How fetal and maternal cell types have co-evolved to enable mammalian placentation poses a unique evolutionary puzzle. Here we integrate and compare single-cell transcriptomes from six species bracketing therian mammal diversity: opossum (a marsupial), Malagasy common tenrec (an afrotherian), mouse and guinea pig (rodents), and macaque and human (primates). We identify a conserved transcriptomic signature of invasive trophoblast across eutherians, probably representing a cell type family that radiated with the evolution of haemochorial placentation. In the maternal stroma, comparative analysis reveals that the endocrine decidual cell evolved from an immunomodulatory predecidual cell type retained in Tenrec and resembling early human decidua. Fetal and maternal cell signalling shows a pronounced tendency towards disambiguation—the exclusive expression of ligands by only one partner—although few ligand–receptor pairs follow an escalatory arms race dynamic. Finally, we reconstruct the uteroplacental cell–cell communication networks of extinct mammalian ancestors, identifying signalling innovations and widespread integration of fetal trophoblast and maternal decidual cells into signalling networks. Together, these results reveal a dynamic history of cell type innovation and co-evolution at the fetal–maternal interface.

Embryo implantation is essential to pregnancy in eutherian (placental) mammals. The cellular changes that enabled invasive implantation to evolve from superficial embryo apposition, the ancestral condition still retained in marsupials, have remained unclear. We generated and compared single-cell transcriptomes from the peri-implantation and non-pregnant uterus of two rodents with invasive implantation, the mouse and the guinea pig, and the opossum, a marsupial lacking invasive implantation. Cross-species analysis of endometrial cell types and ligand-receptor signaling revealed that the opossum blastocyst-stage uterus, despite lacking fetal-maternal contact, shares key features with the eutherian window of receptivity. These include a window in which the epithelial-stromal IHH - PTCH1 axis is active, but the downstream BMP2 -mediated signaling that enables decidualization in eutherians is antagonized through a temporary spike in glandular GREM2 . We propose that a homologous progesterone-responsive endometrial remodeling reaction preceded the evolution of eutherian and marsupial modes of implantation in their respective lineages. Complementary analysis of pre- and post-implantation embryonic transcriptomes from the same species revealed broad conservation of implantation-related functional genes in the blastocyst, as well as a remarkable similarity in ligand production between the mature trophoblast and the luminal epithelium. We suggest that this redundancy between trophectoderm and luminal epithelial signaling was an enabling factor for the evolution of invasive placentation. Together, these findings suggest that eutherian implantation evolved by maternal rewiring of the output of an ancestral window of receptivity-like epithelial-stromal signaling axis toward decidualization, stabilized epithelial loss, and invasion. Significance Statement: Pregnancy in placental mammals begins with implantation and invasion of the embryo, whereas marsupials retain a superficial mode of embryo apposition. How this major evolutionary transition occurred is unknown. Comparing uterine and embryonic single-cell transcriptomes between model rodents and the opossum reveals that the marsupial uterus activates an epithelial-stromal signaling axis canonically associated with receptivity to implantation. In the marsupial uterus, its outputs are opposed by inhibitory signals, whereas in placental mammals, this program promotes uterine decidual transformation and invasion. Early-embryo implantation-related gene expression, in contrast, shows little change across species. These results challenge the view that implantation evolved through the gain of invasive properties by the embryo, and reveal a common developmental foundation from which placentals evolved their novel mode of gestation.

