Anterior positioning of sex chromosomes on the head of human sperm sorted using visible wavelengths.

The human ejaculate contains subpopulations of sperm with distinct properties. Human X-and Y-bearing sperm were separated with fluorescence activated cell sorting. To avoid the use of UV light the quantitative DNA dyes DRAQ5 (R) and Dyecycle (TM) Vybrant (R) Violet were used. Sorting efficiency was similar for both dyes, but lower than what is usually obtained with the classical method involving Hoechst 33342 and UV light (60-70% enrichment, versus 80-90%). A total of 2,739 spermatozoa were evaluated, from seven distinct samples using fluorescence in situ hybridization (FISH) chromosomal probes. No differences were found in sorted and unsorted populations in terms of chromosome positioning, and numeric chromosomal anomalies were not more evident following cell sorting. Furthermore in both sorted and unsorted populations the sex chromosomes were clearly located in the anterior portion of the sperm head, while a control autosome (chromosome 18) showed no such tendency, confirming previous findings. These results suggest that other quantitative DNA dyes may be used for sex chromosome-based human sperm sorting, but with lower efficiency than the standard UV-Hoechst based assay.