Noninvasive preimplantation genetic testing for aneuploidy in spent medium may be more reliable than trophectoderm biopsy

Abstract

Preimplantation genetic testing for aneuploidy (PGT-A) with trophectoderm (TE) biopsy is widely applied in in vitro fertilization (IVF) to identify aneuploid embryos. However, potential safety concerns regarding biopsy and restrictions to only those embryos suitable for biopsy pose limitations. In addition, embryo mosaicism gives rise to false positives and false negatives in PGT-A because the inner cell mass (ICM) cells, which give rise to the fetus, are not tested. Here, we report a critical examination of the efficacy of noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) in the spent culture media of human blastocysts by analyzing the cell-free DNA, which reflects ploidy of both the TE and ICM. Fifty-two frozen donated blastocysts with TE biopsy results were thawed; each of their spent culture medium was collected after 24-h culture and analyzed by next-generation sequencing (NGS). niPGT-A and TE-biopsy PGT-A results were compared with the sequencing results of the corresponding embryos, which were taken as true results for aneuploidy reporting. With removal of all corona-cumulus cells, the false-negative rate (FNR) for niPGT-A was found to be zero. By applying an appropriate threshold for mosaicism, both the positive predictive value (PPV) and specificity for niPGT-A were much higher than TE-biopsy PGT-A. Furthermore, the concordance rates for both embryo ploidy and chromosome copy numbers were higher for niPGT-A than TE-biopsy PGT-A. These results suggest that niPGT-A is less prone to errors associated with embryo mosaicism and is more reliable than TE-biopsy PGT-A.

Keywords: ICSI; culture medium; human blastocyst; noninvasive PGT-A; preimplantation genetic testing.

Conflict of interest statement

Conflict of interest statement: S.L. and X.S.X. are cofounders and shareholders of Yikon Genomics. The other authors declare no conflict of interest.

Figures

Fig. 1.

False positives and false negatives arise from embryo mosaicism in TE biopsy PGT-A.

Fig. 2.

Workflow of sample processing for PGT-A analysis of TE biopsy, embryo, and spent culture media.

Fig. 3.

The CN plots of embryo vs. spent culture medium at a sequencing depth of 0.02×. The chromosomes are shown in alternating red and blue colors. (A) A male euploid embryo with corresponding euploid niPGT-A profile. (B) A male aneuploid embryo with corresponding aneuploid niPGT-A profile. (C) A male euploid embryo with corresponding noisy niPGT-A profile. (D) A male aneuploid embryo with discordant mosaicism in the embryo compared with corresponding niPGT-A profile.

Fig. 4.

FPR and FNR as a function of the percent mosaicism in niPGT-A profiles. Sixty percent of mosaicism was set as the threshold for identifying aneuploidy.