Molecular mechanisms of trophoblast survival: from implantation to birth

Fetal development depends upon a coordinated series of events in both the embryo and in the supporting placenta. The initial event in placentation is appropriate lineage allocation of stem cells followed by the formation of a spheroidal trophoblastic shell surrounding the embryo, facilitating implantation into the uterine stroma and exclusion of oxygenated maternal blood. In mammals, cellular proliferation, differentiation, and death accompany early placental development. Programmed cell death is a critical driving force behind organ sculpturing and eliminating abnormal, misplaced, nonfunctional, or harmful cells in the embryo proper, although very little is known about its physiological function during placental development. This review summarizes current knowledge of the cell death patterns and molecular pathways governing the survival of cells within the blastocyst, with a focus on the trophoblast lineage prior to and after implantation. Particular emphasis is given to human placental development in the context of normal and pathological conditions. As molecular pathways in humans are poorly elucidated, we have also included an overview of pertinent genetic animal models displaying defects in trophoblast survival.     

A functional overlap of plasminogen and MMPs regulates vascularization during placental development

Both plasminogen activators and matrix metalloproteinases (MMPs) have been implicated in a variety of developmental processes in the mouse during embryo implantation and placentation. We show here that pharmacological treatment of plasminogen-deficient mice with the broad spectrum MMP inhibitor galardin leads to a high rate of embryonic lethality. Implantation sites from plasminogen-deficient galardin-treated mice at 7.5 days post coitus (dpc) showed delay in both decidualization and invasion of maternal vessels into the decidua. At 8.5 dpc, half of the embryos were runted and still at the developmental stage of a 7.5 dpc embryo. Most embryos that escaped these initial defects eventually died, probably from defective vascularization and development of the labyrinth layer of the placenta, although a direct role on embryo development cannot be ruled out. These results demonstrate that the combination of MMPs and plasminogen is essential for the proper development of the placenta. Plasminogen deficiency alone and galardin treatment alone had much less effect and there was a pronounced synergism on both placental vascularization and embryonic lethality, indicating a functional overlap between plasminogen and MMPs.     

Adipokines underlie the early origins of obesity and associated metabolic comorbidities in the offspring of women with pregestational obesity

Maternal pregestational obesity is a well-known risk factor for offspring obesity, metabolic syndrome, cardiovascular disease and type 2 diabetes. The mechanisms by which maternal obesity can induce alterations in fetal and later neonatal metabolism are not fully elucidated due to its complexity and multifactorial causes. Two adipokines, leptin and adiponectin, are involved in fetal and postnatal growth trajectories, and both are altered in women with pregestational obesity. The placenta synthesizes leptin, which goes mainly to the maternal circulation and in lesser amount to the developing fetus. Maternal pregestational obesity and hyperleptinemia are associated with placental dysfunction and changes in nutrient transporters which directly affect fetal growth and development. By the other side, the embryo can produce its own leptin from early in development, which is associated to fetal weight and adiposity. Adiponectin, an insulin-sensitizing adipokine, is downregulated in maternal obesity. High molecular weight (HMW) adiponectin is the most abundant form and with most biological actions. In maternal obesity lower total and HMW adiponectin levels have been described in the mother, paralleled with high levels in the umbilical cord. Several studies have found that cord blood adiponectin levels are related with postnatal growth trajectories, and it has been suggested that low adiponectin levels in women with pregestational obesity enhance placental insulin sensitivity and activation of placental amino acid transport systems, supporting fetal overgrowth. The possible mechanisms by which maternal pregestational obesity, focusing in the actions of leptin and adiponectin, affects the fetal development and postnatal growth trajectories in their offspring are discussed.     

Genetic insights into trophoblast differentiation and placental morphogenesis

The placenta is comprised of an inner vascular network covered by an outer epithelium, called trophoblast, all designed to promote the delivery of nutrients to the fetus. Several specialized trophoblast cell subtypes arise during development to promote this function, including cells that invade the uterus to promote maternal blood flow to the implantation site, and other cells that fuse into a syncytium, expand and fold to increase the surface area for efficient transport. Mutation of many genes in mice results in embryonic mortality or fetal growth restriction due to defects in placental development. Several important principles about placental development have emerged from these studies. First, distinct molecular pathways regulate the differentiation of the various trophoblast cell subtypes. Second, trophoblast proliferation, differentiation and morphogenesis are highly regulated by interactions with adjacent cell types. Finally, the specific classes of mutant phenotypes observed in the placenta of knockout mice resemble those seen in humans that are associated with preeclampsia and intrauterine growth restriction.     

