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Departments of Endocrinology (H.F.E.-M., M.L.-R.) and Molecular Genetics (J.L.S.M.), Hospital Ramón y Cajal, Madrid, Spain E-28034
Correspondence: Address all correspondence and requests for reprints to: Héctor F. Escobar-Morreale, M.D., Ph.D., Department of Endocrinology, Hospital Ramón y Cajal, Carretera de Colmenar km 9'1, Madrid E-28034, Spain. E-mail: hescobarm.hrc{at}salud.madrid.org
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Abstract |
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 Top Abstract I. IntroductionII. Evidence Suggesting a...III. Classic Techniques Used...IV. Studies in Pediatric...V. Studies in Hyperandrogenic...VI. Hyperandrogenism, PCOS, and...VII. Explanations for the...VIII. Future Perspective:...IX. SummaryReferences |
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Among others, genomic variants in genes related to the regulation of androgen biosynthesis and function, insulin resistance, and the metabolic syndrome, and proinflammatory genotypes may be involved in the genetic predisposition to functional hyperandrogenism and PCOS.
The elucidation of the molecular genetic basis of these disorders has been burdened by the heterogeneity in the diagnostic criteria used to define PCOS, the limited sample size of the studies conducted to date, and the lack of precision in the identification of ethnic and environmental factors that trigger the development of hyperandrogenic disorders. Progress in this area requires adequately sized multicenter collaborative studies after standardization of the diagnostic criteria used to classify hyperandrogenic patients, in whom modifying environmental factors such as ethnicity, diet, and lifestyle are identified with precision.
In addition to classic molecular genetic techniques such as linkage analysis in the form of a whole-genome scan and large case-control studies, promising genomic and proteomic approaches will be paramount to our understanding of the pathogenesis of functional hyperandrogenism and PCOS, allowing a more precise prevention, diagnosis, and treatment of these prevalent disorders.
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I. Introduction |
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TopAbstractI. IntroductionII. Evidence Suggesting a...III. Classic Techniques Used...IV. Studies in Pediatric...V. Studies in Hyperandrogenic...VI. Hyperandrogenism, PCOS, and...VII. Explanations for the...VIII. Future Perspective:...IX. SummaryReferences |
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The presence of male sexual secondary characteristics in women has been recognized from ancient times, but it was not until 1921 when Achard and Thyers (4) reported the association of hyperandrogenic symptoms with abnormalities in glucose metabolism, highlighting the presence of polycystic ovaries in some of their patients. However, only after the description of seven cases of amenorrhea and bilateral polycystic ovaries by Stein and Leventhal in 1935 (5) was PCOS considered a separate entity that interested clinicians and researchers worldwide.
Although for many years the interest in PCOS has been focused on the cutaneous and reproductive manifestations of this disorder, the recent evidence suggests that metabolic and cardiovascular risk factors cluster in these patients (6, 7, 8). This evidence has renewed research efforts on hyperandrogenism and PCOS, including those directed toward the identification of the genetic and environmental factors involved in the pathogenesis of these prevalent conditions.
At present, there is no consensus on the criteria for the diagnosis of PCOS (Table 1
). Most clinicians and researchers from the United States and from southern Europe use the criteria derived from the conference held at the National Institute of Child Health and Human Development (NICHD) in 1990: clinical and/or biochemical hyperandrogenism, menstrual dysfunction, and exclusion of specific etiologies (9). According to these criteria, the presence of polycystic ovaries on ultrasound examination is not needed for the diagnosis. On the contrary, most specialists from the other European countries, Asia, and Oceania rely mostly on the presence of polycystic ovaries on ultrasound examination for this diagnosis, whereas menstrual dysfunction is not required (10). A recent consensus workshop held in The Netherlands in 2003 under the auspices of the European Society for Human Reproduction and Embryology and the American Society for Reproductive Medicine proposed a revision of the criteria for the diagnosis of PCOS, of which two of the following three would be needed: 1) oligo- and/or anovulation; 2) clinical and/or biochemical signs of hyperandrogenism; and 3) polycystic ovaries, together with the exclusion of other etiologies such as congenital adrenal hyperplasia, androgen-secreting tumors, or Cushing’s syndrome (11, 12).
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-reductase (resulting in increased conversion of testosterone into the more potent androgen dihydrotestosterone), androgen receptor (AR) polymorphisms, and altered local androgen metabolism contribute to the development of hirsutism (13). Functional hyperandrogenism and PCOS cluster in first-degree relatives of patients (14) and are inherited together with insulin resistance and metabolic disorders (15, 16). During the past decades, the inheritance of these disorders has been the subject of intense research (17, 18, 19), but many questions remain unanswered.
The pattern of inheritance is still unknown. Initially, an autosomal dominant model, including premature balding as the male phenotype, was proposed (20, 21), but later studies did not confirm this hypothesis. The heterogeneity of the populations, the large number of candidate genes studied to date (22), and the difficulty inherent to identifying the molecular genetic mechanism leading to a complex metabolic disorder such as PCOS, in which environmental factors play a major role (23, 24), provide an explanation for the fact that the molecular genetic basis of functional hyperandrogenism and PCOS remains largely unknown despite significant efforts.
The purpose of this review is to provide a systematic evaluation of the studies conducted to date in functional hyperandrogenism and PCOS and to suggest priorities and new strategies that may contribute to understanding the pathogenesis of these disorders.
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II. Evidence Suggesting a Genetic Origin for Functional Hyperandrogenism and PCOS |
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TopAbstractI. IntroductionII. Evidence Suggesting a...III. Classic Techniques Used...IV. Studies in Pediatric...V. Studies in Hyperandrogenic...VI. Hyperandrogenism, PCOS, and...VII. Explanations for the...VIII. Future Perspective:...IX. SummaryReferences |
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In addition to hyperandrogenism, insulin resistance clusters in the families of hyperandrogenic women. Legro et al. (33), also using also NICHD criteria for the definition of PCOS, found that 22% of the sisters of patients actually had PCOS, whereas a further 24% of the sisters of these patients presented with hyperandrogenemia and regular menstrual cycles. This bimodal pattern suggested a monogenic defect for hyperandrogenism (33). Additional studies from Legro et al. (34) recently demonstrated that in these families, insulin resistance is associated with hyperandrogenemia rather than with menstrual dysfunction. This suggests that insulin resistance and hyperandrogenemia share the same pathogenic mechanisms, whereas the presence of menstrual dysfunction needed for the diagnosis of PCOS may be only a matter of degree. Therefore, of PCOS sisters, those with PCOS and those presenting with hyperandrogenemia and regular menstrual cycles may actually have functional hyperandrogenism.
