Abstract Study question Does male human sperm exposure to bleomycin in vitro affect their aptitude to breed? Summary answer Bleomycin, an antitumor antibiotic, adversely impacts human male gamete morphology and functionality, thus potentially compromising male fertility. What is known already Bleomycin is a chemotherapeutic agent used to treat a number of tumors that affect males of reproductive age. Hitherto, its effect on human male reproductive capacity alone has not been previously shown. The little information available on this subject relates to in vivo animal studies and supports the feasibility of this study. Study design, size, duration Between November 2022 and June 2023, individuals of reproductive age were enrolled in this study and had their clinical semen analyzed. The remaining ejaculate was supplied by 45 participants with sperm concentration above the World Health Organization (WHO) threshold for scientific purposes. Sperm were isolated from seminal plasma in the ejaculate of each individual, diluted to a concentration of 16 X 106 sz/mL (raw). Participants/materials, setting, methods In vitro incubation was performed in sperm preparation medium (SPM; control) or bleomycin (100 µg/mL; exposed) augmented for two hours at 37oC and 5% CO2. The results of exposed group were compared to raw and control. To evaluate the competence of human sperm, markers such as acrosome and DNA integrity, mitochondrial membrane potential, sperm motility, vitality, and oxidative stress were evaluated. Ultrastructural data from transmission electron microscopy was also obtained and correlated with functional experiments. Main results and the role of chance The participants were all of reproductive age (median age of 36 years old). The raw sperm parameters fulfilled the normozoospermia threshold set by the WHO, 2021. Prior to the current trial, none of the participants had been diagnosed with cancer or had received chemotherapy, radiation therapy, or both. Bleomycin exposure for two hours in vitro significantly (P < 0.001) reduced sperm motility, vitality, and chromatin condensation as compared to raw and control sperm. Moreover, bleomycin significantly (P < 0.001) increased the percentage of sperm with DNA fragmentation (sDNAfrag) and low mitochondrial membrane potential (MMP). Broad strands were significantly (P < 0.001) attained by bleomycin-induced breaks in the sperm DNA (Head staining pattern), which were succeeded by histone-rich areas (PAR-post-acrossomic region staining pattern). Additionally, bleomycin significantly (P < 0.05) delayed the acrossomic response, but had no significantly influence on the generation of extracellular and intracellular reactive oxygen species or in its reconstitution. Modifications in ultrastructural morphology caused by bleomycin corroborate the results obtained by functional tests. Limitations, reasons for caution Bleomycin impact on male fertility competence in vivo remains crucial. Increased sample sizes, including prepubertal subjects, are needed to pinpoint the precise bleomycin’s mechanism to cause oxidative stress, apoptosis, and sDNAfrag. Long-term implications on fertility and reproductive health, besides methods to lessen or offset bleomycin’s effects, should also be pursued. Wider implications of the findings Bleomycin adversely affects male gamete competence in humans. Along with its accompanying lung toxicity, healthcare practitioners should actively monitor and further investigate bleomycin’s gonadotoxicity in males. It is advised that all cancer patients undergoing chemotherapy treatments that contain bleomycin be provided with a fertility preservation plan in the meantime. Trial registration number not applicable
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