Journal: Science Advances

Article Title: Lineage-specific and gestational age–dependent DNA methylation programming orchestrates human placental development

doi: 10.1126/sciadv.aee0560

Figure Lengend Snippet: ( A ) Schematic of development of trophoblasts and workflow for trophoblast isolation. Created in BioRender. Chen, J. (2026) https://BioRender.com/flbmp46 . ICM, inner cell mass; TE, trophectoderm; CTB, cytotrophoblast; EVT, extravillous trophoblast; STB, syncytiotrophoblast. ( B ) Methylation landscape across each developmental stage. The average DNA methylation levels of each developmental stage are calculated on the basis of 300–base pair (bp) tiles. First- or third-trimester CTB or EVT, CTB or EVT from the first- or third-trimester placenta (dots represent three biological replicates). GA, gestational age; dpf, day postfertilization; wks, weeks. ( C ) Snapshots of genome browser view showing DNA methylation levels around POU5F1 and KRT7 gene regions. ( D ) Representative immunofluorescence staining of 5mC (red) with epidermal growth factor receptor (EGFR) (green) in human placentas. Scale bars, 20 μm. ( E ) Analysis of relative intensity of 5mC. Two biological replicates per trimester. A total of 281 first-trimester cells and 370 third-trimester cells were quantified from eight randomly selected fields of view. Data are presented as means ± SD. ( F ) Scatter plot showing comparison of DNA methylation levels between two successive stages. ( G ) DNA methylation dynamics of repeat elements. Yellow, evolutionarily youngest subfamily; blue, evolutionarily oldest subfamily. ( H ) Scatter plot showing the comparison of DNA methylation levels between two successive stages. Blue dots, demethylated regions during EVT differentiation. ( I ) Representative immunofluorescence staining of 5mC (red) with HLA-G (green, EVT marker) in the first-trimester human placenta. Scale bar, 20 μm. ( J ) Analysis of relative intensity of 5mC. A total of 154 HLA-G–negative cells and 161 HLA-G–positive cells were quantified from six randomly selected fields. Data are presented as means ± SD (Student’s t test). *** P < 0.001. ( K ) Box plots showing expression levels and promoter DNA methylation levels of highly expressed genes in EVTs. Significance between the groups was evaluated by Student’s t test.

Article Snippet: The bisulfite-converted DNA was used for generation of sequencing libraries through EpiArt DNA Methylation Library Kit (NE103, Vazyme) according to the manufacturer’s instructions.

Techniques: Isolation, Methylation, DNA Methylation Assay, Immunofluorescence, Staining, Analysis, Comparison, Marker, Expressing

Journal: Science Advances

Article Title: Lineage-specific and gestational age–dependent DNA methylation programming orchestrates human placental development

doi: 10.1126/sciadv.aee0560

Figure Lengend Snippet: ( A ) Principal components analysis (PCA) of DNA methylation showing different developmental trajectories between the embryonic and extraembryonic lineages. EPI, epiblast. ( B ) Bar plots showing DNA methylation levels of embryonic and extraembryonic lineages following implantation. ( C ) Representative immunofluorescence staining of 5mC (green) with α-SMA (red) in the first-trimester fetal heart and 5mC (green) with EpCAM (red) in the first-trimester placenta. Scale bars, 50 μm. ( D ) Pie charts showing the proportion and numbers of five classes of embryonic lineage genes (top) or extraembryonic lineage genes (bottom). ( E ) Heatmaps showing the expression and DNA methylation levels (in promoter and gene body) of five classes of embryonic lineage genes (top) or extraembryonic lineage genes (bottom). The colors represent the normalized expression levels (left) and absolute DNA methylation levels (middle and right). ( F ) Box plots showing the dynamics of DNA methylation in maternal gDMRs (left), placental specific maternal gDMRs (middle), and paternal gDMRs (right) in embryonic and extraembryonic lineages.

Article Snippet: The bisulfite-converted DNA was used for generation of sequencing libraries through EpiArt DNA Methylation Library Kit (NE103, Vazyme) according to the manufacturer’s instructions.

Techniques: Analysis, DNA Methylation Assay, Immunofluorescence, Staining, Expressing

Journal: Science Advances

Article Title: Lineage-specific and gestational age–dependent DNA methylation programming orchestrates human placental development

