Genomic variants in polycystic ovary syndrome

Clinica Chimica Acta 366 (2006) 14 – 26 www.elsevier.com/locate/clinchim

Invited critical review

Genomic variants in polycystic ovary syndrome Manuel Luque-Ramı´rez a,1, Jose´ Luis San Milla´n b, He´ctor F. Escobar-Morreale a,* a

Department of Endocrinology, Hospital Ramo´n y Cajal, Carretera de Colmenar km 9 V1, E-28034 Madrid, Spain b Department Molecular Genetics, Hospital Universitario Ramo´n y Cajal, Madrid, Spain Received 7 September 2005; received in revised form 17 October 2005; accepted 20 October 2005 Available online 7 December 2005

Abstract The polycystic ovary syndrome (PCOS) is a common disorder in premenopausal women, characterized by the presence, among other traits, of hyperandrogenism, insulin resistance, and hyperinsulinism. The familial aggregation of PCOS lead the interest to the molecular genetic basis of this syndrome, especially to the genes encoding proteins involved in androgen synthesis and the regulation of insulin synthesis and action. Considering the relationship between insulin resistance and chronic inflammation, and the clustering of inflammatory markers in PCOS patients, recent studies focused on the involvement of proinflammatory genotypes on the pathogenesis of PCOS. Mounting evidence suggest at present a complex model of inheritance for PCOS, in which predisposing and protecting genomic variants interact with environmental factors such as obesity and a sedentary lifestyle, finally leading to the classic phenotype of this syndrome. Moreover, the association of hyperandrogenism, insulin resistance and chronic inflammation raised the possibility of an increase risk of cardiovascular disease in women suffering from PCOS. In the present review we will summarize the most important findings published to date regarding the molecular genetic mechanisms underlying the association of PCOS with insulin resistance and chronic inflammation, and the possible interaction of these mechanisms with environmental factors. D 2005 Elsevier B.V. All rights reserved. Keywords: Polycystic ovary syndrome; Hyperandrogenism; Insulin resistance; Diabetes; Obesity; Inflammation; Molecular genetics

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . The PCOS genotype from an evolutionary perspective . . . . . . Molecular genetics of the polycystic ovary syndrome . . . . . . . 3.1. Genes related to insulin resistance . . . . . . . . . . . . . 3.2. Hyperandrogenism . . . . . . . . . . . . . . . . . . . . . 3.3. Chronic inflammation and prothrombosis. . . . . . . . . . 4. A unifying hypothesis explaining the results obtained to date (Fig. 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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14 15 15 15 17 19 20 21 21 21

1. Introduction * Corresponding author. Tel./fax: +34 91 336 9029. E-mail address: [email protected] (H.F. Escobar-Morreale). 1 Present Address: Hospital Universitario de La Princesa, Madrid, Spain. 0009-8981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2005.10.017

The polycystic ovary syndrome is a disorder characterized by signs and symptoms of hyperandrogenism and oligo- or amenorrhea, and is frequently associated with

M. Luque-Ramı´rez et al. / Clinica Chimica Acta 366 (2006) 14 – 26

insulin resistance [1,2]. The familial aggregation of the functional hyperandrogenism [3,4] and the metabolic traits associated with PCOS [5 –7] suggested an inherited basis for these disorders. At present PCOS, as occurs with other common metabolic disorders such as type 2 diabetes mellitus, is considered a complex metabolic disorder in terms of inheritance and genetics [8]. These common disorders apparently result from the interaction of several genes that exert minor effects on the phenotype, with environmental disorders that trigger or favor the occurrence of the disorder in a certain subject [8– 10]. To date, most studies pertaining the molecular genetics of PCOS applied a candidate gene approach, using both family-based and case-control studies, and focusing on genes and genomic variants involved in androgen and insulin synthesis, secretion, and action. Based on the evidence supporting an important role of chronic inflammation on insulin resistance, the metabolic syndrome, and cardiovascular disease [11,12], we and others have also studied the possible involvement of inflammatory genotypes and phenotypes in the pathogenesis of PCOS during the past years [13 –21]. As will be seen, most if not all the associations found between PCOS and genomic variants have not been confirmed in populations with different ethnic and geographic backgrounds [8]. This lack of confirmation may be explained by the heterogeneity in the methods applied in the different studies, and especially by the different diagnostic criteria used to define the PCOS phenotype. Yet it is possible that environmental influences involved in the development of PCOS such as diet and lifestyle, which are heavily influenced by ethnicity [10,22 –25], have also played a role in the discrepancies observed between studies conducted in different populations, because the genomic variants associated with PCOS may also vary depending on the different environmental factors involved in its development [8]. In the present review we will summarize the most important findings published to date regarding the molecular genetic mechanisms underlying the association of PCOS with insulin resistance and chronic inflammation, and the possible interaction of these mechanisms with environmental factors. If a more detailed description of the many molecular genetic studies conducted to date is demanded, the reader should consider a more complete review such as Escobar-Morreale et al. [8].

