P-216 Whole genome amplification (WGA) of spent culture media may not be a reliable marker of blastocyst prioritization for transfer
Authors: Defort N , Zrhidri A , Guitard V , Keppi B , Kemeny S , Chiron A , Montagut M , Sanguinet P , Page E , Nogueira D 
Published: July 2024

Abstract

Study question
Does the success or failure of DNA amplification from spent culture media of good-quality blastocysts correlate with their ploidy status and clinical pregnancy outcomes?

Summary answer
Samples with successful DNA amplification exhibited similar rates of euploidy and clinical pregnancy rates compared to spent culture samples with failed DNA amplification.

What is known already
Studies have detected DNA in spent culture media/blastocoelic fluid (BF) from fully developed blastocysts. After amplification, this DNA is sequenced to provide information on their ploidy status. Some authors reported simply detection of amplified DNA as an indicator of blastocyst viability, so that spent culture media/BF with DNA amplification derive from abnormal embryos, and failed DNA amplification from euploid blastocysts. Along with this line of rationale, some authors associated DNA amplification failure with higher clinical pregnancy rates. Reproducibility of such findings is warranted which may stem from various factors, such as culture conditions, amplification methods and statuses of blastocysts analysed.

Study design, size, duration
This preliminary set of results is part of a cohort (nonselective) ongoing study, from which 62 patients participated so far from two tertiary IVF centers during the period of January 2022 until October 2023. Only ICSI patients aged &lt;43-year-old with &gt;6 mature oocytes were included. Single frozen blastocyst transfers were performed blinded to the results of the whole genome amplification (WGA) and/or DNA sequencing and pregnancy outcomes recorded.

Participants/materials, setting, methods
Embryos were singly cultured into 20µl GTL-media in Embryoscope+ incubator (Vitrolife). On day 3, embryos were washed repeated times and placed into new culture dish. Spent media was collected from ≥BL4BB blastocysts (Gardner) on days 5/6 after laser collapse prior vitrification. DNA from culture medium were amplified using MALBAC (Yikon-Genomics). After WGA, DNA was quantified using Qubit 3.0 fluorometer (cut-off of 0.6 ng/µl) and then subjected to next-generation sequencing (NGS) with Illumina MiSeq platform.

Main results and the role of chance
A total of 104 spent culture media samples were analysed from individual blastocysts. About 70% of samples of spent media had amplified DNA and were sequenced further. From those sequenced samples, 55% resulted in euploidy and 45% in abnormal ploidy status.
Embryo transfers took place from blastocysts cultured in 82/104 samples. A total of 36 (44%) clinical pregnancies were obtained from 82 transfers/samples from which, 78% derived from samples with DNA amplification and 22% from samples with failed DNA amplification.
Outcomes of clinical pregnancies were verified for both conditions, samples with DNA amplification or failed DNA amplification. A total of 28/55 (51%) samples with DNA amplification resulted in clinical pregnancies, while only 30% (8/27) of failed DNA amplification samples did so (P = 0.06). Positive predictive values (PPV) and negative predictive values (NPV) of ploidy status of sequenced samples in relation to ongoing pregnancy and live birth rates are ongoing.

Limitations, reasons for caution
The limited sample size.

Wider implications of the findings
The presence of DNA in the spent culture media may offer an attractive alternative as a non-invasive test for aneuploidy. Research is still ongoing and challenging. Nevertheless, truncating the test at DNA amplification phase seems not a reproducible methodology to serve as a criterion to prioritize embryos for transfer.

Trial registration number
This research project is financially supported by Theramex Birth Grant program, a competitive grant supporting IVF research in Europe.