Rosiglitazone-Mediated Activation of PPARγ Restores HO1 Expression in the Human Preeclamptic Placenta.

BACKGROUND: Preeclampsia is a hypertensive disorder of pregnancy characterized by chronic placental ischemia and suppression of proangiogenic proteins, causing oxidative stress, hypertension, and maternal systemic organ damage. The transcription factor, PPARγ (peroxisome proliferator-activated receptor-γ) promotes healthy trophoblast differentiation but is dysregulated in the preeclampsia placenta. Our study identifies the beneficial impact of Rosiglitazone-mediated PPARγ-activation in the stressed preeclampsia placenta. METHODS: We used first trimester placentas, preeclamptic and preterm control placentas, and human trophoblast cell lines to study PPARγ activation. RESULTS: Induction of PPARγ activates cell growth and antioxidative stress pathways, including the gene, heme oxygenase 1 (Hmox1). Protein expression of both PPARγ and HO1 (heme oxygenase 1) are reduced in preeclamptic placentas, but Rosiglitazone restores HO1 signaling in a PPARγ-dependent manner. CONCLUSIONS: Restoring disrupted pathways by PPARγ in preeclampsia offers a potential therapeutic pathway to reverse placental damage, extending pregnancy duration, and reduce maternal sequelae. Future research should aim to understand the full scope of impaired PPARγ signaling in the human placenta and focus on compounds for safe use during pregnancy to prevent severe perinatal morbidity and mortality.

The Potential for Placental Activation of PPARγ to Improve the Angiogenic Profile in Preeclampsia.

Preeclampsia (PE) is one of the most common causes of maternal-fetal morbidity and mortality world-wide. While the underlying causes of PE remain elusive, aberrant trophoblast differentiation and function are thought to cause an imbalance of secreted angiogenic proteins resulting in systemic endothelial dysfunction and organ damage in the mother. The placental dysfunction is also characterized by a reduction of the transcription factor, peroxisome proliferator activated receptor γ (PPARγ) which normally promotes trophoblast differentiation and healthy placental function. This study aimed to understand how placental activation of PPARγ effects the secretion of angiogenic proteins and subsequently endothelial function. To study this, healthy and PE placental tissues were cultured with or without the PPARγ agonist, Rosiglitazone, and a Luminex assay was performed to measure secreted proteins from the placenta. To assess the angiogenic effects of placental activation of PPARγ, human umbilical vein endothelial cells (HUVECs) were cultured with the placental conditioned media and the net angiogenic potential of these cells was measured by a tube formation assay. This is the first study to show PPARγ's beneficial effect on the angiogenic profile in the human preeclamptic placenta through the reduction of anti-angiogenic angiopoietin-2 and soluble endoglin and the upregulation of pro-angiogenic placental growth factor, fibroblast growth factor-2, heparin-binding epidermal growth factor, and follistatin. The changes in the angiogenic profile were supported by the increased angiogenic potential observed in the HUVECs when cultured with conditioned media from rosiglitazone-treated preeclamptic placentas. The restoration of these disrupted pathways by activation of PPARγ in the preeclamptic placenta offers potential to improve placental and endothelial function in PE.

Irisin Protects the Human Placenta from Oxidative Stress and Apoptosis via Activation of the Akt Signaling Pathway.

Irisin is a newly discovered exercise-mediated polypeptide hormone. Irisin levels increase during pregnancy however, women with preeclampsia (PE) have significantly lower levels of Irisin compared to women of healthy pregnancies. Even though many studies suggest a role of Irisin in pregnancy, its function in the human placenta is unclear. In the current study, we aimed to understand key roles of Irisin through its ability to protect against apoptosis is the preeclamptic placenta and in ex vivo and in vitro models of hypoxia/re-oxygenation (H/R) injury. Our studies show that Irisin prevents cell death by reducing pro-apoptotic signaling cascades, reducing cleavage of PARP to induce DNA repair pathways and reducing activity of Caspase 3. Irisin caused an increase in the levels of anti-apoptotic BCL2 to pro-apoptotic BAX and reduced ROS levels in an in vitro model of placental ischemia. Furthermore, we show that Irisin treatment acts through the Akt signaling pathway to prevent apoptosis and enhance cell survival. Our findings provide a novel understanding for the anti-apoptotic and pro-survival properties of Irisin in the human placenta under pathological conditions. This work yields new insights into placental development and disease and points towards intervention strategies for placental insufficiencies, such as PE, by protecting and maintaining placental function through inhibiting hypoxic ischemia-induced apoptosis.

