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A single-cell transcriptome atlas of human euploid and aneuploid blastocysts

Abstract

Aneuploidy is frequently detected in early human embryos as a major cause of early pregnancy failure. However, how aneuploidy affects cellular function remains elusive. Here, we profiled the transcriptomes of 14,908 single cells from 203 human euploid and aneuploid blastocysts involving autosomal and sex chromosomes. Nearly all of the blastocysts contained four lineages. In aneuploid chromosomes, 19.5% ± 1.2% of the expressed genes showed a dosage effect, and 90 dosage-sensitive domains were identified. Aneuploidy leads to prevalent genome-wide transcriptome alterations. Common effects, including apoptosis, were identified, especially in monosomies, partially explaining the lower cell numbers in autosomal monosomies. We further identified lineage-specific effects causing unstable epiblast development in aneuploidies, which was accompanied by the downregulation of TGF-β and FGF signaling, which resulted in insufficient trophectoderm maturation. Our work provides crucial insights into the molecular basis of human aneuploid blastocysts and may shed light on the cellular interaction during blastocyst development.

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Fig. 1: Single-cell transcriptome profiling delineates the emergence of four lineages in human aneuploid blastocysts.
Fig. 2: Chromosome-specific transcriptome alterations in response to aneuploidy.
Fig. 3: Global transcriptome alterations in response to aneuploidy.
Fig. 4: Unstable development of the aneuploid EPI.
Fig. 5: TGF-β and FGF signaling is diminished in aneuploid EPI.
Fig. 6: Gene-dosage imbalances result in cellular stress signatures in aneuploid HYP.
Fig. 7: Characterization of TE in human euploid and aneuploid blastocysts.
Fig. 8: TE maturation is insufficient in aneuploid blastocysts.

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Data availability

Raw and processed data are available in the CNGB database (CNSA: https://db.cngb.org/cnsa) with accession number: CNP0000909 and GSA-Human database (https://ngdc.cncb.ac.cn/gsa-human) with accession number: HRA007085. Public datasets from other studies can be accessed in the Gene Expression Omnibus (GEO) or European Bioinformatics Institute (EMBL-EBI) repository under the following accession numbers: the scRNA-seq data of human pre- and post-implantation embryos are from GSE36552, E-MTAB-3929, GSE136447, GSE66507, and GSE130289; the scRNA-seq data of naïve and primed hESCs are from GSE177689; and the single-cell methylation data of human embryos are from GSE81233. Source data are provided with this paper.

Code availability

Scripts associated with data processing and graphic presentation are available at https://github.com/single-cell-BGI/Aneuploid_embryo, and can also be found in the Zenodo archive at https://doi.org/10.5281/zenodo.10804888 (ref. 85).

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Acknowledgements

We thank Chuanyu Liu, Xiaoyu Wei and Yanan Xing who performed testing and assisted with experiments, and Miguel A. Esteban and Lei Han for helpful comments. This project received funding from the National Key R&D Program of China (2022YFC2702904 to L.L. and F.G.), the National Natural Science Foundation of China (81974230 to G. Lin and 82001556 to L.L.), the Huxiang Young Talents project (2021RC3120 to L.L.), the Natural Science Foundation of Hunan Province (2023JJ10084 and 2021JJ41092 to L.L.).

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G. Lin and Z.S. conceived the project. L.L., Q.W. and Y.G. performed the experiments, including the collection and culture of embryos, collection of single cells and single-cell sequencing data generation with the help of Y.A., Q.D., P.X., C.C., X.C., Q.Z. and J. Lu. S.W. performed bioinformatic analyses with the assistance of J. Li. F.C., L. Liu., H.Y., J.W., X.X., Y.H., F.G., L.H. and G. Lu provided insightful advice and helpful comments on the work. L.L. and S.W. wrote the manuscript with feedback from all authors.

Corresponding authors

Correspondence to Zhouchun Shang or Ge Lin.

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Extended data

Extended Data Fig. 1 Overview of single-cell transcriptome profiling for human euploid and aneuploid blastocysts.

a, Stacked bar plot displaying the number of cells that passed quality control for each type of chromosomal aneuploidy and euploidy. b, Distribution of the number of usable fragments (left) and detected genes (middle), as well as the fraction of mitochondrial transcripts (right) in each type of chromosomal aneuploidy and euploidy. c, As in (b), but for the combination of trisomies or monosomies. d, Bar plots showing the proportion of genomic components for detected genes in each type of chromosomal aneuploidy and euploidy. e, UMAP plot showing the cell information regarding cluster, sequencing platform, gender, and karyotype.

