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.

Reading the palimpsest of cell interactions: What questions may we ask of the data?

Biological function depends on the composition and structure of the organism, the latter describing the organization of interactions between parts. While cells in multicellular organisms are capable of a remarkable degree of autonomy, most functions do require cell communication: the coordination of functions (growth, differentiation, and apoptosis), the compartmentalization of cellular processes, and the integration of cells into higher levels of structural organization. A wealth of data on putative cell interactions has become available, yet its biological interpretation depends on our expectations about the structure of interaction networks. Here, we attempt to formulate basic questions to ask when interpreting cell interaction data. We build on the understanding that cells fulfill two general functions: the integrity-maintaining and the organismal service function. We derive the expected patterns of cell interactions considering two intertwined aspects: the functional and the evolutionary. Based on these, we propose guidelines for analysis and interpretation of transcriptional cell-interactome data.

A common allele increases endometrial Wnt4 expression, with antagonistic implications for pregnancy, reproductive cancers, and endometriosis.

The common human SNP rs3820282 is associated with multiple phenotypes including gestational length and likelihood of endometriosis and cancer, presenting a paradigmatic pleiotropic variant. Deleterious pleiotropic mutations cause the co-occurrence of disorders either within individuals, or across population. When adverse and advantageous effects are combined, pleiotropy can maintain high population frequencies of deleterious alleles. To reveal the causal molecular mechanisms of this pleiotropic SNP, we introduced this substitution into the mouse genome by CRISPR/Cas 9. Previous work showed that rs3820282 introduces a high-affinity estrogen receptor alpha-binding site at the Wnt4 locus. Here, we show that this mutation upregulates Wnt4 transcription in endometrial stroma, following the preovulatory estrogen peak. Effects on uterine transcription include downregulation of epithelial proliferation and induction of progesterone-regulated pro-implantation genes. We propose that these changes increase uterine permissiveness to embryo invasion, whereas they decrease resistance to invasion by cancer and endometriotic foci in other estrogen-responsive tissues.

The value of broad taxonomic comparisons in evolutionary medicine: Disease is not a trait but a state of a trait!

In this short paper, we argue that there is a fundamental connection between the medical sciences and evolutionary biology as both are sciences of biological variation. Medicine studies pathological variation among humans (and domestic animals in veterinary medicine) and evolutionary biology studies variation within and among species in general. A key principle of evolutionary biology is that genetic differences among species have arisen first from mutations originating within populations. This implies a mechanistic continuity between variation among individuals within a species and variation between species. This fact motivates research that seeks to leverage comparisons among species to unravel the genetic basis of human disease vulnerabilities. This view also implies that genetically caused diseases can be understood as extreme states of an underlying trait, that is, an axis of variation, rather than distinct traits, as often assumed in GWAS studies. We illustrate these points with a number of examples as diverse as anatomical birth defects, cranio-facial variation, preeclampsia and vulnerability to metastatic cancer.

Female Genital Variation Far Exceeds that of Male Genitalia: A Review of Comparative Anatomy of Clitoris and the Female Lower Reproductive Tract in Theria.

A review of the literature on the anatomy of the lower female genital tract in therian mammals reveals, contrary to the general perception, a large amount of inter-specific variation. Variation in female external genitalia is anatomically more radical than that in the male genitalia. It includes the absence of whole anatomical units, like the cervix in many Xenarthra, or the absence of the urogenital sinus (UGS), as well as the complete spatial separation of the external clitoral parts from the genital canal (either vagina or UGS). A preliminary phylogenetic analysis shows two patterns. Some morphs are unique to early branching clades, like the absence of the cervix, while others arose multiple times independently, like the flattening out or loss of the UGS, or the extreme elongation of the clitoris. Based on available information, the ancestral eutherian configuration of the external female genitalia included a cervix, a single vaginal segment, a tubular UGS, and an unperforated clitoris close to the entrance of the genital canal. The evidence for either bilobed or unitary glandes clitorides is ambivalent. Despite the wealth of information available, many gaps in knowledge remain and will require a community-wide effort to come to a more robust model of female genital evolutionary patterns.

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.

Endogenous retroviruses drive species-specific germline transcriptomes in mammals.

Gene regulation in the germline ensures the production of high-quality gametes, long-term maintenance of the species and speciation. Male germline transcriptomes undergo dynamic changes after the mitosis-to-meiosis transition and have been subject to evolutionary divergence among mammals. However, the mechanisms underlying germline regulatory divergence remain undetermined. Here, we show that endogenous retroviruses (ERVs) influence species-specific germline transcriptomes. After the mitosis-to-meiosis transition in male mice, specific ERVs function as active enhancers to drive germline genes, including a mouse-specific gene set, and bear binding motifs for critical regulators of spermatogenesis, such as A-MYB. This raises the possibility that a genome-wide transposition of ERVs rewired germline gene expression in a species-specific manner. Of note, independently evolved ERVs are associated with the expression of human-specific germline genes, demonstrating the prevalence of ERV-driven mechanisms in mammals. Together, we propose that ERVs fine-tune species-specific transcriptomes in the mammalian germline.

