Oxidative stress contributes to hypermethylation of Histone H3 lysine 9 in placental trophoblasts from preeclamptic pregnancies.

Background and objective: Aberrant epigenetic regulation and increased oxidative stress in the placenta play a significant role in placental pathophysiology and fetal programming in preeclampsia, a hypertensive disorder in human pregnancy. The purpose of the study is to investigate if hypermethylation of histone H3K9 occurs in placental trophoblasts from preeclampsia. Methods: Trophoblasts were isolated and cultured from 14 placentas, 7 from normotensive pregnant women and 7 from preeclamptic pregnancies. Methylated H3K9 expression and antioxidant superoxide dismutase expression were determined by Western blot. We also examined consequences of oxidative stress and the downstream effects of histone methyltransferase inhibition on H3K9 expression associated with antioxidant CuZn-SOD and Mn-SOD expression in placental trophoblasts. Results: We found that expression of mono-, di-, and tri-methylation of histone H3 lysine 9 (H3K9me1, H3K9me2 and H3K9me3) was significantly increased, p<0.01, which correlated with downregulation of antioxidant superoxide dismutase CuZn-SOD and Mn-SOD expression, in trophoblasts from preeclamptic placentas compared to those from uncomplicated control placentas. We further demonstrated hypoxia could promote histone H3K9 methylation in placental trophoblasts, and hypoxia-induced upregulation of H3K9me1, H3K9me2 and H3K9me3 expression was reversible when hypoxic condition was removed. In addition, we also uncovered that inhibition of methyltransferase not only prevented hypoxia-induced upregulation of H3K9me1, H3K9me2 and H3K9me3 expression, but also abolished hypoxia-induced downregulation of CuZn-SOD and Mn-SOD expression in placental trophoblasts. Conclusions: These findings are noteworthy and provide further evidence that increased oxidative stress in the intrauterine environment is likely a mechanism to induce aberrant histone modification in placental trophoblasts in preeclampsia. Moreover, CuZn-SOD and Mn-SOD expression/activity are possibly H3K9 methylation-dependent in placental trophoblasts, which further suggest that oxidative stress and aberrant histone modification have significant impact on placental trophoblasts/fetal programming in preeclampsia.

Investigation of dosimetric characteristics of radiochromic film in response to alpha particles emitted from Americium-241.

BACKGROUND: In radiotherapy, it is essential to deliver prescribed doses to tumors while minimizing damage to surrounding healthy tissue. Accurate measurements of absorbed dose are required for this purpose. Gafchromic® external beam therapy (EBT) radiochromic films have been widely used in radiotherapy. While the dosimetric characteristics of the EBT3 model film have been extensively studied for photon and charged particle beams (protons, electrons, and carbon ions), little research has been done on α $\alpha$ -particle dosimetry. α $\alpha$ -emitting radionuclides have gained popularity in cancer treatment due to their high linear energy transfer, short range in tissue, and ability to spare surrounding organs at risk, thereby delivering a more localized dose distribution to the tumor. Therefore, a dose-calibration film protocol for α $\alpha$ -particles is required. PURPOSE: This study aimed to develop a dose-calibration protocol for the α $\alpha$ -particle emitting radionuclide 241Am, using Monte Carlo (MC) simulations and measurements with unlaminated EBT3 films. METHODS: In this study, a MC-based user code was developed using the Geant4 simulation toolkit to model and simulate an 241Am source and an unlaminated EBT3 film. Two simulations were performed: one with voxelized geometries of the EBT3 active volume composition and the other using water. The dose rate was calculated within a region of interest in the voxelized geometries. Unlaminated EBT3 film pieces were irradiated with the 241Am source at various exposure times inside a black box. Film irradiations were compared to a 6-MV photon beam from a Varian TrueBeam machine. The simulated dose rate was used to convert the exposure times into absorbed doses to water, describing a radiochromic-film-based reference dosimetry protocol for α $\alpha$ -particles. The irradiated films were scanned and through an in-house Python script, the normalized pixel values from the green-color channel of scanned film images were analyzed. RESULTS: The 241Am energy spectra obtained from the simulations were in good agreement with IAEA and NIST databases, having differences < $<$ 0.516% for the emitted γ $\gamma$ -rays and produced characteristic x-rays and < $<$ 0.006% for the α $\alpha$ -particles. Due to the short range of α $\alpha$ -particles, there was no energy deposition in the voxels outside the active 241Am source region projected onto the film surface. Thus, the total dose rate within the voxels covering the source was 0.847 ± $\pm$ 0.003 Gy/min within the sensitive layer of the film (LiPCDA) and 0.847 ± $\pm$ 0.004 Gy/min in water, indicating that the active volume can be considered water equivalent for the 241Am beam quality. A novel approach was employed in α $\alpha$ -film dosimetry using an exponential fit for the green channel, which showed promising results by reducing the uncertainty in dose estimation within 5%. Although the statistical analysis did not reveal significant differences between the 6-MV photon beam and the α $\alpha$ calibration curves, the dose-response curves exhibited the expected behavior. CONCLUSIONS: The developed MC user code simulated the experimental setup for α $\alpha$ -dosimetry using radiochromic film with acceptable uncertainty. Unlaminated EBT3 film is suitable for the dosimetry of α $\alpha$ -radiation at low doses and can be used in conjunction with other unlaminated GafChromic® films for quality assurance and research purposes.