Preeclampsia: current understanding of the molecular basis of vascular dysfunction

Preeclampsia is a pregnancy-specific disorder characterised by hypertension and proteinuria occurring after the 20th week of gestation. Delivery of the placenta results in resolution of the condition, implicating the placenta as a central culprit in the pathogenesis of preeclampsia. In preeclampsia, an inadequate placental trophoblast invasion of the maternal uterine spiral arteries results in poor placental perfusion, leading to placental ischaemia. This could result in release of factors into the maternal circulation that cause widespread activation or dysfunction of the maternal endothelium. Factors in the maternal circulation might induce oxidative stress and/or elicit an inflammatory response in the maternal endothelium, resulting in the altered expression of several genes involved in the regulation of vascular tone. This review addresses the potential circulating factors and the molecular mechanisms involved in the alteration of vascular function that occurs in preeclampsia.     

Bisphenol A Exposure Disrupts Genomic Imprinting in the Mouse

Exposure to endocrine disruptors is associated with developmental defects. One compound of concern, to which humans are widely exposed, is bisphenol A (BPA). In model organisms, BPA exposure is linked to metabolic disorders, infertility, cancer, and behavior anomalies. Recently, BPA exposure has been linked to DNA methylation changes, indicating that epigenetic mechanisms may be relevant. We investigated effects of exposure on genomic imprinting in the mouse as imprinted genes are regulated by differential DNA methylation and aberrant imprinting disrupts fetal, placental, and postnatal development. Through allele-specific and quantitative real-time PCR analysis, we demonstrated that maternal BPA exposure during late stages of oocyte development and early stages of embryonic development significantly disrupted imprinted gene expression in embryonic day (E) 9.5 and 12.5 embryos and placentas. The affected genes included Snrpn, Ube3a, Igf2, Kcnq1ot1, Cdkn1c, and Ascl2; mutations and aberrant regulation of these genes are associated with imprinting disorders in humans. Furthermore, the majority of affected genes were expressed abnormally in the placenta. DNA methylation studies showed that BPA exposure significantly altered the methylation levels of differentially methylated regions (DMRs) including the Snrpn imprinting control region (ICR) and Igf2 DMR1. Moreover, exposure significantly reduced genome-wide methylation levels in the placenta, but not the embryo. Histological and immunohistochemical examinations revealed that these epigenetic defects were associated with abnormal placental development. In contrast to this early exposure paradigm, exposure outside of the epigenetic reprogramming window did not cause significant imprinting perturbations. Our data suggest that early exposure to common environmental compounds has the potential to disrupt fetal and postnatal health through epigenetic changes in the embryo and abnormal development of the placenta.     

Mek1 and Mek2 functions in the formation of the blood placental barrier

The ERK/MAPK signaling pathway is involved in several cellular functions. Inactivation in mice of genes encoding members of this pathway is often associated with embryonic death resulting from abnormal placental development. The placenta is essential for nutritional and gaseous exchanges between maternal and embryonic circulations, as well as for the removal of metabolic wastes. These exchanges take place without direct contact between the two circulations. In mice, the hematoplacental barrier consists in a triple layer of trophoblast cells and endothelial cells of the embryo. MEK1 and MEK2 are double specificity serine-threonine/tyrosine kinases responsible for the activation of ERK1 and ERK2. Mek1 inactivation results in placental anomalies due to trophoblast cell proliferation and differentiation defects leading to severe delays in the development of placenta and causing the death of the embryo. Although Mek2(-/-) mutant mice survived without any apparent phenotype, double heterozygous Mek1(+/-)Mek2(+/-) mutants die during gestation from placental malformations. Together, these data emphasize the crucial role of the ERK/MAPK cascade in the formation of extraembryonic structures.     