doi: 10.1126/sciadv.aee0560

Figure Lengend Snippet: ( A ) Box plot depicting the first-trimester hypermethylation (157,612 regions) dynamics. ( B ) Box plot depicting the third-trimester hypermethylation (18,424 regions) dynamics. ( C ) Box plots showing DNA methylation levels of gene body and promoter in the first-trimester CTB. Yellow, first-trimester hypermethylated genes (10,199); blue, third-trimester hypermethylated genes (1082). Significance between the groups was evaluated by Student’s t test. ( D ) Gene ontology (GO) enrichment analysis for the first-trimester hypermethylated genes. ( E ) Box plots showing DNA methylation levels of gene body and promoter in the third-trimester CTB. Significance between groups was evaluated by Student’s t test. ( F ) GO enrichment analysis for the third-trimester hypermethylated genes. ( G ) Odds ratio of the peak overlap between histone modifications and the first- or third-trimester hypermethylated regions. ( H ) Box plot showing H3K27me3 levels for two different hypermethylated regions in CTBs. Significance between the groups was evaluated by Student’s t test. ( I ) Gene set enrichment analysis (GSEA) showing the significant up-regulation of the third-trimester hypermethylated genes [involved in (F)] in H3K27me3 reduced hTSCs compared to control hTSCs. ( J ) qRT-PCR analysis of representative third-trimester hypermethylated genes. ( K ) Dynamic changes of H3K27me3 signals around the TSS. ( L ) Schematic workflow to test cross-talk with differentiated endometrial stromal cells (DESCs). Conditioned medium (CM) is collected from induced EVTs (iEVTs) on day 3. ( M ) qRT-PCR analysis in DESCs. COL1A1 , COL1A2 , COL6A2 , MMP14 , and MMP9 are extracellular matrix (ECM) genes. PTGS2 gene is up-regulated for uterine contraction in late gestation. ( N ) qRT-PCR analysis in iEVTs. For qRT-PCR analysis above, GAPDH is set as an endogenous control. Data are presented as means ± SD (three biological replicates; multiple t test). * P < 0.05; ** P < 0.01; *** P < 0.001. ns, not significant. h, hours.

Article Snippet: The bisulfite-converted DNA was used for generation of sequencing libraries through EpiArt DNA Methylation Library Kit (NE103, Vazyme) according to the manufacturer’s instructions.

Techniques: DNA Methylation Assay, Analysis, Control, Quantitative RT-PCR, Significance Assay

Journal: Science Advances

Article Title: Lineage-specific and gestational age–dependent DNA methylation programming orchestrates human placental development

doi: 10.1126/sciadv.aee0560

Figure Lengend Snippet: ( A ) Left, Western blot assessing the relative abundance of H3K9me3 modification in lysates of freshly collected first- and third-trimester placentas. Right, quantification of H3K9me3 level relative to the H3 loading control. Data are presented as means ± SD (Student’s t test). * P < 0.05. ( B ) Enrichment profile showing normalized H3K9me3 reads at hypermethylated (methylation level ≥0.75) or hypomethylated (methylation level <0.25) 300-bp tiles ±2 kb in each stage. ( C ) Average DNA methylation levels of regions with (blue) and without (yellow) H3K9me3 marks. ( D ) Bar plot showing the proportion of LTRs occupied by H3K9me3 between two successive stages. ( E ) Heatmaps showing the dynamics of H3K9me3, DNA methylation, and expression on LTRs marked by lost or gained H3K9me3 peaks in CTB from the first to third trimesters. The colors represent the log 2 -transformed H3K9me3 signal scaled by row (left), the absolute DNA methylation levels and changes (middle), and FPKM values (right). ( F ) The number of LTR families in BSH- and PSH-marked LTRs.

Article Snippet: The bisulfite-converted DNA was used for generation of sequencing libraries through EpiArt DNA Methylation Library Kit (NE103, Vazyme) according to the manufacturer’s instructions.

Techniques: Western Blot, Modification, Control, Methylation, DNA Methylation Assay, Expressing, Transformation Assay

Journal: Science Advances

Article Title: Lineage-specific and gestational age–dependent DNA methylation programming orchestrates human placental development

doi: 10.1126/sciadv.aee0560

Figure Lengend Snippet: ( A ) Pie chart showing the proportion and number of methylated1 and escapee1 5-kb bins from TE to the first-trimester CTB. ( B ) Pie chart showing the proportion and number of methylated2 and escapee2 5-kb bins from the first- to third-trimester CTB. ( C ) Box plots showing the DNA methylation, H3K9me3, H3K9ac, and expression levels of genes and repeats undergoing methylation (genes, 25,044; repeats, 3,799,552) or escapee (genes, 10,225; repeats, 232,372) in the first-trimester CTB from TE to the first trimester. Significance between the groups was evaluated by Student’s t test. ( D ) GO enrichment analysis for methylated1 and escapee1 genes from TE to the first-trimester CTB. ( E ) qRT-PCR analysis of representative methylated1 or escapee1 genes in human embryonic stem cell (hESC), hTSC, and the first-trimester CTB. Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) is set as an endogenous control. Data are presented as means ± SD (three biological replicates). ( F ) Box plots showing the DNA methylation, H3K9me3, H3K9ac, and expression levels of genes and repeats undergoing methylation (genes, 22,098; repeats, 2,847,005) or escapee (genes, 15,384; repeats, 1,217,495) in the third-trimester CTB from the first to third trimesters. Significance between the groups was evaluated by Student’s t test.

