JBRA Assist. Reprod. 2025;29(Suppl 1):68-68
POSTER PRESENTATION
doi: 10.5935/1518-0557.20250120
1Amplexa Genetics, Odense, Denmark
2Department of Fertility, Herlev and Gentofte Hospital, Copenhagen University Hospital, Herlev, Denmark
3IVF-Syd, Fredericia, Denmark
Objective: Preimplantation genetic tests using biopsies from the trophectoderm (TE) have long been the industry standard, and while they have increased successful pregnancy rates, they present clear limitations. Cell-specific bias during biopsy can result in false negatives or false positives, combined with the potential risk of harming the embryo and inaccurate representation due to the TE’s differentiation into the placenta. These factors highlight the need for a safer and more accurate technique. Non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) using cell-free DNA (cfDNA) from spent culture medium (SCM) is a promising alternative to traditional TE biopsy-based methods but lacks robust data to support its superiority. This study aimed to assess the concordance between cfDNA profiles and those from whole blastocysts, TE biopsies, and the inner cell mass (ICM) in day 6 blastocysts previously diagnosed with aneuploidies via day 5 TE biopsy. We evaluated whether cfDNA offers a more comprehensive representation of the embryo’s genomic status and examined the impact of embryonic mosaicism on genetic screening accuracy.
Methods: Previously discarded day 6 blastocysts diagnosed with aneuploidy from day 5 trophectoderm biopsy-based PGT-A were divided into four sample types:
1. Whole blastocyst lysate
2. Trophectoderm biopsy
3. Inner cell mass
4. Spent culture medium containing cell-free DNA
DNA was extracted and amplified using a random-primer binding whole genome amplification setup, and subsequently indexed, followed by next-generation sequencing (NGS). Chromosome copy number variation (CCNV) analysis was conducted across all samples using a suitable software, comparing aneuploidy and mosaicism patterns. Concordance was evaluated based on chromosomal aberration overlap between cfDNA and the other sample types, with the inner cell mass considered the closest proxy for the embryonic genome.
Results: cfDNA obtained from spent culture medium showed higher concordance with both whole blastocyst and inner cell mass chromosomal profiles than trophectoderm biopsies, indicating that cfDNA offers a more comprehensive and representative view of the embryo’s genomic landscape. This likely reflects its origin in apoptotic and necrotic cellular turnover, capturing genetic material shed from both the trophectoderm and inner cell mass. In contrast, trophectoderm biopsies demonstrated greater variability in concordance, often influenced by sampling bias and localized mosaicism that can lead to misinterpretation of the embryo’s actual chromosomal status. In several instances, trophectoderm biopsy results indicated aneuploidy, while both the cfDNA and inner cell mass profiles revealed a euploid genome, highlighting the limitations of relying solely on trophectoderm-based testing. Mosaicism was identified in all sample types, emphasizing the inherent complexity of embryonic chromosomal assessment. In certain cases, cfDNA exhibited intermediate chromosomal copy number values, consistent with mosaicism either confined to the trophectoderm or shared between the trophectoderm and inner cell mass, reinforcing the need for cautious interpretation of results from both invasive and non-invasive methods.
Conclusion: This study provides strong evidence that cell-free DNA from spent culture medium offers a more representative and accurate genomic profile of the embryo compared to conventional trophectoderm biopsy. With higher concordance to both the inner cell mass and whole blastocyst, cfDNA-based non-invasive PGT-A presents a promising approach that preserves embryonic integrity while capturing a broader genomic overview. While mosaicism remains a significant interpretive challenge in both invasive and non-invasive methods-including with the introduction of AI tools for interpretation-the ability of cfDNA to reflect multiple embryonic compartments may help reduce sampling bias and improve diagnostic reliability. These findings support the growing role of non-invasive PGT-A in embryo selection, offering a safer and potentially more informative screening method for clinical use in IVF.