Induction of the PPARγ (Peroxisome Proliferator-Activated Receptor γ)-GCM1 (Glial Cell Missing 1) Syncytialization Axis Reduces sFLT1 (Soluble fms-Like Tyrosine Kinase 1) in the Preeclamptic Placenta.

[Figure: see text].

Placental Regulation of Energy Homeostasis During Human Pregnancy.

Successful pregnancies rely on sufficient energy and nutrient supply, which require the mother to metabolically adapt to support fetal needs. The placenta has a critical role in this process, as this specialized organ produces hormones and peptides that regulate fetal and maternal metabolism. The ability for the mother to metabolically adapt to support the fetus depends on maternal prepregnancy health. Two-thirds of pregnancies in the United States involve obese or overweight women at the time of conception. This poses significant risks for the infant and mother by disrupting metabolic changes that would normally occur during pregnancy. Despite well characterized functions of placental hormones, there is scarce knowledge surrounding placental endocrine regulation of maternal metabolic trends in pathological pregnancies. In this review, we discuss current efforts to close this gap of knowledge and highlight areas where more research is needed. As the intrauterine environment predetermines the health and wellbeing of the offspring in later life, adequate metabolic control is essential for a successful pregnancy outcome. Understanding how placental hormones contribute to aberrant metabolic adaptations in pathological pregnancies may unveil disease mechanisms and provide methods for better identification and treatment. Studies discussed in this review were identified through PubMed searches between the years of 1966 to the present. We investigated studies of normal pregnancy and metabolic disorders in pregnancy that focused on energy requirements during pregnancy, endocrine regulation of glucose metabolism and insulin resistance, cholesterol and lipid metabolism, and placental hormone regulation.

The Role of NFκB in Healthy and Preeclamptic Placenta: Trophoblasts in the Spotlight.

The NFκB protein family regulates numerous pathways within the cell-including inflammation, hypoxia, angiogenesis and oxidative stress-all of which are implicated in placental development. The placenta is a critical organ that develops during pregnancy that primarily functions to supply and transport the nutrients required for fetal growth and development. Abnormal placental development can be observed in numerous disorders during pregnancy, including fetal growth restriction, miscarriage, and preeclampsia (PE). NFκB is highly expressed in the placentas of women with PE, however its contributions to the syndrome are not fully understood. In this review we discuss the molecular actions and related pathways of NFκB in the placenta and highlight areas of research that need attention.

Irisin induces trophoblast differentiation via AMPK activation in the human placenta.

Irisin, an adipokine, regulates differentiation and phenotype in various cell types including myocytes, adipocytes, and osteoblasts. Circulating irisin concentration increases throughout human pregnancy. In pregnancy disorders such as preeclampsia and gestational diabetes mellitus, circulating irisin levels are reduced compared to healthy controls. To date, there are no data on the role and molecular function of irisin in the human placenta or its contribution to pathophysiology. Aberrant trophoblast differentiation is involved in the pathophysiology of preeclampsia. The current study aimed to assess the molecular effects of irisin on trophoblast differentiation and function. First-trimester placental explants were cultured and treated with low (10 nM) and high (50 nM) physiological doses of irisin. Treatment with irisin dose-dependently increased both in vitro placental outgrowth (on Matrigel™) and trophoblast cell-cell fusion. Adenosine monophosphate-activated protein kinase (AMPK) signaling, an important regulator of cellular energy homeostasis that is involved in trophoblast differentiation and pathology, was subsequently investigated. Here, irisin exposure induced placental AMPK activation. To determine the effects of irisin on trophoblast differentiation, two trophoblast-like cell lines, HTR-8/SVneo and BeWo, were treated with irisin and/or a specific AMPK inhibitor (Compound C). Irisin-induced AMPK phosphorylation in HTR-8/SVneo cells. Additionally, as part of the differentiation process, integrin switching from α6 to α1 occurred as well as increased invasiveness. Overall, irisin promoted differentiation in villous and extravillous cell-based models via AMPK pathway activation. These findings provide evidence that exposure to irisin promotes differentiation and improves trophoblast functions in the human placenta that are affected in abnormal placentation.

Endocervical trophoblast for interrogating the fetal genome and assessing pregnancy health at five weeks.

