Villous Cytotrophoblast Research Articles

Fermitin family homolog-2 (FERMT2) is highly expressed in human placental villi and modulates trophoblast invasion

BackgroundIntegrins are transmembrane receptors that mediate cell–extracellular matrix (ECM) and cell-cell adhesion and trophoblast cells undergo changes in integrin expression as they differentiate. However, the mechanism(s) of integrin activation leading to integrin-mediated signaling in trophoblast cell differentiation is unknown. The Fermitin family proteins are integrin activators that help mediate integrin-mediated signaling, but have never been studied in detail within the human placenta. Thus, we examined the spatiotemporal pattern of expression of Fermitin family homolog-2 (FERMT2) in human chorionic villi throughout gestation and its role in trophoblast-substrate adhesion and invasion.MethodsPlacental villous tissue was obtained from patients undergoing elective terminations by dilatation and curettage at weeks 8–12 (n = 10), weeks 13–14 (n = 8), as well as from term deliveries at weeks 37–40 (n = 6). Tissues were fixed, processed and sections utilized for immunofluorescence analysis of FERMT2 expression during gestation. Additionally, HTR8-SVneo human trophoblast cells were transfected by electroporation with FERMT2-specific siRNAs or non-targeting siRNAs (control) and used in cell-substrate adhesion as well as invasion assays.ResultsFERMT2 was more commonly expressed in the basal domain of villous cytotrophoblast cells and prominently localized around the periphery of individual extravillous trophoblast cells. siRNA-mediated knockdown of FERMT2 in HTR8-SVneo cells resulted in significantly decreased trophoblast-substrate attachment (p < 0.05) as well as significantly decreased trophoblast invasion (p < 0.05) relative to control cells.ConclusionsThe detection of FERMT2 throughout extravillous trophoblast columns and the results of invasion assays demonstrated that this protein is likely an important regulator of integrin activation in extravillous cells to modulate migration and invasion.

48. ELABELA and placental development in pregnancy and preeclampsia

The peptide hormone ELABELA (ELA) was discovered in 2013 and found to be essential for zebrafish cardiovascular development through activation of the Apelin receptor (APLNR). In humans, ELA has been found to be expressed in adult kidney and prostate, and in embryonic pluripotent stem cells where it serves as a growth factor, but signals via a yet unidentified second receptor. To further investigate the contribution of ELA to mammalian development ELA knockout mice were generated. The knockout embryos showed clear cardiovascular defects, but also the placentas were affected, which may lead to preeclampsia-like symptoms in ELA knockout mothers. Indeed, the mice showed significantly increased blood pressure and proteinuria. Remarkably, infusion of synthetic ELA was able to entirely rescue blood pressure, proteinuria, and increase fetal birth weight. The above findings let to studying ELA in human placentation and preeclampsia (PE). ELA was localized in villous cytotrophoblasts and, as expected for a hormone, in the syncytiotrophoblast layer of first trimester placental tissues. High expression was also observed in invading extravillous trophoblasts. In trophoblast cell lines ELA addition significantly increased the invasive capacity of these cells as measured in transwell invasion assays. In first trimester placental explant cultures ELA changed the morphology of the extravillous outgrowth and decreased trophoblast proliferation. Interestingly, preliminary results suggest that these downstream effects of ELA are not initiated by ELA binding to the Apelin receptor, therefore the second unknown receptor must be considered instead. Finally, circulating ELA in plasma of healthy and PE pregnancies measured at multiple time points in gestation did not appear to be associated with PE development. However, ELA’s peptide properties were found to interfere with the initial measurements done. These technical issues therefore first need to be resolved before definite conclusions can be drawn on the applicability of ELA as potential PE biomarker.

Genomic Profiling of BDE-47 Effects on Human Placental Cytotrophoblasts.

