Villous Cytotrophoblast Research Articles

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.

The Hippo-dependent transcriptional co-activator YAP controls differentiation of first trimester villous cytotrophoblasts

The Hippo-dependent transcriptional co-activator YAP controls differentiation of first trimester villous cytotrophoblasts

Human placenta expresses both peripheral and neuronal isoform of tryptophan hydroxylase

The role of placental serotonin has been an active topic of research notably because of its crucial role in brain development. However, which cell types synthesize serotonin in human placenta remains unknown. Moreover, it is not known if the two tryptophan hydroxylase isoforms (TPH1 and TPH2), the rate-limiting enzymes in serotonin biosynthesis, are expressed in placenta. Human placentas were obtained in first trimester or at term, and trophoblast cells were isolated and purified using a magnetic cell sorter and placed in primary culture. The tissue sublocalization of each TPH was determined by immunohistochemistry. TPH expression in primary villous trophoblasts was determined by PCR and immunoblotting, and serotonin secretion by LC-MS/MS. Villous cytotrophoblasts, syncytiotrophoblast, fetal capillaries, extravillous cytotrophoblasts, and decidual cells co-expressed TPH1 and TPH2. Moreover, mRNA and protein levels of both TPHs were detected in human primary trophoblast as well as in mouse placental tissues. Finally, human trophoblast cells were shown to produce serotonin de novo. This study demonstrates that both TPH1 and TPH2 are expressed in human and mouse placenta throughout pregnancy and helps to better understand the placental serotonin system, which is crucial for healthy pregnancy and fetal development. It is therefore important to further understand regulation of the placental serotonin system and how its disruption during pregnancy may impact the developing fetus and subsequent child programming.

The use of a unique co-culture model of fetoplacental steroidogenesis as a screening tool for endocrine disruptors: The effects of neonicotinoids on aromatase activity and hormone production

Estrogen biosynthesis during pregnancy is dependent on the collaboration between the fetus producing the androgen precursors, and the placenta expressing the enzyme aromatase (CYP19). Disruption of estrogen production by contaminants may result in serious pregnancy outcomes. We used our recently developed in vitro co-culture model of fetoplacental steroidogenesis to screen the effects of three neonicotinoid insecticides on the catalytic activity of aromatase and the production of steroid hormones. A co-culture of H295R human adrenocortical carcinoma cells with fetal characteristics and BeWo human choriocarcinoma cells which display characteristics of the villous cytotrophoblast was exposed for 24h to various concentrations of three neonicotinoids: thiacloprid, thiamethoxam and imidacloprid. Aromatase catalytic activity was determined in both cell lines using the tritiated water-release assay. Hormone production was measured by ELISA. The three neonicotinoids induced aromatase activity in our fetoplacental co-culture and concordingly, estradiol and estrone production were increased. In contrast, estriol production was strongly inhibited by the neonicotinoids. All three pesticides induced the expression of CYP3A7 in H295R cells, and this induction was reversed by co-treatment of H295R cells with exogenous estriol. CYP3A7 is normally expressed in fetal liver and is a key enzyme involved in estriol synthesis. We suggest that neonicotinoids are metabolized by CYP3A7, thus impeding the 16α-hydroxylation of fetal DHEA(-sulfate), which is normally converted to estriol by placental aromatase. We successfully used the fetoplacental co-culture as a physiologically relevant tool to highlight the potential effects of neonicotinoids on estrogen production, aromatase activity and CYP3A7 expression during pregnancy.

Increased methylation and decreased expression of homeobox genes TLX1, HOXA10 and DLX5 in human placenta are associated with trophoblast differentiation

Homeobox genes regulate embryonic and placental development, and are widely expressed in the human placenta, but their regulatory control by DNA methylation is unclear. DNA methylation analysis was performed on human placentae from first, second and third trimesters to determine methylation patterns of homeobox gene promoters across gestation. Most homeobox genes were hypo-methylated throughout gestation, suggesting that DNA methylation is not the primary mechanism involved in regulating HOX genes expression in the placenta. Nevertheless, several genes showed variable methylation patterns across gestation, with a general trend towards an increase in methylation over gestation. Three genes (TLX1, HOXA10 and DLX5) showed inverse gains of methylation with decreasing mRNA expression throughout pregnancy, supporting a role for DNA methylation in their regulation. Proteins encoded by these genes were primarily localised to the syncytiotrophoblast layer, and showed decreased expression later in gestation. siRNA mediated downregulation of DLX5, TLX1 and HOXA10 in primary term villous cytotrophoblast resulted in decreased proliferation and increased expression of differentiation markers, including ERVW-1. Our data suggest that loss of DLX5, TLX1 and HOXA10 expression in late gestation is required for proper placental differentiation and function.

