Background: Sperm cryopreservation, crucial for assisted re- productive technologies (ART), often leads to cellular damage due to reactive oxygen species (ROS) during the freeze-thaw process. Asthenozoospermia, defined by sperm motility below 40%, is a common cause of male infertility. Sperm from as- thenozoospermic men are more vulnerable to cryo-damage due to higher ROS levels. This damage, including oxidative stress, DNA fragmentation, and acrosomal dysfunction, affects sperm quality and fertilization. This study aimed to assess melatonin's protective effects in reducing ROS, DNA fragmentation, and spontaneous acrosome reactions. Materials and Methods: Semen samples were collected from thirty asthenozoospermic men between November 2023 and May 2024 at the Amir al-Momenin Infertility Center, Arak, Iran. Samples were divided into three groups: fresh (control), freeze, and freeze + melatonin (1 mM). A rapid freezing proto- col with human sperm freezing medium was used. Intracellular ROS was measured using a ROS kit (KiaZist, Iran) by flow cytometry. DNA fragmentation was assessed using the Halo- sperm kit (Ideh Varzan Farda, Iran), and acrosome reaction was evaluated using FITC-PSA staining (Sigma, USA). Data were analyzed using the Repeated Measures Analysis method. Results: Compared to the control group, the freeze group showed significantly increased mean ROS levels, DNA frag- mentation, and acrosome reaction (P=0.010). Melatonin-treat- ed samples exhibited a significant reduction in mean ROS (P=0.005), percentage DNA fragmentation (P=0.015), and acrosome reaction (P=0.004) compared to the freeze group. Conclusion: The results indicate that the addition of melatonin during sperm cryopreservation significantly reduces oxidative stress, preserves DNA integrity, and stabilizes the acrosome in asthenozoospermic sperm. These findings highlight the poten- tial role of melatonin as a protective agent to improve the ef- ficiency of ART.
