Gestational Age-matched Control Placentas Research Articles

Leptin-Mediated Induction of IL-6 Expression in Hofbauer Cells Contributes to Preeclampsia Pathogenesis.

Leptin plays a crucial role in regulating energy homoeostasis, neuroendocrine function, metabolism, and immune and inflammatory responses. The adipose tissue is a main source of leptin, but during pregnancy, leptin is also secreted primarily by the placenta. Circulating leptin levels peak during the second trimester of human pregnancy and fall after labor. Several studies indicated a strong association between elevated placental leptin levels and preeclampsia (PE) pathogenesis and elevated serum interleukin-6 (IL-6) levels in PE patients. Therefore, we hypothesized that a local increase in placental leptin production induces IL-6 production in Hofbauer cells (HBCs) to contribute to PE-associated inflammation. We first investigated HBCs-specific IL-6 and leptin receptor (LEPR) expression and compared their immunoreactivity in PE vs. gestational age-matched control placentas. Subsequently, we examined the in vitro regulation of IL-6 as well as the phosphorylation levels of intracellular signaling proteins STAT3, STAT5, NF-κB, and ERK1/2 by increasing recombinant human leptin concentrations (10 to 1000 ng/mL) in primary cultured HBCs. Lastly, HBC cultures were incubated with leptin ± specific inhibitors of STAT3 or STAT5, or p65 NF-κB or ERK1/2 MAPK signaling cascades to determine relevant cascade(s) involved in leptin-mediated IL-6 regulation. Immunohistochemistry revealed ~three- and ~five-fold increases in IL-6 and LEPR expression, respectively, in HBCs from PE placentas. In vitro analysis indicated that leptin treatment in HBCs stimulate IL-6 in a concentration-dependent manner both at the transcriptional and secretory levels (p < 0.05). Moreover, leptin-treated HBC cultures displayed significantly increased phosphorylation levels of STAT5, p65 NF-κB, and ERK1/2 MAPK and pre-incubation of HBCs with a specific ERK1/2 MAPK inhibitor blocked leptin-induced IL-6 expression. Our in situ results show that HBCs contribute to the pathogenesis of PE by elevating IL-6 expression, and in vitro results indicate that induction of IL-6 expression in HBCs is primarily leptin-mediated. While HBCs display an anti-inflammatory phenotype in normal placentas, elevated levels of leptin may transform HBCs into a pro-inflammatory phenotype by activating ERK1/2 MAPK to augment IL-6 expression.

Cellular and molecular atlas of the placenta from a COVID-19 pregnant woman infected at midgestation highlights the defective impacts on foetal health.

ObjectivesThe impacts of the current COVID‐19 pandemic on maternal and foetal health are enormous and of serious concern. However, the influence of SARS‐CoV‐2 infection at early‐to‐mid gestation on maternal and foetal health remains unclear.Materials and methodsHere, we report the follow‐up study of a pregnant woman of her whole infective course of SARS‐CoV‐2, from asymptomatic infection at gestational week 20 to mild and then severe illness state, and finally cured at Week 24. Following caesarean section due to incomplete uterine rupture at Week 28, histological examinations on the placenta and foetal tissues as well as single‐cell RNA sequencing (scRNA‐seq) for the placenta were performed.ResultsCompared with the gestational age‐matched control placentas, the placenta from this COVID‐19 case exhibited more syncytial knots and lowered expression of syncytiotrophoblast‐related genes. The scRNA‐seq analysis demonstrated impaired trophoblast differentiation, activation of antiviral and inflammatory CD8 T cells, as well as the tight association of increased inflammatory responses in the placenta with complement over‐activation in macrophages. In addition, levels of several inflammatory factors increased in the placenta and foetal blood.ConclusionThese findings illustrate a systematic cellular and molecular signature of placental insufficiency and immune activation at the maternal–foetal interface that may be attributed to SARS‐CoV‐2 infection at the midgestation stage, which highly suggests the extensive care for maternal and foetal outcomes in pregnant women suffering from COVID‐19.

