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.     

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.     

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.     

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.     

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.     

Effect of oxygen on the expression of renin–angiotensin system components in a human trophoblast cell line

During the first trimester, normal placental development occurs in a low oxygen environment that is known to stimulate angiogenesis via upregulation of vascular endothelial growth factor (VEGF). Expression of the placental renin–angiotensin system (RAS) is highest in early pregnancy. While the RAS and oxygen both stimulate angiogenesis, how they interact within the placenta is unknown. We postulated that low oxygen increases expression of the proangiogenic RAS pathway and that this is associated with increased VEGF in a first trimester human trophoblast cell line (HTR-8/SVneo). HTR-8/SVneo cells were cultured in one of three oxygen tensions (1%, 5% and 20%). RAS and VEGF mRNA expression were determined by qPCR. Prorenin, angiotensin converting enzyme (ACE) and VEGF protein levels in the supernatant, as well as prorenin and ACE in cell lysates, were measured using ELISAs. Low oxygen significantly increased the expression of both angiotensin II type 1 receptor (AGTR1) and VEGF (both P < 0.05). There was a positive correlation between AGTR1 and VEGF expression at low oxygen (r = 0.64, P < 0.005). Corresponding increases in VEGF protein were observed with low oxygen (P < 0.05). Despite no change in ACE1 mRNA expression, ACE levels in the supernatant increased with low oxygen (1% and 5%, P < 0.05). Expression of other RAS components did not change. Low oxygen increased AGTR1 and VEGF expression, as well as ACE and VEGF protein levels, suggesting that the proangiogenic RAS pathway is activated. This highlights a potential role for the placental RAS in mediating the proangiogenic effects of low oxygen in placental development.     

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.     

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.     

Distinct effects of short- and long-term type 1 diabetes to the placental extracellular matrix and fetal development in mice

PurposeWe have previously shown that the development of complications in the early pregnant decidua and myometrium in mice correlates with diabetes progression. In the current study, we investigated the influence of diabetes progression on the placental extracellular matrix (ECM) and on fetal development at the end of pregnancy.MethodsAlloxan-induced type 1 diabetic female mice were bred either 30–50 days after diabetes induction (D) or 90-110D. Fetal and placental weights were registered at the 19th day of pregnancy together with analysis of gene expression, deposition and turnover of the placental ECM.ResultsThe short-term diabetic group (30-50D) showed elevated embryonic losses and underweight fetuses (89%) with normal weight placentas. In contrast, the long-term group (90-110D) had increased malformations/fetal deaths and underweight fetuses (42%) and heavy placentas (50%). Normal-weight fetuses from the long-term group had placentas with either regular weight and fetal/placental weight ratio or increased weight and low fetal/placental weight ratio. Furthermore, the placentas of the short-term group showed alterations in the synthesis and deposition of collagen types I and V and in the activity of MMP2 whereas placentas of the 90-110D group presented alterations in collagen type III and V and MMP9.ConclusionsDiabetes progression promoted distinct outcomes in pregnancy. Modifications of both synthesis and turnover of ECM occurred even before changes of placental weight were detected. Adjustment of fetal/placental weight ratio or placental enlargement restored normal growth in part of the fetuses from the long-term group.     

Potential clinical applications of placental stem cells for use in fetal therapy of birth defects

Placental stem cells are of growing interest for a variety of clinical applications due to their multipotency and ready availability from otherwise frequently discarded biomaterial. Stem cells derived from the placenta have been investigated in a number of disease processes, including wound healing, ischemic heart disease, autoimmune disorders, and chronic lung or liver injury. Fetal intervention for structural congenital defects, such as spina bifida, has rapidly progressed as a field due to advances in maternal-fetal medicine and improving surgical techniques. In utero treatment of structural, as well as non-structural, congenital disorders with cell-based therapies is of particular interest given the immunologic immaturity and immunotolerant environment of the developing fetus. A comprehensive literature review was performed to assess the potential utilization of placenta-derived stem cells for in utero treatment of congenital disorders. Most studies are still in the preclinical phase, utilizing animal models of common congenital disorders. Future research endeavors may include autologous transplantation, gene transfers, induced pluripotent stem cells, or cell-free therapies derived from the stem cell secretome. Though much work still needs to be done, placental stem cells are a promising therapeutic agent for fetal intervention for congenital disease.     

