Marita Pall

Association of Androgen Receptor CAG Repeat Polymorphism and Polycystic Ovary Syndrome

CONTEXT Genetically determined heightened androgen sensitivity may influence the phenotype of polycystic ovary syndrome (PCOS). To date, studies of the androgen receptor exon 1 polymorphic CAG repeat have produced conflicting results in PCOS. OBJECTIVE We tested the hypothesis that a lower number of CAG repeats is associated with increased odds of PCOS. We also compared X-chromosome inactivation between cases and controls. DESIGN Women with and without PCOS were genotyped for the CAG repeat and assessed for X-chromosome methylation. Association analyses were performed. SETTING Subjects were recruited from the reproductive endocrinology clinic at the University of Alabama at Birmingham; controls were recruited from the surrounding community. Genotyping took place at Cedars-Sinai Medical Center in Los Angeles. PARTICIPANTS Participants included 330 women with PCOS and 289 controls (77% white, 23% black). MAIN MEASUREMENTS Androgen receptor genotype, X-chromosome methylation, and phenotyping for PCOS were measured. RESULTS A smaller biallelic mean of CAG repeats was associated with increased odds of PCOS. X-chromosome inactivation was not different comparing cases with controls; however, in the subset with nonrandom inactivation, the chromosome bearing the shorter CAG allele was preferentially active in PCOS women. CONCLUSIONS Association of shorter CAG repeats with PCOS is consistent with in vitro functional studies demonstrating higher activity of androgen receptors expressed from alleles with fewer CAG repeats, suggesting inherited alteration in androgen sensitivity may contribute to PCOS. In some women, such heightened sensitivity may also result from preferential expression of androgen receptors with shorter alleles. Thus, genetic and epigenetic changes may be involved in the pathogenesis of PCOS.

The phenotype of hirsute women: a comparison of polycystic ovary syndrome and 21-hydroxylase-deficient nonclassic adrenal hyperplasia.

OBJECTIVE To test the hypothesis that women with polycystic ovary syndrome (PCOS) are distinguishable from those with 21-hydroxylase-deficient nonclassic adrenal hyperplasia on the basis of having polycystic ovaries and metabolic dysfunction. DESIGN Prospective observational. SETTING Tertiary care center. PATIENT(S) Fifty-two lean and 54 obese women with PCOS according to the 1990 National Institutes of Health criteria, 23 women with nonclassic adrenal hyperplasia, and 27 controls. INTERVENTION(S) History and physical examination, blood sampling, ovarian sonography, oral glucose tolerance, and acute adrenocorticotropin stimulation testing. MAIN OUTCOME MEASURE(S) The frequency of clinical, biochemical, and metabolic features. RESULT(S) Women with PCOS had a higher frequency of oligomenorrhea or amenorrhea than those with nonclassic adrenal hyperplasia. Mean androstenedione and DHEAS levels were highest in nonclassic adrenal hyperplasia. The degree of metabolic dysfunction was greatest in obese women with PCOS; women with nonclassic adrenal hyperplasia and lean women with PCOS did not differ in degree of metabolic dysfunction. Women with nonclassic adrenal hyperplasia had a lower prevalence of polycystic ovaries than those with PCOS. The proportion of patients with an LH/FSH ratio >2 was greater in women with PCOS, compared with those with nonclassic adrenal hyperplasia. Basal 17-hydroxyprogesterone levels >2 ng/mL were found in 87%, 25%, 20%, and 7% of women with nonclassic adrenal hyperplasia, lean women with PCOS, obese women with PCOS, and controls, respectively. CONCLUSION(S) Nonclassic adrenal hyperplasia should be excluded in all women presenting with hirsutism, with use of a basal follicular phase 17-hydroxyprogesterone level, regardless of the presence of polycystic ovaries or metabolic dysfunction; however, women with nonclassic adrenal hyperplasia have a higher prevalence of normal ovulation and lower likelihood of having an LH/FSH ratio >2 or polycystic ovaries.

