Association of Polycystic Ovary Syndrome and Adiponectin Gene Polymorphisms

Background: Polycystic Ovary Syndrome (PCOS) is a common gynecological endocrine disorders; which characterized by hyperandrogenism, and anovulation with increased risks of infertility. Adiponectin is a protein specifically and abundantly expressed in adipocytes. Adiponectin Gene Polymorphisms (SNPs) influence adiponectin levels associated with risk for obesity, Insulin Resistance (IR) and T2DM. We aimed to investigate the relation between PCOS and adiponectin gene polymorphism affecting the metabolic disturbance. Materials: Sixty women (Group 1) with PCOS were selected, had presented at gynecological-department of Minia university hospital with fertility problems. Forty healthy women (Group 2) matched with age and with regular menses and without hyperandrogenemia volunteered as controls. Serum level of F.S.H, L.H, total testosterone and glucose were measured. Adiponectin was measured by commercial immunoassays. AMH was measured by (Human AMH ELIZA kit) and insulin was measured by ELIZA, then the glucose/insulin ratio was calculated. Genomic DNA was isolated from peripheral blood leukocytes. The polymorphism was typed according to the curve obtained by real time PCR. Results: The results showed low significant value of adiponcectin and glucose-insulin ratio was with PCOS, whereas FSH, LH, prolactin, total testosterone and AMH were higher in PCOS. Adiponectin, AMH, total testosterone, LH levels and glucose insulin ratio between PCOS and control group showed significant differences. While, there were no significant differences in case of FSH and prolactin. Plasma adiponectin concentration was positively correlated with insulin-stimulated glucose disposal with a significant of 0.003. The genotyping distributions of TG, GG and TT in women with PCOS are 22 (37%), 19 (31.5%) and 19 (31.5%), respectively. The correlation of adiponcetin and genotyping is significant P = 0.001. Conclusion: Prevalence of adiponectin gene polymorphism is higher in PCOS, with significant correlation with the glucose/insulin ratio.


Background
It is known that PCOS is associated with increased risks of infertility, impaired glucose tolerance, type 2 diabetes mellitus, and metabolic syndrome [1]. While 6%-17% of reproductive-age women worldwide suffer PCOS [2,3] some ethnicities, such as South Asian, have higher incidence rates [4]. Etiological studies demonstrated significance of genetic susceptibility for PCOS pathogenesis [5]. Central adiposity play an important role in the insulin resistance of the metabolic syndrome via deregulated production of various adipocytederived cytokines and proteins (adipocytokines), including tumor necrosis factor plasminogen activator inhibitor-1, leptin, resistin, and adiponectin [6]. Adiponectin, which is a known protein secreted exclusively by differentiated adipocytes and circulates in large amounts in humans [7]. Adiponectin (ADIPOQ) is the most common gene product in adipose tissue [8,9]. Adiponectin is encoded by Adipocyte, C1q, and Collagen Domain Containing (ACDC), which is located on chromosome 3 at q27. Genome-wide scans have revealed a susceptibility locus at 3q27 for Type 2 Diabetes (T2D), Coronary Artery Disease (CAD), obesity, and metabolic syndrome [10,11]. Several Adiponectin Gene (ADIPOQ) Single Nucleotide Polymorphisms (SNPs) influence adiponectin levels and associated with incidence of obesity, insulin resistance (IR), T2DM, and CVD [12]. The adiponectin gene consists of three exons and two introns spanning a 17-kb region [13]. Sequence polymorphisms identified in humans and investigated for their possible association of insulin resistance indexes and circulating adiponectin concentrations [11,[14][15][16]. Most studies have focused on two polymorphisms, a silent T-to-G substitution in exon 2 (45T-G) and a G-to-T substitution in intron 2 (276G-T). These polymorphisms associated with obesity, insulin resistance, and the risk of type 2 diabetes [17][18][19][20]. Furthermore, these two polymorphisms were selected because of their high frequencies in all investigated population, while other reported polymorphisms were rare. The 45T-G polymorphism was related to 4-androstenedione concentrations in PCOS [21]. Therefore, we have investigated the relation between PCOS and axon polymorphism of the adiponect in gene, and the effect of this relation on metabolic disturbance.

Materials and Methods
Group I: Sixty women with PCOS were selected, had all presented at our gynecological department of Minia university hospital from April 2015 to January 2016 with oligomenorrhea or fertility problems. None of the women had galactorrhea, or any systemic disease that could possibly affect their reproductive physiology. Any medication could interfere with the normal function of the hypothalamic-pituitary-gonadal axis. Control group (Group 2) was selected from volunteered healthy forty women, who matched with age, with regular menses and without hyperandrogenemia. All women in the study were genetically unrelated. Before the study, blood samples were drawn from each patient between 08.00 and 08.30 a.m., after an 8 h fast for determination of hormone, adiponectin and glucose levels. A standard 75 g Oral Glucose Tolerance Test (OGTT) and the insulin response to oral glucose loading were performed between 08.30 and 10.30 h, after 10-12 h of fasting. Glucose tolerance was evaluated using the criteria of the American Diabetes Association. For all women, blood samples were collected; part of them were put in EDTA tubes stored at -80°C for genetic analysis, and the remaining blood centrifuged directly and the serum was withdrawn and stored at -80°C for estimation of F.S.H, L.H, testosterone, adiponectin and Anti-Mullarian Hormone (AMH).
For all women (Groups 1 and 2), serum level of F.S.H, L.H, total testosterone and glucose were estimated in the laboratory unit in Minia university hospital. Adiponectin was measured by commercial immunoassays (Human Adiponectin RIA Kit, Linco Research, St. Charles, MO, USA) with intra-and inter-assay coefficients of variation below 10%. AMH was measured by commercial immunoassays (Human AMH ELIZA kit, US Biological life sciences, U.S.A) and insulin was measured by ELIZA (Human ELIZA kits, Thermo Fisher Scientific), then a glucose/insulin ratio was calculated.

