Normal human pregnancy is considered a state of enhanced oxidative stress. In pregnancy, it plays important roles in embryo development, implantation, placental development and function, fetal ...development, and labor. However, pathologic pregnancies, including gestational diabetes mellitus (GDM), are associated with a heightened level of oxidative stress, owing to both overproduction of free radicals and/or a defect in the antioxidant defenses. This has important implications on the mother, placental function, and fetal well-being. Animal models of diabetes have confirmed the important role of oxidative stress in the etiology of congenital malformations; the relative immaturity of the antioxidant system facilitates the exposure of embryos and fetuses to the damaging effects of oxidative stress. Of note, there are only a few clinical studies evaluating the potential beneficial effects of antioxidants in GDM. Thus, whether or not increased antioxidant intake can reduce the complications of GDM in both mother and fetus needs to be explored. This review provides an overview and updated data on our current understanding of the complications associated with oxidative changes in GDM.
The insulin/insulin‐like growth factor (IGF) system regulates fetal and placental growth and development. In maternal diabetes, components of this system including insulin, IGF1, IGF2 and various ...IGF‐binding proteins are deregulated in the maternal or fetal circulation, or in the placenta. The placenta expresses considerable amounts of insulin and IGF1 receptors at distinct locations on both placental surfaces. This makes the insulin and the IGF1 receptor accessible to fetal and/or maternal insulin, IGF1 and IGF2. Unlike the receptor for IGF1, the insulin receptor undergoes a gestational change in expression site from the trophoblast at the beginning of pregnancy to the endothelium at term. Insulin and IGFs are implicated in the receptor‐mediated regulation of placental growth and transport, trophoblast invasion and placental angiogenesis. The dysregulation of the growth factors and their receptors may be involved in placental and fetal changes observed in diabetes, i.e. enhanced placental and fetal growth, placental hypervascularization and higher levels of fetal plasma amino acids.
Human umbilical vein and artery endothelial cells (HUVEC; HUAEC), placental endothelial cells (fpAEC), and endothelial colony-forming cells (ECFC) from cord blood are a widely used model for ...researching placental vascular development, fetal and placental endothelial function, and the effect of adverse conditions in pregnancy thereon. However, placental vascular development and angiogenesis start in the first weeks of gestation, and adverse conditions in pregnancy may also affect endothelial function before term, suggesting that endothelial cells from early pregnancy may respond differently. Thus, we established a novel, gentle flow-through method to isolate pure human umbilical endothelial cells from first trimester (FTUEC). FTUEC were characterized and their phenotype was compared to the umbilical endothelium in situ as well as to other fetal endothelial cell models from term of gestation, i.e. HUVEC, fpAEC, ECFC. FTUEC possess a CD34-positive, juvenile endothelial phenotype, and can be expanded and passaged. We regard FTUEC as a valuable tool to study developmental processes as well as the effect of adverse insults in pregnancy in vitro.
The placenta is an endocrine fetal organ, which secretes a plethora of steroid- and proteo-hormones, metabolic proteins, growth factors, and cytokines in order to adapt maternal physiology to ...pregnancy. Central to the growth of the fetus is the supply with nutrients, foremost with glucose. Therefore, during pregnancy, maternal insulin resistance arises, which elevates maternal blood glucose levels, and consequently ensures an adequate glucose supply for the developing fetus. At the same time, maternal β-cell mass and function increase to compensate for the higher insulin demand. These adaptations are also regulated by the endocrine function of the placenta. Excessive insulin resistance or the inability to increase insulin production accordingly disrupts physiological modulation of pregnancy mediated glucose metabolism and may cause maternal gestational diabetes (GDM). A growing body of evidence suggests that this adaptation of maternal glucose metabolism differs between pregnancies carrying a girl vs. pregnancies carrying a boy. Moreover, the risk of developing GDM differs depending on the sex of the fetus. Sex differences in placenta derived hormones and bioactive proteins, which adapt and modulate maternal glucose metabolism, are likely to contribute to this sexual dimorphism. This review provides an overview on the adaptation and maladaptation of maternal glucose metabolism by placenta-derived factors, and highlights sex differences in this regulatory network.
Hofbauer cells (HBCs) are macrophages of the feto-placental unit. Despite the general view that these cells have an anti-inflammatory M2 phenotype, recent studies have claimed that pregnancy ...pathologies-e.g., gestational diabetes mellitus (GDM)-cause a switch from an M2 to an M1 pro-inflammatory phenotype in HBCs. The pilot-study presented here challenges this claim, showing that HBCs maintain anti-inflammatory properties in spite of the hyperglycemic, low-grade inflammatory environment of GDM.
