Mouse prolyl oligopeptidase plays a role in trophoblast stem cell differentiation into trophoblast giant cell and spongiotrophoblast

IntroductionProlyl oligopeptidase (prolyl endopeptidase, Prep), a multifunctional protease hydrolyzing -Pro-X- peptide bonds, is highly expressed in the mouse placenta, but the function during development is not known. We explored the possibility of Prep's involvement in placental differentiation.MethodsWe cultured trophoblast stem cells (TSCs) derived from the E6.5 mouse embryo and investigated the detailed expression pattern of Prep during their differentiation. Prep-specific inhibitors were added to the TSC culture, and the effect on the differentiation was assessed by microscopic observation and the expression of marker gene for each placental cell.ResultsDuring TSC differentiation for 6 days, Prep was constantly detected at mRNA, protein, and activity levels, and the protein was found mainly in the cytoplasm. The addition of 30 μM and 10 μM SUAM-14746, a Prep-specific inhibitor, effectively inhibited the differentiation into spongiotrophoblasts (SpTs) and trophoblast giant cells (TGCs), while the TSC viability was not affected. 5 μM SUAM-14746 impaired the differentiation into SpTs, and 1 μM SUAM-14746 exhibited no effects. Another Prep-specific inhibitor, KYP-2047, did not affect the differentiation. We confirmed efficient inhibition of Prep enzymatic activity in TSCs by both inhibitors.ConclusionThe dose-dependent effect of SUAM-14746 on TSCs suggests that Prep plays an important role in the differentiation into SpTs and TGCs in the mouse placenta.     

Increased placental trophoblast inclusions in placenta accreta

IntroductionTrophoblast inclusions (TIs) are often found in placentas of genetically abnormal gestations. Although best documented in placentas from molar pregnancies and chromosomal aneuploidy, TIs are also associated with more subtle genetic abnormalities, and possibly autism. Less than 3% of non-aneuploid, non-accreta placentas have TIs. We hypothesize that placental genetics may play a role in the development of placenta accreta and aim to study TIs as a potential surrogate indicator of abnormal placental genetics.MethodsForty cases of placenta accreta in the third trimester were identified in a search of the medical records at one institution. Forty two third trimester control placentas were identified by a review of consecutively received single gestation placentas with no known genetic abnormalities and no diagnosis of placenta accreta.ResultsForty percent of cases with placenta accreta demonstrated TIs compared to 2.4% of controls. More invasive placenta accretas (increta and percreta) were more likely to demonstrate TIs than accreta (47% versus 20%). Prior cesarean delivery was more likely in accreta patients than controls (67% versus 9.5%).DiscussionPlacenta accreta is thought to be the result of damage to the endometrium predisposing to abnormal decidualization and invasive trophoblast growth into the myometrium. However, the etiology of accreta is incompletely understood with accreta frequently occurring in women without predisposing factors and failing to occur in predisposed patients.ConclusionThis study has shown that TIs are present at increased rates in cases of PA. Further studies are needed to discern what underlying pathogenic mechanisms are in common between abnormal placentation and the formation of TIs.     

YAP mediates human decidualization of the uterine endometrial stromal cells

IntroductionThe decidualization of uterine endometrial stromal cells (ESCs) is critical for the successful establishment and maintenance of pregnancy and involves extensive cell proliferation and differentiation. A newly established signaling pathway, the Hippo/Yes-associated protein (YAP) pathway, plays a critical role in these proliferation processes. Our previous study demonstrated that YAP is expressed in human ESCs. However, its role in decidualization remains unclear. The objective of the present study was to explore the role of YAP in the decidualization of human ESCs.MethodsThe expression of YAP was first investigated in the endometrium of non-pregnant women and in the decidua of pregnant women. The role of YAP was investigated by transfecting ESCs with mRNA silencing constructs and observing the negative effects of this action upon decidualization induced in vitro.ResultsOur results revealed that the expression of YAP was higher in decidual cells from early pregnant decidua compared with ESCs from non-pregnant endometrium. The expression levels of YAP and TEA domain 1 (TEAD1) were both increased in ESCs during in vitro decidualization and the knockdown of YAP in ESCs caused negative effects upon decidualization in vitro.DiscussionOur study suggests that YAP is upregulated in human decidual cells compared with ESCs and influences the decidualization of ESCs.     

