Fluid shear stress induces a shift from glycolytic to amino acid pathway in human trophoblasts.

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.

Early human trophoblast development: from morphology to function.

Human pregnancy depends on the proper development of the embryo prior to implantation and the implantation of the embryo into the uterine wall. During the pre-implantation phase, formation of the morula is followed by internalization of blastomeres that differentiate into the pluripotent inner cell mass lineage, while the cells on the surface undergo polarization and differentiate into the trophectoderm of the blastocyst. The trophectoderm mediates apposition and adhesion of the blastocyst to the uterine epithelium. These processes lead to a stable contact between embryonic and maternal tissues, resulting in the formation of a new organ, the placenta. During implantation, the trophectoderm cells start to differentiate and form the basis for multiple specialized trophoblast subpopulations, all of which fulfilling specific key functions in placentation. They either differentiate into polar cells serving typical epithelial functions, or into apolar invasive cells that adapt the uterine wall to progressing pregnancy. The composition of these trophoblast subpopulations is crucial for human placenta development and alterations are suggested to result in placenta-associated pregnancy pathologies. This review article focuses on what is known about very early processes in human reproduction and emphasizes on morphological and functional aspects of early trophoblast differentiation and subpopulations.

(Dis)similarities between the Decidual and Tumor Microenvironment.

Placenta-specific trophoblast and tumor cells exhibit many common characteristics. Trophoblast cells invade maternal tissues while being tolerated by the maternal immune system. Similarly, tumor cells can invade surrounding tissues and escape the immune system. Importantly, both trophoblast and tumor cells are supported by an abetting microenvironment, which influences invasion, angiogenesis, and immune tolerance/evasion, among others. However, in contrast to tumor cells, the metabolic, proliferative, migrative, and invasive states of trophoblast cells are under tight regulatory control. In this review, we provide an overview of similarities and dissimilarities in regulatory processes that drive trophoblast and tumor cell fate, particularly focusing on the role of the abetting microenvironments.

The fate of human SUSD2+ endometrial mesenchymal stem cells during decidualization.

Regeneration of the endometrial stromal compartment in premenopausal women is likely maintained by the perivascular endometrial mesenchymal stem/stromal cells (eMSC) expressing sushi domain containing 2 (SUSD2). The fate of SUSD2+ eMSC during pregnancy and their role in decidualization is not fully known. The aim of our study was to determine the effect of progesterone on the stemness of the SUSD2+ eMSC isolated from non-pregnant uterine samples. Secondary objectives were to characterize the functional capacity including differentiation and clonogenicity assays of SUSD2+ eMSC isolated from decidua at full term and compare it to the capacity of those isolated from non-pregnant uterine samples. Progesterone treatment induced changes in the decidual gene expression profile in non-pregnant SUSD2+ eMSC. Data analysis of a publicly available single cell RNA-seq data set revealed differential expression of several mesenchymal and epithelial signature genes between the SUSD2+ eMSC and the decidual stromal cells, suggesting mesenchymal-to-epithelial transition occurs during decidualization. Histological analysis revealed a significantly lower abundance of SUSD2+ eMSC in 1st trimester and full term samples compared to non-pregnant samples, p = 0.0296 and 0.005, respectively. The differentiation and the colony forming capacity did not differ significantly between the cells isolated from non-pregnant and pregnant uterine samples. Our results suggest that SUSD2+ eMSC undergo decidualization in vitro, while maintaining MSC plasma membrane phenotype. Human eMSC seem to play an important role in the course of endometrial decidualization and embryo implantation. Pregnancy reduced the abundance of SUSD2+ eMSC, however eMSC function remains intact.

Automated Quantitative Image Evaluation of Antigen Retrieval Methods for 17 Antibodies in Placentation and Implantation Diagnostic and Research.

Immunostaining in clinical routine and research highly depends on standardized staining methods and quantitative image analyses. We qualitatively and quantitatively compared antigen retrieval methods (no pretreatment, pretreatment with pepsin, and heat-induced pretreatment with pH 6 or pH 9) for 17 antibodies relevant for placenta and implantation diagnostics and research. Using our newly established, comprehensive automated quantitative image analysis approach, fluorescent signal intensities were evaluated. Automated quantitative image analysis found that 9 out of 17 antibodies needed antigen retrieval to show positive staining. Heat induction proved to be the most efficient form of antigen retrieval. Eight markers stained positive after pepsin digestion, with ß-hCG and vWF showing enhanced staining intensities. To avoid the misinterpretation of quantitative image data, the qualitative aspect should always be considered. Results from native placental tissue were compared with sections of a placental invasion model based on thermo-sensitive scaffolds. Immunostaining on placentas in vitro leads to new insights into fetal development and maternal pathophysiological pathways, as pregnant women are justifiably excluded from clinical studies. Thus, there is a clear need for the assessment of reliable immunofluorescent staining and pretreatment methods. Our evaluation offers a powerful tool for antibody and pretreatment selection in placental research providing objective and precise results.

