P-596 Preimplantation genetic testing without invasive embryo biopsy

Abstract

Study question Can non-invasive preimplantation genetic testing (niPGT) using spent culture medium (SCM) accurately detect monogenic disorders and the meiotic/mitotic origin of aneuploidies? Summary answer Our approach enables concurrent genome-wide haplotyping and copy number profiling, enabling the detection of monogenic disorders and aneuploidies with the ability to distinguish meiotic/mitotic aberrations. What is known already Preimplantation genetic testing (PGT) is typically performed through invasive embryo biopsy to detect aneuploidies and monogenic disorders. SCM contains cell-free DNA, offering a non-invasive alternative for embryo testing. However, existing methods mainly focus on aneuploidy detection and fail to determine the meiotic or mitotic origin of chromosomal abnormalities, limiting clinical applicability. Distinguishing between these errors is crucial, as meiotic errors occur during gametogenesis and affect the entire embryo, whereas mitotic errors arise post-fertilization and lead to mosaicism. A non-invasive method capable of accurately detecting genetic disorders and aneuploidies while identifying their segregational origin would advance reproductive care. Study design, size, duration In an exploratory retrospective study, 90 SCM samples were analyzed from 53 blastocysts affected by monogenic disorders from 14 families enrolled in diagnostic PGT. SCM was obtained after embryo transfer to a biopsy dish to avoid interference. The study aimed to identify and develop the optimal experimental protocol and computational method for niPGT, respectively. The resulting haplotypes and copy number profiles were compared to invasive biopsy results. Participants/materials, setting, methods SCM samples from 53 blastocyst were split into halves, yielding 90 samples. Five whole-genome amplification protocols were tested to determine the best method for niPGT. Whole-genome sequencing was performed, followed by haplarithmisis-based haplotyping to reconstruct genome-wide haplotypes. Quality metrics, including allele drop-in, drop-out, and parental contamination, were assessed. Copy number profiles were generated, and results were compared to biopsy outcomes to evaluate diagnostic concordance, haplotype accuracy, and the ability to distinguish meiotic from mitotic aberrations. Main results and the role of chance Parallel genome-wide haplotyping and copy number profiling of cfDNA derived from SCM, enabled the identification of monogenic aberrations and aneuploidies. In 74 samples, the sex identified from SCM matched the biopsy results, while 16 showed mismatches, suggesting (maternal) contamination. High-quality embryos assessed with the optimal amplification method had a median haplotype concordance of 92.5% ([89.83-95.27], n = 15). Notably, all meiotic aberrations (n = 3) detected via biopsy were also observed in SCM, demonstrating niPGT’s ability to detect inherited chromosomal abnormalities. In contrast, mitotic aberrations were inconsistently detected, likely due to embryo mosaicism or variable DNA shedding into SCM. These results indicate that niPGT can reliably detect monogenic disorders and meiotic aneuploidies but may be discordant for mitotic errors, which is inherent to mosaicism. Limitations, reasons for caution While niPGT successfully detects meiotic aberrations and monogenic disorders, contamination remains a major challenge. Mitotic errors are inconsistently detected due to mosaicism caused by chromosomal instability. Further validation in larger cohorts is necessary to refine methods and improve reliability before widespread clinical implementation. Wider implications of the findings By eliminating the need for invasive biopsy, our method could transform reproductive medicine, offering a new paradigm for embryo selection. Future improvements in contamination control will further advance the clinical applicability of niPGT. This approach enables non-invasive genetic testing and could be extended to other areas of personalized medicine. Trial registration number No