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

Abstract

To determine the ratio of X- to Y-bearing human spermatozoa in fractions isolated from discontinuous albumin gradients. 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. Hospital-based university department. Healthy donors with normal semen characteristics. The proportions of haploid cells (X or Y) and cells with two sex chromosomes (XX, YY, or XY) were determined. 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. Discontinuous albumin gradients do not enrich Y-bearing sperm as previously reported.