Sex determination by simultaneous application of polymerase chain reaction and fluorescent in situ hybridization on the same blastomere of a pre-embryo

Objective: To describe an investigation of the feasibility of carrying out polymerase chain reaction (PCR) and fluorescent in situ hybridization on the same single human cell for sex determination. Design: To develop protocol for a clinical diagnostic test in preclinical trials. Setting: Infertile human volunteers in a clinical environment. Patients: Polyploid embryos were obtained from patients treated by IVF at the Advanced Institute of Fertility. Interventions: Seventeen blastomeres biopsied from human polyploid embryos were analyzed for sex determination by simultaneous application of PCR and fluorescent in situ hybridization. Main Outcome Measure: Presence of an X- or X and Y-chromosome band after agarose gel electrophoresis of PCR products and presence of an X- or X and Y-chromosome fluorescent probe signal after fluorescent in situ hybridization following PCR analysis. Results: By PCR, all 17 blastomeres were amplified and, by fluorescent in situ hybridization, 12 (70.6%) blastomeres produced signals that were consistent with PCR results. Two blastomeres showed only X signals, although by PCR they had both X and Y-chromosome bands. Conclusions: The sequential use of PCR and fluorescent in situ hybridization on the same blastomere can be applied to improve the accuracy of sex determination before fresh ET.