Assessing Sperm Selection Techniques: The Impact on Cell Viability Using Flow Cytometry

Abstract

Objective: 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. Post-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 the 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.