Use DNA fingerprinting to identify and confirm triploid embryos in whole genome next generation sequencing (NGS) preimplantation genetic screen (PGS)

Abstract

Whole genome next generation sequencing (NGS) for preimplantation genetic screen (PGS) has higher sensitivity and accuracy in detecting unbalanced translocation and partial aneuploid than targeted NGS. Whole genome NGS also detect some triploid, the most common polyploidy, if the sex chromosomes are unbalanced (69, XXY, and 69, XYY). Unlike targeted NGS, whole genome NGS unusually does not detect triploid 69, XXX, neither does it provide additional data to confirm triploid 69, XXY and 69, XYY identified in PGS. Here we tested using DNA fingerprinting in combination with whole genome NGS to identify and confirm sex chromosome balanced and unbalanced triploid in PGS. Feasibility and validation study. The feasibility is tested on of 5 known triploid cell lines and 5 known diploid cell lines. 24 fingerprinting loci were tested. Further validation and accuracy were tested on 16 blastocyst biopsy samples whole genome amplification product. All 16 samples had whole genome NGS for PGS and were potential triploid samples. 15 of them were identified by unbalanced sex chromosomes for PGS. 1 of them showed 46, XX for PGS but is suspected to be 69, XXX triploid because of the enlarged nuclei observed by the embryologist. DNA fingerprinting was performed on the 16 PGS samples to identify and confirm the triploid embryos. FISH was performed to confirm the DNA fingerprinting result. Homozygous or heterozygous loci (24 of 24) in DNA fingerprinting were detected for all 5 known diploid cells lines, confirming diploid. For the 5 known triploid cell lines, 5 to 10 out of the 24 DNA fingerprinting loci has 3 genotypes, confirming triploid. Among the 15 potential triploid samples identified by PGS, 13 showed triploid in DNA fingerprinting, with 3 to 11 loci having 3 genotypes; 2 showed diploid in DNA fingerprinting. The enlarged nuclei sample with 46, XX for PGS also showed triploid in DNA fingerprinting, with 7 out of 24 loci having 3 genotypes. The 16 embryos FISH result confirmed the DNA fingerprinting result, demonstrating DNA fingerprinting is accurate in identifying and confirming triploid in PGS sample. DNA fingerprinting test on whole genome amplified PGS samples is a simple and accurate method to examine triploid status of embryos. It can confirm triploid indicated by embryo morphology. Triploid identification in PGS based on unbalance sex chromosomes can be difficult and not reliable, especially when embryo DNA quality is compromised. In this situation DNA fingerprinting can identify and confirm the real triploid embryos. Used in combination with whole genome NGS, DNA fingerprinting allows patients to benefit from the advantages of the whole genome NGS, and at the same time, identifying the triploid embryos to reduce pregnancy loss.