Evaluation of Toll-like receptor 3 (TLR3) signaling pathway genes and its genetic polymorphisms in ectopic and eutopic endometrium of women with endometriosis

Background: It has been proven that innate and adaptive immune responses dysregulation has an undeniable role in endometriosis. Toll-like receptors (TLRs, as members of the innate immune system) are expressed in the human endometrium and their aberrant regulation and expression are involved in the pathogenesis of endometrial diseases.Objective: This study aimed at evaluation of TLR3 signaling pathway genes and its genetic changes in women with endometriosis.Materials and Methods: The case-control study included 176 patients: 83 cases with endometriosis and 93 healthy fertile women. Blood samples were collected from all subjects and polymerase chain reaction (PCR) was performed in blood-derived DNA for detection of single nucleotide polymorphisms (SNP) of TLR3. The PCR products were sequenced by Macrogen Company (South Korea). Also, endometriotic lesions from the ectopic ovarian endometrioma (EC) and eutopic endometrial (EU) biopsies from the same subjects (n=20) were obtained from endometriosis women, as well as endometrium from healthy women (n=16, as control group, CE) in the proliferative phase of menstrual cycle (days 5-14). Quantitative PCR was performed for determination of mRNA expression level of TLR3 signaling pathway genes (TLR3, TICAM, NF-kB1A, CXCL10, IRF3, IFN-B1, IL-6 and IL-8 genes) in tissue samples. Also, serum protein levels of TLR3, IFN-β, IL-6 and IL-8 were determined using Enzyme Linked Immunosorbent Assay (ELISA).Results: The mRNA gene expression levels of TLR3, NF-kB1A, IFN-B1, IRF3, TICAM1, IL-6 and IL-8 were significantly higher in Eutopic endometrium compared to ectopic ones and also compared to CE (p<0.05 or p<0.01). There was no significant difference in CXCL10 expression between EU vs. CE whereas its mRNA expression was significantly higher in EU than ectopic ones. We examined the 1.7 kb length of TLR3 gene. The results showed two SNPs including one (rs3775291) which causes amino acid substitutions Leu > Phe and a synonymous (Phe=) SNP (rs3775290). Results from PCR and gene sequencing showed that SNPs frequency was not significantly different between patients and controls. Serum protein levels of TLR3, IFN-β, IL-6 and IL-8 were significantly increased in patients with endometriosis in comparison to the control group (p<0.05).Conclusion: In this study, moreover to elevation of IL-6 and IL-8 cytokines, significant and clear changes was observed in the mRNA expression of other genes in TLR3 cascade in diseased eutopic endometrium, demonstrating that EU similarly to EC was in an intensive inflammatory state. Interestingly, the results showed that expression of aforementioned genes have significant difference between ectopic and eutopic endometrium of endometriosis patients, suggesting that fundamental alterations in the concept of immune response in eutopic endometrium may lead to its activation and escapes from apoptosis. These changes maybe have potential contribution to misplaced implantation of endometrium, and then ectopic tissues become stable to a certain degree from the immunological point of view. Also, the results showed that two SNPs (rs3775291 and rs3775290) in TLR3 gene have not impact on its mRNA expression in endometriotic endometrium.

کلیدواژه ها:

Endometriosis ، Toll like receptor 3 ، Polymorphism ، Signaling pathway