Nature - 2019.08.29

Letter reSeArCH

DNA methylation level of EPI markedly increased from 26.1% at day

6 to 60.0% at day 10. The median DNA methylation level of TE also

increased from 23.5% at day 6 to 46.3% at day 10, an increase that is

not as strong as that with the EPI (Fig. 4a). Notably, although the DNA

methylation level of PE (27.0%) at day 6 is comparable to that of EPI

(26.1%), it increased only to 33.2% at day 8 (compared to 49.9% in

EPI), and 36.8% at day 10 (compared to 60.0% in EPI). The PE unex-

pectedly revealed much slower DNA re-methylation dynamics during

implantation compared to the EPI, even though both cell lineages are

derived from the inner cell mass. These results suggest that the embryos

initiated considerable re-methylation of the DNA shortly after the blas-

tocyst stage, and that the three major lineages not only had different

gene-expression signatures but also showed markedly distinct and

asynchronous DNA re-methylation patterns.

The patterns of hypermethylated gene body and hypomethylated pro-

moter regions were shared in all of the three lineages (Extended Data

Fig. 8i), which is similar to the patterns in pre-implantation embryos^22.

Further analysis of re-methylated genomic regions in all three lineages

showed that DNA re-methylation has clear genomic element-specific

and cell-lineage-specific features during implantation (Fig. 4b).

We found that 241 genes showed increased levels of methylation at

their promoter regions, potentially silencing their expression during

implantation (Extended Data Fig. 10b), and each lineage carried spe-

cifically methylated genes (Fig. 4c). For instance, the promoters of

POU5F1 (also known as OCT4) and NANOG, master regulators of

pluripotency, were specifically methylated in TE cells, but not in EPI

and PE cells at day 8 (Fig. 4c). By contrast, the promoters of devel-

opment genes in the TE^23 ,^24 , such as MMP26, PSG7 and ELF5, were

specifically methylated in EPI and PE cells but not in TE cells at day 8

(Fig. 4c and Extended Data Fig. 10c). Similarly, the promoters of PE

markers^25 ,^26 such as APOA1 and CPN1 were specifically methylated in

EPI and TE cells but not in PE cells at day 8 (Fig. 4c, d). These findings

indicate that DNA methylation might have an important role in reg-

ulating the expression of lineage-specific genes and maintaining the

segregated cell fates of different lineages.

We reconstituted the transcriptome and DNA methylome land-

scapes of human implantation at single-cell resolution and uncov-

ered key developmental events, the molecular dynamics of which was

previously unresolved. Although many potential differences may exist

between in vivo and in vitro implantation systems, this study provides

a potential basis for the development of better strategies to mimic

this unique process in vitro. A better understanding of the implanta-

tion process will also provide valuable information—such as lineage-

specific transcription-factor networks and signalling pathway charac-

teristics (Supplementary Methods)—for the derivation and directed

differentiation of pluripotent stem cells in vitro, which could be a

potentially invaluable resource for reproductive and regenerative

medicine.

Reporting summary

Further information on research design is available in the Nature Research

Reporting Summary linked to this paper.

Data availability

The single-cell RNA-sequencing data have been deposited in the GEO

(accession number GSE109555). The single-cell MALBAC whole-genome

sequencing, full-length RNA-sequencing and Trio-seq2 data have been depos-

ited in the European Genome-phenome Archive (EGA; https://www.ebi.ac.uk/

ega/) with accession number EGAS00001003443. The whole-genome sequencing

data of the paternal sperm and maternal peripheral blood were from a previous

publication (EGAS00001002987). The data deposited and made public are compli-

ant with the regulations of Ministry of Science and Technology of China.

Code availability

Scripts of the main steps of the analysis are provided at https://github.com/WRui/

Post_Implantation. Other R scripts associated with graphic presentation are avail-

able from the corresponding authors on reasonable request.

Online content

Any methods, additional references, Nature Research reporting summaries, source

data, extended data, supplementary information, acknowledgements, peer review

information; and details of author contributions and competing interests are

available at https://doi.org/10.1038/s41586-019-1500-0.

Received: 5 February 2018; Accepted: 15 July 2019;

Published online 21 August 2019.

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DNA methylation level (%)

60

6810 12

50

40

30

((nn = 26) = 22)

(n = 5)

(n = 14)

(n = 14)

(n = 4)

(n = 10)(n = 10)(n = 2)

(n = 8)

(n = 15)

EPI

TE

PE

a

20

–1.0

–0.5

0

0.5

1.0

Day 8

Day 6

EPI PE TE

cdAPOA1

PSG7

ERVFRD-1

EPI PE TE EPI PE TE EPI PE TE

Day 6 Day 8 Day 10

LEFTY1

POU5F1

NANOG

APOA1

FOXH1

MMP26

CPN1

NR2F2

b

−2.0

0

0.1

CGI

Promoter (HCP)

Promoter

SVA

Promoter (LCP)Promoter (ICP)

ERVKLINE-1

Intragenic

ExonLINEERV1Intron

ERVL−MaLR

LTRALR

Intergenic

ERVL

AluSINE

LINE-2

Enhancer

MIR

Lineage

Epi

PE

TE

Day

Fig. 4 | Lineage-specific dynamics of the DNA methylome in human
peri-implantation embryos. a, The global DNA methylation levels
across different developmental stages for each lineage. b, Distribution
of the relative enrichment of increased methylation tiles from day 6 to
day 8 at various genomic features, 1,512,276 (EPI), 310,255 (PE) and
1,140,524 (TE) tiles (300 bp) were de novo-methylated in each lineage.
The re-methylated genomic regions were strongly enriched in enhancers
and mammalian-wide interspersed repetitive (MIR) regions but depleted
in the promoters and CpG islands (CGI) in all lineages. The EPI and
PE lineages shared re-methylation enrichment of long interspersed
nuclear element-2 (LINE-2) retroelements and intergenic regions. For
PE, re-methylated genomic regions were strongly enriched for several
families of repeat elements (for example, alpha satellites (ALR) and long
terminal repeats (LTR)). For TE, re-methylated genomic regions were
strongly enriched for intragenic regions—both exons and introns. This
showed that DNA re-methylation has strong genomic-element-specific
and clear developmental-lineage-specific features during implantation.
Alu, Alu element; ERVs, endogenous retroviruses (ERVK, ERVL); HCP,
high CpG density promoter; ICP, intermediate CpG density promoter;
LCP, low CpG density promoter; MaLR, mammalian LTR; SINE, short
interspersed nuclear elements; SVA, short interspersed element–variable
number tandem repeat–Alu. c, The average DNA methylation levels for
differentially methylated genes at promoter regions (transcription
start sites included the upstream 250  bp and downstream 250  bp).
d, DNA methylation levels of representative loci for lineage-specific genes
at promoter regions. Each column represents one read. Red represents
methylated CpG sites, blue represents unmethylated CpG sites and white
represents undetected sites. Only reads that covered at least five CpG sites
are shown in the heat map.

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