Placental regulation of maternal-fetal interactions and brain development

A variety prenatal insults are associated with the incidence of neurodevelopmental disorders such as schizophrenia, autism and cerebral palsy. While the precise mechanisms underlying how transient gestational challenges can lead to later life dysfunctions are largely unknown, the placenta is likely to play a key role. The literal interface between maternal and fetal cells resides in the placenta, and disruptions to the maternal or intrauterine environment are necessarily conveyed to the developing embryo via the placenta. Placental cells bear the responsibility of promoting maternal tolerance of the semiallogeneic fetus and regulating selective permeability of nutrients, gases, and antibodies, while still providing physiological protection of the embryo from adversity. The placenta's critical role in modulating immune protection and the availability of nutrients and endocrine factors to the offspring implicates its involvement in autoimmunity, growth restriction and hypoxia, all factors associated with the development of neurological complications. In this review, we summarize primary maternal-fetal interactions that occur in the placenta and describe pathways by which maternal insults can impair these processes and disrupt fetal brain development. We also review emerging evidence for placental dysfunction in the prenatal programming of neurodevelopmental disorders. ? 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012.     

Important aspects of placental-specific gene transfer

The placenta is a unique and highly complex organ that develops only during pregnancy and is essential for growth and survival of the developing fetus. The placenta provides the vital exchange of gases and wastes, the necessary nutrients for fetal development, acts as immune barrier that protects against maternal rejection, and produces numerous hormones and growth factors that promote fetal maturity to regulate pregnancy until parturition. Abnormal placental development is a major underlying cause of pregnancy-associated disorders that often result in preterm birth. Defects in placental stem cell propagation, growth, and differentiation are the major factors that affect embryonic and fetal well-being and dramatically increase the risk of pregnancy complications. Understanding the processes that regulate placentation is important in determining the underlying factors behind abnormal placental development. The ability to manipulate genes in a placenta-specific manner provides a unique tool to analyze development and eliminates potentially confounding results that can occur with traditional gene knockouts. Trophoblast stem cells and mouse embryos are not overly amenable to traditional gene transfer techniques. Most viral vectors, however, have a low infection rate and often lead to mosaic transgenesis. Although the traditional method of embryo transfer is intrauterine surgical implantation, the methodology reported here, combining lentiviral blastocyst infection and nonsurgical embryo transfer, leads to highly efficient and placental-specific gene transfer. Numerous advantages of our optimized procedures include increased investigator safety, a reduction in animal stress, rapid and noninvasive embryo transfer, and higher a rate of pregnancy and live birth.     

Maternal diet modulates placenta growth and gene expression in a mouse model of diabetic pregnancy

Unfavorable maternal diet during pregnancy can predispose the offspring to diseases later in life, such as hypertension, metabolic syndrome, and obesity. However, the molecular basis for this phenomenon of "developmental programming" is poorly understood. We have recently shown that a diet nutritionally optimized for pregnancy can nevertheless be harmful in the context of diabetic pregnancy in the mouse, associated with a high incidence of neural tube defects and intrauterine growth restriction. We hypothesized that placental abnormalities may contribute to impaired fetal growth in these pregnancies, and therefore investigated the role of maternal diet in the placenta. LabDiet 5015 diet was associated with reduced placental growth, commencing at midgestation, when compared to pregnancies in which the diabetic dam was fed LabDiet 5001 maintenance chow. Furthermore, by quantitative RT-PCR we identify 34 genes whose expression in placenta at midgestation is modulated by diet, diabetes, or both, establishing biomarkers for gene-environment interactions in the placenta. These results implicate maternal diet as an important factor in pregnancy complications and suggest that the early phases of placenta development could be a critical time window for developmental origins of adult disease.     

Hormonal regulation of the Menkes and Wilson copper-transporting ATPases in human placental Jeg-3 cells

Copper deficiency during pregnancy results in early embryonic death and foetal structural abnormalities including skeletal, pulmonary and cardiovascular defects. During pregnancy, copper is transported from the maternal circulation to the foetus by mechanisms which have not been clearly elucidated. Two copper-transporting ATPases, Menkes (ATP7A; MNK) and Wilson (ATP7B; WND), are expressed in the placenta and both are involved in placental copper transport, as copper accumulates in the placenta in both Menkes and Wilson disease. The regulatory mechanisms of MNK and WND and their exact role in the placenta are unknown. Using a differentiated polarized Jeg-3 cell culture model of placental trophoblasts, MNK and WND were shown to be expressed within these cells. Distinct roles for MNK and WND are suggested on the basis of their opposing responses to insulin. Insulin and oestrogen increased both MNK mRNA and protein levels, altered the localization of MNK towards the basolateral membrane in a copper-independent manner, and increased the transport of copper across this membrane. In contrast, levels of WND were decreased in response to insulin, and the protein was located in a tight perinuclear region, with a corresponding decrease in copper efflux across the apical membrane. These results are consistent with a model of copper transport in the placenta in which MNK delivers copper to the foetus and WND returns excess copper to the maternal circulation. Insulin and oestrogen stimulate copper transport to the foetus by increasing the expression of MNK and reducing the expression of WND. These data show for the first time that MNK and WND are differentially regulated by the hormones insulin and oestrogen in human placental cells.     