How fetal and maternal cell types have co-evolved to enable mammalian placentation poses a unique evolutionary puzzle. Here, we present a multi-species atlas integrating single-cell transcriptomes from six species bracketing therian mammal diversity. We find that invasive trophoblasts share a gene-expression signature across eutherians, and evidence that endocrine decidual cells evolved stepwise from an immunomodulatory cell type retained in Tenrec with affinity to human decidua of menstruation. We recover evolutionary patterns in ligand-receptor signaling: fetal and maternal cells show a pronounced tendency towards disambiguation, but a predicted arms race dynamic between them is limited. We reconstruct cell communication networks of extinct mammalian ancestors, finding strong integration of fetal trophoblast into maternal networks. Together, our results reveal a dynamic history of cell type and signaling evolution. Synopsis: The fetal-maternal interface is one of the most intense loci of cell-cell signaling in the human body. Invasion of cells from the fetal placenta into the uterus, and the corresponding transformation of maternal tissues called decidualization, first evolved in the stem lineage of eutherian mammals( 1 , 2 ). Single-cell studies of the human fetal-maternal interface have provided new insight into the cell type diversity and cell-cell interactions governing this chimeric organ( 3–5 ). However, the fetal-maternal interface is also one of the most rapidly evolving, and hence most diverse, characters among mammals( 6 ), and an evolutionary analysis is missing. Here, we present and compare single-cell data from the fetal-maternal interface of species bracketing key events in mammal phylogeny: a marsupial (opossum, Monodelphis domestica ), the afrotherian Tenrec ecaudatus, and four Euarchontoglires - guinea pig and mouse (Rodentia) together with recent macaque and human data (primates) ( 4 , 5 , 7 ). We infer cell type homologies, identify a gene-expression signature of eutherian invasive trophoblast conserved over 99 million years, and discover a predecidual cell in the tenrec which suggests stepwise evolution of the decidual stromal cell. We reconstruct ancestral cell signaling networks, revealing the integration of fetal cell types into the interface. Finally, we test two long-standing theoretical predictions, the disambiguation hypothesis( 8 ) and escalation hypothesis( 9 ), at transcriptome-wide scale, finding divergence between fetal and maternal signaling repertoires but arms race dynamics restricted to a small subset of ligand-receptor pairs. In so doing, we trace the co-evolutionary history of cell types and their signaling across mammalian viviparity.

A hallmark of eutherian pregnancy is its dependency on elevated progesterone levels for its entire length. This fact substantially affected the evolution of pregnancy and even the life history of eutherians. Progesterone synthesis by the ovarian corpus luteum, however, long predates all origins of vertebrate viviparity, which requires reconsidering the role of the corpus luteum and progesterone before and beyond placental mammals. Pregnancy originated repeatedly by co-opting the ancestral luteal phase of the ovarian cycle. Therefore, it represents a physiological equivalent and “serial homolog” of the nonpregnant luteal phase. Because of this relationship, we argue that understanding the origin and evolution of pregnancy can substantially benefit from examining the evolution and regulation of the corpus luteum during nonpregnancy. In the first part of this paper, we investigate what is known about the evolution of the corpus luteum and its changing regulation and function across vertebrates, covering both pregnant and nonpregnant cycles wherever possible. Studying pregnancy in the context of the nonpregnant cycle reveals the key evolutionary innovation that facilitated the characteristically long eutherian pregnancies: the regulatory decoupling of the pregnant and nonpregnant cycle, that is, the evolution of maternal recognition of pregnancy. Only after the two cycles became separately modifiable, could gestation length increase without deleteriously extending the time to the next fertile phase. In this review, we argue that, assuming selection for body size increase, the origin of MRP led to a punctuated increase of gestation length, which could imply that we overestimate pregnancy length in the eutherian ancestor when using conventional phylogenetic inference methods. Mechanistically, decoupling between cycles means that the progesterone-dominated phase in pregnancy is extended beyond that of nonpregnancy. In eutherians, this is achieved by extending the lifespan of progesterone-producing corpus luteum, providing additional corpora lutea, extraovarian sources of progesterone, or a combination of these. Alternatively, it can also be accomplished by shortening the nonpregnant cycle. The differences in underlying mechanisms for decoupling among the major eutherian lineages suggest that they originated independently, which leads us to conclude that eutherian gestation consists of parts that are not homologous across eutherians. Consequently, eutherian gestation length is not a homologous trait that can be productively compared across species directly. Finally, we find no evidence for an increase in gestational length due to maternal-fetal conflict. Rather, as long appreciated, body size explains a major portion of the variation in gestation length in eutherians. This suggests that the increase in eutherian gestation length was driven by the need to accommodate an increasing body size, a well-documented trend (Cope’s rule), once independent evolution of pregnant and non-pregnant cycles became possible.