome-wide screens of PCOS, and all reported genetic studies have used a candidate gene approach, in which genes are selected for analysis based on known pathophysiology. Plausible candidate genes for PCOS include those encoding proteins involved in steroid hormone biosynthesis, gonadotropin secretion or action, obesity and energy regulation, and insulin action [33]. Most studies have examined the association between PCOS and its intermediate phenotypes, such as androgen levels, PCO or markers of insulin resistance, and candidate gene variants in cases compared with control populations or within groups of PCOS women, to assess genotype– phenotype correlations [10,34]. There have been few linkage and family-based association studies of PCOS because both require large samples of families with two or more affected relatives for linkage analyses. These studies are not confounded by population stratification because they are performed in related individuals [35]. False positive results are still problematic and statistical adjustments should be made for testing multiple alleles [36]. Non parametric linkage analyses that make no assumption about the mode of inheritance are appropriate because PCOS is probably a complex genetic disorder [9,10]. The phenotypes that have been used in these family
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studies have been the PCO/premature male pattern balding phenotype in a series of , 20 families from the UK and the PCOS/hyperandrogenemia phenotype in studies from USA [33,37]. Very few candidate genes or chromosomal regions have been confirmed in two or more independent studies and therefore remain of particular interest given the limitations of these genetic analyses. The gene encoding P450 cholesterol side chain cleavage (CYP11a) has been linked to PCO/premature male pattern balding phenotype in PCOS families [38]. A family-based association study supported this finding by demonstrating a nominally significant association between a marker in the region of the CYP11a gene and the PCOS/hyperandrogenemia phenotype [33]. Association studies have suggested that a variant in the promoter of this gene is associated with circulating testosterone levels in women with PCOS [39]. The variable number tandem repeats (VNTR) locus upstream of the gene encoding insulin (INS), which might regulate INS expression, has been linked to the PCO/premature male pattern balding phenotype [37]. Family-based studies have also supported the association of the INS gene VNTR with this phenotype and with insulin levels in affected women [37]. Two genotype– phenotype studies have found an association with a variant in the gene encoding insulin receptor substrate-1 (IRS1) and parameters of insulin action in women with PCOS [40,41]. However, all of these studies have been constrained by small sample sizes. Evidence has been found to link the gene encoding follistatin (FST) to the PCOS/hyperandrogenemia phenotype in an affected sibling pair analysis [33]. However, further studies failed to identify variants in the FST gene associated with this phenotype, suggesting that follistatin does not play a major role in the pathogenesis of PCOS [42]. Prenatal
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A marker locus on chromosome 19p located near the gene encoding the insulin receptor (INSR) has been found that shows both association and linkage with the PCOS/hyperandrogenemia phenotype [33] (Urbanek, M. et al. (2002) Polycystic ovary syndrome candidate gene region on chromosome 19p13. Presented at the 84th Annual Meeting of the Endocrine Society, San Francisco, California, USA). An independent case– control association study supported this finding [43]. The marker maps to a 1 Mb region that is centromeric to the INSR gene, and linkage disequilibrium is usually maintained only over much smaller regions [33]. Thus, it is probable that a gene other than INSR accounts for the association with this marker. It remains possible that such a gene is still involved in insulin action. Alternatively, this gene might be related to other biological pathways in PCOS. There are several known genes in this region: (1) SCYA25 [44], which encodes a thymus-expressed cytokine; (2) MAP2K7 [45], which encodes a mitogen-activated serine/threonine kinase that activates c-Jun N-terminal kinase in response to activation by growth factors, cytokines and stress; and (3) RETN, which encodes resistin, a recently identified cytokine that is found in adipocytes, is downregulated by thiazolidinediones and induces insulin resistance in rodents [46]. No evidence was found for the association of variants in the RETN gene with hyperandrogenemia, obesity or insulin resistance in PCOS families [47]. Metabolism and reproduction – Syndrome XX In summary, over the past 25 years it has become clear that hyperandrogenemia and anovulation are common findings in women with diverse insulin resistant states, ranging from the syndromes of extreme insulin resistance because of insulin receptor mutations, to lipoatrophic diabetes and PCOS [1]. Increased androgen production might also be
