Ability to detect aneuploidy from cell free DNA collected from media is dependent on the stage of development of the embryo

Abstract

The aim of this study was to assess the ability of cell free DNA isolated from spent culture media to detect blastocyst aneuploidy, using media collected at discrete windows of development. Retrospective NGS analysis of spent culture media for embryo cultures commencing on days 3, 4 or 5 and collected after incubation for 24 to 48h. Blinded media samples (4μl) were collected from 178 embryos from 96 patients (mean age 37.0) and amplified by WGA (either PicoPLEX or DOPlify). Samples that successfully amplified were sequenced and classified as euploid or aneuploid and the sex determined. The media result was then compared to the clinical biopsy result from the same embryo. Concordance was assessed using SPSS and was stratified for the day of culture media collection. 94% of media samples successfully amplified. Starting the embryo incubation in fresh media on day 4 provided excellent embryo ploidy concordance rates of >95% and 100% sex chromosome concordance irrespective of whether the media was collected at 24 or 48h.However, when the incubation window was earlier from day 3 to day 5, the concordance rate of autosomes was only 65.4%. Lack of concordance was primarily due to aneuploidy false negatives from the culture media. Interestingly, in 58.3% of aneuploidy embryos classified from the media in this window, the same chromosome(s) was involved in the biopsy result; however where the biopsy had indicated a gain or loss, the opposite was determined from the culture media. In contrast, the incidence of mismatched aneuploidy (eg trisomy in the biopsy, monosomy in the culture media) reduced to 12.5% in the later window (incubation from d4 or d5) samples. Sexing the embryos from day 3 to day 5 media samples also showed low sensitivity for detection of males, with 21.4% of males misdiagnosed as females as determined by the lack of detection of Y chromosome specific sequences from our standard bioinformatics workflow. Our data suggests that although cell free DNA can be amplified from most media samples, the accuracy of the screening was substantially improved by delaying the start of incubation until day 4. This may increase the concentration of embryonic DNA in the media, reduce the contamination from maternal sources, presumably cumulus cells, and reduce the likelihood of detection of polar body DNA.