L-Proline supplementation preserves sperm function and chromatin integrity in asthenozoospermic patients during cryopreservation

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BACKGROUND: Asthenozoospermia is a major cause of male infertility, accounting for approximately 18% of infertility cases. L-Proline, a natural antioxidant and osmoprotectant, has gained attention for its potential applications in semen cryopreservation. This study aimed to evaluate the beneficial effects of L-proline on sperm quality in samples from asthenozoospermic patients during cryopreservation. RESULTS: This in vitro experimental study utilized semen samples from 30 men (aged 27-40 years) diagnosed with asthenozoospermia. Each sample was divided into three aliquots for cryopreservation: (1) a control group using a standard unsupplemented freezing medium; (2) a group supplemented with 2 mM L-proline; and (3) a group supplemented with 4 mM L-proline. Following thawing, samples were assessed for sperm count, motility, viability, morphology, and chromatin quality. Additionally, levels of malondialdehyde (MDA), total antioxidant capacity (TAC), and nitric oxide (NO) were assessed. Supplementation with 2 mM L-proline resulted in significantly higher post-thaw progressive motility, viability, and chromatin integrity compared to the control group (p < 0.05). These functional improvements were associated with a more favorable antioxidant status, evidenced by significantly lower levels of MDA and NO and higher levels of TAC (p < 0.05). While the 4 mM L-proline group showed some protection, the effects were less pronounced than those observed with the 2 mM concentration. CONCLUSIONS: The results suggest that adding 2 mM L-proline into the freezing medium effectively protects human sperm quality and chromatin integrity in asthenozoospermic samples by mitigating cryopreservation-induced nitro-oxidative stress. This strategy holds promise for improving assisted reproductive technology (ART) outcomes in men with asthenozoospermia.

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