P 9 Involvement of B-cell lymphoma 6 in the fusion process of trophoblastic cells of the placenta

Introduction Preeclampsia (PE) is one of the leading causes of maternal and perinatal mortality and morbidity and despite intensive research its pathogenesis is not totally understood. In a previous study, we evaluated the gene expression of placental samples from PE patients and controls using a self-designed gene array that targeted critical signaling pathways. Interestingly, the gene of B-cell lymphoma 6 (BCL6), a transcriptional repressor and key regulator of B-lymphocyte development, is increased in preeclamptic placentas. It is expressed in villous cytotrophoblasts (vCTBs), promotes proliferation and survival of trophoblastic cells and is a promising target to decipher the molecular mechanisms behind the pathogenesis of PE in more detail. Objectives The cell-cell fusion of mononuclear vCTBs forms the continuous syncytiotrophoblast (STB) layer. This process is critical for the placental development and is known to be deregulated in preeclamptic placenta. In this work, we have investigated the potential role of BCL6 in the fusion process of trophoblastic cells. Materials and methods Various molecular biological methods were used for this study including Western blotting, quantitative real-time PCR, immunofluorescence staining and ELISA to explore the function of BCL6 in the fusion process of trophoblastic cell lines and vCTBs isolated from term placentas. Cell lines were stimulated with forskolin to induce cell fusion. Results We show that BCL6 is present throughout the whole fusion process. We also demonstrate that the fusion capability of the investigated cells is affected by BCL6, as knockdown of BCL6 results in a considerable enhanced fusion rate of BeWo cells. Furthermore, increased mRNA expression of fusion-related proteins is also observable in JEG-3 cells as well as vCTBs. Conversely, stable overexpression of BCL6 impairs the process of fusion in BeWo cells. Conclusion Our data indicate that an accurately regulated expression of BCL6 is important for proper differentiation and successful syncytialization of trophoblasts in the placenta. Increased BCL6 impairs trophoblast fusion, contributing to the development of PE. Further work is required to detail the molecular mechanisms whereby BCL6 affects the fusion. It also warrants investigations in vivo and in vitro to clarify the correlation between elevated BCL6 and deregulated cytokines, hypoxia and oxidative stress in the preeclamptic placenta.

Expression pattern and phosphorylation status of Smad2/3 in different subtypes of human first trimester trophoblast

IntroductionTGF-β superfamily members are thought to play a pivotal role in placental development and differentiation. However, their downstream effectors, the Smad transcription factors, have been poorly investigated in human trophoblasts. MethodsExpression and localisation of the canonical TGF-β targets Smad2/3 and their regulators (Smad4 and Smad7) were investigated in first trimester placenta and purified cytotrophoblast (CTB) subtypes using immunofluorescence, western blotting and qPCR. Canonical and non-canonical activation was analysed in nuclear/cytoplasmic extracts of trophoblast subtypes as well as in tissue sections using antibodies against Smad2/3, phosphorylated either at the C-terminus (pSmad2C/3C) or in their linker regions (pSmad2L/3L). Smad phosphorylation was also examined in differentiating extravillous trophoblasts (EVTs) in the absence or presence of decidual stromal cell (DSC)-conditioned medium. ResultsSmad2, Smad4 and Smad7 protein were uniformly expressed between 6th and 12th week placentae and the different isolated CTB subtypes. Activated pSmad2L was mainly detected in nuclei and cytoplasm of villous CTBs, whereas pSmad2C was absent from these cells. In contrast, pSmad2C could be detected in the cytoplasm of cell column trophoblasts and in the cytoplasm/nuclei of EVTs. Smad3 and its phosphorylated forms pSmad3C and pSmad3L specifically localised to EVT nuclei. During EVT differentiation autocrine activation of pSmad2C/3C and pSmad3L was observed. DSC-conditioned medium further increased Smad2/3 phosphorylation in EVTs. DiscussionThe lack of pSmad2C in villous CTBs suggests that other mitogens than TGF-β could promote Smad2 linker phosphorylation under homeostatic conditions. Whereas autocrine signalling activates Smad2/3 in differentiating EVTs, paracrine factors contribute to Smad phosphorylation in these cells.

