Placental Villous Tissue Research Articles

Increased placental macrophages and a pro-inflammatory profile in placentas and maternal serum in infants with a decreased growth rate in the third trimester of pregnancy.

There is growing evidence for the role of placental inflammation in the pathophysiology of pregnancy complications including fetal growth restriction (FGR). This study aimed to characterize the inflammatory profile in the maternal circulation and the placenta of infants who were growth restricted and those that were small for gestational age (SGA). Placental villous tissue and maternal serum were obtained from pregnancies where infants were SGA at birth or who had a decreasing growth rate (≥25 centiles) across the third trimester. Immunohistochemical and histological analyses of placental samples were conducted for macrophage number, alongside vascular and cell turnover analysis. Inflammatory profile was analyzed in maternal and placental compartments via ELISAs and multiplex assays. There were significantly more CD163+ macrophages in placentas of infants with a decreased growth rate compared to controls, but not in SGA infants (median 8.6/ nuclei vs 3.8 and 2.9, P=.008 and P=.003, respectively). Uric acid (P=.0007) and IL-8 (P=.0008) were increased in placentas, and S100A8 (P<.0002) was increased in maternal serum of infants with decreased growth rate. No changes in the maternal serum or placental lysates of SGA infants were observed. The evidence of an altered inflammatory profile in infants with a decreasing growth rate, but not in those that were born SGA, provides further evidence that inflammation plays a role in true FGR. It remains unclear whether the increased placental macrophages occur as a direct result, or as a consequence of the pro-inflammatory environment observed in fetal growth restriction.

Nitric oxide metabolism in the human placenta during aberrant maternal inflammation.

Nitric oxide (NO) is a gasotransmitter with important physiological and pathophysiological roles in pregnancy. There is limited information available about the sources and metabolism of NO and its bioactive metabolites (NOx) in both normal and complicated pregnancies. The present study characterized and quantified endogenous NOx in human and mouse placenta following determination of the stability of exogenous NOx in placental homogenates. NOx have differential stability in placental homogenates. NO and iron nitrosyl species (FeNOs), are relatively unstable in placental homogenates from normal placentas. Exogenous NO, nitrite and nitrosothiols react with placental homogenates to form iron nitrosyl complexes. FeNOs were also detected endogenously in mouse and human placenta. NOx levels in placental villous tissue are increased in fetal growth restriction vs. placentas from women with normal pregnancies, particularly in fetal growth restriction associated with pre-eclampsia. Villitis was not associated, however, with an increase in NOx levels in either normotensive or pre-eclamptic placentas. The results call for further investigation of FeNOs in normal and complicated pregnancies. Nitric oxide (NO) is a gasotransmitter with important roles in pregnancy under both physiological and pathophysiological conditions. Although products of NO metabolism (NOx) also have significant bioactivity, little is known about the role of NO and NOx under conditions of aberrant placental inflammation during pregnancy. An ozone-based chemiluminescence approach was used to investigate the stability and metabolic fate of NOx in human placental homogenates from uncomplicated pregnancies in healthy mothers compared to that in placental tissue from normotensive and pre-eclamptic pregnancies complicated with fetal growth restriction (FGR) with and without villitis of unknown aetiology. We hypothesized that placental NOx would be increased in FGR vs. normal tissue, and be further increased in villitis vs. non-villitis placentas. Findings indicate that nitrate, nitrite and nitrosothiols, but not NO or iron nitrosyl species (FeNOs), are relatively stable in placental homogenates from normal placentas, and that NO, nitrite and nitrosothiols react with placental homogenates to form iron nitrosyl complexes. Furthermore, NOx levels in placental villous tissue are increased in FGR vs. placentas from women with normal pregnancies, particularly in FGR associated with pre-eclampsia. However, in contrast to our hypothesis, villitis was not associated with an increase in NOx levels in either normotensive or pre-eclamptic placentas. Our results also strongly support the involvement of FeNOs in both mouse and human placenta, and call for their further study as a critical mechanistic link between pre-eclampsia and fetal growth restriction.

