Sex determination of human preimplantation embryo using nested polymerase chain reaction

Objective: Using nested polymerase chain reaction (PCR) to perform preimplantation gender diagnosis. Methods: One (or two) lymphocyte and blastomere (n = 50/group) were collected and prepared under the following conditions: (1) water only (H₂O); (2) freeze-thaw liquid nitrogen, then boiling; (3) potassium hydroxide/dithiotheriol, heated to 65°C, followed by acid neutralization (KOH). Cells were analyzed by PCR using nested primers amplification with amelogenin gene. Results: The amplification rate and allele dropout (ADO) rate for male lymphocytes by the three methods were 83%, 94%, 95% and 24%, 12%, 4%, respectively. Using two cells per reaction did not increase the amplification rate for the KOH method. Conclusion: The KOH method for DNA preparation is superior to the other methods evaluated. Dual blastomere biopsy and independent blastomere analysis may improve preimplantation diagnostic reliability.