Despite gradual legislative efforts to phase out flame retardants (FRs) from the marketplace, polybrominated diphenyl ethers (PBDEs) are still widely detected in human maternal and fetal tissues, eg, placenta, due to their continued global application in consumer goods and inherent biological persistence. Recent studies in rodents and human placental cell lines suggest that PBDEs directly cause placental toxicity. During pregnancy, trophoblasts play key roles in uterine invasion, vascular remodeling, and anchoring of the placenta-fetal unit to the mother. Thus, to study the potential consequences of PBDE exposures on human placental development, we used an in vitro model: primary villous cytotrophoblasts (CTBs). Following exposures, the endpoints that were evaluated included cytotoxicity, function (migration, invasion), the transcriptome, and the methylome. In a concentration-dependent manner, common PBDE congeners, BDE-47 and -99, significantly reduced cell viability and increased death. Upon exposures to sub-cytotoxic concentrations (≤ 5 µM), we observed BDE-47 accumulation in CTBs with limited evidence of metabolism. At a functional level, BDE-47 hindered the ability of CTBs to migrate and invade. Transcriptomic analyses of BDE-47 effects suggested concentration-dependent changes in gene expression, involving stress pathways, eg, inflammation and lipid/cholesterol metabolism as well as processes underlying trophoblast fate, eg, differentiation, migration, and vascular morphogenesis. In parallel assessments, BDE-47 induced low-level global increases in methylation of CpG islands, including a subset that were proximal to genes with roles in cell adhesion/migration. Thus, using a primary human CTB model, we showed that PBDEs induced alterations at cellular and molecular levels, which could adversely impact placental development.

Melatonin modulates autophagy and inflammation protecting human placental trophoblast from hypoxia/reoxygenation.

Melatonin has been proposed as a possible treatment for the deleterious effects of hypoxia/reoxygenation (H/R), such as autophagy, inflammation, and apoptosis. Pathological pregnancies, such as preeclampsia, are associated with placental H/R, and decreased placental melatonin synthesis as well as lower melatonin levels in the placenta and maternal plasma. However, the effects of exogenous melatonin on inflammation and autophagy induced by pregnancy complications associated with H/R await investigation. This study aimed to determine as to whether melatonin protects human primary villous trophoblasts against H/R-induced autophagy, inflammation, and apoptosis. Human primary villous cytotrophoblasts were isolated and immunopurified from normal term placentas. These cells were then exposed or not to 1mmol/L melatonin for 72hour in normoxia (8% O2 ), thereby inducing differentiation into syncytiotrophoblast that was then exposed to H/R (0.5% O2 , for 4hour) or normoxia. H/R decreased endogenous melatonin synthesis (by 68%) and interleukin (IL)-10 levels (by 72%), coupled to increased tumor necrosis factor (TNF) (by 114%), IL-6 (by 55%), and NFκB (by 399%), compared to normoxia. Melatonin treatment reversed the H/R effect, restoring IL-10, TNF, and IL-6 levels to those of the normoxia condition. Melatonin, as well as NFκB inhibition, enhanced autophagy activation, consequently increasing syncytiotrophoblast survival in H/R conditions. This study suggests that H/R, which is present in pregnancy complications, inhibits endogenous melatonin production, thereby contributing to reduced syncytiotrophoblast viability. Results indicate that exogenous melatonin treatment may afford protection against H/R-induced damage, thereby enhancing placental cell survival, and contributing to improved fetal outcomes.

Expression and Regulation of Retinoic Acid Receptor Responders in the Human Placenta.

