Fragmentomic features of cell-free DNA in human spent blastocyst medium and their potential for non-invasive embryo ploidy assessment.

PURPOSE: To characterize the fragmentomic features of cell-free DNA (cfDNA) in spent blastocyst medium (SBM) and their association with embryonic chromosomal status. METHODS: One hundred fifty SBM samples were obtained from 43 preimplantation genetic testing for aneuploidy (PGT-A) cycles in 39 patients. Fragmentomic features of SBM cfDNA, including fragment size, fragment end, end-motif, and nucleosome occupancy, were compared between euploid and aneuploid embryos. RESULTS: SBM cfDNA exhibited characteristic nucleosomal and 10-bp periodicity in its fragment-size profile. Fragment-end analysis revealed a pronounced G-end preference, and end-motif deconvolution suggested an oxidative stress-associated cleavage pattern. Coverage analysis around transcription start sites (TSSs) showed that SBM cfDNA promoter coverage was associated with gene expression, with higher coverage at TSS regions of lowly expressed genes than at those of highly expressed genes. TSS coverage profiles also differed between euploid and aneuploid embryos, with differentially covered genes enriched in biological processes related to embryonic development and cytokinesis. An exploratory TSS coverage-based prediction model showed good discrimination, reasonable calibration and a higher estimated net benefit in this cohort. CONCLUSIONS: This study advances our understanding of the origin and fragmentation patterns of SBM cfDNA. The distinct promoter coverage profiles in SBM cfDNA from embryos with different chromosomal statuses support the development of a fragmentomics-based approach for non-invasive embryo assessment. Further studies in larger independent cohorts are needed to confirm the robustness and generalizability of these findings.