Sperm motility is crucial for male fertility, allowing sperm to reach and fertilize the oocyte. Asthenozoospermia, marked by reduced motility, leads to infertility via oxidative stress, DNA damage, and mitochondrial dysfunc- tion. Freeze-thawing, used in assisted reproductive technologies (ART), damages sperm. This study examined papaverine’s antioxidant effects on improving asthenozoospermic sperm quality during freeze-thawing. Thirty semen samples from asthenozoospermic patients were divided into three groups: Control (fresh), Freeze (cryo- protectant, 30-min incubation), and Freeze + Papaverine (cryoprotectant + 100 μ M papaverine, 30-min incu- bation). Sperm parameters, DNA fragmentation, TNF- α , IL-10, reactive oxygen species (ROS), malondialdehyde (MDA), total antioxidant capacity (TAC), antioxidant enzyme activities (SOD, CAT, GSH), plasma membrane integrity, mitochondrial membrane potential (MMP), and Bcl-2, Bax, and Caspase-3 expression at gene and protein levels were analyzed in all groups. In the Freeze group, sperm parameters, antioxidant enzyme levels, plasma membrane integrity, MMP, acrosomal integrity, levels of IL-10, and Bcl-2 expression significantly decreased (p < 0.05–0.001) compared to controls. In contrast, ROS, MDA, TNF- α , DNA fragmentation, and Bax and Caspase-3 expression significantly increased (p < 0.05–0.001). However, in the Freeze + Papaverine group, all these factors, except acrosomal integrity, showed significant improvement compared to the Freeze group. This study suggests that treating asthenozoospermic sperm with papaverine before freezing improves post-thaw sperm quality by enhancing motility, viability, plasma membrane integrity, and MMP, while reducing oxidative stress, inflammation, and apoptosis—potentially contributing to improved outcomes in ART.