Placental Endocrine Activity: Adaptation and Disruption of Maternal Glucose Metabolism in Pregnancy and the Influence of Fetal Sex.

The placenta is an endocrine fetal organ, which secretes a plethora of steroid- and proteo-hormones, metabolic proteins, growth factors, and cytokines in order to adapt maternal physiology to pregnancy. Central to the growth of the fetus is the supply with nutrients, foremost with glucose. Therefore, during pregnancy, maternal insulin resistance arises, which elevates maternal blood glucose levels, and consequently ensures an adequate glucose supply for the developing fetus. At the same time, maternal ß-cell mass and function increase to compensate for the higher insulin demand. These adaptations are also regulated by the endocrine function of the placenta. Excessive insulin resistance or the inability to increase insulin production accordingly disrupts physiological modulation of pregnancy mediated glucose metabolism and may cause maternal gestational diabetes (GDM). A growing body of evidence suggests that this adaptation of maternal glucose metabolism differs between pregnancies carrying a girl vs. pregnancies carrying a boy. Moreover, the risk of developing GDM differs depending on the sex of the fetus. Sex differences in placenta derived hormones and bioactive proteins, which adapt and modulate maternal glucose metabolism, are likely to contribute to this sexual dimorphism. This review provides an overview on the adaptation and maladaptation of maternal glucose metabolism by placenta-derived factors, and highlights sex differences in this regulatory network.

Platelet-derived factors impair placental chorionic gonadotropin beta-subunit synthesis.

During histiotrophic nutrition of the embryo, maternal platelets may be the first circulating maternal cells that find their way into the placental intervillous space through narrow intertrophoblastic gaps within the plugs of spiral arteries. Activation of platelets at the maternal-fetal interface can influence trophoblast behavior and has been implicated in serious pregnancy pathologies. Here, we show that platelet-derived factors impaired expression and secretion of the human chorionic gonadotropin beta-subunit (ßhCG) in human first trimester placental explants and the trophoblast cell line BeWo. Impaired ßhCG synthesis was not the consequence of hampered morphological differentiation, as assessed by analysis of differentiation-associated genes and electron microscopy. Platelet-derived factors did not affect intracellular cAMP levels and phosphorylation of CREB, but activated Smad3 and its downstream-target plasminogen activator inhibitor (PAI)-1 in forskolin-induced BeWo cell differentiation. While TGF-ß type I receptor inhibitor SB431542 did not restore impaired ßhCG production in response to platelet-derived factors, Smad3 inhibitor SIS3 interfered with CREB activation, suggesting an interaction of cAMP/CREB and Smad3 signaling. Sequestration of transcription co-activators CBP/p300, known to bind both CREB and Smad3, may limit ßhCG production, since CBP/p300 inhibitor C646 significantly restricted its forskolin-induced upregulation. In conclusion, our study suggests that degranulation of maternal platelets at the early maternal-fetal interface can impair placental ßhCG production, without substantially affecting morphological and biochemical differentiation of villous trophoblasts. KEY MESSAGES: Maternal platelets can be detected on the surface of the placental villi and in intercellular gaps of trophoblast cell columns from gestational week 5 onwards. Platelet-derived factors impair hCG synthesis in human first trimester placenta. Platelet-derived factors activate Smad3 in trophoblasts. Smad3 inhibitor SIS3 interferes with forskolin-induced CREB signaling. Sequestration of CBP/p300 by activated Smad3 may limit placental hCG production.

Maternal Platelets­Friend or Foe of the Human Placenta?