Genotype analysis
Isolation of genomic DNA was carried out from peripheral blood leukocytes of women with PCOS (Group 1) and the controls (Group 2). The extraction was done as the followings: Samples were collected in sterile tubes; then DNA was extracted using the QIAamp Min elute kit protocol in automated mode by a Qiacube instrument. Real Time PCR was used according to literature (OD-0002-02) from Life River. The amplification of genomic DNA was done using the following primers: F5-GAATGAGACTCTGCTGAGATGG and R5-

Statistical analysis
Analyses were performed using the Statistical Package for Social Sciences (SPSS version 18.0). Hormonal data were analyzed with durations of disease and with age using Factorial experiment with completely randomized design with three factors, and were analyzed between groups using Factorial experiment with completely randomized design with two factors. Data were represented as mean ± SE. Bivariate correlations were performed using the Pearson correlation coefficient (P): a P value of <0.05 was considered statistically significant.

Results
The demographic data of our case group were as follows. The mean and standard deviation of age were 24.2167+3.63641. Descriptive analysis in both groups in Table  1 shows that the adiponectin concentrations and glucose/ insulin ratios were significantly lower with PCOS (Group 1), whereas FSH, LH, prolactin, total testosterone and AMH were higher in PCOS. There were significant differences in adiponectin, AMH, total testosterone, glucose insulin ratio between Group 1 and control group (Group 2). On the other hand, there were no significant differences in case of FSH and prolactin. 72% of cases of PCOS came from rural areas, while 28% came from urban area ( Table 2). Plasma adiponectin concentration was positively correlated with glucose insulin ratio with a significance of 0.003 ( Table 3). The genotyping distributions of TG, GG and TT in women with Group 1 are 22 (37%), 19 (31.5%) and 19 (31.5%), respectively ( Table 4). They differ from the same quantities in Group 2: 3 (7.5%), 7 (17.5%) and 30 (75%). Finally, our results show that the correlation of adiponcetin and genotyping of both groups is of significant value (P = 0.001, Table 5).

Discussion
In our study, it was noted that serum adiponectin levels of the phenotypes of PCOS were significantly lower than control women (Group 2). That might be attributed to the difference in hyperandrogenism of PCOS women and the body fat distribution and insulin resistance as well. Accordingly, the decreasing in serum adiponectin levels of PCOS patients may be related to any of these variables. Serum adiponectin plays an important role in the pathogenesis of insulin resistance and consequently it might deduce that hypoadiponectinemia would contribute in insulin resistance of PCOS women [22].

Archives of Clinical Microbiology
ISSN 1989-8436 Vol.7 No.3:20 Our results agreed with Spain study of seventy-six PCOS patients and 40 non-hyper androgenic women related to BMI and the degree of obesity. Free testosterone levels, age and abdominal adiposity, irrespective of the degree of obesity, were found as the major determinants of hypoadiponectinemia. Reported results support the hypothesis that hyperandrogenism might indirectly lead to insulin resistance in women, by inducing abdominal adiposity and possibly decreasing adiponectin in PCOS patients [23].
Therefore the possible dysfunction of adipose tissue is the main reason of insulin resistance in PCOS [23]. A study by Elbers et al. demonstrated that hyperandrogenism lower adeponectin level and increase insulin resistance in PCOS women [24]. Several studies have shown the higher incidence of adiponectin gene polymorphisms in PCOS subjects. Possible genetic mechanisms could explain the variations in adiponectin levels in patients with PCOS [25]. On contrary of our results, Mohan et al. revealed a completely similar genotype and allele frequency distribution of SNP in the exon 245 position of the adiponectin gene between the PCOS and control groups. Similarly, a study performed in Greek women, no significant difference was detected between 45T→G polymorphism frequencies in the PCOS and control groups, and this polymorphism at position 45T→G had not been associated with a risk for development of PCOS [26,27]. Escobar-Morreale et al. [28] showed that the adiponectin gene polymorphism, 45T→G, is not associated with patients with PCOS. In another Greek trial, the TT genotype and T allele at the 45 position was detected at a lower frequency, while the TG genotype and G allele were detected at a higher frequency in obese women with PCOS compared to those of normal weight (agreed with our results); however, these differences were not statistically significant [29].    Besides that, it was also reported [29] that the SNPs, 45G 15G (T/G) in the ADIPOQ gene are associated with PCOS. Haap et al. [30] found a higher prevalence of 45T→G polymorphism in the adiponectin gene in women with PCOS compared to controls. The 45T→G polymorphism was associated with an increased risk of type 2 DM in a Japanese study [31], which reported a strong association of 45T→G polymorphism with obesity and insulin resistance. Insulin resistance plays a role in type 2 DM pathogenesis. Patients with PCOS have a higher risk of type 2 DM. The commonality between type 2 DM and PCOS pathogenesis is insulin resistance. Some studies suggested that adiponectin gene polymorphisms may be related to type 2 DM [32]. Previous studies examined the association of these and other adiponectin gene variations with type 2 DM and other components of metabolic syndrome. In our study, there was significant statistical difference in genotype distribution and allele frequencies in PCOS compared to the control group. PCOS subjects were found to have a higher risk of type 2 DM and CVD than healthy women.

Conclusion
We concluded that there is higher prevalence of adiponectin gene polymorphism in cases of PCOS with significant correlation with glucose insulin ratio.