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Fig. 3. Polycystic ovary syndrome (PCOS) has morbidities across the life span of the individual. Girls with premature adrenarche and insulin resistance have evidence for intrauterine growth retardation and are at increased risk for developing PCOS [57,58]. Menstrual irregularity, hirsutism and acne are the major reproductive morbidities in adolescent girls with PCOS. Infertility and endometrial neoplasia are additional complications of PCOS during the reproductive years. Premenopausal women with PCOS have a seven times greater risk for type 2 diabetes mellitus. Women with PCOS frequently have dyslipidemia and other surrogate markers of cardiovascular disease risk. Reproduced with permission from Andrea Dunaif. http://tem.trends.com
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present in women with upper body obesity and insulin resistance. Hyperinsulinemia, secondary to insulin resistance, plays an important role in the pathogenesis of this association by amplifying androgen production via the type 1 IGF receptor in the mutant insulin receptor syndromes or by its cognate receptor in PCOS [1]. However, recent studies indicate that additional factors contribute to this association. Genetic alteration of key components of the insulin signal-transduction pathway, the insulin receptor and IRS-2, results in insulin resistance, anovulation and obesity in mice [48,49]. Because this occurs with targeted disruption of the brain insulin receptor, it suggests that central nervous system insulin signaling is essential for normal reproductive function [48,49]. Nevertheless, not all insulin resistant women have reproductive disturbances and vice versa [1]. Family studies suggest that there is a genetic susceptibility to hyperandrogenemia and insulin resistance in PCOS [16]. Consistent with this hypothesis, there is evidence for linkage and association of marker locus near the insulin receptor with the hyperandrogenemia phenotype [33]. Yet there appear to be differences in ovulation in affected sisters who have similar degrees of hyperandrogenemia and insulin resistance, a finding that suggests that there are additional environmental or genetic factors that modify the expression of the phenotype [31]. The role of androgens in the pathogenesis of PCOS had been discounted because in the adult androgens produce only modest decreases in insulin sensitivity [50]. However, prenatal androgen exposure can produce reproductive and metabolic features of PCOS [51] Androgens can also induce insulin resistance in female rats, and these effects are seen in adulthood after transient neonatal testosterone administration [52]. Taken together, these studies suggest that programming by androgens during development could contribute to the adult PCOS phenotype. The source of these intrauterine androgens remains unknown because maternal androgens are usually completely aromatized by the placenta and do not result in fetal androgen excess [53]. Amniotic fluid androgen levels have been increased in the female offspring of diabetic mothers indicating that the fetal ovary and/or adrenal can produce androgens in the absence of inherited defects in steroidogenesis [54]. The reproductive and metabolic morbidities of PCOS make it an important women’s health problem, with medical consequences across the life-span of the affected individual (Fig. 3). The metabolic syndrome, the constellation of insulin resistance, hypertension, dyslipidemia and visceral adiposity (Box 1), is now recognized as a major risk factor for diabetes and for cardiovascular disease [55,56]. In recognition of the fact that reproductive abnormalities are often part of the syndrome when it occurs in Box 1. Syndrome XX Insulin resistance or type 2 diabetes Dyslipidemia Visceral adiposity Hypertension Anovulation and hyperandrogenemia http://tem.trends.com
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premenopausal women, we propose that in this context, the constellation be christened ‘Syndrome XX’. Acknowledgements This work was supported by NIH grants U54 HD34449 and P50 HD44405 to Andrea Dunaif.