Retinoic acid (RA) signaling through its receptors (RARA, RARB, RARG, and the retinoic X receptor RXRA) is essential for healthy placental and fetal development. An important group of genes regulated by RA are the RA receptor responders (RARRES1, 2, and 3). We set out to analyze their expression and regulation in healthy and pathologically altered placentas of preeclampsia (PE) and intrauterine growth restriction (IUGR) as well as in trophoblast cell lines. We performed immunohistochemical staining on placental sections and analyzed gene expression by real-time polymerase chain reaction. Additionally, we performed cell culture experiments and stimulated Swan71 and Jeg-3 cells with different RA derivates and 2'-deoxy-5-azacytidine (AZA) to induce DNA demethylation. RARRES1, 2, and 3 and RARA, RARB, RARG, and RXRA are expressed in the extravillous part of the placenta. RARRES1, RARA, RARG, and RXRA were additionally detected in villous cytotrophoblasts. RARRES gene expression was induced via activation of RARA, RARB, and RARG in trophoblast cells. RARRES1 was overexpressed in villous trophoblasts and the syncytiotrophoblast from PE placentas, but not in IUGR without PE. Promoter methylation was detectable for RARRES1 and RARB based on their sensitivity toward AZA treatment of trophoblast cell lines. RARRES1, 2 and 3 are expressed in the functional compartments of the human placenta and can be regulated by RA. We hypothesize that the epigenetic suppression of trophoblast RARRES1 and RARB expression and the upregulation of RARRES1 in PE trophoblast cells suggest an involvement of environmental factors (eg, maternal vitamin A intake) in the pathogenesis of this pregnancy complication.

HLA-DR is aberrantly expressed at feto-maternal interface in pre-eclampsia

In normal pregnancy, villous cytotrophoblast and syncytiotrophoblast do not express HLA Class I and Class II molecules, while invasive extravillous trophoblast only express class I HLA-C and the atypical class Ib antigens, HLA-G, -E and -F. Inadequate maternal tolerance of invasive trophoblast has been proposed as a possible immunologic trigger of poor trophoblast invasion and subsequent occurrence of pre-eclampsia. This study aimed to investigate possible aberrant expression of class II HLA-DR on placentae and syncytiotrophoblast-derived extracellular vesicles (STEVs), obtained by dual placental perfusion, from pre-eclampsia (n = 23) and normal pregnant (n = 14) women. Here we demonstrate that HLA-DR can be detected in syncytiotrophoblast from a significant proportion of pre-eclampsia but not control placentae. HLA-DR was also observed, by flow cytometry, on STEVs and associated with placental alkaline phosphatase to validate their placental origin. HLA-DR positive syncytiotrophoblast was detected in placental biopsies from pre-eclampsia but not normal control cases, using immunohistochemistry. The HLA may be fetal or maternal origin. In the latter case a possible mechanism of acquisition is trogocytosis.

Morphological and ultrastructural changes in the placenta of the diabetic pregnant Egyptian women

Diabetes mellitus (DM) is a chronic metabolic disease in which the body fails to produce enough insulin or increased tissue resistance to insulin. The diabetes may have profound effects on placental development and function. This study was designed to detect the placental changes in pregnancy associated with DM comparing these changes with normal placenta. The study was carried out on sixty full-term placentae; divided into three equal groups; control group (group I): placentae of normal pregnancy, uncontrolled diabetes (group II): placentae from pregnant women whose blood glucose is poorly controlled during pregnancy. Controlled diabetes (group III): includes placentae from diabetic women whose blood glucose is controlled during pregnancy. The placentae from group II tend to be heavier and exhibited immaturity of villi, villous edema, fibrosis, excessive syncytial knots formation and infarctions. In addition to, fibrinoid necrosis, increased thickness of vasculosyncytial membrane, syncytial basement membrane, microvillous abnormalities and vascular endothelial changes were demonstrated. The syncytial multivesicular knots were present in placentae of group II. The nuclei within these syncytial knots display condensed chromatin, either dispersed throughout the nucleus or in the form of dense peripheral clumps with and numerous cytoplasmic vacuoles. The syncytial basement membrane showed focal areas of increase in its thickness and irregularity. Villous cytotrophoblasts showed increased number and activity in the form of numerous secretory granules, abundant dilated RER, larger distorted mitochondria. Villous vessels showed various degrees of abnormalities in the form of endothelial cell enlargement, folding, thickening and protrusion of their luminal surfaces into vascular lumen making it narrower in caliber. In placentae of group III, most of these abnormalities decreased. In most of placentae of group III, the VSM appeared nearly normal in thickness and showed nearly normal composition of one layer of syncytiotrophoblastic cells, one layer of smooth, regular capillary endothelium and the space between them. Mild microvillous abnormalities were noted in few placentae as they appeared short and blunted with mild decrease in their number per micron. The electron picture of syncytial knots appeared nearly normal containing aggregations of small, condensed hyperchromatic nuclei, minimal vacuoles could be seen in the cytoplasm of syncytial knots. Syncytial basement membrane appeared regular and nearly normal in its thickness and composition coming in direct contact with fetal blood capillaries but mild abnormalities were noted in the basement membrane in few placentae as increased its thickness and deposition of fibers or fibrinoid. Regarding cytotrophoblasts in the terminal villi of placentae with controlled diabetes, these cells appeared nearly normal. They were scattered beneath the syncytium and were active containing mitochondria, rough endoplasmic reticulum, free ribosomes and a large nucleus with fine dispersed chromatin. The vascular ultrastructural pattern in terminal villi of placentae of this group showed no significant abnormalities and was normally distributed in the villous tree. The luminal surface of the vascular endothelium appeared regular smooth in the majority of placentae of this group. The endothelial cells appeared connected to each other with tight junctions. It could be concluded that whether if long-term diabetes is controlled or not, placentae of diabetic mother showed a variety of significant histological structural changes seen more frequently than in the placentae of pregnant women without diabetes.