2. The PCOS genotype from an evolutionary perspective The high prevalence of PCOS in all Western populations studied to date, in the range of 6 –7% [26 –28], suggest that the genes involved in the pathogenesis of this disorder might have been selected during evolution because a survival advantage of the hyperandrogenic phenotype [8], as has been proposed for other complex metabolic disorders such as type 2 diabetes [29].

15

For ages, human beings were nomads and hunters, suffered prolonged periods of food shortage, and gestation and birth were one of the most important causes of morbimortality in women. In this hostile environment, hyperandrogenism and insulin resistance possibly favored survival [30,31]. On the one hand, androgen excess caused an assertive behavior, and resulted in a relative infertility in affected women, decreasing the birth rate and increasing the interval between pregnancies, thereby favoring maternal and infant survival. On the other, insulin resistance facilitated a thrifty shift of intermediate metabolism to provide the brain with enough glucose for its correct functioning, and contributed to weight gain and increased fuel storage in fat tissue in the rare periods in which food was available. The sudden change in the lifestyle occurred during the last century in most Western Countries, where access to food is not restricted anymore, persons seldom exercise and the life-expectancy has increased markedly because of the improvement in public hygiene and healthcare, turned these previously beneficial mechanisms in a considerable disadvantage. Carriers of hyperandrogenic and insulin resistant genotypes are at present prone to the development of obesity and the metabolic syndrome, finally leading to atherosclerosis and cardiovascular disease [29 – 31].

3. Molecular genetics of the polycystic ovary syndrome Considering that almost all the molecular genetics studies of PCOS conducted to date have used a candidate gene approach, we will review these studies by grouping them depending on the rationale justifying its possible relationship with this disorder. 3.1. Genes related to insulin resistance Insulin resistance and hyperinsulinemia are frequent findings in lean and obese PCOS patients [32 – 36]. The mechanisms by which hyperinsulinemia induces hyperandrogenism are summarized in Table 1 [2]. Among others, insulin may increase the secretion and pulsatility of gonadotropins [37]; the sensitivity of the adrenal androgen secretion to ACTH [38]; the ovarian androgen secretion in response to LH stimulation [39]; the ovarian and adrenal Table 1 Mechanisms involved in the enhancement of androgen synthesis and action by insulin Increased synthesis and pulsatility of LH Increased adrenal sensitivity to ACTH Increased theca cell sensitivity to LH Increased adrenal and ovarian 17a-hydroxylase/17,20-lyase activity Reduced IGFBP-1 levels Upregulation of ovarian IGF-I receptors Ovarian enlargement and cyst development in synergy with LH and bhCG in animal studies Inhibition of hepatic SHBG synthesis