Evidence of kidney injury in preeclampsia: Increased maternal and urinary levels of NGAL and KIM-1 and their enhanced expression in proximal tubule epithelial cells.

Background and objective: Proteinuria and glomerular endotheliosis are characteristics of glomerular injury in preeclampsia, a hypertensive disorder in human pregnancy. Neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) are biomarkers of acute/chronic renal tubule injury. To determine if tubule injury occurs in preeclampsia, we determined maternal plasma and urine NGAL and KIM-1 levels and evaluated NGAL and KIM-1 expression in kidney biopsy specimens from women with preeclampsia. Methods: Prenatal and postpartum maternal blood and urinary samples were collected from three groups of pregnant women: normal pregnancy (n = 100), preeclampsia (n = 83), and pregnancy complicated with chronic hypertension (n = 20). Plasma and urine levels of NGAL and KIM-1 were measured by ELISA. Kidney biopsy tissue sections from patients with preeclampsia (n = 5) were obtained from Pathology Archives and processed to determine NGAL and KIM-1 expression by immunostaining and high kidney solution images were assessed by electron microscopy (EM). Results: Prenatal plasma and urine levels of NGAL and KIM-1 were significantly higher in preeclamptic than in normal controls, p < 0.01. In normal pregnancy, both plasma and urine levels of NGAL and KIM-1 at 24-48 h after delivery and 6-8 weeks postpartum were relatively comparable to that of antenatal levels. In preeclampsia, urine, but not plasma, NGAL levels were reduced at 6-8 weeks postpartum compared to the antenatal levels, p < 0.05. Although maternal and urine KIM-1 levels were reduced at 6-8 weeks postpartum compared to the antenatal levels in preeclampsia, the levels were still higher than those in normal pregnancy. Positive expression of NGAL and KIM-1 was detected in proximal tubule epithelial cells in kidney tissue specimens from preeclampsia but not in non-pregnancy controls. EM examination showed glomerular and tubular injury in preeclampsia. Conclusion: Our findings of increased maternal levels and urine secretion of NGAL and KIM-1, along with the upregulation of NGAL and KIM-1 expression in tubular epithelial cells in preeclampsia, provide plausible evidence that tubular injury exists in preeclampsia. The higher postpartum NGAL and KIM-1 levels in preeclamptic pregnancies indicate that tubular injury would not resolve within 2-3 months after delivery and suggest that proper follow-up and management of kidney function in women with preeclampsia would be necessary to reduce chronic kidney diseases in those women later in life.

Group B Streptococcus Rectovaginal Colonization and Resistance Patterns in HIV-Positive Compared to HIV-Negative Pregnant Patients.

