Alterations in endometrial programming leads to recurrent in vitro fertilization failures

IRIGOYEN M, CASTAGNOLA L, GORI.S., GRASSO E, GNOCCHI D., MARTINEZ G., PÉREZ LEIRÓS C AND RAMHORST R.

Congreso; European Society Human Reproduction and Embriology (ESHRE); 2023

Alterations in endometrial programming leads to recurrent in vitro fertilization failuresIrigoyen M.¤, Castagnola L.§, Gori.§ S., Grasso E.§, Gnocchi D.¤, Martinez G.¤, Pérez Leirós C.§ and Ramhorst R.§§ CONICET. Universidad de Buenos Aires, Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN). Laboratorio de Inmunofarmacología y Laboratorio de Inmunología Reproductiva de la Fertilidad. Buenos Aires, Argentina.¤ Fertilis Medicina Reproductiva, Buenos Aires, ArgentinaStudy question: Could patients with recurrent implantation failures (RIF) display alterations on the decidualization program conditioning decidual cells’ functions and the inflammatory response during peri-implantation period?Summary answer: Endometrium from RIF-patients displays alterations into the decidualization program and into the induction of a physiological-inflammation preventing the attachment and cell-migration for embryo implantation.What is known already: The decidualization program starts on each menstrual cycle and implies not only phenotypical changes on the endometrial stromal cells, but also in their secretory profile. Moreover, this process induces a physiological and sterile inflammatory response associated with the production of inflammatory mediators as IL-1b. At the same time, several molecules associate with receptivity are modulate increasing the expression of adhesion molecules and metolopretinases, while decrease MUC-1 to allow embryo attachment and implantation. Even nowadays different techniques could identify a competent embryo and the window of implantation, RIF could not reach implantation and the mechanisms involve have not been elucidated yet.Study design, size, duration: First a bioinformatic analysis was performed using standarized pathways involved in angiogenesis, placentation, decidualization, inflammation and immune regulation from public data bases (Reactome, Gene Ontology Biological Process, WikiPathways, KEGG). Based on arrays that compare gene expression in endometrial biopsies from RIF or fertile women, we selected those that connect two or more processes and we validate their modulation in endometrial samples. Functional processes as migration and inflammatory production were also evaluated in primary cultures.Participants/materials, setting, methods: Endometrial samples were obtained from fertile and RIF-patients during the secretoyphase. The Investigation and Ethics Committee from San Isidro, Argentina approved this study. RT-qPCR was performed to test decidualization markers: IGFBP1, PRL, PGR; Inflammation: Il-1b, NLRP3, PTGS1; Angiogenesis imbalance CXCL14, ARG2, VEGFA; adhesion molecules: ITGA8 and MUC1. Il-1b production was also quantified by Flow Cytometry. Wound healing assay was performed in human endometrial stromal cells cell line and primary culture of in endometrial RIF patients.Main results and the role of chance: First, we performed bioinformatic analysis based on standardized pathways in public data bases. We focused on genes that connected and were modulated in processes involved in implantation, angiogenesis, placentation, decidualization, inflammation and immune regulation. Then, we validated expression of 15 genes with highest score in biopsies from RIF or fertile women taken on LH+7. We found decreased IGFBP1 expression, early decidualization marker, and the progesterone receptor, suggesting alterations in the decidualization program. Since inflammation is associated with decidualization, we evaluated IL1b pathway. RIF biopsies had reduced expression of NLRP3, associated with inflammasome assembly, accompanied by a reduction in PTGS1 and IL-1b production in comparison with endometrial samples from fertile women. Moreover, when we evaluated genes involved in processes regulated by inflammation, we observed increased expression of MUC1, avoiding blastocyst adhesion, and decreased ITGA8 preventing its attachment accompanied by decreased MMP9 levels, inhibiting trophoblastic invasion. Also, we observed an imbalance between pro and anti angiogenic factors (VGEFA vs CXCL14). Finally, we isolated and cultivated stromal cells from RIF samples to analyze migration ability, process that mediates blastocyst inclusion into the decidua, by wound healing assay. These primary cultures showed a differential migration pattern compared with decidualized stromal cells.Limitations, reasons for caution: The present results were obtained using endometrial samples form RIF-patients and Fertile obtained during the secretroy phase of the menstrual cycle. Even the samples represent the endoemtriaum after in vivo decidualization, further studies are necessary to elucidate whether these mechanisms operate similarly in vivo.Wider implications of the findings: Decidualization process induces a physiological and sterile inflammatory response associated with the modulation of several adhesion molecules and MMP to allow the attachement and embryo implantation. However, this initial inflammatory response is differentially induces in RIF patients in comparison with fertile women, suggesting its relevance as a potential pharmacological target.