DNA methylation is a crucial layer of epigenetic regulation during mammalian embryonic development1,2,3. Although the DNA methylome of early human embryos has been analyzed4,5,6, some of the key features have not been addressed thus far. Here we performed single-cell DNA methylome sequencing for human preimplantation embryos and found that tens of thousands of genomic loci exhibited de novo DNA methylation. This finding indicates that genome-wide DNA methylation reprogramming during preimplantation development is a dynamic balance between strong global demethylation and drastic focused remethylation. Furthermore, demethylation of the paternal genome is much faster and thorough than that of the maternal genome. From the two-cell to the postimplantation stage, methylation of the paternal genome is consistently lower than that of the maternal genome. We also show that the genetic lineage of early blastomeres can be traced by DNA methylation analysis. Our work paves the way for deciphering the secrets of DNA methylation reprogramming in early human embryos.
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We thank W. Guo for his insightful discussion. L.Y., J.Q., and F.T. were supported by grants from the National Natural Science Foundation of China (81561138005, 31230047, 31522034, 31571544, 81521002, and 31625018) and the National Basic Research Program of China (2014CB943200 and 2017YFA0102702). J.Q. and F.T. were also supported by a grant from the Beijing Municipal Science and Technology Commission (D151100002415000). L.Y. was supported by a grant from the National High-Technology Research and Development Program (2015AA020407). The work was supported by the Beijing Advanced Innovation Center for Genomics at Peking University.
Integrated supplementary information
a, Morphologies of oocytes and human early embryos at different developmental stages. Scale bar, 100 μm. b–f, The polar body and nuclear region of MII oocytes were biopsied by laser-assisted micromanipulation. Scale bar, 100 μm. b, Bright-field image of an MII oocyte. c, Staining with Hoechst 33342 dye to confirm the absence of genomic contaminants and reveal the nuclear region (arrow) and the first polar body (triangle) of the MII oocyte. d, Bright-field image of the aspiration of a polar body of an MII oocyte (triangle). e, Bright-field image of aspiration of the nuclear region of an MII oocyte (arrow). f, Hoechst 33342 staining showing aspiration of the nuclear region of an MII oocyte (arrow). g, Heat map showing the pairwise correlations of euploid single cells from different developmental stages. Unsupervised hierarchical clustering indicated that there were at least three major clusters, representing the sperm and male pronuclei (cluster I), GV and MII oocytes and female pronuclei (cluster II), and the cells from cleavage-stage embryos (cluster III), corresponding to the hypermethylation, intermediate methylation, and hypomethylation features of the different cell types.
Supplementary Figure 2 General quality control and sequencing statistics of the single-cell DNA methylome sequencing samples.
a, The number of CpG sites with at least one-, three-, and fivefold coverage across the single-cell samples. The “Mixed” label represents blastocyst cells that we did not separate manually into the ICM and TE (but directly dissociated the whole blastocyst into single-cell suspension and randomly picked single cells from them). The box plot represents the median, 25% and 75% quantiles; whiskers indicate 1.5 times the IQR (interquartile range). b, The CpG coverage (1×) at each developmental stage and the CpG coverage of single-cell and bulk samples at the same stages were merged together. c, Box plot showing the percentage of the digitized CpG methylation output in the single-cell DNA methylome sequencing samples; greater than 90% of CpG sites at each developmental stage had a methylation level of either fully methylated or unmethylated. The box plot represents the median, 25% and 75% quantiles; whiskers indicate 1.5 times the IQR. d, The general quality controls of all samples across different developmental stages; aneuploid samples and samples with low sequencing quality were excluded from subsequent analyses. Low-quality samples were determined by performing pairwise correlation analysis of all single cells at the same developmental stage. e, Pairwise comparison at different developmental stages showing the heterogeneity across intra- and inter-embryos; eight-cell stage embryo and morula were excluded from this analysis owing to the inadequate number of samples; n.s., not significant (Student's t test). The box plot represents the median, 25% and 75% quantiles; whiskers indicate 1.5 times the IQR.