The finding of an increased prevalence of insulin resistance in families of PCOS patients has been replicated in the Turkish population. Yildiz et al. (35) recently studied 102 relatives of 52 PCOS patients defined by NICHD criteria and found that, compared with different population-based control groups matched for sex, age, and pre- or postmenopausal status with the relatives of PCOS patients, insulin resistance and disorders of carbohydrate metabolism were more frequent in the mothers, sisters, and brothers of PCOS patients, and that the mothers and sisters of PCOS women had increased serum androgen levels compared with the controls.
B. Male phenotype
The lack of a clearly defined male phenotype in families of PCOS patients has burdened the progress in the search for the genetic origin of functional hyperandrogenism and PCOS. Although initial studies suggested that male pattern premature balding was the male equivalent for PCOS (20, 29, 31, 36, 37), these findings have not been universally confirmed (38). More recently, increased serum dehydroepiandrosterone sulfate concentrations in the brothers of PCOS patients described above (39) and insulin resistance in the fathers and brothers of PCOS women (35) have been proposed as the male phenotype in PCOS families. However, there was a considerable overlap between male PCOS relatives and controls in serum dehydroepiandrosterone sulfate levels and indexes of insulin resistance, and additional studies are needed to establish the actual usefulness of these abnormalities as markers of the syndrome in male relatives of PCOS patients.
C. Twin studies
There are very few data regarding PCOS in twins. Jahanfar et al. (40) studied 34 (19 monozygotic and 15 dizygotic) twin pairs, in which PCOS diagnosis was based on ultrasonographic and biochemical findings. Although 11 pairs were discordant for the presence of polycystic ovaries, model-fitting analysis suggested that fasting insulin level, serum androstanediol glucuronide, and body mass index were significantly influenced by genetic factors (40). A later report of this study suggested a genetic origin for an unfavorable lipid profile, especially for increased circulating concentrations of lipoprotein (a), but only in twin pairs concordant for polycystic ovaries (41). These data point to a genetic component in the metabolic abnormalities associated with PCOS but suggest a polygenic etiology in which environmental influences play a significant role.
D. Environmental and other confounding factors
Familial aggregation of hyperandrogenism and PCOS strongly suggest a genetic origin for these disorders, but another possibility is that clustering of PCOS within families results from nongenetic inheritance related to certain environmental factors that are present in the affected families, and not in the families of unaffected women.
As exemplified in Fig. 1, insults during pregnancy may induce intrauterine growth retardation, which has been proposed to induce a thrifty phenotype in small for gestational age babies. These babies are predisposed to suffer from insulin resistance, which may result in hypertension, glucose intolerance, adrenal axis hyperactivity with relative cortisol excess, functional hyperandrogenism, and PCOS (42) later in life, especially when these individuals are exposed to environmental factors such as a sedentary lifestyle and a diet rich in saturated fat. These environmental factors may cluster in certain families because exercising and dieting are heavily influenced by parental habits.
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Yet it is important to highlight, as shown in Fig. 1
, that if small for gestational age babies have healthy habits, insulin resistance and its consequences might be ameliorated, and, at least in theory, their fetuses will not be exposed to an unfavorable metabolic milieu during pregnancy, preventing nongenetic inheritance of these conditions. Nevertheless, intrauterine growth retardation may also be influenced by genetic variants, and the most probable scenario is that of an interaction between predisposing genetic abnormalities with unfavorable environmental conditions. Of note, even the influence of intrauterine growth restriction on the development of PCOS is debatable (43, 44, 45), considering that the presence of polycystic ovaries also has been related to above-average birth weight in babies born from obese, androgenized mothers (43, 44, 45).
There are very few data on the influence of environmental factors on the development of hyperandrogenism and PCOS, other than the triggering role for obesity on the development of these disorders (46). Although the prevalence of PCOS is similar in different countries, ethnic factors influence the clinical manifestations of the syndrome. In Caucasian premenopausal women, the reported prevalences of PCOS defined by NICHD criteria ranged from 4.7% in Alabama (47) to 6.5% in Spain (48) and 6.8% in the Greek island of Lesbos (49). Moreover, the prevalence in African-American women from Alabama (3.4%) was comparable with that in Caucasian women (47).
However, ethnicity influences the manifestations of PCOS. Carmina et al. (50) compared the clinical histories, physical examinations, ovarian morphology by ultrasound, insulin sensitivity, serum gonadotropin, and steroid profiles in 25 Japanese, 25 Italian, and 25 Hispanic-American women with PCOS. The three groups were homogeneous in terms of serum testosterone and adrenal androgen concentrations, insulin resistance, and polycystic ovaries, but Japanese women were less obese and did not present with hirsutism (50).
Dunaif et al. (51) found that insulin resistance is higher in Hispanic-Caribbean women with PCOS defined by NICHD criteria compared with Caucasian patients and with control women paired for age, weight, body composition, and ethnicity. Williamson et al. (52) recently reported a higher incidence of obesity, dyslipidemia, and infertility, as well as a higher degree of insulin resistance in Maori and Pacific Islander PCOS patients, defined by ultrasonography and clinical symptoms, compared with European patients. In the United Kingdom, the prevalence of polycystic ovaries and type 2 diabetes is increased in Indian subcontinent Asian women (53). Compared with Caucasian women, PCOS patients of southeastern Asian origin presented with oligomenorrhea and were diagnosed at younger ages; hirsutism, acne, acanthosis nigricans, and subfertility were more prevalent, and patients were more insulin resistant (54).
These ethnic differences in the clinical presentation of PCOS may be related to environmental factors such as diet, exercise, and lifestyle. Of note, even the content of polyunsaturated or monounsaturated fatty acids in the diet may influence the metabolic manifestations of the syndrome (55). Thus, because of differences in the environmental factors triggering the development of PCOS, the genes contributing to PCOS patients may also be different, depending on the population studied. These considerations may also apply to the discrepant results of previous studies regarding the association of genomic variants with PCOS.
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III. Classic Techniques Used in Molecular Genetic Studies |
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TopAbstractI. IntroductionII. Evidence Suggesting a...III. Classic Techniques Used...IV. Studies in Pediatric...V. Studies in Hyperandrogenic...VI. Hyperandrogenism, PCOS, and...VII. Explanations for the...VIII. Future Perspective:...IX. SummaryReferences |
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A. Linkage analysis
Linkage analysis is a family-based molecular genetic technique that has permitted the identification of the genetic abnormalities leading to many monogenic Mendelian disorders (56). However, most complex metabolic disorders result from the interaction between multiple genes and environmental factors, making their identification much more difficult than in disorders with a Mendelian pattern of inheritance (57, 58, 59, 60, 61, 62, 63).