Porphyromonas gingivalis induces IL-8 and IFN-gamma secretion and apoptosis in human extravillous trophoblast derived HTR8/SVneo cells via activation of ERK1/2 and p38 signaling pathways

IntroductionPreterm birth is a major cause for infant mortality and morbidity. A large number of studies have suggested a link between periodontal disease and preterm birth. The purpose of this study was to investigate the interaction between a periodontopathic bacterium Porphyromonas gingivalis and human extravillous trophoblast derived HTR8/SVneo cells.MethodsProduction of cytokines in HTR8 cells was measured via ELISA. Annexin V/PI flow cytometry was performed to assess apoptosis. Protein expression was measured by western blot. Specific pharmacological inhibitors were used to inactivate relevant signaling pathways (p38 MAPK, SB203580; ERK1/2, U0126; JNK, SP600125; NF-κB, JSH-23) to determine their roles in inflammation and apoptosis.ResultsHTR8 cells released significant amounts of IL-8 and IFN-γ during exposure to P. gingivalis. Meanwhile, the percentages of both early and late apoptotic cells increased significantly in response to P. gingivalis. The most significant effect on inflammation was found using SB203580 and U0126, followed by SP600125 and JSH-23. Moreover, U0126 and SB203580 both partially but significantly suppressed P. gingivalis-induced apoptosis, with a large effect by U0126. Additionally, both heat-killed P. gingivalis and P. gingivalis lipopolysaccharide significantly induced IL-8 production.ConclusionP. gingivalis induces inflammation and apoptosis in HTR8 cells, and we demonstrated for the first time that activation of ERK1/2 and p38 MAPK pathways participates in P. gingivalis-induced inflammation and apoptosis. The abnormal regulation of inflammation and apoptosis in human trophoblasts by P. gingivalis infection may give new insights into how maternal periodontal disease and periodontal pathogens might be linked to preterm birth.     

Massage therapy alone and in combination with meditation for breast cancer patients undergoing autologous tissue reconstruction: A randomized pilot study

This study explored whether massage combined with meditation is more helpful than massage alone for women recovering from autologous tissue reconstruction after mastectomy for breast cancer. Forty patients were randomly assigned to either massage therapy or massage plus meditation on postoperative days 1 through 3. Outcome measures were 1) visual analog scale (VAS) scores for stress, anxiety, relaxation, insomnia, alertness, fatigue, tension, pain, mood, and energy, and 2) Perceived Stress Scale-14 scores. Nineteen patients in each group finished the study. Preintervention and postintervention mean total VAS scores improved significantly in both groups (P < .001), but no significant difference occurred between groups.     

Increased trophoblast inclusions in placentas from prematurely born infants: A potential marker of risk for preterm neurodevelopmental outcomes

Trophoblast inclusions (TIs) are placental abnormalities of the trophoblast bilayer. Present in 2–8% of full-term placentas, they are associated with poor neurodevelopment, including autism. Although previously unstudied, examination of chorionic villi from 108 preterm births revealed a ∼4 fold increase in the frequency of TIs (30.5%). Frequency of TIs was inversely related to gestational age (GA); 43% of placentas <30 weeks and 20% of placentas ≥32 weeks had TIs (χ2 = 4.41, p = 0.036). This increased prevalence in preterm infants suggests that TIs may indicate adverse intrauterine processes or undetected genetic abnormalities and could identify infants at risk for poor neurodevelopment.     

