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

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Abstract
Purpose: 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.