Abstract
Human in vitro fertilized embryos exhibit low developmental capabilities, and the mechanisms that underlie embryonic arrest remain unclear. Here using a single-cell multi-omics sequencing approach, we simultaneously analysed alterations in the transcriptome, chromatin accessibility and the DNA methylome in human embryonic arrest due to unexplained reasons. Arrested embryos displayed transcriptome disorders, including a distorted microtubule cytoskeleton, increased genomic instability and impaired glycolysis, which were coordinated with multiple epigenetic reprogramming defects. We identified Aurora A kinase (AURKA) repression as a cause of embryonic arrest. Mechanistically, arrested embryos induced through AURKA inhibition resembled the reprogramming abnormalities of natural embryonic arrest in terms of the transcriptome, the DNA methylome, chromatin accessibility and H3K4me3 modifications. Mitosis-independent sequential activation of the zygotic genome in arrested embryos showed that YY1 contributed to human major zygotic genome activation. Collectively, our study decodes the reprogramming abnormalities and mechanisms of human embryonic arrest and the key regulators of zygotic genome activation.
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Data availability
All raw sequencing data of human embryos reported in this study have been deposited into the Genome Sequence Archive (GSA) for human database with the accession number HRA003366. All raw sequencing data of mouse embryos reported in this study have been deposited into the GSA database with the accession number CRA012505. All processed data of human embryos generated in this study have been deposited into the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE247678. All processed data of mouse embryos generated in this study have been deposited in the OMIX database (China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences) with accession number OMIX004867. Previously published data that were re-analysed in this study are available under the following accession codes from the GEO database: GSE36552 and GSE133856 (scRNA-seq datasets of human early embryos); GSE100272 (scCOOL-seq datasets of human early embryos); GSE124718 (H3K4me3 ChIP–seq datasets of human 4-cell embryos and 8-cell embryos, then converted to hg38 reference using UCSC liftOver tool)92; GSM803513 (ChIP–seq datasets of YY1 in H1 hESCs); GSM3738360 (ChIP–seq datasets of KLF4 in naive hESCs); GSM2026849 (ChIP–seq datasets of ZFP42 in HEK 293T cells); GSE76495 (RNA-seq data of YY1 in HEK 293T cells); and GSE214608 (RNA-seq data of wild type HEK293 cells). For Extended Data Fig. 3b and Supplementary Fig. 4b, raw data were downloaded from the GEO (identifier PRJNA603589 (single-embryo RNA-seq datasets of TUBB8 mutant human arrested zygote)) and were processed according to the methods of the original article4, and the European Bioinformatics Institute (identifier E-MTAB-3929 (scRNA-seq datasets of day 5 blastocyst))105. Further information of resources is available from the lead contact Lin Li on reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank X. Zhao from Southern Medical University and G. Chang from Shenzhen University Medical School for suggestions and discussion. This study was supported by grants from the National Key R&D Program of China (2021YFA1102700 to Lin Li), the National Natural Science Foundation of China (82101745 and 32070833 to Lin Li, 81871211 to Lei Li, 81901566 to R.H. and 81971452 to J.L), the Natural Science Funds for Distinguished Young Scholar of Guangdong province (2022B1515020110 to Lin Li), and the Key-Area Research and Development Program of Guangdong Province, Modernization of Chinese medicine in Lingnan (2020B1111100011 to Lin Li).
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Lin Li conceived and supervised the project. T.W., J.F. and J.P. facilitated its designs. N.T., X.Z., Y.B., X.K., J.L., L.Z. and Lei Li collected the human arrested and 3PN embryos. J.P. performed library constructions of the single-cell/embryo multi-omics sequencing data. X.Z. performed the siRNA microinjection experiments in human early embryos. J.P., R.H., Zhihao Wang, L.W., Y.B., X.Q., Zimeng Wang, C.L. and W.Z. performed the other experiments. T.W. and J.F. conducted the bioinformatics analyses. Lin Li, T.W., J.F. and J.P. interpreted the data. Lin Li, T.W., J.F. and J.P. wrote the paper with assistance from all the authors.