OBJECTIVE: The objective of our study is to determine if human immunodeficiency virus (HIV)-positive pregnant patients have a higher rate of group B streptococcus (GBS) rectovaginal colonization compared with HIV-negative pregnant patients. STUDY DESIGN: Our study is a multi-site retrospective study performed at Ochsner Louisiana State University-Health Shreveport and Monroe campuses including patients who delivered between December 2011and June 2019. Rates of GBS rectovaginal colonization between HIV-positive pregnant patients were compared with a control group of HIV-negative patients. The control group was age and race matched in a 2:1 fashion. The primary outcome was to investigate rates of GBS rectovaginal colonization. Secondary outcomes included GBS culture antibiotic sensitivities, presence of GBS urinary tract infection, GBS positivity based on HIV viral load, and GBS positivity based on new vs established diagnosis of HIV. Continuous data were analyzed using an unpaired t-test, and categorical data were analyzed using a Chi-squared test. The probability level of <0.05 was set as statistically significant. RESULTS: A total of 225 patients were included in the final analysis, 75 HIV-positive and 150 HIV-negative controls. Demographic differences were noted. HIV-positive patients were more likely to deliver preterm and were more likely to deliver via cesarean section. Our primary outcome showed no significant differences in incidence of GBS colonization between HIV-positive patients and control group (n = 31, 41.3% vs n = 46, 30.6%, p = 0.136). Antibiotic resistance patterns showed no significant difference between the two groups. There were no significant differences in GBS positivity based on HIV viral load. CONCLUSION: Our study does not show a statistically significant difference in the incidence of GBS colonization between HIV-positive patients and HIV-negative controls. KEY POINTS: · HIV-positive pregnant patients do not have an increased risk of GBS rectovaginal colonization.. · HIV-positive pregnant patients have similar rates of GBS colonization regardless of viral load.. · GBS antibiotic sensitivities are similar in HIV-positive and HIV-negative pregnant patients..

Aberrant endothelial expression of hnRNPC1/C2 and VDR and reduced maternal vitamin D levels in women with preeclampsia.

Vitamin D deficiency is a widespread health problem globally and vitamin D deficiency/ insufficiency in pregnancy is a risk factor for preeclampsia, a hypertensive disorder in human pregnancy. Vitamin D elicits its biological effects through binding to its receptor VDR. In the present study, we determined maternal vascular expression of VDR and hnRNPC1/C2, a native repressor of VDR, in subcutaneous adipose tissue from women with normal pregnancy and preeclampsia. Maternal antenatal and postnatal vitamin D levels were measured. We found that hnRNPC1/C2 expression was markedly increased, while VDR expression was markedly reduced, in maternal vessel endothelium and smooth muscle cells from women with preeclampsia compared to that from normal pregnant controls. Reduced VDR expression was relevant to low maternal antenatal and postnatal vitamin D levels in women with preeclampsia. Using human umbilical vein endothelial cells (HUVECs) as an endothelial model, we further investigated the role of hnRNPC1/C2-mediated VDR expression in endothelial cells, and tested effect of hnRNPC1/C2 inhibition on endothelial response to bioactive vitamin D, 1,25(OH)2D3. Our results showed that inhibition of hnRNPC1/C2 by hnRNPC1/C2 siRNA resulted in not only an increase in endothelial VDR expression, but further improved endothelial response to 1,25(OH)2D3. These findings indicate that aberrant hnRNPC1/C2 expression may contribute to reduced vascular expression of VDR in women with preeclampsia and suggest that hnRNPC1/C2 could be a target for improving vascular endothelial cell response to vitamin D.

Cell-type specific distribution and activation of type I IFN pathway molecules at the placental maternal-fetal interface in response to COVID-19 infection.

Background and objective: COVID-19 infection in pregnancy significantly increases risks of adverse pregnancy outcomes. However, little is known how the innate immunity at the placental maternal-fetal interface responds to COVID-19 infection. Type I IFN cytokines are recognized as a key component of the innate immune response against viral infection. In this study, we specifically evaluated expression of IFN antiviral signaling molecules in placentas from women infected with COVID-19 during pregnancy. Methods: Expression of IFN activation signaling pathway molecules, including cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), interferon regulatory factor 3 (IRF3), Toll-like receptor 7 (TLR7), mitochondrial antiviral-signaling protein (MAVS), and IFNß were determined in formalin-fixed paraffin embedded (FFPE) placental tissue sections (villous and fetal membrane) by immunostaining. A total of 20 placentas were examined, 12 from COVID-19 patients and 8 from non-COVID-19 controls. Patient demographics, clinical data, and placental pathology report were acquired via EPIC medical record review. Results: Except BMI and placental weight, there was no statistical difference between COVID and non-COVID groups in maternal age, gestational age at delivery, gravity/parity, delivery mode, and newborn gender and weight. In COVID-exposed group, the main pathological characteristics in the placental disc are maternal and fetal vascular malperfusion and chronic inflammation. Compared to non-COVID controls, expression of IFN activation pathway molecules were all upregulated with distinct cell-type specific distribution in COVID-exposed placentas: STING in villous and decidual stromal cells; IRF3 in cytotrophoblasts (CTs) and extra-villous trophoblasts (EVTs); and TLR7 and MAVS in syncytiotrophoblasts (STs), CTs, and EVTs. Upregulation of STING, MAVS and TLR7 was also seen in fetal endothelial cells. Conclusions: STING, IRF3, TLR7, and MAVS are key viral sensing molecules that regulate type I IFN production. Type I IFNs are potent antiviral cytokines to impair and eradicate viral replication in infected cells. The finding of cell-type specific distribution and activation of these innate antiviral molecules at the placental maternal-fetal interface provide plausible evidence that type I IFN pathway molecules may play critical roles against SARS-CoV-2 infection in the placenta. Our findings also suggest that placental maternal-fetal interface has a well-defined antiviral defense system to protect the developing fetus from SARS-CoV-2 infection.

