Normal Human Term Placenta Research Articles

ODP434 Maternally-Derived Pancreatic Extracellular Vesicle Encompassed miRNAs Influence Placental Development in Pregnancies Complicated by Gestational Diabetes

Abstract Pregnancies affected by gestational diabetes (GDM) commonly result in large-for-gestational-age (LGA) infants; these infants have an increased risk of developing cardiometabolic complications compared to appropriate-for-gestational-age (AGA) infants. The mechanisms responsible are unclear but GDM/LGA is associated with altered placental development/function. We have recently reported altered levels of extracellular vesicle (EV) encapsulated miRNAs, including miR-375 (a pancreatic-specific miRNA) in maternal serum prior to the onset of LGA. We propose that the EVs containing miR-375 are produced by maternal pancreas, and that increased miR-375 in maternal circulation contributes to LGA by influencing placental development/function. Human placental tissue was collected from GDM pregnancies that delivered AGA (n=14) or LGA infants (n=10). Levels of mature miR-375 and primary transcript (pri-miR-375) were measured by QPCR. Mature miR-375 was increased in GDM-LGA (4.74-fold; p<0. 01) compared to GDM AGA placentae. Mature miR-375, but not pri-miR-375, was detected in human placenta suggesting that miR-375 is not produced in the placenta but instead is transported, potentially via EVs in maternal circulation. Intact human pancreatic islets (20,000 IEQ/patient) were obtained through the Integrated Islet Distribution Program (IIDP) from female donors of reproductive age. Islets were cultured in EV-depleted media containing 5.5mM (normoglycemia) or 7mM (mild hyperglycaemia) glucose for 3 days. Islet purity and viability were confirmed using dithizone incorporation and trypan blue exclusion, respectively. Media was collected at 24-hour intervals, pooled from all time points, concentrated and EVs were isolated by size exclusion chromatography. Confirmation of the presence of EVs was confirmed by electron microscopy (typical cup-shape morphology of EVs), nanoparticle tracking analysis (NTA; mean size of 83.5-163.2nm; concentration of 1.28×109-1.82×1010 particles/ml), Western blotting (EV-enriched proteins). QPCR confirmed that mature miR-375 was present in islet-derived EVs and revealed that levels were altered following culture in 7mM glucose compared to 5.5mM. Islet-derived EVs were labelled with maleimide-488 and uptake into explants of normal human term placenta was confirmed using fluorescent microscopy=3). miR-375 was overexpressed in normal human placental explants (by 30-fold, p<0. 05; n=6) using specific mimics (100nM for 72 hours) and TMT mass spectrometry was performed to assess the impact of miR-375 overexpression on the placental proteome. Functional enrichment analysis (FEA) was performed on differentially expressed proteins (DEP; P<0. 05; -0.41≥log2foldchange≥0.58) using over representation analysis and Ingenuity Pathway Analysis. This revealed that DEGs were significantly enriched in pathways associated with placental growth, mitochondrial function and glucose metabolism. Our data demonstrates that miR-375 is increased in the placenta in GDM/LGA and that this influences placental development/function. miR-375 does not appear to be produced in the placenta but instead, is likely released in EVs from maternal pancreas under conditions of hyperglycaemia and transported to the placenta, via maternal circulation. This provides insight into the potential mechanisms contributing to LGA in pregnancies complicated by GDM. Presentation: No date and time listed

Proteome profiling of human placenta reveals developmental stage-dependent alterations in protein signature

