Quantification of Cell-Free DNA in Normal and Complicated Pregnancies: Overcoming Biological and Technical Issues

The characterization of cell-free DNA (cfDNA) originating from placental trophoblast in maternal plasma provides a powerful tool for non-invasive diagnosis of fetal and obstetrical complications. Due to its placental origin, the specific epigenetic features of this DNA (commonly known as cell-free fetal DNA) can be utilized in creating universal ‘fetal’ markers in maternal plasma, thus overcoming the limitations of gender- or rhesus-specific ones. The goal of this study was to compare the performance of relevant approaches and assays evaluating the amount of cfDNA in maternal plasma throughout gestation (7.2–39.5 weeks). Two fetal- or placental- specific duplex assays (RPP30/SRY and RASSF1A/β-Actin) were applied using two technologies, real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). Both methods revealed similar performance parameters within the studied dynamic range. Data obtained using qPCR and ddPCR for these assays were positively correlated (total cfDNA (RPP30): R = 0.57, p = 0.001/placental cfDNA (SRY): R = 0.85, p<0.0001; placental cfDNA (RASSF1A): R = 0.75, p<0.0001). There was a significant correlation in SRY and RASSF1A results measured with qPCR (R = 0.68, p = 0.013) and ddPCR (R = 0.56, p = 0.039). Different approaches also gave comparable results with regard to the correlation of the placental cfDNA concentration with gestational age and pathological outcome. We conclude that ddPCR is a practical approach, adaptable to existing qPCR assays and well suited for analysis of cell-free DNA in plasma. However, it may need further optimization to surpass the performance of qPCR.     

Placental Glucose Transfer: A Human In Vivo Study

ObjectivesThe placental transfer of nutrients is influenced by maternal metabolic state, placenta function and fetal demands. Human in vivo studies of this interplay are scarce and challenging. We aimed to establish a method to study placental nutrient transfer in humans. Focusing on glucose, we tested a hypothesis that maternal glucose concentrations and uteroplacental arterio-venous difference (reflecting maternal supply) determines the fetal venous-arterial glucose difference (reflecting fetal consumption).MethodsCross-sectional in vivo study of 40 healthy women with uncomplicated term pregnancies undergoing planned caesarean section. Glucose and insulin were measured in plasma from maternal and fetal sides of the placenta, at the incoming (radial artery and umbilical vein) and outgoing vessels (uterine vein and umbilical artery).ResultsThere were significant mean (SD) uteroplacental arterio-venous 0.29 (0.23) mmol/L and fetal venous-arterial 0.38 (0.31) mmol/L glucose differences. The transplacental maternal-fetal glucose gradient was 1.22 (0.42) mmol/L. The maternal arterial glucose concentration was correlated to the fetal venous glucose concentration (r = 0.86, p<0.001), but not to the fetal venous-arterial glucose difference. The uteroplacental arterio-venous glucose difference was neither correlated to the level of glucose in the umbilical vein, nor fetal venous-arterial glucose difference. The maternal-fetal gradient was correlated to fetal venous-arterial glucose difference (r = 0.8, p<0.001) and the glucose concentration in the umbilical artery (r = −0.45, p = 0.004). Glucose and insulin concentrations were correlated in the mother (r = 0.52, p = 0.001), but not significantly in the fetus. We found no significant correlation between maternal and fetal insulin values.ConclusionsWe did not find a relation between indicators of maternal glucose supply and the fetal venous-arterial glucose difference. Our findings indicate that the maternal-fetal glucose gradient is significantly influenced by the fetal venous-arterial difference and not merely dependent on maternal glucose concentration or the arterio-venous difference on the maternal side of the placenta.     

Localized Fetomaternal Hyperglycemia: Spatial and Kinetic Definition by Positron Emission Tomography

Background

Complex but common maternal diseases such as diabetes and obesity contribute to adverse fetal outcomes. Understanding of the mechanisms involved is hampered by difficulty in isolating individual elements of complex maternal states in vivo. We approached this problem in the context of maternal diabetes and sought an approach to expose the developing fetus in vivo to isolated hyperglycemia in the pregnant rat.

Methodology and Principal Findings

We hypothesized that glucose infused into the arterial supply of one uterine horn would more highly expose fetuses in the ipsilateral versus contralateral uterine horn. To test this, the glucose tracer [¹⁸F]fluorodeoxyglucose (FDG) was infused via the left uterine artery. Regional glucose uptake into maternal tissues and fetuses was quantified using positron emission tomography (PET). Upon infusion, FDG accumulation began in the left-sided placentae, subsequently spreading to the fetuses. Over two hours after completion of the infusion, FDG accumulation was significantly greater in left compared to right uterine horn fetuses, favoring the left by 1.9±0.1 and 2.8±0.3 fold under fasted and hyperinsulinemic conditions (p<10⁻¹¹ n = 32-35 and p<10⁻¹² n = 27–45) respectively. By contrast, centrally administered [³H]-2-deoxyglucose accumulated equally between the fetuses of the two uterine horns. Induction of significant hyperglycemia (10³ mg/dL) localized to the left uterine artery was sustained for at least 48 hours while maternal euglycemia was maintained.