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41 Ehrmann, D.A. et al. (2002) Relationship of insulin receptor substrate-1 and -2 genotypes to phenotypic features of polycystic ovary syndrome. J. Clin. Endocrinol. Metab. 87, 4297 – 4300 42 Urbanek, M. et al. (2000) Allelic variants of the follistatin gene in polycystic ovary syndrome. J. Clin. Endocrinol. Metab. 85, 4455– 4461 43 Tucci, S. et al. (2001) Evidence for association of polycystic ovary syndrome in Caucasian women with a marker at the insulin receptor gene locus. J. Clin. Endocrinol. Metab. 86, 446 – 449 44 Nomiyama, H. et al. (1998) The human CC chemokine TECK (SCYA25) maps to chromosome 19p13.2. Genomics 51, 311 – 312 45 Foltz, I.N. et al. (1998) Human mitogen-activated protein kinase kinase 7 (MKK7) is a highly conserved c-Jun N-terminal kinase/stressactivated protein kinase (JNK/SAPK) activated by environmental stresses and physiological stimuli. J. Biol. Chem. 273, 9344– 9351 46 Steppan, C.M. et al. (2001) The hormone resistin links obesity to diabetes. Nature 409, 307– 312 47 Urbanek, M. et al. (2003) Variation in resistin gene promoter not associated with polycystic ovary syndrome. Diabetes 52, 214 – 217 48 Burks, D.J. et al. (2000) IRS-2 pathways integrate female reproduction and energy homeostasis. Nature 407, 377– 382 49 Bruning, J.C. et al. (2000) Role of brain insulin receptor in control of body weight and reproduction. Science 289, 2122– 2125 50 Moghetti, P. et al. (1996) The insulin resistance in women with hyperandrogenism is partially reversed by antiandrogen treatment: evidence that androgens impair insulin action in women. J. Clin. Endocrinol. Metab. 81, 952– 960 51 Dumesic, D.A. et al. (1997) Prenatal exposure of female rhesus monkeys to testosterone propionate increases serum luteinizing hormone levels in adulthood. Fertil. Steril. 67, 155 – 163 52 Nilsson, C. et al. (1998) Imprinting of female offspring with testosterone results in insulin resistance and changes in body fat distribution at adult age in rats. J. Clin. Invest. 101, 74 – 78 53 Joshi, R. and Dunaif, A. (1995) Ovarian disorders of pregnancy. Endocrinol. Metab. Clin. North Am. 24, 153– 169 54 Barbieri, R.L. et al. (1986) Elevated concentrations of the beta-subunit of human chorionic gonadotropin and testosterone in the amniotic fluid of gestations of diabetic mothers. Am. J. Obstet. Gynecol. 154, 1039– 1043 55 Reusch, J.E. (2002) Current concepts in insulin resistance, type 2 diabetes mellitus, and the metabolic syndrome. Am. J. Cardiol. 90, 19G – 26G 56 Executive summary of the third report of the national cholesterol education program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel III), (2001) JAMA. 285, 2486– 2497 57 Ibanez, L. et al. (2000) Precocious pubarche in girls and the development of androgen excess. J. Pediatr. Endocrinol. Metab. 13, 1261– 1263 58 Ibanez, L. et al. (2000) Recognition of a new association: reduced fetal growth, precocious pubarche, hyperinsulinism and ovarian dysfunction. Ann. Endocrinol. 61, 141– 142
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Polycystic ovary syndrome: Syndrome XX? Susan Sam and Andrea Dunaif Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University, Chicago, IL 60611, USA
Polycystic ovary syndrome (PCOS) is now recognized as an important metabolic and reproductive disorder. It is associated with substantial defects in insulin action and secretion that confer a markedly increased risk for type 2 diabetes mellitus. Insulin resistance modifies reproductive function both by the direct actions of insulin on steroidogenesis and by disruption of insulin signaling pathways in the central nervous system. These insights have led to a new therapy for PCOS with insulin-sensitizing agents. Hyperandrogenemia and insulin resistance cluster in PCOS families, consistent with a genetic susceptibility to these abnormalities. There is evidence for both linkage and association of the hyperandrogenemia phenotype with an allele of a marker locus on chromosome 19, in the region of the gene encoding the insulin receptor. Polycystic ovary syndrome (PCOS) is among the most common endocrine disorders in premenopausal women, and is characterized by increased androgen production and disordered gonadotropin secretion [1]. It is now clear that PCOS is associated with profound insulin resistance and pancreatic b-cell dysfuntion, defects that confer a substantially increased risk for glucose intolerance [2– 5]. Indeed, PCOS is a leading risk factor for impaired glucose tolerance and type 2 diabetes mellitus (T2DM) in adolescent girls as it is in premenopausal women [6,7]. Women with PCOS might also have an increased risk for cardiovascular disease [8]. Investigation of these metabolic abnormalities has revealed that insulin resistance plays an important role in the pathogenesis of the reproductive disturbances of PCOS (Fig. 1). Familial clustering of PCOS is well documented and it is a complex genetic disease reflecting the interplay of genetic and environmental factors [9,10].