Human pregnancy relies on hemochorial placentation, including implantation of the blastocyst and deep invasion of fetal trophoblast cells into maternal uterine blood vessels, enabling direct contact of maternal blood with placental villi. Hemochorial placentation requires fast and reliable hemostasis to guarantee survival of the mother, but also for the neonates. During human pregnancy, maternal platelet count decreases gradually from first, to second, and third trimester. In addition to hemodilution, accelerated platelet sequestration and consumption in the placental circulation may contribute to a decline of platelet count throughout gestation. Local stasis, turbulences, or damage of the syncytiotrophoblast layer can activate maternal platelets within the placental intervillous space and result in formation of fibrin-type fibrinoid. Perivillous fibrinoid is a regular constituent of the normal placenta which is considered to be an important regulator of intervillous hemodynamics, as well as having a role in shaping the developing villous trees. However, exaggerated activation of platelets at the maternal-fetal interface can provoke inflammasome activation in the placental trophoblast, and enhance formation of circulating platelet-monocyte aggregates, resulting in sterile inflammation of the placenta and a systemic inflammatory response in the mother. Hence, the degree of activation determines whether maternal platelets are a friend or foe of the human placenta. Exaggerated activation of maternal platelets can either directly cause or propagate the disease process in placenta-associated pregnancy pathologies, such as preeclampsia.

Histological processing of un-/cellularized thermosensitive electrospun scaffolds.

Histological processing of thermosensitive electrospun poly(ε-caprolactone)/poly(L-lactide) (PCL/PLA) scaffolds fails, as poly(ε-caprolactone) (PCL) is characterized by its low-melting temperature (Tm = 60 °C). Here, we present an optimized low-temperature preparation method for the histological processing of un-/cellularized thermosensitive PCL/PLA scaffolds.Our study is aimed at the establishment of an optimized dehydration and low-melting-point paraffin-embedding method of electrospun PCL/PLA scaffolds (un-/cellularized). Furthermore, we compared this method with (a) automatized dehydration and standard paraffin embedding, (b) gelatin embedding followed by automatized dehydration and standard paraffin embedding, (c) cryofixation, and (d) acrylic resin embedding methods. We investigated pepsin and proteinase K antigen retrieval for their efficiency in epitope demasking at low temperatures and evaluated protocols for immunohistochemistry and immunofluorescence for cytokeratin 7 (CK7) and in situ padlock probe technology for beta actin (ACTB). Optimized dehydration and low-melting-point paraffin embedding preserved the PCL/PLA scaffold, as the diameter and structure of its fibers were unchanged. Cells attached to the PCL/PLA scaffolds showed limited alterations in size and morphology compared to control. Epitope demasking by enzymatic pepsin digestion and immunostaining of CK7 displayed an invasion of attached cells into the scaffold. Expression of ACTB and CK7 was shown by a combination of mRNA-based in situ padlock probe technology and immunofluorescence. In contrast, gelatin stabilization followed by standard paraffin embedding led to an overall shrinkage and melting of fibers, and therefore, no further analysis was possible. Acrylic resin embedding and cyrofixation caused fiber structures that were nearly unchanged in size and diameter. However, acrylic resin-embedded scaffolds are limited to 3 µm sections, whereas cyrofixation led to a reduction of the cell size by 14% compared to low-melting paraffin embedding. The combination of low-melting-point paraffin embedding and pepsin digestion as an antigen retrieval method offers a successful opportunity for histological investigations in thermosensitive specimens.

Human trophoblast invasion: new and unexpected routes and functions.

Until recently, trophoblast invasion during human placentation was characterized by and restricted to invasion into uterine connective tissues and the uterine spiral arteries. The latter was explained to connect the arteries to the intervillous space of the placenta and to guarantee the blood supply of the mother to the placenta. Today, this picture has dramatically changed. Invasion of endoglandular trophoblast into uterine glands, already starting at the time of implantation, enables histiotrophic nutrition of the embryo prior to perfusion of the placenta with maternal blood. This is followed by invasion of endovenous trophoblasts into uterine veins to guarantee the drainage of fluids from the placenta back into the maternal circulation throughout pregnancy. In addition, invasion of endolymphatic trophoblasts into the lymph vessels of the uterus has been described. Only then, invasion of endoarterial trophoblasts into spiral arteries takes place, enabling hemotrophic nutrition of the fetus starting with the second trimester of pregnancy. This new knowledge paves the way to identify changes that may occur in pathological pregnancies, from tubal pregnancies to recurrent spontaneous abortions.

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.

Implantation and extravillous trophoblast invasion: From rare archival specimens to modern biobanking.

