STUDY QUESTION:Are maternal levels of moderate-to-vigorous physical activity (MVPA) and sedentary time (ST) in obese pregnant women associated with placental structural adaptations for facilitating oxygen delivery to the fetus? SUMMARY ANSWER:Higher maternal MVPA and ST are associated with a higher density of villi, a proxy measure of placental surface area for oxygen delivery to the fetus, without further added placental vessels. WHAT IS KNOWN ALREADY:Physical activity during pregnancy intermittently reduces uterine blood flow, potentially limiting placental and fetal oxygen supply. The placenta can mount several adaptive responses, including enlargement of the surface area of villi and/or feto-placental vessels to accommodate fetal needs. Early research on the morphology and growth of the placenta with exercise interventions has shown inconsistencies and is lacking, particularly in non-lean pregnant women. STUDY DESIGN, SIZE, DURATION:This study is a secondary longitudinal analysis of the vitamin D and lifestyle intervention for gestational diabetes prevention (DALI) randomized controlled trial. The prospective study was conducted between 2012 and 2015 in nine European countries at 11 different sites. In this analysis, 92 pregnant women with a BMI ≥ 29 kg/m2 were combined into one cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS:MVPA and percentage of time spent sedentary (% ST) were measured with accelerometers during gestation. Placental sections were immunostained for endothelial cell-specific CD34. Artificial intelligence (AI)-based stereology assessed villous density, number, and cross-sectional area of vessels on whole-slide images and in selected regions comprising peripheral villi only, where the majority of vascular adaptations occur. Expression of pro- and anti-angiogenic factors was quantified using molecular counting analysis. MAIN RESULTS AND THE ROLE OF CHANCE:In multivariable regression, higher levels of maternal MVPA (min/day) were associated with a higher density of villi in both whole-slide images (beta 0.12; 95% CI 0.05, 0.2) and selected regions (0.17; CI 0.07, 0.26). Unexpectedly, ST was also positively associated with density of villi (0.23; CI 0.04, 0.43). MVPA and ST were not associated with vessel count/mm2 villous area, vessel area, or pro- and anti-angiogenic factor mRNA expression. All estimates and statistical significance of the sensitivity analyses excluding smokers, women who developed gestational diabetes or pre-eclampsia and/or pregnancy-induced hypertension were similar in the main analysis. LIMITATIONS, REASONS FOR CAUTION:The placenta is a complex organ undergoing dynamic changes. While various adjustments were made to account for different maternal contributing factors, in addition to the outcome measures, various other factors could impact oxygen delivery to the fetus. WIDER IMPLICATIONS OF THE FINDINGS:For the first time, we evaluated the association between placental structures quantified using an AI-based approach with objectively measured physical activity and ST at multiple time points in pregnant women with obesity. The observed adaptations contribute to the advancement of our understanding of the hemodynamics and adaptations of the placental unit in response to MVPA and ST. However, our results might not be generalizable to lean pregnant women. STUDY FUNDING/COMPETING INTEREST(S):The DALI project has received funding from the European Community's 7th Framework Program (FP7/2007-2013) under grant agreement no. 242187. The funders had no role in study design, collection of data, analyses, writing of the article, or the decision to submit it for publication. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER:ISRCTN70595832.

BACKGROUND:The human placenta, a tissue with a lifespan limited to the period of pregnancy, is exposed to varying shear rates by maternal blood perfusion depending on the stage of development. In this study, we aimed to investigate the effects of fluidic shear stress on the human trophoblast transcriptome and metabolism.RESULTS:Based on a trophoblast cell line cultured in a fluidic flow system, changes caused by shear stress were analyzed and compared to static conditions. RNA sequencing and bioinformatics analysis revealed an altered transcriptome and enriched gene ontology terms associated with amino acid and mitochondrial metabolism. A decreased GLUT1 expression and reduced glucose uptake, together with downregulated expression of key glycolytic rate-limiting enzymes, hexokinase 2 and phosphofructokinase 1 was observed. Altered mitochondrial ATP levels and mass spectrometry data, suggested a shift in energy production from glycolysis towards mitochondrial oxidative phosphorylation. This shift in energy production could be supported by increased expression of glutamic-oxaloacetic transaminase variants in response to shear stress as well as under low glucose availability or after silencing of GLUT1. The shift towards amino acid metabolic pathways could be supported by significantly altered amino acid levels, like glutamic acid, cysteine and serine. Downregulation of GLUT1 and glycolytic rate-limiting enzymes, with concomitant upregulation of glutamic-oxaloacetic transaminase 2 was confirmed in first trimester placental explants cultured under fluidic flow. In contrast, high fluid shear stress decreased glutamic-oxaloacetic transaminase 2 expression in term placental explants when compared to low flow rates. Placental tissue from pregnancies with intrauterine growth restriction are exposed to high shear rates and showed also decreased glutamic-oxaloacetic transaminase 2, while GLUT1 was unchanged and glycolytic rate-limiting enzymes showed a trend to be upregulated. The results were generated by using qPCR, immunoblots, quantification of immunofluorescent pictures, padlock probe hybridization, mass spectrometry and FRET-based measurement.CONCLUSION:Our study suggests that onset of uteroplacental blood flow is accompanied by a shift from a predominant glycolytic- to an alternative amino acid converting metabolism in the villous trophoblast. Rheological changes with excessive fluidic shear stress at the placental surface, may disrupt this alternative amino acid pathway in the syncytiotrophoblast and could contribute to intrauterine growth restriction.