CCN3 Signaling Is Differently Regulated in Placental Diseases Preeclampsia and Abnormally Invasive Placenta.

ObjectivesAn adequate development of the placenta includes trophoblast differentiation with the processes of trophoblast migration, invasion, cellular senescence and apoptosis which are all crucial to establishing a successful pregnancy. Altered placental development and function lead to placental diseases such as preeclampsia (PE) which is mainly characterized by insufficient trophoblast invasion and abnormally invasive placenta (AIP) disorders (Placenta accreta, increta, or percreta) which are characterized by excessive trophoblast invasion. Both of them will cause maternal and fetal morbidity/mortality. However, the etiology of these diseases is still unclear. Our previous study has shown that the matricellular protein nephroblastoma overexpressed (NOV, CCN3) induces G0/G1 cell cycle arrest, drives trophoblast cells into senescence and activates FAK and Akt kinases resulting in reduced cell proliferation and enhanced migration capability of the human trophoblast cell line SGHPL-5. The present study focuses on whether CCN3 can alter cell cycle-regulated pathways associated with trophoblast senescence and invasion activity in pathological versus gestational age-matched control placentas.MethodsCell cycle regulator proteins were investigated by immunoblotting and qPCR. For localization of CCN3, p16, p21, and Cyclin D1 proteins, co-immunohistochemistry was performed.ResultsIn early-onset PE placentas, CCN3 was expressed at a significantly lower level compared to gestational age-matched controls. The decrease of CCN3 level is associated with an increase in p53, Cyclin E1 and pRb protein expression, whereas the level of cleaved Notch-1, p21, Cyclin D1, pFAK, pAKT, and pmTOR protein decreased. In term AIP placentas, the expression of CCN3 was significantly increased compared to matched term controls. This increase was correlated to an increase in p53, p16, p21, Cyclin D1, cleaved Notch-1, pFAK, pAkt, and pmTOR whereas pRb was significantly decreased. However, in late PE and early AIP placentas, no significant differences in CCN3, p16, p21, Cyclin D1, p53, and cleaved Notch-1 expression were found when matched to appropriate controls.ConclusionsCCN3 expression levels are correlated to markers of cell cycle arrest oppositely in PE and AIP by activating the FAK/AKT pathway in AIP or down-regulating in PE. This may be one mechanism to explain the different pathological features of placental diseases, PE and AIP.

Overexpression of the aryl hydrocarbon receptor nuclear translocator partially rescues fetoplacental angiogenesis in severe fetal growth restriction.

Pregnancies complicated by severe fetal growth restriction with abnormal umbilical artery Doppler velocimetry (FGRadv) are at substantial risk for adverse perinatal and long-term outcomes. Impaired angiogenesis of the placental vasculature in these pregnancies results in a sparse, poorly branched vascular tree, which structurally contributes to the abnormally elevated fetoplacental vascular resistance that is clinically manifested by absent or reversed umbilical artery Doppler indices. Previous studies have shown that aryl hydrocarbon receptor nuclear translocator (ARNT) is a key mediator of proper placental angiogenesis, and within placental endothelial cells (ECs) from human FGRadv pregnancies, low expression of ARNT leads to decreased vascular endothelial growth factor A (VEGFA) expression and deficient tube formation. Thus, the aim of the present study was to determine the effect of VEGFA administration or ARNT overexpression on angiogenic potential of FGRadv ECs. ECs were isolated and cultured from FGRadv or gestational age-matched control placentas and subjected to either vehicle vs VEGFA treatment or transduction with adenoviral-CMV (ad-CMV) vs adenoviral-ARNT (ad-ARNT) constructs. They were then assessed via wound scratch and tube formation assays. We found that VEGFA administration nominally improved FGRadv EC migration (P<0.01) and tube formation (P<0.05). ARNT overexpression led to significantly enhanced ARNT expression in FGRadv ECs (P<0.01), to a level similar to control ECs. Despite this, FGRadv EC migration (P<0.05) and tube formation (P<0.05) were still only partially rescued. This suggests that although ARNT does play a role in fetoplacental EC migration, other factors in addition to ARNT are likely also important in placental angiogenesis.

