Received October 21, 2025
Accepted October 24, 2025

Objective: Busulfan, a chemotherapeutic alkylating agent, induces azoospermia by generating reactive oxygen species (ROS), thereby impairing spermatogenesis and steroidogenesis, and threatening fertility in cancer patients. Chlorogenic acid (CGA), a polyphenolic compound with antioxidant and anti-inflammatory properties, may protect against such reproductive toxicity. This study evaluated the effects of CGA on testicular function in a rat model of busulfan-induced azoospermia, with emphasis on spermatogenesis, steroidogenesis, and testicular architecture. Methods: Twenty-four adult male Wistar rats were randomly assigned to four groups: control, CGA (50 mg/kg, oral, for 60 days), busulfan (BUS, 40 mg/kg, single intraperitoneal dose), and BUS+CGA. Testicular function was assessed using stereological analyses (testis weight,; seminiferous tubule and germinal epithelium volumes,; germ and somatic cell counts), sperm parameters, serum hormone levels, oxidative stress markers (total antioxidant capacity [TAC], and malondialdehyde [MDA]), and expression of steroidogenic genes (STAR, CYP11A1, and HSD17B3) by qRT-PCR. Results: Busulfan markedly reduced testis weight, sperm quality, testosterone, TAC, and expression of steroidogenic genes, while increasing MDA and sperm abnormalities (p<0.05). CGA co-administration significantly improved these parameters, restoring gene expression and TAC toward control levels, reducing MDA, and enhancing testosterone, sperm count, morphology, and testicular histoarchitecture. However, sperm motility and testicular volume remained below control values (p<0.05). The CGA-alone group showed no significant differences from controls. Conclusion: CGA mitigates busulfan-induced azoospermia by enhancing antioxidant defenses, and improving spermatogenesis, steroidogenesis, and testicular morphology. These findings suggest that CGA may serve as a potential complementary therapy for fertility preservation in patients undergoing chemotherapy, warranting further mechanistic and clinical investigation.