Endometrial Decidualization: The Primary Driver of Pregnancy Health.

Interventions to prevent pregnancy complications have been largely unsuccessful. We suggest this is because the foundation for a healthy pregnancy is laid prior to the establishment of the pregnancy at the time of endometrial decidualization. Humans are one of only a few mammalian viviparous species in which decidualization begins during the latter half of each menstrual cycle and is therefore independent of the conceptus. Failure to adequately prepare (decidualize) the endometrium hormonally, biochemically, and immunologically in anticipation of the approaching blastocyst-including the downregulation of genes involved in the pro- inflammatory response and resisting tissue invasion along with the increased expression of genes that promote angiogenesis, foster immune tolerance, and facilitate tissue invasion-leads to abnormal implantation/placentation and ultimately to adverse pregnancy outcome. We hypothesize, therefore, that the primary driver of pregnancy health is the quality of the soil, not the seed.

Author Correction: Evolution of placental invasion and cancer metastasis are causally linked.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pregnant Females as Historical Individuals: An Insight From the Philosophy of Evo-Devo.

Criticisms of the "container" model of pregnancy picturing female and embryo as separate entities multiply in various philosophical and scientific contexts during the last decades. In this paper, we examine how this model underlies received views of pregnancy in evolutionary biology, in the characterization of the transition from oviparity to viviparity in mammals and in the selectionist explanations of pregnancy as an evolutionary strategy. In contrast, recent evo-devo studies on eutherian reproduction, including the role of inflammation and new maternal cell types, gather evidence in favor of considering pregnancy as an evolved relational novelty. Our thesis is that from this perspective we can identify the emergence of a new historical individual in evolution. In evo-devo, historical units are conceptualized as evolved entities which fulfill two main criteria, their continuous persistence and their non-exchangeability. As pregnancy can be individuated in this way, we contend that pregnant females are historical individuals. We argue that historical individuality differs from, and coexists with, other views of biological individuality as applied to pregnancy (the physiological, the evolutionary and the ecological one), but brings forward an important new insight which might help dissolve misguided conceptions.

Evolution of the human pelvis and obstructed labor: new explanations of an old obstetrical dilemma.

Without cesarean delivery, obstructed labor can result in maternal and fetal injuries or even death given a disproportion in size between the fetus and the maternal birth canal. The precise frequency of obstructed labor is difficult to estimate because of the widespread use of cesarean delivery for indications other than proven cephalopelvic disproportion, but it has been estimated that at least 1 million mothers per year are affected by this disorder worldwide. Why is the fit between the fetus and the maternal pelvis so tight? Why did evolution not lead to a greater safety margin, as in other primates? Here we review current research and suggest new hypotheses on the evolution of human childbirth and pelvic morphology. In 1960, Washburn suggested that this obstetrical dilemma arose because the human pelvis is an evolutionary compromise between two functions, bipedal gait and childbirth. However, recent biomechanical and kinematic studies indicate that pelvic width does not considerably affect the efficiency of bipedal gait and thus is unlikely to have constrained the evolution of a wider birth canal. Instead, bipedalism may have primarily constrained the flexibility of the pubic symphysis during pregnancy, which opens much wider in most mammals with large fetuses than in humans. We argue that the birth canal is mainly constrained by the trade-off between 2 pregnancy-related functions: while a narrow pelvis is disadvantageous for childbirth, it offers better support for the weight exerted by the viscera and the large human fetus during the long gestation period. We discuss the implications of this hypothesis for understanding pelvic floor dysfunction. Furthermore, we propose that selection for a narrow pelvis has also acted in males because of the role of pelvic floor musculature in erectile function. Finally, we review the cliff-edge model of obstetric selection to explain why evolution cannot completely eliminate cephalopelvic disproportion. This model also predicts that the regular application of life-saving cesarean delivery has evolutionarily increased rates of cephalopelvic disproportion already. We address how evolutionary models contribute to understanding and decision making in obstetrics and gynecology as well as in devising health care policies.

Eutherian-Specific Gene TRIML2 Attenuates Inflammation in the Evolution of Placentation.