Preimplantation factor is an anti-apoptotic effector in human trophoblasts involving p53 signaling pathway.

From the earliest stages of gestation, embryonic–maternal interaction has a key role in a successful pregnancy. Various factors present during gestation may significantly influence this type of juxta/paracrine interaction. PreImplantation Factor (PIF) is a recently identified factor with activity at the fetomaternal interface. PIF is secreted by viable embryos and directly controls placental development by increasing the invasive capacity of human extravillous trophoblasts (EVTs). To further specify PIF's role in the human placenta, we analyzed the genome-wide expression profile of the EVT in the presence of a synthetic PIF analog (sPIF). We found that sPIF exposure altered several pathways related to p53 signaling, survival and the immune response. Functional assays revealed that sPIF acts through the p53 pathway to reduce both early and late trophoblast apoptosis. More precisely, sPIF (i) decreases the phosphorylation of p53 at Ser-15, (ii) enhances the B-cell lymphoma-2 (BCL2) expression and (iii) reduces the BCL2-associated X protein (BAX) and BCL2 homologous antagonist killer (BAK) mRNA expression levels. Furthermore, invalidation experiments of TP53 allowed us to demonstrate that PIF's effects on placental apoptosis seemed to be essentially mediated by this gene. We have clearly shown that p53 and sPIF pathways could interact in human trophoblast and thus promotes cell survival. Furthermore, sPIF was found to regulate a gene network related to immune tolerance in the EVT, which emphasizes the beneficial effect of this peptide on the human placenta. Finally, the PIF protein levels in placentas from pregnancies affected by preeclampsia or intra-uterine growth restriction were significantly lower than in gestational age-matched control placentas. Taken as a whole, our results suggest that sPIF protects the EVT's functional status through a variety of mechanisms. Clinical application of sPIF in the treatment of disorders of early pregnancy can be envisioned.

The proprotein convertase furin is required for trophoblast syncytialization

The multinucleated syncytial trophoblast, which forms the outermost layer of the placenta and serves multiple functions, is differentiated from and maintained by cytotrophoblast cell fusion. Deficiencies in syncytial trophoblast differentiation or maintenance likely contribute to intrauterine growth restriction and pre-eclampsia, two common gestational diseases. The cellular and molecular mechanisms governing trophoblast syncytialization are poorly understood. We report here that the proprotein convertase furin is highly expressed in syncytial trophoblast in the first trimester human placentas, and expression of furin in the syncytiotrophoblast is significantly lower in the placentas from pre-eclamptic patients as compared with their gestational age-matched control placentas. Using multiple experimental models including induced fusion of choriocarcinoma BeWo cells and spontaneous fusion of primary cultured cytotrophoblast cells or placental explants, we demonstrate that cytotrophoblast cell fusion and syncytialization are accompanied by furin expression. Furin-specific siRNAs or inhibitors inhibit cell fusion in BeWo cells, as well as trophoblast syncytialization in human placental explants. Furthermore, type 1 IGF receptor (IGF1R) is indicated in this study as a substrate of furin, and processing of IGF1R by furin is an essential mechanism for syncytialization. Finally, using lentivirus-mediated RNAi targeting to mouse trophectoderm, we demonstrate that furin function is required for the development of syncytiotrophoblast structure in the labyrinth layer, as well as for normal embryonic development.