Prenatal testing for fetal genetic traits and risk of obstetrical complications is essential for maternal-fetal healthcare. The migration of extravillous trophoblast (EVT) cells from the placenta into the reproductive tract and accumulation in the cervix offers an exciting avenue for prenatal testing and monitoring placental function. These cells are obtained with a cervical cytobrush, a routine relatively safe clinical procedure during pregnancy, according to published studies and our own observations. Trophoblast retrieval and isolation from the cervix (TRIC) obtains hundreds of fetal cells with >90% purity as early as five weeks of gestation. TRIC can provide DNA for fetal genotyping by targeted next-generation sequencing with single-nucleotide resolution. Previously, we found that known protein biomarkers are dysregulated in EVT cells obtained by TRIC in the first trimester from women who miscarry or later develop intrauterine growth restriction or preeclampsia. We have now optimized methods to stabilize RNA during TRIC for subsequent isolation and analysis of trophoblast gene expression. Here, we report transcriptomics analysis demonstrating that the expression profile of TRIC-isolated trophoblast cells was distinct from that of maternal cervical cells and included genes associated with the EVT phenotype and invasion. Because EVT cells are responsible for remodeling the maternal arteries and their failure is associated with pregnancy disorders, their molecular profiles could reflect maternal risk, as well as mechanisms underlying these disorders. The use of TRIC to analyze EVT genomes, transcriptomes and proteomes during ongoing pregnancies could provide new tools for anticipating and managing both fetal genetic and maternal obstetric disorders.

Rosiglitazone blocks first trimester in-vitro placental injury caused by NF-κB-mediated inflammation.

Increased inflammation and abnormal placentation are common features of a wide spectrum of pregnancy-related disorders such as intra uterine growth restriction, preeclampsia and preterm birth. The inflammatory response of the human placenta has been mostly investigated in relation to cytokine release, but the direct molecular consequences on trophoblast differentiation have not been investigated. This study measured the general effects of LPS on both extravillous and villous trophoblast physiology, and the involvement of the transcription factors PPARγ and NF-κB, specifically using 1st trimester explants and HTR-8/ SVneo cell line models. While both proteins are known for their roles in inflammatory pathways, PPARγ has been identified as an important molecule in trophoblast differentiation, suggesting its potential role in mediating a crosstalk between inflammation and trophoblast differentiation. Here, LPS (1 µg/ml) exposure of first trimester placental villous explants resulted in secretion of inflammatory cytokines, induction of apoptosis and reduction in trophoblast cell proliferation. Additionally, LPS significantly reduced expression of the trophoblast differentiation proteins GCM1 and ß-hCG, and increased invasion of the extravillous trophoblast. Activation of PPARγ by Rosiglitazone (10 µM) reversed the LPS-mediated effects on inflammatory cytokine release, trophoblast apoptosis and proliferation compared to controls. Lastly, markers of trophoblast differentiation and invasion reverted to control levels upon activation of PPARγ and concomitant inhibition of NF-κB (either by Rosiglitazone or NF-κB specific inhibitor), revealing a new role for NF-κB in trophoblast invasion. This study reveals a novel PPARγ - NF-κB axis that coordinates inflammatory and differentiation pathways in the human placenta. The ability to reverse trophoblast-associated inflammation with Rosiglitazone offers promise that the PPARγ - NF-κB pathway could one day provide a therapeutic target for placental dysfunction associated with both inflammation and abnormal trophoblast differentiation.

Stress Forces First Lineage Differentiation of Mouse Embryonic Stem Cells; Validation of a High-Throughput Screen for Toxicant Stress.

