Preeclampsia Placentas Research Articles

Restore Veto Property in Low Dose Aspirin/ASA Treated Preeclampsia Placenta Mesenchymal Stem Cells: Insights Into ASA-mediated Clinical Response*

Preeclampsia (PE) is a pregnancy complication characterized by elevated blood pressure, proteinuria, and other laboratory abnormalities. PE affects 2–8% of pregnancies globally and can lead to preterm birth and other complications for the mother and fetus. Successful pregnancy depends on the ability of the mother’s immune system to tolerate the allogeneic fetus. However, in PE, this immune tolerance is exacerbated by inflammation. Transforming growth factor β (TGF-β) is important to retain an immune balance in healthy pregnancy. In PE, TGF-β level is reduced, with an imbalance of the T-cell subset pool to favor inflammation. Omics studies by our group reported an increase in TGF-β signaling when PE-derived placenta mesenchymal stem cells (P-MSCs) were treated with low dose aspirin (ASA). This correlated with increased cycling quiescence and epigenetic changes, resembling healthy P-MSCs. This study tested the hypothesis that ASA could restore the veto property of P-MSCs to mitigate inflammation. ASA (10 mM) treated P-MSCs from PE and healthy placentas increased TGFβ1 and its receptor. The ASA treated MSCs, when added as third-party cells to a one-way mixed lymphocyte reaction, suppressed T-cell proliferation. Prediction studies with omics data indicated that ASA-mediated TGFβ signaling could explain ASA-induced blunting of cell apoptosis. Together, the findings support ASA-mediated expression of TGFβ1 and its receptor on P-MSCs from PE to restore the ability to be licensed as immune suppressor to mitigate PE inflammation. The findings provide new insight into the benefit of ASA treatment for PE.

MicroRNAs in the Pathogenesis of Preeclampsia-A Case-Control In Silico Analysis.

Preeclampsia (PE) occurs in 5% to 7% of all pregnancies, and the PE that results from abnormal placentation acts as a primary cause of maternal and neonatal morbidity and mortality. The objective of this secondary analysis was to elucidate the pathogenesis of PE by probing protein-protein interactions from in silico analysis of transcriptomes between PE and normal placenta from Gene Expression Omnibus (GSE149812). The pathogenesis of PE is apparently determined by associations of miRNA molecules and their target genes and the degree of changes in their expressions with irregularities in the functions of hemostasis, vascular systems, and inflammatory processes at the fetal-maternal interface. These irregularities ultimately lead to impaired placental growth and hypoxic injuries, generally manifesting as placental insufficiency. These differentially expressed miRNAs or genes in placental tissue and/or in blood can serve as novel diagnostic and therapeutic biomarkers.

Exogenous Hydrogen Sulfide Prevents Necroptosis by Inhibiting p38MAPK Pathway Activation in JEG-3 Trophoblast Cells: A Role in Preeclampsia

Objectives: Necroptosis, a form of programmed cell death, can occur in the placenta of patients with preeclampsia (PE). Hydrogen sulfide (H2S) can inhibit necroptosis of human umbilical vein endothelial cells under the high glucose-induced injury. Whether H2S can protect trophoblasts against necroptosis underlying PE has not been elucidated. This study aimed to explore the protective role of H2S in trophoblast cells against necroptosis underlying PE. Design: This is an in vitro experimental study. Participants: A total of 10 pregnant women with severe PE and 10 matched control normotensive pregnant women were included. The placenta tissues were extracted from participators. The human JEG-3 trophoblasts were commercially available. Methods: The expression and localization of necrotic proteins were assayed in human placenta samples, and the effect of necrotic cell death on the proliferation and apoptosis of human JEG-3 trophoblasts was evaluated. The component expressions of inflammatory cytokine and p38MAPK signaling pathway were measured in samples pretreated with or without NaHS (H2S donor) and SB203580 (p38 inhibitor). Results: RIPA1, RIPA3, and p-p38 levels were significantly higher in PE placental tissue, whereas cystathionine β-synthase expression was decreased. In JEG-3 trophoblasts, necroptosis increased apoptotic cell numbers, suppressed cell proliferation, increased inflammatory cytokine expression, and increased p38MAPK activation, which can be prevented by NaHS. Limitations: In the present study, we did not provide sufficient evidence that necroptosis was a part of the pathogenesis of PE. Conclusions: We proposed the putative role of necroptosis in early-onset PE, reflected by the blockage of caspase-8/3 and increased expression of RIPA1 and RIPA3 in PE placenta tissues. Furthermore, we demonstrated that exogenous H2S protected cytotrophoblasts against ceramide-induced necroptosis via the p38MAPK pathway.

