Next generation sequencing-based aneuploidy screening improves detection of low-level mosaicism in human embryos

Aneuploidy screening of embryos at blastocyst stage can be jeopardized by the presence of chromosomal mosaicism, which is a phenomenon characterized by the presence a mixture of diploid and aneuploid cell lines in embryos. Although its significance for implantation and the developmental potential of embryos is still unclear, it is reasonable to assume that mosaicism is likely to influence IVF success rates. In this study we investigated whether next generation sequencing (NGS) technologies applied for preimplatation genetic screening (PGS) purposes have the potential to improve mosaicism detection in human embryos over conventional methods. This study was organized into three steps. The first involved mixing experiments with different ratios of euploid and aneuploid single cells, to mimic chromosomal mosaicims. The aim was to determine the minimum ratio of aneuploid to euploid cells that is needed to detect a copy number variation (CNV) by NGS. The second step was a retrospective blinded assessment of whole genome amplification (WGA) products, selected from previously performed clinical PGS cycles, including embryos with mosaic aneuploidy results. The third consisted in a prospective trial involving a blind parallel evaluation, with both NGS and array comparative genomic hybridization (aCGH) techniques. Reconstitution experiments were performed using different ratio of euploid and aneuploid single cells and WGA products. A total of 192 blastocysts were assayed by the NGS-based PGS protocol in the prospective study and 52 blastocysts in the retrospective evaluation. Sequencing was performed using MiSeq instrument. CNV analysis was accomplished by BlueFuse software. Reconstitution experiments results demonstrated that chromosomal mosaicism can be consistently identified by NGS, with the lower representation of detectable aneuploid cells being at 16% level. In addition, NGS was able to detect 86/86(100%) embryos with mosaicism, accounting for a total of 225 chromosomes involved by mosaicism. Paired comparison between NGS and aCGH showed 100% concordant results. This study demonstrates that NGS technique has a substantially increased sensitivity for detection low level mosaicism, three times higher compared to the resolution limits of the conventional methods, usually ranging from 35 to 50%. As a consequence, NGS may represent a more robust method for aneuploidy screening of embryos at blastocyst stage.