Mouse Embryonic Stem Cells (mESCs) are unique in their self-renewal and pluripotency. Hypothetically, mESCs model gestational stress effects or stresses of in vitro fertilization/assisted reproductive technologies or drug/environmental exposures that endanger embryos. Testing mESCs stress responses should diminish and expedite in vivo embryo screening. Transgenic mESCs for green fluorescent protein (GFP) reporters of differentiation use the promoter for platelet-derived growth factor receptor (Pdgfr)a driving GFP expression to monitor hyperosmotic stress-forced mESC proliferation decrease (stunting), and differentiation increase that further stunts mESC population growth. In differentiating mESCs Pdgfra marks the first-lineage extraembryonic primitive endoderm (ExEndo). Hyperosmotic stress forces mESC differentiation gain (Pdgfra-GFP) in monolayer or three-dimensional embryoid bodies. Despite culture with potency-maintaining leukemia inhibitory factor (LIF), stress forces ExEndo as assayed using microplate readers and validated by coexpression of Pdgfra-GFP, Disabled 2 (Dab2), and laminin by immunofluorescence and GFP protein and Dab2 by immunoblot. In agreement with previous reports, Rex1 and Oct4 loss was inversely proportional to increased Pdgfra-GFP mESC after treatment with high hyperosmotic sorbitol despite LIF. The increase in subpopulations of Pdgfra-GFP+ cells>background at ∼23% was similar to the previously reported ∼25% increase in Rex1-red fluorescent protein (RFP)-negative subpopulation at matched high sorbitol doses. By microplate reader, there is a ∼7-11-fold increase in GFP at a high nonmorbid and a morbid dose despite LIF, compared with LIF alone. By flow cytometry (FACS), the subpopulation of Pdgfra-GFP+ cells>background increases ∼8-16-fold at these doses. Taken together, the microplate, FACS, immunoblot, and immunofluorescence data suggest that retinoic acid or hyperosmotic stress forces dose-dependent differentiation whether LIF is present or not and this is negatively correlated with and possibly compensates for stress-forced diminished ESC population expansion and potency loss.

Rosiglitazone augments antioxidant response in the human trophoblast and prevents apoptosis†.

Insufficient perfusion of the trophoblast by maternal blood is associated with an increased generation of reactive oxygen species and complications of the placenta. In this study, we first examined whether rosiglitazone, an agonist of the peroxisome proliferator-activated receptor-γ (PPARγ), protects the human trophoblast from oxidative injury by regulating key antioxidant proteins, catalase (CAT) and the superoxide dismutases (SOD1 and SOD2). In first trimester placental explants, localization of CAT was limited to cytotrophoblasts, whereas SOD1 was expressed in both the cyto- and syncytiotrophoblasts. In first trimester placental explants, hypoxia decreased the expression of both SOD1 and SOD2, and increased apoptosis. Treatment with rosiglitazone dose-dependently upregulated anti-oxidative CAT and SOD2, and rescued hypoxic injury in first trimester villous explants and JEG-3 cells, strongly suggesting the involvement of the PPARγ in regulating their expressions. Rosiglitazone facilitated transcription activity of PPARγ, and enhanced promotor binding, increased transcriptional activity at the CAT promoter, and elevated protein expression/activity. Treatment of hypoxic JEG-3 cells with rosiglitazone resulted in mitochondrial membrane potential increase and a reduction of caspase 9 and caspase 3 activity which is consistent with improved cell survival. To complement PPARγ activation data, we also utilized the antagonist (SR-202) and siRNA to suppress PPARγ expression and demonstrate the specific role of PPARγ in reducing ROS and oxidative stress. Ex vivo examination of term human placenta revealed lower expression of antioxidant proteins in pathologic compared to healthy placental tissues, which could be rescued by rosiglitazone, indicating that rosiglitazone can improve survival of the trophoblast under pathological conditions. These findings provide evidence that the PPARγ pathway directly influences cellular antioxidants production and the pathophysiology of placental oxidative stress.

Trophoblast retrieval and isolation from the cervix: origins of cervical trophoblasts and their potential value for risk assessment of ongoing pregnancies.