Conclusions and Significance

This approach exposes selected fetuses to localized hyperglycemia in vivo, minimizing exposure of the mother and thus secondary effects. Additionally, a set of less exposed internal control fetuses are maintained for comparison, allowing direct study of the in vivo fetal effects of isolated hyperglycemia. Broadly, this approach can be extended to study a variety of maternal-sided perturbations suspected to directly affect fetal health.     

Reduction of Maternal Mortality with Highly Active Antiretroviral Therapy in a Large Cohort of HIV-Infected Pregnant Women in Malawi and Mozambique

Background

HIV infection is a major contributor to maternal mortality in resource-limited settings. The Drug Resource Enhancement Against AIDS and Malnutrition Programme has been promoting HAART use during pregnancy and postpartum for Prevention-of-mother-to-child-HIV transmission (PMTCT) irrespective of maternal CD4 cell counts since 2002.

Methods

Records for all HIV+ pregnancies followed in Mozambique and Malawi from 6/2002 to 6/2010 were reviewed. The cohort was comprised by pregnancies where women were referred for PMTCT and started HAART during prenatal care (n = 8172, group 1) and pregnancies where women were referred on established HAART (n = 1978, group 2).

Results

10,150 pregnancies were followed. Median (IQR) baseline values were age 26 years (IQR:23–30), CD4 count 392 cells/mm³ (IQR:258–563), Viral Load log₁₀ 3.9 (IQR:3.2–4.4), BMI 23.4 (IQR:21.5–25.7), Hemoglobin 10.0 (IQR: 9.0–11.0). 101 maternal deaths (0.99%) occurred during pregnancy to 6 weeks postpartum: 87 (1.1%) in group 1 and 14 (0.7%) in group 2. Mortality was 1.3% in women with <than 350 CD4 cells/mm³ and 0.7% in women with greater than 350 CD4s cells/mm³ [OR = 1.9 (CL 1.3–2.9) p = 0.001]. Mortality was higher in patients with shorter antenatal HAART: 22/991 (2.2%) if less than 30 days and 79/9159 (0.9%) if 31 days or greater [OR = 2.6 (CL 1.6–4.2) p<0.001]. By multivariate analysis, shorter antenatal HAART (p<0.001), baseline values for CD4 cell count (p = 0.012), hemoglobin (p = 0.02), and BMI (p<0.001) were associated with mortality. Four years later, survival was 92% for women with shorter antenatal HAART and 98% for women on established therapy prior to pregnancy, p = 0.001.

Conclusions

Antiretrovirals for PMTCT purposes have significant impact on maternal mortality as do CD4 counts and nutritional status. In resource-limited settings, PMTCT programs should provide universal HAART to all HIV+ pregnant women given its impact in prevention of maternal death.     

Transgenic Increase in N-3/N-6 Fatty Acid Ratio Reduces Maternal Obesity-Associated Inflammation and Limits Adverse Developmental Programming in Mice

Maternal and pediatric obesity has risen dramatically over recent years, and is a known predictor of adverse long-term metabolic outcomes in offspring. However, which particular aspects of obese pregnancy promote such outcomes is less clear. While maternal obesity increases both maternal and placental inflammation, it is still unknown whether this is a dominant mechanism in fetal metabolic programming. In this study, we utilized the Fat-1 transgenic mouse to test whether increasing the maternal n-3/n-6 tissue fatty acid ratio could reduce the consequences of maternal obesity-associated inflammation and thereby mitigate downstream developmental programming. Eight-week-old WT or hemizygous Fat-1 C57BL/6J female mice were placed on a high-fat diet (HFD) or control diet (CD) for 8 weeks prior to mating with WT chow-fed males. Only WT offspring from Fat-1 mothers were analyzed. WT-HFD mothers demonstrated increased markers of infiltrating adipose tissue macrophages (P<0.02), and a striking increase in 12 serum pro-inflammatory cytokines (P<0.05), while Fat1-HFD mothers remained similar to WT-CD mothers, despite equal weight gain. E18.5 Fetuses from WT-HFD mothers had larger placentas (P<0.02), as well as increased placenta and fetal liver TG deposition (P<0.01 and P<0.02, respectively) and increased placental LPL TG-hydrolase activity (P<0.02), which correlated with degree of maternal insulin resistance (r = 0.59, P<0.02). The placentas and fetal livers from Fat1-HFD mothers were protected from this excess placental growth and fetal-placental lipid deposition. Importantly, maternal protection from excess inflammation corresponded with improved metabolic outcomes in adult WT offspring. While the offspring from WT-HFD mothers weaned onto CD demonstrated increased weight gain (P<0.05), body and liver fat (P<0.05 and P<0.001, respectively), and whole body insulin resistance (P<0.05), these were prevented in WT offspring from Fat1-HFD mothers. Our results suggest that reducing excess maternal inflammation may be a promising target for preventing adverse fetal metabolic outcomes in pregnancies complicated by maternal obesity.     