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Extended data
Extended Data Fig. 1 The developmental characteristics and cell morphologies of human IVF embryos.
a. Bar graph showing developmental outcomes of 610 human 2PN embryos underwent IVF procedure. Human arrested embryos are those without further development for another 2 days at the zygote and cleavage stages. Embryos that were developmentally delayed, fragmented or dead were excluded. b. Bar graph showing the number of human embryos arrested at different developmental stages from a. c. Representative bright-field images of human normal and arrested embryos at different developmental stages. Scale bars, 20 μm. d. Chromatin accessibility around the promoter regions (TSS ± 2 kb) in human arrested embryos at each stage. Lines are colored according to developmental stage. TSS, transcription start site. e. The average DNA methylation level along the intragenic regions and their flanking regions in human arrested embryos at each stage. Lines are colored according to the developmental stage. TES, transcription end site. f, g. PCA projections of human normal and arrested embryos at each stage with RNA-part data from single-cell multi-omics sequencing, integrated with the cells from Yan et al.19 and Leng et al.20 In f, Single cells are colored according to data source. In g, single cells are colored according to stage. h. Diffusion map constructing the putative trajectory of human normal and arrested embryos at each stage with RNA-part data from single-cell multi-omics sequencing, integrated with the cells from Yan et al.19 and Leng et al.20. Single cells were colored according to data source.
Extended Data Fig. 2 The reprogramming defects in human arrested embryos.
a. The transcriptional activity (mean expression levels of all genes in each single cell) in human normal and arrested embryos. The number of biologically independent samples is 10, 10, 11, 10, 9 for human normal zygote to morula; 18, 13, 8, 7 for human arrested zygote to 8-cell embryos. b. The number of DEGs between human arrested and normal embryos. c. Hierarchical clustering of human normal and arrested embryos using RNA-part data from single-cell multi-omics sequencing. d. The number of arrested-specific hyper-DMRs (red) and arrested-specific hypo-DMRs (blue) located within promoter regions between human arrested and normal embryos. DMRs, differentially methylated regions. e. Lollipop graphs showing the proportion of arrested-specific hypo-DMRs in each stage of human arrested embryos overlapped with hypo-DMRs between consecutive stages in human normal embryos. f. The number of normal-specific NDRs (red) and arrested-specific NDRs (purple) between human arrested and normal embryos. NDRs, nucleosome-depleted regions. g. The expression levels of CTCF in human normal and arrested embryos. The number of biologically independent samples is 9, 10, 10, 11, 10, 9 for human normal MII to morula; 18, 13, 8, 7 for human arrested zygote to 8-cell embryos. h. RT-qPCR results showing the relative expression levels of CTCF in human normal (3PN) and arrested 2-cell or 8-cell embryos (2PN). Data are represented as mean ± SEM (3, 5, 4, 3 biologically independent samples); two-sided Mann-Whitney U test. In g, h, error bars represent mean ± SEM. i. The average expression levels of major ZGA genes in human normal 8-cell embryos (left) and human arrested zygotes to 8-cell embryos (right) that located within or outside topologically associating domains (TADs) identified from Hi-C data in human early embryos from Chen et al.22. In a, i, each box represents median, 25% and 75% quartiles; whiskers indicate 1.5 times the interquartile range; unpaired two-sided Student’s t-test without adjustment. j. GO terms of up-regulated (left) and down-regulated genes (right) in human arrested embryos compared to human normal embryos with similar morphologies. Hypergeometric test without adjustment.
Extended Data Fig. 3 The maternal-to-zygote transition is disorganized in human arrested embryos.
a. Venn plots showing the overlap between major ZGA genes and up-regulated genes in human arrested zygotes to 4-cell embryos. b. Radar chart showing the average expression levels of representative major ZGA genes in human TUBB8mutant arrested zygotes from Sha et al.4 and human normal zygotes from Yan et al.19 from 2, 3 biologically independent samples; integration of all samples. c. Expression patterns and GO terms of each cluster of up-regulated major ZGA genes in human arrested embryos. Left, heatmap showing k-means clustering (n = 4) of major ZGA genes that are commonly up-regulated in human arrested zygotes to 4-cell embryos. The color key from blue to red indicates the expression level from low to high. Middle, line plots showing the average expression patterns of genes in each cluster. Right, GO terms of genes in each cluster. In a, c, hypergeometric test without adjustment. d-f. Line plots showing the expression levels of d. SVA, e. representative TEs and f. M-decay genes in human normal and arrested embryos at each stage. TEs, transposable elements. CPM, count-per-million. The number of biologically independent samples is 9, 10, 10, 11, 10, 9 for human normal MII to morula; 18, 13, 8, 7 for human arrested zygote to 8-cell embryos. g, h. g. RT-qPCR results showing the relative expression levels of M-decay gene EIF3K in human normal (3PN) and arrested zygotes or 2-cell embryos (2PN). Each dot represents an embryo. Data are from 4, 4, 6, 3, 5 biologically independent samples. h. RT-qPCR results showing the relative expression levels of M-decay genes PABPC1L, TLE6, ZP2 in human normal (3PN) and arrested zygotes (2PN). Data are from 4, 4, 6 biologically independent samples. In c-h, error bars represent the mean ± SEM. In g, h, two-sided Mann-Whitney U test.
