The multifaceted role of GCM1 during trophoblast differentiation in the human placenta.

Remodeling of the uterine vasculature by invasive extravillous trophoblasts (EVTs) is a critical aspect of human placentation. Insufficient EVT invasion can lead to severe obstetrical complications like preeclampsia, intrauterine growth restriction, and preterm birth. Glial cells missing-1 (GCM1) is a transcription factor that is crucial for proper placentation in mice, and is highly expressed in human syncytiotrophoblast (ST) and EVTs. GCM1 is classically considered a master regulator of ST formation, but little is known about its contribution to the development and function of EVTs. Therefore, in this study we test the hypothesis that GCM1 is a critical regulator of both EVT and ST development and function. We show that GCM1 is highly expressed in human trophoblast stem (TS) cells differentiated into either ST or EVTs. Knockdown of GCM1 in TS cells hindered differentiation into both ST and EVT pathways. When placed in ST media, GCM1-knockdown cells formed small, unstable clusters; when placed in EVT media, cells had altered morphology and transcript profiles resembling cells trapped in an intermediate state between CT and EVT, and invasive capacity through matrix was reduced. RNA sequencing analysis of GCM1-deficient TS cells revealed downregulation of EVT-associated genes and enrichment in transcripts related to WNT signaling, which was linked to decreased expression of the EVT master regulator ASCL2 and the WNT antagonist NOTUM. Our findings reveal an essential role of GCM1 during ST and EVT development, and suggest that GCM1 regulates differentiation of human TS cells into EVTs by inducing expression of ASCL2 and NOTUM.

Genome-Wide Analysis of Hypoxia-Inducible Factor Binding Reveals Targets Implicated in Impaired Human Placental Syncytiotrophoblast Formation under Low Oxygen.

Preeclampsia (PE) is a common and serious complication of pregnancy with no cure except premature delivery. The root cause of PE is improper development of the placenta-the temporary organ supporting fetal growth and development. Continuous formation of the multinucleated syncytiotrophoblast (STB) layer via differentiation and fusion of cytotrophoblasts (CTBs) is vital for healthy placentation and is impaired in preeclamptic pregnancies. In PE, there is reduced/intermittent placental perfusion, likely resulting in a persistently low O2 environment. Low O2 inhibits differentiation and fusion of CTBs into STB and may thus contribute to PE pathogenesis; however, the underlying mechanisms are unknown. Because low O2 activates a transcription factor complex in cells known as the hypoxia-inducible factor (HIF), the objective of this study was to investigate whether HIF signaling inhibits STB formation by regulating genes required for this process. Culture of primary CTBs, the CTB-like cell line BeWo, and human trophoblast stem cells under low O2 reduced cell fusion and differentiation into STB. Knockdown of aryl hydrocarbon receptor nuclear translocator (a key component of the HIF complex) in BeWo cells restored syncytialization and expression of STB-associated genes under different O2 levels. Chromatin immunoprecipitation sequencing facilitated the identification of global aryl hydrocarbon receptor nuclear translocator/HIF binding sites, including several near genes implicated in STB development, such as ERVH48-1 and BHLHE40, providing new insights into mechanisms underlying pregnancy diseases linked to poor placental O2 supply.

How trophoblasts fuse: an in-depth look into placental syncytiotrophoblast formation.

In humans, cell fusion is restricted to only a few cell types under normal conditions. In the placenta, cell fusion is a critical process for generating syncytiotrophoblast: the giant multinucleated trophoblast lineage containing billions of nuclei within an interconnected cytoplasm that forms the primary interface separating maternal blood from fetal tissue. The unique morphology of syncytiotrophoblast ensures that nutrients and gases can be efficiently transferred between maternal and fetal tissue while simultaneously restricting entry of potentially damaging substances and maternal immune cells through intercellular junctions. To maintain integrity of the syncytiotrophoblast layer, underlying cytotrophoblast progenitor cells terminate their capability for self-renewal, upregulate expression of genes needed for differentiation, and then fuse into the overlying syncytium. These processes are disrupted in a variety of obstetric complications, underscoring the importance of proper syncytiotrophoblast formation for pregnancy health. Herein, an overview of key mechanisms underlying human trophoblast fusion and syncytiotrophoblast development is discussed.

Antiviral Inflammation during Early Pregnancy Reduces Placental and Fetal Growth Trajectories.

