Comparison of sperm characteristics following single or double-layer density gradient centrifugation

Abstract Background The present study aimed to understand whether single and double-layer density gradient systems provide differential sperm preparation outcomes. The prospective comparative study included semen samples from 25 normozoospermic and 25 non-normozoospermic men. Each sample was equally split and processed using single- and double-layer density gradient centrifugation, and the recovered spermatozoa were assessed for basic sperm characteristics, kinematics, and DNA integrity. The outcomes were compared between the two gradient systems in both groups. Results Sperm motility, kinematics, mitochondrial membrane potential, and DNA integrity did not differ significantly in sperm fractions recovered using single- and double-layer density gradient systems in both normozoospermic and non-normozoospermic ejaculates. In normozoospermic ejaculates, total motility (mean difference, MD 2.1%, 95% CI -2.5 to 6.9; p = 0.36), mitochondrial membrane potential (MD -0.7, 95% CI -6.0 to 4.5, p = 0.77) and DNA integrity (MD -0.0, 95% -3.2 to 3.1; p = 0.98) was not statistically different between the gradient systems immediately post sperm selection. Similar findings were observed in non-normozoospermic ejaculates for total motility (MD 5.0%, 95% CI -4.9 to 14.9; p = 0.31), mitochondrial membrane potential (MD -3.3, 95% CI -18.1 to 11.5; p = 0.65) and DNA integrity (MD -3.0, 95% CI -7.6 to 1.6; p = 0.19). The findings remain unaltered after 6 h of incubation. Conclusion Single-layer density gradient centrifugation effectively selects competent spermatozoa from both normozoospermic and non-normozoospermic samples, highlighting its potential as a technically simple and cost-effective approach for sperm processing.