Cross-omics association of small extracellular vesicle miRNAs and metabolites in seminal plasma of idiopathic male infertility.

BACKGROUND: Idiopathic male infertility (IMI) accounts for 30%-40% of male infertility cases, yet the molecular networks associated with seminal plasma dysfunction and non-invasive biomarkers remain largely unclear. This study aimed to preliminarily explore the transcriptomic and metabolomic alterations in IMI seminal plasma through cross-omics integration, and to screen for exploratory candidate discriminators among the metabolites. METHODS: A total of 30 IMI patients and 30 normozoospermic controls were recruited for untargeted UHPLC-MS/MS metabolomics of seminal plasma. Candidate differential metabolites were screened and subjected to KEGG and Gene Set Enrichment Analysis (GSEA); ROC analysis with bootstrap internal validation was conducted on candidate differential metabolites to explore candidate discriminators. For miRNA sequencing, 10 subjects were randomly selected from each group to isolate and identify seminal plasma small extracellular vesicles (sp-sEVs) following International Society for Extracellular Vesicles (ISEV) standards. Subsequent bioinformatic analyses included miRNA differential expression screening, target gene prediction, GO/KEGG enrichment, protein-protein interaction (PPI) network construction, and pairwise Pearson correlation analysis between dysregulated miRNAs and differential metabolites within the nested subgroup. RESULTS: We identified 68 and 152 candidate differential metabolites under negative and positive electrospray ionization modes, respectively. Metabolite perturbations were predominantly enriched in unsaturated fatty acid biosynthesis, whereas the arginine and proline metabolism pathway was globally suppressed in IMI patients. The phosphatidic acid derivative PA(i‑15:0/18:1(12Z)‑2OH(9,10)) showed the best discriminatory potential among all screened metabolites, with a AUC of 0.822 (95% CI: 0.716-0.929). miRNA sequencing uncovered 128 differentially expressed miRNAs (62 upregulated, 66 downregulated) in IMI sp-sEVs. Functional enrichment of miRNA target genes highlighted perturbed protein phosphorylation and intracellular signal transduction, with the phospholipase D (PLD) signaling pathway as the most statistically significant pathway; PIK3CA, PIK3R1, and AKT1 were suggested to be core hub genes within the PLD network. Correlation analysis suggested that upregulated miRNAs (hsa-miR-141-5p, hsa-miR-28-5p) were positively correlated with up-regulated N-cis-11,14-eicosadienoyl ethanolamine in seminal plasma. CONCLUSION: Coordinated alterations between sp-sEV miRNAs and seminal plasma metabolites were observed in IMI. Several candidate metabolites showed discriminatory value in this single cohort, but require validation. The PLD pathway may be involved, though causality remains unconfirmed.