Dissecting Rubella Placental Infection in an In Vitro Trophoblast Model.

Vertical transmission of rubella virus (RuV) occurs at a high rate during the first trimester of pregnancy. The modes of vertical transmission including the response of trophoblasts to RuV are not well understood. Here, RuV-trophoblast interaction was studied in the BeWo trophoblast cell line. Analysis included early and late time-point kinetics of virus infection rate and the antiviral innate immune response at mRNA and protein level. BeWo characteristics were addressed through metabolic activity by extracellular flux analysis and syncytiotrophoblast formation through incubation with forskolin. We found that RuV infection of BeWo led to profuse type III interferon (IFN) production. Transfecting trophoblast cells with dsRNA analog induced an increase in the production of type I IFN-ß and type III IFNs; however, this did not occur in RuV-infected BeWo trophoblasts. IFN-ß and to a lesser extent type III IFN-λ1 were inhibitory to RuV. While no significant metabolic alteration was detected, RuV infection reduced the cell number in the monolayer culture in comparison to the mock control and resulted in detached and floating cells. Syncytia formation restricted RuV infection. The use of BeWo as a relevant cell culture model for infection of trophoblasts highlights cytopathogenicity in the absence of a type I IFN response as a pathogenic alteration by RuV.

Preeclampsia-induced alterations in brain and liver gene expression and DNA methylation patterns in fetal mice.

Exposure to pregnancy complications, including preeclampsia (PE), has lifelong influences on offspring's health. We have previously reported that experimental PE, induced in mice by administration of adenoviral sFlt1 at gestational day 8.5 combined with LPS at day 10.5, results in symmetrical growth restriction in female and asymmetrical growth restriction in male offspring. Here, we characterize the molecular phenotype of the fetal brain and liver with respect to gene transcription and DNA methylation at the end of gestation.In fetal brain and liver, expression and DNA methylation of several key regulatory genes is altered by PE exposure, mostly independent of fetal sex. These alterations point toward a decreased gluconeogenesis in the liver and stimulated neurogenesis in the brain, potentially affecting long-term brain and liver function. The observed sex-specific growth restriction pattern is not reflected in the molecular data, showing that PE, rather than tissue growth, drives the molecular phenotype of PE-exposed offspring.

An old friend with a new face: YB-1 and its role in healthy pregnancy and pregnancy-associated complications.

By promoting tissue invasion, cell growth and angiogenesis, the Y-box binding protein (YB-1) became famous as multifunctional oncoprotein. However, this designation is telling only part of the story. There is one particular time in life when actual tumorigenic-like processes become undoubtedly welcome, namely pregnancy. It seems therefore reasonable that YB-1 plays also a crucial role in reproduction, and yet this biological aspect of the cold-shock protein has been overlooked for many years. To overcome this limitation, we would like to propose a new perspective on YB-1 and emphasize its pivotal functions in healthy pregnancy and pregnancy-related complications. Moreover, we will discuss findings obtained from cancer research in the light of reproductive events to elucidate the importance of YB-1 at the feto-maternal interface.

Characterization of Three-Dimensional Trophoblast Spheroids: An Alternative Model to Study the Physiological Properties of the Placental Unit.

It was postulated that 3D cell culture models more accurately reflect the complex tissue physiology and morphology in comparison to 2D cell monolayers. Currently, there is a shortage of well-characterized and easily maintainable high-throughput experimental models of the human placenta. Here, we characterized three different 3D cultures (e.g., spheroids) derived from trophoblast cell lines and studied their functionality in comparison to primary fetal trophoblasts and placental tissue. The spheroid growth rates of JEG3, BeWo and HTR8/SVneo cell lines were similar among each other and were significantly larger in comparison to primary trophoblast spheroids. All spheroids exhibited migratory properties and shortest distances were registered for JEG3 spheroids. Even though all spheroids displayed invasive capabilities, only the invasive features of HTR8/SVneo spheroids resulted in specific branching. This was in agreement with the invasive properties of the spheroids obtained from primary trophoblasts. Human chorionic gonadotropin production was highest in JEG3 spheroids and only increased when stimulated with cAMP and forskolin in BeWo, but not HTR8/SVneo spheroids. The gene expression analysis confirmed that 3D trophoblast cell cultures and especially HTR8/SVneo spheroids showed considerable similarities with the gene expression profile of primary placental tissue. This study offers a broad characterization of 3D trophoblast spheroids that, in turn, can help in selecting the best model depending on the scientific question that needs to be answered.