In linkage analysis, markers frequently located at intergenic regions of DNA are studied in multiple members of affected families. The cosegregation of alleles with a disease phenotype is studied, and the linkage of the disease with any marker focuses the search for genomic abnormalities to the DNA regions close to the polymorphic marker segregating with the disorder.
Linkage analysis is based on the meiotic recombination that occurs during ovogenesis and spermatogenesis. The closer two genomic markers are, the more seldom these markers are separated during meiotic recombination. The probability of recombination between two markers is an estimation of the distance between them, and is expressed in centimorgans (cM). Two markers are defined to be 1 cM apart if they become separated by recombination in 1% of meioses. One centimorgan is equivalent on average to 106 bp of physical distance. The markers usually studied in linkage studies include microsatellites and single nucleotide polymorphisms (SNPs).
Classical or parametric linkage analysis requires the specification of a model for the disease or trait a priori, in terms of allelic frequencies, penetrance, and pattern of inheritance, and the result is expressed by the LOD score (64). The LOD score is the base 10 logarithm of the odds that two markers (one being the disease-causing or disease-associated genomic variant) are truly linked, divided by the odds that the observed set of data may result from chance if the markers are unlinked. Usually, significant positive proof of genetic linkage requires a LOD score of 3.0 or greater, corresponding to odds favoring linkage of at least 1000:1 at a given specified recombination frequency. Because the pattern of inheritance of functional hyperandrogenism and PCOS is complex and uncertain, nonparametric or model-free linkage analysis is required to ensure the power to detect linkage (22). In nonparametric linkage analysis, multiple DNA markers are obtained from siblings and, if possible, from their parents, and allele sharing between relatives is investigated (64). Allele sharing is defined as identity-by-state (having the same DNA sequence) or as identity-by-descent (when two alleles come from the same ancestral allele) (64). Nonparametric linkage scores use a similarity statistic defined by the average of the possibilities that relatives are identity-by-state (64). However, the number of meioses assessed, disease penetrance, knowledge of the marker coupling phase, and other relevant variables must be considered before reaching a clinically significant conclusion (65).
There are several factors that may limit the usefulness of linkage studies including, among others, the uncertainty about the pattern of inheritance, a variable penetrance of the genetic defect, the delay in the debut of the disease, possible errors in clinical diagnosis, gene to gene interactions, interaction with environmental factors, and the limited resolution of these techniques. The sample size needed to detect a particular effect varies inversely with the square of the effect (62), and therefore genes that contribute modestly to the phenotype may be missed because of the extremely large sample size needed to demonstrate the association. This is especially important given that PCOS affects women in reproductive age and is associated with reduced fertility, limiting the pedigrees and the generations studied to two (mothers and daughters) in most of the linkage analyses performed to date, resulting in small sample sizes within families (66, 67).
The resolution of linkage analysis may be improved using tests such as affected sibling-pair identity by descent analysis or the transmission disequilibrium test (68), allowing the detection of genes of modest effects that might be otherwise missed by linkage analysis. The transmission disequilibrium test involves genotyping the parents of the proband and the proband, and it determines whether or not the parent heterozygous for the putative allele transmits it more often to their affected children than other allele(s). These tests are frequently used to confirm the linkage already established but may also be useful when used without previous evidence for linkage (69).
Linkage with a polymorphic marker only serves to focus the search of the genomic abnormality associated with the disorder to an area of approximately 1–2 cM, and locating the precise genetic defect may involve studying as many as 30–100 genes in the linked area. After obtaining linkage with a genomic region, the search of the particular genomic abnormality involved can be accomplished by fine-mapping of that region using additional markers, and by a candidate gene approach searching for variants in biologically appealing genes located there. This approach has been facilitated by the growing number of genes identified all across the genome, but frequently the demonstration that a gene has a causative role in a disease requires direct sequencing of the whole gene in affected and nonaffected individuals (70).
B. Case-control studies
These studies look for specific genetic markers or alleles that are more frequent in affected individuals (cases) compared with unaffected subjects from the same population (controls). These studies are focused on association of a genetic variant with a disease in a population and not on the mode of inheritance of a trait (71, 72). Association studies are preferable for finding "susceptibility" loci, low-risk alleles that are often found in relatively high frequencies in the general population, and do not result in robust signals in family-based studies (73). A positive association indicates an increased risk for the disease in subjects carrying the at-risk allele, which may have a causative role in the disorder or be in linkage disequilibrium with the gene actually causing the disease. Population stratification is a major problem in case-control studies, because the higher prevalence of particular alleles in different ethnic groups might bias the results and lead to false-positive associations. Moreover, case-control studies are especially prone to type II errors, that is, ruling out an association that is actually present in the population because of the small sample sizes often used in these studies.
In summary, linkage studies and case-control studies are both valid approaches for the study of the genetics of functional hyperandrogenism and PCOS, provided that the sample sizes of these studies are sufficiently large, the diagnostic criteria used to define affected and nonaffected subjects are clearly defined, and both ethnicity and differences in environmental factors are carefully considered.
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IV. Studies in Pediatric and Adolescent Hyperandrogenism |
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TopAbstractI. IntroductionII. Evidence Suggesting a...III. Classic Techniques Used...IV. Studies in Pediatric...V. Studies in Hyperandrogenic...VI. Hyperandrogenism, PCOS, and...VII. Explanations for the...VIII. Future Perspective:...IX. SummaryReferences |
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The association of low birth weight with the development of hyperinsulinism and type 2 diabetes mellitus has been confirmed in different populations (80, 81), and these children present with an accelerated growth and increased body mass index during adolescence.
Although environmental factors may be an important contributor to the restriction of fetal growth in small for gestational age newborns, genetic factors may also be involved in this association (82). According to this hypothesis, affected fetuses might suffer insulin resistance because of the influence of several unknown genes or genomic variants. Insulin resistance would lead to restriction of intrauterine growth and abnormal fetal vascular development, ultimately leading during adult life to increased vascular resistance and endothelial dysfunction. The compensatory increase in circulating insulin concentrations would contribute to the abdominal deposition of fat, and inflammatory cytokines and other mediators secreted by abdominal adipocytes would perpetuate insulin resistance and endothelial dysfunction, leading to abnormal glucose tolerance, atherosclerosis, and cardiovascular disease (82).
