Ultra-high sensitive C-reactive protein during normal pregnancy and in preeclampsia: a pilot study.

INTRODUCTION: Angiogenic and inflammatory factors have been shown to play an important role in the pathogenesis of preeclampsia. However, there is little information on their interaction. The aims of this study were to investigate the longitudinal pattern of inflammatory markers, such as interleukin-6 (IL-6) and C-reactive protein (CRP) using a novel ultra-high sensitive assay method (uhsCRP), and to explore their relationship with angiogenic factors such as placental growth factor (PLGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and vascular endothelial growth factor (VEGF) in normal pregnancies and pregnancies complicated by preeclampsia. MATERIALS AND METHODS: Serum levels of uhsCRP, IL-6, PLGF, VEGF and s-Flt-1 were longitudinally determined in 16 women with normal, singleton healthy pregnancies at 7-13, 17-22, 27-31 and 37-41 weeks of gestation by ELISA. uhsCRP was measured using a ultra-high sensitivity ELISA test. Serum of women with preeclampsia (n = 15) was available only once, usually in the third trimester of pregnancy. Women with premature rupture of membranes (PROM) or infection such as chorioamnionitis were excluded. Spearman rank correlation, logistic regression, ROC analysis, ANOVA and Mann-Whitney U-test were used for statistical purposes. RESULTS: In normal pregnancies, serum uhsCRP showed a gestational age-dependent increase (r = 0.40; P < 0.001). In women suffering from preeclampsia, uhsCRP levels were higher than in gestational age-matched controls (18010 ±â€Š4763 versus 3026 ±â€Š587 ng/ml; P < 0.001). Similarly, serum IL-6 levels increased throughout pregnancy and correlated with uhsCRP in normal pregnancies and in preeclampsia (n = 64, r = 0.37; P < 0.01 and n = 15, r = 1.00, P < 0.0001). uhsCRP levels were positively correlated with sFlt-1 levels (n = 64, r = 0.34; P < 0.01). CONCLUSION: The increases in uhsCRP (and IL-6) serum levels with advancing gestation indicate a shift towards an inflammatory state during normal pregnancy. The excessive rise in uhsCRP and sFlt-1 in preeclampsia indicate that both may be involved in its pathogenesis. uhsCRP may be useful as an early marker for preeclampsia and studies defining the pattern of its rise throughout pregnancies at risk are urgently needed.

Regulation of human trophoblast GLUT1 glucose transporter by insulin-like growth factor I (IGF-I).

Glucose transport to the fetus across the placenta takes place via glucose transporters in the opposing faces of the barrier layer, the microvillous and basal membranes of the syncytiotrophoblast. While basal membrane content of the GLUT1 glucose transporter appears to be the rate-limiting step in transplacental transport, the factors regulating transporter expression and activity are largely unknown. In view of the many studies showing an association between IGF-I and fetal growth, we investigated the effects of IGF-I on placental glucose transport and GLUT1 transporter expression. Treatment of BeWo choriocarcinoma cells with IGF-I increased cellular GLUT1 protein. There was increased basolateral (but not microvillous) uptake of glucose and increased transepithelial transport of glucose across the BeWo monolayer. Primary syncytial cells treated with IGF-I also demonstrated an increase in GLUT1 protein. Term placental explants treated with IGF-I showed an increase in syncytial basal membrane GLUT1 but microvillous membrane GLUT1 was not affected. The placental dual perfusion model was used to assess the effects of fetally perfused IGF-I on transplacental glucose transport and syncytial GLUT1 content. In control perfusions there was a decrease in transplacental glucose transport over the course of the perfusion, whereas in tissues perfused with IGF-I through the fetal circulation there was no change. Syncytial basal membranes from IGF-I perfused tissues showed an increase in GLUT1 content. These results demonstrate that IGF-I, whether acting via microvillous or basal membrane receptors, increases the basal membrane content of GLUT1 and up-regulates basal membrane transport of glucose, leading to increased transepithelial glucose transport. These observations provide a partial explanation for the mechanism by which IGF-I controls nutrient supply in the regulation of fetal growth.

The impact of metabolic disease associated with metabolic syndrome on human pregnancy.