Abnormal Expression of Genes Involved in Inflammation, Lipid Metabolism, and Wnt Signaling in the Adipose Tissue of Polycystic Ovary Syndrome

CONTEXT Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women. OBJECTIVE Our objective was to compare gene expression pattern in sc abdominal adipose tissue in nonobese PCOS patients vs. body mass index-matched controls. RESEARCH DESIGN AND METHODS Eleven PCOS subjects and 12 controls (body mass index 20-28 kg/m(2)) were recruited. Total RNA was isolated, and gene expression profiling was performed using Affymetrix Human Genome U133 arrays. Differentially expressed genes were classified by gene ontology. Microarray results for selected genes were confirmed by quantitative real-time PCR (RT-qPCR). Frequently sampled iv glucose tolerance tests were used to assess dynamic insulin sensitivity. RESULTS Ninety-six genes were identified with altered expression of at least 2-fold in nonobese PCOS adipose tissues. Inflammatory response genes were significantly down-regulated. RT-qPCR confirmed decreases in expression of IL6 (12.3-fold), CXCL2 (18.3-fold), and SOCS3 (22.6-fold). Lipid metabolism genes associated with insulin resistance were significantly up-regulated, with confirmed increases in DHRS9 (2.5-fold), UCLH1 (2.6-fold), and FADS1 (2.8-fold) expression. Wnt signaling genes (DKK2, JUN, and FOSB) were differentially expressed. RT-qPCR confirmed significant expression changes in DKK2 (1.9-fold increase), JUN (4.1-fold decrease), and FOSB (60-fold decrease). CONCLUSIONS Genes involved in inflammation, lipid metabolism, and Wnt signaling are differentially expressed in nonobese PCOS adipose tissue. Because these genes are known to affect adipogenesis and insulin resistance, we hypothesize that their dysregulation may contribute to the metabolic abnormalities observed in women with PCOS.

The Severity of Menstrual Dysfunction as a Predictor of Insulin Resistance in PCOS

OBJECTIVE The objective of the study was to evaluate the relationship between the severity of menstrual disturbances and the degree of insulin resistance in women with polycystic ovary syndrome (PCOS). DESIGN This was a cross-sectional study. SETTING The study was conducted at a tertiary care academic medical center. PATIENTS Four hundred ninety-four women diagnosed with PCOS by the Rotterdam criteria and 138 eumenorrheic, nonhirsute, control women participated in the study. INTERVENTIONS INTERVENTIONS in the study included history and physical examination and blood sampling. MAIN OUTCOME MEASURE(S) Physical assessment and total and free T, dehydroepiandrosterone sulfate, fasting glucose, and insulin levels and calculated homeostatic model assessment values for insulin resistance (HOMA-IR) were measured. RESULTS Overall, 80% of PCOS subjects included had clinically evident oligomenorrhea. The remainder demonstrated vaginal bleeding intervals of fewer than 35 days (ie, with either polymenorrhea or clinically apparent eumenorrhea). Only 10% of PCOS subjects studied were ovulatory. After adjusting for body mass index, age, and race, all PCOS subjects with menstrual cycles longer than 35 days had significantly higher mean HOMA-IR levels than controls, and those with cycles longer than 3 months had the highest HOMA-IR levels. There was no difference in mean HOMA-IR levels between PCOS with regular vaginal bleeding (apparent eumenorrhea), regardless of whether they were anovulatory or not, or those with cycles fewer than 26 days, when compared with controls. CONCLUSIONS Women with PCOS and overt oligomenorrhea comprise the vast majority of PCOS subjects seen clinically and have significantly more insulin resistance than controls. About 20% of PCOS women seen reported vaginal bleeding intervals of fewer than 35 days in length and did not generally have overt insulin resistance, regardless of whether they were ovulatory or not. Overall, the presence of clinically evident menstrual dysfunction can be used to predict the presence and possibly the degree of insulin resistance in women with PCOS.

Adipocytes from women with polycystic ovary syndrome demonstrate altered phosphorylation and activity of glycogen synthase kinase 3

OBJECTIVE To test the hypothesis that an abnormality in glycogen synthase kinase-3 (GSK3) is a pathogenic factor in polycystic ovary syndrome (PCOS). DESIGN Prospective experimental study (adipocytes). SETTING Tertiary-care academic medical center and teaching hospital. PATIENT(S) Twenty patients with PCOS and 21 healthy control women. INTERVENTION(S) Blood sampling, physical exam, biopsy of SC lower abdominal fat. MAIN OUTCOME MEASURE(S) Glucose transport and protein levels and phosphorylation state of glycogen synthase kinase (GSK)-3alpha and GSK3beta in adipocytes; assessment of GSK3beta activity. RESULT(S) Basal protein levels of glycogen synthase kinase (GSK3alpha and GSK3beta) did not differ between control women and women with PCOS, nor did basal or insulin-stimulated levels of serine phosphorylated GSK3alpha. However, in adipocytes of women with PCOS, insulin stimulation was not associated with increased serine phosphorylation of GSK3beta, in contrast to the case of control women. Tyrosine phosphorylation of GSK3beta also was higher in women with PCOS, compared with in control women. Consistent with the phosphorylation data, GSK3beta activity was elevated in PCOS adipocytes. CONCLUSION(S) These data suggest that GSK3beta is hyperactivated and resistant to down-regulation by insulin in PCOS. By using physiologic approaches, we demonstrated that abnormal GSK3beta regulation is a potential mechanism for the insulin resistance that is seen in some women with PCOS, which may contribute to their development of the syndrome.

