Relationship Between Cell Death Parameters and Changes in MicroRNA-125b Expression During Chlorambucil-Induced Testicular Toxicity and the Protective Potential of Cobalamin

Background: Chemotherapy-related male infertility is a major concern among cancer survivors. Chlorambucil, an alkylating agent, induces oxidative damage that may disrupt the balance between autophagy and apoptosis in testicular tissue. Thus, this study aimed to investigate the role of microRNA-125b (miR-125b) in chlorambucil-induced testicular injury and evaluated the protective effects of cobalamin (vitamin B12). Methods: A total of 32 adult male Wistar rats were randomly assigned to four groups (n = 8): control, chlorambucil (2 mg/kg/day), chlorambucil plus cobalamin (3 μg/kg/day), and cobalamin alone. Histological, spermatological, biochemical, hormonal, and molecular assessments were performed at 30 days of treatment. Results: Chlorambucil administration significantly impaired sperm quality, reduced spermatogonial cell numbers, decreased seminiferous tubule diameter and epithelial height, and lowered Johnsen’s score (p < 0.05). These changes were accompanied by elevated malondialdehyde (MDA) levels, reduced superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, and decreased serum testosterone concentrations. In addition, chlorambucil increased miR-125b, Bax, and caspase-3 expression while suppressing Bcl-2 and autophagy-related genes (Atg5, Atg6, Atg7, Atg8, and Atg12) (p < 0.05). Cobalamin co-treatment significantly mitigated chlorambucil-induced reproductive toxicity, increasing sperm count by approximately 48.3% compared with the chlorambucil-treated group, restoring testosterone levels toward normal, suppressing apoptosis, enhancing the expression of autophagy-related genes, and partially reversing miR-125b upregulation (p < 0.05).Conclusions: Chlorambucil-induced testicular toxicity appears to be mediated, at least in part, through oxidative stress–associated miR-125b dysregulation, promoting apoptosis and suppressing autophagy. Cobalamin exerts significant gonadoprotective effects and may represent a promising therapeutic approach for preserving male reproductive function during chemotherapy.