Two patterns of cytokine production by placental macrophages

IntroductionMacrophages participate in the regulation immune and morphogenetic events in the placenta. However, these roles remain unclear for placental macrophages (Hofbauer cells). The aims of this study were to characterize the consecutive steps of cytokine production (intracellular synthesis and secretion) in placental macrophages in early and late gestation and to compare the secretory profiles of placental macrophages and villous tissue. MethodsMacrophages and villous tissue were isolated from placentas obtained from normal pregnancies at either 9–12 or 38–40 weeks of gestation. Intracellular cytokines were determined by flow cytometry after staining with monoclonal antibodies. Secreted cytokines were quantified by cytometric bead array and ELISA. ResultsTwo patterns of cytokine production were revealed in placental macrophages. Cytokines in the first group (IL-1, IL-6, IL-8, IL-10, TNFα) demonstrated low basal production and were stimulated by bacterial endotoxin. Cytokines in the second group (IL-11, IL-17A, IL-17F, TGF-β, VEGF) were characterized by constitutive production and did not respond to stimulation. Gestational age-dependent changes were observed: basal secretion of TNFα and IL-8 increased whereas IL-11 and IL-17 secretion decreased in third-trimester macrophages compared with the first-trimester cells. Comparison of cytokine production at the cellular and tissue levels suggested the contribution of the placental macrophages both in intraplacental and extraplacental cytokine production. DiscussionIt would be safe to assume that the two patterns of cytokine production, revealed in our study, correspond to two regulatory roles of placental macrophages: “immune” and “morphogenetic”. The inflammatory phenotype of macrophages is attenuated in early gestation and increases with the progression of pregnancy. The cytokines of the first group supposedly contribute to both local and extraplacental levels, whereas the cytokine effects of the second group are more likely confined to the placental tissue.

Mechanism associated with aberrant lncRNA MEG3 expression in gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is a common metabolic condition during pregnancy. Long non-coding RNAs (lncRNAs) have been found to seve critical roles in GDM development; however, the role of lncRNA maternally expressed gene 3 (MEG3) in GDM remains unclear. Therefore, the aim of the present study was to investigate the expression and role of MEG3 in GDM, and to further explore the underlying mechanism. The levels of lncRNA MEG3 in the blood and placental villous tissues of pregnant women with GDM was measured using reverse transcription-quantitative PCR. Bioinformatics analysis and dual luciferase reporter assays were performed to investigate the association between lncRNA MEG3 and microRNA (miR)-345-3p. Transfection was subsequently performed on HTR-8/SVneo cells, a human chorionic trophoblast cell line, to assess the role of lncRNA MEG3 in GDM. In particular, cell viability, cellular migratory/invasive ability and cell apoptosis were analyzed using MTT assay, Transwell assay and flow cytometry, respectively. Compared with pregnant women without GDM, lncRNA MEG3 levels were significantly elevated in the blood and placental villous tissues of GDM pregnant women. miR-345-3p was identified to be a direct target of lncRNA MEG3 using dual luciferase reporter assay, which was found to be reduced in pregnant women with GDM. Further analysis demonstrated that lncRNA MEG3 overexpression significantly inhibited HTR-8/SVneo cell viability, and prevented cell migration and invasion in addition to inducing cell apoptosis. In contrast, lncRNA MEG3 knockdown significantly enhanced HTR-8/SVneo cell viability, promoted cell migration/invasion and reduced cell apoptosis. Inhibiting miR-345-3p expression negated all the observed physiological effects of lncRNA MEG3 knockdown on HTR-8/SVneo cells. In conclusion, lncRNA MEG3 levels were abnormally upregulated in GDM, which participated in the development and progression of GDM by regulating human chorionic trophoblast cell physiology. Therefore, lncRNA MEG3 may be a potential diagnostic and therapeutic target for GDM.

Quality control measures for placental sample purity in DNA methylation array analyses

The purity of tissue samples can affect the accuracy and utility of DNA methylation array analyses. This is particularly important for the placenta which is globally hypomethylated compared to other tissues. Placental villous tissue from early pregnancy terminations can be difficult to separate from non-villous tissue, resulting in potentially inaccurate results. We used several methods to identify mixed placenta samples using DNA methylation array datasets from our laboratory and those contained in the NCBI GEO database, highlighting the importance of determining sample purity during quality control processes.

The miR-27a-3p/USP25 axis participates in the pathogenesis of recurrent miscarriage by inhibiting trophoblast migration and invasion.

