Culture time to optimize embryo cell-free DNA (cfDNA) analysis for frozen-thawed blastocysts undergoing non-invasive preimplantation genetic testing for aneuploidy (niPGT-A)

Objective Cell-free DNA (cfDNA) is released into the spent blastocyst media (spent media) by the embryo. However, optimal timing to determine maximal cfDNA in the case of frozen-thawed blastocysts undergoing non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) remains to be elucidated. In the current study we investigated the ideal time in culture to optimize embryo cell-free DNA (cfDNA) analysis in frozen-thawed blastocysts undergoing niPGT-A. Design In this prospective observational study, 135 spent media and corresponding whole blastocysts were collected from January 2021 through March 2022. Subjects Day-5 frozen-thawed blastocysts were cultured for 8 hours (Day-5 Short) or 24 hours (Day-5 Long), while day-6 frozen-thawed blastocysts were cultured for 8 hours (Day-6 Short). The spent media and whole blastocysts were then collected for further analysis. Spent media and whole blastocysts were amplified using whole genome amplification (WGA) and sequenced using Next Generation Sequencing (NGS). Main outcome measures Informativity and concordance rates between cfDNA in spent media and whole blastocysts DNA were compared according to the different time in culture. Results When comparing time in culture, informativity rates for spent media were significantly higher (p<0.0001) for Day-5 Long and Day-6 Short (>91%) compared to the Day-5 Short group (<60%). A similar trend was observed for cases with and without a previous PGT-A. Regarding blastocyst expansion grade, informativity rates were lower in Day-5 Short, compared to Day-5 Long and Day-6 Short, regardless of expansion degree. This decrease was significant for Gardner grade expansion grade 3 (p=0.0005), 4 (p=0.0366) and 5-6 (p=0.0002). In addition, for a similar time in culture, the grade of expansion did not have an impact on the informativity rates.For concordance rates, no significant differences were observed among the three groups. In all cases concordance rates were 90.5% for Day-5 Short, 93.6% for Day-5 Long and 92.3% for Day-6 Short. No impact of the expansion grade was observed on concordance rates. Conclusion niPGT-A in frozen-thawed blastocysts yields very high concordance rates with whole blastocysts, possibly limiting the need for invasive PGT-A and making it available for a wider range of patients.