Increased placental phospholipase A2 gene expression and free F2-isoprostane levels in response to oxidative stress in preeclampsia

Vasoactive eicosanoids such as thromboxane (TX) A₂ and F₂-isoprostanes (F₂-isoPs) were shown to be increased in the preeclamptic placenta. Only one of the 64 possible isomers of F₂-isoPs derived from the oxidation of arachidonic acid was investigated in the placenta so far. F₂-isoPs are released from membrane phospholipids by phospholipases A₂ (PLA₂) and were shown to act on the TXA₂ receptor (TBXA2R). However, the PLA₂ deregulated in preeclampsia (PE) remains to be determined. In this study, we analyzed the concentrations of six isomers of F₂-isoPs; 8-iso-PGF_2α, 8-iso-15(R)-PGF_2α, 15(R)-PGF_2α, iPF_2α-IV, iPF_2α-VI, 5-iPF_2α-VI and the concentrations of the stable metabolites of TXA₂, TXB_2, by high performance liquid chromatography coupled with tandem mass spectrometry in placentas of PE (n = 17) and normotensive (n = 15) pregnancies according to the biopsy site: peri-insertion or periphery. In the same biopsies, relative mRNA expression of PLA2G2A, PLA2G4A, PLA2G5, PLA2G7, the PLA2 receptor (PLA2R1), the TXA₂ synthase and TBXAR2 were measured by quantitative RT-PCR. We observed similar concentrations of total F₂-isoP isomers between groups whereas higher concentrations (>40%) of free F₂-isoP were observed for all isomers (p ≤ 0.033) in PE than normotensive controls. As expected, we also observed higher placental concentrations of TXB₂ in PE (p = 0.005). Interestingly, we concomitantly found higher mRNA expression of secretory PLA2G2A (p = 0.010), PLA2G5 (p = 0.038) and TBXA2R (p = 0.023) in PE than normotensive placentas. In sum, deregulated PLA₂ could potentially be implicated in freeing F₂-isoP which could participate in local hypertension observed in the PE placenta through the TX pathway.     

The granulocyte colony-stimulating factor (G-CSF) upregulates metalloproteinase-2 and VEGF through PI3K/Akt and Erk1/2 activation in human trophoblast Swan 71 cells

IntroductionAlthough the expression of the granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSFR) in placental tissues suggests that the cytokine could play a role in placental development, the relevance of G-CSF:G-CSFR interaction in trophoblast cells remains to be studied. Thus, the possible functional role of G-CSF was examined in a human trophoblast cell line (Swan 71 cells).Methods and resultsThe expression of G-CSFR was detected by immunocytochemistry and Western blot assays. G-CSF treatment exerted neither a proliferative nor a protective effect on H₂O₂-mediated cell death in trophoblast cells. Gelatin zymography of supernatants collected from G-CSF-treated cells showed an increment of metalloproteinase-2 (MMP-2) activity. We also found higher MMP-2 and VEGF expression levels in conditioned medium from cells exposed to G-CSF. In addition, it was demonstrated that G-CSF induced the activation of PI3K/Akt and Erk1/2 pathways, which in turn activated NF-kB. By using selective pharmacological inhibitors, it was showed that these pathways are mediating the biological effects produced by G-CSF in Swan 71 cells.Discussion and conclusionWe have demonstrated for the first time that G-CSF increases MMP-2 activity and VEGF secretion in Swan 71 cells through activation of PI3K/Akt and Erk signaling pathways, both contributing to the translocation of NF-kB to the nucleus. These data suggest that G-CSF is involved in the regulation of trophoblast function, and should be considered as a locally produced cytokine probably contributing to embryo implantation and the development of a functional placenta.     