Maternal obesity alters brain derived neurotrophic factor (BDNF) signaling in the placenta in a sexually dimorphic manner

IntroductionObesity is a major clinical problem in obstetrics being associated with adverse pregnancy outcomes and fetal programming. Brain derived neurotrophic factor (BDNF), a validated miR-210 target, is necessary for placental development, fetal growth, glucose metabolism, and energy homeostasis. Plasma BDNF levels are reduced in obese individuals; however, placental BDNF has yet to be studied in the context of maternal obesity. In this study, we investigated the effect of maternal obesity and sexual dimorphism on placental BDNF signaling.MethodsBDNF signaling was measured in placentas from lean (pre-pregnancy BMI < 25) and obese (pre-pregnancy BMI>30) women at term without medical complications that delivered via cesarean section without labor. MiRNA-210, BDNF mRNA, proBDNF, and mature BDNF were measured by RT – PCR, ELISA, and Western blot. Downstream signaling via TRKB (BDNF receptor) was measured using Western blot.ResultsMaternal obesity was associated with increased miRNA-210 and decreased BDNF mRNA in placentas from female fetuses, and decreased proBDNF in placentas from male fetuses. We also identified decreased mature BDNF in placentas from male fetuses when compared to female fetuses. Mir-210 expression was negatively correlated with mature BDNF protein. TRKB phosphorylated at tyrosine 817, not tyrosine 515, was increased in placentas from obese women. Maternal obesity was associated with increased phosphorylation of MAPK p38 in placentas from male fetuses, but not phosphorylation of ERK p42/44.DiscussionBDNF regulation is complex and highly regulated. Pre-pregnancy/early maternal obesity adversely affects BDNF/TRKB signaling in the placenta in a sexually dimorphic manner. These data collectively suggest that induction of placental TRKB signaling could ameliorate the placental OB phenotype, thus improving perinatal outcome.     

Endometrial androgen signaling and decidualization regulate trophoblast expansion and invasion in co-culture: A time-lapse study

IntroductionTo elucidate whether trophoblast expansion and invasion are modulated by androgen signaling in an in vitro co-culture model system with decidualizing endometrial stromal cells (ESCs).MethodsWe employed an in vitro co-culture model of early embryo implantation, consisting of human ESCs (EtsT499 cells) and spheroids generated by extravillous trophoblast (EVT) derived HTR8/Svneo. The ESCs were decidualized with 8-bromo-cAMP (8-br-cAMP) in the presence or absence of dihydrotestosterone (DHT) at various concentrations for 5 days before co-culture with EVT spheroids. Trophoblast expansion was monitored by fluorescent time-lapse imaging microscopy. ESCs motility was visualized by using CellTracker™ Orange CMRA fluorescent probe. Apoptosis of ESCs was detected by CellEvent™ Caspase-3/7^® green detection reagent. Invasion assays were performed to quantify EVT invasion through a chemotaxis cell membrane.ResultsExpansion of EVT spheroids was significantly enhanced by decidualized compared to undifferentiated ESCs. This process was further stimulated if ESCs were first decidualized in the presence of DHT. In contrast to decidualized ESCs, undifferentiated cells actively migrated away from expanding EVT spheroids. Invasiveness of EVT toward decidualized ESCs was significantly attenuated in comparison to undifferentiated ESCs. DHT had no effect on EVT invasion. However, an inhibitor of intercellular gap junction communication significantly enhanced EVT invasion towards decidualized ESCs.ConclusionsThese results indicate distinct roles for androgen signaling and gap junction formation in decidual cells in regulating trophoblast expansion and invasion.     

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.