Cadmium exposure activates mitophagy through downregulating thyroid hormone receptor/PGC1α signal in preeclampsia

Gestational cadmium exposure increases the risk of preeclampsia. Placenta mitophagy was activated in preeclampsia. The aim of present study was to explore the mechanism of cadmium-induced mitophagy activation and its association with preeclampsia. Mitophagy markers expression levels were detected by quantitative real-time PCR, Western blot, immunofluorescence and immunochemistry in preeclampsia placenta. JEG3 cells were treated with CdCl2, iopanoic acid (IOP), 3-methyladenine and PGC1α SiRNA to verify mechanism of cadmium-induced mitophagy. Mitophagy marker LC3BII/I and P62 expression were increased and mitochondrial membrane receptor protein TOM20 and FUNDC1 expression were decreased in preeclampsia placenta as compared with that in normotension control. Mitophagy marker LC3BII/I and P62 expression were increased and TOM20 and FUNDC1 expression was decreased in CdCl2-treated JEG3 cells. Meanwhile, mitochondrial biogenesis regulator, PGC1α expression was decreased in preeclampsia and CdCl2-treated JEG3 cells. The expressions of LC3B and P62 were increased and the expressions of TOM20, FUNDC1 and PGC1α were decreased in IOP-treated cell. PGC1α SiRNA transfection led to increased expression of LC3BII/I and P62 and decreased expression of TOM20 and FUNDC1. The expression of sFlt1 was increased in preeclampsia placenta, CdCl2-treated cells, in IOP-treated cells and in PGC1α SiRNA transfected cells. 3-methyladenine treatment protected the increased expression of sFlt1 in CdCl2-treated cells, in IOP-treated cells and in PGC1α SiRNA transfected cells. Meanwhile, co-treatment of cadmium and IOP or PGC1αSiRNA led to a reduce expressions of OPA1, MFN1, MFN2 and FUNDC1 as compared to cadmium-treated, IOP-treated and PGC1α SiRNA-treated cells. These results elucidated that maternal cadmium exposure activated placenta mitophagy through downregulation of thyroid hormone receptor signal mediated decreased expression of PGC1α and was associated with the occurrence of preeclampsia.

MicroRNA analysis of medium/large placenta extracellular vesicles in normal and preeclampsia pregnancies.

Preeclampsia (PE) is a hypertensive disorder of pregnancy, affecting 2%-8% of pregnancies worldwide, and is the leading cause of adverse maternal and fetal outcomes. The disease is characterized by oxidative and cellular stress and widespread endothelial dysfunction. While the precise mechanisms are not entirely understood, the pathogenesis of PE is closely linked to placental dysfunction and, to some extent, syncytiotrophoblast extracellular vesicle release (STB-EVs). These vesicles can be divided into the less well-studied medium/large EVs (220-1,000 nm) released in response to stress and small EVs (<220 nm) released as a component of intercellular communication. The previously described production of m/lSTB-EVs in response to cellular stress combined with the overwhelming occurrence of cellular and oxidative stress in PE prompted us to evaluate the microRNAome of PE m/lSTB-EVs. We hypothesized that the microRNAome profile of m/lSTB-EVs is different in PE compared to normal pregnancy (NP), which might permit the identification of potential circulating biomarkers not previously described in PE. We performed small RNA sequencing on medium/large STB-EVs isolated from PE and NP placentae using dual-lobe ex vivo perfusion. The sequencing data was bioinformatically analyzed to identify differentially regulated microRNAs. Identified microRNAs were validated with quantitative PCR analysis. We completed our analysis by performing an in-silico prediction of STB-EV mechanistic pathways. We identified significant differences between PE and NP in the STB-EVs micro ribonucleic acid (microRNA) profiles. We verified the differential expression of hsa-miR-193b-5p, hsa-miR-324-5p, hsa-miR-652-3p, hsa-miR-3196, hsa-miR-9-5p, hsa-miR-421, and hsa-miR-210-3p in the medium/large STB-EVs. We also confirmed the differential abundance of hsa-miR-9-5p in maternal serum extracellular vesicles (S EVs). In addition, we integrated the results of these microRNAs into the previously published messenger RNA (mRNA) data to better understand the relationship between these biomolecules. We identified a differentially regulated micro-RNA, hsa-miR-9-5p, that may have biomarker potential and uncovered mechanistic pathways that may be important in the pathophysiology of PE.