16

M. Luque-Ramı´rez et al. / Clinica Chimica Acta 366 (2006) 14 – 26

activity of the key enzymes for androgen synthesis, 17ahydroxylase and 17,20-lyase [38,40]. Yet also, hyperinsulinemia favors hyperandrogenism by indirect mechanisms: 1) Insulin decreases insulin-like growth factor binding protein 1 (IGFBP-1), thereby increasing insulin-like growth factor-1 availability in target tissues, and also up-regulate the expression of ovarian insulin-like growth factor-1 receptors [39]; 2) Insulin favors the development of ovarian cysts and ovarian enlargement acting synergistically with hCG and LH in animal models [39]; and 3) Insulin decreases the hepatic synthesis of sexual hormone binding globulin (SHBG), leading to an increase in free androgen concentrations in the circulation [41,42]. Insulin resistance and its associated metabolic disorders aggregate within the families of PCOS patients. Givens et al. [4,43,44] conducted a series of studies suggesting that the prevalence of diabetes, dyslipidemia, hypertension and atherosclerosis was increased in families of women with ovarian hyperandrogenism. More recently, Legro et al. [5] reported a high prevalence of insulin resistance in sisters of PCOS patients, findings that were expanded by the later results of Yildiz et al. [6] showing that this metabolic trait was not restricted to the sisters of these patients, but also to the brothers of this women, and was accompanied by disorders of glucose tolerance. Furthermore, the hyperandrogenic sisters of PCOS patients have an increased incidence of dyslipidemia and the metabolic syndrome [45]. These findings support that genes related to insulin and its actions should be considered candidates to explain the inheritance of PCOS and hyperandrogenism. In PCOS, a primary defect in insulin secretion, indicating pancreatic h-cell dysfunction, has a familial component and is associated with the occurrence of diabetes [46,47]. Accordingly, the insulin gene (INS), located at 11p15.5, has been studied in PCOS. Most studies focused on a variable number of tandem repeats (VNTR) polymorphism (INS VNTR) previously described to influence the genetics of type 1 diabetes [48], consisting in the repeat of 14– 15 bp at 596 from the transcription start site of INS [49]. Depending on the number of repeats, alleles are classified as I, II and III, although class II alleles are rare in Caucasians. Waterworth et al. [50] reported positive linkage and association between homozygosity for class III alleles, PCOS and serum testosterone levels in Caucasian women from the United Kingdom. This results were initially confirmed by others [51], although there were also discrepant studies [52,53]. Very recently, the same authors reporting the initial evidence for association between INS VNTR polymorphism and polycystic ovary syndrome published a rebuttal based on a series of familybased and case-control studies in British and Finnish populations [54]. In conceptual agreement with the presence of insulin resistance in PCOS, initial studies focused on the insulin receptor gene (INSR) located at chromosome 19. Despite the finding of increased phosphorylation of serine residues of

the tyrosin – kinase domain of the insulin receptor both in vitro and in vivo [55,56], no abnormalities have been found in this domain of INSR [57]. More recently, a single nucleotide polymorphism (SNP) consisting in a C/T substitution at the tyrosine kinase domain of INSR in exon 17 has been found in association with PCOS in women from the USA [58], a finding confirmed also in the Chinese population [59]. The possible association of PCOS with another genetic marker located relatively close to the INSR locus, D19S884, resulted in conflicting results between USA and Mediterranean populations [60 –63]. Nevertheless, it must be highlighted that the whole INSR of PCOS patients has been sequenced and no significant abnormalities have been found [64,65]. After insulin binding to the insulin receptor, and the autophosphorylation of the latter, the signal is transmitted by the phosphorylation of the insulin receptor substrates 1 (IRS-1) and 2 (IRS-2). PCOS patients present with a decreased activity of an IRS-1 phosphatydil– inositol 3 kinase (PI3K), apparently related to increased phosphorylation of Ser312 residues. This abnormality is different compared with those found in other disorders of insulin resistance [66]. Several studies have shown that allelic variants in the insulin-receptor substrate genes, IRS-1 Gly972Arg and IRS2 Gly1057Asp, may play a functional role on the insulinresistant component of PCOS [67 –69]. Sir-Petermann et al. [67] reported that the frequency of the IRS-1 Arg972 allele was higher in PCOS patients, compared to normal women, in the Chilean population. El Mkadem et al. [68], studying Caucasian women of European extraction, did not found any difference between PCOS patients and controls in the distribution of IRS-1 Gly972Arg and IRS-2 Gly1057Asp alleles. The IRS-1 Arg972 allele was more prevalent in insulin-resistant PCOS patients compared with non-insulin resistant patients or control subjects [68]. Carriers of IRS-1 Arg972 alleles presented with increased fasting insulin levels and increased insulin resistance measured by homeostasis model assessment (HOMA-IR), compared with subjects homozygous for wild-type alleles [68]. Also, carriers of IRS-2 Asp1057 alleles presented with increased 2-h glucose and insulin levels during an oral glucose tolerance test (OGTT) [68]. Finally, HOMA-IR was higher in carriers of both IRS-1 and IRS-2 variants than in those with IRS-2 mutations only or those with wild-type alleles [68]. On the contrary, Ehrmann et al. [69] in their study of 227 non-diabetic PCOS white and African-American patients, found that non-diabetic subjects carrying one or two Asp1057 alleles of IRS-2 had significantly lower 2-h OGTT glucose levels compared with those homozygous for Gly1057 alleles [69], in sharp contrast with the results of El Mkadem et al. [68]. Our recent results in the Spanish population [70] confirm that these polymorphisms are equally distributed among

