eLife assessment: Sex-biased regulatory changes in the placenta of native highlanders contribute to adaptive fetal development

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Full text Figures and data Peer review Side by side Abstract eLife assessment Introduction Results Discussion Materials and methods Data availability References Article and author information Metrics Abstract Compared with lowlander migrants, native Tibetans have a higher reproductive success at high altitude though the underlying mechanism remains unclear. Here, we compared the transcriptome and histology of full-term placentas between native Tibetans and Han migrants. We found that the placental trophoblast shows the largest expression divergence between Tibetans and Han, and Tibetans show decreased immune response and endoplasmic reticulum stress. Remarkably, we detected a sex-biased expression divergence, where the male-infant placentas show a greater between-population difference than the female-infant placentas. The umbilical cord plays a key role in the sex-biased expression divergence, which is associated with the higher birth weight of the male newborns of Tibetans. We also identified adaptive histological changes in the male-infant placentas of Tibetans, including larger umbilical artery wall and umbilical artery intima and media, and fewer syncytial knots. These findings provide valuable insights into the sex-biased adaptation of human populations, with significant implications for medical and genetic studies of human reproduction. eLife assessment This fundamental study reports differential expression of key genes in full-term placenta between Tibetans and Han Chinese at high elevations, which are more pronounced in the placenta of male fetus than in female fetus. The gene expression data were collected and analyzed using solid and validated methodology, although there is limited support for hypoxia-specific responses due to a lack of low-altitude samples. Several of the placental genes found in this study have been previously reported to show signatures of positive selection in Tibetans, pointing to a potential mechanism of how human populations adapt to high elevation by mitigating the negative effects of low oxygen on fetal growth. The work will be of interest to evolutionary and population geneticists as well as researchers working on human hypoxic response. https://doi.org/10.7554/eLife.89004.5.sa0 Read the peer reviews About eLife assessments Introduction Hypobaric hypoxia at high altitude can restrict fetal growth, resulting in reduced neonatal birth weight (BW) and increased infant mortality (Moore et al., 2001). Due to long-term natural selection at high altitude, native highlanders such as Tibetans have acquired a higher reproductive success than the lowlander migrants (e.g. Han Chinese moving to high altitude), reflected by the higher newborn BW and the lower prenatal and postnatal mortality (Beall et al., 2004; Moore et al., 2011). As expected, Tibetans performed better than Han migrants during fetal development (He et al., 2023; Julian et al., 2009; Moore, 2001; Moore et al., 2011). Previous genomic studies have identified a group of genes potentially contributing to the genetic adaptation of Tibetans to high-altitude environments, and these studies were mostly focused on the blood and cardiopulmonary systems of adult Tibetans (Beall et al., 2010; Bigham et al., 2010; Deng et al., 2019; Peng et al., 2017; Peng et al., 2011; Simonson et al., 2010; Song et al., 2020; Xiang et al., 2013; Xu et al., 2011; Yang et al., 2017; Yi et al., 2010). Although a research reported the association of more pregnancies and a higher survival birth rate based on GWAS analysis of 1008 Nepali Tibetans, no adaptive genetic variant was identified (Jeong et al., 2018). Therefore, whether and how the Tibetan adaptive variants affect their fetal development and eventually improve the reproductive fitness are yet to be explored. The placenta is a temporary organ that connects fetus to the mother's uterus during pregnancy, and it begins to develop around 6–7 days after gestation (Knöfler et al., 2019). As an important organ of maternal-fetal exchange of nutrient, oxygen, and waste, the placenta plays a critical role in fetal growth and development. Previous studies have reported that placental dysfunction can lead to premature birth, abnormal fetal growth, and defects of neurological development (Guttmacher et al., 2014). At the same time, the genetic study reported that placental transcription in utero plays a key role in determining postnatal body size (Peng et al., 2018), and placenta weight is positively correlated with BW, an important trait related to the postnatal survival rate of newborns (Haeussner et al., 2013). Therefore, gene expression profiling of placenta is essential to dissecting the molecular mechanism of the successful reproduction. Recently, a transcriptomic study comparing 91 placental samples (including 47 Tibetans giving birth at high