BACKGROUND: Early during human development, the trophoblast lineage differentiates to commence placentation. Where the placenta contacts the uterine decidua, extravillous trophoblast (EVT) cells differentiate and invade maternal tissues. EVT cells, identified by expression of HLA-G, invade into uterine blood vessels (endovascular EVT), as well as glands (endoglandular EVT), and open such luminal structures towards the intervillous space of the placenta. Endoglandular invasion diverts the contents of uterine glands to the intervillous space, while glands near the margin of the placenta that also contain endoglandular EVT cells open into the reproductive tract. Cells of the trophoblast lineage have thus been recovered from the uterine cavity and endocervical canal. An emerging non-invasive technology [trophoblast retrieval and isolation from the cervix (TRIC)] isolates and examines EVT cells residing in the cervix to explore their origin, biology and relationship to pregnancy and fetal status. OBJECTIVE AND RATIONALE: This review explores the origins and possible uses of trophoblast cells obtained during ongoing pregnancies (weeks 5-20) by TRIC. We hypothesize that endoglandular EVT cells at the margins of the expanding placenta enter the uterine cavity and are carried together with uterine secretion products to the cervix where they can be retrieved from a Papanicolaou (Pap) smear. The advantages of TRIC for investigation of human placentation and prenatal testing will be considered. Evidence from the literature, and from archived in utero placental histological sections, is presented to support these hypotheses. SEARCH METHODS: We used 52 out of 80 publications that appeared between 1966 and 2017 and were found by searching the PubMed and Google Scholar databases. The studies described trophoblast invasion of uterine vessels and glands, as well as trophoblast cells residing in the reproductive tract. This was supplemented with literature on human placental health and disease. OUTCOMES: The literature describes a variety of invasive routes taken by EVT cells at the fetal-maternal interface that could displace them into the reproductive tract. Since the 1970s, investigators have attempted to recover trophoblast cells from the uterus or cervix for prenatal diagnostics. Trophoblast cells from Pap smears obtained at 5-20 weeks of gestation have been purified (>95% ß-hCG positive) by immunomagnetic isolation with nanoparticles linked to anti-HLA-G (TRIC). The isolated cells contain the fetal genome, and have an EVT-like expression profile. Similar EVT-like cells appear in the lumen of uterine glands and can be observed entering the uterine cavity along the margins of the placenta, suggesting that they are the primary source of cervical trophoblast cells. Cells isolated by TRIC can be used to accurately genotype the embryo/fetus by targeted next-generation sequencing. Biomarker protein expression quantified in cervical trophoblast cells after TRIC correlates with subsequent pregnancy loss, pre-eclampsia and fetal growth restriction. A key remaining question is the degree to which EVT cells in the cervix might differ from those in the basal plate and placental bed. WIDER IMPLICATIONS: TRIC could one day provide a method of risk assessment for maternal and fetal disease, and reveal molecular pathways disrupted during the first trimester in EVT cells associated with placental maldevelopment. As perinatal interventions emerge for pregnancy disorders and inherited congenital disorders, TRIC could provide a key diagnostic tool for personalized precision medicine in obstetrics.

Nifedipine Prevents Apoptosis of Alcohol-Exposed First-Trimester Trophoblast Cells.

BACKGROUND: Maternal alcohol abuse leading to fetal alcohol spectrum disorder (FASD) includes fetal growth restriction (FGR). Ethanol (EtOH) induces apoptosis of human placental trophoblast cells, possibly disrupting placentation and contributing to FGR in FASD. EtOH facilitates apoptosis in several embryonic tissues, including human trophoblasts, by raising intracellular Ca2+ . We previously found that acute EtOH exposure increases trophoblast apoptosis due to signaling from both intracellular and extracellular Ca2+ . Therefore, nifedipine, a Ca2+ channel blocker that is commonly administered to treat preeclampsia and preterm labor, was evaluated for cytoprotective properties in trophoblast cells exposed to alcohol. METHODS: Human first-trimester chorionic villous explants and the human trophoblast cell line HTR-8/SVneo (HTR) were pretreated with 12.5 to 50 nM of the Ca2+ channel blocker nifedipine for 1 hour before exposure to 50 mM EtOH for an additional hour. Intracellular Ca2+ concentrations were monitored in real time by epifluorescence microscopy, using fluo-4-AM. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), accumulation of cytoplasmic cytochrome c, and cleavage rates of caspase 3 and caspase 9. RESULTS: The increase in intracellular Ca2+ upon exposure to EtOH in both villous explants and HTR cells was completely blocked (p < 0.05) when pretreated with nifedipine, accompanied by inhibition of EtOH-induced release of cytochrome c, caspase activities, and TUNEL. CONCLUSIONS: This study indicates that nifedipine can interrupt the apoptotic pathway downstream of EtOH exposure and could provide a novel strategy for future interventions in women with fetuses at risk for FASD.

SUMO-4: A novel functional candidate in the human placental protein SUMOylation machinery.