Insufficient invasion ability of trophoblasts might be associated with the development of recurrent miscarriage (RM). Ubiquitin-specific protease 25 (USP25) can regulate the processes of invasion and migration in different types of cancer cells. However, the effect of USP25 on trophoblasts and its roles in the development of RM are unknown. In this study, we first analyzed the USP25 expression in placental villous tissues from RM patients, and then assessed the roles of USP25 in epithelial-to-mesenchymal transition (EMT), invasion and migration of trophoblasts. Furthermore, bioinformatics prediction and luciferase reporter assay were used to explore the mechanism of microRNA on USP25 expression, and regulation of USP25 expression in trophoblasts was assessed following transfection with microRNA mimics or inhibitor. The results showed that the expression of USP25 in the placental villous tissues was downregulated in RM patients. Knockdown of USP25 suppressed the EMT process, the invasion and migration capability of trophoblast cells, while overexpression of USP25 exhibited opposite results. Mechanistically, miR-27a-3p could regulate USP25 expression by binding to the 3'-untranslated region of USP25 in trophoblasts. Quantitative real-time polymerase chain reaction results found the expression of miR-27a-3p were negatively related to USP25 in RM patients. MiR-27a-3p mimics inhibited but miR-27a-3p inhibitor enhanced the migration and invasion capability of trophoblasts. Furthermore, sh-USP25 counteracted the promotion of invasion and migration mediated by the miR-27a-3p inhibitor. Taken together, these data indicate that USP25 downregulation by miR-27a-3p contributes to the EMT process, thereby inhibiting the migration and invasion of trophoblast cells, and these findings might provide potential biomarkers for RM.

FasL on decidual macrophages mediates trophoblast apoptosis: A potential cause of recurrent miscarriage.

Macrophages can induce Fas ligand (FasL)-mediated apoptosis, and the deregulation of apoptosis is known to be associated with recurrent miscarriage (RM). The aim of the present study was to investigate the possible involvement of FasL in macrophage-mediated trophoblast apoptosis and its potential role in RM. Human decidual and placental villous tissues were collected from 81 women (21 for the RM group, 26 for the spontaneous abortion group and 34 for the control group) at 7-9 weeks of gestation. The distribution changes of macrophages and the expression of FasL on macrophages were evaluated by immunohistochemical, immunofluorescence and western blot analyses. A macrophage and trophoblast co-culture model was used to determine the effects of FasL on the apoptosis of trophoblasts. The results indicated that CD86+ macrophage populations in decidual tissues were significantly increased, accompanied by reduced CD163+ macrophages in the abortion and RM groups. Furthermore, the distribution of CD68+ macrophages was also significantly altered in specimens from the abortion and RM groups, and they were observed to have infiltrated into the trophoblast cells. In addition, elevated expression of FasL on CD68+ and CD86+ macrophages in the decidua was observed in the spontaneous abortion and RM groups of patients, and FasL was demonstrated to mediate the induction of trophoblast apoptosis by macrophages in co-culture. These results indicate that the aberration of macrophage-induced FasL-mediated apoptosis may represent one of the causes of RM.

Apelin is a novel regulator of human trophoblast amino acid transport.

Apelin is an insulin-sensitizing hormone increased in abundance with obesity. Apelin and its receptor, APJ, are expressed in the human placenta, but whether apelin regulates placental function in normal body mass index (BMI) and obese pregnant women remains unknown. We hypothesized that apelin stimulates amino acid transport in cultured primary human trophoblast (PHT) cells and that maternal circulating apelin levels are elevated in obese pregnant women delivering large babies. Treating PHT cells with physiological concentrations of the pyroglutamated form [Pyr1]apelin-13 (0.1-10.0 ng/ml) for 24 h dose-dependently increased System A amino acid transport (P < 0.05) but did not affect System L transport activity. Mechanistic target of rapamycin (mTOR), extracellular signal-regulated kinase-1/2 (ERK1/2), and AMP-activated protein kinase-α (AMPKα) signaling were unaffected by apelin (P > 0.05). Plasma apelin was not different in obese women (BMI 35.8 ± 0.7, n = 21) with large babies compared with normal-BMI women (23.1 ± 0.5, n = 16) delivering normal birth weight infants. Apelin was highly expressed in placental villous tissue (20-fold higher vs. adipose), and APJ was present in syncytiotrophoblast microvillous membrane, but neither differed in abundance between normal-BMI and obese women. Phosphorylation (Thr172) of placental AMPKα strongly correlated with microvillous membrane APJ expression (P < 0.01, R = 0.63) but negatively correlated with placental apelin abundance (P < 0.01, R = -0.62). Neither placental APJ nor apelin abundance correlated with maternal BMI, plasma insulin, birth weight, or mTOR or ERK1/2 signaling (P > 0.05). Hence, apelin stimulates trophoblast amino acid uptake, establishing a novel mechanism regulating placental function. We found no evidence that apelin constitutes an endocrine link between maternal obesity and fetal overgrowth.

