Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: protective significance of seminal plasma.

Before the advent of intracytoplasmic sperm injection (ICSI) semen preparation techniques focused on the need to sustain the fertilizing potential of the spermatozoa particularly by reducing oxidative stress. However, for severely oligozoospermic patients treated by ICSI, sperm preparation protocols are used which aim to maximize sperm recovery rather than sperm function. In this study we have examined the impact of different sperm preparation techniques on oxidative stress, sperm motion and DNA integrity. Reactive oxygen species (ROS) generation was monitored using luminol-dependent chemiluminescence, seminal antioxidant activity was assessed using a total reactive antioxidant potential (TRAP) assay while sperm motility and DNA damage were evaluated using computer assisted semen analysis and in-situ nick translation respectively. The results demonstrate a significant increase in the levels of ROS generated by samples prepared by swim-up from a washed pellet compared with spermatozoa isolated directly from seminal plasma. This oxidative stress was associated with a highly significant increase in the level of DNA damage sustained by the spermatozoa while the quality of sperm motility remained largely unchanged. These results suggest that if repeated centifugation protocols are to be used to prepare spermatozoa, strategies should be developed for minimizing collateral DNA damage.