Frequency and epigenetic factors analysis of Treg, Th17 and exhausted T cells in Recurrent Implantation Failure (RIF) patients

Abstract

Recurrent implantation failure (RIF) has many definitions, but usually is defined as the failure to achieve a pregnancy fallowing 2-6 IVF cycles in which more than 10 high grade embryos were transferred to the uterus. Endometrial receptivity is a key determinant of the success of implantation. Immunological abnormalities may be one of the main causes of reproductive failure in RIF. Changes in endometrial leukocyte subpopulations and most of all, in the percentage of uterine natural killer cells (uNK), dendritic cells, and different T lymphocytes during menstrual cycles may have a pivotal role in the implantation process. The objective of the present study was to investigate the peripheral blood flow cytometric staining of regulatory T cell, Th17, exhausted Treg cells and exhausted CD8+ T cells and quantify the expression level of mRNAs and microRNAs extracted from peripheral blood mononuclear cells and isolated T cell subsets. Materials & methods: 40 women with RIF and immunological abnormalities and 50 healthy women as control group participated in the study. We evaluated the pre-conception level of regulatory T cells, Th17, exhausted Treg and exhausted CD8+ T cells in peripheral blood of women with RIF using flow cytometery. The expression level of genes involved in T cell responses including GATA-3, GITR, IRF-4 and T-bet was evaluated by quantitative real time PCR. In addition, microRNAs from PBMCs and isolated T cells were evaluated by quantitative real-time PCR technique. Results: The results showed that Treg cells and exhausted CD8+ T cells were decreased in women with RIF compared with healthy women (p=0.0022, p=0.029, respectively). As expected, a high level of Th17 and exhausted Treg cells were observed in patients (p=0.0048, p=0.0034, respectively). Expression of GATA-3 (p=0.0015) and GITR (p=0.055) which regulate Th2 cell differentiation and are involved in Treg function, respectively, were significantly downregulated in RIFs compared with normal women. Expression of IRF-4 (p=0.0003) and T-bet (p=0.013) were higher in women experiencing IVF failure than normal women. mir-106b-25-93 cluster evaluation, showed a higher rate in PBMCs from RIF patients (P=0.083, 0.03 and 0.013, respectively), while mir-146a and mir-155 were downregulated in RIF subjects (p value=0.01 and 0.0006, respectively). Moreover, we observed significant differences in the expression level of Th17 associated miRNA, mir-326 (p=0.02) in PBMCs from patients.