Correlation of Doppler Velocimetry of Uterine and Umbilical Arteries with Placental Pathology in Pregnancy Associated with Intrauterine Growth Restriction

AbstractTo correlate histomorphology of the placenta with Doppler studies of uterine and umbilical arteries. A comparative observational study conducted on 75 pregnant women divided into 2 groups: Group 1 included 25 women with appropriate for gestational age fetuses. Group 2 included 50 women with FGR. Uterine and umbilical artery Doppler, study of placental pathology and immunohistochemistry of placental villous tissues were evaluated. There was a significant difference between the two study groups regarding both abnormal uterine (0 vs. 58%) and umbilical artery (0 vs. 58%) Doppler (p≤&lt;≤0.001). Syncytial knots≤&gt;≤30% (44 vs. 60%), fibrinoid necrosis≤&gt;≤5% (8 vs. 46.7%), placental infarction≤&gt;≤5% (0 vs. 15%), perivillous fibrinoid deposition≤&gt;≤5% (1.8 vs. 16.7%) (p≤&lt;≤0.001) but not calcifications (56 vs. 60%, p≤=≤0.121) were significantly higher in FGR placentas. A statistically significant (p≤&lt;≤0.001) increase in the expression of VEGF in FGR placentas when compared with normal placentas. Abnormal uterine artery but not umbilical artery Doppler was significantly associated with abnormal placental pathology. Women with both abnormal uterine and umbilical artery Doppler velocimetries were delivered earlier and their babies had lower mean birth and placental weight (p≤&lt;≤0.001). Incidence of abnormal placental pathology was significantly higher in this specific group of FGR pregnancies (p≤&lt;≤0.001). There is high association between abnormal uterine and umbilical artery Doppler and placental pathology in FGR associated pregnancies. Trial Registration NCT03081754.

Mitochondrial-related gene associated to obesity can be modulated by in utero hyperglycemic environment

We investigated whether mitochondrial-related genes and proteins are modulated by hyperglycemia promoted by gestational diabetes (GDM), thereby increasing neonate obesity predisposition. 19 healthy pregnant women, 16 pregnant women with GDM and their respective neonates were enrolled. Additionally, 19 obese and 19 eutrophic adults were recruited as a reference population. Umbilical cord, peripheral blood and placental (villous and decidua) tissues were collected to evaluate SOD2, PPAR-α and PPARGC-1β and their respective protein expressions. Data from the reference population confirmed that the three genes and proteins were overexpressed in blood cells of obese compared to eutrophic subjects. Only SOD2 was found upregulated in placental villous (fetal side) tissue of GDM women. Therefore, our findings showed an interaction between the hyperglycemic environment and SOD2 modulation, but also indicated that none of the three genes is useful as potential biomarkers for obesity development.

The m6A demethylase ALKBH5 controls trophoblast invasion at the maternal-fetal interface by regulating the stability of CYR61 mRNA.

N6-Methyladenosine (m6A) is the most prevalent internal modification in mammalian mRNAs. Although m6A is important in many biological processes, its roles in the placenta are unclear.Methods: Levels of global mRNA m6A methylation and ALKBH5 expression in recurrent miscarriage (RM) patients were determined using quantitative reverse transcription-PCR (qRT-PCR), m6A RNA methylation quantification, and immunohistochemical methods. Using ALKBH5 overexpression and knockdown methods, we determined the role of ALKBH5 in trophoblast invasion at the maternal interface through trophoblasts and an extravillous explant culture experiments. Furthermore, the regulation of CYR61 by ALKBH5 was explored by RNA-sequencing coupled with methylated RNA immunoprecipitation.Results: We found that the level of global mRNA m6A methylation was significantly decreased in placental villous tissue from RM patients, while ALKBH5 expression was specifically unregulated. Furthermore, we demonstrated that ALKBH5 knockdown in human trophoblast promoted trophoblast invasion. Conversely, overexpression of ALKBH5 inhibited cell invasion. ALKBH5 knockdown promoted trophoblast invasion in villous explant culture experiments, while overexpression of ALKBH5 repressed these effects. Furthermore, we clarified that ALKBH5 inhibited trophoblast invasion by regulating CYR61 mRNA stability, and this RNA regulation is m6A dependent. Mechanistic analyses showed that decreased ALKBH5 in trophoblast increased the half-life of CYR61 mRNA and promoted steady-state CYR61 mRNA expression levels.Conclusions: We elucidated the functional roles of ALKBH5 and mRNA m6A methylation in trophoblast and identified a novel RNA regulatory mechanism, providing a basis for further exploration of broad RNA epigenetic regulatory patterns in RM diseases.

