Evaluation of cell-free dna (cfdna) in embryo spent media (esm) and its potential for non-invasive embryo assessment.

The aim of this study was to screen in vitro-fertilized embryos for aneuploidies via analysis of cfDNA in ESM samples using the Embgenix™ ESM Screen Kit to assess factors which could impact the concordance of this approach with preimplantation genetic testing for aneuploidy (PGT-A) from trophectoderm (TE) biopsy. Three sets of ESM samples were analyzed from a total of 95 embryos from two clinical sites. Embryos were cultured until Day 4, when each embryo was washed and transferred to a fresh media droplet and assisted hatching was performed on a subset of embryos. Embryos developed into mature blastocysts between Days 5–7. Prior to TE biopsy, ESM samples were collected and stored at –20°C or –80°C. Care was taken to collect the entire media droplet for each sample with minimal oil carryover, but for a subset of 12 embryos each sample was collected into two tubes so that the first tube contained ∼50% of the sample with minimal oil while the second tube contained the remainder with some oil carryover. cfDNA concentration and fragmentation were assessed for each sample prior to whole-genome amplification and library construction. Libraries were sequenced on an Illumina® MiSeq® and data was analyzed using Embgenix Analysis Software; results that failed quality control were excluded from further analysis. Corresponding ESM and TE biopsy-based PGT-A results were compared to assess the concordance of aneuploidy and sex determination across clinical sites, culture conditions, and collection protocols. ESM calls were categorized as euploid, mosaic (low/high), or aneuploid, while PGT-A results were reported as euploid, mosaic, or aneuploid. Clinical concordances for the three sample sets were 75%, 48%, and 60%, respectively, when mosaic and aneuploid ESM calls were categorized accordingly, and 75%, 57%, and 80%, when mosaic ESM calls were categorized as aneuploid. Sex concordances for the sample sets were 94%, 85%, and 80%, respectively. All 11 sex discordances involved samples called as male by PGT-A and female by ESM analysis, pointing to possible effects of maternal DNA contamination on the ESM results. Consistent with this possibility, 76% of the 42 ESM samples that yielded euploid calls were called female as compared to an frequency of 49% female calls by PGT-A. “Euploid female” ESM calls also accounted for 75% of discordant results involving non-mosaic calls. No differences in concordance were observed for assisted versus non-assisted hatching, and oil carryover did not affect data noisiness or concordance. Differences in concordance between ESM and PGT-A results were observed across sample sets but could not be attributed to factors such as oil carryover or assisted hatching. Additional studies are in progress to further elucidate these differences, but overrepresentation of female calls in the ESM results identifies maternal contamination as a likely driver of discordance and a focal point for refinement of ESM-based approaches.