Downregulation of miR-126-3p expression contributes to increased inflammatory response in placental trophoblasts in preeclampsia.

MiR-126-3p is a prototype of an endothelial miRNA and has protective effects on endothelial cells. However, little is known about the effects of miR-126-3p on placental trophoblasts. In the present study, we tested the hypothesis that aberrant miR-126-3p expression is present in preeclamptic placenta which contributes to increased inflammatory response in trophoblasts. Placentas were obtained immediately after delivery from normotensive and preeclamptic pregnancies. Villous tissue was either fixed with formalin or used for trophoblast isolation. Trophoblast miR-126-3p expression was assessed by in situ hybridization of formalin-fixed tissue sections and by RT-PCR in cultured syncytiotrophoblasts. Culture medium was collected for measurement of IL-6, TNFα, and 8-Isoprostane production by ELISA and total cellular protein was collected for evaluation of HIF1α expression by Western blot. Effects of overexpression of miR-126-3p in trophoblasts on cytokine production were tested by transfection of pre-mir-126, a precursor of miR-126, into primary isolated trophoblasts. We found that downregulation of miR-126-3p expression was associated with increased IL-6 and TNFα production in trophoblasts from preeclamptic placentas vs. normal placentas. Moreover, transient overexpression of miR-126-3p significantly reduced IL-6 and TNFα production in trophoblasts from both normal and preeclamptic placentas. We further found that increase in miR-126-3p expression not only suppressed hypoxia-induced increases in IL-6 and TNFα production, but also attenuated hypoxia-induced increases in HIF1α expression and 8-Isoprostane production in trophoblasts cultured under hypoxic condition. These results provide plausible evidence that downregulation of miR-126-3p expression reduces anti-inflammatory and anti-oxidative stress activities in placental trophoblasts in preeclampsia.

Preeclampsia Status Controls Interleukin-6 and Soluble IL-6 Receptor Release from Neutrophils and Endothelial Cells: Relevance to Increased Inflammatory Responses.

Increased neutrophil-endothelial binding and inflammatory responses are significant pathophysiological events in the maternal vascular system in preeclampsia, a hypertensive disorder in human pregnancy. Interleukin 6 (IL-6) and its soluble receptors (soluble IL-6R (sIL-6R) and soluble gp130 (sgp130)) are critical inflammatory mediators. During pregnancy, maternal IL-6 and sgp130 levels were increased, but sIL-6R levels were decreased, in women with preeclampsia compared to normotensive pregnant women. However, little is known about differences in IL-6, sIL-6R, and sgp130 production by neutrophils and endothelial cells between normal pregnancy and preeclampsia. To study this, we isolated neutrophils and cultured human umbilical vein endothelial cells (HUVECs) from normal and preeclamptic pregnancies. Production of IL-6, sIL-6R, and sgp130 was measured. The role of placental factor(s)-mediated neutrophil production of IL-6, sIL-6R, and sgp130 was also determined by pretreating neutrophils with placental conditioned medium generated from placental villous cultures. We found that IL-6 and sgp130 were mainly produced by endothelial cells, while sIL-6R was mainly produced by neutrophils. Endothelial cells from preeclampsia produced significantly more IL-6 and sgp130, and neutrophils from preeclampsia produced significantly less sIL-6R than normal pregnancy cells. Interestingly, production of IL-6, sIL-6R, and sgp130 were time-dependently increased when neutrophils and endothelial cells were co-cultured. We also found that neutrophils from normal pregnancies produced more IL-6, but less sIL-6R, after being primed by preeclamptic-placental conditioned medium. These results demonstrated that neutrophils and endothelial cells have different capacities in producing IL-6, sIL-6R, and sgp130 between normal pregnancy and preeclampsia. These results also provide evidence that the placenta plays a role in inducing neutrophil activation in preeclampsia.

