Asthenozoospermia is one of the common causes of male infertility and is often associated with increased reactive oxygen species (ROS), oxidative stress, and apoptosis. The freeze-thaw process further enhances ROS production, damaging sperm motility and viability. This experimental study aimed to investigate the protective effect of papaverine, a phosphodiesterase inhibitor, on sperm from asthenozoospermic men during cryopreservation. After liquefaction, semen samples from 30 asthenozoospermic men were divided into three groups: control (fresh), freezing (untreated), and freezing + papaverine (100 µM, 30 min). Sperm motility based on (WHO, 2021), viability (eosin- nigrosin staining), and mitochondrial membrane potential (MMP) (rhodamine-123 staining) were assessed. In addition, malondialdehyde (MDA) level were measured using ELISA, and ROS level were evaluated by flow cytometry. Data were analyzed using repeated measures analysis with Bonferroni post hoc test. Compared to the control group, the freezing group showed significant decrease in the mean percentages of motility, viability, and MMP (p < 0.001), and a significant increase in MDA and ROS levels (p < 0.01). In contrast, the freezing + papaverine group showed significant improvement in the mean percentage of motility, viability, and MMP (p < 0.001), along with a significant reduction in MDA (p < 0.001) and ROS levels (p < 0.05) compared to the freezing group. These findings suggest that papaverine, may enhance sperm quality during the freeze-thaw process in asthenozoospermic men by improving motility, viability, and mitochondrial function, and by reducing oxidative stress markers. Therefore, papaverine may be considered a potential antioxidant candidate in sperm cryopreservation protocols.
