Progress in non-invasive embryo assessment with cell-free nucleic acids in blastocoel fluid

Abstract Study question Can cell-free nucleic acids (cfDNA) from blastocoel fluid (BF) after a trophectoderm (TE) biopsy be used as a non-invasive sample type to assess embryos? Summary answer Both BF and spent media carry comparable, highly fragmented cfDNA and cell-free RNA (cfRNA) which may reveal more about the quality of the embryo. What is known already Preimplantation genetic testing for aneuploidy (PGT-A) is commonly used by IVF clinics to screen embryos for any chromosomal abnormalities during the IVF process. Noninvasive preimplantation genetic testing (ni-PGT) is an emerging category of tools within the field of assisted reproductive technology. Currently, niPGT products have been developed for embryo aneuploidy detection using cfDNA released by the embryo into the spent media. Embryo spent media (ESM) carries cfDNA, which has the potential to be applied to evaluate the quality of the embryo. Previous studies have demonstrated that cfDNA in ESM is highly fragmented to the nucleosome level. Study design, size, duration The quality and quantity of cell free nucleic acid from forty BF-conditioned media (BFCM) samples were analyzed in this study. Embgenix™ Analysis Software was used to analyze and interpret sequencing data. For each sample, the concentration and fragmentation of cfDNA were examined and compared using generalized linear mixed models. To assess RNA quality at a picogram level, reverse-transcribed and amplified cDNA was used to represent the quality of cfRNA in BFCM. Participants/materials, setting, methods Embryos were washed and placed in individual biopsy media droplets for TE biopsy. After biopsy, blastocysts were collapsed, releasing BF into the droplet. The entire droplet was collected as BFCM. The Embgenix ESM Screen Kit was used to prepare sequencing libraries. The SMART-Seq® Single Cell Kit was used to evaluate the RNA quality. The BFCM cfDNA results were also compared to spent media profile that had already been obtained from a different cohort of embryos. Main results and the role of chance Amounts of cfDNA fragmentation detected in BFCM samples was comparable to ESM. Whole-genome amplification (WGA) yields correlated to the amount of cfDNA input but not the degree of cfDNA fragmentation. Informative sequencing reads and computed copy number variations (CNV) plot noise, as evaluated by distribution of log2 ratio spread (DLRS), also showed correlation and stable performance down to ∼2 pg of cfDNA input. We extracted detectable cfRNA from BFCM and created cDNA for RNA quality analysis. Our model demonstrates a significant correlation between cDNA length and RNA quality metrics, with a low DV200 of cfRNA from BFCM samples. The quantity and quality of cfDNA released into spent culture media by a developing embryo are comparable to the cfDNA released into BFCM upon TE biopsy, indicating that BFCM has potential as a noninvasive method for assessing the ploidy status of embryos. WGA yield, informative reads, and DLRS of BFCM samples were likewise comparable to data previously acquired using spent media samples. However, the severely fragmented cfRNA in these samples illustrate the inherent instability of RNA and the difficulty of utilizing RNA analysis to determine embryo health. Assays compatible with small RNA or microRNA (miRNA) are more appropriate for RNA-seq of BFCM. Limitations, reasons for caution Different clinics use varying methods for embryo culture and biopsy; the data reported here is from a single research center. The characteristic of samples may vary between clinics. Our assay estimates RNA quality using amplified cDNA; nonetheless, discrepancies between fragmented RNA and amplified cDNA may exist. Wider implications of the findings In PGT-A, BFCM samples are routinely discarded. Our findings imply BFCM samples contain considerable levels of cell-free nucleic acids which could also be used to screen embryo quality. BFCM samples, therefore, could be analyzed in conjunction with PGT-A to select and/or rank embryos for implantation. Trial registration number Not applicable