Liposomes as Gene Delivery Vectors for Human Placental Cells.

Nanomedicine as a therapeutic approach for pregnancy-related diseases could offer improved treatments for the mother while avoiding side effects for the fetus. In this study, we evaluated the potential of liposomes as carriers for small interfering RNAs to placental cells. Three neutral formulations carrying rhodamine-labelled siRNAs were evaluated on an in vitro model, i.e., human primary villous cytotrophoblasts. siRNA internalization rate from lipoplexes were compared to the one in the presence of the lipofectamine reagent and assessed by confocal microscopy. Results showed cellular internalization of nucleic acid with all three formulations, based on two cationic lipids, either DMAPAP or CSL-3. Moreover, incubation with DMAPAP+AA provided a rate of labelled cells as high as with lipofectamine (53 ± 15% and 44 ± 12%, respectively) while being more biocompatible. The proportion of cells which internalized siRNA were similar when using DMAPAP/DDSTU (16 ± 5%) and CSL-3 (22 ± 5%). This work highlights that liposomes could be a promising approach for gene therapy dedicated to pregnant patients.

Effects of selective serotonin-reuptake inhibitors (SSRIs) on human villous trophoblasts syncytialization

Selective serotonin-reuptake inhibitors (SSRIs) are the most commonly prescribed antidepressants during pregnancy. The human placenta is a highly specialized organ supporting normal growth and development of the fetus. Therefore, this study aims to analyze the effects of SSRIs on villous cytotrophoblasts cells, using BeWo cells and human placental trophoblast cells in primary culture. The SSRIs fluoxetine and its metabolite norfluoxetine, sertraline and venlafaxine did not affect BeWo cell proliferation and viability, nor the percentage of M30-positive (apoptotic) primary trophoblast cells. None of the SSRIs affected basal or forskolin-stimulated BeWo cell fusion, whereas sertraline and venlafaxine increased the fusion of primary villous trophoblasts. Sertraline and venlafaxine also modified human chorionic gonadotropin beta (β-hCG) secretion by BeWo cells, whereas none of the SSRIs affected β-hCG secretion in primary trophoblasts. Norfluoxetine increased CGB (chorionic gonadotropin beta) and GJA1 (gap junction protein alpha 1) levels of gene expression (biomarkers of syncytialization) in BeWo cells, whereas in primary trophoblasts none of the SSRIs tested affected the expression of these genes. This study shows that SSRIs affect villous trophoblast syncytialization in a structure- and concentration-dependent manner and suggests that certain SSRIs may compromise placental health. In addition, it highlights the importance of using primary trophoblast cells instead of “trophoblast -like” cell lines to assess the effects of medications on human villous trophoblast function.