Extended Data Fig. 2 Cell number distribution in euploidy and aneuploidies across four lineages.

Number of cells per blastocyst in each type of chromosomal aneuploidy and euploidy. Data are presented as the mean ± SEM. The number of embryos in each karyotype is shown in Supplementary Table 1. Asterisks indicate statistical significance (unpaired two-tailed Wilcoxon rank-sum test), compared to euploidy, *P < 0.05, **P < 0.01, *** P < 0.001.

Extended Data Fig. 3 CNV landscapes of human euploid and aneuploid blastocysts by the scRNA-seq dataset.

a, Heatmap showing the inferred CNVs for each individual cell from all blastocysts based on single-cell RNA-seq data. Cells were grouped by individual blastocysts and ordered by the karyotypes from PGT. b, Bar plots representing the number of blastocysts for trisomy, monosomy, mosaicism, and euploidy based on CNV inference. c, Bar plots showing the fraction of inferred karyotypes for mosaic blastocysts (upper panel) and the corresponding EPI (bottom panel). The name of the blastocyst represents the karyotype determined by PGT. d, Heatmap showing large-scale CNVs of individual cells from the representative mosaic blastocyst based on single-cell RNA-seq data. e, Bar plots showing the proportion of the four lineages for inferred aneuploid (Inferred Ane) and euploid (Inferred E) cells in mosaicisms.

Extended Data Fig. 4 Characterization of dosage effects on gene expression.

a, Boxplots showing the fraction of transcript abundance for the indicated chromosome in euploidy, the corresponding trisomy and monosomy, as well as other chromosomal trisomies and monosomies. T and M represent other chromosomal trisomies and monosomies, respectively. The dashed lines denote the 0.5-fold, 1-fold, and 1.5-fold of means for the fraction of transcript abundance of the indicated chromosome in euploidy. b, Stacked bar plot showing the fraction of genes carried on aneuploid chromosomes with different responses to aneuploidy. Not significant: Nonsignificant genes, cis-DCGs: Dosage compensation genes, cis-DEGs: Differentially expressed genes proportional to the copy number of the chromosome, cis-DRGs: Differentially expressed genes that reverted to the copy number of the chromosome. c, Bar plots showing the number of cis-DEGs in each pair of complementary trisomic and monosomic chromosome. d, Stacked bar plot showing the fraction of cis-DCGs with different correlation between DNA methylation change and DNA copy numbers. Positive: cis-DCGs with DMRs exhibit hypomethylation in monosomies and hypermethylation in trisomies, Negative: cis-DCGs with DMRs exhibit hypermethylation in monosomies and hypomethylation in trisomies, Not significant: cis-DCGs do not exhibit significant DMRs, Undetected: cis-DCGs that do not detect DMRs in basal regulatory domain. e, Genome browser track of representative regions showing the DNA methylation levels of selected cis-DCGs in euploidy and aneuploidy.

Extended Data Fig. 5 Scoring the general transcriptional alterations in trisomic and monosomic blastocysts.

a–d, Boxplots showing the score of enriched functions regarding apoptosis, stress response, amino acid metabolic process, as well as antigen processing and presentation in each type of chromosomal aneuploidy and euploidy. The dashed lines denote the mean scores for euploidy.

Extended Data Fig. 6 Lineage scores in euploid and aneuploid blastocysts.

a–d, Boxplots showing the lineage scores of single cells for corresponding lineages in each type of chromosomal aneuploidy and euploidy. The dashed lines denote the mean scores for euploidy in the corresponding lineages.

Extended Data Fig. 7 Characterization of subclusters of EPI in human euploid and aneuploid blastocysts.

a, Stacked bar plot displaying the proportion of subclusters of EPI in each type of chromosomal aneuploidy and euploidy. Only the karyotypes with more than two EPI cells are shown. b, Bar plots showing the fraction of cells in each type of chromosomal aneuploidy and euploidy for subclusters of EPI. c, The distribution of EPI along the pseudotime in each type of chromosomal aneuploidy and euploidy. Cells are colored according to the subclusters. d, Boxplots showing normalized expression levels of lineage-specific markers in subclusters (n = 34 EPI1, 55 EPI2, 440 EPI3, 31 EPI4) of EPI and euploid lineages (n = 68 HYP, 344 polar TE, 802 mural TE).