Many viruses are detrimental to pregnancy and negatively affect fetal growth and development. What is not well understood is how virus-induced inflammation impacts fetal-placental growth and developmental trajectories, particularly when inflammation occurs in early pregnancy during nascent placental and embryo development. To address this issue, we simulated a systemic virus exposure in early pregnant rats (gestational day 8.5) by administering the viral dsRNA mimic polyinosinic:polycytidylic acid (PolyI:C). Maternal exposure to PolyI:C induced a potent antiviral response and hypoxia in the early pregnant uterus, containing the primordial placenta and embryo. Maternal PolyI:C exposure was associated with decreased expression of the maternally imprinted genes Mest, Sfrp2, and Dlk1, which encode proteins critical for placental growth. Exposure of pregnant dams to PolyI:C during early pregnancy reduced fetal growth trajectories throughout gestation, concomitant with smaller placentas, and altered placental structure at midgestation. No detectable changes in placental hemodynamics were observed, as determined by ultrasound biomicroscopy. An antiviral response was not evident in rat trophoblast stem (TS) cells following exposure to PolyI:C, or to certain PolyI:C-induced cytokines including IL-6. However, TS cells expressed high levels of type I IFNR subunits (Ifnar1 and Ifnar2) and responded to IFN-⍺ by increasing expression of IFN-stimulated genes and decreasing expression of genes associated with the TS stem state, including Mest IFN-⍺ also impaired the differentiation capacity of TS cells. These results suggest that an antiviral inflammatory response in the conceptus during early pregnancy impacts TS cell developmental potential and causes latent placental development and reduced fetal growth.

Omics Approaches to Study Formation and Function of Human Placental Syncytiotrophoblast.

Proper development of the placenta is vital for pregnancy success. The placenta regulates exchange of nutrients and gases between maternal and fetal blood and produces hormones essential to maintain pregnancy. The placental cell lineage primarily responsible for performing these functions is a multinucleated entity called syncytiotrophoblast. Syncytiotrophoblast is continuously replenished throughout pregnancy by fusion of underlying progenitor cells called cytotrophoblasts. Dysregulated syncytiotrophoblast formation disrupts the integrity of the placental exchange surface, which can be detrimental to maternal and fetal health. Moreover, various factors produced by syncytiotrophoblast enter into maternal circulation, where they profoundly impact maternal physiology and are promising diagnostic indicators of pregnancy health. Despite the multifunctional importance of syncytiotrophoblast for pregnancy success, there is still much to learn about how its formation is regulated in normal and diseased states. 'Omics' approaches are gaining traction in many fields to provide a more holistic perspective of cell, tissue, and organ function. Herein, we review human syncytiotrophoblast development and current model systems used for its study, discuss how 'omics' strategies have been used to provide multidimensional insights into its formation and function, and highlight limitations of current platforms as well as consider future avenues for exploration.

Maternal Iron Deficiency Alters Trophoblast Differentiation and Placental Development in Rat Pregnancy.

Iron deficiency, which occurs when iron demands chronically exceed intake, is prevalent in pregnant women. Iron deficiency during pregnancy poses major risks for the baby, including fetal growth restriction and long-term health complications. The placenta serves as the interface between a pregnant mother and her baby, and it ensures adequate nutrient provisions for the fetus. Thus, maternal iron deficiency may impact fetal growth and development by altering placental function. We used a rat model of diet-induced iron deficiency to investigate changes in placental growth and development. Pregnant Sprague-Dawley rats were fed either a low-iron or iron-replete diet starting 2 weeks before mating. Compared with controls, both maternal and fetal hemoglobin were reduced in dams fed low-iron diets. Iron deficiency decreased fetal liver and body weight, but not brain, heart, or kidney weight. Placental weight was increased in iron deficiency, due primarily to expansion of the placental junctional zone. The stimulatory effect of iron deficiency on junctional zone development was recapitulated in vitro, as exposure of rat trophoblast stem cells to the iron chelator deferoxamine increased differentiation toward junctional zone trophoblast subtypes. Gene expression analysis revealed 464 transcripts changed at least 1.5-fold (P < 0.05) in placentas from iron-deficient dams, including altered expression of genes associated with oxygen transport and lipoprotein metabolism. Expression of genes associated with iron homeostasis was unchanged despite differences in levels of their encoded proteins. Our findings reveal robust changes in placentation during maternal iron deficiency, which could contribute to the increased risk of fetal distress in these pregnancies.