Connexin Hemichannel Blockade Is Neuroprotective after Asphyxia in Preterm Fetal Sheep

Asphyxia around the time of preterm birth is associated with neurodevelopmental disability. In this study, we tested the hypothesis that blockade of connexin hemichannels would improve recovery of brain activity and reduce cell loss after asphyxia in preterm fetal sheep. Asphyxia was induced by 25 min of complete umbilical cord occlusion in preterm fetal sheep (103–104 d gestational age). Connexin hemichannels were blocked by intracerebroventricular infusion of mimetic peptide starting 90 min after asphyxia at a concentration of 50 µM/h for one hour followed by 50 µM/24 hour for 24 hours (occlusion-peptide group, n = 6) or vehicle infusion for controls (occlusion-vehicle group, n = 7). Peptide infusion was associated with earlier recovery of electroencephalographic power after asphyxia compared to occlusion-vehicle (p<0.05), with reduced neuronal loss in the caudate and putamen (p<0.05), but not in the hippocampus. In the intragyral and periventricular white matter, peptide administration was associated with an increase in total oligodendrocyte numbers (p<0.05) and immature/mature oligodendrocytes compared to occlusion-vehicle (p<0.05), with a significant increase in proliferation (p<0.05). Connexin hemichannel blockade was neuroprotective and reduced oligodendrocyte death and improved recovery of oligodendrocyte maturation in preterm fetuses after asphyxia.     

Maternal Vitamin D Status in Preeclampsia: Seasonal Changes Are Not Influenced by Placental Gene Expression of Vitamin D Metabolizing Enzymes

Preeclampsia, a hypertensive disorder in pregnancy develops in 2–8% of pregnancies worldwide. Winter season and vitamin D deficiency have been associated with its onset.

Objective

To investigate the influence of season on maternal vitamin D status and placental vitamin D metabolism.

Methods

25-OH vitamin D and 1,25-(OH)₂ vitamin D were measured in maternal serum obtained during the winter or summer months from 63 pregnant women at delivery (43 healthy, 20 preeclampsia). In a subgroup, mRNA expression of CYP24A1 (24-hydroxylase), CYP27B1 (1α-hydroxylase) and VDR (vitamin D receptor) were quantified by real time PCR in placental samples of 14 women with normal pregnancies and 13 with preeclampsia.

Results

In patients with preeclampsia,25-OH vitamin D levels were lower, but differed significantly from controls only in summer (18.21±17.1 vs 49.2±29.2 ng/mL, P<0.001), whereas 1,25-(OH)₂ vitamin D levels were significantly lower only in winter (291±217 vs 612.3±455 pmol/mL, P<0.05). A two-factorial analysis of variance produced a statistically significant model (P<0.0001) with an effect of season (P<0.01) and preeclampsia (P = 0.01) on maternal 25-OH vitamin D levels, as well as a significant interaction between the two variables (P = 0.02). Placental gene expression of CYP24A1, CYP27B1, and VDR did not differ between groups or seasons. A negative correlation between placental gene expression of CYP24A1 and CYP27B1 was observed only in healthy controls (r = −0.81, P<0.0001).

Summary

Patients with preeclampsia displayed lower vitamin D serum levels in response to seasonal changes.The regulation of placental CYP24A1, but not of the VDR or CYP27B1 might be altered in preeclampsia.     

Fasting Enhances TRAIL-Mediated Liver Natural Killer Cell Activity via HSP70 Upregulation