516: Global transcriptomic analysis of sibling placentas in twin gestations complicated by preeclampsia

Preeclampsia (PE) is a disease of incompletely understood etiology that is the leading contributor to iatrogenic preterm birth (PTB). Our objective was to identify if PE in dichorionic pregnancies is characterized by similarities or dissimilarities in the transcriptome profile of the two placentas. Total RNA was extracted from 60 placental villous tissue samples and subjected to RNA-seq using the Illumina platform. Our study was designed to include two analytical stages: In the first stage placental samples (n=18) originated from singleton pregnancies with unambiguous clinical phenotypes: 1) severe features early-onset PE (sPE, n=8, GA: 30±1 wks); 2) idiopathic preterm birth (n=5, GA: 32±1wks) and 3) term non-labor (n=5, GA: 39±1wks). These samples were used to extract a generic transcriptomic signature of PE placenta. In the second stage placentas of 16 twin pregnancies classified based on chorionicity and PE diagnosis were included as follows: di-di nonPE (n=7, GA: 32±1 wks), mono-di nonPE (n=2, GA: 32±1 wks), di-di PE (n=5, GA: 33±2 wks) and mono-di PE (n=2, GA: 33±3 wks). Two separate biopsies were sequenced from monochorionic placentas. An additional set of singleton PE placentas (n=10, GA: 33±1 wks) was sequenced interspaced among the twin placentas to control for batch effects. Principal components analysis (PCA) and statistical modeling for similarity and dissimilarity between sibling placentas were applied. Our first stage analysis extracted a subset of 613 transcripts differentially expressed between sPE and non-PE singleton placentas. This molecular classifier showed linear separability among first stage samples independent of GA at birth. When applied to twin gestation placentas the classifier revealed several characteristics: 1) In the non-PE cases, biopsies from monochorionic placentas had more similar expression patterns of the genes composing the PE signature than dichorionic placentas which were more scattered in the PCA space (Fig. A); 2) Monochorionic nonPE placentas have closer proximity to the PE space compared to singleton nonPE placentas; 3) Dichorionic sibling placentas of pregnancies complicated by PE were the most discordant (Fig. B). In dichorionic pregnancies complicated by PE, the disease affects each placenta individually. Conversely, PE in twins could be the result of a single diseased placenta.

Expression of indoleamine oxygenase (IDO) on placental villous and uterus endometrial tissue

Expression of indoleamine oxygenase (IDO) on placental villous and uterus endometrial tissue

A single-cell survey of the human first-trimester placenta and decidua.

The placenta and decidua interact dynamically to enable embryonic and fetal development. Here, we report single-cell RNA sequencing of 14,341 and 6754 cells from first-trimester human placental villous and decidual tissues, respectively. Bioinformatic analysis identified major cell types, many known and some subtypes previously unknown in placental villi and decidual context. Further detailed analysis revealed proliferating subpopulations, enrichment of cell type-specific transcription factors, and putative intercellular communication in the fetomaternal microenvironment. This study provides a blueprint to further the understanding of the roles of these cells in the placenta and decidua for maintenance of early gestation as well as pathogenesis in pregnancy-related disorders.

Macrophages are a source of IL-17 in the human placenta.