GnRH pulse frequency selectively increases LH release [11]. The raised LH levels enhance thecal androgen production, and these androgens are incompletely aromatized into estrogens by the granulosa cells, because of arrested follicular development as a consequence of lowlevel cyclic FSH release. The so-called vicious cycle of PCOS is created, in which disordered gonadotropin secretion begets increased ovarian androgen production, which in turn alters gonadal steroid feedback, perpetuating disordered gonadotropin release (Fig. 1) [1]. Whether the primary defect is in the regulation of gonadotropin secretion or of gonadal steroidogenesis has been debated for decades. Adrenal androgen production is also frequently increased in PCOS [12]. This finding might reflect a common defect in ovarian and adrenal androgen biosynthesis because adrenocorticotropin hormone (ACTH) release is not increased [13]. Increasing evidence suggests that there is a primary defect in ovarian steroidogenesis in PCOS. PCOS theca cells show increased activity of multiple steroidogenic enzymes, resulting in raised androgen production, both Insulin resistance
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Reproductive abnormalities in PCOS The cardinal reproductive features of PCOS are increased androgen production and disordered gonadotropin secretion. Both luteinizing hormone (LH) pulse frequency and amplitude are increased, whereas follicle-stimulating hormone (FSH) levels remain tonically in the midfollicular range [11]. The frequency of gonadotropin-releasing hormone (GnRH) release is increased secondary to decreased sensitivity of the GnRH pulse generator to the negative feedback effects of estradiol and progesterone. This increased Corresponding author: A. Dunaif ([email protected]).
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Fig. 1. The frequency of GnRH release is increased in polycystic ovary syndrome (PCOS), leading to a selective increase in LH relative to FSH secretion. PCOS theca cells have increased activity of multiple steroidogenic enzymes, resulting in raised androgen (A) production, both basally and in response to LH. There is decreased intraovarian aromatization of androgen to estradiol (E2) because follicular maturation is arrested as a result of relatively decreased FSH release. Insulin directly stimulates theca cell steroidogenesis in synergy with LH via its cognate receptor or, in cases of extreme insulin resistance, via the type 1 IGF receptor (IGFR). Abbreviations: FSH, follicle-stimulating hormone, GnRH, gonadotropin-releasing hormone, IGF, insulin-like growth factor, LH, luteinizing hormone. Reproduced with permission from Andrea Dunaif.