MUC1 Expression Is Elevated in Severe Preeclamptic Placentas and Suppresses Trophoblast Cell Invasion via β1-Integrin Signaling

Preeclampsia is a pregnancy-specific disorder that features insufficient extravillous trophoblast (EVT) invasion. We have previously shown that MUC1 expression in human placenta increases with gestational age and inhibits choriocarcinoma cell invasion. Here, we studied whether MUC1 expression in preeclamptic placentas is dysregulated and the mechanism of EVT invasion regulated by MUC1. MUC1 expression in severe preeclamptic placentas and gestational age-matched control placentas was analyzed by real-time RT-PCR, Western blot analysis, and immunohistochemistry. The effects of MUC1 expression on cell-matrix adhesion, invasion, and cell signaling were studied in HTR8/SVneo EVT cells. We found that MUC1 mRNA and MUC1 protein were significantly up-regulated in severe preeclamptic placentas when compared with the gestational age-matched control placentas. Immunohistochemical analyses showed increased expression of MUC1 in the syncytiotrophoblast and EVT of severe preeclamptic placentas. In addition, MUC1 overexpression suppressed cell-matrix adhesion and invasion of EVT cells. Importantly, our data showed that MUC1 overexpression inhibited β1-integrin activity and phosphorylation of focal adhesion kinase, whereas the surface expression of β1-integrin was not significantly changed. Our findings suggest that MUC1 is overexpressed in severe preeclamptic placentas and that MUC1 overexpression suppresses EVT invasion mainly via modulating β1-integrin signaling.

Regulation of Interleukin-6 Expression in Human Decidual Cells and Its Potential Role in Chorioamnionitis

Chorioamnionitis frequently precedes both genital tract and placental inflammation and is both a primary cause of maternal morbidity and a major antecedent of preterm premature rupture of the membranes (PPROM) as well as preterm delivery (PTD). In most cases of chorioamnionitis, neutrophils dominate the decidua. In a subset of these cases, a predominance of monocytes is uniquely associated with both neonatal intraventricular hemorrhage and death. The multifunctional cytokine, interleukin-6, promotes local monocyte dominance via several mechanisms. In this study, immunostaining of placental sections revealed significantly higher interleukin-6 HSCOREs in decidual cells (DCs) but not in interstitial trophoblasts, in chorioamnionitis versus gestational age-matched control placentas (P < 0.05). In confluent leukocyte-free term DCs, secreted interleukin-6 levels in incubations with estradiol-17β were increased 2500-fold by IL-1β (P < 0.05). This up-regulation was inhibited by more than 50% in parallel incubations that included medroxyprogesterone acetate (n = 12, P < 0.05). Western blotting data confirmed these enzyme-linked immunosorbent assay results; quantitative RT-PCR findings demonstrated corresponding changes in interleukin-6 mRNA levels. Specific inhibitors of signaling for both nuclear factor-κB activation and p38-mitogen-activated protein kinase, but not for protein kinase C, significantly decreased IL-1β-enhanced interleukin-6 expression levels in cultured DCs. In conclusion, in situ and in vitro results indicate that significantly enhanced interleukin-6 expression levels in DCs during chorioamnionitis could be pivotal in skewing decidual monocyte differentiation to macrophages.

Activator Protein-2 Impairs the Invasion of a Human Extravillous Trophoblast Cell Line

The reduced migration/invasion of extravillous trophoblasts (EVTs) is a key feature of the genesis of preeclampsia. We and others previously reported that transcriptional factors activator protein-2 (AP-2) alpha and AP-2gamma act as suppressors of tumor invasion. The present study examined the expressions of AP-2alpha and AP-2gamma in preeclamptic placenta vs. control placenta and investigated their effect on the function of EVTs. The expressions of AP-2alpha and AP-2gamma were elevated in the preeclamptic placentas in comparison with the gestational age-matched control placentas. Their expressions also increased in EVTs of the preeclamptic placentas. Thereafter, we transfected AP-2alpha or AP-2gamma into human EVT cell line, HTR-8/SVneo. The overexpression of AP-2alpha or AP-2gamma decreased the migratory and invasive abilities in HTR-8/SVneo cells. This was followed by the reduction of protease activated receptor-1 and matrix metalloproteinases and a significant induction of plasminogen activator inhibitor-1 and the tissue inhibitor of metalloproteinase-1. AP-2alpha and AP-2gamma were weakly expressed in the cultured EVTs and HTR-8/SVneo cells, whereas they were induced by TNF-alpha, which increases in preeclamptic placenta and impairs trophoblast invasion. In the presence of TNF-alpha, the invasion of the HTR-8/SVneo cells was partially restored by a blocking of AP-2 induction using small interfering RNA of AP-2. The present data suggest that AP-2 may suppress trophoblast migration and invasion, thus leading to a shallow placentation in preeclampsia.