Thrombosis during pregnancy: Risks,prevention, and treatment for mother and fetus—harvesting the power of omic technology,biomarkers and in vitro or in vivo models to facilitate the treatment of thrombosis

Maternal thromboembolism and a spectrum of placenta‐mediated complications including the pre‐eclampsia syndromes, fetal growth restriction, fetal loss, and abruption manifest a shared etiopathogenesis and predisposing risk factors. Furthermore, these maternal and fetal complications are often linked to subsequent maternal health consequences that comprise the metabolic syndrome, namely, thromboembolism, chronic hypertension, and type II diabetes. Traditionally, several lines of evidence have linked vasoconstriction, excessive thrombosis and inflammation, and impaired trophoblast invasion at the uteroplacental interface as hallmark features of the placental complications. “Omic” technologies and biomarker development have been largely based upon advances in vascular biology, improved understanding of the molecular basis and biochemical pathways responsible for the clinically relevant diseases, and increasingly robust large cohort and/or registry based studies. Advances in understanding of innate and adaptive immunity appear to play an important role in several pregnancy complications. Strategies aimed at improving prediction of these pregnancy complications are often incorporating hemodynamic blood flow data using non‐invasive imaging technologies of the utero‐placental and maternal circulations early in pregnancy. Some evidence suggests that a multiple marker approach will yield the best performing prediction tools, which may then in turn offer the possibility of early intervention to prevent or ameliorate these pregnancy complications. Prediction of maternal cardiovascular and non‐cardiovascular consequences following pregnancy represents an important area of future research, which may have significant public health consequences not only for cardiovascular disease, but also for a variety of other disorders, such as autoimmune and neurodegenerative diseases. Birth Defects Research (Part C) 105:209–225, 2015. © 2015 Wiley Periodicals, Inc.     

Hormonal regulation of placental nitric oxide and pathogenesis of pre-eclampsia

The placenta is central to foetal growth and development in mammalian pregnancy. Compromised placental function (as found in pre-eclampsia) often results in life-threatening situations for both mother and foetus. The nitric-oxide (NO) signalling cascade is important for placental function, in particular for the development of the vascular network and for maintaining vascular tone. This pathway seems to be regulated by multiple hormonal signals. Emerging evidence suggests that pathogenic mechanisms that are involved in abnormal placental function target specific molecules, such as hormone receptors, that regulate NO release and have subsequent dramatic consequences. Here, we discuss the current knowledge of NO function in the placenta, its hormonal regulation in normal pregnancy and in the pathophysiology of pre-eclampsia, its potential pathogenic mechanisms and possible use as a therapeutic target.     

Requirement for Map2k1 (Mek1) in extra-embryonic ectoderm during placentogenesis

Map2k1(-/-) embryos die at mid-gestation from abnormal development and hypovascularization of the placenta. We now show that this phenotype is associated with a decreased labyrinth cell proliferation and an augmented cell apoptosis. Although the activation of MAP2K1 and MAP2K2 is widespread in the labyrinthine region, MAPK1 and MAPK3 activation is restricted to the cells lining the maternal sinuses, suggesting an important role for the ERK/MAPK cascade in these cells. In Map2k1(-/-) placenta, ERK/MAPK cascade activation is perturbed. Abnormal localization of the syncytiotrophoblasts is also observed in Map2k1(-/-) placenta, even though this cell lineage is specified at the correct time during placentogenesis. The placental phenotype can be rescued in tetraploid experiments. In addition, Map2k1-specific deletion in the embryo leads to normal embryo development and to the birth of viable Map2k1(-/-) mice. Altogether, these data enlighten the essential role of Map2k1 in extra-embryonic ectoderm during placentogenesis. In the embryo, the Map2k1 gene function appears dispensable.     