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basally and in response to LH [14,15]. This phenotype persists in theca cells propagated in longterm culture, which is suggestive of a genetically determined trait [14,15]. Consistent with this hypothesis, family studies indicate that hyperandrogenemia is the major reproductive phenotype in brothers and sisters of women with PCOS [16,17]. Recent human and animal studies provide several mechanisms by which a primary defect resulting in hyperandrogenemia could cause PCOS. First, the decreased sensitivity of the GnRH pulse generator to gonadal steroid negative feedback appears to be a consequence of raised circulating androgen levels, because androgen receptor blockade with flutamide abolishes this defect in PCOS [18]. Second, studies of rhesus monkeys suggest that prenatal androgen exposure produces many of the features characteristic of the PCOS phenotype in adult females, such as increased LH secretion, ovarian hyperandrogenism, central obesity and defective insulin secretion [19]. Third, permanent alterations in LH secretion are demonstrable in women who were exposed to excess androgens during in utero development, such as women with congenital adrenal hyperplasia or with neonatal androgensecreting neoplasms [12]. Metabolic abnormalities in PCOS Insulin resistance is a prominent feature of PCOS, independent of obesity [1 –5]. Many but not all women with PCOS are insulin resistant. Obesity and PCOS have an additive deleterious effect on insulin sensitivity [2]. The molecular mechanisms of this defect differ from those in other common insulin resistant conditions, such as T2 DM and obesity, suggesting that PCOS-related insulin resistance has a distinct genetic etiology [1]. Studies of PCOS adipocytes suggest that there is a post-binding defect in insulin receptor-mediated signal transduction, and this observation has recently been confirmed in skeletal muscle, the major site of insulin-mediated glucose uptake (Fig. 2) [1,20]. Studies of insulin receptors isolated from PCOScultured skin fibroblasts suggested that the signaling defect results from a decrease in insulin receptor tyrosine kinase activity, secondary to a constitutive increase in receptor serine phosphorylation [1]. Mixing and immunopurification studies suggest that a factor extrinsic to the insulin receptor is responsible for the serine phosphorylation [1]. Recent studies have suggested that a serine kinase accounts for this finding by demonstrating that a non-specific serine kinase inhibitor normalizes insulin receptor tyrosine autophosphorylation in cultured skin fibroblasts from PCOS patients [21]. The signaling defect produces selective insulin resistance, affecting metabolic but not mitogenic actions of insulin [22]. Insulin acts through its cognate receptor, in synergy with LH, to stimulate theca cell steroidogenesis in PCOS (Fig. 1) [23]. Hence, it is possible that the selective insulin resistance of PCOS accounts for the continued actions of insulin on steroidogenesis, in spite of defects in insulin-mediated glucose metabolism. Insulin also contributes to increased adrenal androgen secretion in PCOS, in part by enhancing adrenal sensitivity to ACTH [24]. Ek and colleagues have identified fat depot-specific abnormalities in the regulation of lipolysis in PCOS [25]. http://tem.trends.com
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Fig. 2. Insulin resistance in some polycystic ovary syndrome (PCOS) tissues (e.g. skin fibroblasts) results from the constitutive serine phosphorylation of the insulin receptor secondary to an as yet unidentified serine kinase. The insulin resistance is selective, affecting the metabolic but not mitogenic actions of insulin. Serine phosphorylation of P450c17 (CYP 17), the rate-limiting enzyme for androgen biosynthesis, increases its activity. It is possible that the same serine kinase phosphorylates the insulin receptor, producing insulin resistance, and P450c17, producing hyperandrogenemia. Cytokines and free fatty acids (FFA) can activate intracellular serine kinases and might play a role in the pathogenesis of PCOS. It is possible that the serine kinase phosphorylates downstream signaling molecules, such as insulin receptor substrates (IRS) 1 or 2, further compromising insulin signaling. Minus symbols (2) denote serine phosphorylation, which inhibits signal transduction. Reproduced with permission from Andrea Dunaif.