Spatiotemporal distribution of small ubiquitin-like modifiers during human placental development and in response to oxidative and inflammatory stress.

Key points The post‐translational modification of target proteins by SUMOylation occurs in response to stressful stimuli in a variety of organ systems.Small ubiquitin‐like modifier (SUMO) isoforms 1–4 have recently been identified in the human placenta, and are upregulated in the major obstetrical complication of pre‐eclampsia.This is the first study to characterize the spatiotemporal distribution of SUMO isoforms and their targets during placental development across gestation and in response to stress induced by pre‐eclampsia and chorioamnionitis.Keratins were identified as major targets of placental SUMOylation. The interaction with SUMOs and cytoskeletal filaments provides evidence for SUMOylation possibly contributing to underlying dysfunctional trophoblast turnover, which is a hallmark feature of pre‐eclampsia.Further understanding the role of individual SUMO isoforms and SUMOylation underlying placental dysfunction may provide a target for a novel therapeutic candidate as an approach for treating pre‐eclampsia complicated with placental pathology. SUMOylation is a dynamic, reversible post‐translational modification that regulates cellular protein stability and localization. SUMOylation occurs in response to various stressors, including hypoxia and inflammation, features common in the obstetrical condition of pre‐eclampsia. SUMO isoforms 1–4 have recently been identified in the human placenta, but less is known about their role in response to pre‐eclamptic stress. We hypothesized that SUMOylation components have a unique spatiotemporal distribution during placental development and that their subcellular localization can be further modulated by extra‐cellular stressors. Placental SUMO expression was examined across gestation. First‐trimester human placental explants and JAR cells were subjected to hypoxia or TNF‐α cytokine, and subcellular translocation of SUMOs was monitored. SUMOylation target proteins were elucidated using mass spectrometry and proximity ligation assay. Placental SUMO‐1 and SUMO‐4 were restricted to villous cytotrophoblast cells in first trimester and syncytium by term, while SUMO‐2/3 staining was evenly distributed throughout the trophoblast across gestation. In placental villous explants, oxidative stress induced hyperSUMOylation of SUMO‐1 and SUMO‐4 in the syncytial cytoplasm, whereas SUMO‐2/3 nuclear expression increased. Oxidative stress also upregulated cytoplasmic SUMO‐1 and SUMO‐4 protein expression (P < 0.05), similar to pre‐eclamptic placentas. Keratins were identified as major targets of placental SUMOylation. Oxidative stress increased the cytokeratin‐7 to SUMO‐1 and SUMO‐4 interactions, while inflammatory stress increased its interaction with SUMO‐2/3. Overall, SUMOs display a unique spatiotemporal distribution in normal human placental development. Our data indicate SUMOylation in pre‐eclampsia, which may impair the stability of cytoskeleton filaments and thus promote trophoblast shedding into the maternal circulation in this condition.

RNA-Seq identifies genes whose proteins are transformative in the differentiation of cytotrophoblast to syncytiotrophoblast, in human primary villous and BeWo trophoblasts

The fusion of villous cytotrophoblasts into the multinucleated syncytiotrophoblast is critical for the essential functions of the mammalian placenta. Using RNA-Seq gene expression and quantitative protein expression, we identified genes and their cognate proteins which are coordinately up- or down-regulated in two cellular models of cytotrophoblast to syncytiotrophoblast development, human primary villous and human BeWo cytotrophoblasts. These include hCGβ, TREML2, PAM, CRIP2, INHA, FLRG, SERPINF1, C17orf96, KRT17 and SAA1. These findings provide avenues for further understanding the mechanisms underlying mammalian placental synctiotrophoblast development.