Upregulation of METTL3 expression and m6A RNA methylation in placental trophoblasts in preeclampsia.

INTRODUCTION: N6-methyladenosine (m6A) has been recognized as one of the most abundant and functionally relevant modifications of RNAs and plays critical roles in biological and pathological processes. Placental trophoblast dysfunction significantly contributes to the pathogenesis of preeclampsia. The present study aimed to determine if altered m6A expression occurs in placental trophoblasts in preeclampsia. Expression of m6A methyltransferase (methyltransferase like 3 (METTL3)), m6A demethylases (fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5)), and m6A reader protein, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC1/C2), were also examined. METHODS: A total of 43 placentas (20 normal term, 5 normotensive preterm, and 18 preeclamptic) were used in the study. Expression of m6A, METTL3, FTO, ALKBH5, and hnRNPC1/C2 were examined by immunostaining in villous tissue sections and/or by Western blot of total cellular protein in trophoblasts isolated from normotensive and preeclamptic placentas. Total RNA extracted from trophoblasts was used to measure m6A RNA methylation. Effects of METTL3 on m6A RNA methylation and hnRNPC1/C2 expression were assessed by transfection of METTL3 siRNA in trophoblasts from preeclamptic placentas. RESULTS: Expression of m6A and m6A RNA methylation were significantly increased in trophoblasts from preeclamptic vs. normotensive placentas, p < 0.05. Expression of METTL3 and hnRNPC1/C2, but not FTO and ALKBH5, was significantly upregulated in trophoblasts from preeclamptic vs. normotensive placentas, p < 0.01. Transfection of METTL3 siRNA significantly reduced the level of m6A RNA methylation and hnRNPC1/C2 expression in trophoblasts from preeclamptic placentas, p < 0.05. CONCLUSION: The finding of increased METTL3 expression and m6A RNA methylation associated with increased hnRNPC1/C2 expression provides a new posttranscriptional mechanism that aberrant m6A modification may contribute to trophoblast dysfunction in preeclampsia.

Upregulation of histone H3K9 methylation in fetal endothelial cells from preeclamptic pregnancies.

Adverse intrauterine environment has been considered a predisposing factor for fetal programming in preeclampsia. Using human umbilical vein endothelial cells (HUVECs), we specifically explored if aberrant histone methylation occurs in fetal endothelial cells in preeclampsia. Strikingly, we found that increased di-, and tri-methylation of histone H3 lysine 9 (H3K9me2 and H3K9me3) expression were associated with upregulation of methyltransferase G9a and downregulation of endothelial nitric oxide synthase and CuZn-SOD expression in preeclamptic HUVECs. We further demonstrated that hypoxia-induced hypermethylation of H3K9 and reduced CuZn-SOD expression mimicked what were seen in preeclamptic HUVECs and inhibition of G9a could attenuate these hypoxia-induced adverse events. Our study was the first to identify hypermethylation status in fetal endothelial cells in preeclampsia, which provides plausible evidence that increased oxidative stress in the intrauterine environment is likely a mechanism to induce aberrant histone modification in fetal endothelial cells which may have a significant impact on fetal programming in preeclampsia.

Report of AAPM Task Group 235 Radiochromic Film Dosimetry: An Update to TG-55.

The use of radiochromic film (RCF) dosimetry in radiation therapy is extensive due to its high level of achievable accuracy for a wide range of dose values and its suitability under a variety of measurement conditions. However, since the publication of the 1998 AAPM Task Group 55, Report No. 63 on RCF dosimetry, the chemistry, composition, and readout systems for RCFs have evolved steadily. There are several challenges in using the new RCFs, readout systems and validation of the results depending on their applications. Accurate RCF dosimetry requires understanding of RCF selection, handling and calibration methods, calibration curves, dose conversion methods, correction methodologies as well as selection, operation and quality assurance (QA) programs of the readout systems. Acquiring this level of knowledge is not straight forward, even for some experienced users. This Task Group report addresses these issues and provides a basic understanding of available RCF models, dosimetric characteristics and properties, advantages and limitations, configurations, and overall elemental compositions of the RCFs that have changed over the past 20 yr. In addition, this report provides specific guidelines for data processing and analysis schemes and correction methodologies for clinical applications in radiation therapy.

