Integrated multi-omics analysis reveals epithelial-derived metabolic and adhesive dysregulation in the endometrium of patients with recurrent implantation failure

BACKGROUND: Recurrent implantation failure (RIF) is a significant clinical challenge in assisted reproduction, yet its underlying molecular mechanisms remain poorly understood. This study aimed to characterize and integrate the metabolomic, proteomic, and transcriptomic profiles of the RIF endometrium to identify novel pathophysiological networks and potential diagnostic biomarkers. METHODS: We performed an integrated multi-omics analysis on endometrial tissues from fertile controls and RIF patients during the window of implantation. Metabolomic profiles were assessed using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteomic changes were quantified using data-independent acquisition (DIA)-based quantitative proteomics. Transcriptomic alterations were analyzed using bulk and single-cell RNA sequencing (scRNA-seq), complemented by spatial metabolomics to localize metabolic shifts. Bioinformatics analyses, including pathway enrichment and gene-metabolite network construction, were used to integrate the datasets. RESULTS: Distinct endometrial metabolomic profiles were observed in RIF patients compared to controls, with dysregulation in amino acid pathways (e.g., tyrosine, phenylalanine, tryptophan, histidine metabolism) and glycerophospholipid disruptions. Key differential metabolites, including 4-hydroxyphenylpyruvic acid, α-ketoglutaric acid, and indole-3-carboxaldehyde, showed high diagnostic potential. Proteomics identified 282 differentially expressed proteins, highlighting impaired extracellular matrix-receptor interactions, cell adhesion, and amino acid transport, with hubs such as fibronectin and integrins. Single-cell analysis localized these deficits primarily to the luminal and glandular epithelial cells. Spatial metabolomics further displayed disrupted amino acid distribution. Integrated multi-omics analysis revealed significant disruptions in amino acid and energy metabolism pathways in RIF. L-Glutamic acid served as a central metabolic hub connecting critical proteins (SLC1A1, ARG2, TGFB2) and genes, with pyruvate carboxylase (PC) linking glycolysis to the citrate cycle. CONCLUSIONS: This integrated multi-omics approach elucidates the complex molecular crosstalk in the non-receptive endometrium. The findings suggest that epithelial metabolic insufficiency and adhesive defects contribute to RIF, offering potential biomarkers and therapeutic targets to improve implantation success.