Arun R. Chavan

Genetic Associations with Gestational Duration and Spontaneous Preterm Birth

BACKGROUND Despite evidence that genetic factors contribute to the duration of gestation and the risk of preterm birth, robust associations with genetic variants have not been identified. We used large data sets that included the gestational duration to determine possible genetic associations. METHODS We performed a genomewide association study in a discovery set of samples obtained from 43,568 women of European ancestry using gestational duration as a continuous trait and term or preterm (<37 weeks) birth as a dichotomous outcome. We used samples from three Nordic data sets (involving a total of 8643 women) to test for replication of genomic loci that had significant genomewide association (P<5.0×10‐8) or an association with suggestive significance (P<1.0×10‐6) in the discovery set. RESULTS In the discovery and replication data sets, four loci (EBF1, EEFSEC, AGTR2, and WNT4) were significantly associated with gestational duration. Functional analysis showed that an implicated variant in WNT4 alters the binding of the estrogen receptor. The association between variants in ADCY5 and RAP2C and gestational duration had suggestive significance in the discovery set and significant evidence of association in the replication sets; these variants also showed genomewide significance in a joint analysis. Common variants in EBF1, EEFSEC, and AGTR2 showed association with preterm birth with genomewide significance. An analysis of mother–infant dyads suggested that these variants act at the level of the maternal genome. CONCLUSIONS In this genomewide association study, we found that variants at the EBF1, EEFSEC, AGTR2, WNT4, ADCY5, and RAP2C loci were associated with gestational duration and variants at the EBF1, EEFSEC, and AGTR2 loci with preterm birth. Previously established roles of these genes in uterine development, maternal nutrition, and vascular control support their mechanistic involvement. (Funded by the March of Dimes and others.)

Embryo implantation evolved from an ancestral inflammatory attachment reaction

Significance Our data suggest that implantation in eutherians is derived from an ancestral inflammatory reaction to embryo attachment in the therian ancestor. These results explain the paradoxical role of inflammation at the beginning and the end of pregnancy in humans: Inflammation is necessary for implantation and parturition, but for most of pregnancy, inflammation threatens the continuation of pregnancy. We argue that the role of inflammation during implantation is an ancestral response to the embryo as a foreign body. By changing the way investigators think about implantation, we expect this research to contribute to new ways to study and treat implantation disorders, the most vulnerable step of assisted reproductive technology, in women. The molecular changes that support implantation in eutherian mammals are necessary to establish pregnancy. In marsupials, pregnancy is relatively short, and although a placenta does form, it is present for only a few days before parturition. However, morphological changes in the uterus of marsupials at term mimic those that occur during implantation in humans and mice. We investigated the molecular similarity between term pregnancy in the marsupials and implantation in eutherian mammals using the gray short-tailed opossum (Monodelphis domestica) as a model. Transcriptomic analysis shows that term pregnancy in the opossum is characterized by an inflammatory response consistent with implantation in humans and mice. This immune response is temporally correlated with the loss of the eggshell, and we used immunohistochemistry to report that this reaction occurs at the materno–fetal interface. We demonstrate that key markers of implantation, including Heparin binding EGF-like growth factor and Mucin 1, exhibit expression and localization profiles consistent with the pattern observed during implantation in eutherian mammals. Finally, we show that there are transcriptome-wide similarities between the opossum attachment reaction and implantation in rabbits and humans. Our data suggest that the implantation reaction that occurs in eutherians is derived from an attachment reaction in the ancestral therian mammal which, in the opossum, leads directly to parturition. Finally, we argue that the ability to shift from an inflammatory attachment reaction to a noninflammatory period of pregnancy was a key innovation in eutherian mammals that allowed an extended period of intimate placentation.

Single-cell transcriptomics of the human placenta: inferring the cell communication network of the maternal-fetal interface