Sexual dimorphism in activation of placental autophagy in obese women with evidence for fetal programming from a placenta-specific mouse model

The incidence of maternal obesity and its co-morbidities (diabetes, cardiovascular disease) continues to increase at an alarming rate, with major public health implications. In utero exposure to maternal obesity has been associated with development of cardiovascular and metabolic diseases in the offspring as a result of developmental programming. The placenta regulates maternal-fetal metabolism and shows significant changes in its function with maternal obesity. Autophagy is a cell-survival process, which is responsible for the degradation of damaged organelles and misfolded proteins. Here we show an activation of autophagosomal formation and autophagosome-lysosome fusion in placentas of males but not females from overweight (OW) and obese (OB) women vs. normal weight (NW) women. However, total autophagic activity in these placentas appeared to be decreased as it showed an increase in SQSTM1/p62 and a decrease in lysosomal biogenesis. A mouse model with a targeted deletion of the essential autophagy gene Atg7 in placental tissue showed significant placental abnormalities comparable to those seen in human placenta with maternal obesity. These included a decrease in expression of mitochondrial genes and antioxidants, and decreased lysosomal biogenesis. Strikingly, the knockout mice were developmentally programmed as they showed an increased sensitivity to high-fat diet-induced obesity, hyperglycemia, hyperinsulinemia, increased adiposity, and cardiac remodeling. In summary, our results indicate a sexual dimorphism in placental autophagy in response to maternal obesity. We also show that autophagy plays an important role in placental function and that inhibition of placental autophagy programs the offspring to obesity, and to metabolic and cardiovascular diseases.     

Human pregnancy: the role of chemokine networks at the fetal-maternal interface

Chemokines are multifunctional molecules initially described as having a role in leukocyte trafficking and later found to participate in developmental processes such as differentiation and directed migration. Similar events occur in pregnancy during development of the fetal-maternal interface, where there is extensive leukocyte trafficking and tissue morphogenesis, and this is accompanied by abundant chemokine expression. The relationship between chemokines, leukocytes and placental development is beginning to be delineated. During pregnancy a specialised population of maternal leukocytes infiltrates the implantation site. These leukocytes are thought to sustain the delicate balance between protecting the developing embryo/fetus and tolerating its hemiallogeneic tissues. A network of chemokine expression by both fetal and maternal components in the pregnant uterus functions in establishing this leukocyte population. Intriguingly, experiments investigating immune cell recruitment revealed the additional possibility that chemokines influence aspects of placental development. Specifically, cytotrophoblasts, the effector cells of the placenta, express chemokine receptors that can bind ligands found at key locations, implicating chemokines as regulators of cytotrophoblast differentiation and migration. Thus, as in other systems, at the fetal-maternal interface chemokines might regulate multiple functions.     

Extracellular nucleic acids in maternal circulation as potential biomarkers for placental insufficiency

Since the placenta is being continuously remodeled during normal placental development, extracellular nucleic acids of both fetal and placental origin, packed into either trophoblast-derived apoptotic bodies or shedding syncytiotrophoblast microparticles, may be detected in maternal circulation during the course of normal gestation. Placental-insufficiency-related pregnancy complications have been shown to be associated with excessive placental trophoblast apoptosis and shedding of placenta debris. Recent advances in the field are reviewed with a focus on the diagnostic potential of particular molecular biomarkers and their eventual implementation in the currently used predictive and diagnostic algorithms for placental-insufficiency-related pregnancy complications.     

How to study placental vascular development?

Both exogenous and endogenous factors during pregnancy may impact placental vascular development and cause different malformations of placental vessels. In humans, consequences of abnormal vascular development have been associated with different pregnancy-related pathologies ranging from miscarriage to intrauterine growth restriction or preeclampsia. Pregnancy-associated exposure to bacterial or viral infections or pharmacologic or toxic agents may also influence vascular development of the placenta and lead to preterm labor and delivery. Several steps of vascular adaptation on both the fetal and maternal side are necessary and include such events as uterine vasodilation, remodeling by extravillous trophoblast, as well as vasculogenesis and angiogenesis within the placenta. Ubiquitous as well as pregnancy-specific angiogenic factors are involved. Morphologic and stereologic approaches, as well as experiments in established laboratory animals, cannot be applied to large domestic animals or humans without hesitation. Thus, further studies into the different aspects of this process will require an appropriate in vitro model of placental vascular development. Reflecting the core of placental vascular development, the in vitro model should facilitate the interactions between trophoblast and stromal cells with endothelial progenitor cells. The effects of viral or bacterial infection as well as pharmacologic or toxic agents may be studied more closely in the process. This report reviews major aspects of vascular development in the placenta and describes the establishment of a three-dimensional in vitro model of human placental vascular development.