YB-1 Is Altered in Pregnancy-Associated Disorders and Affects Trophoblast in Vitro Properties via Alternation of Multiple Molecular Traits.

Cold shock Y-box binding protein-1 (YB-1) coordinates several molecular processes between the nucleus and the cytoplasm and plays a crucial role in cell function. Moreover, it is involved in cancer progression, invasion, and metastasis. As trophoblast cells share similar characteristics with cancer cells, we hypothesized that YB-1 might also be necessary for trophoblast functionality. In samples of patients with intrauterine growth restriction, YB-1 mRNA levels were decreased, while they were increased in preeclampsia and unchanged in spontaneous abortions when compared to normal pregnant controls. Studies with overexpression and downregulation of YB-1 were performed to assess the key trophoblast processes in two trophoblast cell lines HTR8/SVneo and JEG3. Overexpression of YB-1 or exposure of trophoblast cells to recombinant YB-1 caused enhanced proliferation, while knockdown of YB-1 lead to proliferative disadvantage in JEG3 or HTR8/SVneo cells. The invasion and migration properties were affected at different degrees among the trophoblast cell lines. Trophoblast expression of genes mediating migration, invasion, apoptosis, and inflammation was altered upon YB-1 downregulation. Moreover, IL-6 secretion was excessively increased in HTR8/SVneo. Ultimately, YB-1 directly binds to NF-κB enhancer mark in HTR8/SVneo cells. Our data show that YB-1 protein is important for trophoblast cell functioning and, when downregulated, leads to trophoblast disadvantage that at least in part is mediated by NF-κB.

Mid-gestation low-dose LPS administration results in female-specific excessive weight gain upon a western style diet in mouse offspring.

Gestational complications, including preeclampsia and gestational diabetes, have long-term adverse consequences for offspring's metabolic and cardiovascular health. A low-grade systemic inflammatory response is likely mediating this. Here, we examine the consequences of LPS-induced gestational inflammation on offspring's health in adulthood. LPS was administered to pregnant C57Bl/6J mice on gestational day 10.5. Maternal plasma metabolomics showed oxidative stress, remaining for at least 5 days after LPS administration, likely mediating the consequences for the offspring. From weaning on, all offspring was fed a control diet; from 12 to 24 weeks of age, half of the offspring received a western-style diet (WSD). The combination of LPS-exposure and WSD resulted in hyperphagia and increased body weight and body fat mass in the female offspring. This was accompanied by changes in glucose tolerance, leptin and insulin levels and gene expression in liver and adipose tissue. In the hypothalamus, expression of genes involved in food intake regulation was slightly changed. We speculate that altered food intake behaviour is a result of dysregulation of hypothalamic signalling. Our results add to understanding of how maternal inflammation can mediate long-term health consequences for the offspring. This is relevant to many gestational complications with a pro-inflammatory reaction in place.

Innate and Adaptive Immune Responses in HELLP Syndrome.

