P-513 Non-invasive PGT (niPGT) of vitrified blastocysts cultured for 24 hours after thawing
Authors: Takeda S , Watanabe H , Kida Y , Kondo F , Kitasaka H , Fukunaga N , Asada Y 
Published: July 2024

Abstract

Study question
Is niPGT on culture medium exposed for 24 hours to vitrified-thawed blastocysts useful?

Summary answer
niPGT using a culture medium from vitrified blastocysts cultured for 24hours after thawing showed a high concordance rate with the PGTa results.

What is known already
Conventional PGTa, which is widely practiced around the world, requires specialized techniques and involves embryonic trophectoderm (TE) biopsy. Recently, niPGT using cell free DNA released from embryos into culture medium has been reported as an alternative to conventional PGT, and has been found to correlate well with TE-biopsy analysis results (Carmen. Rubio, 2020). Although there have been numerous reports of niPGT accuracy based on the timing of culture medium collection, there have been few comparisons of its accuracy based on different time points of medium collection from the same embryo.

Study design, size, duration
From March to April 2023, 128 vitrified pronuclear stage embryos from 33 patients with consent for research use, were thawed and cultured, and from Day 4, they were individually cultured for over 40 hours. 60 embryos that reached blastocyst by Day 6 were vitrified, and the spent culture medium (SCM1) was collected. The vitrified blastocysts were later thawed and cultured individually for 24 hours, and then the embryos and spent culture medium (SCM2) were collected.

Participants/materials, setting, methods
Embryos were cultured in a 30 μl droplet until Day 4 and a 10 μl droplet for individual culture. Culture medium was collected and embryos were subjected to chromosomal analysis by NGS. We compared the results of niPGT taken from the same embryos on days 4- 6 (SCM1) and after thawing of vitrified blastocysts (SCM2), with PGTa analysis of the embryos.

Main results and the role of chance
Among all media samples analyzed, results were obtained for 80.0% (48/60) SCM1 and 96.7% (58/60) SCM2 samples.
The ploidy concordance rate was 81.3% (39/48) for SCM1 and 98.3% (57/58) for SCM2.
The overall concordance rate, defined as the combined rate of the total concordance for all chromosomes and the partial concordance for some chromosomes was 72.9% (35/48) for SCM1 and 94.8% (55/58) for SCM2 respectively. Significant differences were identified in the informative sample rate, the ploidy concordance rate, and the overall concordance rate(p &lt; 0.05) between SCM1 and SCM2.
Fisher’s exact test was used for statistical analysis.

Limitations, reasons for caution
In this study, embryo culture was continued to day 6, regardless of morphology of the embryo at day 5, which differs from the usual clinical culture process. Additionally, the timing of sample collection differed between the day 4-6 culture medium sample and that for a blastocyst.

Wider implications of the findings
The results demonstrated that accuracy of ploidy status was higher in the SCM2 compared to the SCM1 sample, suggesting that analysis of thawed vitrified blastocyst medium maybe a more precise predictor and therefore a non-invasive alternative to conventional PGTa.

Trial registration number
not applicable