Abstract
BACKGROUND: Male infertility (MI) impacts about one in seven couples globally, involving complex gene-environment interactions. Environmental pollutants may disrupt spermatogenesis via mitochondrial dysfunction, but key targets and mechanisms are unclear. Integrating multi-omics with computational toxicology offers a novel strategy to decipher these interactions. METHODS: We integrated blood transcriptomics from MI patients, GWAS summary statistics, and QTL data (methylation, expression, protein). MI-related mitochondrial genes were identified through differential expression analysis, followed by enrichment and PPI network analysis. SMR approach was employed to assess genetic causality between molecular levels of genes and MI risk, using cis-QTLs as instrumental variables and applying the HEIDI test (p < 0.05) to distinguish pleiotropy from linkage. The candidate gene FIS1 was functionally validated in vitro by siRNA knockdown in GC-1 spg cells. Reverse network toxicology was then used to screen environmental pollutants potentially targeting FIS1, with candidate compounds selected from the CTD based on unidirectional inhibitory effects on FIS1, high bioaccumulation potential (Log P > 5), and predicted toxicity (mutagenicity, cytotoxicity, or endocrine disruption). Binding affinity was evaluated via molecular docking. RESULTS: We identified 232 dysregulated mitochondrial genes in MI. SMR analysis revealed that FIS1 showed a consistent, significant protective association with MI risk across three molecular levels: DNA methylation (e.g., site cg19802458), gene expression, and plasma protein abundance. Clinical samples confirmed downregulated FIS1 expression in MI patients. In vitro, FIS1 knockdown in spermatogonial cells reduced mitochondrial membrane potential, elevated reactive oxygen species, decreased antioxidant enzyme activity, and significantly inhibited proliferation. Reverse toxicology screened six environmental pollutants predicted to target FIS1, including di (2-ethylhexyl) phthalate, bisphenol S, aflatoxin B1, and benzo [a]pyrene. Molecular docking confirmed stable binding of all six compounds to the FIS1 protein (ΔG < -5.0 kcal/mol), suggesting a direct mechanism for disrupting mitochondrial function. CONCLUSION: By integrating multi-omics and computational toxicology, this study validates FIS1's causal protective role in male infertility, reveals its multi-level regulation, and predicts six targeting pollutants with preliminary experimental evidence. This framework offers new insights into gene-environment interactions and establishes a foundation for biomarker development and targeted interventions.