Dynamic maternal and fetal Notch activity and expression in placentation

IntroductionMurine placentation requires trophoblast Notch2, while the Notch ligand, JAGGED1, is reduced in invasive trophoblasts from women with preeclampsia. However, the placental cells with active Notch signaling and expression of other Notch proteins and ligands in placentation have yet to be defined. We sought to identify endothelial cell and trophoblast subtypes with canonical Notch signaling in the decidua and placenta and correlate this to expression of Notch proteins and ligands.MethodsNotch reporter transgenic mice were used to define canonical Notch activity and immunofluorescence staining performed to characterize expression of Notch1, 2, 3, 4 and ligands, Delta-like 4 (Dll4) and Jagged1 (Jag1) during early placentation and in the mature placenta.ResultsNotch signaling is active in maternal and fetal endothelial cells and trophoblasts during early placentation and in the mature placenta. Dll4, Jag1, Notch1, and Notch4 are expressed in maternal vasculature in the decidua. Dll4, Jag1 and Notch1 are expressed in fetal vasculature in the labyrinth. Dll4, Notch2 and Notch4 are co-expressed in the ectoplacental cone. Notch2 and Notch4 are expressed in parietal-trophoblast giant cells and junctional zone trophoblasts with active canonical Notch signaling and in labyrinthine syncytiotrophoblasts and sinusoidal-trophoblast giant cells.DiscussionCanonical Notch activity and distinct expression patterns for Notch proteins and ligands was evident in endothelium and trophoblasts, suggesting Notch1, Notch2, Notch4, Dll4, and Jag1 have distinct and overlapping functions in placentation. Characterization of Notch signaling defects in existing mouse models of preeclampsia may shed light on the role of Notch in developing the preeclampsia phenotype.     

Placental concentrations of bisphenol A and birth weight from births in the Southeastern U.S.

IntroductionBisphenol A (BPA) is a weakly estrogenic compound that has been detected in a wide variety of food products and biological matrices (saliva, blood, urine, etc). Despite the potential risk of human exposure to BPA, little information exists concerning maternal and fetal exposure to BPA during pregnancy. The aim of this study is to evaluate the correlation between placental BPA concentration, infant birth weight and calculated birth weight centile, and several other maternal and infant parameters.MethodsPlacental sample were collected from 200 subjects. BPA levels were measured by isotope dilution GC–MS. Additional maternal and infant data were gathered from medical charts and were potential correlates with placental BPA levels.ResultsPlacental BPA concentrations ranged from 4.4 ng/g to 273.9 ng/g in oven-dried tissue (average 103.4 ± 61.8 ng/g). There was a significant negative correlation between calculated birth weight centile and levels of placental BPA (p < 0.05). Low birth weight and small for gestational age infants also had significantly greater placental BPA concentrations as compared to normal weight infants and average/large for gestational age infants. Infants born to African American mothers also had greater placental BPA concentrations as compared to infants born to Hispanic mothers.DiscussionPlacental BPA concentrations are correlated with the growth potential of the fetus and may play a role in reduced fetal growth.     

The effect of yoga training on enhancement of Adrenocorticotropic hormone (ACTH) and cortisol levels in female patients with multiple sclerosis

The effect of 8 weeks yoga training on cortisol and Adrenocorticotropic hormone (ACTH) levels in female patients with Multiple Sclerosis (MS) is examined. Twenty four MS female patients with Expanded Disability Status Scale (EDSS) 1 to 5.5 participated in this study as the subject. The participants were divided into control (n = 10) or training group (n = 14) randomly. Training group performed 90 min yoga training per session, 3 days a week for 8 weeks. Assessments include body composition measurement and blood sampling 48 h before first session and 48 h after the intervention. The results demonstrated that ACTH increased and cortisol decreased compared to the control group (P < 0.05); In conclusion, it seems that yoga training modulates ACTH level in concomitant with reduction in cortisol level in female patients with MS.     

Ly49 knockdown in mice results in aberrant uterine crypt formation and impaired blastocyst implantation

Genetic knockdown (KD) of the mouse Ly49 receptor family is reported to result in infertility despite the presence of zona-enclosed blastocysts in the uterus. Ly49 receptors regulate leukocyte functions particularly Natural Killer (NK) cell functions and are analogous to human killer immunoglobulin-like receptors (KIRs). Histological analyses of gd3.5–4.5 B6.Ly49^KD uteri identified hatched but retarded blastocysts with pyknotic nuclei, aberrant endometrial crypt formation and impaired uterine lumen closure accompanied by a lack of primary decidualization These data support peri-implantation roles for leukocytes expressing the Ly49 receptor repertoire and may give insight into KIR-based regulation of human infertility.     

