Role Of TNF-α And AR Gene In Polycystic Ovary Syndrome

INTRODUCTION Polycystic ovary syndrome (PCOS) is an endocrine and metabolism disorder with heterogeneity, which is characterized by hyperandrogenaemia (HA) and chronic anovulation. Family aggregation suggested that genetic factor play an important role in the development of PCOS.Association with TNF-a gene-308G/A polymorphism and PCOS were preformed in some studies by researchers. No studies were preformed in TNF-a exon region polymorphism and PCOS. Regarding the role of AR variants in PCOS, interest has focused on the CAG repeat polymorphism in exon 1 of the AR gene, and the possible effect of CAG repeated polymorphism in PCOS is controversial. The rs6152G/A polymorphism in exon 1 of the AR gene is associated with male premature baldness (MPB), which is a characteristic of males in PCOS families. However, none of the studies evaluated the role of rs6152G/A in PCOS. PCOS patients were revealed the peripheral blood pro-inflammatory factors such as TNF-a level increased, implying the presence of chronic inflammation in women with PCOS, the mechanism is completely unclear.In this study, analysis of TNF-a gene promoter-308G/A, exon 1 rs3179060C/A and exon3 rs4645843C/T polymorphisms and androgen receptor (AR) gene exonl rs6152G/A and CAG repeat polymorphisms were conducted in PCOS and control groups. The investigation was preformed that whether this polymorphisms were related to increased testosterone in patients with PCOS. Role of TNF-a gene in chronic inflammation in polycystic ovary syndrome was measured. The part molecular mechanism of PCOS can be clarified by this study, which can also be targeted for further clinical diagnosis and treatment and for opening new avenues for the treatment of PCOS.AIMS To determine the relationship between HA in patients with PCOS and gene polymorphisms of TNF-a gene promoter-308G/A, rs3179060C/A and rs4645843C/T.STUDY POPULATION AND METHODS 130 PCOS patients of Han Chinese racial origin were selected from the Reproductive & Genetic Hospital of CITIC-XIANGYA in China from June of 2006 to February of 2008. The diagnosis of PCOS was based on the criteria included in the 2003 American Society for Reproductive Medicine/European Society for Human Reproduction and Embryology (ASRM/ESHRE criteria) consensus meeting guidelines (2 out of 3):(i) oligo-ovulation and/or anovulation, (ii) clinical (hirsutism) and/or biochemical signs of hyperandrogenism (hyperandrogenemia); (iii) polycystic ovaries by vaginal ultrasound, excluding from other diseases such as congenital adrenal hyperplasia, androgen-secreting tumors, Cushing's syndrome, thyroid dysfunction, hyperprolactinaemia, late-onset 21-hydroxylase deficiency, could be defined as PCOS. In this study, the subjects were selected as following criteria:polycystic ovaries (follicle>12 in Unilateral andbilateral ovaries) and higher serum testosterone(T≥0.7 ng/ml), or poly cystic ovaries and menstrual cycle longer than 37 days, and excluded other disorder. The 175 infertile women with tubal factor or male infertility served as the control group at the same period. A normal menstrual cycle was an additional inclusion criterion in the control women. Selected control individuals had no structural abnormalities of the uterus or ovaries, family hypertension, cardiovascular diseases and diabetes. All subjects were referred to our department as candidates for in vitro fertilization because of infertility.3ml peripheral blood from every subject was drawn on days 2 to 4 of the menstrual cycle or anytime during amenorrhea period. Then 300ul whole blood was taken and anticoagulation was performed with trisodium citrate. The blood DNA was extracted using commercial DNA purification Kits. Serum of the other blood sample was isolated for sex hormone analysis.TNF-a-308G/A polymorphism was detected by polymerase chain reaction-restriction fragment long polymorphism (PCR-RFLP); the polymorphism of TNF-a rs4645843C/T and rs3179060C/A was determined by PCR-On/off Switch mediated by Exo+ Polymerases and 3' phosphorothioate-modified primers with single nucleotide polymorphism to the 3' terminus, and the distribution of genotype frequencies and allele frequencies were compared between two groups and the association between the polymorphisms and hyperandrogenaemia was analyzed in PCOS patients.RESULTS1. Genotype frequencies of GG, GA, AA of TNF-αpromotor-308G/A polymorphism in control group and that in PCOS patients the were respectively 83.4%,15.4%,1.2% and 90.8%,8.4%,0.8%; G, A allele frequencies were respectively 91.1%,8.9% and95.0%,5.0%. No significance was observed in between groups (P=0.196,0.069, respectively).2. Genotype frequencies of CC, CA, AA of rs3179060C/A polymorphism in PCOS patients and that in the control group were respectively 58.4%,23.1%,18.5% and72.0%,17.7%,10.3%; C, A allele frequencies were respectively70.0%,30.0% and 80.9%,19.1%. The statistically significant differences of genotype frequencies and allele frequencies were observed in between groups (P=0.035,0.002, respectively).3. Genotype frequencies of CC, CT, TT of rs4645843C/T polymorphism in PCOS patients and that in the control group were respectively 34.6%,58.5%,6.9% and 43.4%,53.7%,2.9%; C, T allele frequencies were respectively 63.8%,36.2% and 70.3%,29.7%. No significant differences of genotype frequencies and allele frequencies were observed in between groups (P=0.108,0.093, respectively).4. There were no correlation among the three polymorphisms of TNF-agene and increased testosterone levels of patients with PCOS.CONCLUSIONS1. TNF-a rs3179060C/A polymorphism was related to PCOS women of Han Chinese racial