GenX-associated molecular signatures overlap with testicular aging and male infertility: a multi-omics integration analysis.

Spermatids exhibited the highest aging sensitivity among testicular cell types in single-cell RNA sequencing analysis.

• scRNA-seq data from human testicular aging dataset GSE254315 were analyzed to evaluate cell-type-specific aging sensitivity
• Spermatids showed the highest aging sensitivity compared to other testicular cell types
• Progressive decline in intercellular communication was observed in aging testicular tissue
• Cell-type-specific aging sensitivity was assessed using a GenX-Aging gene set constructed by intersecting GenX-related genes with aging-associated genes

Four hub genes—SOD1, XRCC5, FOXO3, and POLB—demonstrated diagnostic value for male infertility.

• Hub genes were identified by integrating male infertility transcriptomic datasets GSE45885 and GSE45887
• LASSO regression combined with SVM-RFE were applied to identify the hub genes
• The four hub genes were identified from the intersection of GenX-related, aging-associated, and male infertility transcriptomic signatures
• These genes were validated by RT-qPCR in GenX-exposed rat testicular tissues

RT-qPCR in GenX-exposed rat testicular tissues confirmed differential expression of the four hub genes, with SOD1, XRCC5, and FOXO3 upregulated and POLB downregulated.

• RT-qPCR validation was performed in GenX-exposed rat testicular tissues
• SOD1, XRCC5, and FOXO3 were upregulated in GenX-exposed rat testicular tissues
• POLB was downregulated in GenX-exposed rat testicular tissues
• RT-qPCR results confirmed the computational predictions derived from multi-omics integration analysis

Functional enrichment analysis implicated FoxO signaling, cellular senescence, and DNA repair pathways as key mechanisms in GenX-associated testicular toxicity.

• Functional enrichment was performed on the GenX-Aging gene set and hub genes
• FoxO signaling pathway was among the enriched pathways, consistent with FOXO3 being a hub gene
• Cellular senescence and DNA repair pathways were also implicated
• Oxidative stress and genome maintenance were identified as key pathological mechanisms

Molecular docking confirmed favorable binding interactions between GenX (HFPO-DA) and the four hub proteins.

• Molecular docking was performed for GenX against SOD1, XRCC5, FOXO3, and POLB proteins
• Favorable GenX-protein binding interactions were confirmed for all four hub proteins
• Molecular docking results supported the hypothesis that GenX directly interacts with proteins involved in oxidative stress and DNA repair

GenX-related genes were overlapped with aging-associated genes to construct a GenX-Aging gene set used for downstream multi-omics integration.

• GenX (HFPO-DA) is described as a short-chain per- and polyfluoroalkyl substance (PFAS) substitute implicated in testicular toxicity
• GenX-related genes were intersected with aging-associated genes to construct the GenX-Aging gene set
• This gene set was then used in scRNA-seq analysis, transcriptomic integration, and machine learning-based hub gene identification
• The approach integrated multiple omics layers including single-cell transcriptomics and bulk transcriptomics from male infertility datasets