• Protective role of papaverine against freeze-thaw-induced damage in sperm of asthenozoospermic men: Regulation of apoptosis-related genes
  • Zahra Azizi,1,* Malek Soleimani Mehranjani,2 Seyed Mohammad Ali Shariatzadeh,3 Nazila Najdi,4 Atena Sadat Azimi,5
    1. Department of Biology, Faculty of Science, Arak University, Arak, Iran
    2. Department of Biology, Faculty of Science, Arak University, Arak, Iran
    3. Department of Biology, Faculty of Science, Arak University, Arak, Iran
    4. Department of Obstetrics and Gynecology, School of Medicine, Arak University of Medical Sciences, Arak, Iran.


  • Introduction: Asthenozoospermia is one of the most common causes of male infertility and is often associated with elevated reactive oxygen species (ROS), oxidative stress, and apoptosis. The freeze-thaw process further enhances ROS production, which can trigger mitochondrial dysfunction and activation of apoptotic pathways, ultimately leading to sperm cell death. Papaverine, an isoquinoline alkaloid and non-specific phosphodiesterase inhibitor, has been widely used as a smooth muscle relaxant. By inhibiting phosphodiesterase activity, papaverine increases intracellular cAMP and cGMP levels, thereby improving cellular signaling and energy metabolism. Recent evidence suggests that papaverine may exert antioxidant, anti-apoptotic, and cytoprotective effects, making it a potential candidate to protect sperm cells against cryo-induced damage. This experimental study aimed to investigate the protective effect of papaverine on sperm from asthenozoospermic men during cryopreservation.
  • Methods: Semen samples were obtained from 30 asthenozoospermic men at the Amir-AL-Momenin Infertility Treatment Center, Arak, Iran, between November 2023 and May 2024. In this experimental in vitro study, each sample was divided into three groups: control (fresh semen), freeze (cryoprotectant only), and freeze + papaverine (cryoprotectant + 100 µM papaverine). The expression of Bcl-2, Bax, and Caspase-3 at both gene and protein levels was assessed using RT-qPCR and immunofluorescence. Data were analyzed using repeated measures analysis by the Bonferroni post hoc test.
  • Results: Compared with the control group, the freeze group showed a significant decrease in Bcl-2 expression and an increase in Bax and Caspase-3 expression at both the gene and protein levels (p < 0.05). In contrast, the freeze + papaverine group exhibited a significant increase in Bcl-2 expression and a decrease in Bax and Caspase-3 expression (p < 0.05).
  • Conclusion: Our study demonstrated that papaverine significantly mitigates freeze-thaw-induced apoptosis in sperm cells by downregulating pro-apoptotic genes (Bax and Caspase-3) and upregulating the anti-apoptotic gene Bcl-2. Excessive ROS generated during the freeze–thaw process can activate apoptotic pathways, thereby impairing sperm function. By regulating the expression of apoptosis-related genes, papaverine appears to protect sperm cells from cryodamage and may represent a promising strategy to preserve sperm quality in asthenozoospermic men.
  • Keywords: Asthenozoospermia, Papaverine, Sperm cryopreservation, Apoptosis, Reactive oxygen species (ROS).