Validation of next-generation sequencing for comprehensive chromosome screening of embryos

Abstract

Massively parallel genome sequencing, also known as next-generation sequencing (NGS), is the latest approach for preimplantation genetic diagnosis. The purpose of this study was to determine whether NGS can accurately detect aneuploidy in human embryos. Low coverage genome sequencing was applied to trophectoderm biopsies of embryos at the blastocyst stage of development. Sensitivity and specificity of NGS was determined by comparison of results with a previously validated platform, array-comparative genomic hybridization (aCGH). In total, 156 samples (116 were blindly assessed) were tested: 40 samples were re-biopsies of blastocysts where the original biopsy specimen was previously tested for aCGH; four samples were re-biopsies of single blastomeres from embryos previously biopsied at the cleavage stage and tested using aCGH; 18 samples were single cells derived from well-characterized cell lines; 94 samples were whole-genome amplification products from embryo biopsies taken from previous preimplantation genetic screening cycles analysed using aCGH. Per embryo, NGS sensitivity was 100% (no false negatives), and 100% specificity (no false positives). Per chromosome, NGS concordance was 99.20%. With more improvement, NGS will allow the simultaneous diagnosis of single gene disorders and aneuploidy, and may have the potential to provide more detailed insight into other aspects of embryo viability.

Evidence for feasibility of fetal trophoblastic cell‐based noninvasive prenatal testing

“…While optimization of WGA methods could improve our copy number resolution, this 2.7 Mb deletion is well below the limits of resolution for currently available plasma‐based NIPT. The experience with various forms of pre‐implantation genetic screening on single cells or small pools of cells suggests the technical feasibility of such an approach . There is evidence that single nuclei can be analyzed in batches of 48 or 96 using NGS with copy number detection at a resolution of 54 kb in 5–6 days, but this resolution has not been shown with single cells subjected to fixation and permeabilization.…”

Comment on: Gleicher N et al., 2016. Reprod biol endocrinol Sep 5;14(1)

“…NGS-diagnosed euploid trophectoderm samples have been reanalyzed in at least 124 embryos showing 100% euploidy concordance [2–4]. Gleicher, et al excluded our 280 normal deliveries from the denominator in their extrapolation of our data secondary to the possibility that these infants were derived from mosaic embryos [1].…”

Euploidy Rates in Donor Egg Cycles Significantly Differ Between Fertility Centers

“…However, embryos with a diagnosis of degraded DNA were excluded from the analysis based on the possibility that poor technique could lead to this outcome. Array CGH has been extensively validated in single-cell biopsies as well as multi-cell blastocyst biopsies and in both instances, the misdiagnosis rate was below 2% (Colls et al, 2012;Gutiérrez-Mateo et al, 2011;Capalbo et al, 2013;Kung et al, 2015).…”