Organismal function is, to a great extent, determined by interactions among their fundamental building blocks, the cells. In this work, we studied the cell-cell interactome of fetal placental trophoblast cells and maternal endometrial stromal cells, using single-cell transcriptomics. The placental interface mediates the interaction between two semiallogenic individuals, the mother and the fetus, and is thus the epitome of cell interactions. To study these, we inferred the cell-cell interactome by assessing the gene expression of receptor-ligand pairs across cell types. We find a highly cell-type-specific expression of G-protein-coupled receptors, implying that ligand-receptor profiles could be a reliable tool for cell type identification. Furthermore, we find that uterine decidual cells represent a cell-cell interaction hub with a large number of potential incoming and outgoing signals. Decidual cells differentiate from their precursors, the endometrial stromal fibroblasts, during uterine preparation for pregnancy. We show that decidualization (even in vitro) enhances the ability to communicate with the fetus, as most of the receptors and ligands up-regulated during decidualization have their counterpart expressed in trophoblast cells. Among the signals transmitted, growth factors and immune signals dominate, and suggest a delicate balance of enhancing and suppressive signals. Finally, this study provides a rich resource of gene expression profiles of term intravillous and extravillous trophoblasts, including the transcriptome of the multinucleated syncytiotrophoblast.

What was the ancestral function of decidual stromal cells? A model for the evolution of eutherian pregnancy.

In human and mouse, decidual stromal cells (DSC) are necessary for the establishment (implantation) and the maintenance of pregnancy by preventing inflammation and the immune rejection of the semi-allograft conceptus. DSC originated along the stem lineage of eutherian mammals, coincidental with the origin of invasive placentation. Surprisingly, in many eutherian lineages decidual cells are lost after the implantation phase of pregnancy, making it unlikely that DSC are necessary for the maintenance of pregnancy in these animals. In order to understand this variation, we review the literature on the fetal-maternal interface in all major eutherian clades Euarchontoglires, Laurasiatheria, Xenarthra and Afrotheria, as well as the literature about the ancestral eutherian species. We conclude that maintaining pregnancy may not be a shared derived function of DSC among all eutherian mammals. Rather, we propose that DSC originated to manage the inflammatory reaction associated with invasive implantation. We envision that this happened in a stem eutherian that had invasive placenta but still a short gestation. We further propose that extended gestation evolved independently in the major eutherian clades explaining why the major lineages of eutherian mammals differ with respect to the mechanisms maintaining pregnancy.

The transcriptomic evolution of mammalian pregnancy: gene expression innovations in endometrial stromal fibroblasts

The endometrial stromal fibroblast (ESF) is a cell type present in the uterine lining of therian mammals. In the stem lineage of eutherian mammals, ESF acquired the ability to differentiate into decidual cells in order to allow embryo implantation. We call the latter cell type “neo-ESF” in contrast to “paleo-ESF” which is homologous to eutherian ESF but is not able to decidualize. In this study, we compare the transcriptomes of ESF from six therian species: Opossum (Monodelphis domestica; paleo-ESF), mink, rat, rabbit, human (all neo-ESF), and cow (secondarily nondecidualizing neo-ESF). We find evidence for strong stabilizing selection on transcriptome composition suggesting that the expression of approximately 5,600 genes is maintained by natural selection. The evolution of neo-ESF from paleo-ESF involved the following gene expression changes: Loss of expression of genes related to inflammation and immune response, lower expression of genes opposing tissue invasion, increased markers for proliferation as well as the recruitment of FOXM1, a key gene transiently expressed during decidualization. Signaling pathways also evolve rapidly and continue to evolve within eutherian lineages. In the bovine lineage, where invasiveness and decidualization were secondarily lost, we see a re-expression of genes found in opossum, most prominently WISP2, and a loss of gene expression related to angiogenesis. The data from this and previous studies support a scenario, where the proinflammatory paleo-ESF was reprogrammed to express anti-inflammatory genes in response to the inflammatory stimulus coming from the implanting conceptus and thus paving the way for extended, trans-cyclic gestation.

The mammalian decidual cell evolved from a cellular stress response

Among animal species, cell types vary greatly in terms of number and kind. The number of cell types found within an organism differs considerably between species, and cell type diversity is a significant contributor to differences in organismal structure and function. These observations suggest that cell type origination is a significant source of evolutionary novelty. The molecular mechanisms that result in the evolution of novel cell types, however, are poorly understood. Here, we show that a novel cell type of eutherians mammals, the decidual stromal cell (DSC), evolved by rewiring an ancestral cellular stress response. We isolated the precursor cell type of DSCs, endometrial stromal fibroblasts (ESFs), from the opossum Monodelphis domestica. We show that, in opossum ESFs, the majority of decidual core regulatory genes respond to decidualizing signals but do not regulate decidual effector genes. Rather, in opossum ESFs, decidual transcription factors function in apoptotic and oxidative stress response. We propose that rewiring of cellular stress responses was an important mechanism for the evolution of the eutherian decidual cell type.

