Differential impact of four sperm preparation techniques on sperm motility, morphology, DNA fragmentation, acrosome status, oxidative stress, and mitochondrial activity: A prospective study

Suboptimal human semen handling in vitro may induce sperm damage. However, the effects of semen swim-up, pellet swim-up, density gradient, and density gradient followed by SU on sperm motility, morphology, DNA fragmentation, acrosome reaction, intracellular reactive oxygen species, and mitochondrial activity were not fully understood.To study the impact of four sperm preparation techniques on sperm functional parameters.This study was conducted on 60 infertile men with a minimum sperm concentration of 20 × 106 /ml and total sperm motility of ≥30%. Each raw semen sample was divided into four aliquots. Each aliquot was prepared by one of the tested techniques. Various sperm characteristics were assessed before and after sperm preparation.Density gradient and density gradient followed by SU resulted in significantly higher DNA fragmentation percentages compared with semen swim-up (p < 0.001 and p < 0.001, respectively) and pellet swim-up (p < 0.001 and p < 0.001, respectively). Significantly higher percentages of spermatozoa with intact acrosome were detected in semen swim-up (p < 0.001) and pellet swim-up (p < 0.001) compared with raw semen. The percentage of reactive oxygen species-positive spermatozoa was significantly higher after pellet swim-up (p < 0.001), density gradient (p < 0.001), and density gradient followed by SU (p < 0.001) than raw semen. In addition, the percentages of 100% stained midpiece (active mitochondria) were significantly higher in semen swim-up (p < 0.001) and pellet swim-up (p < 0.001) compared with raw semen.To the best of our knowledge, this is the first report comparing the impact of these techniques on various sperm functional parameters. Semen swim-up was more effective than density gradient in selecting better spermatozoa in terms of DNA integrity, reactive oxygen species levels, acrosome status, and mitochondrial activity. Randomized clinical trials comparing these four techniques are required to test their impact on embryo development and pregnancy outcomes.