Role of peroxiredoxin2 downregulation in recurrent miscarriage through regulation of trophoblast proliferation and apoptosis

Peroxiredoxin (Prdx) 2 is an antioxidant protein that utilizes its redox-sensitive cysteine groups to reduce hydrogen peroxide molecules and protect cells against oxidative damage from reactive oxygen species (ROS). However, its function in trophoblasts at the maternal–fetal interface has not been clarified yet. In this study, significantly lower Prdx2 expression was found in the first-trimester villous cytotrophoblasts of patients with recurrent miscarriage (RM) than in cytotrophoblasts from healthy controls. Further, Prdx2 knockdown inhibited proliferation and increased apoptosis of trophoblast cells. The reason for this may be an increase in the level of cellular ROS after knockdown of Prdx2, which may subsequently lead to an increase in the expression of phosphorylated p53 (p-p53) and p38-MAPK/p21. Prdx2 knockdown also impaired the fusion of BeWo cells induced by forskolin. Bioinformatics analysis identified a c-Myc-binding site in the Prdx2 promoter region, and chromatin immunoprecipitation verified that c-Myc directly bound to a site in this locus. Suppression and overexpression of c-Myc resulted in reduction and increase of Prdx2 expression respectively. Furthermore, we demonstrated that c-Myc was downregulated in the first-trimester cytotrophoblasts of patients with RM, and its downregulation is also related with inhibited cell proliferation, increased apoptosis, as well as upregulated p21 expression and p-p53/p53 ratio. Our findings indicate that Prdx2 might have an important role in the regulation of trophoblast proliferation and apoptosis during early pregnancy, and that its expression is mediated by c-Myc. Thus, these two proteins may be involved in the pathogenesis of RM and may represent potential therapeutic targets.

Impact of chlorpyrifos on human villous trophoblasts and chorionic villi

Placental barrier regulates maternal-fetal interchange protecting the baby from damage caused by substances found in the uterine environment or circulating in the vascular system. Organophosphate (OP) pesticides are a paramount group of environmental pollutants used in intensive agriculture for protection against diseases and pests. While many studies have reported an increased risk of pregnancy alterations in pregnant women exposed to OPs, few have analyzed the effects caused by these pesticides in the placenta.Herein, we evaluated the effects of chlorpyrifos (CPF), one of the most widely used OP insecticides, on human placenta using in vitro and ex vivo exposure models. Villous cytotrophoblast cells isolated from normal human term placentas maintained their cell viability, differentiated into syncytiotrophoblast-like structures, and increased the expression of β-hCG, ABCG2, and P-gp in the presence of CPF at concentrations of 10 to 100μM. The same doses of CPF induced marked changes in chorionic villi samples. Indeed, CPF exposure increased stroma cell apoptosis, altered villi matrix composition, basement membrane thickness, and trophoblastic layer integrity. Histomorphological and ultrastructural alterations are compatible with those found in placentas where maternal-placenta injury is chronic and able to impair the placental barrier function and nutrient transport from mother to the fetus.Our study shows that placental ex vivo exposure to CPF produces tissue alterations and suggest that human placenta is a potential target of CPF toxicity. In addition, it highlights the importance of using different models to assess the effects of a toxic on human placenta.

