Asthenozoospermia, characterized by reduced sperm motility, is a major cause of male infertility. Cryopreservation, though essential in assisted reproductive technologies (ART), often worsens oxidative stress in low-motility samples, increasing reactive oxygen species (ROS) and impairing mitochondrial function. Theophylline, which enhances motility by modulating intracellular signaling, may counter oxidative damage during freeze–thaw. This study evaluated the impact of theophylline supplementation on ROS levels and sperm quality in asthenozoospermic men. Semen samples from 30 asthenozoospermic men (Roya Infertility Treatment Centre, Qom, Iran) were divided into three groups: fresh (control), freeze (cryoprotectant only), and freeze + theophylline (0.05 mM). Cryopreservation was performed using a rapid freezing protocol. Plasma membrane integrity was assessed by the hypoosmotic swelling test (HOST), mitochondrial membrane potential (MMP) by Rhodamine 123 staining, and reactive oxygen species (ROS) levels by flow cytometry with DCFH-DA. Data were analyzed using repeated measures analysis, with statistical significance set at p < 0.05. A significant reduction in MMP and plasma membrane integrity, along with elevated intracellular ROS levels, was seen in the freeze group when compared with the control (p < 0.001). Conversely, Theophylline treatment during freezing significantly improved MMP and plasma membrane integrity while reducing ROS levels in the freeze + Theophylline group compared with the freeze group (p < 0.001). Cryopreservation impaired mitochondrial function, membrane integrity, and increased ROS in asthenozoospermic sperm. Theophylline supplementation alleviated these effects, preserving sperm quality and reducing oxidative stress, highlighting its potential as an additive to improve post-thaw outcomes.