M. Luque-Ramı´rez et al. / Clinica Chimica Acta 366 (2006) 14 – 26

PCOS patients and controls but, when considering control subjects and PCOS patients as a whole, IRS-1 Arg972 carriers also presented with increased fasting insulin and insulin resistance measured by homeostasis model assessment compared with subjects homozygous for Gly972 alleles, whereas subjects homozygous for the Gly1057 allele of IRS-2 presented with increased glucose levels during an oral glucose tolerance test compared with carriers of one or two Asp1057 alleles. Therefore, the Gly972Arg in IRS-1 and Gly1057Asp in IRS-2 polymorphisms influence glucose homeostasis in premenopausal women, but not specifically in women with PCOS. Finally, a number of genes related in one way or another to insulin and/or insulin resistance have been also studied in PCOS, with still irreproducible or controversial results. These genes are summarized in Table 2. 3.2. Hyperandrogenism Functional hyperandrogenism, mostly of ovarian origin [1] but with a significant adrenal contribution in as many as 20 –30% of patients [87], is the mainstay of PCOS. As occurs with insulin resistance, there is familial aggregation of hyperandrogenism in PCOS [5,88] and, although the male equivalent for PCOS is still matter of debate [89,90], this finding further suggests that an inherited component contributes to this disorder. The gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate ovarian steroidogenesis and follicular development. Up to 40% of PCOS

17

patients have increased LH levels that may favor androgen synthesis and secretion by ovarian theca cells [91], although the serum levels of gonadotropins, because their pulsatile secretion and broad changes during the menstrual cycle, are inaccurate diagnostic markers of PCOS [1]. Gonadotropins are complex glycoproteins that share a a subunit with thyroid-stimulating hormone, and have a h subunit that confers biological specificity. The Trp8Arg and Ile15Thr variants in the LHh gene decrease LH activity in vivo [92] and have been studied in PCOS: The variant LH might protect obese women against the development of PCOS [93] although this particular result has not been confirmed universally [94]. Other point mutations in LHh and FSHh genes influenced menstrual dysfunction in the Japanese and in the Chinese in some but not all the studies conducted to date [95 – 98]. The key enzyme for adrenal and ovarian androgen synthesis is P450c17a, which has the 17a-hydroxylase and 17,20-lyase activities needed for the synthesis of dehydroepiandrosterone (DHEA) and androstenedione. The gene encoding P450c17a, CYP17, is overexpressed in primary cultures of theca cells from PCOS patients, showing increased mRNA stability [99 – 102]. Further studies of these cultures of theca cells demonstrated that alterations in the mitogen-activated protein kinase pathway [103] and in the repression of the promoter of CYP17 by nuclear factor-1C [104] are involved in the pathogenesis of the excessive ovarian androgen production in these cells, and that retinoic acid, and overexpression of the GATA6 factor as studied by DNA microarrays, may influence CYP17 expression

Table 2 Studies in PCOS involving other insulin-related genomic variants Gene

Polymorphism

Phenotype

Reference

IGF-2 IGF-IR PPAR-c2

ApaI* Trinucleotide repeat. Pro12Ala-

Paraoxonase (PON-1)

108C/T Leu55Met Thr228Ala‘ UCSNP-43,-19,-63 UCSNP-43,-45 UCSNP-44P 45 T/G**

PCOS Increased fasting glucose and insulin resistance Body mass index Lower insulin resistance PCOS Obesity and lower insulin resistance Lower insulin resistance and hirsutism score PCOS Obesity and insulin resistance Obesity PCOS and insulin levels Hirsutism score and idiopathic hirsutism PCOS Androstenedione PCOS Insulin resistance Obesity and insulin resistance Lower adiponectin levels

[71] [71] [72] [73] [74] [75] [76] [71] [71] [71] [77] [78] [79,80] [81] [81] [82] [82] [82]

SORBS1 Calpain-10

Adiponectin

276 G/T**

These associations have not been confirmed in other studies, as follows: *No linkage with PCOS [60]. . No linkage with PCOS [60]. No association with PCOS [71,83] or with circulating adiponectin levels in PCOS patients [84]. ‘ No association with premature pubarche, functional hyperandrogenism or obesity in children and adolescents [85]. P No association with PCOS, insulin levels, idiopathic hirsutism, or functional hyperandrogenism [78,86]. ** No association with PCOS [71].