To determine if placental macrophages (Hofbauer cells) can synthesize and secrete cytokines of the IL-17 family throughout pregnancy and to reveal the patterns of cytokine expression in early and late gestation. Macrophages were isolated from the first-trimester and term placental villous tissues from normal pregnancies. Basal and stimulated intracellular production of IL-17A, IL-17E, IL-17F as well as IL-17A secretion was quantified by flow cytometry and cytometric bead array, respectively. The expression of IL-17 and IL-23 receptors was determined on the surface of the placental macrophages by flow cytometry after antibody staining. In early and late gestation, a substantial proportion of the placental macrophages synthesized IL-17A and IL-17F, but not IL-17E, as determined by intracellular staining of the cytokines. Neither the intracellular production nor the secretion of IL-17 was significantly affected by LPS stimulation and spontaneous labour. The level of secretion decreased slightly but significantly at term. The IL-23 receptor was absent on the surface of cells, whereas variable expression of the IL-17 receptor was observed. Placental macrophages constitutively produced IL-17 at different gestational ages and represent thus a source of this cytokine in the human placenta. Patterns of the cytokine and receptor expression suggest that this cell population may participate in non-immune processes and contribute to the regulation of placental development and function.

Extracellular vesicles generated by placental tissues ex vivo: A transport system for immune mediators and growth factors.

To study the mechanisms of placenta function and the role of extracellular vesicles (EVs) in pregnancy, it is necessary to develop an ex vivo system that retains placental cytoarchitecture and the primary metabolic aspects, in particular the release of EVs and soluble factors. Here, we developed such a system and investigated the pattern of secretion of cytokines, growth factors, and extracellular vesicles by placental villous and amnion tissues ex vivo. Placental villous and amnion explants were cultured for 2weeks at the air/liquid interface and their morphology and the released cytokines and EVs were analyzed. Cytokines were analyzed with multiplexed bead assays, and individual EVs were analyzed with recently developed techniques that involved EV capture with magnetic nanoparticles coupled to anti-EV antibodies and flow cytometry. Ex vivo tissues (i) remained viable and preserved their cytoarchitecture; (ii) maintained secretion of cytokines and growth factors; (iii) released EVs of syncytiotrophoblast and amnion epithelial cell origins that contain cytokines and growth factors. A system of ex vivo placental villous and amnion tissues can be used as an adequate model to study placenta metabolic activity in normal and complicated pregnancies, in particular to characterize EVs by their surface markers and by encapsulated proteins. Establishment and benchmarking the placenta ex vivo system may provide new insight in the functional status of this organ in various placental disorders, particularly regarding the release of EVs and cytokines. Such EVs may have a prognostic value for pregnancy complications.

Proteomics and bioinformatics analysis of altered protein expression in the placental villous tissue from early recurrent miscarriage patients

IntroductionRecurrent miscarriage (RM) affects 5% of women, it has an adverse emotional impact on women. Because of the complexities of early development, the mechanism of recurrent miscarriage is still unclear. We hypothesized that abnormal placenta leads to early recurrent miscarriage (ERM). The aim of this study was to identify ERM associated factors in human placenta villous tissue using proteomics. Investigation of these differences in protein expression in parallel profiling is essential to understand the comprehensive pathophysiological mechanism underlying recurrent miscarriage (RM). MethodsTo gain more insight into mechanisms of recurrent miscarriage (RM), a comparative proteome profile of the human placenta villous tissue in normal and RM pregnancies was analyzed using iTRAQ technology and bioinformatics analysis used by Ingenuity Pathway Analysis (IPA) software. ResultsIn this study, we employed an iTRAQ based proteomics analysis of four placental villous tissues from patients with early recurrent miscarriage (ERM) and four from normal pregnant women. Finally, we identified 2805 proteins and 79,998 peptides between patients with RM and normal matched group. Further analysis identified 314 differentially expressed proteins in placental villous tissue (≥1.3-fold, Student's t-test, p < 0.05); 209 proteins showed the increased expression while 105 proteins showed decreased expression. These 314 proteins were analyzed by Ingenuity Pathway Analysis (IPA) and were found to play important roles in the growth of embryo. Furthermore, network analysis show that Angiotensinogen (AGT), MAPK14 and Prothrombin (F2) are core factors in early embryonic development. We used another 8 independent samples (4 cases and 4 controls) to cross validation of the proteomic data. DiscussionThis study has identified several proteins that are associated with early development, these results may supply new insight into mechanisms behind recurrent miscarriage.

Alterations in the placental methylome with maternal obesity and evidence for metabolic regulation.