Immunoexpression of P53 protein in trophoblastic diseases

Background and objective: Trophoblastic diseases constitute a spectrum of tumors and tumor- like conditions characterized by proliferation of pregnancy associated trophoblastic tissue of progressive malignant potential. This study aimed to assess the value of p53 protein immunoexpression in the diagnosis of hydatidiform molar pregnancy and the differential diagnosis of its subtypes (complete and partial) from abortions. Methods: A cross-sectional study of tissue sections from 68 formalin-fixed, paraffin- embedded specimens of products of conception, including 1st trimester abortion (n=15), partial hydatidiform mole PHM (n=24), complete hydatidiform CHM (n=24) and full term placenta (n=5), all were examined at the Histopathology Department of Maternity Teaching Hospital in Erbil, Iraq during the period of Jan.2013-Jun.2013. Immunohistochemistry was performed using p53 antibody and the standard streptavidin-biotin immunoperoxidase method. The labeling index (number of positive nuclei/total number of nuclei) for villous cytotrophoblasts, syncytiotrophoblasts and stromal cells were evaluated separately. Statistical analysis was carried out by one way ANOVA and Fisher’s exact tests, statistical significance was determined at P ≤0.05. Results: All villous trophoblastic lesions showed higher p53 immunoexpression in all villous components especially cytotrophoblasts, being the highest in complete hydatidiform mole (>50%) and partial hydatidiform mole (>20%). A statistically significant difference was found in immunoexpressins of p53 that was useful in separating abortion from complete hydatidiformmole, P <0.001, and partial hydatidiform mole, P <0.0002. It was also useful in separating between the complete hydatidiform mole and partial hydatidiform mole (P <0.001). Conclusion: p53 immunoexpression was valuable in differentiation between molar and non molar pregnancies and between subtypes of molar pregnancies.

The Placenta in Pre-Eclampsia: Association of Histology with Umbilical Artery Doppler Velocimetry

Objectives: To study the association of umbilical artery Doppler velocimetry with histological changes of the placenta in pre-eclampsia. Method: This was an observational study, conducted at two tertiary care centers in Sri Lanka between 2009 and 2010. A total of fifty placentae were studied; forty from women with pre-eclampsia and ten from normotensive women with uncomplicated pregnancies. Twenty pre-eclamptic patients with abnormal umbilical artery Doppler velocimetry were recruited in group 1. Group 2 included twenty gestational age-matched, pre-eclamptic patients with normal umbilical artery Doppler velocimetry. Ultra sound scan for umbilical artery Doppler was carried out to obtain the pulsatility index (PI) and resistance index (RI) approximately 24 hours prior to delivery. Five randomly selected, haematoxylin and eosin-stained sections from the maternal surface of each placenta were examined under high power (magnification 40) of light microscopy. Hundred terminal villi in each slide were examined to identify the presence of four histological features; syncytial knots, proliferative villous cytotrophoblast cells, thickening of sub-trophoblastic basement membrane, and villous hypovascularity. Results:All four histological features were present in a significantly higher proportion of placentae from pre-eclamptic patients (P 34 weeks). In hypertensive placentae, all four histological features were present in comparatively higher proportions in Group 1 (abnormal umbilical artery Doppler velocimetry) compared to Group 2 (normal umbilical artery Doppler velocimetry). But statistical significance was observed only invillous cytotrophoblast proliferation (P = 0.005), and villous hypovascularity (P = 0.002). Conclusion: The placental histological changes are significantly increased in pre-eclampsia. Further deterioration of placental histology is associated with abnormal umbilical artery Doppler velocimetry.

Transcriptional Dynamics of Cultured Human Villous Cytotrophoblasts.

During human pregnancy, cytotrophoblasts (CTBs) play key roles in uterine invasion, vascular remodeling, and anchoring of the feto-placental unit. Due to the challenges associated with studying human placentation in utero, cultured primary villous CTBs are used as a model of the differentiation pathway that leads to invasion of the uterine wall. In vitro, CTBs emulate in vivo cell behaviors, such as migration, aggregation, and substrate penetration. Although some of the molecular features related to these cell behaviors have been described, the underlying mechanisms, at a global level, remain undefined at midgestation. Thus, in this study, we characterized second-trimester CTB differentiation/invasion in vitro, correlating the major morphological transitions with the transcriptional changes that occurred at these steps. After plating on Matrigel as individual cells, CTBs migrated toward each other and formed multicellular aggregates. In parallel, using a microarray approach, we observed differentially expressed (DE) genes across time, which were enriched for numerous functions, including cell migration, vascular remodeling, morphogenesis, cell communication, and inflammatory signaling. DE genes encoded several molecules that we and others previously linked to critical CTB function in vivo, suggesting that the novel DE molecules we discovered played important roles. Immunolocalization confirmed that CTBs in situ gave a signal for two of the most highly expressed genes in vitro. In summary, we characterized, at a global level, the temporal dynamics of primary human CTB gene expression in culture. These data will enable future analyses of various types of in vitro perturbations-for example, modeling disease processes and environmental exposures.

