Efficiency of polymerase chain reaction assay for cystic fibriosis in single human blastomeres according to the presence or absence of nuclei.

OBJECTIVE: To amplify by polymerase chain reaction (PCR) assay the region of the most common mutation of cystic fibrosis (CF) in human blastomeres. DESIGN: Blastomeres were isolated from two- to eight-cell tripronucleate embryos. The nuclear status of blastomeres was recorded by light microscopy (LM) and by fluorescence microscopy after vital labeling with the fluorochrome Hoechst 33342 (H-33342; Sigma, Brussels, Belgium). In each blastomere the region around the delta F508 mutation site was amplified by two PCRs with nested primers. SETTING: Research units of the Centres for Reproductive Medicine and Medical Genetics of the Dutch-speaking Free University of Brussels, Belgium. RESULTS: The presence of a nucleus by LM in 118 of 160 blastomeres was always confirmed by fluorescence microscopy, and in 10 additional blastomeres the nucleus was only visible by fluorescence microscopy. In the PCR assay all blanks were negative and in nucleate blastomeres (assessed by LM) the amplification rate was 96%. After staining with Hoechst dye the percentage of amplification was 71% or 91% if PCR was performed 2 hours or 20 hours after coloration. CONCLUSION: Efficient preimplantation diagnosis for CF on blastomeres requires assessment of nuclear status by LM and vital staining on blastomeres that are anucleate by LM.