The Prevalence of Chronic Deciduitis in Cases of Preterm Labor without Clinical Chorioamnionitis

Preterm labor is a major cause of perinatal mortality and morbidity, and in approximately 30% of cases a clinical cause is not identified. Acute chorioamnionitis is found histologically in a significant percentage of placentas from preterm deliveries, and the mother is often asymptomatic. Although such subclinical acute chorioamnionitis is known to play a role in preterm labor, this study explores the hypothesis that chronic deciduitis with plasma cells is seen more frequently in cases of preterm labor than in control placentas. Thirty-nine singleton placentas from patients with idiopathic preterm labor were examined microscopically and compared in a blinded fashion with 39 gestational age-matched control placentas. Cases of clinical acute chorioamnionitis and known chronic maternal diseases were excluded. Thirty-nine control singleton placentas were obtained from patients undergoing induction of labor for fetal structural abnormalities, excluding aneuploidy. The presence or absence of acute chorioamnionitis, acute fetal inflammatory response, chronic deciduitis, chronic villitis, infarction, and decidual vasculopathy was noted. Immunohistochemical staining was undertaken to further define leukocyte subtypes. Forty-one percent of cases and 15% of controls showed chronic deciduitis (P = 0.022). Forty-six percent of cases and 18% of controls showed histologic acute chorioamnionitis (P = 0.015). There were 8 cases demonstrating acute fetal inflammatory response but only 1 control (P = 0.029). Little difference was seen in the distribution of lymphocyte subsets between cases and control placentas. Our findings suggest that chronic deciduitis plays a role in the etiology of some cases of preterm labor.

Preeclampsia-Related Inflammatory Cytokines Regulate Interleukin-6 Expression in Human Decidual Cells

Preeclampsia, a common pregnancy disorder associated with an increase in systemic inflammation, is the leading cause of maternal and fetal morbidity and mortality throughout the world. It is associated with shallow extravillous trophoblast invasion of the decidua, leading to uteroplacental blood flow that is inadequate for the developing fetal-placental unit. In preeclamptic women, interleukin-6 (IL-6) levels in plasma, but not placenta, are elevated, prompting evaluation of the decidua as a potential source of this excess, circulating IL-6. The current study found significantly higher immunohistochemical staining for IL-6 in decidual cells from preeclamptic versus preterm, gestational age-matched control placentas. Pro-inflammatory cytokines associated with the genesis of preeclampsia (i.e., tumor necrosis factor-alpha and interleukin-1beta) enhanced IL-6 mRNA levels and increased secreted IL-6 levels in first trimester leukocyte-free decidual cell incubations, as measured by real time quantitative RT-PCR, ELISA, and Western blotting. Therefore, decidual cell-derived IL-6 may contribute to excess circulating IL-6 levels that can promote both endothelial cell dysfunction (and subsequent vascular dysfunction) and the pathogenesis of preeclampsia whereas locally elevated IL-6 levels may contribute to an excess of decidual macrophages implicated in shallow extravillous trophoblast invasion of the decidua.

