Evaluation of Blastocelic Fluid, Trophectoderm and Inner Cell Mass for Chromosome Analysis Using Next-Generation Sequencing

The utility of BF as a source of DNA for non-invasive PGS has been previously studied. It has been reported that BF ploidy analyzed by array-comparative genomic hybridization (aCGH) corresponds with the ploidy measured in TE and the whole embryo. However, there is little positive evidence indicating that BF is useful for PGS. We evaluate blastocoelic fluid, trophectoderm and inner cell mass for chromosome analysis using next-generation sequencing. Twenty, vitrified-warmed blastocysts donated for research (donor age: 26-42 years) were used for the present study. BF, TE and ICM were biopsied from each blastocyst and DNA analyzed by Next Generation Sequencing (NGS). The results from BF samples were compared with the corresponding TE and ICM samples. Warmed day 5/6 blastocysts were cultured and a BF biopsy collected when blastocysts had expanded to more than 160μm in diameter. After BF biopsy and blastocysts re-expansion, ICM was separated from TE by laser pulses. NGS was used to determine concordance rates for whole chromosome copy number between these three samples. The amplification rate of BF samples after whole genome amplification (WGA) was 60% (12/20). Fifteen of 20 (75.0%) of the ICM samples were 100% concordant and two samples were partially concordant with (cases where the ploidy condition was confirmed, but not all single chromosomes corresponded) TE samples from the same embryos. Three blastocysts were discordant between ICM and TE. The full concordance rate between BF and ICM was 25.0% (3/12) and between BF and TE was 8.3% (1/12). The partial concordance rate between BF and ICM was 16.7% (2/12) and between BF and TE was 25% (3/12). The discordance rates were 58.3% (7/12) and 66.7% (8/12) respectively. Five out of 6 BF samples (83%) from blastocysts were aneuploid or mosaic whereas corresponding ICM and TE were characterised as euploid. The chromosomal status of BF samples shows low concordance with ICM and TE samples from the same blastocysts. BF is not a valid source of DNA for chromosomal testing.