Maternal obesity induced by a ‘cafeteria’ diet in the rat does not increase inflammation in maternal,placental or fetal tissues in late gestation

IntroductionObesity during pregnancy can cause serious complications for maternal and infant health. While this has often been attributed to increased inflammation during obese pregnancy, human and animal studies exhibit variable results with respect to the inflammatory status of the mother, placenta and fetus. Cafeteria (CAF) feeding induces more inflammation than standard high-fat feeding in non-pregnant animal models. This study investigated whether maternal obesity induced by a CAF diet increases maternal, fetal or placental inflammation.MethodsMaternal obesity was established in rats by 8 weeks of pre-pregnancy CAF feeding. Maternal plasma inflammatory markers (IL-1β, IL-6, IL-10, IL-12p40, MCP1, GRO/KC, MIP-2 and TNFα) and expression of inflammatory genes (Tnfα, Il-6, Il-1β, Tlr2, Tlr4, Cox2 and Emr1) in maternal, placental and fetal tissues were measured at day 21 of gestation.ResultsDespite CAF animals having 63% more central body fat than controls at day 21 of gestation, plasma inflammatory markers were not increased; indeed, levels of IL-6, IL-12p40 and MIP2 were reduced slightly. Similarly, inflammatory gene expression remained largely unaffected by CAF feeding, except for slight reductions to Tlr4 and Emr1 expression in CAF maternal adipose tissue, and reduced Tlr4 expression in male labyrinth zone (LZ). The junctional zone (JZ) displayed increased Il-6 expression in CAF animals when fetal sexes were combined, but no inflammatory genes were affected by the CAF diet in fetal liver.ConclusionsMaternal obesity induced by a CAF diet before and during pregnancy does not increase the inflammatory status of the mother, placenta or fetus in late gestation.     

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.     

Abnormal labyrinthine zone in the Hectd1-null placenta

IntroductionThe labyrinthine zone of the placenta is where exchange of nutrients and waste occurs between maternal and fetal circulations. Proper development of the placental labyrinth is essential for successful growth of the developing fetus and abnormalities in placental development are associated with intrauterine growth restriction (IUGR), preeclampsia and fetal demise. Our previous studies demonstrate that Hectd1 is essential for development of the junctional and labyrinthine zones of the placenta. Here we further characterize labyrinthine zone defects in the Hectd1 mutant placenta.MethodsThe structure of the mutant placenta was compared to wildtype littermates using histological methods. The expression of cell type specific markers was examined by immunohistochemistry and in situ hybridization.ResultsHectd1 is expressed in the labyrinthine zone throughout development and the protein is enriched in syncytiotrophoblast layer type I cells (SynT-I) and Sinusoidal Trophoblast Giant cells (S-TGCs) in the mature placenta. Mutation of Hectd1 results in pale placentas with frequent hemorrhages along with gross abnormalities in the structure of the labyrinthine zone including a smaller overall volume and a poorly elaborated fetal vasculature that contain fewer fetal blood cells. Examination of molecular markers of labyrinthine trophoblast cell types reveals increased Dlx3 positive cells and Syna positive SynT-I cells, along with decreased Hand1 and Ctsq positive sinusoidal trophoblast giant cells (S-TGCs).DiscussionTogether these defects indicate that Hectd1 is required for development of the labyrinthine zonethe mouse placenta.     

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.     

Mini-dose long gonadotropin-releasing hormone (GnRH) agonist versus agonist flare stimulation protocol for in vitro fertilization poor responders

ObjectivesTo compare 2 stimulation protocols, mini-dose long gonadotropin releasing hormone (GnRH) agonist versus agonist flare for in vitro fertilization poor responders.DesignProspective comparative nonrandomized clinical trial.SettingDr. Samir Abasss IVF center, Jeddah, Kingdom of Saudi Arabia from april 2012 to December 2012 on 50 women undergoing IVF/ICSI fulfilling the criteria of poor responders.Material and methodsPatients were allocated into 2 groups, group 1 (n = 25) received mini-dose long agonist and group 2 (n = 25) received agonist flare protocol.Main outcomeNumber of oocytes retrieved (primary outcome), duration of stimulation (days), peak E2 level on the day of hCG injection, number of fertilized oocytes, number of transferred embryos and pregnancy rate/cycle.ResultsBoth groups were comparable regarding age, body mass index and duration of infertility (years). The difference in basal FSH and duration of stimulation (days) does not reach statistical significance (p value 0.833 and 0.373 respectively). There was a high statistical difference between both groups regarding peak E2 on day of hCG injection, number of oocytes retrieved, number of fertilized oocytes, number of transferred embryos; which is higher in the mini-dose agonist group (p value 0.00).Pregnancy rate/cycle was higher in the mini-dose agonist group (9/25 vs. 6/25) however this difference does not reach statistical significance (p value 0.355) which may be attributed to small sample size or advanced maternal age.ConclusionMini-dose long GnRHa stimulation protocol appears to be more beneficial for poor responders than GnRHa agonist flare.     