Extended Data Fig. 4 DNA hyper-methylation (intragenic regions) and excessive open chromatins link with up-regulated major ZGA genes in human arrested embryos.
a, f. Relative enrichment analysis of a. DMRs, f. differential NDRs between human normal and arrested embryos in different genomic regions. b. The expression levels of genes associated with arrested-specific hypo-DMRs and hyper-DMRs (promoter) in human arrested embryos. c. Heatmap showing the expression levels of DEGs in human arrested embryos compared with human normal embryos (left). Pie charts showing the proportion of DEGs associated with DMRs (promoter or intragenic, right). d, e. The relationship between gene expression and DNA methylation level of DMRs (intragenic) at d. zygote and e. 2-cell stages of major ZGA genes. Up-regulated major ZGA genes with significant changes in DNA methylation levels are labeled in red. g. The spearman correlation coefficients between the normalized chromatin accessibility (core promoter regions) and gene expression levels. The number of biologically independent samples is 20, 20, 22, 22 for human normal zygote to 8-cell embryos; 15, 13, 7, 6 biologically independent samples for human arrested ones. Error bars represent mean ± SEM. h. The average expression levels of genes associated with arrested-specific proximal-merged NDRs (mNDRs) and that associated with normal-specific proximal mNDRs in human arrested embryos. In b, h, data are from 10, 10, 11, 12 biologically independent samples for human normal zygote to 8-cell embryos; 15, 13, 7, 6 biologically independent samples for human arrested ones; each box represents the median, 25% and 75% quartiles; whiskers indicate 1.5 times the interquartile range; unpaired two-sided Student’s t-tests without adjustment. i. Heatmap showing the proximal NDRs that were specifically open in human normal or arrested embryos (left). Pie charts showing the proportion of DEGs associated with arrested or normal-specific mNDRs (proximal, right). j, k. The association between major ZGA genes and differential NDRs (proximal) in j. human arrested zygotes and k. 2-cell embryos. Red dots represent up-regulated major ZGA genes in human arrested embryos occupied with arrested-specific NDRs (proximal). In d, e, j, k, representative major ZGA genes are labeled in purple.
Extended Data Fig. 5 KDM5 or H3K4me3 erasure regulates human zygotic genome activation.
a. Line plot showing the average expression levels of KDM5B in human normal MII to morula and arrested embryos. The number of biologically independent samples is 9, 10, 10, 11, 10, 9 for human normal MII to morula; 18, 13, 8, 7 for human arrested zygote to 8-cell embryos. Error bars represent the mean ± SEM. b. RT-qPCR results showing the relative expression levels of KDM5B in human normal (3PN) and arrested zygotes (2PN). Data are represented as mean ± SEM from 4, 4, 6 biologically independent samples; two-sided Mann-Whitney U test. c. Flowchart of the experimental scheme for KDM5 inhibitor treatment of human zygote (3PN). Human 8-cell embryos (3PN) were collected on day 3 post in vitro fertilization (IVF). d. Immunofluorescence of H3K4me3 in human 8-cell embryos (developed from 3PN zygotes) from control and KDM5 inhibited group. Data are from 7, 10 biologically independent samples. Scale bar, 20 μm. Data are represented as mean ± SEM; unpaired two-sided Student’s t-test. e. Boxplots showing the expression levels of TEs in human control and KDM5 inhibited 8-cell embryos (developed from 3PN zygote). Each dot represents an embryo. Data are from 4, 5 biologically independent samples. Each box represents the median, 25% and 75% quartiles; whiskers indicate 1.5 times the interquartile range. Unpaired two-sided Student’s t-test. TEs, transposable elements. CPM, count-per million. f-h. Genome browser views showing the normalized chromatin accessibility of major ZGA genes accompanied with H3K4me3 decreases from human normal 4-cell (broad peak) to normal 8-cell (narrow peak) embryos in human normal and arrested f. zygotes, g. 2-cell embryos and h. 4-cell embryos. The expression levels of major ZGA genes are shown at right. Black bars represent GCH sites with normalized chromatin accessibility less than 1/10 of the normalized data range (but are detected). Bar plot under ChIP-seq signals represents peaks of H3K4me3 from Xia et al.13.
