{"paper_id":"266dbf6a-7d9d-4818-af51-6179ae22618f","body_text":"Polycystic ovary syndrome (PCOS) is the most common endocrine disorder, and its\nclinical features include hirsutism, infertility, acne, alopecia, oligo-anovulation,\nand metabolic abnormalities such as insulin resistance, excessive weight or obesity,\ntype 2 diabetes, dyslipidemia, and an increased risk of cardiovascular disease \n 1 , 2 \n .\nDysregulation of ovarian angiogenesis contributes to abnormal follicular development\nin women with PCOS. This alteration may contribute to the ovarian features of PCOS,\nsuch as abnormal follicular development, increase in the number of small follicles,\nand failure in the selection of the dominant follicle, with anovulation and cyst formation \n 3 \n .\nModifications in the vascular endothelial growth factor (VEGF) family are associated\nwith the ovarian angiogenesis \n 3 \n . In the ovary, this gene is expressed in theca cells, granulosa lutein cells,\nand interstitial tissues and may be involved in the physiological regulation of\novarian angiogenesis, in a manner that suggests a role of this growth factor in both\ncyclic angiogenesis and regulation of vascular permeability, both critical for\novarian folliculogenesis and for normal reproductive function \n 4 \n . The VEGF gene is located in the chromosome region 6p21.3 \n 5 \n . Single-nucleotide polymorphisms (SNPs) have been observed in the promoter,\nintronic, and untranslated regions of the VEGF gene, and several studies have\nsuggested VEGF gene polymorphisms may be associated with PCOS risk \n 6 , 7 , 8 \n .\nVEGF rs833061 T and rs2010963 G alleles appear to correlate with altered VEGF\nexpression levels. The increased levels of VEGF have been reported in PCOS6. The\nrs2010963 and rs833061 SNPs VEGF have recently been investigated in five independent\nstudies6-10. One of them evaluated the association of VEGFA SNPs (nine tested\nvariants) with altered VEGF secretion level and PCOS among ethnically matched\ncontrol women. This study showed that VEGF levels in rs833061 genotypes were\nsignificantly higher in PCOS9. The other study also published in 2019 investigated\nSNPs rs2010963 and rs833061 and showed that the first one may be associated with the\nrisk of PCOS in Chinese women10. The studies published in 20206-8 are of the\nmeta-analysis type and confirm associations of polymorphisms in the VEGF gene with\nsusceptibility to PCOS, with emphasis on rs20109637,8.\nThe rs2010963 polymorphism had been described as C→T exchange at nucleotide position\n936, in the 3ʹ-UTR of the VEGF gene, and was associated with lower VEGF plasma levels \n 11 \n . Therefore, the rs833061 is located in the promoter region and has been\nassociated with increased VEGF expression levels \n 12 \n .\nIn the literature, there are several studies on polymorphisms in the VEGF gene in\npatients with PCOS from different populations; however, no research was conducted on\nthese two polymorphisms in Brazilian women.\nThe objective of this study was to investigate the association of the VEGF\npolymorphisms rs201093 and rs833061 and to identify the frequency of haplotypes with\nthe risk of developing PCOS in women compared with control group.\n\nThis study was approved by the Research Ethics Committee of the Federal\nUniversity of Triângulo Mineiro (UFTM), protocol 1796, and all participants\nsigned an informed consent form.\nParticipants’ inclusion in the study occurred in the period from 2012 to 2016.\nThis case-control study included 102 patients with a clinical diagnosis of PCOS\nand 108 control women. The PCOS diagnosis was based on Rotterdam criteria, and\nthe patients who visited the Endocrinology and Gynecology Outpatient Clinic of\nthe UFTM were selected. In the control group, women at reproductive age who had\nno history of hyperandrogenism, menstrual dysfunction, infertility, or\nsonographic sign of PCOS, and those who sought medical care for gynecological\nroutine were selected for the study. All participants answered a questionnaire\nabout the risk factors.\nGenomic DNA from the peripheral blood was extracted by a salting-out method.