altitudes) found that the genes related to autophagy and tricarboxylic acid cycle showed significant up-regulation in Tibetans. However, no lowlander migrants living at high altitude (>2500 m) were included in this study (Wu et al., 2022). In addition, it is known that the intrauterine growth rate of the male fetus is faster than that of the female fetus, and this growth rate difference leads to a higher proportion of preeclampsia, premature delivery, and intrauterine growth restriction of the male fetus (Clifton, 2010). The between-sex difference of fetal development was explained by the higher transport efficiency, but weaker reserve capacity of the male fetal placenta (Eriksson et al., 2010). Consistently, we recently reported a male-biased pattern of BW seasonality in Tibetans and Han migrants living at high altitude. Compared to the female fetuses, the male fetuses are more sensitive to hypobaric hypoxia, reflected by their more serious BW reduction and relatively lower survival rates (He et al., 2022). However, whether the placenta plays a key role in the observed sex-biased sensitivity to hypoxia during fetal development at high altitude remains elusive. In this study, we sampled 69 full-term placental tissues, including 35 native Tibetans and 34 Han migrants living at the same altitude (an elevation of 3650 m). Seven tissue layers of the placenta were dissected and analyzed in detail. With the use of RNA-seq, we generated a systematic map of placental transcriptomes of indigenous and migrant populations living at high altitude. Markedly, we found a male-biased transcriptomic divergence between Tibetans and Han migrants, and the gene expression pattern of the fetal umbilical cord (UC) may affect the BW of the male newborns. We also observed adaptive histological changes in the Tibetan male-infant placentas, providing further evidence on the sex-biased adaptation of fetal development. Results Gene expression profiles of placentas of native Tibetans and Han migrants To understand the transcriptomic patterns of placentas of Tibetans and Han migrants at high altitude, we collected full-term (>37 weeks of gestation) placental samples from 35 Tibetans (16 male newborns and 19 female newborns) and 34 Han migrants (21 male newborns and 13 female newborns) with singleton birth (Figure 1). These Tibetan and Han migrant mothers live at the same high altitude (Lhasa, Tibet Autonomous Region, China; elevation = 3650 m), and had experienced their entire pregnancy at this altitude. The population ancestry of the subjects was confirmed by self-claim, and further validated by the genome-sequencing data (Materials and methods, Figure 1—figure supplement 1). Figure 1 with 2 supplements see all Download asset Open asset 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). We first compared the newborn traits between the Tibetan group and the Han group. As expected, the average BW of the Tibetan newborns is significantly higher than that of the Han newborns (p=0.0121, unpaired Student's t-test), a clear indication of a better fetal development in Tibetans, while there are no significant differences in placental weight (PW) and placental volume (PLV) (Figure 1—figure supplement 2). Given the placenta is a highly heterogeneous organs underlying functional and histological specializations in discrete anatomical parts (Sood et al., 2006), we dissected each placenta into seven tissue layers according to its anatomic structure (from fetal side to maternal side), including umbilical cord (UC), amnion (AN), chorion (CN), chorionic plate (CP), villus of fetal (VF), villus of intermediate (VI), and villus of maternal (VM), and we conducted RNA-seq of 483 placental samples (69 placentas × 7 layers) (Figure 1A), In total, ~12 billion short reads were generated. After applying a stringent quality control (QC) (Materials and methods), we kept 448 placental transcriptome data, and each sample contains ~27 million reads on average (sample details in Supplementary file 1a). In addition, we obtained the genome-wide variants of these mothers and their newborns from our previous studies (He et al., 2023; Zheng et al., 2023). As the placenta is a maternal-fetal mosaic organ, we first determined the maternal-fetal compositions of each dissected layer using the genomic and transcriptomic data (see Materials and methods for details). The results show that UC and VF are 100% fetal origin, so are VM, VI, CP, and AN (>95%) with tiny proportions of maternal origin. By contrast, CN is mostly maternal origin (70%) (Figure 1B). This pattern is consistent with the anatomic structure of the placenta (Sood et al., 2006). Consequently, the RNA-seq data of placenta mostly represent the transcriptomes of the newborns, except for the CN reflecting the mixed transcriptomes of mothers and newborns. The principal component analysis (PCA) indicates that the placental samples cluster by layers instead of population groups, suggesting that the transcriptomic difference between placental