IntroductionPlacenta is a complex organ that plays a significant role in the maintenance of pregnancy health. It is a dynamic organ that undergoes dramatic changes in growth and development at different stages of gestation. In the first-trimester, the conceptus develops in a low oxygen environment that favors organogenesis in the embryo and cell proliferation and angiogenesis in the placenta; later in pregnancy, higher oxygen concentration is required to support the rapid growth of the fetus. This oxygen transition, which appears unique to the human placenta, must be finely tuned through successive rounds of protein signature alterations. This study compares placental proteome in normal first-trimester (FT) and term human placentas (TP).MethodsNormal human first-trimester and term placental samples were collected and differentially expressed proteins were identified using two-dimensional liquid chromatography-tandem mass spectrometry.ResultsDespite the overall similarities, 120 proteins were differently expressed in first and term placentas. Out of these, 72 were up-regulated and 48 were down-regulated in the first when compared with the full term placentas. Twenty out of 120 differently expressed proteins were sequenced, among them seven showed increased (GRP78, PDIA3, ENOA, ECH1, PRDX4, ERP29, ECHM), eleven decreased (TRFE, ALBU, K2C1, ACTG, CSH2, PRDX2, FABP5, HBG1, FABP4, K2C8, K1C9) expression in first-trimester compared to the full-term placentas and two proteins exclusively expressed in first-trimester placentas (MESD, MYDGF).ConclusionAccording to Reactome and PANTHER softwares, these proteins were mostly involved in response to chemical stimulus and stress, regulation of biological quality, programmed cell death, hemostatic and catabolic processes, protein folding, cellular oxidant detoxification, coagulation and retina homeostasis. Elucidation of alteration in protein signature during placental development would provide researchers with a better understanding of the critical biological processes of placentogenesis and delineate proteins involved in regulation of placental function during development.

Micro-CT and histological investigation of the spatial pattern of feto-placental vascular density

IntroductionThere are considerable variations in villous morphology within a normal placenta. However, whether there is a reproducible spatial pattern of variation in villous vascular density is not known. Micro-CT provides three-dimensional volume imaging with spatial resolution down to the micrometre scale. In this study, we applied Micro-CT and histological analysis to investigate the degree of heterogeneity of vascularisation within the placenta. MethodTen term placentas were collected at elective caesarean section, perfused with contrast agent and imaged whole with Micro-CT. Eight full depth tissue blocks were then taken from each placenta and imaged. Sections were taken for histological analysis. Data was analysed to investigate vascular fill, and vascular density in relation to location from cord insertion to placental edge at each scale. ResultsWhole placental imaging revealed no spatially consistent difference in villous vessel density within the main placental tissue, although there was a great degree of heterogeneity. Both block imaging and histological analysis found a large degree of heterogeneity of vascular density within placentas, but no strong correlation between villous vascular density and block location (rs = 0.066, p = 0.7 block imaging, rs = 0.06, p = 0.6 histological analysis). DiscussionThis work presents a novel method for imaging the human placenta vascular tree using multiscale Micro-CT imaging. It demonstrates that there is a large degree of variation in vascular density throughout normal term human placentas. The three-dimensional data created by this technique could be used, with more advanced computer analysis, to further investigate the structure of the vascular tree.

In Vitro Human Placental Studies to Support Adenovirus-Mediated VEGF-DΔNΔC Maternal Gene Therapy for the Treatment of Severe Early-Onset Fetal Growth Restriction.

Severe fetal growth restriction (FGR) affects 1 in 500 pregnancies, is untreatable, and causes serious neonatal morbidity and death. Reduced uterine blood flow (UBF) is one cause. Transduction of uterine arteries in normal and FGR animal models using an adenovirus (Ad) encoding VEGF isoforms increases UBF and improves fetal growth in utero. Understanding potential adverse consequences of this therapy before first-in-woman clinical application is essential. The aims of this study were to determine whether Ad.VEGF-DΔNΔC (1) transfers across the human placental barrier and (2) affects human placental morphology, permeability and primary indicators of placental function, and trophoblast integrity. Villous explants from normal term human placentas were treated with Ad.VEGF-DΔNΔC (5 × 107-10 virus particles [vp]/mL), or virus formulation buffer (FB). Villous structural integrity (hematoxylin and eosin staining) and tissue accessibility (LacZ immunostaining) were determined. Markers of endocrine function (human chorionic gonadotropin [hCG] secretion) and cell death (lactate dehydrogenase [LDH] release) were assayed. Lobules from normal and FGR pregnancies underwent ex vivo dual perfusion with exposure to 5 × 1010 vp/mL Ad.VEGF-DΔNΔC or FB. Perfusion resistance, para-cellular permeability, hCG, alkaline phosphatase, and LDH release were measured. Ad.VEGF-DΔNΔC transfer across the placental barrier was assessed by quantitative polymerase chain reaction in DNA extracted from fetal-side venous perfusate, and by immunohistochemistry in fixed tissue. Villous explant structural integrity and hCG secretion was maintained at all Ad.VEGF-DΔNΔC doses. Ad.VEGF-DΔNΔC perfusion revealed no effect on placental permeability, fetoplacental vascular resistance, hCG secretion, or alkaline phosphatase release, but there was a minor elevation in maternal-side LDH release. Viral vector tissue access in both explant and perfused models was minimal, and the vector was rarely detected in the fetal venous perfusate and at low titer. Ad.VEGF-DΔNΔC did not markedly affect human placental integrity and function in vitro. There was limited tissue access and transfer of vector across the placental barrier. Except for a minor elevation in LDH release, these test data did not reveal any toxic effects of Ad.VEGF-DΔNΔC on the human placenta.

