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

Human semen is heterogeneous in quality, not only between males but also within a single ejaculate. Differences in quality are evident, both when examining the classical parameters of sperm number, motility and morphology and in the integrity of the sperm nucleus. The aim of this study was to determine the efficiency of the PureSperm(R), Percoll(R) and swim-up preparation techniques to eliminate spermatozoa with nuclear anomalies. Semen samples were collected, washed and one part of the semen spread on a slide, the remainder was prepared using the swim-up, PureSperm(R) or Percoll(R) techniques. Spermatozoa from different fractions were fixed on slides and assessed. Sperm 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 Percoll(R)). Spermatozoa prepared using PureSperm(R) (n = 35) and Percoll(R) (n = 37) were also examined for the presence of endogenous DNA nicks. Good quality spermatozoa should not possess DNA nicks and not stain (i.e. fluoresce) with CMA(3). When prepared using the swim-up technique the spermatozoa recovered showed no significant improvement with the CMA(3) staining. When spermatozoa 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) and Percoll(R) techniques can enrich the sperm population by separating out those with nicked DNA and with poorly condensed chromatin.