PD-L1 Expression in Premalignant and Malignant Trophoblasts From Gestational Trophoblastic Diseases Is Ubiquitous and Independent of Clinical Outcomes.

Recently reported expression of programmed cell death 1 ligand 1 (PD-L1) in gestational trophoblastic diseases (GTDs) suggests that the immune tolerance of pregnancy might be hijacked during neoplastic process. We assessed PD-L1 protein expression in premalignant and malignant GTD lesions and analyzed associations with disease severity and chemotherapy outcomes. We included 83 GTD whole-tissue sections from 76 patients in different treatment settings. PD-L1 protein expression was assessed with immunohistochemistry in each trophoblast subtype with the Allred total score (ATS), which combines intensity and proportion expression on a 0- to 8-point scale. Peritumoral immune infiltrate was scored on hematoxylin-eosin-safran-stained slides. PD-L1 expression was ubiquitous and strong in all GTD trophoblast subtypes. For invasive moles, ATS scores were maximal at 8 in 100%, 100%, and 75% of syncytiotrophoblast, villous cytotrophoblast, and extravillous cytotrophoblast specimens, respectively. For choriocarcinomas, ATS was 8 in 80% of specimens. Immune infiltrates were moderate to severe in 61%, 100%, and 100% of peritumoral zones of choriocarcinoma, epithelioid trophoblastic tumor, and invasive moles, respectively. Because of the homogeneous pathological findings, no significant differences in PD-L1 expression profiles or peritumoral immune infiltrates were found regarding FIGO (International Federation of Gynecology Obstetrics) prognostic score, fatal outcome, or chemosensitivity. We confirm that PD-L1 is constitutively expressed in all GTD premalignant and malignant trophoblast subtypes, independently from FIGO score, chemoresistance, or fatal outcomes, thereby suggesting a crucial role for PD-L1 in the development and tolerance of GTD. Assessment of anti-PD-L1 drug in GTD patients has been activated.

VariableDAXXgene methylation is a common feature of placental trophoblast differentiation, preeclampsia, and response to hypoxia

Placental functioning relies on the appropriate differentiation of progenitor villous cytotrophoblasts (CTBs) into extravillous cytotrophoblasts (EVCTs), including invasive EVCTs, and the multinucleated syncytiotrophoblast (ST) layer. This is accompanied by a general move away from a proliferative, immature phenotype. Genome-scale expression studies have provided valuable insight into genes that are associated with the shift to both an invasive EVCT and ST phenotype, whereas genome-scale DNA methylation analysis has shown that differentiation to ST involves widespread methylation shifts, which are counteracted by low oxygen. In the current study, we sought to identify DNA methylation variation that is associated with transition from CTB to ST in vitro and from a noninvasive to invasive EVCT phenotype after culture on Matrigel. Of the several hundred differentially methylated regions that were identified in each comparison, the majority showed a loss of methylation with differentiation. This included a large differentially methylated region (DMR) in the gene body of death domain-associated protein 6 (DAXX ), which lost methylation during both CTB syncytialization to ST and EVCT differentiation to invasive EVCT. Comparison to publicly available methylation array data identified the same DMR as among the most consistently differentially methylated genes in placental samples from preeclampsia pregnancies. Of interest, in vitro culture of CTB or ST in low oxygen increases methylation in the same region, which correlates with delayed differentiation. Analysis of combined epigenomics signatures confirmed DAXX DMR as a likely regulatory element, and direct gene expression analysis identified a positive association between methylation at this site and DAXX expression levels. The widespread dynamic nature of DAXX methylation in association with trophoblast differentiation and placenta-associated pathologies is consistent with an important role for this gene in proper placental development and function.-Novakovic, B., Evain-Brion, D., Murthi, P., Fournier, T., Saffery, R. Variable DAXX gene methylation is a common feature of placental trophoblast differentiation, preeclampsia, and response to hypoxia.