origin, but had no obvious relationship with serum androgen level, which suggested that the polymorphism may play some role in the pathogenesis of PCOS but showed little effect on hyperandrogenaemia.2. Neither-308G/A polymorphism or rs4645843C/T polymorphism was related to PCOS or hyperandrogenaemia, which suggested that they were no major cause of PCOS. AIMS To determine the association of hyperandrogenaemia in PCOS patients of Han Chinese racial region people with CAG repeated polymorphism and rs6152G/A polymorphism.STUDY POPULATION Two hundred and twenty-four (224) PCOS patients (aged 21-38 years) were recruited from the Reproductive and Genetic Hospital of CITIC-XIANGYA in China from June 2006 to October 2008. The PCOS patients were referred to our department as candidates for in vitro fertilization (IVF) because of infertility. The diagnosis of PCOS was based on the standards recommend in the 2003 American Society for Reproductive Medicine/European Society for Human Reproduction and Embryology (ASRM/ESHRE criteria) consensus meeting guidelines. A total of 223 infertile women (aged 20-39 years) with tubal factor or male infertility served as the control group during this period and were evaluated consecutively, exhibiting no menstrual cycle irregularities, no clinical or biochemical hyperandrogenism and without PCO. Recruited control individuals had no structural abnormalities of the uterus or ovaries, as confirmed by vaginal ultrasound or laparoscopy at the same period.METHODS AR gene CAG microsatellite polymorphism of 224 PCOS patients and 223 control patients was analyzed by sequencing and the compared with distribution of AR genotype and allele frequencies in PCOS and control patients. And the relation of AR gene CAG repeated polymorphism and hyperandrogenaemia in PCOS was investigated. Polymerase chain reaction-restriction fragment long polymorphism (PCR-RFLP) was used to detect the rs6152G/A polymorphism, to compare the rs6152G/A genotype and allele frequencies distribution between groups, as well as to analyze its association with hyperandrogenaemia in PCOS.RESULTS1. Both G and A alleles and GG,GA and AA three genotypes in AR exon 1 rs6152G/A were observed.2. G and A allele frequencies in the PCOS and the control group were 88.4%,11.6% and 92.8%,7.2%, respectively. GG and GA+AA genotype frequencies in the PCOS are respectively 82.6% and 17.4%, while that in the control group were 89.2% and 10.8%, respectively. The significance was observed both PCOS and control groups in genotype frequencies (P=0.043); and it was the same significance as for the allele distribution in the two groups (P=0.023), as well.3. Patients carrying the rs6152A allele had a 1.608-fold greater risk of developing polycystic ovary syndrome compared with rs6152GG homozygotes (OR=1.608, CI=1.008-2.597, P<0.05).4. Compared with control groups, there was a trend for a lower mean CAG biallelic length among PCOS patients compared with control group (22.65±2.5 vs.23.09±2.1). The frequency of short CAG(n<22) was significantly higher in PCOS compared with control groups (56.25% vs 29.14%, P＜0.001).5. In PCOS, there were a statistically significantly shorter mean total biallelic average (22.3±2.5 vs.23.9±2.2, P=0.008) and long (24.3±1.4 vs.25.9±1.6, P=0.05) biallelic lengths in patients with T less than 0.7ng/ml compared with those whose T was more than 0.7ng/ml.6. The testosterone were correlated with the CAG(n<22), CAG(n=22)and CAG(n>22) by ANOVA in PCOS patients(F=3.121,P=0.048).CONCLUSIONSThe individuals carrying the rs6152A allele had significantly higher susceptibility to polycystic ovary syndrome than those that were GG homozygotes.2. The CAG polymorphism of Chinese racial origin was significantly correlated with PCOS and hyperandrogenaemia, implying CAG polymorphism may play an important role in the development of polycystic ovary syndrome and hyperandrogenaemia.AIMS To evaluate the role of of TNF-a in the chronic inflammation in PCOS.METHODS The study population consisted of 30 reproductive-age women with PCOS (thirty lean, thirty obese) and 30 age-and body mass-matched controls (thirty lean, thirty obese) from the Reproductive & Genetic Hospital of CITIC-XIANGYA in China from June 2006 to October 2008. Blood and follicular fluid samples were collected and then blood serum and mononuclear cells were isolated. Follicular fluid and granular cells were isolated. TNF-a concentration in serum was detected via ELISA. And RNA, total protein and nucleoprotein were extracted in mononuclear cells. TNF-a mRNA expression in mononuclear cells and IRS1 and IRS2 mRNA expresssion in granular cells were detected by RT-PCR. NFκB and IκB protein expression were analyzed by Western blot. Nuclear-bound NFκB was analysed by electrophoresis mobility shift assay.RESULTS Compared with lean control, TNF-a concentration in serum was higher in PCOS patients. Compared with lean control, TNF-a mRNA expression, NFκB protein expression and nuclear-bound NFκB in mononuclear cells were significantly higher in PCOS groups (p<0.05) and IRS1 mRNA expression was significantly down-regulated(p<0.05) and IRS2 mRNA expression did not significantly vary in granular cells(P>0.05). IκB protein expression was significantly down-regulated in PCOS patients than in the control lean group (p<0.05).CONCLUSIONSTNF-a serum concentration, TNF-a mRNA expression, NFκB protein expression and nuclear-bound NFκB were increased in PCOS patients, which imply the presence of chronic low-grade inflammation in PCOS women.In PCOS group, IRS1 expression significantly down-regulated in granulosa cells may revealed the insulin resistance by mediated-inflammatory in PCOS.