Les raisons de l'élévation de l'hCG dans le sérum de mères porteuses d'un fœtus trisomique

Human chorionic gonadotropin (hCG) is increased in maternal serum at 14–18 weeks of trisomy 21 (T21)-affected pregnancy, despite low placental hCG synthesis. We sought an explanation for this paradox. We first observed that in T21-affected pregnancies, maternal serum hCG levels peaked at around 10 weeks and then followed the same pattern throughout pregnancy as in controls, albeit at a higher (2.2-fold) level. We isolated cytotrophoblasts from 29 T21-affected placentas (12–25 weeks) and 13 gestational age-matched control placentas and cultured them for 3 days. In this large serie we confirmed that in the culture medium of trophoblasts isolated from T21 placentas, hCG secretion was significantly lower ( P < 0.003) than in controls, in contrast to the high hCG in maternal serum of the same patients. In corresponding culture medium, hCG was abnormally glycosylated, highly acidic (pHi = 4.5) and weakly bioactive (38% of control) as determined using the Leydig cell model. In conclusion, T21 trophoblast cells produced hCG that was weakly bioactive and abnormally glycosylated but whose maternal clearance seemed unaltered.

Trophoblast Production of a Weakly Bioactive Human Chorionic Gonadotropin in Trisomy 21-Affected Pregnancy

Total human chorionic gonadotropin (hCG) is high in maternal serum at 14-18 wk of trisomy 21 (T21)-affected pregnancy, despite low placental hCG synthesis. We sought an explanation for this paradox. We first observed that, in T21-affected pregnancies, maternal serum hCG levels peaked at around 10 wk and then followed the same pattern throughout pregnancy as in controls, albeit at a higher (2.2-fold) level. After delivery, hCG clearance was not significantly different from that in controls. We isolated cytotrophoblasts from 29 T21-affected placentas (12-25 wk) and 13 gestational age-matched control placentas and cultured them for 3 d. In this large series, we confirmed that, in the culture medium of trophoblasts isolated from T21 placentas, hCG secretion was significantly lower (P < 0.003) than in controls, in contrast to the high hCG in maternal serum of the same patients. In T21 cultured trophoblasts, transcripts of sialyltransferase-1 and fucosyltransferase-1 were abnormally high. In corresponding culture medium, hCG was abnormally glycosylated; highly acidic [isoelectric points (pHi) = 4.5] as shown by isoelectric focusing, immunoblotting, and lectin binding; and weakly bioactive (46% of control) as determined using the Leydig cell model. In conclusion, T21 trophoblast cells produced hCG that was weakly bioactive and abnormally glycosylated but whose maternal clearance was unaltered.

Intrauterine growth restriction with absent end-diastolic flow velocity in the umbilical artery is associated with maldevelopment of the placental terminal villous tree

OBJECTIVE: Our purpose was to evaluate the structure of placental terminal villi and their capillaries in pregnancies complicated by intrauterine growth restriction with absent end-diastolic flow velocity in the umbilical artery. STUDY DESIGN: Glutaraldehyde-perfusion-fixed villous tissue and a plastic cast of the vessels in at least two cotyledons were prepared from 10 cases with intrauterine growth restriction and 9 gestational age-matched control placentas. The structure and dimensions of 20 terminal capillary loops per cast were determined by scanning electron microscopic examination, and their appearances were correlated with the peripheral villi of the perfusion-fixed villous tissue. RESULTS: Capillary loops in the growth-restricted cases were sparse in number and significantly longer than in the control cases (218 μm [72] vs 137 μm [30], mean and SD, p < 0.05). They exhibited fewer branches (4.0 [1.9] per loop vs 6.1 [2.2], p < 0.05) and a majority of loops were uncoiled (79% vs 18%, p < 0.05). The villous tissues from the growth-restricted cases demonstrated elongated villi, consistent with the cast findings. The trophoblast surface was wrinkled and in some areas covered by fibrin plaques. CONCLUSIONS: The terminal villous compartment of the placenta appears to be maldeveloped in preterm intrauterine growth restriction pregnancies where absent end-diastolic flow velocity is demonstrated in the umbilical artery before delivery. These findings are consistent with an increase in fetoplacental vascular impedance at the capillary level and may account for the impaired gas and nutrient transfer in this disorder. (Am J Obstet Gynecol 1996;175:1534-42.)