Effects of Endogenous Androgens and Abdominal Fat Distribution on the Interrelationship Between Insulin and Non-Insulin-Mediated Glucose Uptake in Females

BACKGROUND Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism and insulin resistance. Glucose disposal occurs via noninsulin-mediated glucose uptake (NIMGU) and insulin-mediated glucose uptake (IMGU). It is unknown whether in PCOS NIMGU increases to compensate for declining IMGU and whether androgens and fat distribution influence this relationship. OBJECTIVES The objective of the study was to compare in women with PCOS and controls the interrelationship between NIMGU [ie, glucose effectiveness (Sg)] and IMGU [ie, the insulin sensitivity index (Si)] and the role of androgens and fat distribution. PARTICIPANTS Twenty-eight PCOS (by National Institutes of Health 1990 criteria) and 28 control (age, race, and body mass index matched) women were prospectively studied. A subset of 16 PCOS subjects and 16 matched controls also underwent abdominal computed tomography. MAIN OUTCOME MEASURES Glucose disposal (by a frequently sampled iv glucose tolerance test), circulating androgens, and abdominal fat distribution [by waist to hip ratio and visceral (VAT) and sc (SAT) adipose tissue content] were measured. RESULTS PCOS women had lower mean Si and similar Sg and abdominal fat distribution compared with controls. PCOS women with Si below the PCOS median (more insulin resistant) had a lower mean Sg than controls with Si above the control median (more insulin sensitive). In PCOS only, body mass index, free T, modified Ferriman-Gallwey score, and waist to hip ratio independently predicted Sg, whereas Si did not. In PCOS, VAT and SAT independently and negatively predicted Si and Sg, respectively. CONCLUSION The decreased IMGU in PCOS is not accompanied by a compensatory increase in NIMGU or associated with excessive VAT accumulation. Increased general obesity, SAT, and hyperandrogenism are primary predictors of the deterioration of NIMGU in PCOS.

Steroidogenic Regulatory Factor FOS Is Underexpressed in Polycystic Ovary Syndrome (PCOS) Adipose Tissue and Genetically Associated with PCOS Susceptibility

CONTEXT Polycystic ovary syndrome (PCOS) is a heterogeneous common genetic disorder characterized by hyperandrogenemia and insulin resistance. Alterations in gene expression profiles of the ovary and adipose tissue identified the candidate gene FBJ murine osteosarcoma viral oncogene homolog (FOS) for further investigation of expression changes in metabolic tissues and genetic studies. OBJECTIVE The objective of the study was to confirm the underexpression of the FOS gene in sc adipose and determine whether variants in this gene are risk factors for PCOS. DESIGN RT-PCR was performed in sc fat from women with and without PCOS. Genotyping of single-nucleotide polymorphisms in the FOS locus was performed to test for association with PCOS. SETTING The study was conducted at a tertiary care academic institution. PARTICIPANTS Twenty-two PCOS and 13 control subjects were recruited for gene expression studies. We assembled a discovery genotyping cohort of 354 cases and 161 controls and a replication cohort of 476 cases and 315 controls, all of whom were Caucasian. MAIN MEASUREMENTS Gene expression by quantitative real-time RT-PCR, FOS genotype, and PCOS status were measured. RESULTS FOS expression was confirmed to be reduced in PCOS adipose tissue. Three single-nucleotide polymorphisms were significantly associated with PCOS in the discovery cohort (rs8006998, P = 0.0031; rs8013918, P = 0.0006; rs8013942, P = 0.0087). rs8006998 was also associated with PCOS in the replication cohort (P = 0.013). CONCLUSIONS Differential gene expression in sc fat and genetic association at the FOS locus in PCOS subjects implicates a role for this transcription factor in PCOS. FOS dysfunction may be a common factor between hyperandrogenism and insulin resistance.

Basal metabolic rate in women with PCOS compared to eumenorrheic controls.