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[105,106]. Moreover, increased phoshorylation of serine residues of P450c17a, as described for the INSR, has been also proposed to explain the increased enzyme activity [107 –109]. However unfortunately, the search for mutations and genomic variants in CYP17 associated with PCOS has been mostly negative [110 – 117]. The rate-limiting step in adrenal and ovarian steroidogenesis is the conversion of cholesterol into pregnenolone by the enzyme P450scc, after introduction of cholesterol into the mitochondria by the sterodogenic acute regulatory protein (StAR). P450scc is encoded by a gene termed CYP11A, and its expression is increased in the theca cell cultures from PCOS patients described above [99,100]. The CYP11A promoter contains a VNTR consisting in 4, 6, 8 or 9 repeats of the TTTTA pentanucleotide in Caucasians. Gharani et al. [118] reported that the absence of the more common 4 repeats allele was associated with PCOS and serum testosterone levels in Caucasians, and this was initially followed by confirmatory reports by two independent groups [119,120] but not by others [60,121]. Moreover, our search in a group of hyperandrogenic women for mutations in the complete CYP11A, as well as in the SF-1, DAX-1 and StAR genes which are also involved in the regulation of CYP11A, yielded negative results [122]. Very recently, as happened with the association of PCOS with the INS VNTR [50], the UK group originally reporting the positive association of PCOS with the CYP11A VNTR published a rebuttal, based on their studies conducted in the UK and Finnish populations, concluding that the associations between CYP11A promoter variation and androgenrelated phenotypes had been substantially overestimated in previous studies. Although initial studies of the adrenal responses to ACTH in hyperandrogenic women suggested a role for heterozygous mutations and nonclassic deficiencies in the genes encoding other steroidogenic enzymes such as 21ahydroxylase (CYP21) or 3h-hydroxysteroid dehydrogenase (HSD3B2) [123,124], at present there is mounting evidence against a significant role of these genes in PCOS [60,125 –127]. Because its central role in the generation of testosterone from androstenedione, the gene encoding 17h-hydroxysteroid dehydrogenase type III (HSD17B3) has been considered a candidate gene, but no association with PCOS has been reported to date [60,128]. Recent studies considered candidate genes those encoding 11h-hydroxysteroid dehydrogenase type 1 (HSD11B1) and hexose-6-phosphate dehydrogenase (H6PD), because of their apparent involvement in the inheritance of cortisonereductase deficiency (CRD). This is a very rare disorder in which affected women present with a PCOS-like clinical picture, and is characterized by a decreased conversion of cortisone into cortisol –cortisone reduction—that can be clinically diagnosed by the finding of very low urinary ratios of tetrahydrocortisol plus allo-tetrahydrocortisol to

tetrahydrocortisone, usually below 0.05 [129]. The widely accepted hypothesis, although not supported by direct experimental evidence, is that the reduced generation of cortisol in target tissues leads to stimulation of the pituitary –adrenal axis, resulting in adrenal hyperandrogenism and PCOS-like symptoms [129]. Draper et al. [130] proposed a bigenic triallelic model of inheritance for CRD involving certain variants in the HSD11B1 and H6PD genes. According to their hypothesis, this occurs because the mutations in HSD11B1 result in reduced enzyme expression, and because the mutations in H6PD impair the generation of NADPH within the lumen of the endoplasmic reticulum, because NADPH might be essential in driving 11ß-HSD1 oxo-reductase activity [130]. The study of the families of three cases of CRD showed that affected individuals were characterized by the concurrent presence of a homozygous and a heterozygous mutation in exon 5 of H6PD (R453Q or 620ins29bp621 variants) and in intron 3 of HSD11B1 (83557insA or 83597T/G variants) [130], and concluded that both genes should be considered candidate genes for PCOS given the resemblances of CRD with this syndrome. However, subsequent studies, including one from our group, cast doubt about this model being the actual cause of CRD, given that these bigenic triallelic genotypes were present in both healthy and PCOS women in prevalences much higher that those of CRD, and these women had completely normal ratios of urinary cortisol metabolites [131,132]. Furthermore, in our study the H6PD variant had a protective role against hyperandrogenism, given that it was more frequent in healthy women compared with PCOS patients, and was associated with amelioration of adrenal hyperactivity [131]. The actual mechanisms underlying these associations remain to be established. But aside from studies focused on factors involved in the synthesis of androgens, molecular genetic studies of PCOS also included factors involved in androgen transport to target tissues, and their local metabolism and action. The major transporter of androgen in serum is sex hormone-binding globulin (SHBG), which regulates the amount of testosterone available to target tissues. Initial reports of hyperandrogenism related to a single-nucleotide deletion within exon 8 of the SHBG gene, producing a reading-frame shift within the codon for E326 and a premature termination codon, raised expectations about the possible role of this particular mutation in the pathogenesis of PCOS, yet it was seldom found in unselected PCOS patients [133]. More recently, two different groups of researchers have described association of a (TAAAA)n VNTR polymorphism in the promoter of the SHBG gene (which is in strong disequilibrium linkage with a Asp327Asn SNP in exon 8) with hirsutism, PCOS and delayed menarche [134 –136]. The variant allele of the Asp327Asn polymorphism was also associated with higher SHBG levels, supporting a functional role for the SHBG polymorphisms in hyperandrogenic disorders [135].