Two Distinct Myeloid Subsets at the Term Human Fetal-Maternal Interface.

During pregnancy, immune cells infiltrate the placenta at different stages of fetal development. NK cells and macrophages are the most predominant cell types. These immune cells play pleiotropic roles, as they control spiral artery remodeling to ensure appropriate blood supply and maintain long-term tolerance to a true allograft; yet, they must be able to mount appropriate immune defenses to pathogens that may threaten the fetus. Whether the same cell type accomplishes all these tasks or if there are dedicated subsets remains controversial. Here, we identify and characterize two distinct subsets of myeloid cells that differ in their pro-inflammatory/regulatory capacity. While one subset predominantly produces the immune-modulating cytokine IL-10, the second subset has superior capacity to secrete pro-inflammatory mediators, such as IL-1β and IL-6. The putative regulatory myeloid cells also express high levels of inhibitory receptors and their ligands, including programmed cell death 1 (PD1) ligands. Importantly, a large fraction of CD8 and CD4 cells in normal term human placenta are PD1 positive, suggesting that the PD1/PD1 ligands axis might be critical to maintain tolerance during pregnancy.

Impact of chlorpyrifos on human villous trophoblasts and chorionic villi

Placental barrier regulates maternal-fetal interchange protecting the baby from damage caused by substances found in the uterine environment or circulating in the vascular system. Organophosphate (OP) pesticides are a paramount group of environmental pollutants used in intensive agriculture for protection against diseases and pests. While many studies have reported an increased risk of pregnancy alterations in pregnant women exposed to OPs, few have analyzed the effects caused by these pesticides in the placenta.Herein, we evaluated the effects of chlorpyrifos (CPF), one of the most widely used OP insecticides, on human placenta using in vitro and ex vivo exposure models. Villous cytotrophoblast cells isolated from normal human term placentas maintained their cell viability, differentiated into syncytiotrophoblast-like structures, and increased the expression of β-hCG, ABCG2, and P-gp in the presence of CPF at concentrations of 10 to 100μM. The same doses of CPF induced marked changes in chorionic villi samples. Indeed, CPF exposure increased stroma cell apoptosis, altered villi matrix composition, basement membrane thickness, and trophoblastic layer integrity. Histomorphological and ultrastructural alterations are compatible with those found in placentas where maternal-placenta injury is chronic and able to impair the placental barrier function and nutrient transport from mother to the fetus.Our study shows that placental ex vivo exposure to CPF produces tissue alterations and suggest that human placenta is a potential target of CPF toxicity. In addition, it highlights the importance of using different models to assess the effects of a toxic on human placenta.

First trimester infection with Listeria monocytogenes is characterized by decidual and placental inflammation in pregnant cynomolgus macaques

First trimester infection with Listeria monocytogenes is characterized by decidual and placental inflammation in pregnant cynomolgus macaques

Expression of Mammalian Target of Rapamycin and Downstream Targets in Normal and Gestational Diabetic Human Term Placenta.