Prolonged Fetal Heart Rate Decelerations in Labor: Can We Reduce Unplanned Primary Cesarean Sections in This Group?

INTRODUCTION: Non-reassuring fetal tracing is the second leading cause of primary cesarean delivery in the United States. Prolonged fetal heart rate decelerations are non-reassuring fetal heart rate characteristics, which do not uniformly predict poor fetal outcome but can prompt obstetricians to proceed with cesarean delivery. The objective of this manuscript is to identify a strategy to reduce the primary cesarean section rate in patients with prolonged fetal heart rate decelerations in labor. METHODS: This is a retrospective cohort study over a 5-year period at an academic medical center, including patients undergoing primary cesarean section following labor induction, augmentation, or spontaneous labor who were noted to have prolonged fetal heart rate deceleration(s) in the 1 h prior to the time of delivery. Two groups were compared: "crash" cesarean sections versus "emergent" cesarean sections. The primary outcome was if fetal heart tones were rechecked in the operating room prior to cesarean section incision. Secondary outcomes included maternal-fetal monitoring versus Doppler fetal heart tones in the operating room, return to baseline noted in the operating room, fetal outcomes, fetal monitoring characteristics, and anesthesia type between crash versus emergent groups. RESULTS: Of 1969 term singleton cesarean sections, 119 patients met our inclusion criteria (emergent group n = 80) (crash group n = 39), which accounted for 13.9% of all primary cesarean sections during the study period. The emergent group had a significantly higher rate of reassessment of fetal heart tones in the operating room n = 61 (76.2%) versus the crash group n = 15 (38.4%) (p ≤ 0.0001). There were no statistically significant differences regarding fetal outcomes between the two groups. The crash group had a higher rate of category 1 fetal heart rate tracing prior to the prolonged deceleration, a longer median prolonged deceleration, and a deeper median nadir of the prolonged deceleration; these differences were statistically significant. The prolonged-to-delivery interval was significantly shorter in the crash group (median = 15 min) than tin he emergent group (median = 33 min) (p ≤ 0.0001). The crash group also had a higher rate of general anesthesia (n = 11, 28.2%) than the emergent group (n = 6, 7.5%) (p = 0.002). The crash group was specifically investigated. Of the 15 patients with fetal heart tones rechecked in the crash group, 7 had returned to baseline in the operating room, but underwent cesarean section without fetal monitoring. CONCLUSION: Our results indicate that the practice of placing patients on fetal monitor upon arrival to the operating room prior to performing crash cesarean delivery could reduce the rate of primary cesarean deliveries performed for prolonged decelerations in labor. When fetal heart tones have returned to baseline upon arrival in the operating room, the decision to proceed with cesarean delivery can be reconsidered. However, many clinical factors must be taken into consideration, and the decision to proceed is ultimately at the discretion of the obstetrics provider.

Prospective, randomized, double-blind, placebo-controlled evaluation of the Pharmacokinetics, Safety and Efficacy of Recombinant Antithrombin Versus Placebo in Preterm Preeclampsia.

BACKGROUND: Despite expectant management, preeclampsia remote from term usually results in preterm delivery. Antithrombin, which displays antiinflammatory and anticoagulant properties, may have a therapeutic role in treating preterm preeclampsia, a disorder characterized by endothelial dysfunction, inflammation, and activation of the coagulation system. OBJECTIVE: This randomized, placebo-controlled clinical trial aimed to evaluate whether intravenous recombinant human antithrombin could prolong gestation and therefore improve maternal and fetal outcomes. STUDY DESIGN: We performed a double-blind, placebo-controlled trial at 23 hospitals. Women were eligible if they had a singleton pregnancy, early-onset or superimposed preeclampsia at 23 0/7 to 30 0/7 weeks' gestation, and planned expectant management. In addition to standard therapy, patients were randomized to receive either recombinant human antithrombin 250 mg loading dose followed by a continuous infusion of 2000 mg per 24 hours or an identical saline infusion until delivery. The primary outcome was days gained from randomization until delivery. The secondary outcome was composite neonatal morbidity score. A total of 120 women were randomized. RESULTS: There was no difference in median gestational age at enrollment (27.3 weeks' gestation for the recombinant human antithrombin group [range, 23.1-30.0] and 27.6 weeks' gestation for the placebo group [range, 23.0-30.0]; P=.67). There were no differences in median increase in days gained (5.0 in the recombinant human antithrombin group [range, 0-75] and 6.0 for the placebo group [range, 0-85]; P=.95). There were no differences between groups in composite neonatal morbidity scores or in maternal complications. No safety issues related to recombinant human antithrombin were noted in this study, despite the achievement of supraphysiological antithrombin concentrations. CONCLUSION: The administration of recombinant human antithrombin in preterm preeclampsia neither prolonged pregnancy nor improved neonatal or maternal outcomes.