Supplementary Figure 3 The copy number variation landscapes of human early embryos deduced from the DNA methylome dataset.
a, The normal sate (light blue), gain (red), and loss (cyan) of autosomes at different developmental stages. The “Mixed” label represents blastocyst cells that we did not separate manually into the ICM and TE (but directly dissociated the whole blastocyst into single-cell suspension and randomly picked single cells from them). b, Several representative examples of euploid and aneuploid single-cell samples; chromosomes with an aberrant copy number are highlighted in the purple boxes. c,d, Histograms of the statistics regarding the number of embryos (c) or single cells (d) with CNVs on each chromosome.
a, Density distribution of DNA methylation for ICM samples grouped by chromosome copy number. b, Density distribution of DNA methylation for TE samples grouped by chromosome copy number. c, Box plots show the DNA methylation distribution of 35 individual TE cells isolated from the same embryo and grouped by chromosome copy number; P values were calculated by t test. The box plot represents the median, 25% and 75% quantiles; whiskers indicate 1.5 times the IQR.
a, The representative regions that were de novo methylated from the earlier stage to the later stage. White open circles represent unmethylated CpG dinucleotides, whereas black filled circles represent methylated CpG dinucleotides. b, Histogram showing the numbers of increasing (magenta) and decreasing (cyan) CpG sites across consecutive stages. c, Enrichment analysis of de novo–methylated tiles on different genomic elements from the early male to mid-pronuclear stage and the four- to eight-cell stage (hypergeometric test).
Supplementary Figure 6 Select representative loci showing the hypermethylated maternal genome and hypomethylated paternal genome at different developmental stages.
White open circles represent unmethylated CpG dinucleotides, whereas black filled circles represent methylated CpG dinucleotides. Heterozygous SNPs were used to trace the parental genome.
Supplementary Figure 7 Parental-specific methylation and allele-specific expression in blastocyst-stage embryos.
Scatterplot showing ASE (x axis) and parental-specific methylation at the promoter regions of the corresponding genes (y axis), both presented as maternal scores minus paternal scores. The histogram shows the frequency distribution of methylation (M – P) indicating hypermethylation of the maternal genome. Genes showed both parental-specific methylation at promoters and ASE was labeled in blue. Genes labeled more than once were presented by different embryos (n = 4). ICM samples were displayed in a, and TE samples are displayed in b.
a, Length distributions of sperm-specific (red) and MII oocyte–specific (blue) DMRs. b, Gene ontology analysis of the genes with promoters located in the gamete-specific DMRs. c,d, Two representative regions showing the longest sperm-specific DMRs (c) and one representative maternal imprinting region, PEG3 (d) (red, CpG sites with methylation greater than 50%; blue, CpG sites with methylation level less than 50%). e,f, Enrichment analysis of the sperm-specific (e) and MII oocyte–specific (f) DMRs on different genomic elements (hypergeometric test).
Supplementary Figure 9 DNA methylation of all 31 known imprinting regions in preimplantation euploid cells and postimplantation tissues Heat map showing the DNA methylation of all 31 known imprinting regions in both preimplantation euploid cells and postimplantation tissues.
Colors indicate methylation levels from low (cyan) to high (red). White indicates missing (undetected) values. The “Mixed” label represents blastocyst cells that we did not separate manually into the ICM and TE (but directly dissociated the whole blastocyst into single-cell suspension and randomly picked single cells from them).
Supplementary Figure 10 Schematic of the chromosome-level DNA methylation discrepancies from the zygotes to four-cell embryos.
Three distribution types of DNA methylation for one genomic region in each cell from the zygote to four-cell embryos, indicating either “high–low” or “intermediate–intermediate” DNA methylation patterns for the two sibling cells in a four-cell embryo from a mother's cell at the two-cell stage.
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