Oxidative stress markers and sperm structural integrity regarding semen processing methods followed by cryopreservation.

Abstract

Background: Seminal cryopreservation causes significant sperm damage, prompting research into alternative preservation methods. This study aimed to compare the effects of washing and density gradient processing-both involving seminal plasma removal-on oxidative damage, acrosomal, and mitochondrial integrity.

Materials and methods: Seminal samples from 26 normozoospermic patients were divided into three parts: one containing seminal plasma, the second processed by washing, and the third processed by selection through a density gradient. The samples were cryopreserved for at least 2 weeks. reactive oxygen species (ROS) production (measured using the DCF probe), plasma membrane peroxidation (BODIPY probe), acrosome integrity (fluorescein isothiocyanate-labeled/prostate-specific antigen [FITC/PSA] probe), lipid disorder (merocyanine 540 probe), and mitochondrial potential (JC1 probe) were evaluated through flow cytometry before cryopreservation and after thawing.

Results: Density gradient processing reduced ROS production compared to washing (p < 0.05). No increase in the ROS production was observed after thawing. In the fresh condition, both density gradient and washed samples showed a lower proportion of oxidized cells compared to raw samples (p < 0.001). Acrosomal damage was significantly higher in washed samples compared to other groups in fresh samples (p < 0.001). Cryopreservation caused acrosomal damage only in raw samples (p < 0.001), with gradient samples maintaining greater acrosomal integrity after thawing. Plasma membrane instability was lower in the gradient of the fresh group compared to raw or washed samples (p < 0.001). Cryopreservation induced plasma membrane destabilization in processed samples but not in samples frozen with seminal plasma. After thawing, plasma membrane destabilization was lower in the gradient group compared to raw and washed samples (p < 0.05 and p < 0.001). Cells in the gradient group showed higher mitochondrial potential (p < 0.001), followed by washed samples compared to raw samples (p < 0.01). Mitochondrial membrane potential was significantly impaired in all three processes, with no differences among the groups after thawing.

Conclusion: Cryopreservation significantly damages the acrosome and mitochondria of spermatozoa. In normozoospermic patients, the gradient method selected higher-quality spermatozoa compared to other processes, preserving functional advantages after thawing.