Reply to Liu: Inflammation before implantation both in evolution and development

In a paper in PNAS (1) we show that, in opossums, endometrial inflammation follows attachment of the embryo to the uterine wall. We argue that this is similar to what occurred in the first live-bearing mammals, and that inflammatory signaling seen at implantation in eutherians evolved from this attachment-induced inflammation. In a letter on our paper, Liu (2) presents evidence of endometrial inflammatory gene expression in mice before embryonic attachment. Liu infers that inflammatory signaling at implantation cannot be a consequence of embryonic attachment because inflammation precedes implantation in mice and humans. Liu alternatively proposes that inflammation during implantation in eutherians has evolved because the embryo coopted L-selectin expression, allowing it to “act” like a leukocyte and facilitate endometrial infiltration. This is an interesting hypothesis, because it may explain why inflammatory … [↵][1]1To whom correspondence should be addressed. Email: oliver.griffith{at}yale.edu. [1]: #xref-corresp-1-1

Evolution of Gene Expression in the Uterine Cervix related to Steroid Signaling: Conserved features in the regulation of cervical ripening

The uterine cervix is the boundary structure between the uterus and the vagina and is key for the maintenance of pregnancy and timing of parturition. Here we report on a comparative transcriptomic study of the cervix of four placental mammals, mouse, guinea pig, rabbit and armadillo, and one marsupial, opossum. Our aim is to investigate the evolution of cervical gene expression as related to putative mechanisms for functional progesterone withdrawal. Our findings are: 1) The patterns of gene expression in eutherian (placental) mammals are consistent with the notion that an increase in the E/P4 signaling ratio is critical for cervical ripening. How the increased E/P4 ratio is achieved, however, is variable between species. 2) None of the genes related to steroid signaling, that are modulated in eutherian species, change expression during opossum gestation. 3) A tendency for decreased expression of progesterone receptor co-activators (NCOA1, -2 and -3, and CREBBP) towards term is a shared derived feature of eutherians. This suggests that parturition is associated with broad scale histone de-acetylation. Western-blotting on mouse cervix confirmed large scale histone de-acetylation in labor. This finding may have important implications for the control of premature cervical ripening and prevention of preterm birth in humans.

Evolution of embryo implantation was enabled by the origin of decidual cells in eutherian mammals

Embryo implantation is the first step in the establishment of pregnancy in eutherian (Placental) mammals. Although viviparity evolved prior to the common ancestor of marsupials and eutherian mammals (therian ancestor), implantation is unique to eutherians. The ancestral therian pregnancy likely involved a short phase of attachment between the fetal and maternal tissues followed by parturition rather than implantation, similar to the mode of pregnancy found in marsupials such as the opossum. Embryo implantation in eutherian mammals as well as embryo attachment in opossum, induce a homologous inflammatory response in the uterus. Here, we elucidate the evolutionary mechanism by which the ancestral inflammatory fetal-maternal attachment was transformed into the process of implantation. We performed a comparative transcriptomic and immunohistochemical study of the gravid and non-gravid uteri of two eutherian mammals, armadillo (Dasypus novemcinctus) and hyrax (Procavia capensis); a marsupial outgroup, opossum (Monodelphis domestica); and compared it to previously published data on rabbit (Oryctolagus cuniculus). This taxon sampling allows inference of the eutherian ancestral state. Our results show that in the eutherian lineage, the ancestral inflammatory response was domesticated by suppressing a detrimental component viz. signaling by the cytokine IL17A, while retaining components that are beneficial to placentation, viz. angiogenesis, vascular permeability, remodeling of extracellular matrix. IL17A mediates recruitment of neutrophils to inflamed mucosal tissues, which, if unchecked, can damage the uterus as well as the embryo and lead to expulsion of the fetus. We hypothesized that the uterine decidual stromal cells, which evolved coincidentally with embryo implantation, evolved, in part, to prevent IL17A-mediated neutrophil infiltration. We tested a prediction of this hypothesis in vitro, and showed that decidual stromal cells can suppress differentiation of human naïve T cells into IL17A-producing Th17 cells. Together, these results provide a mechanistic understanding of early stages of the evolution of the eutherian mode of pregnancy, and also identify a potentially ancestral function of an evolutionary novelty, the decidual stromal cell-type.