Background: Asthenozoospermia, characterized by reduced or complete loss of sperm motility, is a prevalent cause of male infertility. Sperm cryopreservation, vital for assisted reproductive technologies, induces cryoinjury, leading to structural and functional sperm impairment through elevated oxidative stress, DNA fragmentation, and apoptosis. Excessive reactive oxygen species (ROS) generation during freeze-thaw cycles heightens oxidative damage, particularly in asthenozoospermic men, reducing fertility potential. Papaverine (PPV), a powerful antioxidant, effectively reduces oxidative damage, with prior studies showing its protective effects on cryopreserved normozoospermic men. Objective: In this study, the effects of PPV on sperm biochemical factors and intracellular ROS levels were investigated in asthenozoospermic men during cryopreservation. Materials and Methods: Semen samples from 30 asthenozoospermic men, obtained at the Amir-AL- Momenin Infertility Treatment Center, Arak, Iran, between November 2023 and May 2024, were examined in this experimental in vitro study. Each sample was divided into 3 groups: control (fresh semen), freeze (cryoprotectant only), and freeze + PPV (cryoprotectant + 100 µM PPV). Cryopreservation involved a standardized sperm freezing medium and a rapid vitrification technique. Sperm malondialdehyde (MDA), total antioxidant capacity (TAC), and antioxidant enzymes catalase, glutathione, superoxide dismutase, were measured via enzyme-linked immunosorbent assay. Intracellular ROS levels were assessed using the DCFH-DA fluorescent probe (Kiazist ROS Assay Kit, Iran) with a partec pas flow cytometer. Data were analyzed using FlowJo™ software, and results were expressed as mean fluorescence intensity. Results: The freeze group exhibited significantly reduced mean levels of glutathione, catalase, superoxide dismutase and TAC (all p < 0.001) and elevated mean MDA and ROS levels (p < 0.001) compared to the control group. Conversely, the freeze + PPV group demonstrated significantly enhanced antioxidant enzyme levels and TAC (p < 0.001 for all), along with reduced MDA (p < 0.001) and ROS (p = 0.040) levels, compared to the freeze group. Conclusion: This study demonstrated that the addition of PPV to the sperm freezing medium effectively reduces oxidative stress and intracellular ROS levels in cryopreserved sperm in asthenozoospermic men. These findings suggest that PPV has the potential to mitigate freeze-thaw-induced damage and improve outcomes in assisted reproductive technology.