MiR-518b Enhances Human Trophoblast Cell Proliferation Through Targeting Rap1b and Activating Ras-MAPK Signal

Preeclampsia is a pregnancy-specific complication defined as newly onset gestational hypertension and proteinuria. Deficiency in placental development is considered as the predominant cause of preeclampsia. Our previous study found that the expression of miR-518b increased significantly in the preeclamptic placentas, indicating the potential participation of this small RNA in the occurrence of preeclampsia. In this study, data analysis using multiple databases predicted Rap1b as a candidate target of miR-518b. An evident decrease in Rap1b expression was observed in preeclamptic placentas when compared with the control placentas, which was negatively correlated with the level of miR-518b. Based on the data of in situ hybridization and immunohistochemistry showing that Rap1b exhibited similar localization with miR-518b in villous cytotrophoblast cells and column trophoblasts, we further explored their function in regulating trophoblast cell proliferation. In HTR8/SVneo cells, exogenous transfection of miR-518b reduced the expression of Rap1b, and dual-luciferase reporter assay validated Rap1b as the direct target of miR-518b. The small RNA could increase the BrdU incorporation and the ratio of cells at S phase, and enhance the phosphorylation of Raf-1 and ERK1/2. Such growth-promoting effect could be efficiently reversed by Rap1b overexpression. The data indicate that miR-518b can promote trophoblast cell proliferation via Rap1b–Ras–MAPK pathway, and the aberrant upregulation of miR-518b in preeclamptic placenta may contribute to the excessive trophoblast proliferation. The study provides new evidence to further understand the etiology of preeclampsia.

Profile of CYP19A1 mRNA expression and aromatase activity during syncytialization of primary human villous trophoblast cells at term

Estrogen production by the human villous trophoblast is dependent on the biosynthetic enzyme aromatase (CYP19; CYP19A1) and is crucial for successful placental development and pregnancy outcome. Using villous cytotrophoblast cells (vCTs) freshly isolated from normal term placenta, we characterized the promoter-specific expression of CYP19A1 mRNA (derived from promoters I.1, I.4, I.8 or total transcript) and aromatase activity during villous trophoblast syncytialization. CYP19A1 mRNA levels and aromatase activity in vCTs reached a maximum after about 48 h of culture. The cAMP inducer forskolin (10 μM) and protein kinase C stimulant phorbol myristate acetate (1 μM) increased CYP19A1 mRNA levels by 1.8- and 1.6-fold, respectively, as well as inducing aromatase catalytic activity. Dexamethasone (100 nM) and vascular endothelial growth factor (5 ng/mL) decreased CYP19A1 mRNA levels, while having no effect on aromatase activity. Our results emphasize the importance of not solely studying CYP19A1 regulation and function at the mRNA level but also considering posttranslational mechanisms that alter the final catalytic activity of aromatase.

Derivation of Human Trophoblast Stem Cells

Trophoblast cells play an essential role in the interactions between the fetus and mother. Mouse trophoblast stem (TS) cells have been derived and used as the best invitro model for molecular and functional analysis of mouse trophoblast lineages, but attempts to derive human TS cells have so far been unsuccessful. Here we show that activation of Wingless/Integrated (Wnt) and EGF and inhibition of TGF-β, histone deacetylase (HDAC), and Rho-associated protein kinase (ROCK) enable long-term culture of human villous cytotrophoblast (CT) cells. The resulting cell lines have the capacity to give rise to the three major trophoblast lineages, which show transcriptomes similar to those of the corresponding primary trophoblast cells. Importantly, equivalent cell lines can be derived from human blastocysts. Our data strongly suggest that the CT- and blastocyst-derived cell lines are human TS cells, which will provide a powerful tool to study human trophoblast development and function.

Isolation and Purification of Villous Cytotrophoblast Cells from Term Human Placenta.

The placenta is a key element during pregnancy for the health of the fetus and the mother, which justifies why placental studies are so important. One of the best models for placental studies is the primary cell culture of cytotrophoblast cells from human term placentas. In this chapter, we will detail firstly the isolation of cytotrophoblast cells, with tissue preparation, digestion, Percoll gradient, and cell freezing, and secondly the cell immunopurification and seeding.

