CCL2/CCR2 axis suggests a role in bridging obesity-related adipose inflammation to Sertoli cell injury in asthenozoospermia: Integrated multi-omics, machine learning and in vitro validation.

This study established an integrated framework combining multi-omics analysis, machine learning (ML), single-cell RNA sequencing (scRNA-seq), molecular docking, and in vitro validation to investigate potential mechanisms and therapeutic targets of obesity-associated asthenozoospermia (AZS). Based on transcriptomic data from the Gene Expression Omnibus (GEO), differential expression analysis and weighted gene co-expression network analysis (WGCNA) identified 37 candidate genes enriched in cytokine-cytokine receptor interactions, tumor necrosis factor (TNF) signaling, and chemokine-related inflammatory pathways. Protein-protein interaction (PPI) analysis indicated CTSS as a hub gene. Among 113 ML models using 12 algorithms, glmBoost + LDA performed best and prioritized CCL2, CCL4, EDEM3, PHF3, and SLC38A2 as core biomarkers; Shapley additive explanations (SHAP) assessed their predictive contributions. Gene set enrichment analysis (GSEA), immune infiltration, and scRNA-seq analyses identified adipose tissue macrophages as a major cellular source of Ccl2/CCL2. However, as the scRNA-seq data were derived from mouse adipose tissue rather than human testis, this finding primarily serves to generate the hypothesis that macrophage-derived CCL2/CCR2 signaling may, in theory, contribute to testicular microenvironment disruption and Sertoli cell injury through inflammatory amplification and intercellular communication, rather than providing direct in vivo evidence of such cross-tissue transmission. Molecular docking, cellular thermal shift assay (CETSA), and TM4 cell experiments showed that palmatine modulated the CCL2/CCR2 axis and suppressed MAPK/NF-κB signaling, alleviating inflammation, apoptosis, and blood-testis barrier (BTB) injury. Collectively, the CCL2/CCR2 axis is suggested to act as a pathological bridge in the context of obesity-associated adipose inflammation and male reproductive dysfunction, with palmatine showing potential as a candidate compound for further mechanistic and translational investigation. These findings provide a testable hypothesis for future in vivo studies to determine whether the CCL2/CCR2 axis plays a causal role in obesity-associated AZS.