P–527 Embryonic cell-free DNA (cfDNA) in spent culture medium for aneuploidy screening and its concordance with trophoectoderm biopsy in PGT-A cycles

Abstract

Abstract Study question Is embryonic cfDNA detectable in blastocyst spent culture medium; can its analysis via Next Generation Sequencing compare with trophoctoderm biopsy results of the respective embryos? Summary answer Embryonic cfDNA is detected in blastocyst spent culture medium (BCM) and gives comparable aneuploidy rates with trophoectoderm biopsy in PGT-A cycles. What is known already Currently, PGT- A involves the use of invasive techniques to obtain embryonic DNA, with significant technical and ethical limitations. Recently, spent culture medium (SCM) has been proposed as an alternative source of embryonic DNA. Studies have reported the detection of cfDNA in SCM and highlighted the diagnostic potential of non invasive PGT (niPGT) for assessing the genetic status of preimplantation embryos. Moreover, invasive PGT-A can lead to genetic misdiagnosis in case of mosaic embryos, while niPGT-A may be more represantative of the whole embryonic chromosome status. However, the reliability of this approach for clinical applications needs to be further determined. Study design, size, duration These are preliminary data from an observational study conducted in the period 2019–2020. 40 embryos from 13 patients, undergoing PGT-A, were analyzed. Trophoectoderm biopsies (TEB) and respective SCMs from individually cultured embryos were analyzed by Next Generation Sequencing NGS. The results from trophoectoderm biopsies and SCMs of the respective embryos were compared. Participants/materials, setting, methods The embryos were cultured individually in 10μl drops, from day 3 to the blastocyst stage (day5/6). On day 5/6, TEB was performed and the corresponding BCM was collected and stored at –80 °C, until analysed with NGS. Data were analysed with McNemar’s test and ROC analysis. The results were considered significant when P < 0.05. 95% Confidence intervals (95%CI) were calculated. Main results and the role of chance The amplification rate, for embryonic cfDNA from BCM samples collected from embryos cultured for 48–72 hours after day 3, was 100%. DNA concentration in each sample after whole genome amplification (WGA) ranged between 2500–30000 ng/ml for TEB and 2000–20400 ng/ml for BCM. Respective blank medium negative controls associated with each sample that underwent WGA showed no amplification in all cases. The trophoectoderm biopsy showed aneuploidy at a percentage of 61% (95% CI: 43–78%), vs. BCM aneuploidy at a percentage of 55% (95% CI: 37–72%). The overall agreement BCM vs. TE biopsy, from samples taken from the same embryo, was 27/33, 81.8% (95% CI: 68–96%). McNemar test: p = 0.687, non-significant. The aneuploidy agreement was 88.9% (sensitivity) and the euploidy agreement was 73.3% (specificity). In ROC analysis, AUC was 82.3% (95% CI 66.9–97.8). In 4 BCM samples detected euploidy, while TE biopsy showed embryo monosomes, possibly due to mosaicism. 7 samples were excluded due to low quality cfDNA. Of the 33 samples, 7 were male (XY), according to both TE biopsy and BCM analysis, a fact that confirms the safety of the method, as it shows no contamination by maternal DNA. Limitations, reasons for caution The study is limited by the small sample size. To become the niPGT reliable, several steps must be optimized: DNA collection methods, DNA amplification and downstream techniques for analysis. Also, the analysis of discarded whole blastocysts as a gold standard control may determine the method’s reliability. Wider implications of the findings: Non-invasive Preimplantation Genetic Testing (niPGT), is a promishing alternative that may give an accurate and reliable option of detecting euploid human embryos, also dealing with the problem of mosaicism in trophodectoderm biopsies. Further technical refinement is needed to perfect niPGT, so that it can be used in routine clinical practice. Trial registration number N/A