However, the analysis of the influence of genomic variants on the association of birth weight and insulin resistance has yielded conflicting results. Dunger et al. (83) analyzed a variable number of tandem repeats (VNTR) polymorphism in the insulin gene (INS) at 11p15.5. This VNTR polymorphism consists of a repeated sequence of 14 to 15 bp (ACAGGGGTGTGGGG), and is located at –596 bp of the start of transcription site (84). Among Caucasians, these alleles have been typed as class I (small, with 28–44 repeats, frequency approximately 70%), class II (intermediate, rare), and class III (large, with 138–159 repeats, frequency approximately 30%). The VNTR polymorphism in the INS promoter regulates the transcriptional rate of the gene (85) and probably that of the gene encoding IGF-II (84, 85, 86, 87, 88, 89). Newborns homozygous for class III alleles, which are those associated with increased transcription of the INS, presented with increased head circumference, length, and weight at birth as compared with babies homozygous for class I alleles, possibly favoring newborn survival. On the contrary, data from the same researchers suggested that the maternally inherited 16,189 mitochondrial DNA variant, which increases the risk for type 2 diabetes mellitus, was associated with restrained fetal growth, thereby favoring maternal survival by decreasing birth-related morbidity and mortality (90). Therefore, other genetic and environmental factors are possibly involved in the relationship between birth weight and the development of insulin resistance and type 2 diabetes mellitus in adulthood.
Given the association of insulin resistance with functional hyperandrogenism, Ibáñez et al. (91) have proposed that low birth weight is related to the development of premature pubarche, and of functional hyperandrogenism during adolescence, insulin resistance being the underlying disorder common to both conditions. Retrospective analysis of a cohort of patients presenting with premature pubarche showed, especially in those girls who developed hyperinsulinemia and functional ovarian hyperandrogenism at adolescence, a lower birth weight compared with a control group, suggesting that low birth weight, premature pubarche, and functional hyperandrogenism are different manifestations of a unique disorder originating during prenatal life (92, 93, 94, 95), but becoming clinically apparent peripubertally (42).
Additional analysis of this cohort showed that adolescent girls who had anovulation and higher androgen levels were those with lower birth weight (96) and also confirmed the association of adrenal androgen excess, low birth weight, premature pubarche, and hyperinsulinemia (97).
In conceptual agreement, an exaggerated adrenal response to adrenocorticotropin stimulation, similar to that described in women with functional hyperandrogenism and PCOS, has been found in girls with premature pubarche, suggesting that this disorder can be considered an early manifestation of the syndrome (98). However, the relationship of low birth weight with hyperandrogenism and PCOS has not been universally confirmed. Jaquet et al. (99) compared a large population of young women born with intrauterine growth retardation with age-matched healthy controls, and although low birth weight was associated with insulin resistance, androgen levels were similar in both groups of women. Moreover, birth weight had no influence on the presence of hyperandrogenic symptoms suggestive of PCOS in a large cohort of Finnish women recently reported (100).
Given the association of functional hyperandrogenism and insulin resistance in children and adolescents, several genomic variants related to insulin resistance and the metabolic syndrome have been studied in these patients, mostly by the group of Ibánez in Spain and the group of Witchel in Pittsburgh.
The VNTR locus at the INS has been studied in girls with a history of premature pubarche (101). Although class I and class III alleles were equally distributed in patients and controls, patients carrying class I alleles presented with lower birth weight and lower insulin sensitivity compared with patients homozygous for class III alleles (101).
The common Gly972Arg variant in the gene encoding the insulin receptor substrate 1 (IRS-1), which has been shown to influence insulin resistance and glucose tolerance in adult PCOS patients (102, 103), has also been studied in adolescents (104). The frequencies of heterozygosity for the Gly972 allele were 31% among girls with a history of premature pubarche, 40% among girls with hyperinsulinemic ovarian hyperandrogenism, and only 19% among healthy control subjects. Carriers of Gly972 alleles presented with decreased sex hormone-binding levels (104).
The human homolog for the sorbin and SH3-domain-containing-1 gene (SORBS1), which encodes for an important signaling molecule in insulin-stimulated glucose uptake in the mouse, might play a role in human disorders with insulin resistance. The Ala228 allele of the Thr228Ala polymorphism of SORBS1 is a protective factor for both obesity and diabetes (105). However, alleles of the Thr228Ala polymorphism of SORBS1 were equally distributed in a multiethnic group of healthy adolescents compared with those presenting with premature pubarche and/or functional hyperandrogenism (106).
The Arg64 allele of the Trp64Arg polymorphism in the ß3-adrenergic receptor gene is associated with abdominal obesity and resistance to insulin and may contribute to the early onset of type 2 diabetes mellitus (107). However, Witchel et al. (108) found no differences in the distribution of Trp64Arg alleles in the girls presenting with premature pubarche and/or functional hyperandrogenism described above compared with healthy adults, and neither was an influence of this polymorphism on the body mass index of these girls. Similarly, the study of the common Pro12Ala polymorphism in the gene encoding the peroxisome proliferator-activated receptor-
2 (PPAR-2), which influences insulin sensitivity in Caucasians (109), has been studied in children with premature pubarche and in adolescent hyperandrogenic girls, but no association was found with any of the PPAR-2 alleles and hyperandrogenism (110).
In addition to genes related to insulin resistance, those encoding for the steroidogenic enzymes involved in androgen biosynthesis and the genes encoding several molecules involved in androgen metabolism and action have been considered candidate genes to explain premature pubarche and adolescent hyperandrogenism.
Witchel and colleagues from Pittsburgh conducted a series of studies focused on the steroidogenic enzymes involved in androgen synthesis, which are shared by the adrenal and the ovaries. These authors have found that children with premature pubarche and adolescent girls with hyperandrogenism were heterozygous for mutations in CYP21, the gene encoding for 21-hydroxylase, and in the gene encoding 3ß-hydroxysteroid-dehydrogenase (HSD3B2), more frequently than nonhyperandrogenic controls (111, 112, 113). In these patients, CYP17 mutations were ruled out as the cause of androgen excess (114). However, because congenital adrenal hyperplasia is an autosomal recessive disease requiring mutations in both alleles–in homozygosity or double heterozygosity–of CYP21 or of HSD3B2, it is unclear how carrying only one defective allele of these genes in these clinically symptomatic heterozygotes contributes to the hyperandrogenic phenotype (113). Moreover, defective CYP21 and HSD3B2 alleles were found only in a minority of these patients, and the authors concluded that other genomic variants were also involved in the pathogenesis of these disorders (111, 112, 113).
To further explore this possibility, Witchel et al. (115) evaluated the prevalence of the common variants and mutations in CYP21, HSD3B2, IRS-1, ß3-adrenergic receptor gene described above, and the glucocorticoid receptor gene within their series of children with premature pubarche and adolescent hyperandrogenism. They found that the prevalence of these variants and mutations was clearly increased when compared with 15 healthy controls, suggesting that the occurrence of multiple sequence variants in these genes might contribute to the development of hyperandrogenism.
