Recycling of a single human blastomere fixed on a microscopic slide for sexing and diagnosis of specific mutations by various types of polymerase chain reaction.

Overview

OBJECTIVE: To investigate the suitability of recycling single blastomeres to assess multiple genetic variables for preimplantation genetic diagnosis. DESIGN: Prospective randomized study. SETTING: An academic medical center. PATIENT(S): Patients undergoing IVF-ET. INTERVENTION(S): Blastomeres were disaggregated from donated embryos obtained from patients. MAIN OUTCOME MEASURE(S): Polymerase chain reaction (PCR) amplification products. RESULT(S): Fifty-eight blastomeres individually fixed on slides were separated into four groups. Sequential PCRs (group I, n = 30), primed in situ labeling (PRINS) before five sequential PCRs (group II, n = 10), staining with hematoxylin before performing five sequential PCRs (group III, n = 11) and preamplification of whole DNAs by degenerate oligonucleotide primer (DOP) before performing PCR were executed. The amplification efficiencies of five sequential PCRs were 100%, 100%, 96.6%, 83.3%, 56.7% for group I; 100% 100%, 100%, 80%, 40% for group II; 54.5%, 36.4%, 18.2%, 9.1% for group III; and 100%, 100%, 100%, 100%, 100% for group IV. CONCLUSION(S): Blastomeres fixed for PRINS can be recycled for PCR to obtain more genetic information. Hematoxylin staining appears to increase the incidence of failed amplification. Preamplification of whole genomic DNAs by DOP-PCR appears to facilitate diagnosis with high efficiency.