Nox2 inhibition reduces trophoblast ferroptosis in preeclampsia via the STAT3/GPX4 pathway.

Ferroptosis, a novel mode of cell death characterized by lipid peroxidation and oxidative stress, plays an important role in the pathogenesis of preeclampsia (PE). The aim of this study is to determine the role of Nox2 in the ferroptosis of trophoblast cells, along with the underlying mechanisms. The mRNA and protein levels of Nox2, STAT3, and GPX4 in placental tissues and trophoblast cells were respectively detected by qRT-PCR and western blot analysis. CCK8, transwell invasion and tube formation assays were used to evaluate the function of trophoblast cells. Ferroptosis was evaluated using flow cytometry and the lipid peroxidation assay. Glycolysis and mitochondrial respiration were investigated by detecting the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using Seahorse extracellular flux technology. The t-test or one-way ANOVA was used for statistical analysis. Nox2 was up-regulated while STAT3 and GPX4 were down-regulated in PE placental tissues. Nox2 knockdown inhibited ferroptosis in trophoblast cells, which was shown by enhanced proliferation and invasion, decreased ROS and lipid peroxide levels, and reduced glycolysis and mitochondrial dysfunction. Nox2 negatively correlated with MVD in PE placentas, and Nox2 knockdown restored ferroptosis-inhibited tube formation. Nox2 could interact with STAT3. Inhibiting Nox2 restored ferroptosis-induced alterations in the mRNA and protein levels of STAT3 and GPX4. Nox2 may trigger ferroptosis through the STAT3/GPX4 pathway, subsequently leading to regulation of mitochondrial respiration, transition of glycolysis, and inhibition of placental angiogenesis. Therefore, targeted inhibition of Nox2 is expected to become a new therapeutic target for PE.

Oral origin of the placenta microbiome in pregnant women with preeclampsia

Preeclampsia (PE) is a leading cause of morbidity and mortality in pregnancy with elusive etiology. Patients with PE are thought to be associated with a higher rate of periodontal diseases (PDs) and changes of oral bacteria with targeted PCR techniques. However, few studies have investigated the associations between oral microbiome dysbiosis and secondarily disseminated microbes in the placenta simultaneously in patients with PE. The association between detected microbiome in placenta and systemic inflammation in PE is also unclear. We enrolled 54 pregnant patients with and without PE and PD, and profiled the subgingival and placenta microbiome by the V4 region of 16S rRNA gene sequencing. Systemic inflammatory markers tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), lipopolysaccharide binding protein (LBP), and interleukins 6 and 8 (IL-6 and IL-8) in blood were measured by ELISA. We found that PD significantly increased the risk of PE after adjusting for age and smoking status (OR = 2.26, 95% CI = 1.14–4.48, p = 0.024). A combined group of oral-associated bacteria Veillonella, Fusobacterium, Haemophilus, Granulicatella, Streptococcus, Gemella, and Neisseria in placenta had a significantly higher prevalence in women with PE compared to women without PE (53.8% vs. 19.0%, p = 0.018), with the highest prevalence in patients with both PE and PD (58.8%). The relative abundance of Haemophilus, Veillonella, and Fusobacterium in subgingival samples was significantly higher in patients with PE than those without PE. The relative abundance of Haemophilus in subgingival samples was associated with increased risk of PE (OR = 2.11, 95% CI = 1.11–4.52, p = 0.032). Proinflammatory cytokine analysis showed that PE patients with PD had higher blood IL-8 levels than PE patients without PD (p = 0.026). CRP, LBP, and TNF-α showed no statistical difference in patients with and without PE or PD. Blood IL-6 levels were significantly higher in patients with detectable placenta microbiome compared to those without placenta microbiome (p = 0.028). Together, our data suggest a potential oral origin of the placental microbiota present in patients with PE, and the microbiota detected in placenta is associated with increased IL-6 level in the blood.