Mammalian target of rapamycin (mTOR) signaling serves as a central regulator of cell growth, proliferation, and survival by interacting with various proteins. To date, few studies implicated mTOR in placenta. Human placenta in gestational diabetes mellitus (GDM) shows several alterations including villous immaturity, impaired placental function, and overgrowth. Hence, we aimed to investigate the expression of mTOR, phospho-mTOR (p-mTOR), and the 2 phosphorylated downstream targets of mTOR, ribosomal protein S6 kinase 1 (p-p70S6K), and eukaryotic initiation factor 4E-binding protein 1 (p-4EBP1) in normal term and gestational diabetic human placentas. Immunohistochemistry and Western blot were performed with antibodies against mTOR, p-mTOR, p-p70S6K, and p-4EBP1 (Thr37/46) in normal and diabetic placentas (n = 6 each) and quantified by ImageJ. All mTOR pathway components that we studied were immunolocalized in both normal and diabetic placenta groups. Syncytiotrophoblast and the vascular wall in villi displayed cytoplasmic mTOR and p-mTOR (S2448) immunoreactivities in all placenta samples. However, increased expression of p70S6K in syncytiotrophoblast and p-4EBP1 (Thr37/46) in villous stromal cells was observed in gestational diabetic placentas. Western blot analysis also confirmed the statistically significant increase in p-p70S6K (T389) expression in diabetic placentas. The altered expression of downstream components of mTOR signaling in gestational diabetic placentas suggests an involvement of mTOR activity in the placental pathology of GDM. However, whether increased nutrient transport via this pathway will stimulate fetal and placental overgrowth is still unknown. Although this is a descriptive study, further studies with a functional analysis to highlight the molecular mechanisms underlying this placental pathology are proposed.

Study of Arterial Pattern & Its Correlation with the Shape of Human Placenta by Corrosion Cast Method

Introduction: Placenta is an organ that connects the developing fetus to uterine wall to allow nutrient uptake, waste elimination and gaseous exchange via the mother’s blood supply. Placenta is the most accurate record of prenatal experience. Our aim of study was to see different vascular patterns in normal full term human placenta by corrosion cast method. Material and Methods: 100 normal placentae were collected within one hour of delivery. Normal full term placentas were taken for study and any abnormal placenta was excluded - eg: Bidiscoidal placenta, multidiscoidal placenta, lobed placenta, placenta succenturita, fenesterated placenta, circumvallate placenta, batterdore placenta, furcated placenta, velametous placenta. Results: The findings of the present study bring forth some important facts as follows: there were three type of placental shape -round, oval & irregular. Round type had more incidence than oval & irregular. Dispersed pattern & magisterial pattern was seen more in round placenta. Conclusion: These observations on normal placenta call for further studies on the variations of its vascular pattern & shape in placenta of diseased mother eg. Hypertension, diabetes, anemia etc. Key words: Placenta, Vascular pattern, corrosion cast

Decrease in Sphingomyelin (d18:1/16:0) in Stem Villi and Phosphatidylcholine (16:0/20:4) in Terminal Villi of Human Term Placentas with Pathohistological Maternal Malperfusion.

