Understanding the scope and significance of mosaicism in human preimplantation embryos

Mitotic errors that occur during post-zygotic cell division lead to mosaicism in human embryos. Research suggests a higher incidence of mosaicism in day 3 embryos, suggesting that embryos with complex mitotic-derived aneuploidy are more likely to arrest1. Mosaicism may have been previously overestimated in cleavage stage embryos due to technical limitations (i.e. FISH on single blastomeres). Few studies have evaluated mosaicism throughout embryo development, using NGS-which allows for high resolution copy number variant (CNV) analysis. The purpose of the study was to characterize the degree of mosaicism according to embryo developmental stage, day of development, viability, and morphology. Prospective cohort study Patients who donated fresh embryos, during IVF, from January-June 2016. Embryos were collected from day 3 to 7 and biopsied, with ∼4 cells removed for aneuploidy screening by NGS using the ReproSeq assay. Samples with <100,000 reads and with Median of the Absolute values of all Pairwise Differences (MAPD) >0.3 were excluded. Mosaicism was identified based on intermediate position CNV (between disomy and aneuploidy, 1.2-1.8 or 2.2-2.8). Student's t-test, chi-square and linear regression was used for analysis. Of the 78 embryos (41 blastocyst/9 morulae/29 cleavage), 63 were aneuploid, with 92% displaying varying degrees of mosaicism. The number of mosaic calls did not vary according to embryo stage (p=0.6). Mosaicism was most often absent in blastocysts (26.8% vs. 5.2%, p=0.03). Mosaic calls were not modified by developmental arrested (β =-1.6, p=0.2), number of cells on day 3 (β =0.4, p=0.2), or ICM grade (β =-0.3 p=0.6). Controlling for day of development, mosaicism was increased in blastocysts with poor expansion (β =7.3, p=0.001) and low trophectoderm grade (β =-7.9, p=0.001). Given the consistent level of mosaicism detected from the cleavage to blastocyst stage and the lack of association with developmental arrest, our findings refute the hypothesis that complex mitotic errors lead to negative selection. This is the first report of an association between increasing degree of mosaicism and poor expansion and trophectoderm grade. This finding supports the theory of progressive clonal depletion as a compensatory mechanism against postzygotic mitotic errors, which could translate into limited proliferation and increased apoptosis within the trophectoderm. Future research is required to: 1) overcome technical and biological limitations to the accurate assessment of mosaicism, 2) understand the diverse molecular mechanisms contributing to mitotic error, and 3) define the impact of mitotic errors in preimplantation embryogenesis.