Abstract
Background and Aims: The non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) using cell-free DNA (cfDNA) from the spent culture medium (SCM) has been reported. Nevertheless, the recommendation of niPGT-A in routine IVF clinics has been controversial due to diverse study conditions. To establish a reliable clinical protocol, the critical factors affecting accuracy of niPGT-A assay and clinical outcomes were investigated. Methods: Three hundred and sixty-two blastocysts from 94 patients undergoing IVF between January 2021 and August 2022 were analyzed. In a frozen embryo arm (n=100), the accuracy of niPGT-A and PGT-A using SCM and corresponding trophectoderm (TE) biopsy, respectively, was examined when compared with whole blastocysts as gold standard. In a fresh embryo arm (n=262), the concordance between niPGT-A and PGT-A were analyzed. Results: Comparing different commercial kits, the PicoPLEX Gold showed the highest sequencing quality and accuracy. Through applying an optimized threshold for mosaicism, the overall PPV, NPV, specificity, sensitivity, and accuracy of niPGT-A reached to 92.1%, 88.2%, 75.0%, 96.7%, and 91.3%, respectively. Regarding the fresh embryos, niPGT-A and PGT-A had reached a ploidy concordant rate of 76.5% and sex concordant rate of 90.7%. Applying assisted-hatching effectively elevated the amplification rate ( [Formula: see text] [Formula: see text]0.001), and the accuracy of niPGT-A (91.3%) was significantly better than PGT-A (79.2%, [Formula: see text] =0.033). The longer culture duration improved the ploidy concordance (1-day: 63.6%, 2-day: 73.5%, 3-day: 83.6%). Finally, 21 frozen-thawed embryo transfer cycles were performed and guided by euploid results of the TE biopsy. Live-birth rate was higher in euploid-TE/euploid-SCM (54.4%) than in euploid-TE/aneuploid-SCM (30.0%), and no miscarriages were observed in euploid-TE/euploid-SCM. Conclusions: Our current niPGT-A workflow adopts from the most common clinical setting of embryo culture and shows superior accuracy. Given the nature of niPGT-A that reflects the ploid state of the whole blastocyst, larger scale studies are warranted to support niPGT-A in routine clinical practice.
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