structures is greater than the between-population divergence. This pattern is in line with previous transcriptome of three-layer placentas (CN, AN, and decidua) of Nepali Tibetans, in which the samples were grouped by tissue type instead of ethnicity (Wu et al., 2022). Notably, the four trophoblast layers (CP, VF, VI, and VM) cannot be clearly separated in the PCA map and they form a large cluster. These four layers belong to the chorionic villus, the essential structure in placenta (Wapner and Jackson, 1988). The UC, AN, and CN layers form three separate clusters (Figure 1C). Accordingly, the gene expression heat map analysis generates the same clustering pattern (Figure 1D). These results suggest that different layers of the placenta have distinct gene expression profiles, which can provide detailed regulatory information in understanding the role of placenta in fetal development. VF and VI show the largest expression divergence between Tibetans and Han To gain insights into the differences of gene expression in placenta between Tibetans and Han, we performed differential gene expression analysis of the seven placental layers (see Materials and methods for technical details). In total, we identified 579 differentially expressed genes (DEGs) between Tibetans and Han, accounting for 3.4% of the total number of expressed genes. Our results show that VF and VI have the largest numbers of DEGs between Tibetans and Han (305 DEGs in VI and 173 DEGs in VF), accounting for 82.56% (478/579) of the total DEGs in the placenta. By contrast, the numbers of DEGs in the other five layers are much less (8–46 DEGs) (Figure 2A, Supplementary file 1b). Notably, most of the DEGs (399/579, 68.91%) are detected in only one layer (Figure 2A), an indication of a layer-specific differential expression pattern. The results of GO enrichment analyses show that the mRNA regulation metabolic process is up-regulated and immune response (indicated by the terms of defense response to virus) is down regulated in VF of Tibetans compared to Han (Figure 2B, Supplementary file 1c). In VI, the significant functional terms decreased expression in Tibetans are mostly related to proteins targeting endoplasmic reticulum (ER) (Figure 2B, Supplementary file 1c). Taken together, these results indicate that the differences between Tibetans and Han are mostly unfolded in the VF and VI layers, and Tibetans have less hypoxia-induced immune response and ER stress in the placenta (Elvekrog and Walter, 2015; Plumb et al., 2015; Tenzing et al., 2021). We did not see significant enrichment terms in the other layers due to the small numbers of DEGs. Figure 2 with 1 supplement see all Download asset Open asset 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 placental 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 villus of fetal (VF) and villus of intermediate (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 (n=35) and Han (n=34) in the seven layers of placenta. The significant between-population differences are indicated. The p-value was adjusted by FDR. Adjusted p-value (p): *p<0.05; **p<0.01; ***p<0.001; ns: not significant. For each boxplot, we draw a box from the first quartile to the third quartile. A vertical line goes through the box at the median. The whiskers go from each quartile to the minimum or maximum. (E) The volcano plots of the DEGs in VF and VI, respectively. The top genes are indicated. There are 85 shared DEGs among the placental layers. As expected, most of them are two-layer-shared DEGs between VF and VI (76/85, 89.41%), and the other 9 are multi-layer-shared DEGs (Figure 2C). In particular, there are two four-layer-shared DEGs, and the two involved genes are KCNE1 (potassium voltage-gated channel subfamily E regulatory subunit 1) and AC004057.1. KCNE1 is significantly up-regulated in all four trophoblast layers (CP, VF, VI, and VM) of Tibetans compared to Han (Figure 2D), and it is also the top up-regulated DEG in both VF and VI (Figure 2E). It encodes a potassium ion voltage-gated channel protein. The expression of KCNE1 is reportedly down-regulated in preeclampsia placentas (Mistry et al., 2011), and the in vitro experiment also showed its down-regulation in the cultured cells under hypoxia (Luo et al., 2013). Therefore, the relative up-regulation of KCNE1 in Tibetans is presumably beneficial to the reproductive outcomes at high altitude. The function of AC004057.1 is currently unknown. For the down-regulated genes in Tibetans, the top genes in VF and VI are PADI1 (peptidyl arginine deiminase 1) and TAC3 (tachykinin precursor 3), respectively (Figure 2E). PADI1 is a VF-specific DEG with 3.4-fold lower expression in Tibetans compared to Han (p=0.0001) (Figure 2—figure supplement 1). PADI1 encodes a member of the peptidyl arginine deiminase family enzymes. The expression of PADI1 is triggered by hypoxia and it stimulates PKM2 activity and contributes to the increased glycolysis under hypoxic condition (Coassolo et al., 2021). TAC3 is a