Placental villi play pivotal roles in feto-maternal transportation and phospholipids constitute a major part of the villous membrane. We have been developing and optimizing an imaging system based on a matrix-assisted laser desorption/ionization (MALDI)-based mass spectrometer, which provides clear two-dimensional molecular distribution patterns using highly sensitive mass spectrometry from mixtures of ions generated on tissue surfaces. We recently applied this technology to normal human uncomplicated term placentas and detected the specific distribution of sphingomyelin (SM) (d18:1/16:0) in stem villi and phosphatidylcholine (PC) (16:0/20:4) in terminal villi. In the present study, we applied this technology to nine placentas with maternal or fetal complications, and determined whether a relationship existed between these specific distribution patterns of phospholipid molecules and the six representative pathological findings of placentas, i.e., villitis of unknown etiology (VUE), thrombus, atherosis, chorioamnionitis (CAM), immature terminal villi, and multiple branched terminal villi. In two placentas with the first and second largest total number of positive pathological findings, i.e., five and three positive findings, the specific distribution of SM (d18:1/16:0) in stem villi and PC (16:0/20:4) in terminal villi disappeared. The common pathological findings in these two placentas were atherosis, immature terminal villi, and multiple branched terminal villi, suggesting the possible involvement of the underperfusion of maternal blood into the intervillous space. On the other hand, the number of pathological findings were two or less in the seven other placentas, in which no specific relationships were observed between the differential expression patterns of these two phospholipids in stem and terminal villi and the pathological findings of the placentas; however, the specific distribution pattern of SM (d18:1/16:0) in stem villi disappeared in four placentas, while that of PC (16:0/20:4) in terminal villi was preserved. These results suggested that the absence of the specific distribution of PC (16:0/20:4) in terminal villi, possibly in combination with the absence of SM (d18:1/16:0) in stem villi, was linked to placental morphological changes in response to maternal underperfusion of the placenta.

Saturated fatty acids enhance TLR4 immune pathways in human trophoblasts.

What are the effects of fatty acids on placental inflammatory cytokine with respect to toll-like receptor-4/nuclear factor-kappa B (TLR4/NF-kB)? Exogenous fatty acids induce a pro-inflammatory cytokine response in human placental cells in vitro via activation of TLR4 signaling pathways. The placenta is exposed to changes in circulating maternal fatty acid concentrations throughout pregnancy. Fatty acids are master regulators of innate immune pathways through recruitment of toll-like receptors and activation of cytokine synthesis. Trophoblast cells isolated from 14 normal term human placentas were incubated with long chain fatty acids (FA) of different carbon length and degree of saturation. The expression and secretion of interleukin-6 (IL-6), IL-8 and tumor necrosis factor-alpha (TNF-α) were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Antibodies against TLR4 ligand binding domain, downstream signaling and anti-p65 NFkB-inhibitor were used to characterize the pathways of FA action. General approach used primary human term trophoblast cell culture. Methods and end-points used real-time quantitative PCR, cytokine measurements, immunohistochemistry, western blots. The long chain saturated fatty acids, stearic and palmitic (PA), stimulated the synthesis as well as the release of TNF-α, IL-6 and IL-8 by trophoblast cells (2- to 6-fold, P < 0.001). In contrast, the unsaturated (palmitoleic, oleic, linoleic) acids did not modify cytokine expression significantly. Palmitate-induced inflammatory effects were mediated via TLR4 activation, NF-kB phosphorylation and nuclear translocation. TNF-α protein level was close to the limit of detection in the culture medium even when cells were cultured with PA. These mechanisms open the way to a better understanding of how changes in maternal lipid homeostasis may regulate placental inflammatory status. X.Y. was recipient of fellowship award from West China Second University Hospital, Sichuan University (NIH HD 22965-19). The authors have nothing else to disclose. None.

Propagation of pure fetal and maternal mesenchymal stromal cells from terminal chorionic villi of human term placenta.

Long term propagation of human fetal mesenchymal stromal cells (MSC) in vitro has proven elusive due to limited availability of fetal tissue sources and lack of appropriate methodologies. Here, we have demonstrated the presence of fetal and maternal cells within the tips of terminal chorionic villi (TCV) of normal human term placenta, and we have exploited inherent differences in the adhesive and migratory properties of maternal vs. fetal cells, to establish pure MSC cultures of both cell types. The origin and purity of each culture was confirmed by X-Y chromosome-specific fluorescence in situ hybridization (FISH) and short tandem repeat (STR) genotyping. This is the first demonstration of fetal and maternal cells in the TCV of human term placenta and also of deriving pure fetal MSC cultures from them. The concomitant availability of pure cultures of adult and fetal MSC from one tissue provides a good system to compare genetic and epigenetic differences between adult and fetal MSCs; and also to generate new models of cell based therapies in regenerative medicine.

