| Objective: Endometriosis(EMs) is a common gynecological disorder, associated with pelvic pain and infertility, seriously affect the health and fertility of women of reproductive age. So far the exact pathogenesis is still unclear, but the most widely accepted theory is that viable endometrial tissue fragments are refluxed through the fallopian tubes into the pelvic cavity during retrograde menstruation. For endometriotic fragments to survive and develop into an endometriotic lesion, the ability of invasion and metastasis of the ectopic endometriotic cells play an important role in the establishment of endometriotic lesions. Related studies suggested that endometriotic cells might acquire invasive ability via the epithelial–mesenchymal transition.E-cadherin is a transmembrane glycoprotein that mediates calcium-dependent homophilic cell-cell adhesion and plays a major role in the establishment and maintenance of intercellular adhesion. Reduced expression of E-cadherin is thought to result in dysfunction of the cell-cell junction system and increase the activity of cells, which is a hallmark of the epithelial–mesenchymal transition. Related studies proved that epithelial cells of endometriotic lesions showed less E-cadherin expression than those of menstrual endometrium. And E-cadherin-negative endometriotic epithelial cells are invasive. Therefore, the results of these studies concur with the hypothesis that decreased E-cadherin expression may play an important role in developing endometriosis.Any mutation in the E-cadherin gene can result in aberrant E-cadherin protein expression. So studying on E-cadherin gene contributed to further clarifying the mechanism of EMs. The purpose of this study was to investigate the associations of single nucleotide polymorphisms(SNPs) of the E-cadherin gene with endometriosis.Methods: We conducted a case-control study to analyse the relationship between endometriosis development and the E-cadherin gene single nucleotide polymorphisms.Five milliliters of venous blood was obtained from inpatients with endometriosis and healthy female donors, and stored at 4℃. Genomic DNA was extracted by using proteinase K digestion followed by a salting out procedure. The genomic DNA samples of endometriosis patients and controls were sent to Capitalbio Corporation for DNA sequence analysis and mutation screening. The sequencing regions included the promoter region, exons and 3’-UTR of the E-cadherin gene. In total, 17 SNPs were reported by direct sequencing. After analyzing these SNPs, we selected six candidate SNPs—rs16260, rs28372783, rs1801552, rs1801026, rs8049282, and rs13689. The genotyps of the six selected SNPs was analyzed by polymerase chain reaction-ligation detection reaction(PCR-LDR) method(consisted of 651 women with endometriosis and 655 control subjects).Statistical analysis was performed using SPSS v.13.0 software package, and P<0.05(two-sided) was used as the criterion of statistical significance. Hardy Weinberg Equilibrium(HWE) analysis was performed to compare the genotype frequencies in the controls using the χ2 test. Comparison of age in the study groups was performed using the Student’s t test. Differences in genotype frequencies in the endometriosis cases and controls were assessed using the χ2 test. The linkage disequilibrium coefficients were estimated using the EH linkage disequilibrium software and the 2LD program. The odds ratio(OR) and 95% confidence intervals(CI) for the genotypic-specific risk were calculated using an unconditional logistic regression model and were adjusted accordingly for age. This study further analysed the differences in genotype and allele frequencies of the EMs patients with primary infertility and the patients with a history of fertility and the controls using the χ2 test. And the odds ratio(OR) and 95% confidence intervals(CI) were also calculated.Rusults: 1 The mean age of the patients was 35.12 ± 7.03 years(range, 20–50 years), and the mean age of the controls was 35.58 ± 6.88 years(range, 20–50 years). Comparing the age in two groups using the Student’s t test, and there was no significant differences in two groups(P>0.05). 2 DNA sequencing analysis 2.1 In total, 17 SNPs were reported by direct sequencing; 15 SNPs had been recorded in the NCBI database, while there was no record of two SNPs located in 3’-UTR at +759 and +1445. 2.2 After analyzing all of the 17 SNPs by sequencing, we selected six candidate SNPs — rs16260,rs28372783, rs1801552, rs1801026, rs8049282, and rs13689.2.3 Our sequencing analysis showed that 8 of the 17 SNPs were in the 3’-UTR, and 2LD analysis showed that 6 of those 8 SNPs were in high LD and were located in one haplotype block; however, the rs145920869(3’-UTR+221) and rs74957007(3’-UTR+475) SNPs were singled out. 3 Association between E-cadherin gene SNPs and the risk of developing endometriosis.Analyzing the association between the six selected SNPs and the risk of developing endometriosis. But the results showed that there were no significant differences in the genotype and allele frequencies of the six SNPs among the cases and controls(P>0.05). 4 Association between SNPs and primary infertility in ovarian endometriosisThe EMs patients were divided into infertile and fertile groups, and patients without sexual experience were excluded from this study.Statistical analysis showed that the allele frequencies of the rs8049282 SNP in the EMs patients with primary infertility and those with a history of pregnancy were 91.5%、8.5% and 85.4%、14.6%, there were significantly different between two groups(χ2=6.358, P=0.012). When compared to the C/T+T/T genotype, the C/C genotype could significantly increase the risk of primary infertility in patients with endometriosis, the odds ratio was 2.33(95%CI: 1.36-3.99). Furthermore, the age-adjusted OR was 2.70(95% CI: 1.45-5.00).When compared with the control group, the allele frequencies of the rs8049282 SNP in the EMs patients with primary infertility and controlswere 91.5%、8.5% and 85.2%、14.8%, there were significantly different between two groups(χ2=4.621, P=0.032). When compared to the C/T+T/T genotype, the C/C genotype could significantly increase the risk of primary infertility in patients with endometriosis, the odds ratio was 2.25(95%CI: 1.32-3.83). And the age-adjusted OR was 2.70(95% CI: 1.45-5.00).Furthermore, the allele frequencies of the rs1801552 SNP in the EMs patients with primary infertility and those with a history of pregnancy were 42.5%、57.5% and 35.1%、64.9%, there were significantly different between two groups(χ2=4.621, P=0.032). When compared with the T/T genotype, the C/C genotype could significantly increase the risk of primary infertility in patients with endometriosis, the odds ratio was 1.93(95%CI: 1.07-3.48). But the age-adjusted OR was no longer significant(P>0.05). Statistical results indicated that the allelic frequencies and genotype distribution of the rs16260, rs28372783, rs1801026, and rs13689 SNPs showed no differences.Conclusion:1 SNPs in the promoter, exons, and 3’-UTR of the E-cadherin gene may not significantly affect the risk of developing endometriosis.2 The E-cadherin gene polymorphism rs8049282C>T may associated with the risk of endometriosis-related infertility, the CC genotype may significantly increase the risk of primary infertility in endometriosis patients. |
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