Innate and adaptive immune involvement in hemolysis, elevated liver enzymes and low platelet (HELLP) syndrome is an understudied field, although it is of high clinical importance. This syndrome implies a risk of serious morbidity and mortality to both the mother and the fetus during pregnancy. It was proposed that HELLP syndrome occurs in a circulatory inflammatory milieu, that might in turn participate in a complex interplay between the secreted inflammatory immunomodulators and immune cell surface receptors. Meanwhile, reported immune cell attenuation during HELLP may consequently lead to a prolonged immunoactivation and tissue damage. In this regard, learning more about the immune components of this syndrome should widen the understanding of the HELLP pathophysiology and eventually enable development of novel immune-based therapeutics. This review aims to summarize and discuss the recent and previous findings of the innate and adaptive immune responses during HELLP in order to update the current knowledge of the immune involvement in HELLP pathogenesis.

Human sFLT1 Leads to Severe Changes in Placental Differentiation and Vascularization in a Transgenic hsFLT1/rtTA FGR Mouse Model.

The anti-angiogenic soluble fms-like tyrosine kinase 1 (sFLT1) is one of the candidates in the progression of preeclampsia, often associated with fetal growth restriction (FGR). Therapeutic agents against preeclampsia with/without FGR, as well as adequate transgenic sFLT1 mouse models for testing such agents, are still missing. Much is known about sFLT1-mediated endothelial dysfunction in several tissues; however, the influence of sFLT1 on placental and fetal development is currently unknown. We hypothesize that sFLT1 is involved in the progression of FGR by influencing placental differentiation and vascularization and is a prime candidate for interventional strategies. Therefore, we generated transgenic inducible human sFLT1/reverse tetracycline-controlled transactivator (hsFLT1/rtTA) mice, in which hsFLT1 is ubiquitously overexpressed during pregnancy in dams and according to the genetics in hsFLT1/rtTA homozygous and heterozygous fetuses. Induction of hsFLT1 led to elevated hsFLT1 levels in the serum of dams and on mRNA level in all placentas and hetero-/homozygous fetuses, resulting in FGR in all fetuses at term. The strongest effects in respect to FGR were observed in the hsFLT1/rtTA homozygous fetuses, which exhibited the highest hsFLT1 levels. Only fetal hsFLT1 expression led to impaired placental morphology characterized by reduced placental efficiency, enlarged maternal sinusoids, reduced fetal capillaries, and impaired labyrinthine differentiation, associated with increased apoptosis. Besides impaired placental vascularization, the expression of several transporter systems, such as glucose transporter 1 and 3 (Glut-1; Glut-3); amino acid transporters, solute carrier family 38, member one and two (Slc38a1; Slc38a2); and most severely the fatty acid translocase Cd36 and fatty acid binding protein 3 (Fabp3) was reduced upon hsFLT1 expression, associated with an accumulation of phospholipids in the maternal serum. Moreover, the Vegf pathway showed alterations, resulting in reduced Vegf, Vegfb, and Plgf protein levels and increased Bad and Caspase 9 mRNA levels. We suggest that hsFLT1 exerts an inhibitory influence on placental vascularization by reducing Vegf signaling, which leads to apoptosis in fetal vessels, impairing placental differentiation, and the nutrient exchange function of the labyrinth. These effects were more pronounced when both the dam and the fetus expressed hsFLT1 and ultimately result in FGR and resemble the preeclamptic phenotype in humans.

A double-hit pre-eclampsia model results in sex-specific growth restriction patterns.

Pre-eclampsia is a multifactorial pregnancy-associated disorder characterized by angiogenic dysbalance and systemic inflammation; however, animal models that combine these two pathophysiological conditions are missing. Here, we introduce a novel double-hit pre-eclampsia mouse model that mimics the complex multifactorial conditions present during pre-eclampsia and allows for the investigation of early consequences for the fetus. Adenoviral overexpression of soluble fms-like tyrosine kinase (sFlt-1) and lipopolysaccharide (LPS) administration at mid-gestation in pregnant mice resulted in hypertension and albuminuria comparable to that of the manifestation in humans. A metabolomics analysis revealed that pre-eclamptic dams have increased plasma concentrations of phosphadytilcholines. The fetuses of both sexes were growth restricted; however, in males a brain-sparing effect was seen as compensation for this growth restriction. According to the plasma metabolomics, male fetuses showed changes in amino acid metabolism, while female fetuses showed pronounced alterations in lipid metabolism. Our results show that combined exposure to sFlt-1 and LPS mimics the clinical symptoms of pre-eclampsia and affects fetal growth in a sex-specific manner, with accompanying metabolome changes.

