The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies

Human semen is heterogeneous in quality, not only between males but also wi thin a single ejaculate. Differences in quality are evident, both when exam ining the classical parameters of sperm number, motility and morphology and in the integrity of the sperm nucleus. The aim of this study was to determ ine the efficiency of the PureSperm(R), Percoll(R) and swim-up preparation techniques to eliminate spermatozoa with nuclear anomalies. Semen samples w ere collected, washed and one part of the semen spread on a slide, the rema inder was prepared using the swim-up, PureSperm(R) or Percoll(R) techniques . Spermatozoa from different fractions were fixed on slides and assessed. S perm samples (n) from different men were stained using the chromomycin A(3) (CMA(3)) fluorochrome, which indirectly demonstrates a decreased presence of protamine (n = 31 for swim-up; n = 45 for PureSperm(R); n = 39 for Perco ll(R)). Spermatozoa prepared using PureSperm(R) (n = 35) and Percoll(R) (n = 37) were also examined for the presence of endogenous DNA nicks. Good qua lity spermatozoa should not possess DNA nicks and not stain (i.e. fluoresce ) with CMA(3). When prepared using the swim-up technique the spermatozoa re covered showed no significant improvement with the CMA(3) staining. When sp ermatozoa were prepared using the PureSperm(R) and Percoll(R) techniques, a significant (P < 0.001) decrease in both CMA(3) positivity and DNA strand breakage was observed. These results indicate that both the PureSperm(R) an d Percoll(R) techniques can enrich the sperm population by separating out t hose with nicked DNA and with poorly condensed chromatin.