Isolated subcutaneous abdominal adipocytes are resistant to catecholamine-induced lipolysis, whereas the opposite phenomenon, markedly enhanced sensitivity, is observed in visceral adipocytes from women with PCOS [26]. These abnormalities are independent of obesity. Analogous to the defects in glucose metabolism, the molecular mechanisms of the lipolytic abnormalities in PCOS are unique. In contrast to adrenoceptor-mediated alterations in lipolysis in the metabolic syndrome, in PCOS there is a postreceptor increase in protein kinase A activity [25]. The increase in visceral fat lipolysis could lead to an increase in free fatty acid (FFA) release directly into the portal circulation. Portal FFA levels are major positive modulators of hepatic glucose production [27]. Accordingly, enhanced visceral fat lipolysis could be one mechanism for the increased risk for glucose intolerance in PCOS [28]. Prenatal androgen administration can increase visceral adiposity and alter lipolysis in animals [29]. The possible role of androgens in the pathogenesis of the lipolytic defects in PCOS merits further investigation. Increased serine kinase activity: a unifying hypothesis for PCOS? Serine phosphorylation of the rate-limiting enzyme for androgen biosynthesis, P450c17 (CYP17), increases its activity (Fig. 2) [1]. This observation led to the parsimonious hypothesis that the same kinase was responsible for serine phosphorylation of the insulin receptor, producing insulin resistance, and P450c17-a, producing hyperandrogenism in PCOS [1]. The one study that has tested this hypothesis by transfecting P450c17 into skin fibroblasts
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failed to detect increased serine phosphorylation of P450c17 in PCOS fibroblasts with increased serine kinase activity [30]. Nevertheless, this study only fails to support rather than disprove the serine kinase hypothesis, because fibroblasts are not steroidogenic cells and might have lacked essential cofactors for phosphorylation of P450c17. Further investigation of this hypothesis is clearly warranted, and genes encoding serine kinases are highpriority candidate genes for PCOS. Phenotypes in PCOS families: both men and women are affected Familial clustering of PCOS is well documented, suggesting that there is a genetic susceptibility to the disorder [10,16,17,31]. It was found that , 40% of premenopausal sisters of women with PCOS have elevations of total or biologically available testosterone levels [16]. There are two phenotypes in the affected sisters: (1) raised testosterone levels with irregular menses (classic PCOS); and (2) raised testosterone levels with regular menstrual cycles [16]. Although ovulation was not documented in the sisters with hyperandrogenemia and regular menses, their fecundity was significantly greater than that in sisters with the PCOS phenotype and similar to that seen in unaffected sisters, suggesting that the hyperandrogenemic sisters had ovulatory cycles [16]. Dehydroepiandrosterone sulfate (DHEAS) levels were also raised in affected sisters, suggesting that there was an adrenal component to hyperandrogenemia [16]. A possible candidate gene to account for this finding would be one that regulated both ovarian and adrenal steroidogenesis. Androgen levels were similarly raised in sisters with classic PCOS and those with hyperandrogenemia and regular menses [16]. Sisters with classic PCOS and those with hyperandrogenemia and regular menses both had similarly raised markers of insulin resistance, whereas sisters who had normal circulating androgen levels and menstrual cycles (i.e. unaffected sisters) had no evidence of insulin resistance [31]. This study also demonstrates that hyperandrogenemia and markers of insulin resistance track together in affected sisters, suggesting that these findings might have a common pathogenesis or might be the result of closely linked genes [31]. Furthermore, these findings suggest that using the hyperandrogenemia phenotype in linkage studies will also identify genes associated with insulin resistance. There have been limited studies of male relatives in PCOS families and few of these have used concurrently studied control subjects. Several studies suggested that premature male pattern balding was a male phenotype, but more comprehensive studies of male relatives have questioned this [9]. A large group of brothers of women with PCOS were found to have significantly raised DHEAS levels compared with control men [17]. Furthermore, DHEAS levels were significantly correlated between the brothers and their proband sisters with PCOS, consistent with a heritable trait. Testosterone levels were not raised in brothers, as they were in the proband sisters with PCOS; however, testosterone secretion in men is tightly regulated by hypothalamic – pituitary feedback of androgens [17]. It is also possible that subtle differences in http://tem.trends.com
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testosterone levels escaped detection. The elevation in adrenal androgen DHEAS suggests that affected brothers might have the same putative defect in steroidogenesis as affected sisters do [16]. There was no increased prevalence of balding in the brothers of women with PCOS [17]. The endocrine syndrome and polycystic ovarian morphology The significance of polycystic ovarian morphology has been debated for decades. Polycystic ovaries (PCO) also cluster in families, consistent with a heritable trait, and anovulatory women with PCO can have ovulatory sisters with PCO [9,16]. The probands and their sisters with PCO have similarly raised androgen concentrations (Rush, K. et al. (2002) Serum androgen and insulin concentrations in probands and affected sisters with polycystic ovaries. Presented at the 84th Annual Meeting of the Endocrine Society, San