BACKGROUND: Small ubiquitin-like modifiers (SUMOs) conjugate to proteins post-translationally, thereby affecting target localization, activity and stability. Functional SUMO family members identified in the human placenta include SUMO-1 to SUMO-3, which are elevated in pre-eclampsia. Whether the fourth isoform, SUMO-4, plays a role in placental development and function remains unknown. OBJECTIVES: We tested the hypothesis that SUMO-4 is expressed in the human placenta and demonstrates altered SUMOylation in pre-eclamptic pregnancies. METHODS: SUMO-4 mRNA (qRT-PCR) and protein (Western blot and immunohistochemistry) were measured in Jar cells, BeWo cells, first trimester placental villous explants and placental tissues across normal gestation and in pre-eclampsia. SUMO-4 expression in response to oxidative stress (H2O2: 0, 0.1, 1 and 5mM), as well as, hypoxia-reperfusion (O2: 1%, 8% and 20%) was measured. Lastly, SUMO-4 binding (covalently vs. non-covalently) to target proteins was investigated. RESULTS: SUMO-4 mRNA and protein were unchanged across gestation. SUMO-4 was present in the villous trophoblast layer throughout gestation. SUMO-4 mRNA expression and protein levels were increased ~2.2-fold and ~1.8-fold in pre-eclamptic placentas compared to age-matched controls, respectively (p<0.01). SUMO-4 mRNA and protein expression increased in Jars, BeWos and first trimester placental explants with 5mM H2O2 treatment, as well as with exposure to hypoxia-reperfusion. SUMO-1 to SUMO-3 did not show consistent trends across models. SUMO-4 hyper-SUMOylation was predominantly covalent in nature. CONCLUSIONS: SUMO-4 is expressed in normal placental development. SUMO-4 expression was increased in pre-eclamptic placentas and in models of oxidative stress and hypoxic injury. These data suggests that SUMO-4 hyper-SUMOylation may be a potential post-translational mechanism in the stressed pre-eclamptic placenta.

Quo vadis, trophoblast? Exploring the new ways of an old cell lineage.

Trophoblast cells are the first embryonic lineage to differentiate during human development, and are needed to sustain fetal life through their role in constructing a placenta. As the fetus grows, the trophoblast rapidly expands and further differentiates to produce an extravillous subtype that invades the maternal tissues. Some of the extravillous trophoblast cells find their way into the reproductive tract, and can be safely captured by noninvasive collection from the endocervical canal, similarly to a Pap smear. We are developing a new technology for investigating trophoblast cells residing in the cervix to better understand their development, and to glean information from them about pregnancy status. Trophoblast retrieval and isolation from the cervix (TRIC) efficiently isolates hundreds of trophoblast cells without limitations due to early gestational age, maternal obesity, or uteroplacental insufficiency disorders. Cells that appear to be extravillous trophoblast, based on their molecular phenotype, can be purified from Pap smears obtained between 5 and 20 weeks of gestation, using magnetic nanoparticles coupled to an antibody recognizing HLA-G that they specifically produce. Information about fetal genotype and adverse pregnancy outcomes has been obtained using TRIC, and could one day provide assessment of maternal and fetal risk of disease. As perinatal interventions for placental disorders and inherited congenital disorders emerge, TRIC could provide a key diagnostic tool for personalize precision pregnancy management.

Enhancement of trophoblast differentiation and survival by low molecular weight heparin requires heparin-binding EGF-like growth factor.