Acute starvation, which is frequently observed in clinical practice, sometimes augments the cytolytic activity of natural killer cells against neoplastic cells. In this study, we investigated the molecular mechanisms underlying the enhancement of natural killer cell function by fasting in mice. The total number of liver resident natural killer cells in a unit weight of liver tissue obtained from C57BL/6J mice did not change after a 3-day fast, while the proportions of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)⁺ and CD69⁺ natural killer cells were significantly elevated (n = 7, p <0.01), as determined by flow cytometric analysis. Furthermore, we found that TRAIL⁻ natural killer cells that were adoptively transferred into Rag-2^−/− γ chain^−/− mice could convert into TRAIL⁺ natural killer cells in fasted mice at a higher proportion than in fed mice. Liver natural killer cells also showed high TRAIL-mediated antitumor function in response to 3-day fasting. Since these fasted mice highly expressed heat shock protein 70 (n = 7, p <0.05) in liver tissues, as determined by western blot, the role of this protein in natural killer cell activation was investigated. Treatment of liver lymphocytes with 50 µg/mL of recombinant heat shock protein 70 led to the upregulation of both TRAIL and CD69 in liver natural killer cells (n = 6, p <0.05). In addition, HSP70 neutralization by intraperitoneally injecting an anti- heat shock protein 70 monoclonal antibody into mice prior to fasting led to the downregulation of TRAIL expression (n = 6, p <0.05). These findings indicate that acute fasting enhances TRAIL-mediated liver natural killer cell activity against neoplastic cells through upregulation of heat shock protein 70.     

Relation of DNA Methylation of 5′-CpG Island of ACSL3 to Transplacental Exposure to Airborne Polycyclic Aromatic Hydrocarbons and Childhood Asthma

In a longitudinal cohort of ∼700 children in New York City, the prevalence of asthma (>25%) is among the highest in the US. This high risk may in part be caused by transplacental exposure to traffic-related polycyclic aromatic hydrocarbons (PAHs) but biomarkers informative of PAH-asthma relationships is lacking. We here hypothesized that epigenetic marks associated with transplacental PAH exposure and/or childhood asthma risk could be identified in fetal tissues. Mothers completed personal prenatal air monitoring for PAH exposure determination. Methylation sensitive restriction fingerprinting was used to analyze umbilical cord white blood cell (UCWBC) DNA of 20 cohort children. Over 30 DNA sequences were identified whose methylation status was dependent on the level of maternal PAH exposure. Six sequences were found to be homologous to known genes having one or more 5′-CpG island(s) (5′-CGI). Of these, acyl-CoA synthetase long-chain family member 3 (ACSL3) exhibited the highest concordance between the extent of methylation of its 5′-CGI in UCWBCs and the level of gene expression in matched fetal placental tissues in the initial 20 cohort children. ACSL3 was therefore chosen for further investigation in a larger sample of 56 cohort children. Methylation of the ACSL3 5′-CGI was found to be significantly associated with maternal airborne PAH exposure exceeding 2.41 ng/m³ (OR = 13.8; p<0.001; sensitivity = 75%; specificity = 82%) and with a parental report of asthma symptoms in children prior to age 5 (OR = 3.9; p<0.05). Thus, if validated, methylated ACSL3 5′CGI in UCWBC DNA may be a surrogate endpoint for transplacental PAH exposure and/or a potential biomarker for environmentally-related asthma. This exploratory report provides a new blueprint for the discovery of epigenetic biomarkers relevant to other exposure assessments and/or investigations of exposure-disease relationships in birth cohorts. The results support the emerging theory of early origins of later life disease development.     

Association of E/E′ and NT-proBNP with Renal Function in Patients with Essential Hypertension

Objectives

To evaluate the association of left ventricular (LV) diastolic function and N-terminal pro-brain natriuretic peptide (NT-proBNP) with renal function in essential hypertension.

Methods

LV diastolic function was estimated by the ratio of early diastolic velocities (E) from transmitral inflow to early diastolic velocities (E′) of tissue Doppler at mitral annulus (septal corner); NT-proBNP was measured in 207 hypertensive patients (mean age 56±14 years). The subjects were classified into 3 groups: E/E′≤10 group (n = 48), 10<E/E′≤15 group (n = 109) and E/E′>15 group (n = 50). The renal function was estimated by glomerular filtration rate (GFR) with ^99mTc-DTPA. GFR from 30 to 59 ml/min/1.73 m² was defined as Stage 3 chronic kidney disease (CKD). GFR was also estimated using the modified MDRD equation. Albuminuria was defined by urinary albumin/creatinine ratio (UACR).

Results

GFR was lower and UACR was higher in E/E′ >15 group than in 10< E/E′ ≤15 group or E/E′ ≤10 group (p<0.0001), GFR was significantly negative and UACR was positive correlated with E/E′ and NT-proBNP (p<0.0001). In multivariate stepwise linear analysis, GFR had significant correlation with age (p = 0.001), gender (p = 0.003), E/E′ (p = 0.03), lgNT-proBNP (p = 0.001) and lgUACR (p = 0.01), while eGFR had no significant correlation with E/E′ or lgNT-proBNP. Multivariate logistic regression analysis, adjusted for potential confounding factors, showed that participants in E/E′>15 group were more likely to have Stage 3 CKD compared with those in E/E′≤10 group with an adjusted odds ratio of 8.31 (p = 0.0036).