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The most potent androgen is dihydrotestosterone, which results from the local conversion from testosterone by the enzyme 5a-reductase in target tissues. The activity of 5areductase is increased in ovarian theca cells from PCOS patients [137], and therefore this enzyme has been considered a candidate gene for PCOS and idiopathic hirsutism. However, to date, the study of the genes encoding type 1 and type 2 5a-reductase has not demonstrated any consistent abnormality in hyperandrogenic women. Finally, several studies addressed the possible role of variants in the androgen receptor gene (AR), located in the X-chromosome, in hyperandrogenic disorders. Initial studies suggested that a VNTR consisting in (CAG)n repeats was involved in PCOS and in idiopathic hirsutism. This VNTR polymorphism is located in the transactivation domain of AR, and modulates the activity of the androgen receptor: the longer the CAG track, the lower the AR activity [138]. Initial studies suggested that shorter alleles of the (CAG)n VNTR polymorphism in AR were involved in PCOS and idiopathic hirsutism, either directly [139,140], or by skewed inactivation of the longer Xchromosome allele, favoring the expression of shorter alleles [141,142]. However, we have not been able to demonstrate any influence of the length of AR alleles, or of skewed AR inactivation, in female hyperandrogenism and idiopathic hirsutism [143], and the AR locus was not linked to PCOS in a family-based study conducted in the USA [60]. 3.3. Chronic inflammation and prothrombosis Nowadays, cardiovascular disease is considered a lowgrade chronic inflammatory condition [144,145] and several inflammatory markers have demonstrated their usefulness as predictors of cardiovascular events [146,147]. There are direct correlations between the circulating levels of some of these inflammatory markers and the degree of insulin resistance [12], that reflects the fact that chronic inflammation is one of the mechanisms involved in obesity associated insulin resistance [148]. Considering that both lean and obese PCOS patients are frequently insulin resistant, and that classic and nonclassic cardiovascular risk markers cluster in these women [149], we and others have studied the possible contribution of chronic inflammation to PCOS and functional hyperandrogenism. Supporting this hypothesis, hyperandrogenic women present with increased leukocyte counts [150,151], serum C reactive protein (CRP) concentrations [13,14], serum interleukin 18 concentrations [152], plasma homocystein [153] and ferritin [154] levels, yet it is possible that in some cases these associations actually results from the frequent coexistence of obesity [20,21]. But despite these minor discrepancies in clinical studies, the possibility that proinflammatory genotypes underlay PCOS, as has been demonstrated for other insulin resistant disorders [12], has been supported by the studies conducted to date.

