Fig. 5

Trophoblast dysfunction associated with the DNA methylation change in the DMR near the GNE gene in both AMA and SA. A Venn diagram shows the relationship between AMA-related DMRs and SA-DMRs. Genes near the overlapping DMRs are shown in the frame. The different colors of gene symbols refer to the different genetic elements where DMRs are located. The numeral suffix after a gene symbol refers to the serial number for a specific exon or intron in the reference gene. The bolded gene symbols refer to the four genes near the DMRs shown in B. B Box and dot plot show the distribution of DNA methylation levels of four DMRs near CDK11A, C19orf71, COL5A1, and GNE in the AMA, YMA, and SA groups. The changes between SA and YMA were larger than those between AMA and YMA in these four DMRs. C Column diagram shows the relative gene expression level of GNE determined by qRT-PCR in the AMA (orange) and YMA (pink) groups. Each dot represents the gene expression level of a detected sample. The P value was determined using an unpaired t test (**P < 0.01). D UMAP visualization presents the single-cell clusters of human villus cells reconstructed using the first-trimester placental data [43]. Colors indicate cell types. CTBs, cytotrophoblasts; STBs, syncytiotrophoblasts; EVTs, extravillous trophoblasts; fFBs, fetal fibroblasts; HB, Hofbauer cells; Mycs, maternal macrophages; Endo, endothelial cells; Epi, epithelial cells; dSTCs, decidual stromal cells; TBNKs, the mixed population consisting of T cells, B cells, and natural killer cells. E UMAP visualization displays the expression pattern of GNE. F Western blot analysis of GNE expression levels in HTR8-S/Vneo cells treated with GNE siRNA (15 pmol/L). G Point graph shows the relative migration area in different time points for both the GNE inhibition group (red) and control group (CTRL, blue). The lines refer to the fitting curves. The P value between the AMA and YMA groups was determined using two-way ANOVA (P < 0.0001). H Cell migration assays for HTR8-S/Vneo cells in representative time points (0 h, 12 h, 24 h, 36 h) for CTRL (top) and GNE siRNA (bottom) groups. I A schematic illustration presents the close connection between advanced maternal age and placental dysfunction. AMA-induced abnormal alterations in specific local DNA methylation might disturb the gene expression of corresponding genes (GNE, etc.), which will reduce the cellular capability of villous cells and thus lead to the increased risk of pregnancy disorders, such as spontaneous abortion