Conclusions

LV diastolic function, assessed with E/E′ and NT-proBNP is associated with renal function in essential hypertension.     

Targeting Serum Glucocorticoid-Regulated Kinase-1 in Squamous Cell Carcinoma of the Head and Neck: A Novel Modality of Local Control

Purpose

The inhibition of serum glucocorticoid-regulated kinase-1 (SGK-1) has been found to decrease growth of colon and prostate cancer cells. The purpose of this study is to evaluate the therapeutic effect of SGK-1 inhibition in head and neck squamous cell carcinoma (SCC).

Experimental Design

Human head and neck tumors (HTB41/43) were established in athymic mice. Growth rates between mice treated with vehicle (PBS) injection (group 1, n = 5), SGK-1 Inhibitor GSK 650394 (group 2, n = 6), systemic cisplatin (group 3, n = 6), and a combination of SGK-1 Inhibitor and cisplatin (group 4, n = 6) were compared using repeated measures one-way ANOVA with Newman-Keuls Multiple Comparison Test. Tumor cells were subsequently submitted to further analyses.

Results

At the end of the experiment mean tumor sizes were 122.33+/−105.86, 76.73+/−36.09, 94.52+/−75.92, and 25.76+/−14.89 mm² (mean +/− SD) for groups 1 to 4. Groups 2 and 3 showed decreased tumor growth compared to controls (p<0.001). Group 4 displayed even greater growth suppression (p<0.0001). Importantly, group 4 fared better than group 3 (p<0.001). CD44 expression was reduced in group 2 (p<0.05), and to an even greater extent in groups 3 and 4 (p<0.0025). A trend towards reduction of HER 2 expression was noted in group 4.

Conclusions

SGK-1 inhibition suppresses tumor growth, and in combination with systemic cisplatin exceeds the effect of cisplatin alone. Decreased expression of CD44 and HER 2 implies depletion of tumor stem cells, and less tumorigenicity. SGK-1 inhibition represents a potential modality of local control for palliation in advanced cases.     

Antenatal Dexamethasone after Asphyxia Increases Neural Injury in Preterm Fetal Sheep

Background and Purpose

Maternal glucocorticoid treatment for threatened premature delivery dramatically improves neonatal survival and short-term morbidity; however, its effects on neurodevelopmental outcome are variable. We investigated the effect of maternal glucocorticoid exposure after acute asphyxia on injury in the preterm brain.

Methods

Chronically instrumented singleton fetal sheep at 0.7 of gestation received asphyxia induced by complete umbilical cord occlusion for 25 minutes. 15 minutes after release of occlusion, ewes received a 3 ml i.m. injection of either dexamethasone (12 mg, n = 10) or saline (n = 10). Sheep were killed after 7 days recovery; survival of neurons in the hippocampus and basal ganglia, and oligodendrocytes in periventricular white matter were assessed using an unbiased stereological approach.

Results

Maternal dexamethasone after asphyxia was associated with more severe loss of neurons in the hippocampus (CA3 regions, 290±76 vs 484±98 neurons/mm², mean±SEM, P<0.05) and basal ganglia (putamen, 538±112 vs 814±34 neurons/mm², P<0.05) compared to asphyxia-saline, and with greater loss of both total (913±77 vs 1201±75/mm², P<0.05) and immature/mature myelinating oligodendrocytes in periventricular white matter (66±8 vs 114±12/mm², P<0.05, vs sham controls 165±10/mm², P<0.001). This was associated with transient hyperglycemia (peak 3.5±0.2 vs. 1.4±0.2 mmol/L at 6 h, P<0.05) and reduced suppression of EEG power in the first 24 h after occlusion (maximum −1.5±1.2 dB vs. −5.0±1.4 dB in saline controls, P<0.01), but later onset and fewer overt seizures.

Conclusions

In preterm fetal sheep, exposure to maternal dexamethasone during recovery from asphyxia exacerbated brain damage.     

Determining Prenatal,Early Childhood and Cumulative Long-Term Lead Exposure Using Micro-Spatial Deciduous Dentine Levels

