Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test

Objective: To improve the sperm chromatin dispersion (SCD) test and develop it as a simple kit (Halosperm (R) kit) for the accurate determination of sperm DNA fragmentation using conventional bright-field microscopy. Design: Method development, comparison, and validation. Setting: Medical genetics laboratory, academic biology center, and reproductive medicine centers. Patient(s): Male infertility patients attending the Reproductive Medicine Center. A varicocele patient and a group of nine fertile subjects. Intervention (S): None. Main Outcome Measure(s): [1] The quality of chromatin staining in relaxed sperm nuclear halos and tail preservation; [2] SCD scoring reproducibility; [3] comparison with the sperm chromatin structure assay in 45 samples; [4] frequency of sperm with DNA fragmentation after incubation with increasing doses of the nitric oxide donor sodium nitroprusside and in sperm samples for 9 fertile men, 46 normozoospermic patients, 23 oligoasthenoteratozoospermic patients, and a subject with varicocele. Result(S): The sperm nuclei with DNA fragmentation, either spontaneous or induced, do not produce or show very small halos of DNA loop dispersion after sequential incubation in acid and lysis solution. The improved SCD protocol (Halosperm (R) kit) results in better chromatin preservation, therefore highly contrasted halo images can be accurately assessed using conventional bright-field microscopy after Wright staining. Moreover, unlike in the original SCD procedure, the sperm tails are now preserved, making it possible to unequivocally discriminate sperm from other cell types. The chi(2) test did not detect significant differences in the mean number of sperm cells with fragmented DNA as scored by four different observers. The intraobserver coefficient of variation for the estimated percentage of spermatozoa with fragmented DNA ranged from 6% to 12%. There was good correlation between the SCD and the sperm chromatin structure assay DNA fragmentation index (intraclass correlation coefficient R: 0.85; percent DNA fragmentation index mean difference: 2.16 significantly higher for SCD). Using the Halosperm (R)(R) kit, a dose-dependent increase in sperm DNA damage after sodium nitroprusside incubation was detected. The percentage of sperm cells with fragmented DNA in the fertile group was 16.3 +/- 6.0, in the normozoospermic group, 27.3 +/- 11.7, and in the oligoasthenoteratozoospermic group, 47.3 +/- 17.3. In the varicocele sample, an extremely high degree of nuclear disruption was detected in the population of sperm cells with fragmented DNA. Conclusion(S): The improved SCD test, developed as the Halosperm (R) kit, is a simple, cost effective, rapid, reliable, and accurate procedure, for routinely assessing human sperm DNA fragmentation in the clinical andrology laboratory.