STUDY QUESTION: Does low molecular weight heparin (LMWH) require heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) signaling to induce extravillous trophoblast differentiation and decrease apoptosis during oxidative stress? SUMMARY ANSWER: LMWH increased HBEGF expression and secretion, and HBEGF signaling was required to stimulate trophoblast extravillous differentiation, increase invasion in vitro and reduce trophoblast apoptosis during oxidative stress. WHAT IS KNOWN ALREADY: Abnormal trophoblast differentiation and survival contribute to placental insufficiency syndromes, including preeclampsia and intrauterine growth restriction. Preeclampsia often manifests as a pro-thrombotic state, with unsuccessful transformation of the spiral arteries that reduces oxygen supply and can produce placental infarction. LMWH improves placental function by increasing blood flow. Recent data suggest that the actions of LMWH transcend its anti-coagulative properties, but the molecular mechanism is unknown. There is evidence that LMWH alters the expression of human HBEGF in trophoblast cells, which regulates human trophoblast pathophysiology. HBEGF, itself, is capable of increasing trophoblast survival and invasiveness. STUDY DESIGN, SIZE, DURATION: First-trimester placental explants and the HTR-8/SVneo cell line, established using extravillous trophoblast outgrowths from first-trimester villous explants, were treated in vitro with LMWH to examine the effects on HBEGF signaling and trophoblast function under normal physiological and pathological conditions. A highly specific antagonist of HBEGF and other inhibitors of HBEGF downstream signaling were used to determine the relationship between LMWH treatment and HBEGF. PARTICIPANTS/MATERIALS, SETTING, METHODS: Placental tissues (n = 5) were obtained with IRB approval and patient consent from first-trimester terminations. Placental explants and HTR-8/SVneo cells were cultured on plastic or Matrigel™ and treated with a therapeutic dose of LMWH (Enoxaparin; 10 IU/ml), with or without CRM197, pan Erb-B2 Receptor Tyrosine Kinase (ERBB) inhibitor, anti-ERBB1 or ERBB4 blocking antibodies, or pretreatment of cells with heparitinase I. Extravillous differentiation was assessed by immunocytochemistry to determine the relative levels of integrins α6ß4 and α1ß1. Trophoblast invasiveness was assessed in villous explants by measuring outgrowth from villous tips cultured on Matrigel, and by invasion assays with HTR-8/SVneo cells cultured on Matrigel-coated transwell insert. Placental explants and HTR-8/SVneo cells were exposed to oxidative stress in a hypoxia-reoxygenation (H-R) model, measuring cell death by TUNEL assay, caspase 3 cleavage, and BCL-2α expression. MAIN RESULTS AND THE ROLE OF CHANCE: LMWH induced extravillous differentiation, according to trophoblast invasion assays and integrin (α6ß4-α1ß1) switching. Treatment with LMWH rescued cytotrophoblasts and HTR-8/SVneo cells from apoptosis during exposure to reoxygenation injury, based on TUNEL, caspase 3 cleavage and BCL-2α expression. Experiments using CRM197, ERBB1 and ERBB4 blocking antibodies, pan-ERBB inhibitor and removal of cell surface heparin demonstrated that the effects of LMWH on trophoblast invasion and survival were dependent upon HBEGF signaling. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The primary limitation of this study was the use of only in vitro experiments. Patient demographics from elective terminations were not available. WIDER IMPLICATIONS OF THE FINDINGS: These data provide new insights into the non-coagulation-related aspects of perinatal LMWH treatment in the management of placental insufficiency disorders. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by grants from the National Institutes of Health (HD071408 and HL128628), the March of Dimes, and the W. K. Kellogg Foundation. There were no conflicts or competing interests.

Rosiglitazone Regulates TLR4 and Rescues HO-1 and NRF2 Expression in Myometrial and Decidual Macrophages in Inflammation-Induced Preterm Birth.

INTRODUCTION: Elevated inflammation accounts for approximately 30% of preterm birth (PTB) cases. We previously reported that targeting the peroxisome proliferator-activated receptor gamma (PPARγ) pathway reduced the incidence of PTB in the mouse model of endotoxin-induced PTB. The PPARγ has proven anti-inflammatory functions and its activation via rosiglitazone significantly downregulated the systemic inflammatory response and reduced PTB and stillbirth rate by 30% and 41%, respectively, in our model. Oxidative stress is inseparable from inflammation, and rosiglitazone has a reported antioxidative activity. In the current study, we therefore aimed to evaluate whether rosiglitazone treatment had effects outside of inflammatory pathway, specifically on the antioxidation pathway in our model. METHODS: Pregnant C57BL/6J mice (E16.5) were treated with phosphate-buffered saline (PBS), rosiglitazone (Rosi), lipopolysaccharide (LPS; 10µg in 200µL 1XPBS), or LPS + Rosi (6 hours after the LPS injection). The myometrial and decidual tissues were collected and processed for macrophage isolation using magnetic cell sorting and F4/80+ antibody. Expression levels of antioxidative factors- Nrf2 and Ho-1-along with the LPS receptor Tlr4 were quantified by quantitative polymerase chain reaction. The protein levels were assessed by immunofluorescence staining. RESULTS: Both the decidual and myometrial macrophages from the LPS-treated animals showed significantly lowered expression of Ho-1 and Nrf2 and higher expression of Tlr4 when compared to the PBS control group. The macrophages from the animals in the LPS + Rosi group had significantly elevated expression of Ho-1 and Nrf2 and downregulated expression of Tlr4 when compared to the LPS group. CONCLUSION: Rosiglitazone administration prevents PTB by downregulating inflammation and upregulating antioxidative response.