Hypoxia Directs Human Extravillous Trophoblast Differentiation in a Hypoxia-Inducible Factor–Dependent Manner

Villous cytotrophoblasts are epithelial stem cells of the early human placenta, able to differentiate either into syncytiotrophoblasts in floating chorionic villi or extravillous trophoblasts (EVTs) at the anchoring villi. The signaling pathways regulating differentiation into these two lineages are incompletely understood. The bulk of placental growth and development in the first trimester occurs under low oxygen tension. One major mechanism by which oxygen regulates cellular function is through the hypoxia-inducible factor (HIF), a transcription factor complex stabilized under low oxygen tension to mediate cellular responses, including cell fate decisions. HIF is known to play a role in trophoblast differentiation in rodents; however, its role in human trophoblast differentiation is poorly understood. Using RNA profiling of sorted populations of primary first-trimester trophoblasts, we evaluated the first stage of EVT differentiation, the transition from epidermal growth factor receptor+ villous cytotrophoblasts into human leukocyte antigen-G+ proximal column EVT (pcEVT) and identified hypoxia as a major pcEVT-associated pathway. Using primary cytotrophoblasts, we determined that culture in low oxygen directs differentiation preferentially toward human leukocyte antigen-G+ pcEVT, and that an intact HIF complex is required for this process. Finally, using global RNA profiling, we identified integrin-linked kinase and associated cytoskeletal remodeling and adhesion to be among HIF-dependent pcEVT-associated signaling pathways. Taken together, we propose that oxygen regulates EVT differentiation through HIF-dependent modulation of various cell adhesion and morphology-related pathways.

Integrin linked kinase regulates syncytialization of BeWo trophoblast cells.

During placental development, mononuclear villous cytotrophoblast cells differentiate and fuse with the overlying syncytiotrophoblast. This process requires the dissolution of E-cadherin (CDH1)-containing adherens junctions in cytotrophoblast. Integrin linked kinase (ILK) can downregulate CDH1 through poly (ADP-ribose) polymerase 1 (PARP1) and Snail-1 (SNAI1) during epithelial-mesenchymal transition. ILK is known to be expressed in cytotrophoblast; thus, the role of a potential ILK-PARP1-SNAI1 pathway in aiding trophoblast syncytialization via the downregulation of CDH1 was examined. The spatiotemporal expression of PARP1, SNAI1, and CDH1 were determined in first and early second trimester chorionic villi, term villi, and BeWo cells by immunofluorescence analysis. PARP1 and SNAI1 were highly detectable in villous cytotrophoblast nuclei of human chorionic villi and SNAI1 expression, in particular, also persisted in syncytiotrophoblast. In BeWo cells undergoing syncytialization, PARP1 and SNAI1 increasingly localized to cell nuclei in correlation with decreased CDH1 expression. Using luciferase reporter assays, it was determined that PARP1 and SNAI1 promoter activities were significantly higher in BeWo cells during syncytialization compared to the activities in proliferating cells. Overexpression of wild type or constitutively active ILK also resulted in significantly increased PARP1 and SNAI1 promoter activities while dominant negative ILK overexpression significantly reduced promoter activities. Lastly, siRNA-mediated depletion of ILK expression in BeWo cells undergoing syncytialization resulted in significantly reduced SNAI1 expression and a significant reduction in the incidence of syncytialization correlating with increased CDH1 expression. These results demonstrate that ILK aids trophoblast syncytialization via the downregulation of CDH1, perhaps through an ILK-PARP1-SNAI1 pathway.

Placental DAPK1 and autophagy marker LC3B-II are dysregulated by TNF-\u03b1 in a gestational age-dependent manner

Autophagy, a cell-survival process responsible for degradation of protein aggregates and damaged organelles, is increasingly recognized as another mechanism essential for human placentation. A substantial body of experiments suggests inflammation and oxidative stress as the underlying stimuli for altered placental autophagy, giving rise to placenta dysfunction and pregnancy pathologies. Here, the hypothesis is tested whether or not pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α are able to influence the expression profile of autophagy genes in human first-trimester villous placenta. Autophagy-focused qPCR arrays identified substantial downregulation of death-associated protein kinase 1 (DAPK1) in first-trimester placental explants in response to IL-6 and TNF-α, respectively. Immunohistochemistry of placental explants detected considerable DAPK1 staining in placental macrophages, villous cytotrophoblasts and less intense in the syncytiotrophoblast. Both immunohistochemistry and Western blot showed decreased DAPK1 protein in TNF-α-treated placental explants compared to control. On cellular level, DAPK1 expression decreased in SGHPL-4 trophoblasts in response to TNF-α. Observed changes in the expression profile of autophagy-related genes were reflected by significantly decreased lipidation of autophagy marker microtubule-associated protein light chain 3 beta (LC3B-II) in first trimester placental explants in response to TNF-α. Analysis of TNF-α-treated term placental explants showed decreased DAPK1 protein, whereas in contrast to first-trimester LC3B expression and lipidation increased. Immunohistochemistry of placental tissues from early-onset preeclampsia (PE) showed less DAPK1 staining, when compared to controls. Accordingly, DAPK1 mRNA and protein were decreased in primary trophoblasts isolated from early-onset PE, while LC3B-I and -II were increased. Results from this study suggest that DAPK1, a regulator of apoptosis, autophagy and programmed necrosis, decreases in human placenta in response to elevated maternal TNF-α, irrespective of gestational age. In contrast, TNF-α differentially regulates levels of autophagy marker LC3B in human placenta over gestation.