Finally, the AR and uridine diphosphate-glucuronyltransferase 2B15, an enzyme involved in androgen inactivation, have been considered candidate genes for premature pubarche and adolescent hyperandrogenism. The study of the AR gene CAG repeat polymorphism suggested that shorter AR gene CAG numbers, indicative of increased androgen sensitivity, were associated with premature pubarche (116), whereas the allele frequencies of the D85Y polymorphism in uridine diphosphate-glucuronyltransferase 2B15 gene were similar in patients and in controls (117).
In summary, studies conducted to date in girls with premature pubarche and adolescent hyperandrogenism suggest a polygenic etiology for these disorders, which can be considered predictors of functional hyperandrogenism and PCOS later in life.
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V. Studies in Hyperandrogenic Adults |
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TopAbstractI. IntroductionII. Evidence Suggesting a...III. Classic Techniques Used...IV. Studies in Pediatric...V. Studies in Hyperandrogenic...VI. Hyperandrogenism, PCOS, and...VII. Explanations for the...VIII. Future Perspective:...IX. SummaryReferences |
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A. Genes involved in androgen biosynthesis, transport, and action, and their regulation (Table 3)
1. CYP17.
The limiting step in androgen biosynthesis in the ovary and adrenal gland is an enzyme termed P450c17, which possesses both 17-hydroxylase and 17,20-lyase activities. Therefore, P450c17 may catalyze the conversion of pregnenolone and progesterone into 17-hydroxypregnenolone and 17-hydroxyprogesterone, respectively, and of these steroids into dehydroepiandrosterone and androstenedione, although in humans only the conversion of 
5 steroids has been demonstrated (118).
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was responsible for the enhanced androgen synthesis and secretion. They proposed the increase in serum 17-hydroxyprogesterone in response to the GnRH analog nafarelin as the marker of functional ovarian hyperandrogenism (119). Studies using adrenal stimulation with ACTH and ovarian stimulation and suppression with the long-acting GnRH triptorelin also suggested that most hyperandrogenic women have increased P450c17 activity in the adrenal and ovary (121, 122, 123), and that the increased adrenal P450c17 activity was not influenced by ovarian function (122). Moreover, P450c17 expression and activity are increased in ovarian theca cells from PCOS women, defined by NICHD criteria, compared with those from nonhyperandrogenic controls (124).
Given these findings, the gene encoding the P450c17 enzyme, CYP17, was considered a candidate gene in early studies. CYP17 is located in chromosome 10q24.3 (125, 126), and its promoter contains a T/C SNP at –34 bp from the start of transcription site that might modulate enzyme activity. Some studies suggested that this polymorphism was associated with the presence of polycystic ovaries on ultrasound (125), and PCOS patients homozygous for C alleles of this polymorphism presented with increased serum testosterone levels (127, 128). Other studies failed to confirm these observations, suggesting that this base change is a polymorphism without functional consequences for the development of polycystic ovaries and hyperandrogenism (129, 130, 131). Also, no evidence for linkage or association was found between PCOS, defined by NICHD criteria, and the CYP17 locus in a family-based study that included mostly families of European ancestry from the United States (132). Moreover, no abnormalities were found after single-strand conformational polymorphism analysis of the entire coding region of CYP17 in a small sample of PCOS patients with or without exaggerated 17-hydroxyprogesterone response to a GnRH analog (133), ruling out CYP17 as a major candidate gene for the pathogenesis of PCOS and functional hyperandrogenism. However, posttranscriptional hyperphosphorylation of the serine residues of P450c17 by a defective serine kinase might increase the 17,20-lyase activity of this enzyme, contributing to hyperandrogenism (118, 134). Confirmation of this hypothesis is still pending (135).
2. CYP11A.
Ovarian theca cells from women with PCOS defined by NICHD criteria overexpress all the steroidogenic enzymes involved in androgen biosynthesis (136, 137), and these cells secrete increased amounts of progesterone, 17-hydroxyprogesterone, testosterone, and androstenedione compared with theca cells from nonhyperandrogenic women (136, 138).
The initial step in adrenal and ovarian steroidogenesis is the conversion of cholesterol into progesterone, which is catalyzed by the cholesterol side chain cleavage enzyme. The CYP11A gene, located at 15q24, encodes the cholesterol side chain cleavage enzyme and has therefore been considered a candidate gene for functional hyperandrogenism and PCOS (139). It has been proposed that a VNTR polymorphism, consisting in repeats of a (tttta)n pentanucleotide at –528 bp from the ATG start of translation site in the CYP11A promoter, plays a role in the pathogenesis of PCOS (140). Evidence for linkage with the CYP11A locus was found in 20 pedigrees presenting with PCOS, based mostly on the presence of polycystic ovaries or male pattern premature balding, and the absence of the more common four-repeats allele (this VNTR appears with four, six, eight, and nine repeats in Caucasians) was associated with hirsute PCOS patients and with higher serum testosterone levels (140). Diamanti-Kandarakis et al. (141), using NICHD criteria for the definition of PCOS, confirmed its association with absence of four-repeat alleles in Greek patients. In women from the United States, nine-repeat alleles were more frequent in PCOS patients, defined by oligomenorrhea and polycystic ovaries, and four- and six-repeat alleles were more frequent in controls, but these allelic differences did not influence CYP11A expression in theca cells (142).
Other studies failed to demonstrate linkage with the CYP11A locus in PCOS patients defined by NICHD criteria (132) or association of CYP11A VNTR alleles with functional hyperandrogenism (143). Moreover, in a small sample of hirsute hyperandrogenic patients from Spain (144), no consistent genomic abnormalities have been found in the entire CYP11A coding region, nor in the genes encoding the steroidogenic acute regulatory protein, steroidogenic factor-1, and dosage-sensitive sex reversal-adrenal hypoplasia gene on the X chromosome gene-1 (DAX-1), which are also involved in the first step of steroidogenesis and its regulation. The family-based study cited above failed also to demonstrate linkage or association with the steroidogenic acute regulatory protein (132). Finally, Gaasenbeek et al. (145) recently failed to confirm any influence of CYP11A VNTR alleles on polycystic ovaries and on serum testosterone levels in a series of experiments involving a large number of subjects, concluding that the existence of associations between CYP11A promoter variation and androgen-related phenotypes had been substantially overestimated in previous studies, including their own preliminary report cited above (140).