Background Matrix metalloproteinase 12 (MMP12) is a macrophage-secreted protein that is massively upregulated as a pro-inflammatory factor in metabolic and vascular tissues of mice and humans suffering from cardiometabolic diseases (CMDs). However, the molecular mechanisms explaining the contributions of MMP12 to CMDs are still unclear. Methods We investigated the impact of MMP12 deficiency on CMDs in a mouse model that mimics human disease by simultaneously developing adipose tissue inflammation, insulin resistance, and atherosclerosis. To this end, we generated and characterized low-density lipoprotein receptor (Ldlr)/Mmp12-double knockout (DKO) mice fed a high-fat sucrose- and cholesterol-enriched diet for 16–20 weeks. Results DKO mice showed lower cholesterol and plasma glucose concentrations and improved insulin sensitivity compared with LdlrKO mice. Untargeted proteomic analyses of epididymal white adipose tissue revealed that inflammation- and fibrosis-related pathways were downregulated in DKO mice. In addition, genetic deletion of MMP12 led to alterations in immune cell composition and a reduction in plasma monocyte chemoattractant protein-1 in peripheral blood which indicated decreased low-grade systemic inflammation. Aortic en face analyses and staining of aortic valve sections demonstrated reduced atherosclerotic plaque size and collagen content, which was paralleled by an improved relaxation pattern and endothelial function of the aortic rings and more elastic aortic sections in DKO compared to LdlrKO mice. Shotgun proteomics revealed upregulation of anti-inflammatory and atheroprotective markers in the aortas of DKO mice, further supporting our data. In humans, MMP12 serum concentrations were only weakly associated with clinical and laboratory indicators of CMDs. Conclusion We conclude that the genetic deletion of MMP12 ameliorates obesity-induced low-grade inflammation, white adipose tissue dysfunction, biomechanical properties of the aorta, and the development of atherosclerosis. Therefore, therapeutic strategies targeting MMP12 may represent a promising approach to combat CMDs.

Detecting and quantifying surface densities of placental villi and their vasculature adds important information on the development of the placenta under different exposures and pathological conditions. Today, a larger number of samples and tissue areas can be examined using automated Artificial Intelligence-based approaches. Although each image series calls for a particular approach, sharing the methods will help in facilitating reproducibility and comparability. Here we show the protocol of a software-based quantification of vessels (number and area) in villous tissues of human placentas, based on scanned images of full-size placental sections.

Angiotensin II receptor 1 blockers are commonly used to treat hypertension in women of childbearing age. While the fetotoxic effects of these drugs in the second and third trimesters of pregnancy are well documented, their possible impacts on placenta development in early gestation are unknown. Candesartan, a member of this group, also acts as a peroxisome proliferator-activated receptor gamma (PPARγ) agonist, a key regulator shown to be important for placental development. We have previously shown that trophoblasts do not express the candesartan target–receptor angiotensin II type 1 receptor AGTR1. This study investigated the possible role of candesartan on trophoblastic PPARγ and its hallmark target genes in early gestation. Candesartan did not affect the PPARγ protein expression or nuclear translocation of PPARγ. To mimic extravillous trophoblasts (EVTs) and cytotrophoblast/syncytiotrophoblast (CTB/SCT) responses to candesartan, we used trophoblast cell models BeWo (for CTB/SCT) and SGHPL-4 (EVT) cells as well as placental explants. In vitro, the RT-qPCR analysis showed no effect of candesartan treatment on PPARγ target genes in BeWo or SGHPL-4 cells. Treatment with positive control rosiglitazone, another PPARγ agonist, led to decreased expressions of LEP and PPARG1 in BeWo cells and an increased expression of PPARG1 in SGHPL-4 cells. Our previous data showed early gestation–placental AGTR1 expression in fetal myofibroblasts only. In a CAM assay, AGTR1 was stimulated with angiotensin II and showed increased on-plant vessel outgrowth. These results suggest candesartan does not negatively affect PPARγ or its target genes in human trophoblasts. More likely, candesartan from maternal serum may first act on fetal-placental AGTR1 and influence angiogenesis in the placenta, warranting further research.

