Sampling strategy and gene expression patterns of the placental layers of Tibetans and Han migrants at high altitude.

(A) The strategy of sampling the full-term placentas. The placenta was dissected into seven layers, as shown from the fetal side to the maternal side, are umbilical cord (UC), amnion (AN), chorion (CN), chorionic plate (CP), villus of fetal (VF), villus of intermediate (VI) and villus of maternal (VM). The seven layers are labeled with seven different colors. (B) Analysis of the maternal-fetal origins of the placental layers. (C) The map of principal component analysis (PCA) showing the clustering pattern of placental tissue layers. (D) The heat map of gene expression in the seven placental layers of Tibetans and Han migrants reveals the same clustering pattern seen in the PCA map. The layers are color-coded (see panel-A).

The gene expression differences of the placental layers between Tibetans and Han migrants.

(A) The numbers of differentially expressed genes (DEGs) between Tibetans and Han in each placenta layer; Purple: the number of up-regulated DEGs in Tibetans; Green: the number of down-regulated DEGs in Tibetans; The pie chart indicates the shared DEGs among two and more placental layers. (B) The enriched functional categories (GO terms) of the up-regulated (top) and the down-regulated (bottom) DEGs in VF and VI, respectively. The dashed line denotes the threshold of significant test (adjusted p-value < 0.05). (C) The heat map of the 85 shared DEGs among two and more placental layers. (D) Comparison of the expression levels of KCNE1 between Tibetans and Han in the seven layers of placenta. The significant between-population differences are indicated. adjusted p-value (p): *-p < 0.05; **-p < 0.01; ***-p < 0.001. (E) The volcano plots of the DEGs in VF and VI, respectively. The top genes are indicated.

Sex-biased placental expression divergence between Tibetans and Han migrants.

(A) The DEG numbers of the seven placental layers in the male infants and the female infants, respectively. Left: female (pink); Right: male (blue); Venn chart: the overlapped DEGs between male-infant placentas and female-infant placentas. (B) Bar plot show the difference of gene counts between cross-sex DEGs in native Tibetans and Han migrants. (C) The up- and down-regulated genes in the male infants of Tibetans compared to Han, shown in the volcano plots of UC, AN and VF; The top-5 DEGs are indicated. The example gene DHCR7 is highlighted in red. (D) The enriched functional categories (GO terms) of the up-regulated (purple) and the down-regulated (green) DEGs in UC and AN, respectively. The dashed line denotes the threshold of significant test (adjusted p-value < 0.05).

Gene expression modules in UC of the male infants correlate with neonatal phenotypes.

(A) The heat maps of p-values showing the correlations between gene expression and the newborn traits of the male-infant placentas (left) and the female-infant placentas (right). Inner ring: module name; Outer ring: layers; Red: significantly differential modules (SDMs). (B) The counts of the module-associated DEGs in the seven placental layers of the male infants and the female infants (the upper panels), and their correlations with the newborn traits (the lower panels). (C) The gene co-expression network of Module 8 of the male infants. (D) Gene expression comparisons of two hub genes in UC of the male infants between Tibetans and Han.

Sex-biased histological changes in the male-infant placentas of Tibetans.

(A) Schematic diagram of the sampling strategy in histological analysis. (B)(C) The H&E stained cross-sections of the UC vessels (umbilical vein (UV); panel B; and umbilical artery (UA); panel C). The umbilical vein wall (UVW) and umbilical artery intima and media (UAIM) are highlighted in red. The boxplots show the comparisons of area size of UVW and UAIM between Tibetan and Han male infants (left) or female infants (right). The two umbilical arteries of each umbilical cord were subject to analysis in Panel-C. (D)(E) The H&E stained sections of the placental VI and VF. The arrows denote the syncytial knots. The boxplots show the comparison of the villi numbers or the syncytial knot/villi ratios between Tibetan and Han male infants (left) or female infants (right). Unpaired Student’s t-tests are used to evaluate the significance of difference. *, p-value < 0.05; **, p-value < 0.01; ns, not significant.

The differentially expressed genes in the placenta that underwent positive selection in Tibetans.

(A) The Venn plots show the intersections between the placental DEGs and the 192 Tibetan selection-nominated genes (TSNGs), covering the gender-combined and the gender-separated DEGs. (B) The three DEGs (gender-combined) under positive selection in Tibetans. The upper panels show the allele frequencies of variants with the strongest signals of selection within the gene. Besides of Tibetans and Han Chinese, the data from three other reference populations are also presented, including Japanese (JPT), Europeans (CEU) and Africans (YRI) from the 1000 Genome Project. The solid and hollow bars in red denote the allele frequencies in the published 1,001 Tibetan individuals and 35 Tibetan individuals, respectively, and the solid and hollow bars in blue denote the allele frequencies in the published 103 Han individuals and 34 Han individuals, respectively. The bottom panels show the comparison of the expression levels between Tibetans and Han in the seven layers of placenta. Only the significant between-population differences are indicated. adjusted p-value: *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001. (C) The three DEGs (gender-separated analysis) with signals of positive selection in Tibetans.