3. CYP21.
An exaggerated serum 17-hydroxyprogesterone response to ACTH stimulation is a common finding in women with PCOS or functional hyperandrogenism (121, 146). This finding prompted several groups to study CYP21, which encodes the 21-hydroxylase enzyme catalyzing the conversion of 17-hydroxyprogesterone into 11-deoxycortisol; increased serum 17-hydroxyprogesterone levels are the biochemical marker for 21-hydroxylase deficiency. This type of congenital adrenal hyperplasia is an autosomal recessive disease resulting from homozygosity or double heterozygosity for missense or nonsense mutations in CYP21 and is characterized by hyperandrogenism with or without mineralocorticoid deficiency.
However, whether or not heterozygous CYP21 mutations influence functional hyperandrogenism and PCOS is still matter of debate. As stated above, girls presenting with premature adrenarche and adolescent hyperandrogenism carry CYP21 mutations more frequently than nonhyperandrogenic controls (112). Clinically symptomatic heterozygotes for CYP21 mutations present with a phenotype that resembles that of PCOS (113), but it is unclear whether all carriers of CYP21 mutations have an increased risk of having PCOS. Moreover, in hyperandrogenic women carrying CYP21 mutations there is no clear concordance between the CYP21 genotype and the functional origin of androgen excess (147).
Finally, the presence of polycystic ovaries in ultrasound scans has been associated with increased frequency of DRW6 and decreased DR7 human leukocyte antigen haplotypes, whereas 21-hydroxylase deficiency is associated with the Bw47, B14, or DR1 haplotypes (148). Moreover, in families of patients with 21-hydroxylase deficiency, polycystic ovaries segregate independently from adrenal dysfunction (149).
4. HSD3B2.
This enzyme catalyzes the conversion of 5 steroids into 4 steroids in the adrenal and in the ovary. Mutations in HSD3B2 result in a rare form of classic congenital adrenal hyperplasia, causing various degrees of salt wasting in both sexes and incomplete masculinization of the external genitalia in genetic males because both adrenal and ovarian steroidogenesis are severely impaired (150). Biochemically, this disorder is characterized by a marked increase in serum 17-hydroxypregnenolone and dehydroepiandrosterone concentrations (150).
Mild increases in these steroid precursors, and in the ratio of 5 to 4 steroids, are not infrequent in hyperandrogenic patients, and hyperandrogenic patients presenting with increased 17-hydroxypregnenolone concentrations and/or increased 5 to 4 ratios after adrenal stimulation with cosyntropin were initially considered to have a nonclassic form of HSD3B2 deficiency (151, 152, 153). However, these mild increases in serum 17-hydroxypregnenolone concentrations and in the 5 to 4 ratios were interpreted by others as an exaggerated adrenal response to cosyntropin stimulation without a genetic origin (154). Molecular analysis of HSD3B2 of hyperandrogenic patients presenting with increased 5 steroids and increased 5 to 4 ratios revealed no abnormalities (155, 156), ruling out the existence of a nonclassic form of HSD3B2 deficiency in these women. In conceptual agreement, a marker close to the HSD3B2 locus was not in linkage or association with PCOS, defined by NICHD criteria, in the American family-based study cited above (132).
5. 17ß-Hydroxysteroid dehydrogenases.
17ß-Hydroxysteroid dehydrogenase type III is also known as 17-ketosteroid reductase. This enzyme catalyzes the conversion of androstenedione into testosterone in the testis. Up to 20 mutations in HSD17B3 have been identified as the cause of male pseudohermaphroditism because of testosterone deficiency (157). Pang et al. (158) hypothesized that genetic females with ovarian 17-ketosteroid reductase deficiency would probably have a normal female phenotype at birth and normal pubertal breast development but would present at puberty with virilization and menstrual disorders because of the sudden increase of the ovarian secretion of androstenedione. However, no mutations have been identified in HSD17B3 in Caucasian and African-American women presenting with PCOS defined by NICHD criteria (159). Also, no evidence for linkage or association with the type 1, type 2, and type 3 17ß-hydroxysteroid dehydrogenase loci was found in the American family-based study cited previously (132).
6. Aromatase.
This enzyme, encoded by CYP19, is responsible for the conversion of C19 steroids (androgens) into C18 steroids (estrogens). Aromatase activity may be decreased in granulosa cells and follicles from women with PCOS, and the possible androgen excess resulting from this decreased activity might contribute to abnormal follicle development (160, 161). Although mutations in CYP19 have been associated with multicystic ovaries in a case report, the clinical picture was that of hypergonadotropic hypogonadism (162). Moreover, no evidence for linkage of CYP19 with PCOS was found in studies conducted in the United Kingdom (140) and in the United States (132).
7. 11ß-Hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase.
These enzymes regulate the inactivation of cortisol into cortisone at the tissue level. 11ß-Hydroxysteroid dehydrogenase type 1 converts cortisol into cortisone directly, and endoluminal hexose-6-phosphate dehydrogenase regenerates reduced nicotinamide adenine dinucleotide phosphate in the endoplasmic reticulum, thereby influencing the directionality of 11ß-hydroxysteroid dehydrogenase type 1 activity. Mutations in the genes encoding 11ß-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase have been recently shown to cause cortisone reductase deficiency (163), and because the phenotypic features of affected women resemble those of PCOS, these genes should be considered candidates to explain the pathogenesis of PCOS. However, this possibility has not been explored in any series of patients with functional hyperandrogenism and PCOS to date.
8. Gonadotropins.
In as many as 40% of PCOS patients, LH hypersecretion is present (164). The gene encoding the ß-subunit of LH (LHß), which is responsible for LH specificity, has been explored in PCOS and hyperandrogenic women. Initially, two point mutations, Trp8Arg and Ile15Thr, were identified as the cause of an immunologically abnormal LHß molecule (165). These mutations induce structural changes in the mutant LHß molecule (166) and have a 15% prevalence worldwide, although with significant differences depending on the population studied (167). The resulting LHß molecule has an increased in vitro activity and a decreased in vivo half-life compared with the wild-type molecule (168).
This LHß variant has been associated with increased serum testosterone, estrogen, and SHBG levels in healthy women, but not in PCOS patients (169). Furthermore, the prevalence of this LHß variant is reduced in obese women with PCOS, suggesting a protective role against the development of hyperandrogenic symptoms in these women and a possible usefulness in determining the risk for PCOS in obese women (170, 171). However, no association with PCOS, hyperandrogenism, or serum androgen or estrogen concentrations has been found in other studies (172, 173).
Other variants in the LHß gene might influence hyperandrogenism and PCOS. A Gly102Ser mutation in exon 3 has been related to menstrual disorders in Chinese women (172, 174). Finally, in Japanese patients with ovulatory disorders, several SNPs in the promoter of the LHß gene (–894C/T, –1018G/C, –1036C/A, –1098C/T, and –1423C/T) are more frequent than in normal ovulatory women (175).
