First price poster award at ASRM 2021

We carried out a prospective study to investigate the concordance of cfDNA with the corresponding TE and ICM biopsies. A total of 117 day 6/7 blastocysts underwent TE biopsy and SBM were collected in the PGT-A cycle. These blastocysts were donated for research as part of a clinical trial, and ICM biopsy was performed afterwards. Media collection, TE and ICM biopsies were analyzed from January 2019 to March 2021. Embryos were cultured in routine conditions up to day 4, when embryos were washed, transferred to a new media 10μl culture medium droplet, and cultured for at least a further 48 hours. Culture media were collected before TE biopsy and frozen at -20ºC. Assisted hatching, blastocyst biopsy and vitrification were performed after media collection. All samples were analyzed by NGS using the Ion ReproSeq PGS Kit (ThermoFisher Scientific) using Ion Chef^TM plus the Ion S5 XL Sequencer^TM, with modifications in the amplification protocol for the embryo cfDNA. Customized algorithms were applied to the interpretation of results in TE, ICM and embryo cfDNA.

Informative results were obtained in 98.3% of the TE biopsies (115/117), 92.3% of the cfDNA samples from SBM (108/117), and 93.2% of the ICM (109/117). In combination, the three sample types were informative in 84.6% of the blastocysts (99/117). Considering ICM as those cells that will become the fetus, ploidy concordance with cfDNA was 85.9% (85/99) and with TE was 89.9% (89/99), without statistical differences. False positive rates were similar for cfDNA and TE biopsy (6.1% and 9.1%, respectively), and false negative rates were not significantly different, but higher in cfDNA (8.1%) than in TE (1.0%), due to potential contamination with maternal DNA. Embryo cfDNA ploidy concordance with TE biopsies was 89.9% (89/99). Considering the 14 non-concordant cases of the cfDNA with the ICM, 6 of them corresponded to aneuploid cfDNA and euploid ICM, that could be attributed to the presence of mosaicism in the blastocyst (TE biopsies were also aneuploid in the 6 cases). The remaining 8 non-concordant embryos corresponded to euploid cfDNA and aneuploid ICM, in most of the cases due to the presence of maternal contamination.

Embryo cfDNA detected in SBM correlates with both ICM in 85.9% of the blastocysts, and TE in 89.9% of the blastocysts.

We have shown high concordance rates of embryo cfDNA, not only with TE biopsies, as in previous studies, but also with ICM biopsies, which represent the true chromosomal content of the embryo. The niPGT-A approach would avoid the need of invasive biopsy techniques for embryo aneuploidy testing.

ClinicalTrials.gov. ID NCT03520933