Epigenetic effects of Qixiong Formula on sperm DNA methylation in a rat model of asthenozoospermia.

Background: Asthenozoospermia is a leading cause of male infertility with limited treatment options. Emerging evidence implicates epigenetic alterations, particularly DNA methylation, in its pathogenesis. The Qixiong Formula (QXF) has shown clinical efficacy in improving sperm motility, yet its underlying epigenetic mechanism remains unclear. This study therefore aimed to investigate the therapeutic effects of QXF and its regulation of genome-wide sperm DNA methylation in a rat model of asthenozoospermia. Methods: Asthenozoospermia was induced in male Sprague-Dawley rats via oral administration of ornidazole (400 mg/kg/day) for 28 days. Rats were treated with low, medium, or high doses of QXF. Semen parameters, testicular and epididymal histology, organ coefficients, and liver and kidney function were assessed. Reduced representation bisulfite sequencing (RRBS) was used to profile genome-wide DNA methylation in sperm and identify differentially methylated regions (DMRs), followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Results: QXF significantly improved sperm motility without affecting sperm concentration or systemic toxicity. Histological analysis showed partial recovery of epididymal architecture in QXF-treated rats. RRBS revealed that QXF induced a shift toward global hypomethylation in sperm DNA, reversing the hypermethylation pattern observed in the model group. Enrichment analyses implicated several key signalling pathways in QXF's action, particularly cAMP, cGMP-PKG, and PI3K/Akt pathways, which are known to regulate sperm motility and survival. Conclusions: QXF improves sperm motility and reverses aberrant methylation patterns in a rat model of asthenozoospermia. These findings suggest that QXF exerts its therapeutic effect through dual regulation of DNA methylation and activation of sperm-related signalling pathways, offering new insights into the epigenetic basis of its action.