P-563 The effect of embryo culture time on concordance rates between invasive and non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) in spent culture media (SCM) analysis
Authors: Biricik A , Bianchi V , Lecciso F , Surdo M , Bavaro I , Manno M , Saino V , Timperi P , Cotroneo E , Gigante L , Diano L , Spinella F 
Published: June 2022

Abstract

Study question
To evaluate different embryo culture times (day5 versus day6) to understand the ideal time-frame for the performance of concordance analysis between invasive and non-invasive PGT-A(niPGT-A)

Summary answer
Different levels of ploidy concordance rates were observed between day5 and day6 groups: 72,6% versus 84,8%, respectively

What is known already
The recent data supports that a non-invasive approach for evaluating embryo ploidy status may be an alternative to standard invasive methods. Embryo cell-free DNA(cfDNA) released into culture media during in-vitro embryo development represents the potential source for this analysis. The release of cfDNA from embryos is expected to be directly proportional to embryo culture time and late-stage released cfDNA may be more representative of the embryo. Therefore, it is important to estimate the most effective time frame of the culture that will provide the most conclusive data from spent-culture-media(SCM) without adversely affecting the development of the embryo

Study design, size, duration
A total of 334 SCM from blastocyst stage embryos have been included in this study. The SCM samples were divided in 2 subgroups according to the embryo culture time as day5 (n = 154) and day6 (n = 180). The cfDNA of SCM samples were amplified by whole genome amplification(WGA) and analyzed by next generation sequencing(NGS) in parallel to day5 or day6 trophectodermal(TE) biopsied samples of their corresponding embryos. Ploidy status and concordance were compared between two groups

Participants/materials, setting, methods
Day3 embryos were washed and transferred in 20µl fresh culture media until the biopsy. SCM samples were collected in PCR tubes and stored at -20 °C while embryos were biopsied for standard PGT-A analysis. Both SCM and corresponding TE biopsy samples were amplified by Sureplex(Vitrolife). Then TE and SCM samples were analyzed by next-generation sequencing(NGS) using MiSeq® System (Illumina). Data analysis has been done by Bluefuse Multi Software 4.5(Illumina) for all SCM and TE samples

Main results and the role of chance
A successful DNA amplification rate (&gt;30ng/µl) was obtained in both day5 and day6 groups with 95,5% (147/154) and 99,4% (179/180) respectively. The DNA concentration after WGA was ranging between 30.1-122.5ng/µl and 38.5-123.2ng/µl in day5 and day6 groups respectively. Not conclusive (NC) results including a noisy NGS profile and chaotic chromosome aneuploidies (&gt;5 chromosomes) were excluded in both groups. Therefore, 64,6% (95/147) of SCM samples in day5 group and 81,0% (145/179) of SCM samples in day6 group were conclusive for NGS analysis. The ploidy concordance rate between SCM and TE samples (euploid vs euploid, aneuploid vs aneuploid) was 72,6% (69/95) in day5 and 84,8% (123/145) in day6 group. In day5 group, the false-negative rate was 10.5% (10/95), and false-positive rate was 16.8% (16/95) while in day6 group the false-negative rate was 6,8% (10/145), and false-positive rate was 8,2% (12/145). Sensitivity and speciﬁcity were calculated as 83,6% and 52,9% in day5 group, and 90,9% and 65,7 in day6 group respectively

Limitations, reasons for caution
We have high number of samples in our study, but larger prospective studies may change the significance of ploidy concordance. One of the important issues in SCM analysis is the maternal DNA contamination risk which cannot be revealed always. Therefore the use of molecular markers would increase the reliability

Wider implications of the findings
A non-invasive approach for aneuploidy screening may be an alternative to standard PGT-A procedure with invasive embryo biopsy but the evaluation of ideal culture time is crucial for the performance of niPGT-A method. Our study demonstrates that the late collection time of SCM provides significantly better ploidy concordance rates

Trial registration number
Not applicable