Letter
https://doi.org/10.1038/s41586-019-1500-0
Reconstituting the transcriptome and DNA
methylome landscapes of human implantation
Fan Zhou1,2,7, rui Wang1,2,7, Peng Yuan1,3,7, Yixin ren1,3,7, Yunuo Mao1,2,7, rong Li1,3, Ying Lian1,3, Junsheng Li1,3, Lu Wen1,2,
Liying Yan1,2,3,4, Jie Qiao1,2,3,4,5,6 & Fuchou tang1,2,3,5,6
Implantation is a milestone event during mammalian
embryogenesis. Implantation failure is a considerable cause
of early pregnancy loss in humans^1. Owing to the difficulty of
obtaining human embryos early after implantation in vivo, it
remains unclear how the gene regulatory network and epigenetic
mechanisms control the implantation process. Here, by combining
an in vitro culture system for the development human embryos after
implantation and single-cell multi-omics sequencing technologies,
more than 8,000 individual cells from 65 human peri-implantation
embryos were systematically analysed. Unsupervised dimensionality
reduction and clustering algorithms of the transcriptome data show
stepwise implantation routes for the epiblast, primitive endoderm
and trophectoderm lineages, suggesting robust preparation for
the proper establishment of a mother-to-offspring connection
during implantation. Female embryos showed initiation of
random X chromosome inactivation based on analysis of parental
allele-specific expression of X-chromosome-linked genes during
implantation. Notably, using single-cell triple omics sequencing
analysis, the re-methylation of the genome in cells from the primitive
endoderm lineage was shown to be much slower than in cells of
both epiblast and trophectoderm lineages during the implantation
process, which indicates that there are distinct re-establishment
features in the DNA methylome of the epiblast and primitive
endoderm—even though both lineages are derived from the inner
cell mass. Collectively, our work provides insights into the complex
molecular mechanisms that regulate the implantation of human
embryos, and helps to advance future efforts to understanding early
embryonic development and reproductive medicine.
Human embryonic development starts from a fertilized egg, after
which a free-floating blastocyst is formed, which consists of an outer
trophectoderm (TE) and an inner cell mass. The mature inner cell
mass is composed of the pluripotent epiblast (EPI) covered by a layer(^1) Beijing Advanced Innovation Center for Genomics, Department of Obstetrics and Gynecology, Third Hospital, School of Life Sciences, Peking University, Beijing, China. (^2) Biomedical Pioneering
Innovation Center and Center for Reproductive Medicine, Third Hospital, Peking University, Beijing, China.^3 Key Laboratory of Assisted Reproduction and Key Laboratory of Cell Proliferation
and Differentiation, Ministry of Education, Beijing, China.^4 Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China.^5 Academy for Advanced
Interdisciplinary Studies, Peking University, Beijing, China.^6 Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.^7 These authors contributed equally: Fan Zhou, Rui Wang,
Peng Yuan, Yixin Ren, Yunuo Mao. *e-mail: [email protected]; [email protected]
a
b
Day 5
Day 10
Day 6
Day 11
Day 7
Day 12
Day 8
Day 13
Day 9
Day 14
Blastocyst Attached
TE
c d
Sequencing
scRNA-seq/scTrio-Seq2 Data analysis
Pre-implantation
Post-implantation
2,005 cells
3,508 cells
3,300 cells
1,567 cells
648 cells
Day 6
Day 8
Day 10
Day 12
Day 14 Copy number variationsand X chromosome
inactivation
Normalization and
quality control
Global transcriptional changeLineage identi cation
DEGs between neighbour
developmental stages
TF regulation networks
among lineages
s
...A...CCGTAGC...GATCGA...
...TACGACG...
...ACGTAGC......CGATCGA...
...TACGACG...
Cell-type
identi cation
...A...CCGTAGC...GATCGA...
...TACGACG...
DNA methylation
dynamics
Embryo culture
t-SNE1
t-SNE2Day
6
(^810)
12
EPI
ysTE
PE
–20
20
0
–40 –200 20
t-SNE1
t-SNE2
3,184 single cells 3,184 single cells
–40
–40 –20 020
–20
20
0
–40
TE
STRT-seq/full-length Smart-seq2
STRT-seq (cytoplasm)
scBS-seq (nucleus)
ICM
Fig. 1 | Single-cell RNA-sequencing transcriptome profiling of human
post-implantation embryos. a, Schematic illustration of the strategy for
the collection of single cells, and transcriptome and DNA methylome
analyses used in this study. DEGs, differentially expressed genes; scBS-seq,
single-cell bisulfite sequencing; scRNA-seq, single-cell RNA sequencing;
STRT-seq, single-cell tagged reverse transcription sequencing; TF,
transcription factor. b, Representative bright-field images of human
in vitro cultured embryos until day 14. The blastocysts clearly attached
to the bottom of the culture plate and formed a ring-like structure, with
the EPI cells surrounded by TE cells, at approximately days 7–8. ICM,
inner cell mass. All scale bars, 100 μm. All embryos used in this study are
described in Supplementary Table 1. c, d, Cells from embryos containing
all three major lineages at four representative stages were projected on
to the t-SNE map, enabling the identification of the developmental path
and cell lineage. Cells were identified as EPI, PE, TE and ysTE cells.
Cells (dots) are coloured according to embryonic stages (c) and original
lineage identities (d). c, d, In total, 3,184 single cells were included. c, Day
6, n = 387 cells; day 8, n = 1,525 cells; day 10, n = 1,021 cells; day 12,
n = 251 cells. d, EPI, n = 282 cells; PE, n = 138 cells; TE, n = 2,725 cells;
ysTE, n = 39 cells.
660 | NAtUre | VOL 572 | 29 AUGUSt 2019