Sperm cryopreservation as a routine technique in assisted reproductive technique (ART) laboratories has detrimental effects on spermatozoa. Various methods have been introduced to improve it. The aim of this research was to evaluate the effects of L‐proline supplementation in cryopreservation medium on normozoospermic semen samples. A total of 30 semen samples were collected from normozoospermic men. Cryopreservation media were supplemented with different concentrations of L‐proline (0, 1, 2 and 4 mmol/L). The semen samples were cryopreserved. After thawing, sperm parameters and chromatin integrity (aniline blue (AB), toluidine blue (TB), sperm chromatin dispersion test (SCD) and chromomycin A3 (CMA3)), reactive oxygen species (ROS), and total antioxidant capacity (TAC) and malondialdehyde (MDA) levels were evaluated. A total of 4 mmol/L L‐proline significantly improved progressive motility and viability (p < 0.05). MDA and ROS levels significantly diminished in samples were cryopreserved by 4 mmol/L L‐proline supplemented cryopreservation media (p < 0.001). Also, it significantly increased TAC level. Also, chromatin damages (AB, TB and CMA3) significantly improved in samples were cryopreserved by 4 mmol/L L‐proline supplemented cryopreservation media (p < 0.05). The results support that the usage of L‐proline supplemented cryopreservation media to improve sperm quality after cryopreservation.

L-Proline is a natural anti-oxidative and osmoprotectant agent, playing a versatile role in cell metabolism and physiology. The present study aimed to explore the antioxidant effects of L-Proline on human sperm function during incubation. Thirty healthy, normozoospermic men (27-40 years) were enrolled. Sperm samples were incubated in an unsupplemented sperm medium (control group), or supplemented with L-Proline (1, 2 and 4 mmol/L) to evaluate its effect during 0, 1, 4 and 24 h of incubation. Sperm were assessed in terms of motility, viability, morphology, chromatin and DNA integrity. Moreover, the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and total antioxidant capacity (TAC) were determined in the sperm medium. The results indicated that 2 mmol/L of L-Proline significantly improved the maintenance of sperm motility, viability, normal morphology, chromatin and DNA integrity, and TAC levels compared to the control group during 24 h of incubation (p < 0.05). However, 1 and 4 mmol/L of L-Proline could not significantly preserve sperm parameters, chromatin quality, and antioxidant status during different incubation times compared to the control group (p > 0.05). Collectively, the inclusion of L-Proline (2 mmol/L) in the human sperm medium maintains sperm parameters and chromatin quality probably by modulating the oxidative status.

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