Extended Data Fig. 6 YY1 promotes human zygotic genome activation.
a. Regulatory network of key transcription factors (TFs) that are shared or unique among human arrested zygotes to 4-cell embryos predicted by SCENIC analysis. b. Bar graph showing the number of target genes that are regulated by TFs in human arrested zygotes to 4-cell embryos predicted by SCENIC analysis. Bars are colored according to developmental stage; integration of all samples. c. Line plots showing the average expression levels of KLF4 and ZFP42 in human normal MII to morula and arrested embryos at each stage. The number of biologically independent samples is 9, 10, 10, 11, 10, 9 for human normal MII to morula; 18, 13, 8, 7 for human arrested zygote to 8-cell embryos. d. Venn plot showing the overlap between major ZGA genes and the target genes of KLF4 or ZFP42 identified from public ChIP-seq data in human embryonic stem cells (hESCs)33,34. Hypergeometric test without adjustment. e. Venn plot showing the overlap among target genes that are regulated by YY1, KLF4 or ZFP42 identified from public ChIP-seq datasets in hESCs or HEK 293T cells32,33,34. f. Boxplots showing the average expression levels of major ZGA genes in human 2PN (from Yan et al.19, n = 16) and 3PN 8-cell embryos (n = 16). Data are from 3, 4 biologically independent samples. Each box represents the median, 25% and 75% quartiles; whiskers indicate 1.5 times the interquartile range. Unpaired two-sided Student’s t-test. g. Bar plot showing the expression levels of YY1 among YY1 KD and control human 8-cell embryos (developed from human 3PN zygotes). Each point represents an embryo. Data are from 3, 3, 2 biologically independent samples. In c, g, error bars represent mean ± SEM. h. Genome browser views showing YY1 signals from public ChIP-seq data in hESCs32.
Extended Data Fig. 7 Human and mouse arrested embryos display abnormal microtubule cytoskeleton and mitosis defects.
a. GSEA analysis showed the enriched terms that were down-regulated in human arrested embryos. Circle size: normalized enrichment score (NES); color: FDR. b, c. The average expression levels of b. representative cytoskeletal or mitotic genes, and c. ORC5. Data in b, c are from 9, 10, 10, 11, 10, 9 biologically independent samples for human normal MII to morula; 18, 13, 8, 7 for human arrested zygote to 8-cell embryos. d. RT-qPCR results showing the relative expression levels of ORC5 in human normal (3PN) and arrested zygotes (2PN) from 5, 11 biologically independent samples. e. Immunofluorescence of γ-tubulin (green) co-stained with β-tubulin (red) in human arrested zygotes (2PN, with magnified images showing nucleus). Scale bar, 20 μm. f. GO terms of down-regulated genes in mouse arrested zygotes from MLN8237 treated group. MLN8237, AURKA inhibitor. g. Immunofluorescence and h. statistical analysis of γ-tubulin signal intensity in mouse control (KSOM) and arrested zygotes (7 biologically independent samples). Scale bar, 20 μm. In b-d, h, error bars represent mean ± SEM. i. The average expression levels of representative cytoskeletal and mitotic genes in mouse arrested zygotes and control zygotes. j. Hierarchical clustering of mouse arrested zygotes and control zygotes, early two-cell (E2C) as well as late two-cell (L2C) embryos. k. The average expression levels of major ZGA genes in mouse arrested zygotes and mouse control embryos. Data are from 17, 10, 30, 17 biologically independent samples. l. The overlap between up-regulated genes in mouse arrested zygotes and mouse major ZGA genes. In f, l, hypergeometric test without adjustment. m. The expression levels of representative major ZGA genes in mouse arrested zygotes and control embryos. Data are from 17, 10, 30, 17 biologically independent samples. In k, m, each box represents the median, 25% and 75% quartiles; whiskers indicate 1.5 times the interquartile range. n. Scatter plots of TEs expression in mouse arrested zygotes compared with control zygotes. Blue dots represent differentially expressed TEs with representative ZGA TEs shown in red. In d, h, k, m, n, unpaired two-sided Student’s t-test without adjustment.
Extended Data Fig. 8 Aberrant reprogramming of DNA methylation and chromatin accessibility link with cytoskeleton defects in human arrested embryos.
a. Venn plot showing the overlap of genes associated with arrested-specific hypo-DMRs (promoter) among human arrested zygotes to 8-cell embryos. b. Differences in the DNA methylation level of autophagy and RNA splicing genes (promoter) between human arrested and normal embryos at each stage. Data are from biologically independent samples as indicated in Fig. 1f; integration of all samples. c. Genome browser views showing the DNA methylation levels of cytoskeletal and mitotic related genes in human normal and arrested embryos at each stage. The expression levels of genes are shown (right). The WCG sites detected with DNA methylation lower than 1/10 of the data range (but are detected) are shown as black bars. d. GO terms of genes associated with arrested-specific NDRs (see details in the Method section) and normal-specific NDRs (promoter) in human arrested embryos at each stage. Bars are colored according to developmental stage. Genes in GO terms that are consistent with gene expression levels are colored in red. Hypergeometric test without adjustment.