Placental insufficiency contributes to fatty acid metabolism alterations in aged female mouse offspring.

Intrauterine growth restriction (IUGR) is an accepted risk factor for metabolic disorders in later life, including obesity and type 2 diabetes. The level of metabolic dysregulation can vary between subjects and is dependent on the severity and the type of IUGR insult. Classical IUGR animal models involve nutritional deprivation of the mother or uterine artery ligation. The latter aims to mimic a placental insufficiency, which is the most frequent cause of IUGR. In this study, we investigated whether IUGR attributable to placental insufficiency impacts the glucose and lipid homeostasis at advanced age. Placental insufficiency was achieved by deletion of the transcription factor AP-2y ( Tfap2c), which serves as one of the major trophoblast differentiation regulators. TdelT-IUGR mice were obtained by crossing mice with a floxed Tfap2c allele and mice with Cre recombinase under the control of the Tpbpa promoter. In advanced adulthood (9-12 mo), female and male IUGR mice are respectively 20% and 12% leaner compared with controls. At this age, IUGR mice have unaffected glucose clearance and lipid parameters (cholesterol, triglycerides, and phospholipids) in the liver. However, female IUGR mice have increased plasma free fatty acids (+87%) compared with controls. This is accompanied by increased mRNA levels of fatty acid synthase and endoplasmic reticulum stress markers in white adipose tissue. Taken together, our results suggest that IUGR by placental insufficiency may lead to higher lipogenesis in female mice in advanced adulthood, at least indicated by greater Fasn expression. This effect was sex specific for the aged IUGR females.

Diabetes Mellitus in Pregnancy Leads to Growth Restriction and Epigenetic Modification of the Srebf2 Gene in Rat Fetuses.

Diabetic pregnancy is correlated with increased risk of metabolic and neurological disorders in the offspring putatively mediated epigenetically. Little is known about epigenetic changes already present in fetuses of diabetic pregnancies. We aimed at characterizing the perinatal environment after preexisting maternal diabetes mellitus and at identifying relevant epigenetic changes in the fetus. We focused on the transcription factor Srebf2 (sterol regulatory element binding transcription factor 2), a master gene in regulation of cholesterol metabolism. We tested whether diabetic pregnancy induces epigenetic changes in the Srebf2 promoter and if they become manifest in altered Srebf2 gene expression. We worked with a transgenic rat model of type 2 diabetes mellitus (Tet29) in which the insulin receptor is knocked down by doxycycline-induced RNA interference. Doxycycline was administered preconceptionally to Tet29 and wild-type control rats. Only Tet29 doxycycline dams were hyperglycemic, hyperinsulinemic, and hyperlipidemic. Gene expression was analyzed with quantitative real-time reverse transcriptase polymerase chain reaction and CpG promoter methylation with pyrosequencing. Immunohistochemistry was performed on fetal brains. Fetuses from diabetic Tet29 dams were hyperglycemic and growth restricted at the end of pregnancy. They further displayed decreased liver and brain weight with concomitant decreased microglial activation in the hippocampus in comparison to fetuses of normoglycemic mothers. Importantly, diabetic pregnancy induced CpG hypermethylation of the Srebf2 promoter in the fetal liver and brain, which was associated with decreased Srebf2 gene expression. In conclusion, diabetic and hyperlipidemic pregnancy induces neurological, metabolic, and epigenetic alterations in the rat fetus. Srebf2 is a potential candidate mediating intrauterine environment-driven epigenetic changes and later diabetic offspring health.