Francisco, California, USA) analogous to the finding of sisters with hyperandrogenemia and regular menses. The presence of these ovulatory (hyperandrogenemia and/or PCO alone) and anovulatory (PCOS) reproductive phenotypes within one family suggests that such phenotypes reflect variable expression of the same genetic trait. Most women with PCO have hyperandrogenemia and raised LH levels [1]. Functional ovarian hyperandrogenism can be unmasked with long-acting GnRH analogs in women with PCO who have normal basal androgen levels [32]. Theca cells from women with PCO consistently secrete increased androgens, independent of ovulatory function or circulating androgen levels [14]. Taken together, these findings suggest that PCO are intrinsically abnormal. The family studies suggest that PCO and hyperandrogenemia are heritable traits. We propose that hyperandrogenemia is the biochemical correlate of PCO. Genetic analyses of PCOS There have been no genome-wide screens of PCOS, and all reported genetic studies have used a candidate gene approach, in which genes are selected for analysis based on known pathophysiology. Plausible candidate genes for PCOS include those encoding proteins involved in steroid hormone biosynthesis, gonadotropin secretion or action, obesity and energy regulation, and insulin action [33]. Most studies have examined the association between PCOS and its intermediate phenotypes, such as androgen levels, PCO or markers of insulin resistance, and candidate gene variants in cases compared with control populations or within groups of PCOS women, to assess genotype– phenotype correlations [10,34]. There have been few linkage and family-based association studies of PCOS because both require large samples of families with two or more affected relatives for linkage analyses. These studies are not confounded by population stratification because they are performed in related individuals [35]. False positive results are still problematic and statistical adjustments should be made for testing multiple alleles [36]. Non parametric linkage analyses that make no assumption about the mode of inheritance are appropriate because PCOS is probably a complex genetic disorder [9,10]. The phenotypes that have been used in these family
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studies have been the PCO/premature male pattern balding phenotype in a series of , 20 families from the UK and the PCOS/hyperandrogenemia phenotype in studies from USA [33,37]. Very few candidate genes or chromosomal regions have been confirmed in two or more independent studies and therefore remain of particular interest given the limitations of these genetic analyses. The gene encoding P450 cholesterol side chain cleavage (CYP11a) has been linked to PCO/premature male pattern balding phenotype in PCOS families [38]. A family-based association study supported this finding by demonstrating a nominally significant association between a marker in the region of the CYP11a gene and the PCOS/hyperandrogenemia phenotype [33]. Association studies have suggested that a variant in the promoter of this gene is associated with circulating testosterone levels in women with PCOS [39]. The variable number tandem repeats (VNTR) locus upstream of the gene encoding insulin (INS), which might regulate INS expression, has been linked to the PCO/premature male pattern balding phenotype [37]. Family-based studies have also supported the association of the INS gene VNTR with this phenotype and with insulin levels in affected women [37]. Two genotype– phenotype studies have found an association with a variant in the gene encoding insulin receptor substrate-1 (IRS1) and parameters of insulin action in women with PCOS [40,41]. However, all of these studies have been constrained by small sample sizes. Evidence has been found to link the gene encoding follistatin (FST) to the PCOS/hyperandrogenemia phenotype in an affected sibling pair analysis [33]. However, further studies failed to identify variants in the FST gene associated with this phenotype, suggesting that follistatin does not play a major role in the pathogenesis of PCOS [42]. Prenatal
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A marker locus on chromosome 19p located near the gene encoding the insulin receptor (INSR) has been found that shows both association and linkage with the PCOS/hyperandrogenemia phenotype [33] (Urbanek, M. et al. (2002) Polycystic ovary syndrome candidate gene region on chromosome 19p13. Presented at the 84th Annual Meeting of the Endocrine Society, San Francisco, California, USA). An independent case– control association study supported this finding [43]. The marker maps to a 1 Mb region that is centromeric to the INSR gene, and linkage disequilibrium is usually maintained only over much smaller regions [33]. Thus, it is probable that a gene other than INSR accounts for the association with this marker. It remains possible that such a gene is still involved in insulin action. Alternatively, this gene might be related to other biological pathways in PCOS. There are several known genes in this region: (1) SCYA25 [44], which