Abstract
About 70% of human cleavage stage embryos show chromosomal mosaicism, falling to 20% in blastocysts. Chromosomally mosaic human blastocysts can implant and lead to healthy new-borns with normal karyotypes. Studies in mouse embryos and human gastruloids have shown that aneuploid cells show proteotoxic stress, autophagy and p53 activation and that they are eliminated from the epiblast by apoptosis while being tolerated in the trophectoderm. These observations suggest a selective loss of aneuploid cells from human embryos, but the underlying mechanisms are not yet fully understood. In this study we investigated the cellular consequences of aneuploidy in a total of 125 human blastocysts. RNA-sequencing of trophectoderm cells showed transcriptional signatures of activated p53 pathway and apoptosis, which was proportionate to the level of chromosomal imbalance. Immunostaining corroborated that aneuploidy triggers proteotoxic stress, autophagy, p53-signalling, and apoptosis independent from DNA damage. Total cell numbers were lower in aneuploid embryos, due to a decline both in trophectoderm and in epiblast/primitive endoderm cell numbers. While lower cell numbers in trophectoderm may be attributed to apoptosis, it appeared that aneuploidy impaired the second lineage segregation, particularly primitive endoderm formation. This might be reinforced by retention of NANOG in aneuploid embryos. Our findings might explain why fully aneuploid embryos fail to further develop and we hypothesize that the same mechanisms lead to removal of aneuploid cells from mosaic embryos. This hypothesis needs further study as we did not analyze chromosomal mosaic embryos. Finally, we demonstrated a few differences with previous findings in the mouse, emphasizing the need for human embryo research to understand the consequences of aneuploidy.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
These are the main changes made to the initial manuscript: 1.The RNA-seq data (Figures 1+2) is now FDR corrected and been reanalyzed. This has not affected the initial observations on the activation of p53 and apoptosis in aneuploid human embryos, as well as that the transcriptomic changes are driven by gene dosage effects. 2.We have included the transcriptome analysis of reversine-treated embryos in the supplementary data. 3.For validation of novel findings such as the presence of DNA-damage and the expression of DRAM1 in aneuploid embryos, we now include the stainings of 30 human blastocysts (Figure 3o-t). We found absence of DNA-damage in aneuploid embryos and that DRAM1 is increased in the TE but not the ICM of aneuploid embryos. 4.We re-analyzed the co-expression of CASP8/HSP70 in reversine-embryos as suggested by reviewer 1 and found that both proteins tend to be co-expressed. 5.We have added a new analysis of NANOG expression (Figure 4a,b) of the embryos used in Figure 3o-t and have found retention of NANOG protein in both the TE and ICM. 6.We have added 6 euploid and 4 aneuploid embryos to Figure 4l-s, which support the conclusions on the absence of autophagy activation in the ICM and failure of PrE formation in aneuploid embryos. 7.We have significantly changed the layout of the figures, revised the supplementary tables, added source data files and rewritten the discussion.
Data availability
Data are available from the corresponding author upon reasonable request. Files are stored at the Open Science Framework (OSF) and can be accessed through the identifier: osf.io/tc248. At OSF we provided for Figure 1, 2 and Supplementary Figure 1 the input and output of the GSEA for each comparison. Also, source data files are provided for figures 3 and 4 and Supplementary Figure 2. We also provide the Prism file for all comparisons in which significancy was tested. Raw RNA-sequencing files are not provided due to GDPR guidelines concerning human embryo research. However, count tables generated from the raw data files are included in the source data files.
Acknowledgements
The authors would like to thank Marleen Carlé from the Center for Medical Genetics (UZ Brussel) for assisting during the tubing of trophectoderm samples, Hanne Vlieghe (UCLouvain, Brussels) for the validation of antibodies and the reversine reagent, Wilfried Cools from the Biostatistics and Medical Informatics Group (VUB) for the statistical advice and the members of the BRIGHTcore facility (UZ Brussel) for performing the RNA sequencing. We thank Prof. Rajiv McCoy for the scientific input and data exchange. This study was funded by the Fonds for Scientific Research in Flanders -G017218N-(Fonds Wetenschappelijk Onderzoek – Vlaanderen [FWO]). M.R., E.C.D.D. and C.J. are doctoral fellows at the FWO with grant numbers (1133622N, 1S73521N and 11H9823N, respectively). Y.L. is a predoctoral fellow supported by the China Scholarship Council (CSC)
