P-426 Tissue-resident Pseudomonas aeruginosa impairs endometrial receptivity via T3SS-mediated STAT3 signaling inhibition

Abstract Study question Does Pseudomonas aeruginosa impair endometrial receptivity by inhibiting STAT3 phosphorylation through its type III secretion system (T3SS)? Summary answer Yes, P. aeruginosa utilizes its T3SS to suppress STAT3 phosphorylation in endometrial epithelial cells, disrupting transcriptional activation and reducing receptivity. What is known already Endometrial receptivity is critical for successful embryo implantation, yet approximately 50-60% of IVF cycles fail due to implantation issues. Recurrent implantation failure (RIF) affects 15% of women undergoing IVF, often linked to impaired endometrial receptivity. The window of implantation (WOI) depends on precise transcriptional activation of endometrial epithelial cells, but the mechanisms underlying its dysregulation remain unclear. Emerging evidence suggests that aberrant endometrial microbiota may play a role, but specific pathogens and their mechanisms remain unidentified. Study design, size, duration Single-cell transcriptomics was performed on mid-secretory endometrial samples from 9 RIF patients and 5 controls. Tissue-resident bacteria were identified using the CSI pipeline. The causal role of P. aeruginosa was validated in antibiotic-treated mouse models with bacterial transplantation, combined with FISH, immunofluorescence, and transcriptomic analyses. Participants/materials, setting, methods Endometrial samples were collected from RIF patients and controls. Single-cell RNA sequencing was used to analyze cell types and transcriptional states. P. aeruginosa colonization was assessed using CSI and SAHMI pipelines. Mouse models were used to verify the impact of P. aeruginosa on embryo implantation, STAT3 phosphorylation, and receptivity gene expression. Main results and the role of chance Single-cell transcriptomic analysis of mid-secretory endometrial samples from 9 RIF patients and 5 controls identified six major cell types, with secretory epithelial cells showing disrupted phase transitions in RIF patients. Pseudotime analysis revealed impaired Phase 3 to Phase 4 transition, linked to suppressed STAT3 phosphorylation. P. aeruginosa was significantly enriched in RIF epithelial cells and associated with inhibited STAT3 phosphorylation. Mouse experiments confirmed reduced implantation rates, suppressed STAT3 phosphorylation, and downregulated receptivity genes following P. aeruginosa colonization. Further mechanistic studies indicated that P. aeruginosa utilizes its type III secretion system (T3SS) to inhibit the JAK/STAT pathway, specifically targeting STAT3 phosphorylation. This suppression disrupts transcriptional activation, impairing endometrial receptivity. Additionally, functional enrichment analysis highlighted key pathways affected by P. aeruginosa, including cytokine signaling and cell adhesion, further supporting its role in receptivity impairment. These findings were consistent across multiple analyses, including single-cell RNA sequencing, functional enrichment, and animal models, minimizing the role of chance. The study provides robust evidence that P. aeruginosa, through its T3SS, plays a critical role in reducing endometrial receptivity by interfering with STAT3 signaling, offering a potential therapeutic target for improving IVF outcomes in RIF patients. Limitations, reasons for caution The study’s sample size, though sufficient for single-cell analysis, may limit generalizability. Mouse models, while informative, may not fully replicate human endometrial physiology. Further validation in larger cohorts and mechanistic studies are needed. Wider implications of the findings This study identifies P. aeruginosa as a key pathogen impairing endometrial receptivity through T3SS-mediated STAT3 signaling inhibition, offering new insights into the microbial etiology of RIF. These findings could lead to targeted therapies, such as microbiota modulation or T3SS inhibition, to improve IVF outcomes. Trial registration number No