The inflammatory and metabolic derangements of obesity in pregnant women generate an adverse intrauterine environment, increase pregnancy complications and adverse fetal outcomes and program the fetus for obesity and metabolic syndrome in later life. We hypothesized that epigenetic modifications in placenta including altered DNA methylation/hydroxymethylation may mediate these effects. Term placental villous tissue was collected following cesarean section from lean (prepregnancy BMI<25) or obese (BMI>30) women. Genomic DNA was isolated, methylated and hydroxymethylated DNA immunoprecipitated and hybridized to the NimbleGen 2.1M human DNA methylation array. Intermediate metabolites in placental tissues were measured by HPLC-ESI-MS, ascorbate levels by reverse phase HPLC and gene expression by RT-PCR. Differentially methylated and hydroxymethylated regions occurred across the genome, with a 21% increase in methylated but a 31% decrease in hydroxymethylated regions in obese vs lean groups. Whereas increased methylation and decreased methylation was evident around transcription start sites of multiple genes in the GH/CSH and PSG gene clusters on chromosomes 17 and 19 in other areas there was no relationship. Increased methylation was associated with decreased expression only for some genes in these clusters. Biological pathway analysis revealed the 262 genes which showed reciprocal differential methylation/ hydroxymethylation were enriched for pregnancy, immune response and cell adhesion-linked processes. We found a negative relationship for maternal BMI but a positive relationship for ascorbate with α-ketoglutarate a metabolite that regulates ten eleven translocase (TET) which mediates DNA methylation. We provide evidence for the obese maternal metabolic milieu being linked to an altered DNA methylome that may affect placental gene expression in relation to adverse outcomes.

Rab11 family expression in the human placenta: Localization at the maternal-fetal interface.

Rab proteins are a family of small GTPases involved in a variety of cellular processes. The Rab11 subfamily in particular directs key steps of intracellular functions involving vesicle trafficking of the endosomal recycling pathway. This Rab subfamily works through a series of effector proteins including the Rab11-FIPs (Rab11 Family-Interacting Proteins). While the Rab11 subfamily has been well characterized at the cellular level, its function within human organ systems is still being explored. In an effort to further study these proteins, we conducted a preliminary investigation of a subgroup of endosomal Rab proteins in a range of human cell lines by Western blotting. The results from this analysis indicated that Rab11a, Rab11c(Rab25) and Rab14 were expressed in a wide range of cell lines, including the human placental trophoblastic BeWo cell line. These findings encouraged us to further analyse the localization of these Rabs and their common effector protein, the Rab Coupling Protein (RCP), by immunofluorescence microscopy and to extend this work to normal human placental tissue. The placenta is a highly active exchange interface, facilitating transfer between mother and fetus during pregnancy. As Rab11 proteins are closely involved in transcytosis we hypothesized that the placenta would be an interesting human tissue model system for Rab investigation. By immunofluorescence microscopy, Rab11a, Rab11c(Rab25), Rab14 as well as their common FIP effector RCP showed prominent expression in the placental cell lines. We also identified the expression of these proteins in human placental lysates by Western blot analysis. Further, via fluorescent immunohistochemistry, we noted abundant localization of these proteins within key functional areas of primary human placental tissues, namely the outer syncytial layer of placental villous tissue and the endothelia of fetal blood vessels. Overall these findings highlight the expression of the Rab11 family within the human placenta, with novel localization at the maternal-fetal interface.

Isolation and characterization of trophoblasts from enzymatic explants of human term placenta.

In the present study, we describe a new method of isolation and culture of human villous and extravillous trophoblasts from term placenta. The cultivation of trypsinized placental villous tissue explants, followed by the isolation of cells from outgrowth islets allows for obtaining a cytotrophoblast subpopulation that is free from contamination by other cell types. Compared to other methods, our protocol is mild, simple and effective, does not request costly reagents and provides isolation of the mononuclear cytotrophoblast cell populations free from contamination by other types of placental cells. The isolated cells proliferated and formed a pleomorphic monolayer, where cells fused into a small number of binuclear or polynuclear syncytiotrophoblasts. Isolated cytotrophoblast cells expressed the specific epithelial intermediate filament cytokeratin 7 (CK7), the epithelium-specific cell-cell adhesion molecule E-cadherin and were CD9-, CD45- and vimentin-negative. Cyto- and syncytiotrophoblasts obtained by this method can be used as a model or tool for the fundamental research of differentiation and function of human placental cells, and can provide a new understanding of drug distribution in placenta. Their combination with other in vitro cell models can be useful for studying a variety of other aspects concerning placental functions, which will provide new knowledge for understanding immunology, endocrinology and development of placenta.