The aim of this study was to assess the validity of micro-spatial dentine lead (Pb) levels as a biomarker for accurately estimating exposure timing over the prenatal and early childhood periods and long-term cumulative exposure to Pb. In a prospective pregnancy cohort sub-sample of 85 subjects, we compared dentine Pb levels measured using laser ablation-inductively coupled plasma mass spectrometry with Pb concentrations in maternal blood collected in the second and third trimesters, maternal bone, umbilical cord blood, and childhood serial blood samples collected from the ages of 3 months to ≥6 years. We found that Pb levels (as ²⁰⁸Pb:⁴³Ca) in dentine formed at birth were significantly associated with cord blood Pb (Spearman ρ = 0.69; n = 27; p<0.0001). The association of prenatal dentine Pb with maternal patella Pb (Spearman ρ = 0.48; n = 59; p<0.0001) was stronger than that observed for tibia Pb levels (Spearman ρ = 0.35; n = 41; p<0.03). When assessing postnatal exposure, we found that Pb levels in dentine formed at 3 months were significantly associated with Pb concentrations in children’s blood collected concurrently (Spearman ρ = 0.64; n = 55; p<0.0001). We also found that mean Pb concentrations in secondary dentine (that is formed from root completion to tooth shedding) correlated positively with cumulative blood lead index (Spearman ρ = 0.38; n = 75; p<0.0007). Overall, our results support that micro-spatial measurements of Pb in dentine can be reliably used to reconstruct Pb exposure timing over the prenatal and early childhood periods, and secondary dentine holds the potential to estimate long-term exposure up to the time the tooth is shed.     

In-Vitro Study of the Effect of Anti-Hypertensive Drugs on Placental Hormones and Angiogenic Proteins Synthesis in Pre-Eclampsia

Introduction

Antihypertensive drugs lower the maternal blood pressure in pre-eclampsia (PE) by direct or central vasodilatory mechanisms but little is known about the direct effects of these drugs on placental functions.

Objective

The aim of our study is to evaluate the effect of labetolol, hydralazine, α-methyldopa and pravastatin on the synthesis of placental hormonal and angiogenic proteins know to be altered in PE.

Design

Placental villous explants from late onset PE (n = 3) and normotensive controls (n = 6) were cultured for 3 days at 10 and 20% oxygen (O₂) with variable doses anti-hypertensive drugs. The levels of activin A, inhibin A, human Chorionic Gonadotrophin (hCG), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) were measured in explant culture media on day 1, 2 and 3 using standard immunoassays. Data at day 1 and day 3 were compared.

Results

Spontaneous secretion of sEndoglin and sFlt-1 were higher (p<0.05) in villous explants from PE pregnancies compared to controls. There was a significant time dependant decrease in the secretion of sFlt-1 and sEndoglin in PE cases, which was seen only for sFlt-1 in controls. In both PE cases and controls the placental protein secretions were not affected by varying doses of anti-hypertensive drugs or the different O₂ concentration cultures, except for Activin, A which was significantly (p<0.05) higher in controls at 10% O₂.

Interpretation

Our findings suggest that the changes previously observed in maternal serum hormones and angiogenic proteins level after anti-hypertensive treatment in PE could be due to a systemic effect of the drugs on maternal blood pressure and circulation rather than a direct effect of these drugs on placental biosynthesis and/or secretion.     

The Effect of pH and Ion Channel Modulators on Human Placental Arteries

Chorionic plate arteries (CPA) are located at the maternofetal interface where they are able to respond to local metabolic changes. Unlike many other types of vasculature, the placenta lacks nervous control and requires autoregulation for controlling blood flow. The placental circulation, which is of low-resistance, may become hypoxic easily leading to fetal acidosis and fetal distress however the role of the ion channels in these circumstances is not well-understood. Active potassium channel conductances that are subject to local physicochemical modulation may serve as pathways through which such signals are transduced. The aim of this study was to investigate the modulation of CPA by pH and the channels implicated in these responses using wire myography. CPA were isolated from healthy placentae and pre-contracted with U46619 before testing the effects of extracellular pH using 1 M lactic acid over the pH range 7.4 - 6.4 in the presence of a variety of ion channel modulators. A change from pH 7.4 to 7.2 produced a 29±3% (n = 9) relaxation of CPA which increased to 61±4% at the lowest pH of 6.4. In vessels isolated from placentae of women with pre-eclampsia (n = 6), pH responses were attenuated. L-methionine increased the relaxation to 67±7% (n = 6; p<0.001) at pH 6.4. Similarly the TASK 1/3 blocker zinc chloride (1 mM) gave a maximum relaxation of 72±5% (n = 8; p<0.01) which compared with the relaxation produced by the TREK-1 opener riluzole (75±5%; n = 6). Several other modulators induced no significant changes in vascular responses. Our study confirmed expression of several ion channel subtypes in CPA with our results indicating that extracellular pH within the physiological range has an important role in controlling vasodilatation in the human term placenta.     