Metabolic diseases induced by metabolic syndrome (MS) have been increased during the past two decades. During healthy pregnancy maternal organs and placenta are challenged to adapt to the increasingly physiological changes. In addition to the increasingly proatherogenic MS, pregnant woman develops a high cardiac output, hypercoagulability, increased inflammatory activity and insulin resistance with dyslipidemia. The MS describes a cluster of metabolic changes associated with an impact on the physiology of many organs. While the metabolic syndrome is directly responsible for the development of atherosclerotic cardiovascular disease, additional impact on human pregnancy like preterm delivery with low-birth-weight infants as well as the development of diseases such as diabetes, preeclampsia and hypertension. Recent evidence suggests that MS is originated in fetal life in association with maternal nutrition during pregnancy and fetal programming which apparently increases the susceptibility for MS in children and later life. This review will describe the MS in association with the origin of the emerging diseases during pregnancy such as diabetes, preeclampsia and others. The influence of perinatal environment and maternal diet and smoking on MS as well as the genetic biomarkers of MS will be described.

Role of IgG antibodies in association with placental function and immunologic diseases in human pregnancy.

During human pregnancy, the maternal immune system develops and changes, providing protection for the growing placenta and fetus. These protective changes provide mechanisms allowing two genetically different individuals to interact with each other without allograft rejection. In addition to normal pregnancy, some pregnancies may develop under immunologic diseases, during which specific monitoring and medical treatments are essential. The aim of this current review is to provide information regarding the development of human placental function during pregnancy, the immunology of human pregnancy and the role of the placenta in providing the fetal tissue with antibodies (IgG and its subclasses 1-4), which are required for the passive immunization of the newborn. In addition, the available methods for the determination of placental function will be explored. Furthermore, immunologic diseases observed during pregnancy and the possible therapies for these diseases will be assessed.

Drugs and medicines in pregnancy: the placental disposition of opioids.

Over the past decade therapeutic interventions during pregnancy, including the use of drugs, has increased; for example, the prescription use of opioids and nonopioid analgesics for pain management during pregnancy has increased by about 40%. Additionally, women also self-medicate during pregnancy with opioids, and are treated with methadone and buprenorphine which are used as substitution therapy for substance dependence during pregnancy. A nationally representative survey noted that ~74% of women of reproductive age reported substance use during the previous year, decreasing to ~63% amongst those who were pregnant. Substance dependence was reported by ~20% of women who were not pregnant, and ~15% of those who were pregnant. Understanding the consequences of opioid treatment or dependence during pregnancy requires an investigation of the placental disposition (metabolism and transfer), as well as of the impact of the drugs on the placental function. This review summarizes the research on the placental disposition of opioids and their impact on the placental function.

Nanoparticulate drug delivery in pregnancy: placental passage and fetal exposure.

During the past decade there has been an explosion in the number of nanoparticulate drugs or drug delivery systems being explored, developed and marketed for the treatment and prevention of human diseases. While the potential dangers of drug administration in pregnancy are well known, there are circumstances where the benefits of maternal drug administration are perceived to outweigh the risks to the fetus. Hence, the administration of potentially harmful drugs in pregnancy is surprisingly common. Nanoparticulate delivery systems offer a potential avenue for delivering therapeutics to maternal tissues with minimal risk of incidental fetal exposure, depending on the ability of the nanoparticle in question to cross the placenta. As the conduit to the fetus, the placenta is both a drug target and a drug barrier, as well as a potential target of any toxicity. Limited studies on this topic show considerable uncertainty regarding the transplacental passage of nanoparticles, and our understanding of the criteria that determine transferability is poor. Despite the fact that the toxicity caused by environmental and man-made nanoparticulates has been widely studied in various organ systems, data on the effects of maternal nanoparticle exposure on human fetal tissues are lacking, although studies in rodents indicate that caution is justified. In this review, we examine the evidence relating to the potential toxicity of nanoparticles in pregnancy, the ability of the placenta to take up and transfer nanoparticles to the fetus, and the theoretical benefits and risks of administration of nanoparticle-based therapeutics in pregnancy.

Human placental stem cells: biomedical potential and clinical relevance.