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.     

Vesicular uptake of macromolecules by human placental amniotic epithelial cells

IntroductionStudies in animal models have shown that unidirectional vesicular transport of amniotic fluid across the amnion plays a primary role in regulating amniotic fluid volume. Our objective was to explore vesicle type, vesicular uptake and intracellular distribution of vesicles in human amnion cells using high- and super-resolution fluorescence microscopy.MethodsPlacental amnion was obtained at cesarean section and amnion cells were prepared and cultured. At 20%–50% confluence, the cells were incubated with fluorophore conjugated macromolecules for 1–30 min at 22 °C or 37 °C. Fluorophore labeled macromolecules were selected as markers of receptor-mediated caveolar and clathrin-coated vesicular uptake as well as non-specific endocytosis. After fluorophore treatment, the cells were fixed, imaged and vesicles counted using Imaris^® software.ResultsVesicular uptake displayed first order saturation kinetics with half saturation times averaging 1.3 min at 37 °C compared to 4.9 min at 22 °C, with non-specific endocytotic uptake being more rapid at both temperatures. There was extensive cell-to-cell variability in uptake rate. Under super-resolution microscopy, the pattern of intracellular spatial distribution was distinct for each macromolecule. Co-localization of fluorescently labeled macromolecules was very low at vesicular dimensions.ConclusionsIn human placental amnion cells, 1) vesicular uptake of macromolecules is rapid, consistent with the concept that vesicular transcytosis across the amnion plays a role in the regulation of amniotic fluid volume; 2) uptake is temperature dependent and variable among individual cells; 3) the unique intracellular distributions suggest distinct functions for each vesicle type; 4) non-receptor mediated vesicular uptake may be a primary vesicular uptake mechanism.     

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.     

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.     

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.     

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.     

Tensile strain increased COX-2 expression and PGE2 release leading to weakening of the human amniotic membrane

IntroductionThere is evidence that premature rupture of the fetal membrane at term/preterm is a result of stretch and tissue weakening due to enhanced prostaglandin E₂ (PGE₂) production. However, the effect of tensile strain on inflammatory mediators and the stretch sensitive protein connexin-43 (Cx43) has not been examined. We determined whether the inflammatory environment influenced tissue composition and response of the tissue to tensile strain.MethodsHuman amniotic membranes isolated from the cervix (CAM) or placenta regions (PAM) were examined by second harmonic generation to identify collagen orientation and subjected to tensile testing to failure. In separate experiments, specimens were subjected to cyclic tensile strain (2%, 1 Hz) for 24 h. Specimens were examined for Cx43 by immunofluorescence confocal microscopy and expression of COX-2 and Cx43 by RT-qPCR. PGE₂, collagen, elastin and glycosaminoglycan (GAG) levels were analysed by biochemical assay.ResultsValues for tensile strength were significantly higher in PAM than CAM with mechanical parameters dependent on collagen orientation. Gene expression for Cx43 and COX-2 was enhanced by tensile strain leading to increased PGE₂ release and GAG levels in PAM and CAM when compared to unstrained controls. In contrast, collagen and elastin content was reduced by tensile strain in PAM and CAM.DiscussionFibre orientation has a significant effect on amniotic strength. Tensile strain increased Cx43/COX-2 expression and PGE₂ release resulting in tissue softening mediated by enhanced GAG levels and a reduction in collagen/elastin content.ConclusionA combination of inflammatory and mechanical factors may disrupt amniotic membrane biomechanics and matrix composition.     