VI-VF-shared DEG with both >3-fold changes in the two layers (Tibetans lower than Han, p=0.04 for VI and p=0.04 for VF) (Figure 2—figure supplement 1). TAC3 encodes a member of the tachykinin family of secreted neuropeptides, and its high expression can lead to preeclampsia (Page et al., 2006) and severe fetal growth restriction (Whitehead et al., 2013). Hence, the observed down-regulation of these two genes suggests that compared to the Han migrants, the placenta of Tibetans is more adapted to hypoxic environment, presumably with a low level of hypoxia-induced glycolysis and a reduced risk of fetal growth restriction. Sex-biased expression divergence in the placenta between Tibetans and Han Given the male fetuses are more sensitive to high-altitude hypoxia than the female fetuses as described in our previous report (He et al., 2022), we next analyzed the placental transcriptomes by taking into account the infant gender (Figure 3, Materials and methods). When the gender identity is superimposed onto the PCA map in Figure 1D, we did not see any clustering patterns by genders, implying that the gender-related expression changes do not cause a dramatic profile shift (Figure 3—figure supplement 1). However, surprisingly, we found a striking difference in view of the numbers of DEGs (Tibetans vs. Han), where the male-fetus placentas have many more DEGs than the female-fetus placentas (759 genes vs. 31 genes), and there is no intersection between them (Figure 3A). This between-sex difference is also reflected by the shared DEGs between the gender-combined DEG set and the gender-separated DEG sets, where the male set has 128 shared DEGs, but only 5 shared DEGs in the female set (Figure 3—figure supplement 1), suggesting that the male-fetus placentas contribute more to the detected between-population expression divergence. Figure 3 with 3 supplements see all Download asset Open asset Sex-biased placental expression divergence between Tibetans and Han migrants. (A) The differentially expressed gene (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 shows 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 umbilical cord (UC), amnion (AN), and villus of fetal (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). Of the 759 male-fetus DEGs, 728 DEGs are detected in one layer. There are 25 two-layer-shared DEGs, 4 three-layer-shared DEGs, and 2 four-layer-shared DEGs (Figure 3—figure supplement 2). By contrast, there is no multi-layer-shared DEGs in the female-fetus placentas. Among the 31 layer-shared DEGs in the male-fetus placentas, 22 DEGs are up-regulated and 9 DEGs are down-regulated in Tibetans. Similar to the above results, there are a large portion of overlap between the layer-shared DGEs of the male-fetus placentas and the gender-combined placentas (Figure 3—figure supplement 2). Layer-wise, in the male-fetus placentas, UC, AN, and VF have the largest numbers of DEGs (396, 177, and 189, respectively, Figure 3A, Supplementary file 1d), accounting for 95.49% (762/798) of all DEGs, while the DEGs in the other layers are much less (<15). By contrast, the great majority of the DEGs in the female-fetus placentas is from UC (23 genes, Supplementary file 1e), while the other layers only have a few DEGs (<5). This DEG pattern is highly different from that seen in the gender-combined result (Figure 2A), again, an indication of a sex-biased expression divergence between Tibetans and Han migrants at high altitude. Similar with the gender-combined result, the great majority (>95% in both males and females) of the DEGs are one-layer DEGs. Given the previous studies have been mostly focused on the functional role of the placental villi (Lorca et al., 2021; Tana et al., 2021; Vaughan et al., 2020), our gender-separated analyses illustrate the importance of UC and AN in fetal development, especially for the male fetus. Markedly, in the gender-combined result, we only detected 31 DEGs in the UC layer, contrasting the large number of DEGs (396 genes) in the male-fetus placentas. Considering 62.63% of DEGs (248/396) with an opposite direction of between-population expression divergence in males and females, respectively (Figure 3—figure supplement 3), we reckon that there might be other factors such as sample size or cell composition affecting the identification of DEGs, which could cancel out the differences in the gender-combined Consistently, the between-sex expression between in a Tibetan male vs. Han male vs. indicates that the UC shows the largest DEG difference between native Tibetans and Han migrants (Figure and Figure 3—figure supplement Consequently, the UC layer shows the most pronounced expression divergence. The sex-biased expression divergence in placenta is by the differential responses of male and female fetuses to hypoxic stress at altitude, as in our previous study (He et al., 2022). In UC, AN, and VF, where the largest numbers of DEGs were detected in the male placentas, the top 5 DEGs are in Figure These genes are involved in immune