Assessment of the separation of X- and Y-bearing sperm on albumin gradients using double-label fluorescence in situ hybridization.

OBJECTIVE: To determine the ratio of X- to Y-bearing human spermatozoa in fractions isolated from discontinuous albumin gradients. DESIGN: The proportions of X- and Y-bearing sperm were determined in neat semen samples (control) and in albumin-separated fractions from the same samples. Two albumin methods were used: a two-layer method (experiment 1) and a three-layer method (experiment 2). X- and Y-bearing sperm were identified simultaneously using chromosome-specific DNA probes and fluorescence in situ hybridization. SETTING: Hospital-based university department. PARTICIPANTS: Healthy donors with normal semen characteristics. MAIN OUTCOME MEASURES: The proportions of haploid cells (X or Y) and cells with two sex chromosomes (XX, YY, or XY) were determined. RESULTS: Labeling efficiencies were > 96% in all samples. Control samples showed a 1:1 ratio of X- to Y-bearing sperm. Fractions isolated on albumin gradients showed a slight, but statistically significant enrichment of X-bearing sperm. This was evident with both albumin methods. CONCLUSIONS: Discontinuous albumin gradients do not enrich Y-bearing sperm as previously reported.