Extravillous trophoblast invasion serves to attach the placenta to the uterus and to enable access to nutrients for the embryo throughout pregnancy - secretions of the uterine glands in the first trimester, maternal blood in the second and third trimester. For assessing extravillous trophoblast invasion, histology (in combination with immunohistochemistry) still plays a major role in placental research. This is especially true for the re-assessment of rare archival specimens from early human implantation sites or placenta in utero with the background of recent knowledge which may help to strengthen current hypotheses. This review summarizes the recently expanded picture of extravillous trophoblast invasion, gives an overview about fundamental archival specimens in placental research, presents new images of archival specimens, gives insights into the latest developments in the field of biobanking and provides insight into the current situation on sample usage in the absence of biobanks. Modern techniques allow expanding our hitherto believed concept of extravillous trophoblast invasion, which is not restricted to spiral arteries: Extravillous trophoblasts also invade into uterine glands and uterine veins and thereby connect all these luminal structures with the intervillous space. All biomedical research dramatically depends on the quality of the assessed biological samples. Hence, researchers should be aware that the time between collection of a sample from a body and the beginning of analysis (pre-analytical phase) may have more impact on the outcome of a study than previously assumed.

Placental DAPK1 and autophagy marker LC3B-II are dysregulated by TNF-α in a gestational age-dependent manner.

Autophagy, a cell-survival process responsible for degradation of protein aggregates and damaged organelles, is increasingly recognized as another mechanism essential for human placentation. A substantial body of experiments suggests inflammation and oxidative stress as the underlying stimuli for altered placental autophagy, giving rise to placenta dysfunction and pregnancy pathologies. Here, the hypothesis is tested whether or not pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α are able to influence the expression profile of autophagy genes in human first-trimester villous placenta. Autophagy-focused qPCR arrays identified substantial downregulation of death-associated protein kinase 1 (DAPK1) in first-trimester placental explants in response to IL-6 and TNF-α, respectively. Immunohistochemistry of placental explants detected considerable DAPK1 staining in placental macrophages, villous cytotrophoblasts and less intense in the syncytiotrophoblast. Both immunohistochemistry and Western blot showed decreased DAPK1 protein in TNF-α-treated placental explants compared to control. On cellular level, DAPK1 expression decreased in SGHPL-4 trophoblasts in response to TNF-α. Observed changes in the expression profile of autophagy-related genes were reflected by significantly decreased lipidation of autophagy marker microtubule-associated protein light chain 3 beta (LC3B-II) in first trimester placental explants in response to TNF-α. Analysis of TNF-α-treated term placental explants showed decreased DAPK1 protein, whereas in contrast to first-trimester LC3B expression and lipidation increased. Immunohistochemistry of placental tissues from early-onset preeclampsia (PE) showed less DAPK1 staining, when compared to controls. Accordingly, DAPK1 mRNA and protein were decreased in primary trophoblasts isolated from early-onset PE, while LC3B-I and -II were increased. Results from this study suggest that DAPK1, a regulator of apoptosis, autophagy and programmed necrosis, decreases in human placenta in response to elevated maternal TNF-α, irrespective of gestational age. In contrast, TNF-α differentially regulates levels of autophagy marker LC3B in human placenta over gestation.

Extravillous trophoblasts invade more than uterine arteries: evidence for the invasion of uterine veins.

During the first trimester of pregnancy, extravillous trophoblasts (EVTs) invade into the decidual interstitium to the first third of the myometrium, thereby anchoring the placenta to the uterus. They also follow the endovascular and endoglandular route of invasion; plug, line and remodel spiral arteries, thus being responsible for the establishment of hemotrophic nutrition with the beginning of the second trimester and invade and open uterine glands toward the intervillous space for a histiotrophic nutrition during the first trimester. The aim of this study was to provide proof that uterine veins are invaded by EVTs similar to uterine arteries and glands in first trimester of pregnancy. Therefore, serial sections from in situ first trimester placenta were immuno-single- and immuno-double-stained to distinguish in a first step between arteries and veins and secondly between invaded and non-invaded vessels. Subsequently, invasion of EVTs into uterine vessels was quantified. Our data show that uterine veins are significantly more invaded by EVTs than uterine arteries (29.2 ± 15.7 %) during early pregnancy. Counted vessel cross sections revealed significantly higher EVT invasion into veins (59.5 ± 7.9 %) compared to arteries (29.2 ± 15.7 %). In the lumen of veins, single EVTs were repeatedly found, beside detached glandular epithelial cells or syncytial fragments. This study allows the expansion of our hitherto postulated concept of EVT invasion during first trimester of pregnancy. We suggest that invasion of EVTs into uterine veins is responsible the draining of waste and blood plasma from the intervillous space during the first trimester of pregnancy.

