Discontinuous Percoll gradients enrich X-bearing human spermatozoa: a study using double-label fluorescence in-situ hybridization.

The aim of this study was to evaluate the efficacy of enriching X-bearing human spermatozoa using 12-step (25-80%) discontinuous Percoll gradients. X- and Y-bearing spermatozoa were simultaneously identified in neat semen (controls) and in 80% Percoll fractions from the same samples using double-label fluorescence in-situ hybridization and chromosome-specific DNA probes. Hybridization and labelling efficiencies of 95-99% were obtained in all samples. The mean ratio of X- to Y-bearing spermatozoa in the controls was 49.0:48.2, whereas there was a significant enrichment (P < 0.0001) of X-bearing spermatozoa in the Percoll fractions (mean X:Y ratio was 55.1:41.1 or 1.35). The ratio varied from 1.1-1.5 in individual Percoll samples. There were no significant differences in the proportions of aneuploid spermatozoa (XX, YY, XY) between the control and Percoll fractions. We conclude that 12-step discontinuous Percoll gradients enrich X-bearing spermatozoa, but the degree of enrichment is insufficient for use in preconceptional sex selection.