HBCs were isolated from placentae of healthy women (
= 5) and women with GDM (
= 6) diagnosed in the second trimester. FACS was used to measure surface markers associated with either M1 or M2 phenotype on the cells. In addition, placental tissue sections were subjected to immune histochemical imaging to assess the phenotype within the tissue context. Supernatant from control and GDM HBCs was collected at defined time points and used in a multiplex ELISA-on-beads approach to assess secretion of cytokines, chemokines, and growth factors. The effect of HBC cell culture supernatant on placental endothelial activation was investigated.
FACS and immune staining showed that, indeed, M2 markers, such as CD206 and CD209, are increased in HBCs isolated from GDM placentae. Also, the M1 marker CD86 was increased, but only by trend. Secretion of numerous cytokines, chemokines and growth factors was not changed; pro-inflammatory interleukin (IL)-1β and IL-6 release form GDM HBC was increased but not significant. Exposure to GDM HBC supernatant did not induce cell adhesion molecules (VCAM-1, selectins, vascular endothelial-cadherin) in placental endothelial cells compared to supernatant from control HBCs, an induction of intracellular adhesion molecule 1 was observed however.
Our study-although performed in a small set of patients-shows that placental macrophages maintain their anti-inflammatory, tissue remodeling M2 phenotype even in pregnancies affected by gestational diabetes. This consistent phenotype might be important for propagation of maternal tolerance toward the fetus and for protection of the fetus from a low-grade inflammatory environment.
IL-33, the most recently discovered member of the IL-1 superfamily and ligand for the transmembrane form of ST2 (ST2L), has been linked to several human pathologies including rheumatoid arthritis, ...asthma, and cardiovascular disease. Deregulated levels of soluble ST2, the natural IL-33 inhibitor, have been reported in sera of preeclamptic patients. However, the role of IL-33 during healthy pregnancy remains elusive. In the current study, IL-33 was detected in the culture supernatants of human placental and decidual macrophages, identifying them as a major source of secreted IL-33 in the uteroplacental unit. Because flow cytometry and immunofluorescence stainings revealed membranous ST2L expression on specific trophoblast populations, we hypothesized that IL-33 stimulates trophoblasts in a paracrine manner. Indeed, BrdU incorporation assays revealed that recombinant human IL-33 significantly increased proliferation of primary trophoblasts as well as of villous cytotrophoblasts and cell column trophoblasts in placental explant cultures. These effects were fully abolished upon addition of soluble ST2. Interestingly, Western blot and immunofluorescence analyses demonstrated that IL-33 activates AKT and ERK1/2 in primary trophoblasts and placental explants. Inhibitors against PI3K (LY294002) and MEK1/2 (UO126) efficiently blocked IL-33-induced proliferation in all model systems used. In summary, with IL-33, we define for the first time, to our knowledge, a macrophage-derived regulator of placental growth during early pregnancy.
Molecular mechanisms underlying sexual dimorphism in mammals, fetal sex influences on intrauterine development, and the sex-biased susceptibility for selected diseases in adulthood are novel areas of ...current research. As importantly, two decades of multifaceted research has established that susceptibility to many adult disorders originates in utero, commonly secondary to the effects of placental dysfunction. We hypothesized that fetal sex influences gene expression and produces functional differences in human placentas. We thus extended previous studies on sexual dimorphism in mammals, which used RNA isolated from whole tissues, to investigate the effects of sex on four cell-phenotypes within a single key tissue, human placental villi. The cells studied included cytotrophoblasts, syncytiotrophoblast, arterial and venous endothelial cells. The cells were isolated from placentas of male or female fetuses and subjected to microarray analysis. We found that fetal sex differentially affected gene expression in a cell-phenotype dependent manner among all four cell-phenotypes. The markedly enriched pathways in males were identified to be signaling pathways for graft-versus-host disease as well as the immune and inflammatory systems that parallel the reported poorer outcome of male fetuses. Our study is the first to compare global gene expression by microarray analysis in purified, characterized, somatic cells from a single human tissue, i.e. placental villi. Importantly, our findings demonstrate that there are cell-phenotype specific, and tissue-specific, sex-biased responses in the human placenta, suggesting fetal sex should be considered as an independent variable in gene expression analysis of human placental villi.
Type 1 diabetes mellitus (T1DM) is associated with reduced fetal growth in early pregnancy, but a contributing role of the placenta has remained elusive. Thus, we investigated whether T1DM alters ...placental development in the first trimester. Using a protein array, the level of 60 cell-cycle-related proteins was determined in human first trimester placental tissue (gestational week 5-11) from control (
= 11) and T1DM pregnancies (
= 12). Primary trophoblasts (gestational week 7-12,
= 32) were incubated in the absence (control) or presence of hyperglycemia (25 mM D-glucose) and hyperosmolarity (5.5 mM D-glucose + 19.5 mM D-mannitol). We quantified the number of viable and dead trophoblasts (CASY Counter) and assessed cell cycle distribution (FACS) and trophoblast invasion using a transwell assay. T1DM was associated with a significant (
< 0.05) downregulation of Ki67 (-26%), chk1 (-25%), and p73 (-26%). The number of viable trophoblasts was reduced under hyperglycemia (-23%) and hyperosmolarity (-18%), whereas trophoblast invasion was increased only under hyperglycemia (+6%). Trophoblast cell death and cell cycle distribution remained unaffected. Collectively, our data demonstrate that hyperglycemia decreases trophoblast proliferation as a potential contributing factor to the reduced placental growth in T1DM in vivo.