Interventional Photoacoustic Imaging of the Human Placenta with Ultrasonic Tracking for Minimally Invasive Fetal Surgeries.

Image guidance plays a central role in minimally invasive fetal surgery such as photocoagulation of inter-twin placental anastomosing vessels to treat twin-to-twin transfusion syndrome (TTTS). Fetoscopic guidance provides insufficient sensitivity for imaging the vasculature that lies beneath the fetal placental surface due to strong light scattering in biological tissues. Incomplete photocoagulation of anastamoses is associated with postoperative complications and higher perinatal mortality. In this study, we investigated the use of multi-spectral photoacoustic (PA) imaging for better visualization of the placental vasculature. Excitation light was delivered with an optical fiber with dimensions that are compatible with the working channel of a fetoscope. Imaging was performed on an ex vivo normal term human placenta collected at Caesarean section birth. The photoacoustically-generated ultrasound signals were received by an external clinical linear array ultrasound imaging probe. A vein under illumination on the fetal placenta surface was visualized with PA imaging, and good correspondence was obtained between the measured PA spectrum and the optical absorption spectrum of deoxygenated blood. The delivery fiber had an attached fiber optic ultrasound sensor positioned directly adjacent to it, so that its spatial position could be tracked by receiving transmissions from the ultrasound imaging probe. This study provides strong indications that PA imaging in combination with ultrasonic tracking could be useful for detecting the human placental vasculature during minimally invasive fetal surgery.

Expression of mTOR and downstream target components in normal and gestational diabetic human term placenta

Our recent findings demonstrated that oxaliplatin entering CNS may directly induce spinal central sensitization, and contribute to the rapid development of CNS-related side effects including acute pain during chemotherapy. However, the mechanism is largely unclear. In the current study, we found that the amplitude of C-fiber-evoked field potentials was significantly increased and the expression of phosphorylated mammalian AMP-activated protein kinase α (AMPKα) was markedly decreased following high frequency stimulation (HFS) or single intraperitoneal injection of oxaliplatin (4 mg/kg). Spinal local application of AMPK agonist metformin (25 μg) prevented the long term potentiation (LTP) induction and the activation of mTOR/p70S6K signal pathway, and significantly attenuated the acute thermal hyperalgesia and mechanical allodynia following single oxaliplatin treatment. Importantly, we found that incubation of low concentration oxaliplatin at dose of 6.6 nM (the detected concentration in CSF following a single intraperitoneal injection of oxaliplatin) also significantly inhibited the AMPKα activation and increased the amplitude of sEPSCs, the number of action potential, and the expression of p-mTOR and p-p70S6K in spinal cord slices. Metformin (25 μg) or rapamycin (2 μg) inhibited the increased excitability of dorsal horn neurons and the decrease of p-AMPKα expression induced by low concentration oxaliplatin incubation. Furthermore, spinal application of AMPK inhibitor compound C (5 μg) induced the spinal LTP, thermal hyperalgesia and mechanical allodynia, and rapamycin attenuated the spinal LTP, the thermal hyperalgesia and mechanical allodynia following oxaliplatin treatment (i.p.). Local application of metformin significantly decreased the mTOR and p70S6K activation induced by tetanus stimulation or oxaliplatin (i.p.). These results suggested that the decreased AMPKα activity via negatively regulating mTOR/p70S6K signal pathway enhanced the synaptic plasticity and contributed to acute pain induced by low concentration of oxaliplatin entering CNS.

The expression of cell cycle related proteins PCNA, Ki67, p27 and p57 in normal and preeclamptic human placentas.