Human Cytomegalovirus-Induces Cytokine Changes in the Placenta with Implications for Adverse Pregnancy Outcomes

Human cytomegalovirus (CMV) infection of the developing fetus can result in adverse pregnancy outcomes including death in utero. Fetal injury results from direct viral cytopathic damage to the CMV-infected fetus, although evidence suggests CMV placental infection may indirectly cause injury to the fetus, possibly via immune dysregulation with placental dysfunction. This study investigated the effects of CMV infection on expression of the chemokine MCP-1 (CCL2) and cytokine TNF-α in placentae from naturally infected stillborn babies, and compared these changes with those found in placental villous explant histocultures acutely infected with CMV ex vivo. Tissue cytokine protein levels were assessed using quantitative immunohistochemistry. CMV-infected placentae from stillborn babies had significantly elevated MCP-1 and TNF-α levels compared with uninfected placentae (p = 0.001 and p = 0.007), which was not observed in placentae infected with other microorganisms (p = 0.62 and p = 0.71) (n = 7 per group). Modelling acute clinical infection using ex vivo placental explant histocultures showed infection with CMV laboratory strain AD169 (0.2 pfu/ml) caused significantly elevated expression of MCP-1 and TNF-α compared with uninfected explants (p = 0.0003 and p<0.0001) (n = 25 per group). Explant infection with wild-type Merlin at a tenfold lower multiplicity of infection (0.02 pfu/ml), caused a significant positive correlation between increased explant infection and upregulation of MCP-1 and TNF-α expression (p = 0.0001 and p = 0.017). Cytokine dysregulation has been associated with adverse outcomes of pregnancy, and can negatively affect placental development and function. These novel findings demonstrate CMV infection modulates the placental immune environment in vivo and in a multicellular ex vivo model, suggesting CMV-induced cytokine modulation as a potential initiator and/or exacerbator of placental and fetal injury.     

Cardiac Mechanics and Ventricular Twist by Three-Dimensional Strain Analysis in Relation to B-Type Natriuretic Peptide as a Clinical Prognosticator for Heart Failure Patients

BackgroundThree dimensional (3D) echocardiography-derived measurements of myocardial deformation and twist have recently advanced as novel clinical tools. However, with the exception of left ventricular ejection fraction and mass quantifications in hypertension and heart failure populations, the prognostic value of such imaging techniques remains largely unexplored.MethodsWe studied 200 subjects (mean age: 60.2±16 years, 54% female, female n = 107) with known hypertension (n = 51), diastolic heart failure (n = 61), or systolic heart failure (n = 30), recruited from heart failure outpatient clinics. Fifty-eight healthy volunteers were used as a control group. All participants underwent 3D-based myocardial deformation and twist analysis (Artida, Toshiba Medical Systems, Tokyo, Japan). We further investigated associations between these measures and brain natriuretic peptide levels and clinical outcomes.ResultsThe global 3D strain measurements of the healthy, hypertension, diastolic heart failure, and systolic heart failure groups were 28.03%, 24.43%, 19.70%, and 11.95%, respectively (all p<0.001). Global twist measurements were estimated to be 9.49°, 9.77°, 8.32°, and 4.56°, respectively. We observed significant differences regarding 3D-derived longitudinal, radial, and global 3D strains between the different disease categories (p<0.05), even when age, gender, BMI and heart rate were matched. In addition, 3D-derived longitudinal, circumferential, and 3D strains were all highly correlated with brain natriuretic peptide levels (p<0.001). At a mean 567.7 days follow-up (25^th–75^th IQR: 197–909 days), poorer 3D-derived longitudinal, radial, and global 3D strain measurements remained independently associated with a higher risk of cardiovascular related death or hospitalization due to heart failure, after adjusting for age, gender, and left ventricular ejection fraction (all p<0.05).Conclusions3D-based strain analysis may be a feasible and useful diagnostic tool for discriminating the extent of myocardial dysfunction. Furthermore, it is able to provide a prognostic value beyond traditional echocardiographic parameters in terms of ejection fraction.     

t-10, c-12 CLA Dietary Supplementation Inhibits Atherosclerotic Lesion Development Despite Adverse Cardiovascular and Hepatic Metabolic Marker Profiles

Animal and human studies have indicated that fatty acids such as the conjugated linoleic acids (CLA) found in milk could potentially alter the risk of developing metabolic disorders including diabetes and cardiovascular disease (CVD). Using susceptible rodent models (apoE^−/− and LDLr^−/− mice) we investigated the interrelationship between mouse strain, dietary conjugated linoleic acids and metabolic markers of CVD. Despite an adverse metabolic risk profile, atherosclerosis (measured directly by lesion area), was significantly reduced with t-10, c-12 CLA and mixed isomer CLA (Mix) supplementation in both apoE^−/− (p<0.05, n = 11) and LDLr^−/− mice (p<0.01, n = 10). Principal component analysis was utilized to delineate the influence of multiple plasma and tissue metabolites on the development of atherosclerosis. Group clustering by dietary supplementation was evident, with the t-10, c-12 CLA supplemented animals having distinct patterns, suggestive of hepatic insulin resistance, regardless of mouse strain. The effect of CLA supplementation on hepatic lipid and fatty acid composition was explored in the LDLr^−/− strain. Dietary supplementation with t-10, c-12 CLA significantly increased liver weight (p<0.05, n = 10), triglyceride (p<0.01, n = 10) and cholesterol ester content (p<0.01, n = 10). Furthermore, t-10, c-12 CLA also increased the ratio of 18∶1 to 18∶0 fatty acid in the liver suggesting an increase in the activity of stearoyl-CoA desaturase. Changes in plasma adiponectin and liver weight with t-10, c-12 CLA supplementation were evident within 3 weeks of initiation of the diet. These observations provide evidence that the individual CLA isomers have divergent mechanisms of action and that t-10, c-12 CLA rapidly changes plasma and liver markers of metabolic syndrome, despite evidence of reduction in atherosclerosis.     

