Assessing sperm selection techniques : the impact on cell viability using flow cytometry

Abstract

Introduction: This study aimed to evaluate the effects of different sperm selection techniques on seminal parameters and structural characteristics using flow cytometry, with a focus on cell viability, reactive oxygen species (ROS) production, and acrosome integrity. Methods: Semen samples from eight normospermic volunteers were subjected to two sperm selection techniques: simple washing and density gradient centrifugation. Following processing, the samples were analyzed using flow cytometry to assess motility, viability, ROS production, lipid peroxidation, the acrosome reaction, and plasma membrane stability. Results: The density gradient method significantly increased total motility (70.75% ± 12.87%) compared with both raw semen (55.63% ± 8.55%) and the washing technique (53.13% ± 4.61%). Additionally, the percentage of live spermatozoa was higher in the density gradient group (71.63% ± 13.72%) than in the washed group (54.50% ± 9.53%). There was no significant difference in ROS production between the techniques; however, the density gradient technique reduced the number of ROS-producing dead cells (0.04% ± 0.06%) compared with the washing technique (0.13% ± 0.23%). There was an increase in the number of cells with a reacted acrosome after density gradient centrifugation (25.79% ± 15.07%) compared with raw semen (8.80% ± 4.28%). Conclusion: Density gradient centrifugation is superior to the washing technique in selecting viable and motile sperm: It significantly improved total motility and viability. Although the density gradient technique did not increase oxidative stress, it was associated with a higher incidence of the acrosome reaction, which may have implications for fertility outcomes.