Polymorphisms in the FSH ß-subunit gene have also been reported to influence PCOS. In Chinese women, homozygosity for a thymine-cytosine substitution in exon 3 (codon 76, TAT to TAC) has been found more frequently in PCOS patients, as defined by oligomenorrhea and polycystic ovaries, compared with nonhyperandrogenic women, especially in obese patients, and correlating with higher serum androgen concentrations (176). In these women, two SNPs in the gene encoding the FSH receptor, Thr307Ala and Ser680Asn, showed similar allele distribution among PCOS patients and controls (177). Moreover, the search for mutations in exons 6, 7, 9, and 10 of the FSH receptor gene, which have been shown previously to inactivate the receptor, yielded negative results in Japanese patients with oligomenorrhea and polycystic ovaries (178). This locus was not in linkage or association with PCOS in a family-based study conducted in the United States (132). Finally, mutations in the gene encoding the GnRH receptor have not been found in PCOS patients, defined by NICHD criteria (179).
9. Dopamine receptor.
Given that dopamine is involved in the hypothalamic control of gonadotropin secretion, the association between polymorphisms in the dopamine receptor and disorders of ovulation and fertility has been studied. Legro et al. (180) reported that SNPs in exons 5 and 6 of the gene encoding the type 2 dopamine receptor influenced serum gonadotropin and prolactin levels, as well as parity and prevalence of miscarriage in women of Hispanic ancestry. In these women, a genomic variant in the type 3 dopamine receptor was associated with hyperandrogenic chronic anovulation and resistance to clomiphene citrate (181), but this association was not confirmed in a large series of non-Hispanic women (182).
10. SHBG.
SHBG regulates the access of testosterone and estradiol to target tissues. Decreased SHBG concentrations are characteristic of hyperandrogenic women, contributing to increased tissue androgen availability (183). Despite the fact that evidence for linkage or association was not found between a marker close to the SHBG locus and PCOS in a family-based study conducted in the United States (132), a missense Pro156Leu mutation in the SHBG gene, resulting in abnormal glycosylation and secretion of a SHGB mutant that retains binding capacity (184), has been detected in a women presenting with severe hyperandrogenism during pregnancy. However, it is seldom found in unselected hyperandrogenic women (184).
Recently, an association between a (TAAAA)n polymorphism in the promoter of the SHBG gene and PCOS has been reported (185). Longer alleles (more than eight repeats) were frequent in Greek PCOS patients, defined by NICHD criteria, whereas nonhyperandrogenic women presented with a higher frequency of shorter alleles (185). Furthermore, in the PCOS group, carriers of the longer allele genotypes had lower SHBG levels than in those with shorter alleles (185). Therefore, this polymorphism might be related to the low SHBG levels characteristic of PCOS, facilitating androgen availability to target tissues. Cousin et al. (186) studied the (TAAAA)n polymorphism in hirsute women and found strong disequilibrium linkage with an Asp327Asn SNP in exon 8 of SHBG, 327Asn alleles being associated with eight-repeat (TAAAA)n alleles, and resulting in increased serum SHBG levels when compared with subjects homozygous for 327Asp alleles. Moreover, longer (TAAAA)n alleles resulted in decreased serum SHBG levels when compared with six-repeat alleles (186), in conceptual agreement with the results in PCOS women described above (185).
11. Glucocorticoid receptor.
Mutations in the glucocorticoid receptor gene result in a compensatory increase in circulating ACTH, resulting in excess secretion of adrenal androgens. Therefore, the glucocorticoid receptor has been studied as a candidate gene for functional hyperandrogenism and PCOS. However, genomic abnormalities and polymorphisms in the glucocorticoid receptor gene do not appear to influence the pathogenesis of functional hyperandrogenism (187, 188) and PCOS (189).
12. Steroid 5-reductase (SRD5A).
This enzyme catalyzes the conversion of testosterone into the more potent androgen dihydrotestosterone. SRD5A1 and SRD5A2 are the two isoforms of the enzyme. Mutations in SRD5A2 result in male pseudohermaphroditism (190, 191). Because total SRD5A activity is increased in polycystic ovaries (192), the genes encoding the SRD5A isoforms can be considered candidate genes for functional hyperandrogenism and PCOS. To date, no data have been published regarding the possible involvement of molecular genetic variants in these genes in hyperandrogenic disorders.
13. AR.
The AR is a member of the superfamily of ligand-activated transcription factors that regulate many biological processes and is encoded by a gene located at Xq11–12 (193, 194). The AR has three functional domains responsible for transactivation, for ligand binding, and for binding to DNA. Exon 1 contains a VNTR polymorphism consisting of (CAG)n repeats, which encodes for a polyglutamine tract in the N-terminal transactivation domain of the AR protein (195).
The number of CAG repeats in the normal population varies between 11 and 31, with 20 repeats being the most frequent (196). The number of CAG repeats is inversely related to the transactivation of the AR and its activity (197). Thus, the decreased number of CAG repeats has been proposed to increase androgen activity at target tissues, favoring hirsutism (198), premature pubarche, and ovarian hyperandrogenism (116) in women, infertility in men (199), as well as androgen-dependent skin disorders in both men and women (200). A decreased number of AR gene CAG repeats has been suggested to explain the normal serum androgen levels found in some women with polycystic ovaries, infertility, and oligomenorrhea, in whom the hyperandrogenic symptoms would result from the intrinsic increase in the AR activity (201).
One of the two X chromosomes in every cell of a woman undergoes inactivation, a process that involves methylation of DNA and occurs in a random fashion. Skewed inactivation of X chromosome with the larger CAG repeat, favoring expression of the shorter allele, has been proposed to play a role for idiopathic hirsutism and PCOS (202, 203). However, we have shown that both the number of CAG repeats and the prevalence of skewed X chromosome inactivation were equally distributed in hirsute patients with or without hyperandrogenemia and in healthy women (204). Moreover, no evidence for linkage or association with PCOS was found with the AR locus in the family-based study conducted in the United States cited above (132).
14. Aldosterone synthetase.
The renin-angiotensin system may be hyperactive in women with PCOS (205, 206). Recently, a –344T/C SNP in the promoter of the gene encoding aldosterone synthetase, CYP11B2, has been proposed to influence the pathogenesis of PCOS, because C alleles are more frequent in these patients than in healthy controls (207). Interestingly, women homozygous for C alleles presented with increased plasma renin activity and increased serum angiotensin II, aldosterone, and testosterone levels compared with women homozygous for T alleles, suggesting that this polymorphism influences the activity of aldosterone synthetase and also contributes to androgen excess (207).