Pregnancy-Related Immune Adaptation Promotes the Emergence of Highly Virulent H1N1 Influenza Virus Strains in Allogenically Pregnant Mice.

Pregnant women are at high risk for severe influenza disease outcomes, yet insights into the underlying mechanisms are limited. Here, we present models of H1N1 infection in syngenic and allogenic pregnant mice; infection in the latter mirrors the severe course of 2009 pandemic influenza in pregnant women. We found that the anti-viral immune response in the pregnant host was significantly restricted as compared to the non-pregnant host. This included a reduced type I interferon response as well as impaired migration of CD8+ T cells into the lung. The multi-faceted failure to mount an anti-viral response in allogenic pregnant mice resulted in a less stringent selective environment that promoted the emergence of 2009 H1N1 virus variants that specifically counteract type I interferon response and mediate increased viral pathogenicity. These insights underscore the importance of influenza vaccination compliance in pregnant women and may open novel therapeutic avenues.

Placental-Specific Overexpression of sFlt-1 Alters Trophoblast Differentiation and Nutrient Transporter Expression in an IUGR Mouse Model.

Since it is known that placental overexpression of the human anti-angiogenic molecule sFlt-1, the main candidate in the progression of preeclampsia, lead to intrauterine growth restriction (IUGR) in mice by lentiviral transduction of mouse blastocysts, we hypothesize that sFlt-1 influence placental morphology and physiology resulting in fetal IUGR. We therefore examined the effect of sFlt-1 on placental morphology and physiology at embryonic day 18.5 with histologic and morphometric analyses, transcript analyses, immunoblotting, and methylation studies. Interestingly, placental overexpression of sFlt-1 leads to IUGR in the fetus and results in lower placental weights. Moreover, we observed altered trophoblast differentiation with reduced expression of IGF2, resulting in a smaller placenta, a smaller labyrinth, and the loss of glycogen cells in the junctional zone. Changes in IGF2 are accompanied by small changes in its DNA methylation, whereas overall DNA methylation is unaffected. In addition, the expression of placental nutrient transporters, such as the glucose diffusion channel Cx26, is decreased. In contrast, the expression of the fatty acid transporter CD36 and the cholesterol transporter ABCA1 is significantly increased. In conclusion, placental sFlt-1 overexpression resulted in a reduction in the differentiation of the spongiotrophoblast into glycogen cells. These findings of a reduced exchange area of the labyrinth and glycogen stores, as well as decreased expression of glucose transporter, could contribute to the intrauterine growth restriction phenotype. All of these factors change the intrauterine availability of nutrients. Thus, we speculate that the alterations triggered by increased anti-angiogenesis strongly affect fetal outcome and programming. J. Cell. Biochem. 118: 1316-1329, 2017. © 2016 Wiley Periodicals, Inc.

Preeclampsia As Modulator of Offspring Health.

A balanced intrauterine homeostasis during pregnancy is crucial for optimal growth and development of the fetus. The intrauterine environment is extremely vulnerable to multisystem pregnancy disorders such as preeclampsia, which can be triggered by various pathophysiological factors, such as angiogenic imbalance, immune responses, and inflammation. The fetus adapts to these conditions by a mechanism known as developmental programming that can lead to increased risk of chronic noncommunicable diseases in later life. This is shown in a substantial number of epidemiological studies that associate preeclampsia with increased onset of cardiovascular and metabolic diseases in the later life of the offspring. Furthermore, animal models based predominantly on one of the pathophysiological mechanism of preeclampsia, for example, angiogenic imbalance, immune response, or inflammation, do address the susceptibility of the preeclamptic offspring to increased maternal blood pressure and disrupted metabolic homeostasis. Accordingly, we extensively reviewed the latest research on the role of preeclampsia on the offspring's metabolism and cardiovascular phenotype. We conclude that future research on the pathophysiological changes during preeclampsia and methods to intervene in the harsh intrauterine environment will be essential for effective therapies.