Clinical Utility of Weekly Laboratory Testing in the Outpatient Management of Preeclampsia and Gestational Hypertension.

Objective To investigate the utility of obtaining weekly laboratory testing in patients managed as an outpatient for gestational hypertension and preeclampsia without severe features. Study Design A multisite retrospective cohort study was performed evaluating preterm women diagnosed with gestational hypertension/preeclampsia managed in an outpatient setting between gestational ages of 23 0/7 and 36 6/7 . Patients were divided into two groups: weekly laboratory evaluation (laboratories group) and a no laboratories group. The primary study outcome was composite maternal morbidity including more than one of the following: development of severe features, HELLP syndrome, eclampsia, placental abruption, maternal intensive care unit admission, or maternal death. Results A total of 204 patients were included in this study, laboratories group ( n = 120) and no laboratories group ( n = 84). The laboratories group was older (28.8 vs. 26.6 years, p = 0.02), had a higher rate of chronic hypertension (44 [36.7%] vs. 17 [20.2%], p = 0.01), and more often experienced the primary composite outcome (53 [44.2%] vs. 24 [28.5%], p = 0.02). No patients in our cohort were delivered for abnormal laboratory values. Conclusion This study found that weekly laboratory testing may have minimal clinical utility in the outpatient management protocol in monitoring patients with mild gestational hypertension or preeclampsia. Delivery was guided by other clinical factors.

Maternal soluble PD-1 levels are significantly increased in women with preeclampsia.

PROBLEM: Programmed cell death-1 (PD-1) and its ligand (PD-L1) have emerged as key players in regulating immune tolerance. Preeclampsia is associated with maladaptation of immune tolerance during pregnancy. This study aimed to determine if maternal soluble PD-1 (sPD-1) and soluble PD-L1 (sPD-L1) levels are altered in preeclampsia. METHOD OF STUDY: Maternal sPD-1 and sPD-L1 levels were measured by ELISA in 172 pregnant women (86 normotensive and 86 preeclampsia). The differences in sPD-1 and sPD-L1 levels between normotensive and preeclamptic pregnant women, <34 vs >34 weeks, and fetal gender differences were assessed. Data were analyzed by unpaired t test or chi-square. A probability level of <.05 was considered statistically significant. RESULTS: Maternal sPD-1 levels were significantly higher in preeclamptic than in normotensive pregnant women, 6262 ± 1860 vs 1134 ± 349 pg/mL, P < .01. sPD-1 levels were not statistically different between <34 and >34 weeks of gestation in both normotensive and preeclamptic groups. sPD-1 levels were relatively higher in mothers with female fetus than with male fetus in the preeclamptic group: 8104 ± 3054 vs 3802 ± 2177 pg/mL, but relatively lower in mothers with female fetus than with male fetus in the normotensive group: 425 ± 134 vs 625 ± 182 pg/mL. Maternal sPD-L1 levels were relatively higher in preeclamptic than in normotensive pregnant women: 143 ± 52 vs 69 ± 13 pg/mL. CONCLUSION: Aberrant sPD-1/sPD-L1 signaling is present in preeclampsia. Whether increased maternal sPD-1 and sPD-L1 levels were associated with fetal gender difference or immune tolerance dissimilarity during pregnancy in women with preeclampsia warrants further investigation.

Downregulation of vitamin D receptor and miR-126-3p expression contributes to increased endothelial inflammatory response in preeclampsia.

