PREIMPLANTATION DIAGNOSIS OF SINGLE-GENE DISORDERS BY 2-STEP OOCYTE GENETIC-ANALYSIS USING FIRST AND 2ND POLAR BODY

Previous work on preimplantation genetic diagnosis (PGD) of single gen e disorders by the first polar body (IPB) analysis has demonstrated th at the genotype of a considerable number of embryos resulting from het erozygous oocytes cannot be predicted without testing their second PB (IIPB). To overcome this limitation we introduce a two-step DNA analys is of oocytes using both IPB and IIPB to identify hemizygous mutation- free oocytes following the second meiotic division. In the application of the approach to PGD of cystic fibrosis (CF) Delta F-508 mutation, sickle cell disease, and hemophilia B, 80 oocytes were studied by both PBs, resulting in the identification and transfer of 32 homozygous no rmal embryos. A follow-up genotyping of 52 embryos, resulting from ooc ytes tested by both IPB and IIPB demonstrated the accuracy of the pred icted genotypes. In addition to a nested PCR analysis of the mutant ge nes in PBs and resulting embryos, simultaneous amplification of differ ent polymorphic markers was performed, demonstrating the reliability o f the two-step polar body analysis of oocytes. (C) 1997 Academic Press .