Extended Data Fig. 9 Human arrested 2-cell embryos present increased intra-embryonic heterogeneity.
a. Heatmap showing the variance of gene expression level in each blastomeres within an embryo in human normal and arrested 2-cell to 8-cell embryos (no more than one blastomere failed quality control within an embryo are allowed for 4-cell and 8-cell embryos). The color key from blue to red indicates the variance from low to high. The number of biologically independent samples is 6, 5, 2 for human normal 2-cell to 8-cell embryos; 4, 7, 3 for human arrested 2-cell to 8-cell embryos. b. GO terms of up-regulated genes between two blastomeres of human arrested 2-cell embryos. Hypergeometric test without adjustment. c. The expression levels of up-regulated genes between two blastomeres of human normal and arrested 2-cell embryos. Blastomeres are colored by transcription activity (blastomeres 1: higher transcription activity; blastomeres 2: lower transcription activity). The dispersion degree along the y-axis can be visualized in each embryo and represents the overall heterogeneity of gene expression levels between two blastomeres within each embryo. Error bars represent mean ± SEM. In a, c, unpaired two-sided Student’s t-test is used for statistical analysis. d, e. iTALK analysis of cell-cell communication between two blastomeres in d. human normal (left) or e. human arrested (right) 2-cell embryos. Data in c-e are from biologically independent samples as indicated in a.
Extended Data Fig. 10 Human arrested embryos display increased genomic instability.
a. Boxplots showing the gene expression levels (left), the DNA methylation level of WCG sites within DMRs (intragenic, middle), and chromatin accessibility of arrested-specific NDRs (promoter, right) in individual cell of human normal and arrested 4-cell embryos. Data are from biologically independent samples as indicated in Fig. 1f (RNA-part data) and Fig. 3b (DNA-part data). Each box represents median, 25% and 75% quartiles; whiskers indicate 1.5 times the interquartile range; one side Wilcoxon test. b. Bar graphs showing the number of CNVs on 22 autosomes in human arrested embryos at each stage. Data are from 10, 10, 11, 12 biologically independent samples for human normal zygote to 8-cell embryos; 15, 13, 7, 6 biologically independent samples for human arrested zygote to 8-cell embryos; integration of all samples. c. Representative examples of euploid and aneuploid cells in intact human arrested embryos. CNVs that are complementary in blastomeres within a complete human embryo are highlighted by a black dotted frame. The CNV patterns were profiled using DNA-part data at 1 M resolution. Windows with normalized count data ≥ 3 or ≤ 1 are marked in green.
Supplementary information
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Supplementary Tables 1–10. Supplementary Table 1: Quality assessment of the single-cell multi-omics data in human arrested embryos. Supplementary Table 2: DEGs in human arrested embryos compared with normal ones (two-sided t-test with Benjamin and Hochberg adjustment). Supplementary Table 3: NDR- and DMR-associated DEGs in human arrested embryos. Supplementary Table 4: Quality assessment of the single-embryo RNA-seq data in human 3PN 8-cell embryos from the control group and the KDOAM25-treated group. Supplementary Table 5: The overlap between upregulated major ZGA genes in human arrested embryos and targeted genes of YY1, KLF4 and ZFP42 with publicly available ChIP–seq data from hESCs or HEK 293T cells. Supplementary Table 6: Quality assessment of the single-embryo multi-omics data in human 3PN 8-cell embryos from the negative control group and the YY1 knockdown group. Supplementary Table 7: Quality assessment of the single cell RNA-seq data in mouse control embryos and arrested zygotes from the AURKA-inhibited group. Supplementary Table 8: DEGs between mouse arrested zygotes from the AURKA-inhibited group and the control group. Supplementary Table 9: Quality assessment of the single-cell multi-omics data in human arrested zygotes from the AURKA-inhibited group and control zygotes. Supplementary Table 10: RT–qPCR primer sequences.
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Wang, T., Peng, J., Fan, J. et al. Single-cell multi-omics profiling of human preimplantation embryos identifies cytoskeletal defects during embryonic arrest. Nat Cell Biol 26, 263–277 (2024). https://doi.org/10.1038/s41556-023-01328-0
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DOI: https://doi.org/10.1038/s41556-023-01328-0