Article Snippet: The bisulfite-converted DNA was used for generation of sequencing libraries through EpiArt DNA Methylation Library Kit (NE103, Vazyme) according to the manufacturer’s instructions.

Techniques: DNA Methylation Assay, Expressing, Methylation, Analysis, Quantitative RT-PCR, Stem Cell, Control, Significance Assay

Journal: Science Advances

Article Title: Lineage-specific and gestational age–dependent DNA methylation programming orchestrates human placental development

doi: 10.1126/sciadv.aee0560

Figure Lengend Snippet: ( A ) Bar plots showing the dynamics of DNA methylation among placentation, aging, and tumor progression. ( B ) Enrichment profile showing DNA methylation levels at CGI ± 5 kb in placentation, aging, and tumor progression. Senescent blood cell and colon cancer cell show a gain of DNA methylation at CGI. Human placental CTB shows a preferential gain at flanking regions of CGI. ( C ) Venn diagram showing the number of overlaps among PMDs identified in newborn blood, first-trimester CTB, and primary colon cancer. ( D ) Enrichment profile showing normalized H3K9me3 reads from first- and third-trimester CTB at somatic PMDs ±2 kb (left), placental PMDs ±2 kb (middle), and tumor PMDs ±2 kb (right). ( E ) Bar plot showing number of hypo- and hypermethylated DMRs during different developmental processes. ( F ) GO enrichment analysis for shared hypermethylated DMR target genes between placentation (from the first- to third-trimester CTB) and aging (from newborn to old). ( G ) A snapshot of the genome browser view showing DNA methylation levels around CDKN2A gene regions. The gene body of CDKN2A gene shows increased methylation deposition during both placentation and aging. ( H ) Representative immunohistochemical labeling showing reduced MKI67 signals in the third-trimester placenta compared with the first-trimester placenta. Scale bars, 200 μm. MKI67 is a marker of cell proliferation. ( I ) GO enrichment analysis for overlapped genes, which are targets for hypermethylated DMRs during placentation (from the first- to third-trimester CTB) but for hypomethylated DMRs during aging (from newborn to old). ( J ) A snapshot of the genome browser view showing DNA methylation levels around IL6R gene regions. IL6R gene shows increased methylation deposition during placentation but decreased methylation deposition during aging.

Article Snippet: The bisulfite-converted DNA was used for generation of sequencing libraries through EpiArt DNA Methylation Library Kit (NE103, Vazyme) according to the manufacturer’s instructions.

Techniques: DNA Methylation Assay, Analysis, Methylation, Immunohistochemical staining, Labeling, Marker, Significance Assay, Cell Differentiation

Journal: Science Advances

Article Title: Lineage-specific and gestational age–dependent DNA methylation programming orchestrates human placental development

doi: 10.1126/sciadv.aee0560

Figure Lengend Snippet: ( A ) qRT-PCR analysis of DNMT1 in lentivirus-mediated knockdown in hTSCs. GAPDH is set as an endogenous control. Data are presented as means ± SD (three biological replicates; Student’s t test). ** P < 0.01. ( B ) Western blot analysis assessing the relative abundance of DNMT1 in hTSCs. GAPDH was used as a loading control. Protein levels were quantified and normalized to those of control hTSCs. ( C ) Representative phase-contrast images of control and DNMT1 -deficient hTSCs. Scale bar, 500 μm. ( D ) Left, representative images of EdU staining in hTSCs. Scale bars, 10 μm. Right, analysis of percentage of EdU-positive cells ( n > 20 single nuclei). ( E ) Flow cytometry analysis of hTSCs stained with PI and annexin V–FITC. ( F ) and ( G ) qRT-PCR analysis of indicated genes in control and DNMT1 -deficient hTSCs. GAPDH is set as an endogenous control. Data are presented as means ± SD (two or three biological replicates; multiple t test). ** P < 0.01; *** P < 0.001. ( H ) Representative phase-contrast images showing progressive loss of self-renewal capacity during serial passage of DNMT1 -deficient hTSCs. Scale bar, 500 μm. ( I ) Bar plots showing the enrichment of DMRs between DNMT1 knockdown and control hTSCs in different genomic regions. ( J ) GO enrichment analysis for genes related to hypomethylated DMRs in DNMT1 -deficient hTSCs. ( K ) GSEA showing the significant activation of STB-associated genes in DNMT1 -deficient hTSCs. ( L ) Box plot showing differential DNA methylation across distinct gene sets between DNMT1 knockdown and control hTSCs. Significance between the groups was evaluated by Student’s t test.

Article Snippet: The bisulfite-converted DNA was used for generation of sequencing libraries through EpiArt DNA Methylation Library Kit (NE103, Vazyme) according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Analysis, Knockdown, Control, Western Blot, Phase Contrast, Staining, Flow Cytometry, Activation Assay, DNA Methylation Assay, Negative Control, Gene Expression