development, cell and all of which are related to fetal development et al., 2017; et al., 2019; et al., Yang et al., 2021). In UC, the enriched GO terms up-regulation of and transcription and down-regulation of in Tibetans (Figure Supplementary file 1c). In AN, Tibetans show a down-regulation in metabolic and process (Figure Supplementary file 1c). These enriched functional categories a more and a reduced risk of hypoxia-induced metabolic which are presumably beneficial to fetal development of the Tibetan male newborns et al., 2017; et al., et al., We did not see significant functional terms in VF file 1c). It is known that hypoxia can lead to of and of ER stress 2006). Our results suggest that compared to native Tibetans, the Han migrants are more to hypoxic reflected by their relative down-regulation of and transcription and this is mostly in the UC layer of the male placentas UC is the key channel of maternal-fetal exchange of oxygen and the other plays an important role in development et al., 2014). We detected the down-regulation of a gene in Tibetans (Figure The can to to in and development et al., of DHCR7 and in will cause a et al., Gene expression changes in the UC are associated with BW of the male newborns As a critical organ for maternal-fetal and oxygen the of the placenta is related to the neonatal We performed gene analysis to the association between gene expression and the The traits BW, gestation fetal and A total of and molecular were obtained from the placentas of the male infants and the female infants, respectively. We a as a significantly differential the gene expression of a is significantly correlated to population divergence (Tibetans vs. Han, and the traits (see Materials and methods for details). there are in but only 4 in (Figure Supplementary file Materials and methods). Markedly, the UC layer of the male infants has 3 including 4 × and 9 × (Figure which are significantly associated with two newborn traits and a of UC in fetal development, especially for the male There are four AN two VF and one VI that are associated with five newborn including and and and and respectively (Figure For the female placentas, four are associated with and respectively (Figure These identified suggest that the difference of regulation of the placental layers between Tibetans and Han migrants do have on the newborn especially on the male Figure 4 with 1 supplement see all Download asset Open asset Gene expression in umbilical cord (UC) of the male infants with neonatal (A) The heat of showing the between gene expression and the newborn traits of the male-infant placentas and the female-infant placentas significantly differential (B) The counts of the differentially expressed genes (DEGs) in the seven placental layers of the male infants and the female infants and their with the newborn traits lower (C) The gene of of the male (D) Gene expression of two genes in UC of the male infants between Tibetans and Han The p-value was adjusted by FDR. Adjusted p-value (p): *p<0.05; ns: not significant. For each boxplot, we draw a box from the first quartile to the third quartile. A vertical line goes through the box at the median. The whiskers go from each quartile to the minimum or maximum. We next at the DEGs in the For the male placentas, there are a of DEGs involved in the By contrast, among the four in the female placentas, there is only one DEG (Figure This pattern is consistent with the male-biased gene expression divergence between Tibetans and In particular, in the UC layer of the male placentas, there are DEGs in and DEGs in of the DEGs in are down-regulated in the UC layer of Tibetans (Figure and this is correlated with BW (Figure and Figure supplement 1), that the down-regulated gene expression in the UC layer of the Tibetan male placentas may contribute to the higher BW of the Tibetan newborns compared to There are seven genes in and all of them are down-regulated in the Tibetan male placentas (Figure For subunit and it is one of the known genes et al., subunit is a gene that encodes the growth of the by hypoxia to cell under hypoxia et al., 2004; Figure Hence, the down-regulation of the great majority of DEGs in suggests that the male fetuses are more sensitive to hypoxic stress than the female fetuses, and compared to native Tibetans, they are more in the Han migrants, which is associated with the higher BW in the adapted compared to the migrants Taken together, the above results indicate that the UC layer plays a key role in the development of the male fetus and is the only DEG in the of the female newborns (Figure The gene expression level of in the VF layer is positively correlated with of the female newborns, an of fetal growth et al., but not the male newborns (Figure supplement 1). Consistently, it is up-regulated in Tibetans, to the higher in Tibetans (He et al., 2023). the expression pattern of in placenta development and growth and 2014). outcomes of the sex-biased expression divergence To further test the of