Human trophoblast stem cells: Real or not real?

Abnormal trophoblast differentiation is the root cause of many placenta-based pregnancy complications, including preeclampsia and fetal growth restriction. Human trophoblast differentiation is difficult to study due to the lack of a stem cell model. Such a multipotent “trophoblast stem” (TS) cell, with the ability to differentiate into all trophoblast subtypes, has been derived from mouse blastocysts, but attempts to derive similar human cells have failed. We consider here several possibilities for the TS cell niche in the human placenta. Aside from discussion of such a niche in the pre-implantation blastocyst, we discuss evidence for these TS cells residing in the post-implantation villous cytotrophoblast layer, or even in the non-trophoblast portions, of the human placenta. It is our hope that recognition of the niche would lead to successful derivation and in vitro establishment of such cells, which could then be disseminated widely to the placental biology community for advancing the field. Availability of self-renewing human TS cells, whose gene expression and environment could be manipulated, will provide a platform, not just for the study of pathophysiology of placental disease, but also for the discovery of diagnostic biomarkers and therapeutic targets for common pregnancy complications.

Transcription Factors That Regulate Trophoblast Development and Function.

The placenta is a transient organ that plays a critical role in sustaining pregnancy and supporting fetal growth and nutrition. The placental epithelium is comprised of trophoblast cells. Trophoblast cells are the first cell type to differentiate during embryogenesis and ultimately diversify into a heterogeneous population of cells specializing in distinct functions essential for placentation. The emergence of the trophoblast lineage and subsequent specialization into distinct trophoblast sublineages is tightly regulated by transcription factors. This chapter will provide an overview of transcription factors that regulate trophoblast development and function. The chapter is divided into three sections. In the first section, a generalized outline of trophoblast ontogeny and a functional description of different trophoblast sublineages will be provided. In the second section, transcription factors involved in emergence of the trophoblast lineage and maintenance of trophoblast stem cells will be discussed. In the third section, transcription factors implicated in the formation and function of villous and extravillous cytotrophoblast lineages will be described.

Features of cytotrophoblast invasion in complete placenta previa and increta

to investigate the characteristics of cytotrophoblast invasion in complete placenta previa and increta. Three groups of placentas and amputated uteri were examined. These were: 1) 10 placentas at 20-22 weeks' gestation after drug-induced abortion; 2) 4 uteri with typical placentation at 34-36 weeks and wall ruptures; 3) 12 uteri with ultrasound-confirmed complete placenta previa and subsequent hysterectomy (at 34-36 weeks.) due to massive bleeding. In all cases, the sections were stained with hematoxylin and eosin, azan by the Mallory's method; immunovisualization of invasive cells with the marker cytokeratin 8 was also used. In Groups 2 and 3, the uterine distribution density of invasive cells was compared in a standard slice area (×200) separately, within the endometrium and myometrium. Complete placenta previa was found to have the following characteristics: 1) all the uteri exhibited focal or diffuse friable, or thick scars after cesarean section; 2) multiple active anchor villi with villous cytotrophoblast layers, which were characteristic of Group 1 placentas and absent in the uteri women of Group 2; 3) bays diagnosed in the basal endometrium with ingrown villi (placenta increta); 4) a morphometrically significant increase in the distribution density of interstitial cytotrophoblast in the endometrium and only a similar trend in the myometrium. Invasive cells did not penetrate into the area of scars. Failure of the second wave of cytotrophoblast invasion was confirmed by incomplete gestational restructuring and partial obliteration of the myometrial radial arteries. Real risks for severe clinical forms of abnormal placentation declare more stringent indications for surgical delivery.