SFRP2 suppresses trophoblast cell migration by inhibiting the Wnt/β‑catenin pathway.

The present study investigates the role of Secreted Frizzled‑Related Protein 2 (SFRP2) in trophoblast cells, a key factor in preeclampsia (PE) progression. Elevated levels of Secreted Frizzled‑Related Protein 1/3/4/5 (SFRP1/3/4/5) are associated with PE, but the role of SFRP2 is unclear. We analyzed SFRP2 expression in PE placental tissue using the GSE10588 dataset and overexpressed SFRP2 in JEG‑3 cells via lentiviral transfection. The viability, migration, apoptosis, and proliferation of SFRP2‑overexpressing JEG‑3 cells were assessed using Cell Counting Kit‑8, Transwell assays, flow cytometry, and EdU staining. Additionally, we evaluated the impact of SFRP2 overexpression on key proteins in the Wnt/β‑catenin pathway and apoptosis markers (Bax, cleaved‑caspase 3, BCL‑2, MMP9, E‑cadherin, Wnt3a, Axin2, CyclinD1, c‑Myc, p‑β‑catenin, β‑catenin, phosphorylated Glycogen Synthase Kinase 3 beta (p‑GSK3β), and GSK3β) through western blotting. Results showed high SFRP2 mRNA and protein expression in PE placenta and JEG‑3 cells post‑transfection. SFRP2 overexpression significantly reduced JEG‑3 cell viability, proliferation, and migration, while increasing apoptosis. It also altered expression levels of Wnt pathway proteins, suggesting SFRP2's potential as a therapeutic target for PE by inhibiting trophoblast cell migration through the Wnt/β‑catenin signaling cascade.

An integral role of mitochondrial function in the pathophysiology of preeclampsia.

Preeclampsia (PE) is associated with high maternal and perinatal morbidity and mortality. The development of effective treatment strategies remains a major challenge due to the limited understanding of the pathogenesis. In this review, we summarize the current understanding of PE research, focusing on the molecular basis of mitochondrial function in normal and PE placentas, and discuss perspectives on future research directions. Mitochondria integrate numerous physiological processes such as energy production, cellular redox homeostasis, mitochondrial dynamics, and mitophagy, a selective autophagic clearance of damaged or dysfunctional mitochondria. Normal placental mitochondria have evolved innovative survival strategies to cope with uncertain environments (e.g., hypoxia and nutrient starvation). Cytotrophoblasts, extravillous trophoblast cells, and syncytiotrophoblasts all have distinct mitochondrial morphology and function. Recent advances in molecular studies on the spatial and temporal changes in normal mitochondrial function are providing valuable insight into PE pathogenesis. In PE placentas, hypoxia-mediated mitochondrial fission may induce activation of mitophagy machinery, leading to increased mitochondrial fragmentation and placental tissue damage over time. Repair mechanisms in mitochondrial function restore placental function, but disruption of compensatory mechanisms can induce apoptotic death of trophoblast cells. Additionally, molecular markers associated with repair or compensatory mechanisms that may influence the development and progression of PE are beginning to be identified. However, contradictory results have been obtained regarding some of the molecules that control mitochondrial biogenesis, dynamics, and mitophagy in PE placentas. In conclusion, understanding how the mitochondrial morphology and function influence cell fate decisions of trophoblast cells is an important issue in normal as well as pathological placentation biology. Research focusing on mitochondrial function will become increasingly important for elucidating the pathogenesis and effective treatment strategies of PE.