The Transcription Factor OVOL2 Represses ID2 and Drives Differentiation of Trophoblast Stem Cells and Placental Development in Mice.

Trophoblasts are the first cell type to be specified during embryogenesis, and they are essential for placental morphogenesis and function. Trophoblast stem (TS) cells are the progenitor cells for all trophoblast lineages; control of TS cell differentiation into distinct trophoblast subtypes is not well understood. Mice lacking the transcription factor OVO-like 2 (OVOL2) fail to produce a functioning placenta, and die around embryonic day 10.5, suggesting that OVOL2 may be critical for trophoblast development. Therefore, our objective was to determine the role of OVOL2 in mouse TS cell fate. We found that OVOL2 was highly expressed in mouse placenta and differentiating TS cells. Placentas and TS cells lacking OVOL2 showed poor trophoblast differentiation potential, including increased expression of stem-state associated genes (Eomes, Esrrb, Id2) and decreased levels of differentiation-associated transcripts (Gcm1, Tpbpa, Prl3b1, Syna). Ectopic OVOL2 expression in TS cells elicited precocious differentiation. OVOL2 bound proximate to the gene encoding inhibitor of differentiation 2 (ID2), a dominant negative helix-loop-helix protein, and directly repressed its activity. Overexpression of ID2 was sufficient to reinforce the TS cell stem state. Our findings reveal a critical role of OVOL2 as a regulator of TS cell differentiation and placental development, in-part by coordinating repression of ID2.

Histone deacetylase 1 and 2 drive differentiation and fusion of progenitor cells in human placental trophoblasts.

Cell fusion occurs when several cells combine to form a multinuclear aggregate (syncytium). In human placenta, a syncytialized trophoblast (syncytiotrophoblast) layer forms the primary interface between maternal and fetal tissue, facilitates nutrient and gas exchange, and produces hormones vital for pregnancy. Syncytiotrophoblast development occurs by differentiation of underlying progenitor cells called cytotrophoblasts, which then fuse into the syncytiotrophoblast layer. Differentiation is associated with chromatin remodeling and specific changes in gene expression mediated, at least in part, by histone acetylation. However, the epigenetic regulation of human cytotrophoblast differentiation and fusion is poorly understood. In this study, we found that human syncytiotrophoblast development was associated with deacetylation of multiple core histone residues. Chromatin immunoprecipitation sequencing revealed chromosomal regions that exhibit dynamic alterations in histone H3 acetylation during differentiation. These include regions containing genes classically associated with cytotrophoblast differentiation (TEAD4, TP63, OVOL1, CGB), as well as near genes with novel regulatory roles in trophoblast development and function, such as LHX4 and SYDE1. Prevention of histone deacetylation using both pharmacological and genetic approaches inhibited trophoblast fusion, supporting a critical role of this process for trophoblast differentiation. Finally, we identified the histone deacetylases (HDACs) HDAC1 and HDAC2 as the critical mediators driving cytotrophoblast differentiation. Collectively, these findings provide novel insights into the epigenetic mechanisms underlying trophoblast fusion during human placental development.

Syndecan-4 regulates extravillous trophoblast migration by coordinating protein kinase C activation.

Extravillous trophoblast (EVT) invasion is an essential component of human placentation. Poor EVT invasion is associated with obstetrical complications including preeclampsia. Integration of cues from the extracellular environment is required for directional EVT invasion, but how EVTs coordinate responses to these cues is not well understood. Syndecan-4 (SDC4) is a transmembrane heparan sulfate proteoglycan that binds to, and modulates the activity of, many extracellular proteins implicated in placental development. Therefore, we determined the functional importance of SDC4 for EVT invasion. We found that SDC4 is expressed by a first trimester EVT line (HTR8), and in EVTs in placenta throughout pregnancy, with higher expression during early pregnancy than at term. Higher expression was also observed in placentas from preeclampsia compared to normotensive pregnancies. SDC4-deficient HTR8 EVTs exhibited reduced migration and Matrigel-based invasion, both under basal conditions and following exposure to basic fibroblast growth factor and heparin-binding epidermal growth factor. SDC4-deficient HTR8 EVTs also showed reduced protein kinase C-alpha (PKCα) and AKT phosphorylation. SDC4 directly bound to activated PKCα in EVTs, and inhibition of PKCα decreased EVT invasion and migration. Our findings reveal an essential role of SDC4 as a regulator of EVT motility, in part through coordination of PKCα activation.