PCOS is associated with obesity and insulin resistance. Efforts have focused on whether an abnormal energy homeostasis contributes to the development of obesity in these patients. There are conflicting results in the literature regarding whether women with PCOS have an altered basal metabolic rate (BMR), thereby leading to difficulties in weight loss. The objective of this study is to compare basal metabolic rate (BMR) in women with PCOS and controls.

Effect of sex steroids and insulin on dehydroepiandrosterone sulfate production by hepatoma G2 cells

OBJECTIVE To test the hypothesis that DHEAS production from DHEA occurs in hepatic cells and that this production is augmented by the presence of sex steroids or insulin. DESIGN In vitro prospective experiment. SETTING Academic medical center. INTERVENTION(S) Hepatoma G2 cells cultured in media supplemented with [1] DHEA (10(-5) mol/L) only, [2] DHEA (10(-5) mol/L) + T (10(-6) mol/L), [3] DHEA (10(-5) mol/L) + E(2) (10(-6) mol/L), [4] DHEA (10(-5) mol/L) + dihydrotestosterone (10(-6) mol/L), [5] DHEA (10(-5) mol/L) + insulin (10 ng/mL), or [6] DHEA (10(-5) mol/L) + insulin (100 ng/mL). MAIN OUTCOME MEASURE(S) Levels of DHEAS in the media were measured at 0, 2, 4, 6, 8, 12, 24, 48, and 72 hours after adding treatments at time-point 0. RESULT(S) Dehydroepiandrosterone sulfate was first detected in the hepatoma G2 cell culture media at 12 hours of incubation. The cumulative production rate of DHEAS increased linearly until 72 hours of incubation. When compared with the effect of treatment with DHEA only, treatment with DHEA plus T, dihydrotestosterone, or E(2) delayed the cumulative DHEAS production; alternatively, the addition of insulin did not alter DHEAS production. CONCLUSION(S) These data suggest that although hepatic cells have the ability of converting DHEA to DHEAS, neither sex steroids nor insulin results in the increased hepatic production of DHEAS.

423 GENETIC VARIATION IN 5α-REDUCTASE INFLUENCES DEVELOPMENT OF POLYCYSTIC OVARY SYNDROME AND SEVERITY OF HIRSUTISM.

The 5α-reductase enzyme converts testosterone to dihydrotestosterone, the most potent androgen in certain tissues such as the hair follicle. Increased activity of this enzyme has been seen in the ovaries of patients with polycystic ovary syndrome (PCOS), the most common endocrinopathy in reproductive-aged women. Increased activity of this enzyme may have important clinical implications in PCOS. For example, 5α-reduced androstenedione inhibits aromatase in the ovaries, reducing estrogen production, a possible cause of follicular arrest and thus polycystic ovaries. Our hypothesis is that genetic variation in the gene for 5α-reductase type 1 (SRD5A1) influences the development of PCOS and the severity of hirsutism. We studied 287 white women with PCOS and 187 controls. PCOS was diagnosed using the 1990 NIH criteria. Hirsutism was quantified using the Ferriman-Gallwey (FG) score, with a score of 6 or greater indicating presence of hirsutism. Seven single nucleotide polymorphisms (SNPs) in SRD5A1 were chosen from the International HapMap project such that they would allow efficient identification of the common SRD5A1 haplotypes (a haplotype is a collection of alleles inherited together on the same chromosome). Genotyping was performed using the 5′-exonuclease reaction. Haplotypes and haplotype blocks were constructed using the program Haploview. Association of haplotypes with PCOS risk and presence of hirsutism was assessed using logistic regression and association with FG score using ANCOVA. SNP rs3797179 (C/C versus C/T+TT) was associated with reduced risk of PCOS (OR 0.4, p = .005). We discovered two haplotype blocks in the SRD5A1 gene, block 1 comprising the first five SNPs, block 2 the last two SNPs. Haplotype T-A (block 2) was associated with a 2.5-fold increased risk of PCOS (p = .004), increased risk of hirsutism (OR 2.6, p = .015), and increased FG score (9.3 vs.8.0, p = .013). Haplotype G-G (block 2) was associated with decreased severity of hirsutism with lower FG score (7.8 vs 9.6, p = .018). Haplotype C-G-G-T-T (block 1) was associated with the presence of hirsutism (OR 2.9, p = .009). In conclusion, variation in the SRD5A1 gene not only influences the risk of developing PCOS but also affects the severity of hirsutism in affected women. This is the first report to examine SRD5A1 as a candidate gene in PCOS. Identification of such underlying genes will lead to an increased understanding of the pathophysiology of this common condition, ultimately contributing to advances in risk prediction, disease prevention, and treatment.