B. Genes involved in insulin resistance and associated disorders (Table 4)
1. Insulin receptor gene (INSR).
The presence of insulin resistance is common in PCOS (3) and is frequent in other hyperandrogenic patients (208, 209, 210). Therefore, the genes encoding the INSR and those encoding several molecules involved in postreceptor signaling have been studied in hyperandrogenic patients.
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- and two ß-subunits, and it is encoded by a gene located at chromosome 19. A marker relatively close (1 cM) to INSR, D19S884, has been reported in association with PCOS, defined by NICHD criteria, using the transmission disequilibrium test in a family-based study (132), and in a case-control study (211), both conducted in the United States. However, this association was not confirmed in PCOS patients from Spain and Italy using a case-control design (212). In conceptual agreement with the latter, the 22 exons of INSR have been sequenced in PCOS patients with negative results (213). INSR contains several polymorphisms, yet most of them are silent polymorphisms or are located in intronic regions and are present with similar frequencies in patients with polycystic ovaries and hyperandrogenism and in controls (214). Recently, Siegel et al. (215) have observed a C/T SNP at the tyrosine kinase domain of INSR associated with PCOS defined by NICHD criteria. This SNP could be a susceptible variant for PCOS, or it could be in linkage disequilibrium with another INSR polymorphism; such an association must be confirmed in future studies.
Studying cultured skin fibroblasts and muscle samples from PCOS patients defined by NICHD criteria, Dunaif et al. (216) found increased INSR serine phosphorylation, which decreases its protein tyrosine kinase activity, in as many as 50% of the cases. Subsequent studies in these patients confirmed in vivo the defect in insulin signaling, using serial skeletal muscle biopsies obtained during euglycemic glucose clamp studies (217). However, the search for mutation in the tyrosine kinase domain of the INSR gene did not show abnormalities other than polymorphisms in exon 17, which were not associated with insulin resistance (218). Therefore, the still unidentified factor responsible for the increased phosphorylation of the serine residues of the INSR appears to be extrinsic to the receptor and might also contribute to the increased serine phosphorylation of P450c17 also found in some PCOS patients (118).
2. IRS-1 and IRS-2.
After insulin binding, autophosphorylation of tyrosine residues results in the activation of the INSR, and tyrosine kinase activity phosphorylates intracellular substrates such as IRS-1 and IRS-2 (3). PCOS women present a defect in insulin receptor signaling characterized by a decreased IRS-1-associated phosphatidylinositol 3-kinase activity (217). Two common SNPs in the genes encoding insulin-receptor substrates, Gly972Arg in IRS-1 and Gly1057Asp in IRS-2, are susceptibility genes for type 2 diabetes mellitus (219, 220); they have been studied in PCOS patients, despite the fact that evidence for linkage or association with PCOS was not found with IRS-1 in a family-based study conducted in the United States (132).
Carriers of Arg972 IRS-1 alleles presented with increased fasting insulin levels compared with women homozygous for Gly972 alleles, whereas carriers of Asp1057 IRS-2 alleles presented with increased glucose and insulin levels 2 h after an oral glucose load and had an increased prevalence of glucose intolerance compared with subjects homozygous for Gly1057 alleles (102). However, a subsequent study in a larger series of PCOS patients showed only the effect of the Gly1057Asp polymorphism in IRS-2 on glucose tolerance, and no effect of the Gly972Arg polymorphism in IRS-1 (103). Surprisingly, the effect was just the opposite found previously, because the 2-h glucose values were actually increased in subjects homozygous for Gly1057 alleles when compared with carriers of Asp1057 alleles (103). Additional studies are needed to confirm the influence of these polymorphisms on glucose tolerance and insulin resistance in PCOS.
3. INS.
The presence of pancreatic ß-cell dysfunction in women presenting with PCOS appears to have a genetic origin (221). Therefore, INS has been studied in women with PCOS and functional hyperandrogenism. Waterworth et al. (222) found that women with menstrual disturbances and/or hirsutism and polycystic ovaries, who were homozygous for class III alleles, were more frequently anovulatory and had increased body mass index and fasting insulin compared with women homozygous for class I alleles. Moreover, class III alleles were associated with symptomatic women (222, 223). Paternal transmission of class III alleles from heterozygous fathers to anovulatory PCOS patients is more frequent than maternal transmission of the allele (222, 223, 224), and class III alleles predisposed these patients to both PCOS and type 2 diabetes mellitus. However, later case-control studies in European Caucasian women, conducted outside the United Kingdom, have failed to reproduce these results (225, 226), and the INS locus was not associated with PCOS in a linkage study in American PCOS patients (132).
4. IGF system.
IGFs, their receptors, binding proteins, and proteases are important for the normal development of the ovary (227). IGFs stimulate ovarian cellular mitosis and steroidogenesis, inhibit apoptosis, and might be related to the development of functional hyperandrogenism and PCOS (228).
No evidence for linkage with PCOS was found for markers close to the genes encoding IGF-I and IGF-binding proteins 1 and 3 in a family study conducted in the United States (132). San Millán et al. (229) recently found an association of PCOS with homozygosity for G alleles of the ApaI polymorphism in IGF-II, but not with a dinucleotide polymorphism in IGF-I, a trinucleotide polymorphism in the IGF-I receptor, or with an ACAA-insertion/deletion polymorphism at the 3' nontranslated region of the IGF-II receptor, previously described (230, 231, 232). G alleles of the ApaI polymorphism in IGF-II have been attributed to increased IGF-II mRNA in leukocytes compared with A alleles (233), and possibly result in increased liver IGF-II expression and secretion (234). Given that IGF-II stimulates adrenal (235, 236) and ovarian (237) androgen secretion, the increased frequency of homozygosity for these alleles might contribute to hyperandrogenism in some PCOS patients, provided we assume that G alleles may increase IGF-II expression at the ovary, as reported for other tissues. In the same case-control study involving PCOS patients cited above (229), we found that subjects homozygous for 90-bp alleles of a trinucleotide repeat polymorphism in the gene encoding IGF-I receptor had increased fasting glucose levels and fasting insulin resistance index compared with subjects carrying 93-bp alleles, but no association of any genotype with PCOS.
5. PPAR-2.
Activation of PPAR- by using the insulin sensitizer drugs, thiazolidinediones, has been one of the most important advances for the treatment of type 2 diabetes mellitus in past years. As described above, insulin resistance is a common finding in hyperandrogenic patients, and thiazolidinediones improve insulin sensitivity, hyperandrogenism, and ovulation in women with PCOS (238, 239, 240, 241, 242, 243).
The Pro12Ala SNP in PPAR-
