ANALYSIS OF UNFERTILIZED OOCYTES SUBJECTED TO INTRACYTOPLASMIC SPERM INJECTION USING 2 ROUNDS OF FLUORESCENCE IN-SITU HYBRIDIZATION AND PROBES TO 5 CHROMOSOMES

Chromosomal aberrations are the major cause of pre-and post-implantati on embryo wastage and some studies suggest that half of all human conc epti have a chromosomal abnormality, Analysis of gametes provides info rmation on the origin of these chromosomal aberrations, The purpose of this study was to develop a reliable multi-probe fluorescence in-situ hybridization (FISH) procedure that would enable us to investigate an euploidy in unfertilized oocytes subjected to intracytoplasmic sperm i njection (ICSI), Oocytes were spread with HCl and Tween 20 solution, a nd then two rounds of triple-probe FISH were performed on each oocyte using directly-labelled centromeric probes: chromosomes 1, 7, 15 (over night hybridization); chromosomes 1, X, Y (2 h hybridization), After t he first round, the slides were counterstained and evaluated, and the positions of FISH signals were recorded. For the second round, the cou nterstain was removed and the second probe cocktail was applied, The c hromosome 1 probe was an internal control for the two hybridization pr ocedures, while the Y chromosome probe was used to detect sperm DNA, T o evaluate the method, a total of 79 oocytes from 27 patients were stu died, Of these, 67 (84.8%) were successfully spread and 97% of these o ocytes exhibited discernible FISH signals, Upon lysis, oocytes exhibit ed one or more DNA fragments (mean 1.9, range 1-3), Of the 65 analysab le oocytes, 17 (26.2%) displayed a normal haploid chromosome constitut ion with paired spots for the two chromatids. A further 23 oocytes (35 .4%) showed an ambiguous chromosome complement due to an abnormal numb er of DNA fragments which may have resulted from loss of DNA during sp reading or to an abnormal oocyte, while 25 oocytes (38.4%) displayed a neuploidy for one or more of the chromosomes studied, In conclusion, t his new approach is a quick and efficient method with which numerical chromosomal abnormalities in human oocytes can be studied; interpretat ion of the patterns of DNA fragments and FISH signals requires further clarification.