Comparative single cell analysis reveals complex patterns of cell type and cell signaling innovations at the fetal-maternal interface.

The decidual-placental interface is one of the most diverse and rapidly evolving tissues in mammals. Its origin as a chimeric fetal-maternal tissue poses a unique evolutionary puzzle. We present single-cell RNA sequencing atlases from the fetal-maternal interfaces of the opossum, a marsupial, the Malagasy common tenrec, an afrotherian with primitive reproductive features, and mouse, guinea pig, and human. Invasive trophoblast shares a common transcriptomic signature across eutherians, which we argue represents a cell type family that radiated following the evolution of hemochorial placentation. We find evidence that the eutherian decidual stromal cell evolved stepwise from a predecidual state retained in Tenrec , followed by a second decidual cell type originating in Boreoeutheria with endocrine characteristics. We reconstruct ligand-receptor signaling to test evolutionary hypotheses at scale. Novel trophoblast and decidual cell types display strong integration into signaling networks compared to other cells. Additionally, we find consistent disambiguation between fetal and maternal signaling. Using phylogenetic analysis, we infer the cell-cell signaling network of the Placental common ancestor, and identify increased rates of signaling evolution in Euarchontoglires. Together, our findings reveal novel cell type identities and cell signaling dynamics at the mammalian fetal-maternal interface.

Single-cell analysis of prostaglandin E2-induced human decidual cell in vitro differentiation: a minimal ancestral deciduogenic signal†.

The decidua is a hallmark of reproduction in many placental mammals. Differentiation of decidual stromal cells is known to be induced by progesterone and the cyclic AMP/protein kinase A (cAMP/PKA) pathway. Several candidates have been identified as the physiological stimulus for adenylyl cyclase activation, but their relative importance remains unclear. To bypass this uncertainty, the standard approach for in vitro experiments uses membrane-permeable cAMP and progestin. We phylogenetically infer that prostaglandin E2 (PGE2) likely was the signal that ancestrally induced decidualization in conjunction with progesterone. This suggests that PGE2 and progestin should be able to activate the core gene regulatory network of decidual cells. To test this prediction, we performed a genome-wide study of gene expression in human endometrial fibroblasts decidualized with PGE2 and progestin. Comparison to a cAMP-based protocol revealed shared activation of core decidual genes and decreased induction of senescence-associated genes. Single-cell transcriptomics of PGE2-mediated decidualization revealed a distinct, early-activated state transitioning to a differentiated decidual state. PGE2-mediated decidualization was found to depend upon progestin-dependent induction of PGE2 receptor 2 (PTGER2) which in turn leads to PKA activation upon PGE2 stimulation. Progesterone-dependent induction of PTGER2 is absent in opossum, an outgroup taxon of placental mammals which is incapable of decidualization. Together, these findings suggest that the origin of decidualization involved the evolution of progesterone-dependent activation of the PGE2/PTGER2/PKA axis, facilitating entry into a PKA-dominant rather than AKT-dominant cellular state. We propose the use of PGE2 for in vitro decidualization as an alternative to 8-Br-cAMP.

Evolution of Embryo Implantation Was Enabled by the Origin of Decidual Stromal Cells in Eutherian Mammals.

Mammalian pregnancy evolved in the therian stem lineage, that is, before the common ancestor of marsupials and eutherian (placental) mammals. Ancestral therian pregnancy likely involved a brief phase of attachment between the fetal and maternal tissues followed by parturition-similar to the situation in most marsupials including the opossum. In all eutherians, however, embryo attachment is followed by implantation, allowing for a stable fetal-maternal interface and an extended gestation. Embryo attachment induces an attachment reaction in the uterus that is homologous to an inflammatory response. Here, we elucidate the evolutionary mechanism by which the ancestral inflammatory response was transformed into embryo implantation in the eutherian lineage. We performed a comparative uterine transcriptomic and immunohistochemical study of three eutherians, armadillo (Dasypus novemcinctus), hyrax (Procavia capensis), and rabbit (Oryctolagus cuniculus); and one marsupial, opossum (Monodelphis domestica). Our results suggest that in the eutherian lineage, the ancestral inflammatory response was domesticated by suppressing one of its modules detrimental to pregnancy, namely, neutrophil recruitment by cytokine IL17A. Further, we propose that this suppression was mediated by decidual stromal cells, a novel cell type in eutherian mammals. We tested a prediction of this model in vitro and showed that decidual stromal cells can suppress the production of IL17A from helper T cells. Together, these results provide a mechanistic understanding of early stages in the evolution of eutherian pregnancy.

