FISH analysis of six chromosomes in unfertilized human oocytes after polarbody removal

Purpose: To develop an improved technique for estimating chromosomal abnorm alities in human oocytes by fluorescence in situ hybridization (FISH) and t o correlate the position of single chromatids with the chromosomal status o f the oocytes. Methods: Oocytes that were at metaphase II about 17-20 hr after inseminatio n or intracytoplasmic sperm injection (ICSI) were treated with pronase so r emove the zona pellucida and polar body (PB) and then spread on slides usin g HCl and Tween 20. Two rounds of FISH were performed using direct-labeled probes. chromosomes 1, 13, 21 (round 1): chromosomes X, 7, 18 (round 2). Results: Of the 63 oocytes from 18 patients (mean age 32 years), 48 (76%) h ad one DNA complement as expected, 9 (14%) had 2 DNA complements, 3 (5%) ga ve incomplete FISH signals, and 3 (5%) Mere nor analyzable. Of the 48 oocyt es with one set of DNA, 48% were haploid, 44% were aneuploid for one or mor e chromosomes, and 8% Mere polyploid. Cte also found an increased frequency of predivision of chromatid bivalents in aneuploid oocytes, especially for chromosome 21. Conclusions: This technique enables simultaneous assessment of six chromoso mes in human oocytes, and therefore can he useful for accurately determinin g the incidence and causes of genetic imbalances in human oocytes and appar ently low fertilization rates.