encodes a thymus-expressed cytokine; (2) MAP2K7 [45], which encodes a mitogen-activated serine/threonine kinase that activates c-Jun N-terminal kinase in response to activation by growth factors, cytokines and stress; and (3) RETN, which encodes resistin, a recently identified cytokine that is found in adipocytes, is downregulated by thiazolidinediones and induces insulin resistance in rodents [46]. No evidence was found for the association of variants in the RETN gene with hyperandrogenemia, obesity or insulin resistance in PCOS families [47]. Metabolism and reproduction – Syndrome XX In summary, over the past 25 years it has become clear that hyperandrogenemia and anovulation are common findings in women with diverse insulin resistant states, ranging from the syndromes of extreme insulin resistance because of insulin receptor mutations, to lipoatrophic diabetes and PCOS [1]. Increased androgen production might also be
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Fig. 3. Polycystic ovary syndrome (PCOS) has morbidities across the life span of the individual. Girls with premature adrenarche and insulin resistance have evidence for intrauterine growth retardation and are at increased risk for developing PCOS [57,58]. Menstrual irregularity, hirsutism and acne are the major reproductive morbidities in adolescent girls with PCOS. Infertility and endometrial neoplasia are additional complications of PCOS during the reproductive years. Premenopausal women with PCOS have a seven times greater risk for type 2 diabetes mellitus. Women with PCOS frequently have dyslipidemia and other surrogate markers of cardiovascular disease risk. Reproduced with permission from Andrea Dunaif. http://tem.trends.com
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present in women with upper body obesity and insulin resistance. Hyperinsulinemia, secondary to insulin resistance, plays an important role in the pathogenesis of this association by amplifying androgen production via the type 1 IGF receptor in the mutant insulin receptor syndromes or by its cognate receptor in PCOS [1]. However, recent studies indicate that additional factors contribute to this association. Genetic alteration of key components of the insulin signal-transduction pathway, the insulin receptor and IRS-2, results in insulin resistance, anovulation and obesity in mice [48,49]. Because this occurs with targeted disruption of the brain insulin receptor, it suggests that central nervous system insulin signaling is essential for normal reproductive function [48,49]. Nevertheless, not all insulin resistant women have reproductive disturbances and vice versa [1]. Family studies suggest that there is a genetic susceptibility to hyperandrogenemia and insulin resistance in PCOS [16]. Consistent with this hypothesis, there is evidence for linkage and association of marker locus near the insulin receptor with the hyperandrogenemia phenotype [33]. Yet there appear to be differences in ovulation in affected sisters who have similar degrees of hyperandrogenemia and insulin resistance, a finding that suggests that there are additional environmental or genetic factors that modify the expression of the phenotype [31]. The role of androgens in the pathogenesis of PCOS had been discounted because in the adult androgens produce only modest decreases in insulin sensitivity [50]. However, prenatal androgen exposure can produce reproductive and metabolic features of PCOS [51] Androgens can also induce insulin resistance in female rats, and these effects are seen in adulthood after transient neonatal testosterone administration [52]. Taken together, these studies suggest that programming by androgens during development could contribute to the adult PCOS phenotype. The source of these intrauterine androgens remains unknown because maternal androgens are usually completely aromatized by the placenta and do not result in fetal androgen excess [53]. Amniotic fluid androgen levels have been increased in the female offspring of diabetic mothers indicating that the fetal ovary and/or adrenal can produce androgens in the absence of inherited defects in steroidogenesis [54]. The reproductive and metabolic morbidities of PCOS make it an important women’s health problem, with medical consequences across the life-span of the affected individual (Fig. 3). The metabolic syndrome, the constellation of insulin resistance, hypertension, dyslipidemia and visceral adiposity (Box 1), is now recognized as a major risk factor for diabetes and for cardiovascular disease [55,56]. In recognition of the fact that reproductive abnormalities are often part of the syndrome when it occurs in Box 1. Syndrome XX Insulin resistance or type 2 diabetes Dyslipidemia Visceral adiposity Hypertension Anovulation and hyperandrogenemia http://tem.trends.com
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premenopausal women, we propose that in this context, the constellation be christened ‘Syndrome XX’. Acknowledgements This work was supported by NIH grants U54 HD34449 and P50 HD44405 to Andrea Dunaif.
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