Extended Data Fig. 8 TGF-β and FGF signaling pathways are required for pluripotency progression in human early embryos.

a, Heatmap depicting the expression dynamic of ligands during human pre-implantation development. b, Heatmap showing the log(fold change) of the average expression level of the ligands in each type of chromosomal aneuploid EPI, as compared to euploid EPI. P values are based on Tukey’s tests in GLMM and adjusted by the BH algorithm (Methods). * Adjusted P value < 0.05. c, Boxplots showing normalized expression levels of the ligands in euploid, trisomic, and monosomic EPI across subclusters, EPI1 n = 5 (T)/29 (M), EPI2 n = 7 (T)/48 (M), EPI3 n = 86 (E)/206 (T)/148 (M), EPI4 n = 16 (T)/15 (M), unpaired two-tailed Wilcoxon rank-sum test was used to determine P values. d, Immunostaining of human blastocysts at 6 dpf cultured in either control medium, A8301 (TGF-βi) or PD (FGFi). Scale bars: 50 μm. Number of embryos: control (n = 3), TGF-βi (n = 5), and FGFi (n = 5). e, Fluorescence quantification of NANOG-positive cells in embryos from (d). Data are presented as the mean ± s.d. Each dot represents individual cells. Control vs. TGF-βi: 51.6 vs. 24.6, P = 9.9 × 10−17; Control vs. FGFi: 51.6 vs. 29.7, P = 7.2 × 10−13 (unpaired two-tailed Student’s t-test). Number of cells: control (n = 29), TGF-βi (n = 32), and FGFi (n = 38). f, Immunostaining of human monosomies at 8 dpf cultured in control medium (n = 2) or in ACTIVIN A and FGF4 (n = 2). Scale bars: 20 μm.

Extended Data Fig. 9 TGF-β and FGF signaling pathways are required for TE maturation in early human embryos.

a, The distribution of TE along the pseudotime in each type of chromosomal aneuploidy and euploidy. TE from each pair of complementary trisomy and monosomy are grouped, with euploidy used as a control. b, Immunostaining of human blastocysts at 6 dpf cultured in either control medium, A8301 (TGF-βi) or PD (FGFi). Scale bars: 50 μm. Number of embryos: control (n = 5), TGF-βi (n = 3), and FGFi (n = 3). c, Percentage of NR2F2-positive cells in embryos from (b). Data are presented as the mean ± s.d. Number of embryos: control (n = 5), TGF-βi (n = 3), and FGFi (n = 3). d, Immunostaining of human monosomies at 8 dpf cultured in control medium or in ACTIVIN A with FGF4. Euploidy was used as a positive control. Scale bars: 20 μm. e, Percentage of NR2F2-positive cells in embryos from (d). Data are presented as the mean ± s.d. Number of embryos: euploidy (n = 2), monosomy (n = 2), and monosomy + ACTIVIN A + FGF4 (n = 2).

Supplementary information

Reporting Summary

Supplementary Table 1

Supplementary Table 1: Information of each euploid and aneuploid blastocyst in this project and related basic information of single cells. Supplementary Table 2: The summary of cis-genes for each aneuploidy; statistical analysis of enriched bands; the number of trans-DEGs directly regulated by cis-TF; and list of frequent DEGs. Supplementary Table 3: DNA methylation levels of selected cis-DCGs related to Extended Data Fig. 4d. Supplementary Table 4: The list of putative intercellular communications between the ligands of EPI and the receptors of embryonic lineages in euploidy related to Fig. 5b. Supplementary Table 5: The list of differentially expressed ligands between aneuploid and euploid EPI related to Extended Data Fig. 8b. Supplementary Table 6: Antibodies. Details of primary and secondary antibodies used for this study, related to Figs. 1e, 3i, 4g and h, 5e and i, 6f, 7f, 8b, and Extended Data Fig. 8d and f, and 9b and d.

Source data

Source Data Fig. 1

Statistical source data for Fig. 1c–f and Extended Data Fig. 2.

Source Data Fig. 2

Statistical source data for Fig. 2a,c and Extended Data Fig. 4a,b.

Source Data Fig. 3

Statistical source data for Fig. 3a–c,f,g,i and k.

Source Data Fig. 4

Statistical source data for Fig. 4e,f and i.

Source Data Fig. 5

Meta data of scRNA-seq for TGF-βi/FGFi blastocysts (day 6) and statistical source data for Fig. 5g,h j,k and Extended Data Fig. 8e.

Source Data Fig. 6

Statistical source data for Fig. 6e,h–k.

Source Data Fig. 7

Statistical source data for Fig. 7c and g.

Source Data Fig. 8

Statistical source data for Fig. 8c,d.

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Wang, S., Leng, L., Wang, Q. et al. A single-cell transcriptome atlas of human euploid and aneuploid blastocysts. Nat Genet (2024). https://doi.org/10.1038/s41588-024-01788-6

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