Abstract Oxygen is necessary for life yet too much or too little oxygen is toxic to cells. The oxygen tension in the maternal plasma bathing placental villi is <20 mm Hg until 10–12 weeks’ gestation, rising to 40–80 mm Hg and remaining in this range throughout the second and third trimesters. Maldevelopment of the maternal spiral arteries in the first trimester predisposes to placental dysfunction and sub-optimal pregnancy outcomes in the second half of pregnancy. Although low oxygen at the site of early placental development is the norm, controversy is intense when investigators interpret how defective transformation of spiral arteries leads to placental dysfunction during the second and third trimesters. Moreover, debate rages as to what oxygen concentrations should be considered normal and abnormal for use in vitro to model villous responses in vivo. The placenta may be injured in the second half of pregnancy by hypoxia, but recent evidence shows that ischemia with reoxygenation and mechanical damage due to high flow contributes to the placental dysfunction of diverse pregnancy disorders. We overview normal and pathologic development of the placenta, consider variables that influence experiments in vitro , and discuss the hotly debated question of what in vitro oxygen percentage reflects the normal and abnormal oxygen concentrations that occur in vivo . We then describe our studies that show cultured villous trophoblasts undergo apoptosis and autophagy with phenotype-related differences in response to hypoxia.
Background: The trophoblast compartment of the placenta comprises various subpopulations with distinct functions. They interact among each other by secreted signals thus forming autocrine or paracrine regulatory loops. We established a first trimester trophoblast cell line (ACH-3P) by fusion of primary human first trimester trophoblasts (week 12 of gestation) with a human choriocarcinoma cell line (AC1-1). Results: Expression of trophoblast markers (cytokeratin-7, integrins, matrix metalloproteinases), invasion abilities and transcriptome of ACH-3P closely resembled primary trophoblasts. Morphology, cytogenetics and doubling time was similar to the parental AC1-1 cells. The different subpopulations of trophoblasts e. g., villous and extravillous trophoblasts also exist in ACH-3P cells and can be immuno-separated by HLA-G surface expression. HLA-G positive ACH-3P display pseudopodia and a stronger expression of extravillous trophoblast markers. Higher expression of insulin-like growth factor II receptor and human chorionic gonadotropin represents the basis for the known autocrine stimulation of extravillous trophoblasts. Conclusion: We conclude that ACH-3P represent a tool to investigate interaction of syngeneic trophoblast subpopulations. These cells are particularly suited for studies into autocrine and paracrine regulation of various aspects of trophoblast function. As an example a novel effect of TNF-alpha on matrix metalloproteinase 15 in HLA-G positive ACH-3P and explants was found.
Abstract Placental stress has been implicated in the pathophysiology of complications of pregnancy, including growth restriction and pre-eclampsia. Initially, attention focused on oxidative stress, but recently mitochondrial and endoplasmic reticulum stress have been identified. Complex molecular interactions exist among these different forms of stress, making it unlikely that any occurs in isolation. In part, this is due to close physiological connections between the two organelles principally involved, mitochondria and the endoplasmic reticulum (ER), mediated through Ca2+ signalling. Here, we review the involvement of the mitochondria-ER unit in the generation of stress within the trophoblast, and consider consequences for obstetric outcome. Mild stress may induce adaptive responses, including upregulation of antioxidant defences and autophagy, while moderate levels may affect stem cell behaviour and reduce cell proliferation, contributing to the growth-restricted phenotype. High levels of stress can stimulate release of pro-inflammatory cytokines and anti-angiogenic factors, increasing the risk of pre-eclampsia. In addition, chronic stress may promote senescence of the trophoblast, which in other cell types leads to a pro-inflammatory senescence-associated secretory phenotype. Evidence from rodents suggests that a degree of trophoblastic stress develops with increasing gestational age in normal pregnancies. The increase in maternal concentrations of soluble fms-like tyrosine kinase-1 (sFlt-1) and reduction in placental growth factor (PlGF) suggest the same may occur in the human, starting around 30 weeks of pregnancy. Placental malperfusion, or co-existing maternal conditions, such as diabetes, will exacerbate that stress. Amelioration of trophoblastic stress should remain a research priority, but will be difficult due to the complexity of the molecular pathways involved.
Abstract At the tips of anchoring villi, cytotrophoblast (CTB) proliferation leads to a process of multilayering in which cells lose their attachment to the villous basement membrane and develop into columns, within which they adhere to one another using desmosomes, with associated intermediate filament bundles. Non-desmosomal cadherins, tight junction proteins and other adhesion molecules are also present, suggesting that actin-associated adhesions contribute to placental anchorage. In the distal columns, cell–cell interactions diminish, cells upregulate β1 integrins and bind to a provisional fibrinoid extracellular matrix, eventually detaching to migrate into the decidual stroma and myometrium, where interstitial and endovascular extravillous trophoblast (EVT) populations show distinct repertoires of adhesion molecules.
