Nicholas J. Swann

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.

FISH Analysis of Six Chromosomes in Unfertilized Human Oocytes After Polar Body Removal

AbstractPurpose: To develop an improved technique for estimatingchromosomal abnormalities in human oocytes byfluorescence in situ hybridization (FISH) and to correlate theposition of single chromatids with the chromosomal status ofthe oocytes. Methods: Oocytes that were at metaphase II about17–20 hr after insemination or intracytoplasmic sperm injection(ICSI) were treated with pronase to remove the zonapellucida and polar body (PB) and then spread on slides usingHCl and Tween 20. Two rounds of FISH were performedusing direct-labeled probes: chromosomes 1, 13, 21 (round1); chromosomes X, 7, 18 (round 2). Results: Of the 63 oocytes from 18 patients (mean age,32 years), 48 (76%) had one DNA complement as expected, 9(14%) had 2 DNA complements, 3 (5%) gave incomplete FISHsignals, and 3 (5%) were not analyzable. Of the 48 oocyteswith one set of DNA, 48% were haploid, 44% were aneuploidfor one or more chromosomes, and 8% were polyploid. Wealso found an increased frequency of predivision of chromatidbivalents in aneuploid oocytes, especially for chromosome 21. Conclusions: This technique enables simultaneousassessment of six chromosomes in human oocytes, and thereforecan be useful for accurately determining the incidence andcauses of genetic imbalances in human oocytes andapparently low fertilization rates.