Cryopreservation of human sperm, essential for ART and fertility preservation, induces damage through cold shock and oxidative stress, impairing membranes, mitochondria, and fertilization capacity. Selenium, a potent antioxidant trace element, maintains redox balance and sperm stability. This study evaluated selenium’s effects on ROS, mitochondrial membrane potential, and plasma membrane integrity in sperm from asthenozoospermic men during the freeze–thaw process. Semen samples from 30 asthenozoospermic men (Roya Infertility Centre, Qom, Iran) were assigned to fresh (control), freeze (cryoprotectant only), and freeze + selenium (2 µg/mL) groups. Cryopreservation was performed using rapid freezing with commercial medium. After thawing, plasma membrane integrity was measured by hypoosmotic swelling test (HOST), mitochondrial membrane potential (MMP) by Rhodamine 123 staining, and intracellular ROS by flow cytometry with DCFH-DA. Data were analyzed using repeated measures, with significance at p < 0.05. A significant reduction in mean MMP and plasma membrane integrity, along with an increase in intracellular ROS levels, was observed in the freezing group compared with the control (p < 0.001). In contrast, supplementation with selenium during freezing increased MMP and plasma membrane integrity and significantly reduced ROS levels in the selenium + freeze group compared with the freezing group (p < 0.001). Cryopreservation of human sperm caused oxidative stress and structural damage, reducing function. Selenium supplementation during freeze– thaw decreased ROS, preserved mitochondrial activity, and improved membrane integrity, highlighting selenium as a promising supplement to enhance post-thaw sperm quality and mitigate cryodamage.