Pentoxifylline inhibits lipopolysaccharide-induced inflammatory mediators in human second trimester placenta explants

BackgroundIntrauterine infection and inflammation during pregnancy, which leads to up-regulation of inflammatory cytokines and prostaglandin synthesis, has been implicated in the pathogenesis of preterm delivery and other pregnancy complications. Effective preventive and therapeutic strategies to reduce these outcomes are lacking to date. Pentoxifylline (PTX) is a non-specific phosphodiesterase inhibitor which raises intracellular cyclic adenosine monophosphate and decreases production of pro-inflammatory mediators while enhancing anti-inflammatory cytokines. We hypothesized that pentoxifylline will decrease lipopolysaccharide (LPS)-induced pro-inflammatory cytokines production in human placental explants.MethodsPlacental explants derived from normal second trimester human placentas were treated with PTX, stimulated with LPS and cultured at 37 °C in 5% CO₂. Conditioned media were assayed for pro- and anti-inflammatory mediators with multiplex immunoassays or ELISA, and explant tissues for mRNA with real time PCR. Means of PTX-treated and untreated samples were compared using paired t tests and Wilcoxon-signed rank tests.ResultsPTX preferentially inhibited placental expression and production of LPS-induced pro-inflammatory cytokines including TNF-α (25461 vs. 1908 pg/ml, p < 0.001), IL-1β (2921 vs. 1067 pg/ml, p < 0.001) and IFN-γ (2190 vs 427 pg/ml, p < 0.001) with relative preservation of anti-inflammatory mediators. The suppressive effects on LPS-induced placental inflammation were independent of the timing of PTX administration in relation to LPS-induced stimulation.ConclusionOur study suggests that PTX attenuates the LPS-induced pro-inflammatory milieu in human placental explants. We speculate that PTX may have utility as a candidate anti-inflammatory agent for prophylaxis and/or treatment of human placental inflammation.     

Autophagy in the placenta of women with hypertensive disorders in pregnancy

IntroductionAutophagy has not been studied extensively in the human placenta. This study was performed to determine whether autophagy is increased in the placentas of women with hypertensive disorders in pregnancy compared to normotensive pregnancies.MethodsLC3-II and p62 protein expression were examined by quantitative Western blotting analysis in 40 placentas from women not experiencing labor pains. The 40 placentas were from 13, 8, and 19 women with preeclampsia, gestational hypertension, and normal pregnancy, respectively. Hypertensive disorders in pregnancy included preeclampsia and gestational hypertension.ResultsLC3-II expression was significantly increased, while that of p62 was significantly reduced in 21 placentas of women with hypertensive disorders compared to those with normal blood pressure irrespective of the presence or absence of fetal growth restriction (FGR). LC3-II expression was also significantly increased in 13 placentas of women with preeclampsia irrespective of the presence or absence of FGR.DiscussionThe results of this study suggested that autophagy is active in the placenta of hypertensive disorders even in the absence of FGR.     

Genetic studies of gestational duration and preterm birth

The fine control of birth timing is important to human survival and evolution. A key challenge in studying the mechanisms underlying the regulation of human birth timing is that human parturition is a unique to human event — animal models provide only limited information. The duration of gestation or the risk of preterm birth is a complex human trait under genetic control from both maternal and fetal genomes. Genomic discoveries through genome-wide association (GWA) studies would implicate relevant genes and pathways. Similar to other complex human traits, gestational duration is likely to be influenced by numerous genetic variants of small effect size. The detection of these small-effect genetic variants requires very large sample sizes. In addition, several practical and analytical challenges, in particular the involvement of both maternal and fetal genomes, further complicate the genetic studies of gestational duration and other pregnancy phenotypes. Despite these challenges, large-scale GWA studies have already identified several genomic loci associated with gestational duration or the risk of preterm birth. These genomic discoveries have revealed novel insights about the biology of human birth timing. Expanding genomic discoveries in larger datasets by more refined analytical approaches, together with the functional analysis of the identified genomic loci, will collectively elucidate the biological processes underlying the control of human birth timing.