O-225 May embryonic cfDNA in the culture media play a role as a non-invasive assessment of aneuploidy in PGT
Authors: Rubio LLuesa C 
Published: June 2023

Abstract
Non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) in spent blastocyst media (SBM) is a promising technique that has the potential to revolutionize current preimplantation genetic testing procedures. This technique is based in the analysis of the cell-free DNA (cfDNA) released by the embryo during the latest stages of preimplantation development. It allows for the detection of chromosomal abnormalities in embryos without the need for invasive procedures that can harm the embryo (reviewed in Navarro-Sánchez et al., 2022).
Our group among others has conducted different studies in this field, and the early clinical application of the cfDNA analysis in the culture medium has been proposed as a screening tool, giving a priority score for embryo transfer based on the media results (Rubio et al., 2019, 2020). In the final analysis of our multicenter study in day-6 and day-7 blastocysts, we have been able to compare the chromosomal results of the SBM with trophectoderm (TE) biopsies in 2,187 samples, and with the inner cell mass ICM in 230 blastocysts. In this study, the analysis of the cfDNA showed a concordance rate of 87% with the ICM biopsy, which is currently considered the gold standard for PGT-A. However, it is important to continue evaluating the factors that may affect the informativity and concordance rates of cfDNA-based assays, such as the culture day when medium is collected, contamination with external and/or cumulus cell DNA, and previous manipulation of the embryos. Multivariate analysis has shown that the longer the time in culture, the higher the informativity rate, and female age had the greatest impact on concordance rates with TE biopsies. Regarding technical aspects, the SBMs with higher number of reads after sequencing resulted in higher concordance rates, reflecting the importance of analysing samples with sufficient DNA quantity. Finally, the predictive value in terms of concordance with TE biopsies was higher for aneuploid blastocyst. No significant independent correlation was observed for embryo quality.
The improvements in IVF lab protocols and the use of time-lapse and short incubation protocols for vitrified-thawed blastocysts are also noteworthy, as they may further increase the concordance rates of cfDNA-based assays with blastocysts. In fact, concordance in previously vitrified-thawed blastocysts as result as high as 90%.
Regarding clinical outcomes, several groups have evaluated non-invasive PGT-A with transfer of euploid embryos showing good results. In a pilot study with only 7 SETs in couples with PGT-A or PGT-SR indication, 5 pregnancies were achieved resulting in 5 livebirths (Xu et al., 2016). In a second study, in couples with recurrent spontaneous abortion or repeated implantation failure, 50 transfers of euploid media were performed resulting in a 58% clinical pregnancy rate, with 27 healthy babies born (Fang et al., 2019). Similar results were described in good prognosis patients &lt;38 years of age with ongoing pregnancy rates comparable to PGT-A (61.5%), and higher than conventional IVF or ICSI (48.5%) (Franco et al., 2021).
Overall, the high concordance rates with trophectoderm biopsies and inner cell mass biopsies are encouraging, and the clinical outcomes with transfer of euploid embryos are promising. It is exciting to see the progress in non-invasive preimplantation genetic testing using cfDNA in culture medium, and it will be interesting to see how this technology evolves and its potential role as a screening or diagnostic tool in the future.