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Title: Repeated implantation failure is associated with increased Th17/Treg cell ratio, during the secretory phase of the human endometrium.

Repeated implantation failure (RIF) is a significant limiting factor in assisted reproduction. Chronic endometrial inflammation has been noted in RIF women, therefore we sought to investigate the potential association of endometrial Th17/Treg ratio and endometrial inflammation in these cases. Endometrial pipelle biopsies were obtained from volunteers, 29 women with RIF (failure to achieve pregnancy following at least 3 transfers of high-grade embryos in IVF-cycles) and 27 fertile women (at least one child) in total, at the secretory phase of the menstrual cycle. Using tissues from 17 fertile and 18 RIF endometrial samples, stromal and immune cells were isolated and flow cytometry analysis was performed to determine Th17 and CD4+ CD25high FOXP3+ cell populations in endometrial stromal cell suspensions. Another group of tissues from 10 fertile and 11 RIF samples were used for mRNA expression levels of Treg and Th17-cell transcription factors, FOXP3 and RORγt respectively. Endometrial inflammatory mediators' mRNA expression was also analyzed. A statistically significant increase in protein flow cytometry analysis of Th17/Treg ratio (p ≤ 0.05) as well as a reduction in absolute Treg cells in the endometrium (p ≤ 0.05) was noted in women with RIF. Additionally, RNA analysis on the same set of women indicated RORγt/FOXP3 significantly increased in women with RIF compared to fertile ones (p ≤ 0.05). Finally, women with RIF exhibited significantly (p ≤ 0.05) elevated mRNA levels of pro-inflammatory mediators (ΤΝF-a, ΙL-6, IL-8 and CCl2). Women with RIF exhibit elevated Th17/Treg ratio, mostly due to endometrial Treg depletion, as well as a pro-inflammatory state in the endometrium.

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