Associations between the levels of soluble (pro)renin receptor in maternal and umbilical cord blood and hypertensive disorder of pregnancy

IntroductionThe prorenin (PR) receptor [(P)RR] contributes to the regulation of the tissue renin-angiotensin system (RAS) and Wnt signaling, which is involved in embryogenesis and the pathological progression of malignant tumors and diabetes mellitus. Placental (P)RR is significantly upregulated in placental tissues from preeclamptic women. However, because it cannot be examined during pregnancy, the chronological relationship between the acceleration of tissue RAS and the disease state of hypertensive disorder of pregnancy (HDP) has not been reported. In this study, we examined whether chronological changes in placental tissue RAS can be assessed by measuring soluble (P)RR [s(P)RR].MethodsWe obtained maternal and umbilical cord blood samples from 517 pregnant women (441 singleton and 76 twin pregnancies). The concentrations of s(P)RR and prorenin (PR) were measured using enzyme-linked immunosorbent assays.ResultsMultivariate analysis showed that maternal serum s(P)RR levels were significantly higher in patients with HDP or fetal growth restriction (FGR) and were positively correlated with serum PR levels. Furthermore, the maternal s(P)RR level was significantly higher in HDP with severe hypertension and after the onset of HDP. However, maternal s(P)RR levels were not affected by the severity of proteinuria. Serum s(P)RR levels in umbilical cord blood of singleton pregnancies were significantly correlated with gestational week at delivery and PR level.DiscussionMaternal serum s(P)RR concentrations may reflect acceleration of tissue RAS in the placenta and blood pressure severity; however, the umbilical serum s(P)RR concentration was not affected by maternal HDP.     

Different expression of placental pyruvate kinase in normal,preeclamptic and intrauterine growth restriction pregnancies

IntroductionPreeclampsia (PE) and intrauterine growth restriction (IUGR) are two diseases that affect pregnant women and their unborn children. These diseases cause low birth weight, pre-term delivery, and neurological and cardiovascular disorders in babies. Combined they account for 20% of preterm deliveries. Pyruvate kinase M2 (PKM2) is a metabolism enzyme found in developing embryonic and cancer tissues. Our objective is to determine the expression of PKM2 in human PE and IUGR compared to normal pregnancies. Understanding expression of PKM2 in PE and IUGR could help us to better understand the mechanisms and find treatments for PE and IUGR.MethodsHuman placental tissues were obtained for PKM2 determination and analyzed by immunohistochemistry, Western blot, and a pyruvate assay. Placental samples were homogenized and cytoplasmic and nuclear proteins were extracted for Western blot analysis.ResultsPreeclampsia samples had elevated levels of p-PKM2, p-ERK, and ERK in the cytoplasm. Beta-catenin and lactose dehydrogenase (LDH) were also elevated in preeclampsia placenta samples.Discussion and conclusionWe conclude that PKM2 is expressed in normal, PE and IUGR pregnancies. Also, that this expression is increased in the PE placenta at delivery. These results suggest placental metabolism through PKM2 could play a role in human preeclampsia.     

Impact of early- and late-onset preeclampsia on features of placental and newborn vascular health

IntroductionOffspring exposed to preeclampsia (PE) show an increased risk of cardiovascular disease in adulthood. We hypothesize that this is mediated by a disturbed vascular development of the placenta, umbilical cord and fetus. Therefore, we investigated associations between early-onset PE (EOPE), late-onset PE (LOPE) and features of placental and newborn vascular health.MethodsWe performed a nested case-control study in The Rotterdam Periconceptional Cohort, including 30 PE pregnancies (15 EOPE, 15 LOPE) and 218 control pregnancies (164 uncomplicated controls, 54 complicated controls including 28 fetal growth restriction, 26 preterm birth) and assessed macroscopic and histomorphometric outcomes of the placenta and umbilical cord.ResultsA significant association was observed between PE and a smaller umbilical vein area and wall thickness, independent of gestational age and birth weight. In EOPE we observed significant associations with a lower weight, length and width of the placenta, length of the umbilical cord, and thickness and wall area of the umbilical vein and artery. These associations attenuated after gestational age and birth weight adjustment. In LOPE a significant association with a larger placental width and smaller umbilical vein wall thickness was shown, independent of gestational age and birth weight.DiscussionOur study suggests that PE is associated with a smaller umbilical cord vein area and wall thickness, independent of gestational age and birth weight, which may serve as a proxy of disturbed cardiovascular development in the newborn. Follow-up studies are needed to ultimately predict and lower the risk of cardiovascular disease in offspring exposed to PE.     

Identification of novel genetic markers for mouse yolk sac cells by using microarray analyses

The mouse embryonic yolk sac consists of a visceral yolk sac (VYS) and parietal yolk sac (PYS), and may function as a materno-fetal exchange system for nutrients and wastes, and physical protector for the embryo/fetus. The present study was undertaken to characterize gene expression of the VYS and PYS endodermal cells, and to identify their novel genetic markers from microarray data. Apoa4, Lrp2, Fxyd2, Slc34a3 and Entpd2 were predominantly expressed in VYS epithelial cells. Gkn2 and Pga5 were selected as markers for PYS cells. These genetic markers may be useful for characterization of murine yolk sacs during development.