MicroRNA-195-5p Attenuates Pregnancy-Induced Hypertension by Inhibiting Oxidative Stress via OTX1/MAPK Signaling Pathway.

Pregnancy-induced hypertension (PIH) is a hypertensive disorder during pregnancy and can induce perinatal death of human infants. MicroRNA (miR)-195-5p was validated to display low expression in severe preeclampsia placentas, but the role of miR-195-5p in pregnancy-induced hypertension (PIH) has not been investigated. The study emphasized on the functions and mechanism of miR-195-5p in PIH. A reduced uterine perfusion pressure (RUPP) rat model was established to mimic PIH in vivo. Adenovirus (Ad)-miR-195-5p agomir and/or Ad-OTX1 were further injected into some model rats. RT-qPCR was conducted to assess the expression of miR-195-5p and orthodenticle homeobox 1 (OTX1) in rat placental tissues, the isolated aortic endothelial cells (AECs), and in serum samples of PIH patients. Western blot analysis was implemented to measure the protein levels of OTX1, VEGFA, and key factors involved in the MAPK signaling pathway. The concentrations of oxidative stress markers (superoxide dismutase, catalase, and lipid hydroperoxide) in AECs and placental tissues of RUPP rats were measured by corresponding kits. The binding relation between miR-195-5p and OTX1 was verified using the dual-luciferase reporter assay. Hematoxylin-eosin staining was conducted to evaluate the pathological features of rat placental tissues. MiR-195-5p was downregulated, while OTX1 was upregulated in rat placental tissues and human serum samples of PIH patients. MiR-195-5p could target OTX1 and inversely regulate OTX1 expression in AECs and rat placental tissues. In addition, miR-195-5p can negatively regulate VEGFA level. Furthermore, miR-195-5p inactivates oxidative stress and the MAPK signaling by downregulating OTX1 in AECs. In vivo experiments revealed that OTX1 overexpression reversed the protective effect of miR-195-5p overexpression on placental damage and oxidative stress. MiR-195-5p alleviates PIH by inhibiting oxidative stress via targeting OTX1 and inactivating MAPK signaling.

Sex-Specific Dysregulation of Placental Lipid Metabolism in Preeclampsia.

Preeclampsia (PE) is a hypertensive disorder of pregnancy associated with adverse maternal and fetal outcomes. While placental dysfunction is implicated in PE pathogenesis, the impact of PE on placental lipid metabolism and its potential sexual dimorphism remains poorly understood. We conducted a comprehensive analysis of term placentas from PE and normotensive pregnancies with male and female fetuses. Lipid profiles were quantified using mass spectrometry, and mRNA expression of genes involved in fatty acid oxidation, esterification, and transport was assessed using qPCR. Placentas from PE pregnancies exhibited elevated lipid levels, with male placentas showing a more pronounced increase in triacylglycerols, cholesteryl esters, and free cholesterol compared to female placentas. Gene expression analysis revealed sexually dimorphic alterations, with male PE placentas exhibiting upregulation of genes involved in fatty acid uptake, oxidation, and esterification, while female PE placentas showed a more complex response with both upregulation and downregulation of certain genes. Notably, peroxisomal fatty acid oxidation was upregulated in male PE placentas but suppressed in female PE placentas. Our findings reveal sexually dimorphic alterations in placental lipid metabolism in PE, suggesting that male placentas may be more vulnerable to lipotoxicity. These insights may have implications for understanding the pathogenesis of PE and developing sex-specific interventions to improve maternal and fetal outcomes.

100 Artificial intelligence (machine learning) and molecular classification of sibling twin (TW) placentas in preeclampsia (PE).

100 Artificial intelligence (machine learning) and molecular classification of sibling twin (TW) placentas in preeclampsia (PE).

Disaccharide compositional analysis of chondroitin sulphate using WAX HILIC-MS with pre-column procainamide labelling; application to the placenta in pre-eclampsia.