P57 and Ki-67 expression in hydropic abortion and molar pregnancy

Background The histopathological diagnosis and differentiation between partial mole (PM) and complete mole (CM) bears difficulties. Hence, additional methods rather than morphology can be useful to reach the proper diagnosis. Immunohistochemical (IHC) analysis using antibodies such as p57 and Ki-67 can be used to solve this problem. This study was done to investigate the role of p57 and Ki-67 IHC in distinguishing between CM, PM, and hydropic abortion (HA). Patients and methods Twenty cases of molar pregnancies – 10 (CM) and 10 (PM) – and 10 HA diagnosed on histopathological criteria were studied histopathologically along with IHC using antibodies against p57 and Ki-67. The cases were collected in a retrospective manner from Pathology Lab, Faculty of Medicine, Mansoura University from January 2015 to December 2015. Results We found that CM demonstrated enlarged villi with circumferential trophoblastic hyperplasia. Villous stromal cells and cytotrophoblast were negative for p57. PM demonstrated hydropic, irregularly shaped villi with scalloped contours, trophoblastic inclusions, and mild circumferential trophoblastic hyperplasia. Villous stromal cells and cytotrophoblast were diffusely positive for p57. In all 10 CM cases, the Ki-67 proliferation index was relatively high (>26% of cells) with complete absence of p57 immunostaining in the cytotrophoblasts and villous stromal cells in comparison with PM cases with statistical significance (P Conclusion Our study further confirms the valuable role of p57 in the diagnosis of CM augmented by the traditional histopathological criteria. Ki-67 has a limited value in differentiating between some cases of PM and CM. The best possible use of Ki-67 is to differentiate between molar and nonmolar pregnancy along with other histological parameters, especially trophoblastic proliferation and villous enlargement.

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNFα), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNFα regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNFα signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta.

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNFα), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNFα regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNFα signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Use of GATA3 and TWIST1 Immunofluorescence Staining to Assess In Vitro Syncytial Fusion Index.

In human placenta, the multinucleated syncytiotrophoblast (ST) allows all the exchanges between the maternal and fetal circulation and is also the site of placental hormonal functions. Absence or disturbances of ST formation are associated with a defect or pathologies of pregnancy such as preeclampsia (PE) and intrauterine growth retardation (IUGR). All along pregnancy, the ST is regenerated by fusion of underlying mononucleated villous cytotrophoblasts (VCT). The protocol described here provides details on how GATA3 or TWIST1 immunostaining and analysis can be used to easily assess the in vitro differentiation of human placental cytotrophoblast.

The early pregnancy placenta foreshadows DNA methylation alterations of solid tumors

ABSTRACTThe placenta relies on phenotypes that are characteristic of cancer to successfully implant the embryo in the uterus during early pregnancy. Notably, it has to invade its host tissues, promote angiogenesis—while surviving hypoxia—, and escape the immune system. Similarities in DNA methylation patterns between the placenta and cancers suggest that common epigenetic mechanisms may be involved in regulating these behaviors. We show here that megabase-scale patterns of hypomethylation distinguish first from third trimester chorionic villi in the placenta, and that these patterns mirror those that distinguish many tumors from corresponding normal tissues. We confirmed these findings in villous cytotrophoblasts isolated from the placenta and identified a time window at the end of the first trimester, when these cells come into contact with maternal blood, as the likely time period for the methylome alterations. Furthermore, the large genomic regions affected by these patterns of hypomethylation encompass genes involved in pathways related to epithelial-mesenchymal transition, immune response, and inflammation. Analyses of expression profiles corresponding to genes in these hypomethylated regions in colon adenocarcinoma tumors point to networks of differentially expressed genes previously implicated in carcinogenesis and placentogenesis, where nuclear factor kappa B is a key hub. Taken together, our results suggest the existence of epigenetic switches involving large-scale changes of methylation in the placenta during pregnancy and in tumors during neoplastic transformation. The characterization of such epigenetic switches might lead to the identification of biomarkers and drug targets in oncology as well as in obstetrics and gynecology.