It Takes Two to Tango: Including a Female Perspective in Reproductive Biology.

Like many scientific disciplines, the field of reproductive biology is subject to biases in terminology and research foci. For example, females are often described as coy and passive players in reproductive behaviors and are termed "promiscuous" if they engage in extra-pair copulations. Males on the other hand are viewed as actively holding territories and fighting with other males. Males are termed "multiply mating" if they mate with multiple females. Similarly, textbooks often illustrate meiosis as it occurs in males but not females. This edition of Integrative and Comparative Biology (ICB) includes a series of papers that focus on reproduction from the female perspective. These papers represent a subset of the work presented in our symposium and complementary sessions on female reproductive biology. In this round table discussion, we use a question and answer format to leverage the diverse perspectives and voices involved with the symposium in an exploration of theoretical, cultural, pedagogical, and scientific issues related to the study of female biology. We hope this dialog will provide a stepping-stone toward moving reproductive science and teaching to a more inclusive and objective framework.

The Primacy of Maternal Innovations to the Evolution of Embryo Implantation.

Embryo implantation is a hallmark of the female reproductive biology of eutherian (placental) mammals and does not exist in a sustainable form in any other vertebrate group. Implantation is the initial process that leads to a sustained fetal-maternal unit engendering a complex functional relationship between the mother and the embryo/fetus. The nature of this relationship is often portrayed as one of conflict between an aggressive embryo and a passive or defensive maternal organism. Recent progress in elucidating the evolutionary origin of eutherian pregnancy leads to a different picture. The emerging scenario suggests that the very initial stages in the evolution of embryo implantation required evolutionary changes to the maternal physiology which modified an ancestral generic mucosal inflammation in response to the presence of the embryo into an active embedding process. This "female-first" evolutionary scenario also explains the role of endometrial receptivity in human pregnancy. On the marsupial side, where in most animals the fetal-maternal interaction is short and does not lead to a long term sustainable placentation, the relationship is mutual. In these mammals, uterine inflammation is followed by parturition in short order. The inflammatory signaling pathways, however, are cooperative, i.e., they are performed by both the fetus and the mother and therefore we call this relationship "cooperative inflammation." Based on these discoveries we reconceive the narrative of the maternal-fetal relationship.

Cooperative inflammation: The recruitment of inflammatory signaling in marsupial and eutherian pregnancy.

The evolution of viviparity in therian mammals, i.e. marsupials and "placental" mammals, occurred by retention of the conceptus in the female reproductive tract and precocious "hatching" from the shell coat. Both eutherian embryo implantation and the opossum embryo attachment reaction are evolutionarily derived from and homologous to a defensive inflammatory process induced after shell coat hatching. However, both lineages, marsupials and placental mammals, have modified the inflammatory response substantially. We review the induction, maintenance, and effects of inflammation throughout pregnancy, with special attention to the role of prostaglandins and the mucosal inflammatory response, both of which likely had roles in early mammalian viviparity. We propose that the key step was not only suppression of the inflammatory response after implantation in placental mammals, but also the transfer of the inflammatory cell-cell communication network to a different set of cell types than in generic inflammation. To support this conclusion we discuss evidence that pro-inflammatory signal production in the opossum is not limited to maternal cells, as expected in bona fide defensive inflammation, but also includes fetal tissues, in a process we term cooperative inflammation. The ways in which the inflammatory reaction was independently modified in these two lineages helps explain major life history differences between extant marsupials and eutherians.