Emerging evidence shows that different adult tissues of the human body harbour small amounts of adult mesenchymal stem cells (MSCs) which have the potential to give a rise to all cell types of the specific tissue in which they reside. These cells were found in human adult bone marrow, muscle, fat, brain, cartilage, dental pulp, etc. Depending on the type of host tissue as well as age and state of the donor's health, the plasticity of MSCs can vary considerably. Previously accomplished research efforts have shown that adult MSCs have a broad therapeutic potential due to their ability to differentiate towards multiple adult cell types under appropriate conditions. This unique property characterizes adult MSCs as potential candidates for biomedical and clinical applications. In the last decade human extra-embryonic tissues (placenta, amniotic membranes, and umbilical cord) as well as human placental fluids (amniotic fluid and umbilical cord blood), were also defined as sources of MSCs, which represents a great interest for the biomedical applications. Human placenta is a feto-maternal organ which works as a natural barrier between mother and foetus and playing a crucial role for the nutrition and immune-tolerance during the whole period of the fetal development. Anatomically, the placenta can be divided in four regions: amniotic epithelial, amniotic mesenchymal, chorionic mesenchymal and chorionic trophoblastic. Immunomodulatory properties of human placenta as well as the clinical applicability of such placental parts as amniotic membranes for wound healing, treatment of burns, and reconstruction of the oral cavity, will be described. Due to the specific structure and functions of placenta, human extra-embryonic MSCs represent stem cell types which combine some properties of pluripotent embryonic stem cells with other properties of multipotent mesenchymal stem cells. Due to the close ontogenic relationship to embryonic stem cells, MSCs derived from different placental regions as well as from amniotic fluid and umbilical cord blood have immunoprivileged characteristics, posses a broader plasticity, and proliferate faster than adult MSCs. Moreover, the human placenta is normally discarded after birth, and cells can be isolated avoiding any ethical concerns. In this review we summarize and discuss different aspects of development, ontogenetic properties, plasticity, and immunomodulatory characteristics of MSCs from different placental regions as well as from amniotic fluid and umbilical cord blood. In addition the potential of placental MSCs for biomedical and clinical applications are discussed.

In vitro studies of ferric carboxymaltose on placental permeability using the dual perfusion model of human placenta.

An in vitro perfusion model of human placenta was used to study the transplacental passage of iron applied in the form of the drug compound ferric carboxymaltose (FCM) which had been radio-labelled with 59Fe. In four placental perfusion experiments, two simulated circuits for the maternal and fetal sides of the placenta were set up with two experimental phases each lasting 3 h. FCM was added to the maternal circuit at the beginning of each phase to a final iron concentration of 11 mM, which is at least 10 times higher than the maximal predicted level in blood after an administration of 200 mg iron as FCM. The effects of adding transferrin at a physiological concentration of 1.67 mg/ ml were also tested. The concentration profiles of 59Fe showed a 10% decrease within the first 30 min of perfusion on the maternal side. Thereafter the radioactivity levels remained unchanged. The addition of transferrin had no effect on the tissue uptake of 59Fe-FCM. No transferred iron radioactivity could be detected in the fetal circuit. Despite a loss of approximately 10% of the radio-labelled iron observed on the maternal side, only 0.5-2% of the radioactivity was detected in the placental tissue after perfusion. No free iron could be detected at the end of perfusion on the maternal side using ultrafiltration or acid precipitation methods. In addition, the production of transferrin receptor remained unchanged, with similar concentrations in placental tissue before and after perfusion. No effects of FCM on placental viability were observed in terms of energy metabolism (glucose consumption and lactate production), hormone release or placental permeability (assessed by the transfer rates of creatinine and antipyrine). However, two additional observations were made: firstly, a significant reduction in the rate of cell death compared to control conditions was observed in the presence of FCM; secondly, the integrity of the fetal capillary system was improved on the fetal side of the perfusion system. It is concluded that the iron compound FCM does not cross the placenta and may increase the integrity of placental tissue (at least under in vitro conditions), but this latter observation needs further investigation.

Barrier capacity of human placenta for nanosized materials.

BACKGROUND: Humans have been exposed to fine and ultrafine particles throughout their history. Since the Industrial Revolution, sources, doses, and types of nanoparticles have changed dramatically. In the last decade, the rapidly developing field of nanotechnology has led to an increase of engineered nanoparticles with novel physical and chemical properties. Regardless of whether this exposure is unintended or not, a careful assessment of possible adverse effects is needed. A large number of projects have been carried out to assess the consequences of combustion-derived or engineered nanoparticle exposure on human health. In recent years there has been a growing concern about the possible health influence of exposure to air pollutants during pregnancy, hence an implicit concern about potential risk for nanoparticle exposure in utero. Previous work has not addressed the question of whether nanoparticles may cross the placenta. OBJECTIVE: In this study we investigated whether particles can cross the placental barrier and affect the fetus. METHODS: We used the ex vivo human placental perfusion model to investigate whether nanoparticles can cross this barrier and whether this process is size dependent. Fluorescently labeled polystyrene beads with diameters of 50, 80, 240, and 500 nm were chosen as model particles. RESULTS: We showed that fluorescent polystyrene particles with diameter up to 240 nm were taken up by the placenta and were able to cross the placental barrier without affecting the viability of the placental explant. CONCLUSIONS: The findings suggest that nanomaterials have the potential for transplacental transfer and underscore the need for further nanotoxicologic studies on this important organ system.