Effects of Subclinical Hypothyroidism on Maternal and Perinatal Outcomes during Pregnancy: A Single-Center Cohort Study of a Chinese Population

Objective

Adverse maternal outcomes and perinatal complications are closely associated with overt maternal hypothyroidism, but whether these complications occur in women with subclinical hypothyroidism (SCH) during pregnancy remains controversial. The aim of this study was to evaluate the effects of SCH on maternal and perinatal outcomes during pregnancy.

Methods

A prospective study of data from 8012 pregnant women (371 women with SCH, 7641 euthyroid women) was performed. Maternal serum samples were collected in different trimesters to examine thyroid hormone concentrations. SCH was defined as a thyroid stimulating hormone concentration exceeding the trimester-specific reference value with a normal free thyroxine concentration. The occurrence of maternal outcomes, including gestational hypertension (GH), gestational diabetes mellitus, placenta previa, placental abruption, prelabor rupture of membranes (PROM), and premature delivery; and perinatal outcomes, including intrauterine growth restriction (IUGR), fetal distress, low birth weight (LBW; live birth weight ≤2500 g), stillbirth, and malformation, was recorded. Logistic regression with adjustment for confounding demographic and medical factors was used to determine the risks of adverse outcomes in patients with SCH.

Results

Compared with euthyroid status, SCH was associated with higher rates of GH (1.819% vs. 3.504%, P = 0.020; χ ² = 7.345; odds ratio (OR), 2.243; 95% confidence interval (CI), 1.251–4.024), PROM (4.973% vs. 8.625%, P = 0.002; χ ² = 72.102; adjusted OR, 6.014; 95% CI, 3.975–9.099), IUGR (1.008% vs. 2.965%, <0.001; χ ² = 13.272; adjusted OR, 3.336; 95% CI, 1.745–6.377), and LBW (1.885% vs. 4.582%, P<0.001; χ ² = 13.558; adjusted OR, 2.919; 95% CI, 1.650–5.163).

Conclusions

The results of this study indicate that pregnant women with SCH had increased risks of GH and PROM, and their fetuses and infants had increased risks of IUGR and LBW. Thus, routine maternal thyroid function testing is necessary to improve maternal and perinatal outcomes.     

Deficiency in Clonogenic Endometrial Mesenchymal Stem Cells in Obese Women with Reproductive Failure – a Pilot Study

Objectives

The mechanisms of obesity associated reproductive complications remain poorly understood. Endometrial mesenchymal stem-cells are critical for cyclic renewal and uterine function. Recently, W5C5⁺ cells, with high clonogenicity, capable of producing endometrial stroma in vivo, have been described. We sought to investigate the abundance and cloning efficiency of W5C5⁺ and W5C5⁻ endometrial cells in relation to Body Mass Index, age and reproductive outcome.

Design

W5C5⁺ and W5C5⁻ cells were purified from mid-luteal endometrial biopsies (n = 54) by magnetic bead separation and subjected to in vitro colony-forming assays.

Results

First trimester pregnancy losses were significantly higher in obese subjects (n = 12) compared to overweight (n = 20) and subjects with normal Body Mass Index (n = 22) (P<0.05, P<0.01, respectively). W5C5⁺ cells (%) were significantly lower in obese subjects compared to subjects with normal Body Mass Index (P<0.05). W5C5⁺ cloning efficiency was significantly lower in obese subjects compared to overweight and subjects with normal Body Mass Index (P<0.05, respectively). W5C5⁻ cloning efficiency was significantly lower in obese subjects compared to subjects with normal Body Mass Index (P<0.05). Body Mass Index was significantly negatively correlated with W5C5⁺ cloning efficiency and W5C5⁻ cloning efficiency (P<0.01, respectively), and positively correlated with first trimester loss (P<0.01). We found no significant results with age (P>0.05).

Conclusions

Our observations suggest that the regenerative capacity and plasticity of the endometrium of obese women is suboptimal, which in turn may account for the increased risk of reproductive complications associated with obesity.