19

Most studies about inflammatory mediators in insulin resistant disorders addressed the role of tumor necrosis factor-a (TNF-a). TNF-a increases insulin resistance by inducing serine phosphorylation of IRS-1, which is then converted into an inhibitor of the INSR tyrosine kinase activity [155]. In addition, TNF-a has important reproductive effects, including the stimulation of steroidogenesis and the proliferation of theca cells in animal models [156,157] as well as favoring ovarian apoptosis and anovulation [158]. Considering that insulin resistance, increased ovarian steroidogenesis and anovulation are characteristic of PCOS, TNF-a has been considered as a candidate to explain the pathogenesis of this disorder. Initial reports of increased TNF-a levels in PCOS patients [159] have not been confirmed lately [20]. Moreover, PCOS is not associated in case-control studies with common variants in the TNF-a gene [16,160,161], although carriers of 308A alleles of the 308G/A variant present with increased basal and leuprolide-stimulated serum androgens and 17-hydroxyprogesterone levels, which are markers of functional ovarian hyperandrogenism in PCOS patients [91]. The metabolic effects of TNF-a result from its binding to two different receptors (TNFRs), type 1 receptor which mediates the actions of TNF-a, and type 2 receptor that potentiates its effects [162,163]. The soluble fraction of these TNF-a receptors may be measured in serum. Type 2 TNFR concentrations are increased in obesity and insulin resistant disorders, and are influenced by common variants in the TNFR2 gene (TNFRSF1B) [12,164,165]. We have recently reported that the methionine 196 arginine polymorphism in exon 6 of TNFRSF1B is associated with hyperandrogenism and PCOS in Spanish and Italian women [18], further suggesting the involvement of the TNF-a system in PCOS. The most endocrine of cytokines is interleukin 6 (IL-6), because it can exert effects on distant tissues after circulating in blood [166]. IL-6 is synthesized and secreted in adipose tissue, and its serum levels are increased in obesity and insulin resistant disorders [12]. The effects of IL-6 are mediated by binding to membrane receptor composed by two subunits: the a subunit binds IL-6 and the ß subunit, also termed gp130, is a signal transducer that acts through the Janus-kinase system [167]. Although serum IL-6 and gp130 levels are not increased in PCOS patients independently of obesity [19 –21], polymorphisms in the genes encoding these proteins have been found in association with this disorder. The IL-6 gene promoter contains two SNPs, 597G/A and 174G/C that are in nearly complete disequilibrium linkage [17]. We have recently shown that G alleles of both polymorphisms are more common in hyperandrogenic patients compared with healthy women, and that the variant alleles protected against the increase in IL-6 levels induced by obesity, and against the development of hyperandrogenism [17]. The latter has been confirmed by an independent study conducted in the German population [21], but not in Austrians in whom the

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response to treatment with the insulin sensitizers metformin and troglitazone [174,176], possibly reflecting the high prevalence of insulin resistance and obesity in these women [177]. However, PCOS patients do not present with increased frequency mutations know to enhance thrombosis [170], and the possible association of PCOS with the 675 4G/5G insertion polymorphism in the 5Vregulatory region of PAI-1 gene, which influences the expression and activity of the enzyme and influences insulin resistance disorders [178], is still debated [71,175,179,180].

variant 174C allele was associated with having increased body mass index and serum testosterone levels [168]. Regarding the gp130 gene, common alleles of the Gly148Arg polymorphism are associated with PCOS whereas variant alleles protected against the adrenal hyperactivity frequently found in this patients [19], further suggesting that common genomic variants in the genes encoding the IL-6 system play a modifying role in the PCOS phenotype. Aside from the evidence suggesting a chronic inflammatory milieu in women with PCOS, these patients have a decreased global fibrinolytic capacity [169] and suffer a prothrombotic state [170], factors that might contribute to an increase in cardiovascular risk and a high miscarriage rate in this disorder [171]. The plasminogen activator inhibitor-1 (PAI-1) is widely used as a marker of thrombophilia and correlates with insulin resistance [172], given that insulin, and also inflammatory cytokines such as TNF-a, stimulate its secretion in adipose tissue [173]. Plasma levels of PAI-1 are increased in PCOS patients [174,175] and decrease in

N

+ PITUITARY

The findings described above suggest a complex pathophysiologic scenario for PCOS. Central to the syndrome is a dysregulation of androgen synthesis in the ovary, and probably in the adrenal glands, resulting from an increased activity of several steroidogenic enzymes [91], although the genetic abnormalities underlying this dysregu-

GnRH

HYPOTHALAMUS

E

4. A unifying hypothesis explaining the results obtained to date (Fig. 1)

( ↑pulse frequency and amplitude)