Multipotent mesenchymal stem cells from human placenta: critical parameters for isolation and maintenance of stemness after isolation.

OBJECTIVE: This study was undertaken to isolate and characterize multipotent mesenchymal stem cells from term human placenta (placenta-derived mesenchymal stem cells, PD-MSCs). STUDY DESIGN: Sequential enzymatic digestion was used to isolate PD-MSCs in which trypsin removes the trophoblast layer, followed by collagenase treatment of remaining placental tissue. Karyotype, phenotype, growth kinetics, and differentiability of PD-MSC isolates from collagenase digests were analyzed. RESULTS: PD-MSC isolation was successful in 14 of 17 cases. Karyotyping of PD-MSC isolates from deliveries with a male fetus revealed that these cells are of maternal origin. Flow cytometry and immunocytochemistry confirmed the mesenchymal stem cell phenotype. Proliferation rates of PD-MSCs remained constantly high up to passage 20. These cells could be differentiated toward mesodermal lineage in vitro up to passage 20. Nonconfluent culture was critical to maintain the MSC stemness during long-term culture. CONCLUSION: Term placenta constitutes a rich, very reliable source of maternal mesenchymal stem cells that remain differentiable, even at high passage numbers.

The impact of cocaine and heroin on the placental transfer of methadone.

BACKGROUND: Methadone is the therapeutic agent of choice for the treatment of opiate addiction in pregnancy. The co-consumption (heroin, cocaine) which may influence the effects of methadone is frequent. Therefore, the impact of cocaine and heroin on the placental transfer of methadone and the placental tissue was investigated under in vitro conditions. METHODS: Placentae (n = 24) were ex-vivo perfused with medium (m) (control, n = 6), m plus methadone (n = 6), m plus methadone and cocaine (n = 6) or m plus methadone and heroin (n = 6). Placental functionality parameters like antipyrine permeability, glucose consumption, lactate production, hormone production (hCG and leptin), microparticles release and the expression of P-glycoprotein were analysed. RESULTS: Methadone accumulated in placental tissue. Methadone alone decreased the transfer of antipyrine from 0.60 +/- 0.07 to 0.50 +/- 0.06 (fetal/maternal ratio, mean +/- SD, P < 0.01), whereas the combination with cocaine or heroin increased it (0.56 +/- 0.08 to 0.68 +/- 0.13, P = 0.03 and 0.58 +/- 0.21 to 0.71 +/- 0.24; P = 0.18). Microparticles (MPs) released from syncytiotrophoblast into maternal circuit increased by 30% after cocaine or heroin (P < 0.05) and the expression of P-glycoprotein in the tissue increased by >or= 49% after any drug (P < 0.05). All other measured parameters did not show any significant effect when methadone was combined with cocaine or heroine. CONCLUSION: The combination of cocaine or heroin with methadone increase antipyrine permeability. Changes of MPs resemble findings seen in oxidative stress of syncytiotrophoblast.

Engineered polyelectrolyte multilayer substrates for adhesion, proliferation, and differentiation of human mesenchymal stem cells.

Polyelectrolyte multilayer coatings have emerged as substrates to control cellular behavior, but interactions with human multipotent mesenchymal stromal cells (MSCs) have not been studied. We looked at layer-by-layer coatings of cationic poly-L-lysine (PLL) and anionic hyaluronic acid (HA) as substrates for MSCs of placenta and adipose tissue. This system allows for modulation of thickness (number of deposition cycles), stiffness (chemical cross-linking of bulk layer), and adhesiveness (fibronectin (FN) interface). Native, as-built PLL/HA multilayer coatings were poorly adhesive for MSCs despite spectroscopy-confirmed high surface density of pre-adsorbed FN. Stratification of cross-linked PLL/HA multilayers of different stiffnesses revealed that multilayers modified with a high cross-linking regimen became efficient substrates for MSC adhesion and proliferation. MSCs on cross-linked multilayers grew to confluence. Using comparative confocal microscopy analysis of PLL/HA multilayers with physically adsorbed versus chemically coupled FN, we demonstrated that cross-linking strongly influenced FN surface distribution, leading to denser presentation of adhesion sites for cells. The covalent affixation of FN promoted focal adhesion formation and was critical to maintaining densely grown MSC cultures over weeks for their differentiation. Multilayer-bound MSCs were capable of differentiating into osteocytes and chondrocytes upon culture with induction factors. Together, cross-linked, FN-terminated PLL/HA multilayers provide a versatile platform for studies of human MSCs for biotechnological or therapeutic applications.