Critical roles for DNA methylation in embryonic development are well established, but less is known about its roles during trophoblast development, the extraembryonic lineage that gives rise to the placenta. We dissected the role of DNA methylation in trophoblast development by performing mRNA and DNA methylation profiling of mutants. We find that oocyte-derived methylation plays a major role in regulating trophoblast development but that imprinting of the key placental regulator is only partially responsible for these effects. We have identified several methylation-regulated genes associated with trophoblast differentiation that are involved in cell adhesion and migration, potentially affecting trophoblast invasion. Specifically, trophoblast-specific DNA methylation is linked to the silencing of , a Polycomb Repressive Complex 1 protein that drives loss of cell adhesion in methylation-deficient trophoblast. Our results reveal that maternal DNA methylation controls multiple differentiation-related and physiological processes in trophoblast via both imprinting-dependent and -independent mechanisms. Branco et al. dissect the role of DNA methylation in mouse trophoblast development through genome-wide profiling of methylation-deficient mutants. DNA methylation marks carried over from the oocyte play a major role in trophoblast development and cell adhesion, which is partly dependent on silencing of the Polycomb gene .
Abstract In the placental villus, cells attach to basement membrane via integrin α6β4 and adhere both laterally and apically to their neighbours. The most prominent adhesive specialisation seen using the electron microscope is the desmosome, which connects cytotrophoblast cells (CTB) laterally and also contributes to the attachment of CTB to the overlying syncytium. However, numerous cadherins and other junctional proteins are also present in the corresponding plasma membrane domains, indicating a multiplicity of adhesive interactions. Integrins, tight junction components and cadherins are all found in the syncytial microvillous membrane, perhaps reflecting its ability to form intersyncytial bridges. There is a wide gulf to be filled between molecular anatomy and functional studies, with much to be learned about the role of adhesion molecules in regulating villous epithelial integrity, homeostasis and growth.
Objective To examine the expression pattern of biomarker proteins in extravillous trophoblast (EVT) cells obtained noninvasively by trophoblast retrieval and isolation from the cervix (TRIC) in patients with early pregnancy loss compared with control patients with uncomplicated term delivery. Design Case-control study. Setting Academic medical center. Patient(s) Women with either early pregnancy loss (EPL, n = 10) or an uncomplicated term delivery (N = 10). Intervention(s) Endocervical specimens obtained from ongoing pregnancies at gestational ages of 5–10 weeks to generate an archive of EVT cells isolated by TRIC, with medical records examined to select specimens matched for gestational age at the time of endocervical sampling. Main Outcome Measure(s) Known serum biomarkers for adverse pregnancy outcome that are expressed by EVT cells were evaluated by semiquantitative immunocytochemistry, using antibodies against endoglin (ENG), FMS-like tyrosine kinase-1 (FLT-1), α-fetoprotein (AFP), pregnancy-associated plasma protein-A (PAPP-A), galectin-13 (LGALS13), galectin-14 (LGALS14), and placental growth factor (PGF). Result(s) The EVT purity was over 95% in all specimens, based on chorionic gonadotropin expression; however, the number of EVT cells obtained was significantly lower in women with EPL than the control group. There was a statistically significant elevation of AFP, ENG, and FLT-1, and statistically significant reduction of PAPP-A, LGALS14, and PGF in the EPL group compared with controls. Conclusion(s) In this pilot study, EVT cells isolated by TRIC early in gestation exhibited altered protein expression patterns before an EPL compared with uncomplicated term pregnancies.
Abstract During human pregnancy the semi-allogeneic/allogeneic fetal graft is normally accepted by the mother's immune system. Initially the contact between maternal and fetal cells is restricted to the decidua but during the 2nd trimester it is extended to the entire body. Two contrary requirements influence the extent of invasion of extravillous fetal trophoblast cells (EVT) in the maternal decidua: anchorage of the placenta to ensure fetal nutrition and protection of the uterine wall against over-invasion. To establish the crucial balance between tolerance of the EVT and its limitation, recognition of the semi-allogeneic/allogeneic fetal cell by maternal leukocytes is prerequisite. A key mechanism to limit EVT invasion is induction of EVT apoptosis. Apoptotic bodies are phagocytosed by antigen-presenting cells (APC). Peptides from apoptotic cells are presented by APC cells and induce an antigen-specific tolerance against the foreign antigens on EVT cells. These pathways, including up-regulation of the expression of IDO, IFNγ and CTLA-4 as well as the induction of Tregulatory cells, are general immunological mechanisms which have developed to maintain peripheral tolerance to self-antigens. Together these data suggest that the mother extends her “definition of self” for 9 months on the foreign antigens of the fetus.