Improved concordance rates for aneuploidy detection in spent culture media compared to trophectoderm biopsies: a step forward towards non-invasive preimplantation genetic testing (niPGT-A)

Study question: Is the embryonic cell-free DNA in the spent culture media representative of the chromosomal constitution of the blastocysts? Summary answer: Increased knowledge on the dynamics of embryonic cellfree DNA has improved aneuploidy detection in blastocysts by the analysis of spent culture media. What is known already: Current techniques used for preimplantation genetic testing of aneuploidies (PGT-A) enable the analysis of the full chromosome content of a single cell with high sensitivity and specificity. Recent studies have reported the existence of embryonic cell-free DNA, opening a new era of possibilities for niPGT-A. However, the percentages of informative samples vary widely. These discrepancies, could reflect the existence of mosaicism and presence of DNA from granulosa cells or polar bodies in the spent culture media. Thus, efforts should focus on identifying embryonic cell-free DNA and rule out maternal DNA contamination to translate this non-invasive technology into clinical use. Study design, size, duration: This is a pilot prospective study that includes analysis of 64 samples of spent culture media from embryos included in a PGTA program. These samples correspond to 22 patients with ages ranging from 35-45 years (39.1 ± 2.5), with advanced maternal age, recurrent miscarriage, or repetitive implantation failure as PGT-A indications. Collection and analysis of samples were carried out from November 2017 to January 2018. Analysis of both types of samples from each embryo was performed blindly. Participants/materials, setting, methods: In the PGT-A cycle, chromosome diagnosis from each blastocyst stage embryo was obtained from the trophectoderm biopsy and the spent culture media. Both, spent culture media and trophectoderm biopsies have been analysed by Next Generation Sequencing (NGS). NGS was performed using the Ion ReproSeq PGS Kit (ThermoFisher Scientific) using Ion ChefTM plus the Ion S5TM XL SequencerTM. For the analysis of the spent culture media, the standard protocol has been modified to improve amplification efficiency. Main results and the role of chance: Informative results were obtained from all 64 trophectoderm biopsies and from 60 spent culture media (93.7% successful amplification in spent culture media). Concordance rate between trophectoderm biopsies and spent culture media in terms of blastocyst diagnosis as aneuploid or euploid was 75.0% (45/60). Concordance rate for autosomes was 80.0% (48/60). Considering the 15 discrepancies, 3 of them corresponded to euploid diagnosis in both types of samples, but with discordant gender (5% gender mismatch); 11 discrepancies were classified as false positives, with the spent culture media diagnosed as aneuploid and the trophectoderm biopsy as euploid (18.3% false positive rate); and only 2 discrepancies were false negatives, with spent culture media diagnosed as euploid and trophectoderm biopsy as aneuploid (3.3% false negative rate). In 4 of the false positive samples, the spent culture media was diagnosed as chaotic (40% of the false positives), with multiple chromosomes altered in a pattern reflecting low input of DNA or poor DNA quality. In the 2 false negative samples, the result in the trophectoderm biopsy was a single uniform monosomy in one case and, a single low-degree mosaic monosomy in the other one, raising the question of which sample better represents full blastocyst chromosome content. Limitations, reasons for caution: This is a study with limited number of samples. There is a need to fully discard maternal DNA traces in spent culture media, to understand the impact of mosaicism in the accuracy of the diagnosis in PGT-A and niPGT-A and, to unravel why some embryos release more DNA than others. Wider implications of the findings: In this study, the improvements in methodology and concordance rates prompt us to foresee the possibility of including niPGT-A in the clinical practice. The niPGT-A approach would help patient’s access to aneuploidy testing by avoiding the need of invasive biopsy techniques and by reducing the cost of the procedure. Trial registration number: Not applicable.