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Updated: Jun 9, 2026
Human Blastocyst Biopsy and Vitrification
Published on: July 26, 2019
Culture time to optimize embryo cell-free DNA analysis for frozen-thawed blastocysts undergoing noninvasive
Goli Ardestani1, Maria Banti2, Carmen M García-Pascual3
1Boston IVF - IVIRMA Global Research Alliance, Waltham, Massachusetts.
Objective:
To investigate the ideal time in culture to optimize embryo cell-free deoxyribonucleic acid (cfDNA) analysis in frozen-thawed blastocysts undergoing noninvasive preimplantation genetic testing for aneuploidy (PGT-A). Cell-free DNA is released into the spent blastocyst media (spent media) by the embryo. However, the optimal timing to determine maximal cfDNA in the case of frozen-thawed blastocysts undergoing noninvasive PGT-A remains to be elucidated.
Design:
In this prospective observational study, 135 spent media and corresponding whole blastocysts were collected from January 2021 through March 2022.
Setting:
Private fertility clinics.
Patients:
Day-5 frozen-thawed blastocysts were cultured for 8 hours (Day-5 Short) or 24 hours (Day-5 Long), whereas 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 and sequenced using next-generation sequencing.
Main Outcome Measures:
Informativity and concordance rates between cfDNA in spent media and whole blastocyst DNA were compared according to the different times in culture.
Results:
When comparing time in culture, informativity rates for spent media were significantly higher for Day-5 Long and Day-6 Short (>91%) compared with 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 on Day-5 Short compared with Day-5 Long and Day-6 Short, regardless of expansion degree. This decrease was significant for Gardner-grade expansion grades 3, 4, and 5-6. 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 3 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:
Noninvasive PGT-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.
