ASSESSMENT OF THE SEPARATION OF X-BEARING 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: T he proportions of X- and Y-bearing sperm were determined in neat semen samples (control) and in albumin-separated fractions from the same sa mples. 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 fl uorescence in situ hybridization. Setting: Hospital-based university d epartment. Participants: Healthy donors with normal semen characterist ics. Main Outcome Measures: The proportions of haploid cells (X or Y) and cells with two sex chromosomes (XX, YY, or XY) were determined. Re sults: Labeling efficiencies were >96% in all samples. Control samples showed a 1:1 ratio of X- to Y-bearing sperm. Fractions isolated on al bumin gradients showed a slight, but statistically significant enrichm ent of X-bearing sperm. This was evident with both albumin methods. Co nclusions: Discontinuous albumin gradients do not enrich Y-bearing spe rm as previously reported.