Fetal genome profiling at 5 weeks of gestation after noninvasive isolation of trophoblast cells from the endocervical canal.

Single-gene mutations account for more than 6000 diseases, 10% of all pediatric hospital admissions, and 20% of infant deaths. Down syndrome and other aneuploidies occur in more than 0.2% of births worldwide and are on the rise because of advanced reproductive age. Birth defects of genetic origin can be diagnosed in utero after invasive extraction of fetal tissues. Noninvasive testing with circulating cell-free fetal DNA is limited by a low fetal DNA fraction. Both modalities are unavailable until the end of the first trimester. We have isolated intact trophoblast cells from Papanicolaou smears collected noninvasively at 5 to 19 weeks of gestation for next-generation sequencing of fetal DNA. Consecutive matched maternal, placental, and fetal samples (n = 20) were profiled by multiplex targeted DNA sequencing of 59 short tandem repeat and 94 single-nucleotide variant sites across all 24 chromosomes. The data revealed fetal DNA fractions of 85 to 99.9%, with 100% correct fetal haplotyping. This noninvasive platform has the potential to provide comprehensive fetal genomic profiling as early as 5 weeks of gestation.

Altered Biomarkers in Trophoblast Cells Obtained Noninvasively Prior to Clinical Manifestation of Perinatal Disease.

A contributing factor to poor placental perfusion, leading to intrauterine growth restriction and preeclampsia, is the failure of invading extravillous trophoblast (EVT) cells to remodel the maternal uterine arteries during the first and second trimesters of pregnancy. Noninvasive assessment of EVT cells in ongoing pregnancies is possible beginning three weeks after conception, using trophoblast retrieval and isolation from the cervix (TRIC). Seven proteins were semi-quantified by immunofluorescence microscopy in EVT cells obtained between gestational weeks 6 and 20 from pregnancies with normal outcomes (N = 29) and those with intrauterine growth restriction or preeclampsia (N = 12). Significant differences were measured in expression of PAPPA, FLT1, ENG, AFP, PGF, and LGALS14, but not LGALS13 or the lineage marker KRT7. These findings provide for the first time direct evidence of pathology-associated protein dysregulation in EVT cells during early placentation. The TRIC platform provides a novel approach to acquire molecular signatures of EVT cells that can be correlated with pregnancy outcome.

Genome-wide placental DNA methylation analysis of severely growth-discordant monochorionic twins reveals novel epigenetic targets for intrauterine growth restriction.

BACKGROUND: Intrauterine growth restriction (IUGR), which refers to reduced fetal growth in the context of placental insufficiency, is etiologically heterogeneous. IUGR is associated not only with perinatal morbidity and mortality but also with adult-onset disorders, such as cardiovascular disease and diabetes, posing a major health burden. Placental epigenetic dysregulation has been proposed as one mechanism that causes IUGR; however, the spectrum of epigenetic pathophysiological mechanisms leading to IUGR remains to be elucidated. Monozygotic monochorionic twins are particularly affected by IUGR, in the setting of severe discordant growth. Because monozygotic twins have the same genotype at conception and a shared maternal environment, they provide an ideal model system for studying epigenetic dysregulation of the placenta. RESULTS: We compared genome-wide placental DNA methylation patterns of severely growth-discordant twins to identify novel candidate genes for IUGR. Snap-frozen placental samples for eight severely growth-discordant monozygotic monochorionic twin pairs were obtained at delivery from each twin. A high-resolution DNA methylation array platform was used to identify methylation differences between IUGR and normal twins. Our analysis revealed differentially methylated regions in the promoters of eight genes: DECR1, ZNF300, DNAJA4, CCL28, LEPR, HSPA1A/L, GSTO1, and GNE. The largest methylation differences between the two groups were in the promoters of DECR1 and ZNF300. The significance of these group differences was independently validated by bisulfite pyrosequencing, implicating aberrations in fatty acid beta oxidation and transcriptional regulation, respectively. Further analysis of the array data identified methylation changes most prominently affecting the Wnt and cadherin pathways in the IUGR cohort. CONCLUSIONS: Our results suggest that IUGR in monozygotic twins is associated with impairments in lipid metabolism and transcriptional regulation as well as cadherin and Wnt signaling. We show that monozygotic monochorionic twins discordant for growth provide a useful model to study one type of the epigenetic placental dysregulation that drives IUGR.