LH

ADIPOSE TISSUE LHβ

FSH

V I

+

INFLAMMATION

G

TNF-α TNFRSF1B IL-6 PAI-1

E

R O

OVARY

↑ steroidogenic enzymes

↓aromatase activity

N CYP17

O

+

N M

+

E

↑

N

Follicular atresia Cyst development

↓ Progesterone

H6PD

+

ANDROGENS

T

β-CELL, LIVER AND PERIPHERAL TISSUES

ADRENAL

− +

T Y

+

P

INSR; IRS-1; PPARγ2 Calpain-10 Adiponectin SHBG

(TAAAA)n D327N

ANOVULATION

−

E

+ HYPERINSULINISM INSULIN RESISTANCE

PHENOTYPE POLYCYSTIC OVARIES, HYPERANDROGENISM, SUBFERTILITY, ENDOTHELIAL DYSFUNCTION, ATHEROSCLEROSIS

Fig. 1. The interaction of environmental factors that predispose to obesity (diet, exercise) with genomic variants related to hyperandrogenism, insulin resistance and inflammation results in the PCOS phenotype. The central role of the syndrome is played by a dysregulation of ovarian androgen synthesis resulting from an increased activity of steroidogenic enzymes. This hyperandrogenic environment leads to follicular atresia and anovulation, and to the absence of increase in the progesterone levels during luteal phase of the menstrual cycle, thereby increasing the frequency and amplitude of the GnRH pulses. The latter favors an increase in LH levels, stimulating androgen synthesis and secretion by theca cells. The endogenous hyperinsulinism, resulting from the insulin resistance inherent to the syndrome, favors androgen synthesis, and inhibits SHBG production by the liver, increasing circulating free androgen levels. Inflammatory cytokines from adipose tissue stimulate adrenal and ovarian androgen synthesis, and insulin resistance.

M. Luque-Ramı´rez et al. / Clinica Chimica Acta 366 (2006) 14 – 26

lation remain to be established. The hyperandrogenic environment leads to follicular atresia and the polycystic appearance of the ovaries (Fig. 1). Also, anovulation is not followed by the development of the corpus luteum, and the absence of increased progesterone levels during the second phase of the menstrual cycle decreases the negative feedback at the hypothalamic level, thereby increasing the frequency and amplitude of the GnRH pulses [1]. Although other factors such as inhibins might contribute to the abnormal gonadotropin secretory dynamics [181], the increase in GnRH pulses favors an increase in LH levels and a decrease in those of FSH, the former stimulating androgen synthesis and secretion by theca cells. Moreover, the decrease in FSH levels reduces aromatase activity, leading to a further increase in androgen levels because their reduced conversion into estrogens [1]. Insulin resistance, and the resulting endogenous hyperinsulinism, exacerbates the increased capacity of androgen synthesis of PCOS patients, especially in obese women [2]. In each patient, weight gain would interact with genomic variants playing favoring or protective roles on the development of insulin resistance, explaining in part the familial aggregation of insulin resistance within PCOS families. The endogenous hyperinsulinism resulting from insulin resistance favors the ACTH-mediated adrenal and LH-mediated ovarian androgen synthesis and, by inhibiting SHBG synthesis at the liver– a mechanism that may be influenced by genomic variants in the SHBG gene – favors androgen bioavailability to target tissues. And also, insulin might directly influence hypothalamic dysregulation characteristic of PCOS, explaining the essential role of insulin resistance in PCOS. Furthermore, the inflammatory cytokines secreted in adipose tissue because the underlying proinflammatory genotypes, exacerbated by weight gain and abdominal obesity, would also stimulate adrenal and ovarian androgen secretion and insulin resistance. Finally, the interaction of environmental factors intimately related to the development of obesity such as diet and exercise, with genomic variants that predispose to hyperandrogenism, insulin resistance and chronic inflammation, probably results in the PCOS phenotype. And considering that the environmental and genetic factors involved may be different in each woman, this may explain the phenotypic variability observed within PCOS patients, who may present with a constellation of symptoms and signs including hirsutism, acne, alopecia, oligoovulation and subfertility, endometrial hyperplasia and cancer, metabolic disorders such as diabetes and dyslipidemia, atherosclerosis, and an increased cardiovascular risk.

5. Conclusions The study of the molecular genetic basis of PCOS suggests a complex etiology for these disorders, in which environmental factors, heavily influenced by ethnicity,

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would interact with predisposing and protective genomic variants finally leading to the phenotype observed in the patients. The recent advances in the study of the genetics of PCOS have permitted a better understanding of the heterogeneous nature of this frequent disorder, highlighting the need of substantial efforts in the standardization of the methods and diagnostic criteria used to characterize these women. Hopefully, the application of unified diagnostic methods and criteria, and of state-of-the-art modern techniques including genomics, proteomics and pharmacogenetics to the study of PCOS, will provide essential data for the comprehension and management of this fascinating disorder.

Acknowledgments This work was supported by Grants FIS 02/0741 and RGDM 03/212 from the Fondo de Investigacio´n Sanitaria, Instituto de Salud Carlos III, and GR/SAL/0137/2004 from the Consejerı´a de Educacio´n, Comunidad de Madrid, Spain.

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