The trophoblast plug during early pregnancy: a deeper insight.

During the first trimester of pregnancy, foetal endovascular trophoblasts invade into maternal spiral arteries, accumulate and form plugs in the lumen of the vessels. These plugs only allow blood plasma to seep through. Hence, during the first trimester of pregnancy, a first flow of fluids through the placental intervillous space is established, resulting in a physiological oxygen gradient between mother and foetus. The trophoblast plugs block spiral arteries until the beginning of the second trimester (11-14 weeks). In parallel, uterine glands are invaded and opened by endoglandular trophoblasts towards the intervillous space of the placenta, without showing the formation of plugs (Moser et al. in Hum Reprod 25:1127-1136, 2010, Hum Reprod Oxf Engl 30:2747-2757, 2015). This enables histiotrophic nutrition of the embryo prior to onset of maternal blood flow into the placenta. Failure of these endovascular and endoglandular invasion processes may lead to miscarriage or pregnancy disorders such as intrauterine growth restriction (IUGR). After dissolution of the plugs, the onset of maternal blood flow allows maternal blood cells to enter the intervillous space and oxygen concentrations rise up. In this study, we demonstrate for the first time serial cross sections through a trophoblast plug in a first trimester placental bed specimen. Invaded and plugged arteries as well as invaded uterine glands in week 11 of gestation are visualized with specific immunohistochemical double staining techniques. We show that spiral artery plugs appear throughout the placental invasion zone and illustrate erythrocytes stowed due to trophoblast plugs. In addition, we give evidence of the presence of MMP-1 in plugs of invaded spiral arteries. The results reveal a better understanding and a closer insight into the morphological appearance of trophoblast plugs and the consequences for placental and uterine blood flow.

Arterial endothelial cytokines guide extravillous trophoblast invasion towards spiral arteries; an in-vitro study with the trophoblast cell line ACH-3P and female non-uterine endothelial cells.

INTRODUCTION: Invasion of extravillous trophoblasts (EVT) is tightly linked to appropriate cell to cell contact as well as paracrine guidance of EVT by maternal uterine cells, conducted by a variety of locally expressed cytokines. Here we investigated the interaction of the first trimester trophoblast cell line ACH-3P with adult iliac arterial (AEC) and venous endothelial cells (VEC). METHODS: The impact of ACH-3P conditioned medium (Cdm), obtained at 2.5% and 21% oxygen, on endothelial cell viability (LDH-Assay) and network formation (Matrigel-Assay) was tested. We investigated cytokine expression of AEC- and VEC-Cdm and confirmed results with ELISA analysis, and investigated the influence of Cdm on ACH-3P proliferation and invasion. Additionally, direct co-culture experiments with ACH-3P and AEC on Matrigel were performed. A subset of experiments was verified with primary trophoblasts as well as with first trimester placenta in situ specimens. RESULTS: ACH-3P-Cdm significantly enhanced cell viability of AEC and VEC after 72 h. ACH-3P-Cdm at 2.5% oxygen stabilized endothelial network structures in Matrigel up to 24 h, similar to the effect of a direct co-culture of AEC and ACH-3P. AEC and VEC showed a similar pattern of secreted cytokines. However, elevated levels of cytokines secreted by AEC were found for GRO, IL-6, MMP-1 and uPAR. ELISA confirmed elevated concentrations of IL-6 and uPAR in AEC compared to VEC. ACH-3P and primary trophoblasts more likely invaded towards AEC-Cdm than towards VEC-Cdm. Addition of IL-6 to Cdm increased the invasion potential of both cell types. AEC- and VEC-Cdm reduced ACH-3P cell proliferation after 24 h of culture. IL-6 was highly expressed in uterine AEC compared to VEC as visualized by immunohistochemistry. DISCUSSION: The presented results clearly demonstrate that cytokines of both cell types, AEC and trophoblasts, differentially contribute to successful guidance and interaction in the process of trophoblast invasion.

Phospholipid scramblase 1 (PLSCR1) in villous trophoblast of the human placenta.

