Multi-level profiling of the endometrial transcriptome identifies immune dysregulation in recurrent implantation failure

Recurrent implantation failure (RIF) remains a significant clinical challenge in assisted reproductive technology. Its etiology is often attributed to inadequate endometrial receptivity, yet the underlying molecular mechanisms are poorly characterized, leading to a lack of reliable diagnostic biomarkers and targeted treatments. We conducted RNA sequencing on endometrial biopsies obtained during the window of implantation from 9 RIF patients and 13 fertile controls. Differential gene expression analysis was performed, followed by comprehensive functional enrichment analysis (GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), and GSEA (Gene Set Enrichment Analysis)). The expression of key candidate genes was validated using qRT-PCR, Western blotting, and immunohistochemistry. RNA sequencing revealed 103 differentially expressed genes in RIF endometrium. GO and KEGG analyses consistently highlighted significant enrichment in immune-related processes and pathways, particularly acute inflammatory response, leukocyte-mediated immunity, and complement activation. GSEA highlighted strong enrichment of immune pathways including natural killer cell-mediated cytotoxicity and interleukin signaling, which emphasized dysregulation of immune and inflammatory processes. Key upregulated molecules including EDNRB, AACT, REL, USP18, and ANG were validated experimentally, suggesting their potential as biomarkers for RIF. Our study delineates a distinct immune-dysregulated transcriptomic profile in the endometrium of RIF patients, providing crucial insights into the pathogenesis of this condition. The validated genes, EDNRB, AACT, REL, USP18, and ANG, represent promising biomarkers with significant potential for improving the diagnosis and future therapeutic strategies for RIF.