Placenta is a transitional area making many physiological activities between mother and fetus and therefore, it is a critical organ influencing the outcome of pregnancy. Fetal growth is directly related to placental development. Accurate placental development depends on coordinated action of trophoblasts' proliferation, differentiation and invasion. Information on cell cycle related proteins that control these events is limited and how they are affected in preeclampsia is not fully understood yet. Therefore, in this study, in order to understand the role of cell cycle regulators in preeclamptic placentas we aimed to determine the spatio-temporal immunolocalizations of cell cycle regulators in preeclamptic and normal human term placentas. Term placentas were obtained from women diagnosed with preeclampsia and from normal pregnancies with informed consent following cesarean deliveries. Placental samples were stained via immunohistochemistry with PCNA, Ki67, p27, p57, vimentin and cytokeratin 7 antibodies and were examined by light microscopy. PCNA and Ki67 staining intensities significantly increased in villous parts, significantly decreased in basal plates of PE group and did not change in chorionic plates. Staining intensities of cell cycle inhibitors p27 and p57 significantly increased in all parts of preeclamptic placentas compared to control. Placental abnormalities of preeclamptic placentas might be associated with proliferation and cell cycle arrest mechanisms' alterations occurred in preeclampsia.

Expression of G-Protein Subunit α-14 Is Increased in Human Placentas from Preeclamptic Pregnancies

G-proteins mediate cellular function upon interaction with G-protein coupled receptors. Of the 16 mammalian G-protein α subunits identified, G-protein subunit α-11 (GNA11) and -14 (GNA14) have been implicated in modulating hypertension and endothelial function. However, little is known about their expression and roles in human placentas. Here, we examined GNA11 and GNA14 protein expression in first trimester (FT), normal term (NT), and severe preeclamptic (sPE) human placentas as well as in NT human umbilical cords. We found that GNA11 and GNA14 were immunolocalized primarily in trophoblasts, villous stromal cells, and endothelial cells in placentas as well as in endothelial and/or smooth muscle cells of the umbilical cord artery and vein. Western blotting revealed that the GNA14, but not GNA11, protein levels were increased (2.5-2.9 fold; p<0.01) in sPE vs. NT placentas. GNA11 protein was detected only in NT, but not FT, placentas, whereas GNA14 protein levels were increased (7.7-10.6 fold; p<0.01) in NT vs. FT placentas. Thus, GNA11 and GNA14 may mediate the function of several cell types in placentas. Moreover, the high expression of GNA14 in sPE placentas may also imply its importance in sPE pregnancies as in the other hypertension-related disorders.

Immunolocalization of cell cycle proteins (p57, p27, cyclin D3, PCNA and Ki67) in intrauterine growth retardation (IUGR) and normal human term placentas

Placental development involves a series of events that depend on the coordinated action of proliferation, differentiation and invasion of trophoblasts. Studies on cell cycle related proteins controlling these events are fairly limited. It is still not fully determined how placental tissue proliferation is affected by intrauterine growth retardation (IUGR). Information on cell cycle related proteins that control these events is limited and how they are affected in IUGR is not fully understood. The aim of this study was to understand the role of cell cycle regulators in IUGR placentas and to determine the spatio-temporal immunolocalization of these cell cycle regulators in human IUGR and normal term placentas. Placental samples were stained immunohistochemically with PCNA, Ki67, cyclin D3, p27 and p57 antibodies and were examined by light microscopy. In all regions of IUGR placentas, PCNA, Ki67 and cyclin D3 staining intensities were statistically significantly decreased compared to normal controls. p27 staining intensity of the IUGR group was statistically significantly increased in villous parts and chorionic plates in comparison with the normal term placentas. Moreover, p57 staining intensity was statistically significantly increased in all parts of the IUGR group compared to controls. The observed placental abnormalities in IUGR placentas may be associated with arrest mechanisms affecting cell proliferation and cell cycle alterations in IUGR.