Chondroitin sulphate (CS) and dermatan sulphate are negatively charged linear heteropolysaccharides. These glycosaminoglycans (GAG) are involved in cellular signalling via binding to growth factors. CS is expressed in a range of tissue and biological fluids and is highly expressed in the placenta. There is evidence that decorin; a CS proteoglycan is significantly decreased in pre-eclampsia and fetal growth restriction. It is considered that GAG chain composition may influence cellular processes that are altered in pre-eclampsia. The goal of the present study was to develop an LC-MS method with precolumn procainamide labelling for the disaccharide compositional analysis of CS. The method was used to investigate whether the disaccharide composition of placenta-extracted CS is altered in pre-eclampsia. The study revealed differential disaccharide compositions of placental chondroitin sulphate between pre-eclampsia and other pregnancy conditions. This suggests that the method may have diagnostic potential for pregnancy disorders. Furthermore, the findings suggest that CS sulphation might play a significant role in maternal labour.

Higher placental activin-A (ACV-A) and inhibin-A (INH-A) in preeclampsia placenta mother compare to diabetic mother‎

Higher placental activin-A (ACV-A) and inhibin-A (INH-A) in preeclampsia placenta mother compare to diabetic mother‎

Investigation of Bax and Phospho-Tau Protein Expression in Preeclampsia Placenta

Background: Preeclampsia is a pregnancy complication with a high morbidity and mortality rate.BAX is a proapoptotic protein and in recent years, 14-3-3 tau proteins are considered as key regulators of many processes, such as apoptosis. In this study, we investigated the expression of phospho-tau and Bax in preeclampsia apoptosis immunohistochemically. Materials and Methods: 25 healthy and 25 preeclamptic pregnant placentas were included in the study. Placental samples were fixed with 10% neutral buffered formalin. Routine paraffin wax tissue protocol was used. Results: According to histological micrograf, the trophoblastic cells in the villi were normal in the control group. Mild dilatation in blood vessels was seen. Hyalinized and necrotic areas in chorionic villi and an increase in fibrinoid tissue in root villi were detected in preeclampsia sections. Phospho-tau and Bax primary antibodys were used for immunohistochemical evaluation. The sections from of the preeclampsia group were highly positive for syncytiotrophoblasts and villous connective tissue. It was also statistically different from the control group (p&amp;lt;0.05). Conclusions: When we evaluate the results shows that Phospho-tau and Bax may be determinant proteins in the apoptosis pathway of preeclampsia.

Circ_0001861 facilitates trophoblast cell proliferation, migration, invasion and epithelial-mesenchymal transition via the miR-296-5p/forkhead box protein 1 pathway in preeclampsia.

Preeclampsia (PE) is one of the leading causes of maternal mortality and placental trophoblastic disorders. Recent studies reported that circular RNAs (circRNAs) were involved in PE pathogenesis. However, the role of circ_0001861 in PE progression is largely unknown. The RNA expression of circ_0001861, forkhead box protein 1 (FOXP1) and microRNA-296-5p (miR-296-5p) was detected by quantitative real-time polymerase chain reaction (qRT-PCR) assay. Western blot assay was performed to examine the protein levels of FOXP1 and epithelial-mesenchymal transition (EMT) markers. Cell viability, proliferation, migration and invasion were detected by cell counting kit-8, 5-ethynyl-2'-deoxyuridine, and transwell assays. Luciferase reporter assay, RNA pull-down assay, and RNA immunoprecipitation (RIP) assay were conducted to explore the interaction between miR-296-5p and circ_0001861 or FOXP1. Circ_0001861 and FOXP1 were downregulated but miR-296-5p was upregulated in PE placenta. Upregulation of circ_0001861 facilitated trophoblast cell proliferation, migration, invasion and EMT. Mechanistically, circ_0001861 sponged miR-296-5p to elevate FOXP1 expression, thus promoting trophoblast cell progression. The circ_0001861/miR-296-5p/FOXP1 axis plays a critical role in trophoblast cell proliferation, migration, invasion and EMT, which may provide a novel insight into developing potential therapeutic targets for PE.