A crucial factor for effective villous trophoblast fusion in the human placenta is the transient deregulation of plasma membrane phospholipid asymmetry leading to externalization of phosphatidylserine to the outer membrane leaflet. Screening of scramblase family members implicated in the collapse of phospholipid asymmetry revealed that phospholipid scramblase 1 (PLSCR1) is strongly expressed in villous trophoblast. Therefore, we assessed the putative role of PLSCR1 in villous trophoblast fusion. Spatio-temporal analysis in first trimester and term placenta showed abundant expression of PLSCR1 in syncytiotrophoblast, macrophages and endothelial cells, while it was virtually absent in villous cytotrophoblasts. For functional studies, BeWo cells, isolated primary term trophoblasts and first trimester villous explants were used. During forskolin-mediated BeWo cell differentiation, neither PLSCR1 mRNA nor protein levels showed significant changes. In contrast, when primary trophoblasts were stimulated with Br-cAMP, a decrease in PLSCR1 mRNA and protein expression was observed. To elucidate a role for PLSCR1 in syncytialization, we used RNA interference and a chemical scramblase inhibitor, R5421 (ethanedioic acid). Silencing of PLSCR1 using siRNA had no effects while inhibition of scramblase activity by R5421 increased GCM-1 mRNA expression, beta-hCG protein secretion and fusion rates of BeWo cells. In primary trophoblasts and villous explants, no effects of siRNA or R5421 treatment on fusion were detected. This study provides data on PLSCR1 localization and general expression in the human placenta. The data make it tempting to speculate on a role of PLSCR1 in negatively regulating trophoblast fusion.

Trophoblast invasion and oxygenation of the placenta: measurements versus presumptions.

Invasion of extravillous trophoblast into maternal tissues has a profound effect on the oxygenation of the placenta and hence the fetus. The main route of trophoblast invasion is interstitial invasion into the tissues of the decidua and myometrium. From this main route side branches reach the spiral arteries (endovascular trophoblast) as well as the uterine glands (endoglandular trophoblast) to open both structures toward the intervillous space. This enables histiotrophic nutrition in the first trimester and hemotrophic nutrition in the second and third trimesters of pregnancy. Failure of endovascular trophoblast invasion has profound effects on the oxygenation of the placenta. Interestingly, this does not lead to hypoxia as has long been presumed. Rather, all measurements available today point to increased oxygen levels within the placenta in patients with a failure of spiral artery transformation. This should lead to a rethink regarding pathological conditions such as intrauterine growth restriction and preeclampsia.

Fibulin-5 expression in the human placenta.

Fibulin-5 is a secreted extracellular matrix glycoprotein and displays a diverse panel of biological functions, which can be segregated into elastogenic as well as extra-elastogenic functions. While elastogenic functions of fibulin-5 include essential roles in early steps of elastic fibre assembly, extra-elastogenic functions are widespread. Depending on the cell type used, fibulin-5 mediates cell adherence via a subset of integrins, antagonizes angiogenesis and inhibits migration as well as proliferation of endothelial and smooth muscle cells. In this study, we focused on the spatiotemporal expression of fibulin-5 in the human placenta. With progressing gestation, placental fibulin-5 expression increased from first trimester towards term. At term, placental fibulin-5 mRNA expression is lower when compared with other well-vascularized organs such as lung, kidney, heart, uterus and testis. In first trimester, placenta immunohistochemistry localized fibulin-5 in villous cytotrophoblasts and extravillous cytotrophoblasts of the proximal cell column. In term placenta, fibulin-5 was detected in the endothelial basement membrane and adventitia-like regions of vessels in the chorionic plate and stem villi. Cell culture experiments with the villous trophoblast-derived cell line BeWo showed that fibulin-5 expression was downregulated during functional differentiation and intercellular fusion. Moreover, cultivation of BeWo cells under low oxygen conditions impaired intercellular fusion and upregulated fibulin-5 expression. The spatiotemporal shift from the trophoblast compartment in first trimester to the villous vasculature at term suggests a dual role of fibulin-5 in human placental development.

Functional and structural microbial diversity in organic and conventional viticulture: organic farming benefits natural biocontrol agents.

Statistically significant differences in the structure and function of above-ground grapevine-associated microorganisms from organically and conventionally managed vineyards were found. Aureobasidium pullulans, a copper-detoxifying fungus and biocontrol agent, plays a key role in explaining these differences. The black fungus was strongly enriched in the communities of organically managed plants and yielded a higher indigenous antiphytopathogenic potential.