\nGenotyping of the rs2010963 polymorphism was performed in 106 PCOS patients and\n97 controls using polymerase chain reaction restriction fragment length\npolymorphism (PCR-RFLP) analysis.\nThe PCR was carried out in a total volume of 30 μL containing approximately 100\nng genomic DNA, 1× PCR buffer, 1 mM MgCl 2  (25 mM), 2 μM of dNTP, 20\npmol of each primer (F: 5´-CCGACGGCTTGGGGAGATTG-3´; R:\n5´-CGGCGGTCACCCCCAAAAG-3´), 1 U of Taq DNA polymerase, and 5% of glycerol. The\namplification program consisted of an initial denaturation step at 94°C for 10\nmin and 40 cycles (denaturation at 94°C for 45 s, annealing for 45 s at 62°C,\nextension at 72°C for 30 s) and final extension for 10 min at 72°C.\nThe PCR products were digested with 0.2 U of the BsmFI restriction enzyme.\nDigested fragments were analyzed by 10% polyacrylamide gel electrophoresis.\nAfter the restriction enzyme treatment, the CC genotype was visualized as a\nsingle 197 bp fragment; the CG genotype as three fragments of 197, 167, and 30\nbp; and the GG genotype was visualized as two fragments of 167 and 30 bp.\nThe real-time PCR allelic discrimination technique was used to analyze the\nrs833061 polymorphism in 94 patients and 87 controls, on the ABI PRISM 7500\nSequence Detection System (Applied Biosystems) using TaqMan Minor Groove Binder\n(MGB) probes. Primers and probes were designed by Life Technologies (ID:\nC_1647381_10).\nThe chi-square test was used for the statistical analysis of the genotypic and\nallelic distribution of the polymorphisms, as well as to verify the\nHardy-Weinberg equilibrium (HWE). A statistical power of 95% was tested using\nthe G* Power program 3.1.9.2. In addition, a post-hoc test with the total sample\n(n=210) was performed, with an effect size of 0.27 and an alpha significance\nlevel of 0.05. The multiple logistic regression model was used to determine the\neffect of risk factors (e.g., family history, smoking, alcohol consumption, and\nthe presence of polymorphism) in PCOS. The SNPStats program was used for the\nlogistic regression model adjusted for age. The effect of polymorphisms was\nevaluated by the following inheritance models: codominance, dominance,\nrecessive, and overdominance.\nThe haplotype from VEGF gene polymorphisms was inferred using the SNPStats\nprogram, checking the estimated population frequency of the haplotypes.\nThe results were presented in the odds ratio (OR) and 95% confidence interval\n(95%CI), with the level of statistical significance being defined as p<0.05.\nA post-hoc analysis was performed, and the statistical power for association\ntests was found to be 98%.\n\nThe clinical characteristics of the patients showed that 75.8% of the patients did\nnot become pregnant, 36.3% had a family history of PCOS, 58.6% were obese, and about\n60% had clinical characteristics of hyperandrogenism.\nIn the univariate analysis, no statistically significant differences were observed\nbetween the two groups for polymorphisms rs2010963 and rs833061 (χ 2 =0.38,\np=0.83 and χ 2 =0.86, p=0.65, respectively). For the allele frequencies, no\ndifferences were found between the groups (χ 2 =0.18, p=0.67 and\nχ 2 =0.02, p=1.00, respectively).\nThe genotype distributions of the rs2010963 and rs833061 polymorphisms were in\nHardy-Weinberg equilibrium in both patient (χ 2 =2.35; p=0.12;\nχ 2 =0.05; p=0.82) and control (χ 2 =1.05; p=0.31;\nχ 2 =1.04; p=0.31) groups.\nThe genotypes of 90 women with PCOS and 80 controls were adjusted for age according\nto the heritable models and showed no association between the polymorphisms and PCOS\n( Table 1 ).\nOR: odds ratio; CI: confidence interval. Significant p<0.05.\nThe haplotypes that were constructed with the analysis of the two VEGF gene\npolymorphisms were evaluated in this study. All estimated haplotypes had similar\nfrequencies between the two groups.\nThe multiple logistic regression data are shown in  Table 2 , considering the risk factors (e.g., family history, smoking,\nand alcoholism) and the two polymorphisms studied in PCOS patients (n=88) and\ncontrols (n=81). It was evidenced that the family history is more frequent in\npatients with PCOS, smoking is more frequent in controls, and there are no\ndifferences in relation to alcoholism and in the distribution of the rs2010963 and\nrs833061 polymorphisms.