The Association Analysis of Vascular Endothelial Growth Factor -2549 Insertion/ Deletion Variant and Endometriosis Risk.

Table 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. Baseline characteristics data of study population Genotype and allele distributions of -2549 I/D between two groups, and endometriosis risk of VEGF polymorphism The proportion of patients suffering from endometriosis symptoms such as dysmenorrhea was significantly higher in cases in comparison with the control group (P <0.01). A 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 . Table 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).

Our 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 ). Yang 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). Among 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 ). In 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. Buraczynska 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 ). The 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. In conclusion, we did not find a statistically significant association in VEGF alleles frequencies between the endometriosis and non-endometriosis Iranian women. Prospective 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.

Subjects Two 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. Demographic 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. Sample collection, DNA extraction, and VEGF polymorphism evaluation For 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. Genomic 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. The 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. Statistical analysis Data 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. The 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.