P-486 Alterations in endometrial programming lead to recurrent in vitro fertilization failures
Authors: Irigoyen M , Castagnola L , Gori S , Grasso E , Gnocchi D , Cattaneo A , Pérez Leirós C , Ramhorst R 
Published: June 2023

Abstract

Study 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 inflammatory mediators production, such as IL-1β. At the same time, several molecules associated with receptivity are modulated; increasing the expression of adhesion molecules and metalloproteinases, while decreasing MUC-1 to allow embryo attachment and implantation. Even nowadays with different techniques that can identify a competent embryo and the implantation window, RIF-patients cannot reach implantation and the mechanisms involved have not been elucidated yet.

Study design, size, duration
First, a bioinformatic analysis was performed using standardized pathways involved in angiogenesis, placentation, decidualization, inflammation and immune regulation from public databases (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 validated their modulation in endometrial samples. Functional processes, such 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 secretory phase. 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-1β, NLRP3, PTGS1; angiogenesis balance : CXCL14, ARG2, VEGFA; adhesion molecules: ITGA8 and MUC1. IL-1β production was quantified by Flow Cytometry. Wound healing assay was performed in human endometrial stromal cells derived from cell line and RIF patient’s biopsy

Main results and the role of chance
First, we performed bioinformatic analysis based on standardized pathways in public databases. We focused on genes that connected and were modulated in processes involved in implantation, angiogenesis, placentation, decidualization, inflammation and immune regulation. Then, we validated the expression of 15 genes with the highest score in biopsies from RIF or fertile women taken on LH + 7. We found decreased IGFBP1 expression, an early decidualization marker, and the progesterone receptor (p &lt; 0.0001), suggesting alterations in the decidualization program. Since inflammation is associated with decidualization, we evaluated the IL-1β pathway. RIF biopsies had reduced expression of NLRP3, associated with inflammasome assembly, accompanied by a reduction in PTGS1 and IL-1β 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 (p &lt; 0.005). Also, we observed an imbalance between pro and anti-angiogenic factors (VEGFA vs CXCL14). Finally, we isolated and cultivated stromal cells from RIF samples to analyze migration ability, a 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 from RIF patients and fertile women obtained during the secretory phase of the menstrual cycle. Even though the samples represent the endometrium 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 attachment and embryo implantation. However, this initial inflammatory response is differentially induced in RIF patients in comparison with fertile women, suggesting its relevance as a potential pharmacological target.

Trial registration number
This work was funded by the National Agency of Sciences and Technology ANPCyT (PICT 2018-4715 and PICT 2028-2461 to RR) and University of Buenos Aires (UBACyT UBACyT 20020090200034 to RR).