Immunohistochemical distribution of cell cycle proteins p27, p57, cyclin D3, PCNA and Ki67 in normal and diabetic human placentas

The placenta is a regulator organ for many metabolic activities between mother and fetus. Therefore, fetal growth is directly related to the placental development. Placental development is a series of events that depend on the coordinated action of trophoblasts' proliferation, differentiation and invasion. Studies on cell cycle related proteins which control these events are fairly limited. How placental tissue proliferation is affected by diabetes is not exactly known yet. Therefore in this study, the immunohistochemical localizations of cell cycle related proteins like PCNA, Ki67, cyclin D3, p27 and p57 in the differentiation, proliferation and apoptosis mechanisms of normal and diabetic placentas were investigated. Information on cell cycle related proteins that control these events is limited and how they are affected in diabetes mellitus is not fully understood yet. Therefore, in this study, to understand the role of cell cycle regulators in diabetic placentas we aimed to determine the spatio-temporal immunolocalizations of cell cycle regulators in diabetic and normal human term placentas. Term placentas were obtained from diabetic women and from normal pregnancies with informed consent following caesarean deliveries. Placental samples were stained via immunohistochemistry with PCNA, Ki67, cyclin D3, p27 and p57 antibodies and were examined by light microscopy. When compared to control placentas, PCNA, Ki67 and cyclin D3 staining intensities significantly increased in villous parts of diabetes group. Moreover, Ki67 and cyclin D3 stainings also significantly increased in basal plates and chorionic plate respectively. In chorionic plates, p27 and p57 staining intensities significantly decreased in diabetic group. p57 staining also significantly decreased in villous parts of diabetic placentas. Placental abnormalities seen in diabetic placentas could be associated with proliferation and cell cycle arrest mechanisms' alterations occurred in diabetes mellitus.

Characterisation of K+ Channels in Human Fetoplacental Vascular Smooth Muscle Cells

Adequate blood flow through placental chorionic plate resistance arteries (CPAs) is necessary for oxygen and nutrient transfer to the fetus and a successful pregnancy. In non-placental vascular smooth muscle cells (SMCs), K+ channels regulate contraction, vascular tone and blood flow. Previous studies showed that K+ channel modulators alter CPA tone, but did not distinguish between effects on K+ channels in endothelial cells and SMCs. In this study, we developed a preparation of freshly isolated CPASMCs of normal pregnancy and investigated K+ channel expression and function. CPASMCs were isolated from normal human term placentas using enzymatic digestion. Purity and phenotype was confirmed with immunocytochemistry. Whole-cell patch clamp was used to assess K+ channel currents, and mRNA and protein expression was determined in intact CPAs and isolated SMCs with RT-PCR and immunostaining. Isolated SMCs expressed α-actin but not CD31, a marker of endothelial cells. CPASMCs and intact CPAs expressed h-caldesmon and non-muscle myosin heavy chain-2; phenotypic markers of contractile and synthetic SMCs respectively. Whole-cell currents were inhibited by 4-AP, TEA, charybdotoxin and iberiotoxin implicating functional Kv and BKCa channels. 1-EBIO enhanced whole cell currents which were abolished by TRAM-34 and reduced by apamin indicating activation of IKCa and SKCa respectively. BKCa, IKCa and SKCa3 mRNA and/or protein were expressed in CPASMCs and intact CPAs. This study provides the first direct evidence for functional Kv, BKCa, IKCa and SKCa channels in CPASMCs. These cells display a mixed phenotype implicating a dual role for CPASMCs in controlling both fetoplacental vascular resistance and vasculogenesis.

TonEBP and SMIT expression in human placenta

Tonicity-responsive enhancer binding protein (TonEBP) is a signal transcription factor of transporters such as sodium-myo-inositol cotransporter (SMIT), aldose reductase. TonEBP has a variety of functions such as control of intracellular osmolytes and immunomodulating. It is known that TonEBP is abundant in the placenta, but location and function aren't known. The aim of this study is to describe the localization of TonEBP in the placenta. We assayed the immunohistochemistry of TonEBP and performed in situ hybridization of SMIT in normal human full term placenta. In normal human full term placenta, TonEBP was in villous trophoblasts, extravillous trophoblasts and some endothelial cells. The result of the in situ hybridization of SMIT was similar to that of immunohistochemistry of TonEBP. Neither TonEBP nor SMIT was present in TonEBP knockout mouse placenta. This shows TonEBP is a key factor in SMIT transcription. TonEBP may play an important role in transporting of inositol to fetus in placenta.