How Placenta Promotes the Successful Reproduction in High-Altitude Populations: A Transcriptome Comparison between Adaptation and Acclimatization

Abstract

As the best adapted high altitude population, Tibetans feature a relatively high offspring survival rate. Genome-wide studies have identified hundreds of candidate SNPs related to high altitude adaptation of Tibetans, although most of them have unknown functional relevance. To explore the mechanisms behind successful reproduction at high altitudes, we compared the placental transcriptomes of Tibetans, sea level Hans (SLHan), and Han immigrants (ImHan). Among the three populations, placentas from ImHan showed a hyperactive gene expression pattern. Their increased activation demonstrates a hypoxic stress response similar to sea level individuals experiencing hypoxic conditions. Unlike ImHan, Tibetan placentas were characterized by the significant up-regulation of placenta-specific genes, and the activation of autophagy and the tricarboxylic acid (TCA) cycle. Certain conserved hypoxia response functions, including the antioxidant system and angiogenesis, were activated in both ImHan and Tibetans, but mediated by different genes. The coherence of specific transcriptome features linked to possible genetic contribution was observed in Tibetans. Furthermore, we identified a novel Tibetan-specific EPAS1 isoform with a partial deletion at exon six, which may be involved in the adaption to hypoxia through the EPAS1-centred gene network in the placenta. Overall, our results show that the placenta grants successful pregnancies in Tibetans by strengthening the natural functions of the placenta itself. On the other hand, the placenta of ImHan was in an inhabiting time-dependent acclimatization process representing a common hypoxic stress response pattern.

Keywords: Tibetans; acclimatization; adaptation; hypoxia; placenta; transcriptome.

Fig. 1.

Diverse expressions of placental genes in Tibetans, ImHan, and SLHan. (A) Hierarchical clustering of all samples. Notably, the dominant clustering was categorized by tissues: amnion (purple), chorion (ivory), and decidua (light blue); secondarily, samples were grouped by populations; the sex of the fetus had no effect (light blue for boy and pink for girl) on the clustering results, although certain sampling biases may have influenced the clustering patterns in amnion and decidua due to more female samples in the ImHan group. Under each type of tissue, ImHan (orange) show a distinct expression pattern from both SLHan (green) and Tibetans (blue). (B) Lower global Pearson correlation coefficients (Pcc) in ImHan, corresponding to a higher heterogeneity than in SLHan and Tibetans. (C) A significantly higher proportion of highly expressed genes was found in ImHan than in SLHan and Tibetans. (D) Hierarchical clustering of the ImHan generations. High similarity of the transcriptomes was seen in the residents of multiple generations (G ≥ 3, upper in pink). In contrast, significant heterogeneity was found among the first and second generations (G1 and G2, lower in gray). (E) Distinct transcriptome patterns of native and immigrant highlanders. The left panel shows the expression heat map generated by STEM across the three altitudes’ populations. Inflammatory and other immune-related responses were significantly enriched in ImHan (up-right, top 5 by P-values), whereas female pregnancy and related transporting genes were highly expressed in Tibetans (bottom-right, top 5 by P-values).

Fig. 2.

DEGs among SLHan, ImHan, and Tibetans and functional comparison of the most significant DEGs. (A) Numbers of DEGs in pairwise comparison. (B) Significantly overrepresented biological processes (cutoff at top 5) and pathways of the up-regulated DEGs in SLHan (olive), ImHan (orange), and Tibetans (purple). (C) Progressively up-regulated genes upon hypoxia treatment in the HUVECs of SLHan. (D) Venn diagram showing the number of up-regulated genes and overrepresented pathways and processes, generated by kKEGG and GO ORA, in ImHan and HUVECs. (E) DEGs Comparison of Tibetans/SLHan (top), ImHan/SLHan (left), and Tibetans/ImHan (right). (F) Functional enrichment of the Tibetan-specific genes. (G) Comparison between the general placenta’s highly expressed genes and the Tibetan-specific DEGs in chorion (1,053, see text). Most of the 26 placental-specific genes were up-regulated only in Tibetans (bottom). (H) The immunohistochemistry staining of CGB, CSHL1, and PSG6 in syncytiotrophoblast illustrates their up-regulation in Tibetans.

Fig. 3.

Placental expression of EPSA1 in Tibetans. (A) EPAS1 and its interacting genes in the PPI network. A total of 52 genes were extracted from STRING with high confidence (≥ 0.9) as the interacting genes of EPAS1; the edges with |Pcc| ≥ 0.4 are displayed in the network. (B) Pcc distribution between EPAS1 and its interacting genes in the PPI network shows significantly higher Pcc values in Tibetans. (C) Co-factors and regulators of EPAS1 showed strong co-expression with EPAS1 in Tibetans. (D) Most EPAS1 targeted genes were significantly up-regulated in Tibetans. Red indicates the genes specifically expressed in the placenta. (E) Placenta-specific genes showing strong co-expression with EPAS1 in Tibetans. Edges with |Pcc| ≥ 0.4 are displayed in the network. The genes in pink were up-regulated and the genes in green were down-regulated in Tibetans. (F) Splice junction patterns between exons five and six. Light red lines indicate various lengths of splicing. A novel junction 56 bp downstream of the wild-type splicing site of exon six was identified and observed mainly in Tibetans. Stars indicate each of four randomly picked samples with rs150877473 sequenced, gray stars indicate CC, green stars show CG, and red stars illustrate the Tibetan-specific GG genotype (supplementary table 8, Supplementary Material online). (G) Alternative splicing of exon six results in a novel isoform of EPAS1 in Tibetans. (H) Consequence of the alternative splicing on the protein structure of EPAS1. The upper diagram shows the protein structure of wild-type EPAS1, and the bottom diagram predicts the truncated EPAS1 protein structure caused by the alternative splicing. (I) Validation of the alternative splicing event in the HUVECs of SLHan.

Fig. 4.

Differentially activated pathways in highlanders. (A) Activation of the TCA cycle in Tibetans only (left) and activated oxidative phosphorylation in both ImHan and Tibetans (right). Several rate-limiting enzymes from the TCA cycle were up-regulated in Tibetans. (B) Activation of the antioxidant-associated genes in ImHan and Tibetans. The upper row shows the expression of three genes as ROS markers, and the lower row lists the expression of three antioxidant-associated genes. (C) Autophagy was activated in Tibetans only, and proteasome was activated in both ImHan and Tibetans. (D) Highly expressed autophagy-associated genes in Tibetans. The expression of three autophagy marker genes is indicated in the upper row. The lower row shows three autophagy genes mediated by calcium signaling that were highly expressed in Tibetans. (E) A proposed model of the signaling cascades granting successful reproductions in two groups of highlanders. These cascades are illustrated in the bio-processes of energy metabolism (green square), angiogenesis (pink square), and antioxidant-related pathways (blue square, see text). Significantly up-regulated genes and pathways are highlighted with a blue (Tibetans only), pink (ImHan only), or purple (ImHan and Tibetans) frame. The gray frame shows the pathway without significant expression changes. Asterisks mark the genes and pathways that contain Tibetan-specific genetic variants as shown by previous studies as well as our recent results (data not shown).