\nOR: odds ratio; CI: confidence interval; PCOS: polycystic ovary syndrome.\nBold value indicates significant p<0.05.\n\nVEGF alterations characterize numerous pathologies, either with increased, decreased,\nor abnormal angiogenesis. Therefore, it has been suggested that these alterations\nmay be associated with the decreased, or lack of, ovulation rates and with the\nformation of many antral follicles in the PCOS ovaries. According to the literature,\nfurther studies are required to clarify the role of angiogenesis in PCOS and to\ndevelop new potential therapies \n 2 , 3 \n , such as bromocriptine, metformin, and melatonin \n 13 , 14 , 15 \n .\nThe VEGF is the main angiogenic factor that promotes endothelial cell proliferation\nand migration and vascular permeability \n 3 \n . Thus, genetic analysis in the VEGF gene may help clarify the pathogenesis of\nPCOS.\nTo the best of our knowledge, this is the first molecular study to investigate the\nassociation between rs2010963 and rs833061 polymorphisms and PCOS susceptibility in\nBrazilian women. With regard to VEGF, our group evaluated the polymorphisms\nrs3025039, rs1570360, and rs699947 and showed that the polymorphism rs1570360 is\nassociated with PCOS and that the T-G-C haplotype could be associated with\nprotective factors \n 16 \n .\nSeveral VEGF SNPs are associated with various conditions in women, including endometriosis \n 17 , 18 \n , recurrent miscarriage \n 19 \n , and preeclampsia \n 20 \n . These results together suggest that VEGF SNP can contribute to the\npathogenesis of female reproductive diseases.\nIn the sample analyzed, the rs2010963 and rs833061 polymorphisms are not associated\nwith PCOS. The lack of significance in this study may be due to the sample size,\nsample stratification, and ethnic issues related to the Brazilian population.\nHowever, they have been extensively studied with conflicting results ( Table 3 ) \n 9 , 10 , 12 , 21 – 24 \n , probably due to different ethnicities. It is worth mentioning that PCOS is a\nmultifactorial disease and environmental factors and polymorphisms in genes other\nthan VEGF might play an important role in women’s susceptibility to its\noccurrence.\nThere are seven studies that investigated the polymorphisms rs2010963 and rs833061 of\nthe VEGF gene. Three studies did not confirm any association between PCOS and the\npolymorphisms investigated \n 17 , 19 , 20 \n , a result similar to that found in this study. In contrast, other studies\nfound an association of polymorphism rs2010963 \n 17 , 22 , 23 \n  and rs833061 \n 10 \n  with PCOS.\nIn relation to haplotype analysis, two studies showed a relationship between\nhaplotypes and PCOS \n 21 , 24 \n . However, there is no significant difference in the occurrence of the four\nhaplotypes between the controls and cases, in relation to the polymorphisms\nrs2010963 and rs833061 \n 17 \n , which is in agreement with our results.\nA meta-analysis of seven studies showed there is little association between PCOS risk\nand the VEGF gene polymorphisms rs2010963, rs833061, and rs699947 in the general\npopulations, whereas the genotype CC (rs2010963) might decrease the risk of PCOS\namong Asian women \n 8 \n . Another recent meta-analysis included 10 relevant case-control studies,\ninvolving 1347 PCOS cases and 1378 controls. The VEGF rs2010963 polymorphism was\nassociated with decreased PCOS risk in the whole population and the Asian populations \n 9 \n .\nThis study demonstrated that family history is more frequent in patients with PCOS.\nAccording to the literature, multiple familial and twin studies confirmed the role\nof genetics in the etiology of PCOS with high heritability of 70% \n 21 \n .\nThere are some limitations to our study. The serum levels of the VEGF are not\nmeasured. However, it is also worth mentioning that our study so far is the only one\nto evaluate these polymorphisms in the Brazilian population.\n\nThe PCOS patients have similar rates of VEGF polymorphisms rs2010963 and rs833061 on\nthe general population.","source_license":"CC-BY-4.0","license_restricted":false}