Glutathione Peroxidase 4 (GPX4) is a vital antioxidant enzyme that plays a crucial role in protecting cells against oxidative stress and regulating ferroptosis. In spermatozoa, GPX4 localizes to mitochondria, and diminished GPX4 levels have been linked to mitochondrial abnormalities and impaired motility. This study aimed to evaluate GPX4 expression in the semen of infertile men with asthenozoospermia and to elucidate its regulatory role in human ejaculatory spermatozoa.

Eighteen semen samples were analyzed, comprising nine normozoospermic and nine asthenozoospermic specimens. Semen quality was assessed using Computer-Assisted Semen Analysis (CASA), followed by sperm isolation via centrifugation. Exosomes were extracted from the residual seminal plasma. RT-PCR was used to evaluate the expression of GPX4 and TOMM20, while Ago2 protein levels were examined using Western blot analysis. GPX4 knockdown was performed using siRNA, and translational activity in spermatozoa was assessed with a puromycin incorporation assay. Group comparisons were conducted using a two-tailed Student’s t-test.

Asthenozoospermic sperm exhibited significantly lower GPX4 expression compared to normozoospermic counterparts. Similarly, GPX4 levels in seminal plasma exosomes were reduced in asthenozoospermic samples. Ago2 protein was detectable in ejaculatory spermatozoa, suggesting the presence of RNA regulatory mechanisms. GPX4 knockdown resulted in decreased expression of both GPX4 and the mitochondrial marker TOMM20, along with diminished translational activity, suggesting a functional role for GPX4 in spermatozoal protein synthesis and mitochondrial integrity.

GPX4 expression is downregulated in both spermatozoa and seminal exosomes of men with asthenozoospermia. These findings support a dynamic and protective role of GPX4 in maintaining mitochondrial function in human ejaculatory spermatozoa.