{"paper_id":"74049df3-71db-44af-be53-c2813d1037a9","body_text":"Subjects\nTwo hundred forty-four women who were admitted for laparoscopy or laparotomy for gynecological procedures in two university hospitals of Tehran, Iran from April 2013 through September 2015 were considered eligible. The study design was approved by the Endocrinology and Metabolism Research Institute, affiliated to Tehran University of Medical Sciences (TUMS), and written informed consent was obtained from all participants.\nDemographic data, gynecological and obstetrical history such as parity, menstrual cycle, and dysmenorrhea were obtained by interviews during preoperative appointments. Subjects were considered as cases (n=100) if they had visible ectopic implants, and a histologically confirmed diagnosis of endometriosis after their operative findings, according to the presence of endometrial glands or stroma in the lesions. The control group (n=144) consisted of women undergoing laparoscopy or laparotomy for tubal ligation or treatment of benign diseases such as ovarian cysts, myoma, hydrosalpinx, etc., and who had no macroscopic signs of endometriosis at surgery. The patients with no history of malignant disease and who had not received hormonal treatment for at least 6 months from the date of sampling were included in the study.\nSample collection, DNA extraction, and  \n VEGF \n  polymorphism evaluation\nFor DNA extraction, 3 ml of peripheral blood (in EDTA containing vial) was obtained from each study subject at the time of enrollment and stored at -20°C.\nGenomic DNA was separated from peripheral blood leukocytes using phenol protocol. In this study, I/D polymorphism of 18 bp fragment at -2549 position of the promoter region in  VEGF  was selected to investigate. The polymerase chain reaction (PCR) assay was used directly for the detection of this polymorphism.\nThe I/D polymorphism was analyzed using the common set of forward 5'- GCTGAGG ATGGGGCTGACTAGGTA-3' and reverse: 5'-GTTTCTGACCTGGCTATTTCCAGG-3' primers as indicated by Buraczynska et al. ( 15 ). PCR amplification was accomplished in 25 µl final volume containing 1 µl template DNA, 10 µl Red Master Mix (Amplicon, UK), 0.7µl each primer and 12 µl H 2 O. The allele/detection process was performed on a Biorad MJ mini (Singapore) to determine the allelic discernment. Thermal cycling was initiated with the first denaturation step of 6 min at 95°V, followed by 35 cycles of denaturation at 95°C for 30 s and annealing at 72°C for 10 min. The amplification products were segregated by electrophoresis on 2.5% agarose gel stained with SYBR green. For the  VEGF  I/D polymorphism, two bands were observed, a band of 229 bp represents an 18 bp insertion (I allele), and the band of 211 bp represents an 18 bp deletion (D allele). About 5% of the accidentally selected samples were confirmed by sequencing.\nStatistical analysis\nData were analyzed using Statistical Package for Social Sciences software, version 16.0 (SPSS, Chicago, IL). Continuous variables were expressed as mean ± standard deviation (SD), and categorical variables were expressed as number and percentage. Student’s t-test was used for comparing quantitative variables and the Chi-squared test was used to analyze categorical data.\nThe difference of genotype and allele frequencies between endometriosis patients and controls was assessed by regression test, and an odds ratio (OR) with 95% confidence interval (CI) was used as a measure of the strength of association between genotypes and allele frequencies. Probability values less than or equal to 0.05 were considered statistically significant.\n\nTable 1  shows the baseline characteristics of the study population. Both groups were age matched (P =0.17). On the contrary body mass index (BMI), dysmenorrhea, gravidity and infertility were significantly different between the two groups (P<0.01). No significant differences were found with regard to bleeding volume and irregular menses between two groups.\nBaseline characteristics data of study population\nGenotype and allele distributions of -2549 I/D between two groups, and endometriosis risk of VEGF polymorphism\nThe proportion of patients suffering from endometriosis symptoms such as dysmenorrhea was significantly higher in cases in comparison with the control group (P <0.01).\nA total of 244 (100 cases and 144 controls) of the enrolled subjects were successfully genotyped for  VEGF  polymorphism. The genotype and allele frequency of the -2549 I/D  VEGF  gene polymorphism among controls and cases are shown in  Table 2 .\nTable 2  shows the endometriosis risk related to  VEGF  polymorphism. No significant differences were found in allele and genotype distribution of the  VEGF  -2549 I/D polymorphism between cases and controls even after adjustment for age and body mass index (BMI).\n\nOur results do not provide any evidence supporting an association between this  VEGF  polymorphism and endometriosis susceptibility. Previous studies indicated that the DD genotype of -2549 I/D polymorphism has been associated with diabetic nephropathy ( 13 ,  14 ), and peripheral arterial disease in T2DM ( 12 ).\nYang et al. ( 13 ), showed that the D allele and D/D genotype of the  VEGF  may be associated with susceptibility to diabetic nephropathy. The pattern including the 18 bp deletion (D allele) had a 1.95-fold increase in the transcriptional activity compared with the other allele containing the inserted construct (I allele).\nAmong the North Indian population, DD genotype at -2549 region of the  VEGF  gene has been associated with 1.58-fold increased risk of end-stage renal disease (ESRD) ( 11 ).\nIn a study by Fouad et al. ( 20 ), no significant association was perceived between I/D polymorphism in the promoter region of the  VEGF  gene and retinopathy in Egyptian T2DM patients. However, DD genotype of -2549 I/D polymorphism had>2- fold and D allele had >1.5 –fold raised risk in patients with diabetic retinopathy than controls.\nBuraczynska et al. ( 15 ) identified the association between the DD genotype and the D allele with retinopathy but not nephropathy in type 2 diabetic patients. Some reports showed that, this polymorphism at -2549 position of the  VEGF  gene is in complete linkage with -2578 A/C polymorphism ( 21 ). It has been showed that individuals with the -2578A allele have an 18 bp insertion, but those with - 2578 C allele have not ( 13 ).\nThe results of the study by Kapahi et al. ( 8 ) in North India suggested that -2549 I/D polymorphism was associated with susceptibility to breast cancer. On the contrary, some studies did not perceive any association between ID and DD genotypes with recurrent miscarriages in the North Indian population ( 22 ), systemic sclerosis in the European Caucasians ( 23 ), and hepatocellular carcinoma (HCC) in the Chinese Han population ( 24 ). On the other hand, this genotype showed >2- fold risk in patients with prostate cancer ( 18 ). Salvarani et al. ( 19 ) showed that the I allele of -2549 I/D polymorphism was associated with Behcet's disease (BD) in Italian patients.\nIn conclusion, we did not find a statistically significant association in  VEGF  alleles frequencies between the endometriosis and non-endometriosis Iranian women.\nProspective studies in larger endometriotic patient populations, as well as the functional studies, are needed to elucidate the role of the  VEGF  I/D polymorphism in endometriosis disease.","source_license":"CC0","license_restricted":false}