Development of a human model to study homing behavior of immune cells into decidua and placental villi under ex vivo conditions.

PROBLEM: Homing of lymphocytes and NK cells into the decidua and its regulation has been very controversially discussed. Therefore, we aimed to establish an in vivo simulation method for analysis of homing behavior, which might be also useful for other cells such as stem or tumor cells. METHOD OF STUDY: A human term placenta has been perfused with medium to elute blood and then with maternal autologous carboxyfluorescein succinimidyl ester (CFSE)-labeled peripheral blood lymphocytes for 3 hr and rinsed for another 2 hr. Tissue was analysed histologically for detection of labeled cells. Labeled lymphocytes and beads in perfusate have been identified and counted by flow cytometry. RESULTS: At the moment of tissue fixation for histology, the perfusate was free of labeled cells. Labeled perfused lymphocytes have been found adhered and integrated in vessel wall structures, in decidual stroma and as colonies in individual villi. CONCLUSION: Placenta perfusion with a lymphocyte suspension is feasible without plugging the tube system. Time is sufficient for cells to adhere and to migrate into the stroma. Also some villi have been infiltrated which might be caused by inflammatory stimuli. The perfusion system might be useful to test substances for their capacity to influence homing of lymphocytes or other cells.

Placental mesenchymal stem cells as potential autologous graft for pre- and perinatal neuroregeneration.

OBJECTIVE: Mesenchymal stem cells (MSCs) have a broad differentiation potential. We aimed to determine if MSCs are present in fetal membranes and placental tissue and to assess their potential to differentiate into neurogenic and mesodermal lineages. STUDY DESIGN: MSCs isolated from first and third trimester chorion and amnion and first trimester chorionic villi and characterized morphologically and by flourescence-activated cell sorting analysis. Their ability to mature under different culture conditions into various cells of mesodermal and neuroectodermal cell lines was assessed by immuno- and cytochemical staining. RESULTS: Independent of gestational age, cells isolated from fetal membranes and placenta showed typical MSC phenotype (positive for CD166, CD105, CD90, CD73, CD49e, CD44, CD29, CD13, MHC I; negative for CD14, CD34, CD45, MHC II) and were able to differentiate into mesodermal cells expressing cell markers/cytologic staining consistent with mature chondroblasts, osteoblasts, adipocytes, or myocytes and into neuronal cells presenting markers of various stages of maturation. The differentiation pattern was mainly dependent on cell type. CONCLUSION: Mesenchymal cells from chorion, amnion, and villous stroma can be differentiated into neurogenic, chondrogenic, osteogenic, adipogenic, and myogenic lineage. Placental tissue obtained during prenatal chorionic villous sampling or at delivery might be an ideal source for autologous stem cell graft for peripartum neuroregeneration and other clinical issues.

Ex vivo human placenta models: transport of immunoglobulin G and its subclasses.

The comparison between fetal and maternal levels of immunoglobulins indicate that the human placenta during pregnancy develops a specific transport mechanism for IgG in the maternal to fetal direction. There are differences for the four subclasses, with preferential transfer of IgG(1) while the slowest transfer is seen for IgG(2). Under in vitro perfusion conditions of human term placenta IgG, when compared to other proteins, showed a significantly higher transfer rate of its subclasses from the maternal to the fetal side, indicating a specific transport mechanism. There is a preferential transfer rate, highest for IgG(1) and lowest for IgG(2), similar to those observed under in vivo conditions. The similarity in transfer of anti-TT-IgG and tetanus antigen, which was observed under in vivo and in vitro conditions, may suggest the transport as antibody-antigen complex.

Evidence of fetal C-reactive protein urinary excretion in early gestation.

BACKGROUND: C-reactive protein has been measured in amniotic fluid in the second and third trimesters of gestation, and its elevated concentration has been found to be associated with adverse pregnancy outcome. It remains unexplained whether amniotic fluid C-reactive protein is of fetal origin. CASE: We report the measurement of C-reactive protein in fetal urine obtained by transabdominal vescicocentesis in a fetus at 15 weeks' gestation affected by obstructive uropathy. Using an enzyme-linked immunosorbent assay, C-reactive protein was detected at a concentration of 234 ng/mL. CONCLUSION: The fetal kidneys excrete C-reactive protein as early as 15 weeks' gestation.