Evolution of highly invasive placentation in the stem lineage of eutherians and subsequent extension of pregnancy set eutherians apart from other mammals, that is, marsupials with short-lived placentas, and oviparous monotremes. Recent studies suggest that eutherian implantation evolved from marsupial attachment reaction, an inflammatory process induced by the direct contact of fetal placenta with maternal endometrium after the breakdown of the shell coat, and shortly before the onset of parturition. Unique to eutherians, a dramatic downregulation of inflammation after implantation prevents the onset of premature parturition, and is critical for the maintenance of gestation. This downregulation likely involved evolutionary changes on maternal as well as fetal/placental side. Tripartite-motif family-like2 (TRIML2) only exists in eutherian genomes and shows preferential expression in preimplantation embryos, and trophoblast-derived structures, such as chorion and placental disc. Comparative genomic evidence supports that TRIML2 originated from a gene duplication event in the stem lineage of Eutheria that also gave rise to eutherian TRIML1. Compared with TRIML1, TRIML2 lost the catalytic RING domain of E3 ligase. However, only TRIML2 is induced in human choriocarcinoma cell line JEG3 with poly(I:C) treatment to simulate inflammation during viral infection. Its knockdown increases the production of proinflammatory cytokines and reduces trophoblast survival during poly(I:C) stimulation, while its overexpression reduces proinflammatory cytokine production, supporting TRIML2's role as a regulatory inhibitor of the inflammatory pathways in trophoblasts. TRIML2's potential virus-interacting PRY/SPRY domain shows significant signature of selection, suggesting its contribution to the evolution of eutherian-specific inflammation regulation during placentation.

Evolution of placental invasion and cancer metastasis are causally linked.

Among mammals, placental invasion is correlated with vulnerability to malignancy. Animals with more invasive placentation (for example, humans) are more vulnerable to malignancy. To explain this correlation, we propose the hypothesis of 'Evolved Levels of Invasibility' proposing that the evolution of invasibility of stromal tissue affects both placental and cancer invasion. We provide evidence for this using an in vitro model. We find that bovine endometrial and skin fibroblasts are more resistant to invasion than are their human counterparts. Gene expression profiling identified genes with high expression in human but not in bovine fibroblasts. Knocking down a subset of them in human fibroblasts leads to stronger resistance to cancer cell invasion. Identifying the evolutionary determinants of stromal invasibility can provide important insights to develop rational antimetastatic therapeutics.

An experimental test of the ovulatory homolog model of female orgasm.

The ovulatory homolog model of female orgasm posits that the neuro-endocrine mechanisms underlying female orgasm evolved from and are homologous to the mechanisms mediating copulation-induced ovulation in some mammals. This model predicts that pharmacological agents that affect human orgasm, such as fluoxetine, should also affect ovulation in animals with copulation-induced ovulation, such as rabbits. We tested this prediction by treating rabbits with daily doses of fluoxetine for 2 wk and found that fluoxetine treatment reduces the number of ovulations postcopulation by 30%. In a second experiment we tested whether this result was mediated by an effect on the brain or via peripheral serotonin functions. We treated animals with fluoxetine and induced ovulation with a single injection of human chorionic gonadotropin. In this experiment ovulation rate was nominally reduced by only 8%, which is statistically not significant. We conclude that the effect of fluoxetine on copulation-induced ovulation rate supports the ovulatory homolog model of female orgasm, suggesting that female orgasm has very deep evolutionary roots among the early eutherian mammals.

Peptide/Receptor Co-evolution Explains the Lipolytic Function of the Neuropeptide TLQP-21.

Structural and functional diversity of peptides and GPCR result from long evolutionary processes. Even small changes in sequence can alter receptor activation, affecting therapeutic efficacy. We conducted a structure-function relationship study on the neuropeptide TLQP-21, a promising target for obesity, and its complement 3a receptor (C3aR1). After having characterized the TLQP-21/C3aR1 lipolytic mechanism, a homology modeling and molecular dynamics simulation identified the TLQP-21 binding motif and C3aR1 binding site for the human (h) and mouse (m) molecules. mTLQP-21 showed enhanced binding affinity and potency for hC3aR1 compared with hTLQP-21. Consistently, mTLQP-21, but not hTLQP-21, potentiates lipolysis in human adipocytes. These findings led us to uncover five mutations in the C3aR1 binding pocket of the rodent Murinae subfamily that are causal for enhanced calculated affinity and measured potency of TLQP-21. Identifying functionally relevant peptide/receptor co-evolution mechanisms can facilitate the development of innovative pharmacotherapies for obesity and other diseases implicating GPCRs.

Endometrial recognition of pregnancy occurs in the grey short-tailed opossum ( Monodelphis domestica).

