PGT for human blastocysts with potential parental contamination using quantitative parental contamination testing (qPCT): an evidence-based study

Background：There is an urgent clinical need for preimplantation genetic testing (PGT) in frozen conventional human in vitro fertilization (IVF) embryos. However, it is difficult to detect parental contamination from sperm and cumulus cells in frozen conventional human IVF embryos during PGT with current methods. We established a quantification method for the parental contamination test, named qPCT, which ensures the results more reliable and substantially reduces the risk of PGT in frozen conventional IVF embryos.Methords：34 patients who had experienced repeated implantation failure or abortion due to chromosomal abnormalities after embryo transfer in prior routine IVF cycles. A total of 120 surplus frozen blastocysts from them were available for the study. Parental DNA contamination in the biopsied TE cells was detected via qPCT assay during PGT. To analyze the ploidy status of blastocysts, the amplified DNA of TE samples was sequenced using a NextSeq 550 sequencer with a single-ended read length of 55 bp..Result(s): A new quantification method for parental contamination testing (qPCT) in single-cell whole-genome amplification (WGA) products based on allelic ratio analysis was established and validated in an artificial model by comparing 22 results obtained before and after adding different numbers of sperm and cumulus cells to biopsied TE cells. The results of the prospective clinical study of qPCT-PGT-A showed that the maternal contamination rate was 0.83% (1/120) and the risk of paternal contamination was negligible. The euploidy rate in these blastocysts was 47.50% (57/120), and 21 frozen embryo transfer (FET) cycles resulted in ten ongoing clinical pregnancies and four healthy births.Conclusion(s): The evidence we provide in the study shows a low risk of PGT in embryos simultaneously with adhered sperm and cumulus cells. The qPCT assay can be used to detect the risk of potential contamination and ensure the accuracy of PGT results, thereby improving the clinical outcome of IVF.