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative studies of the changes in densities and distributions of such features, an unbiased sampling protocol is vital for reliable results. Here, we present a workflow for the image acquisition of brain samples. The workflow allows systematic uniform random sampling within a defined brain region, and the images can be analyzed using a disector. This technique is much faster than extensive examination of serial sections but still presents a feasible approach to estimate the densities and distributions of ultrastructure features. Before embedding, stained vibratome sections were used as a reference to identify the brain region under investigation, which helped speed up the overall specimen preparation process. This approach was used for comparative studies investigating the effect of an enriched-housing environment on several ultrastructural parameters in the mouse brain. Based on the successful use of the workflow, we adapted it for the purpose of elemental analysis of brain samples. We optimized the protocol in terms of the time of user-interaction. Automating all the time-consuming steps by compiling a script for the open source software SerialEM helps the user to focus on the main work of acquiring the elemental maps. As in the original workflow, we paid attention to the unbiased sampling approach to guarantee reliable results.

Background: The numerical density of synapses and their ultrastructural features are best assessed with electron microscopy. Counting is done within counting frames placed on a pair of sections (disector technique). But this requires that the thin sections are taken from comparable brain regions and the disectors are placed in a uniform random fashion. Small brain areas like the polymorph layer of the mouse dentate gyrus are difficult to encounter, and manually moving the microscope stage for placing the micrographs seems arbitrary.New method: Here the polymorph layer was approximated with 20 mu m thin, Nissl-stained vibratome sections. The subsequent vibratome section was processed for electron microscopy and serially thin sectioned. The microscope stage was moved using a random number generator, placing at least 20 disectors onto a pair of sections. The numerical synapse density, the numerical density of dense-core vesicles, and other ultrastructural features were compared between mice that had been kept in an enriched environment and mice kept under standard housing conditions.Results: Environmental enrichment significantly decreased the numerical density of dense-core vesicles and synaptic cleft widths within the polymorph layer, associated with behavioral improvement in the Morris water maze, a hippocampus-dependent task of spatial learning and memory.Comparison with existing methods: This procedure was easy to handle and enabled us to produce thin sections in small, defined brain areas. Furthermore, placing the disectors with random numbers excluded observer bias.Conclusions: Our procedure provides an uncomplicated way of assessing numerical densities in small brain areas in an unbiased manner. (C) 2014 The Authors. Published by Elsevier B.V.

The pathogenesis of preeclampsia (PE) includes the release of placental factors into the maternal circulation, inducing an inflammatory environment in the mother. One of the factors may be the proinflammatory chemokine fractalkine, which is expressed in the syncytiotrophoblast of human placenta, from where it is released into the maternal circulation by constitutive shedding. We examined whether placental fractalkine is up-regulated in severe early-onset PE and whether the proinflammatory cytokines tumor necrosis factor (TNF)-alpha and IL-6 are able to increase the expression of fractalkine. Gene expression analysis, enzyme-linked immunosorbent assay, and immunohistochemistry consistently showed increased fractalkine expression in placentas from severe early-onset PE, compared to gestational age-matched controls. Expression of a disintegrin and metalloproteinases (ADAMs) 10 and 17, which convert transmembrane fractalkine into the soluble form, was significantly increased in these cases. Incubation of first-trimester placental explants with TNF-alpha provoked a significant increase in fractalkine expression and release of the soluble form, whereas IL-6 had no effect. TNF-alpha-mediated up-regulation of placental fractalkine was reversed in the presence of the aspirin-derivative salicylate, which impaired activation of NF-kappa B p65 in TNF-alpha-treated explants. On the basis of data from placental explants, we suggest that increased maternal TNF-alpha may up-regulate the expression and release of placental fractalkine, which, in turn, may contribute to an exaggerated systemic inflammatory response in PE.