The placental barrier is crucial for the supply of nutrients and oxygen to the developing fetus and is maintained by differentiation and fusion of mononucleated cytotrophoblasts into the ...syncytiotrophoblast, a process only partially understood. Here transcriptome and pathway analyses during differentiation and fusion of cultured trophoblasts yielded p53 signaling as negative upstream regulator and indicated an upregulation of autophagy-related genes. We further showed p53 mRNA and protein levels decreased during trophoblast differentiation. Reciprocally, autophagic flux increased and cytoplasmic LC3B-GFP puncta became more abundant, indicating enhanced autophagic activity. In line, in human first trimester placenta p53 protein mainly localized to the cytotrophoblast, while autophagy marker LC3B as well as late autophagic compartments were predominantly detectable in the syncytiotrophoblast. Importantly, ectopic overexpression of p53 reduced levels of LC3B-II, supporting a negative regulatory role on autophagy in differentiating trophoblasts. This was also shown in primary trophoblasts and human first trimester placental explants, where pharmacological stabilization of p53 decreased LC3B-II levels. In summary our data suggest that differentiation-dependent downregulation of p53 is a prerequisite for activating autophagy in the syncytiotrophoblast.
STUDY QUESTION
Does endothelin-1 (ET-1) regulate matrix metalloproteinase (MMP) 14 and 15 production and invasion of human first trimester trophoblasts?
SUMMARY ANSWER
ET-1 in pathophysiological ...concentrations down-regulates MMP14 and MMP15 expression via endothelin receptor (ETR) type B and decreases trophoblast migration and invasion.
WHAT IS KNOWN ALREADY
MMP14 and MMP15 are involved in trophoblast invasion. Impairment of invasion has been linked to pregnancy complications such as pre-eclampsia (PE). ET-1 is up-regulated in PE.
STUDY DESIGN, SIZE, DURATION
In vitro study using primary human trophoblasts from 50 first trimester placentas (gestational week 7–12).
PARTICIPANTS/MATERIALS, SETTING, METHODS
Trophoblasts were cultured in the absence or presence of 10–100 nM ET-1. MMP14 and MMP15 mRNA and protein were quantified by RT-qPCR and Western blotting, respectively. Selective antagonists for ETRA (BQ-123) or ETRB (BQ-788) were used to identify ETR subtypes involved. Functional ET-1 effects were tested in first trimester chorionic villous explants and transwell invasion assays. The roles of tumor necrosis factor (TNF)-α (25 ng/ml) and oxygen (1%) in ET-1 regulation of MMP14 and 15 expression were assessed by Western blotting.
MAIN RESULTS AND THE ROLE OF CHANCE
ET-1 down-regulated MMP14 and MMP15 mRNA (−21% and −26%, respectively, P < 0.05) and protein levels (–18% and –22%, respectively, P < 0.05). This effect was mediated via ETRB. ET-1 decreased trophoblast outgrowth in placental explants (−24%, P < 0.05) and trophoblast invasion (−26%, P ≤ 0.01). TNF-α enhanced ET-1 mediated MMP15 down-regulation (by 10%, P < 0.05), whereas hypoxia abolished the effect of ET-1 on both MMPs.
LARGE SCALE DATA
N/A.
LIMITATIONS, REASONS FOR CAUTION
Only primary trophoblasts were used in this study. Since trophoblast yield from first trimester placental material is limited, further aspects of MMP14 and 15 regulation could not be characterized. Other anti-invasive factors may be altered by ET-1 in trophoblasts and, thus, contribute to the reduced invasion, but have not been investigated. Oxygen levels similar to those found in the decidua (5–8% O2) were not analyzed in this study.
WIDER IMPLICATIONS OF THE FINDINGS
ET-1 modifies placental function already during the first trimester of pregnancy, the time-window when the placental changes implicated in PE occur. Thus, our results improve the understanding of the placental mechanisms underlying trophoblast invasion and PE.
STUDY FUNDING/COMPETING INTEREST(S)
The study was funded by the Oesterreichische Nationalbank (Anniversary Fund, project number: 14796) and the Herzfelder'sche Familienstiftung (to J.P.; number: 00685). AMM received funding from the Austrian Science Fund FWF (W1241) and the Medical University Graz through the PhD Program Molecular Fundamentals of Inflammation (DK-MOLIN). The authors have no conflict of interest.
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