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.

PLACENTAL MUTATIONAL BURDEN NEGATIVELY CORRELATES WITH GESTATIONAL AGE IN EARLY-ONSET PREECLAMPSIA

Abstract The placenta exists in a hypoxic environment during early pregnancy, making it susceptible to somatic genetic mutations caused by oxidative stress and rapid cell proliferation. Oxidative stress is one of the leading factors involved in cellular aging causing genetic mutations, cellular senescence, and mitochondria dysfunction over time. While it has been demonstrated that healthy placentas accumulate somatic mutations as they develop, we hypothesize that diseased placentas display an accelerated aging phenotype through the accumulation of somatic mutations. To address this, we analyzed RNA-seq data from 84 human placentas throughout gestation from healthy (N=33) and preeclamptic pregnancies (N=51). We identified rare single nucleotide variants, insertions and deletions using GATK Best practices Workflow for RNA-seq short variant discovery using bcbio-nextgen and annotated with VEP and LOFTEE. Mutational burden, defined as the total number of rare mutations per megabase, was calculated after strict quality control and filtering for rare (gnomADg&amp;lt; 0.05) variants using maftools in R. Linear regression models were employed to determine the association between mutational burden and gestational age. Differences in mean values were computed with a pairwise t-test . This study revealed that mutational burden in early-onset preeclampsia placentas is negatively correlated to gestational age (R^2=0.256; p-value=0.032). The early-onset preeclampsia group had a higher mutational burden relative to the other groups (p-value&amp;lt; 0.05). This study revealed a significant correlation of mutational burden with gestational age in early-onset preeclampsia, paving the way for additional investigation of accelerated aging in placentas and its contribution to the development of preeclampsia.

Proteomic analysis of N-glycosylation of the human placenta between preeclampsia and normal pregnancies

Protein N-glycosylation plays critical roles in modulating placental function, but little is known about N-glycoproteins in the human placenta and modifications in preeclampsia (PE). Here, we show a large, site-specific N-glycoproteome profiling study of PE and normal placenta using quantitative N-glycoproteomics based on mass spectrometry. The study identified disease signatures of altered N-glycoproteins and N-glycosylation site occupancy in PE and provided a system-level view of human placental N-glycoproteins and in vivo N-glycosylation sites. The study led to the discovery of a roster of glycoproteins with aberrant N-glycosylation levels associated with PE, including CD34, ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase family member 1), insulin-like growth factor binding protein (IGFBP3), and HYOU1 (hypoxia up-regulated 1). An emerging phenomenon that N-glycosylation is involved in several PE pathways, including cell adhesion molecules, PI3K-Akt signaling, pyrimidine metabolism, and metabolic pathways was revealed by pathway analysis of PE-associated aberrant glycoproteins. After enzymolysis, the proteins in each group were enriched with N-glycosylated peptides by lectin, the glycochain was excised by peptide N-glycosidase F (PNGase F) in H218O, and the glycosylated sites were analyzed by LC-MS/MS to achieve large-scale qualitative and quantitative analysis of N-glycosylated proteins. Our findings highlight the role of N-glycosylation in the pathogenesis of PE and provide new molecular and system-level insights for understanding and treating this disease.

Morfofunctional and Molecular Changes in Placenta and Peripheral Blood in Preeclampsia and Gestational Diabetes Mellitus

Gestational diabetes mellitus (GDM) and preeclampsia (PE) are common pregnancy complications with similar risk factors. Although GDM is associated with PE, the exact mechanism underlying the association is unclear. The objective of this work was to study the morphofunctional and molecular changes in the placenta and peripheral blood in PE and GDM. Local and systemic changes in the production of several placental proteins were assessed along with markers of inflammation and metabolic disorders. Expression of placental lactogen, trophoblastic β1-glycoprotein, placental alpha-1-microglobulin, and proteinase 3 in villi was found to change in complicated pregnancy groups. Similarity of underlying pathogenic mechanisms was demonstrated for PE and GDM.