In human pregnancy, recognition of an embryo within the uterus is essential to support the fetus through gestation. In most marsupials, such as the opossums, pregnancy is shorter than the oestrous cycle and the steroid hormone profile during pregnancy and oestrous cycle are indistinguishable. For these reasons, it was assumed that recognition of pregnancy, as a trait, evolved in the eutherian (placental) stem lineage and independently in wallabies and kangaroos. To investigate whether uterine recognition of pregnancy occurs in species with pregnancy shorter than the oestrous cycle, we examined reproduction in the short-tailed opossum ( Monodelphis domestica), a marsupial with a plesiomorphic mode of pregnancy. We examined the morphological and gene expression changes in the uterus of females in the non-pregnant oestrous cycle and compared these to pregnancy. We found that the presence of an embryo did not alter some aspects of uterine development but increased glandular activity. Transcriptionally, we saw big differences between the uterus of pregnant and cycling animals. These differences included an upregulation of genes involved in transport, inflammation and metabolic-activity in response to the presence of a fetus. Furthermore, transcriptional differences reflected protein level differences in transporter abundance. Our results suggest that while the uterus exhibits programmed changes after ovulation, its transcriptional landscape during pregnancy responds to the presence of a fetus and upregulates genes that may be essential for fetal support. These results are consistent with endometrial recognition of pregnancy occurring in the opossum. While the effects on maternal physiology appear to differ, recognition of pregnancy has now been observed in eutherian mammals, as well as, Australian and American marsupials.

Humans as inverted bats: A comparative approach to the obstetric conundrum.

OBJECTIVES: The narrow human birth canal evolved in response to multiple opposing selective forces on the pelvis. These factors cannot be sufficiently disentangled in humans because of the limited range of relevant variation. Here, we outline a comparative strategy to study the evolution of human childbirth and to test existing hypotheses in primates and other mammals. METHODS: We combined a literature review with comparative analyses of neonatal and female body and brain mass, using three existing datasets. We also present images of bony pelves of a diverse sample of taxa. RESULTS: Bats, certain non-human primates, seals, and most ungulates, including whales, have much larger relative neonatal masses than humans, and they all differ in their anatomical adaptations for childbirth. Bats, as a group, are particularly interesting in this context as they give birth to the relatively largest neonates, and their pelvis is highly dimorphic: Whereas males have a fused symphysis, a ligament bridges a large pubic gap in females. The resulting strong demands on the widened and vulnerable pelvic floor likely are relaxed by roosting head-down. CONCLUSIONS: Parturition has constituted a strong selective force in many non-human placentals. We illustrated how the demands on pelvic morphology resulting from locomotion, pelvic floor stability, childbirth, and perhaps also erectile function in males have been traded off differently in mammals, depending on their locomotion and environment. Exploiting the power of a comparative approach, we present new hypotheses and research directions for resolving the obstetric conundrum in humans.

Predicting evolutionary potential: A numerical test of evolvability measures.

Despite sophisticated mathematical models, the theory of microevolution is mostly treated as a qualitative rather than a quantitative tool. Numerical measures of selection, constraints, and evolutionary potential are often too loosely connected to theory to provide operational predictions of the response to selection. In this paper, we study the ability of a set of operational measures of evolvability and constraint to predict short-term selection responses generated by individual-based simulations. We focus on the effects of selective constraints under which the response in one trait is impeded by stabilizing selection on other traits. The conditional evolvability is a measure of evolutionary potential explicitly developed for this situation. We show that the conditional evolvability successfully predicts rates of evolution in an equilibrium situation, and further that these equilibria are reached with characteristic times that are inversely proportional to the fitness load generated by the constraining characters. Overall, we find that evolvabilities and conditional evolvabilities bracket responses to selection, and that they together can be used to quantify evolutionary potential on time scales where the G-matrix remains relatively constant.

Decidualization of Human Endometrial Stromal Fibroblasts is a Multiphasic Process Involving Distinct Transcriptional Programs.

Decidual stromal cells differentiate from endometrial stromal fibroblasts (ESFs) under the influence of progesterone and cyclic adenosine monophosphate (cAMP) and are essential for implantation and the maintenance of pregnancy. They evolved in the stem lineage of placental (eutherian) mammals coincidental with the evolution of implantation. Here we use the well-established in vitro decidualization protocol to compare early (3 days) and late (8 days) gene transcription patterns in immortalized human ESF. We document extensive, dynamic changes in the early and late decidual cell transcriptomes. The data suggest the existence of an early signal transducer and activator of transcription (STAT) pathway dominated state and a later nuclear factor κB (NFKB) pathway regulated state. Transcription factor expression in both phases is characterized by putative or known progesterone receptor ( PGR) target genes, suggesting that both phases are under progesterone control. Decidualization leads to proliferative quiescence, which is reversible by progesterone withdrawal after 3 days but to a lesser extent after 8 days of decidualization. In contrast, progesterone withdrawal induces cell death at comparable levels after short or long exposure to progestins and cAMP. We conclude that decidualization is characterized by a biphasic gene expression dynamic that likely corresponds to different phases in the establishment of the fetal-maternal interface.