Background: Asthenozoospermia, defined by reduced sperm motility (<42%, WHO 2021), is a primary cause of male infer- tility. Sperm cryopreservation, essential for assisted reproduc- tive technologies (ART), induces cryoinjury through excessive reactive oxygen species (ROS) production during freeze-thaw cycles. This results in oxidative stress, membrane damage, and impaired sperm function, particularly in asthenozoospermic men. This study evaluated the effects of papaverine on sperm acrosome reaction, membrane integrity, and intracellular ROS levels in cryopreserved samples from asthenozoospermic men. Materials and Methods: Semen samples were collected from 30 asthenozoospermic men at the Amir-AL-Momenin Infertil- ity Centre in Arak, Iran, in 2023. The samples were divided into three groups: control (fresh), freeze, and freeze + papa- verine (100 µM). Cryopreservation in the freeze groups was performed using a rapid freezing protocol. Membrane integrity was evaluated using the hypo-osmotic swelling (HOS) test, acrosome reaction was assessed by fluorescein isothiocyanate- labeled Pisum sativum agglutinin (FITC-PSA) staining, and in- tracellular reactive oxygen species (ROS) levels were measured using the DCFH-DA dye and flow cytometry. Data were ana- lyzed statistically using the repeated measures analysis method. Results: Cryopreservation significantly decreased membrane integrity (P<0.001) and increased acrosome reaction (P<0.004) and ROS levels (P<0.001) compared to fresh samples. Treat- ment with papaverine significantly improved membrane integ- rity (P<0.001) and reduced ROS levels (P<0.040) compared to the freeze group. However, papaverine had no significant effect on reducing the rate of acrosome reaction (P > 0.127). Conclusion: Our results indicate that adding papaverine to the cryopreservation medium can protect asthenozoospermic sperm by reducing oxidative stress, minimizing spontaneous acrosome reactions, and improving membrane integrity. These effects highlight papaverine’s potential to enhance sperm qual- ity in assisted reproductive technology (ART).