PROBLEM: To investigate whether downregulation of miR-126-3p and vitamin D receptor (VDR) expression contributes to increased endothelial inflammatory response in preeclampsia. METHODS OF STUDY: Maternal vessel miR-126-3p expression was assessed by in situ hybridization. VDR expression and VCAM-1 expression were determined by immunostaining. Subcutaneous adipose tissue sections from normotensive and preeclamptic pregnant women were used. HUVECs from normotensive deliveries were used to test anti-inflammatory effects of vitamin D and miR-126-3p in endothelial cells (ECs) treated with TNFα in vitro. 1,25(OH)2 D3 was used as bioactive vitamin D. Transient overexpression of miR-126-3p in ECs was induced by transfection of pre-mir-126 precursor. Endothelial VCAM-1 and SOCS-3 expression or production was determined by Western blotting or by ELISA, respectively. RESULTS: Reduced VDR and miR-126-3p expression, but increased VCAM-1 expression, was observed in maternal vessel endothelium in tissue sections from women with preeclampsia compared to normotensive pregnant controls. Transient overexpression of miR-126-3p not only attenuated upregulation of VCAM-1 expression and production, but also preserved downregulation of SOCS-3 expression, induced by TNFα in ECs. VDR expression and miR-126-3p expression were significantly upregulated in cells treated with 1,25(OH)2 D3 , but not in cells transfected with VDR siRNA. CONCLUSION: Downregulation of VDR and miR-126-3p expression was associated with upregulation of VCAM-1 expression in systemic vessel endothelium in preeclampsia. The finding of increased anti-inflammatory property by 1,25(OH)2 D3 through promotion of VDR and miR-126-3p expression in ECs provide plausible evidence that vitamin D deficiency and downregulation of VDR expression could contribute to increased inflammatory phenotypic changes in maternal vasculature in preeclampsia.

Histone deacetylase inhibition disturbs the balance between ACE and chymase expression in endothelial cells: a potential mechanism of chymase activation in preeclampsia.

Chymase is a major angiotensin-converting enzyme (ACE)-independent angiotensin convertase, and its expression is upregulated in the maternal vascular endothelium in preeclampsia, a hypertensive disorder in human pregnancy. Increased chymase-dependent angiotensin II generation has been reported in several cardiovascular diseases, including atherosclerosis and aneurysmal lesions. However, it remains unclear how chymase is activated. Histone modification is an important regulatory mechanism that controls gene expression. In this study, using a chymase overexpression cell model, we investigated the mechanisms of chymase activation to test our hypothesis that histone acetylation could promote endothelial chymase expression. Human umbilical vein endothelial cells were transfected with the chymase gene. Trichostatin A was used to inhibit histone deacetylases (HDACs). The expression levels of chymase, ACE, and HDACs were determined by western blotting. Our results showed that ACE was strongly expressed in control cells, but was significantly downregulated in cells transfected to express chymase. Strikingly, we also found that HDAC inhibition resulted in a dose-dependent increase in chymase expression but a dose-dependent decrease in ACE expression in cells transfected with the chymase gene. HDAC inhibition was confirmed by the decreased expression of HDAC1 and HDAC6 in cells treated with trichostatin A. Increased chymase expression associated with reduced histone deacetylase expression was further confirmed by immunostaining of subcutaneous adipose sections from women with preeclampsia. We conclude that aberrant HDAC expression/activity could disturb the balance between ACE and chymase expression in endothelial cells. Our results support the clinical importance of chymase as a new pharmacological target for cardiovascular disorders.

Loss of slit protein nephrin is associated with reduced antioxidant superoxide dismutase expression in podocytes shed from women with preeclampsia.

Recent findings of podocyte shedding/podocyturia highlight the central significance of podocyte injury in preeclampsia, a hypertensive disorder unique to human pregnancy. To test the hypothesis that oxidative stress contributes to kidney podocyte injury in preeclampsia, we specifically examined expression and distribution of antioxidant CuZn-SOD with nephrin and podoplanin in shed podocytes from women with preeclampsia. Human podocyte AB 8/13 cells served as control. We found that CuZn-SOD was localized at the front/outreach region of nephrin at the cell periphery (foot process areas) in control podocytes and expression of CuZn-SOD, nephrin, and podoplanin were all dislocated or lost in shed podocytes from preeclamptic patients. We further tested oxidative stress-induced nephrin shedding in podocytes, in which AB 8/13 podocytes were cultured under lowered oxygen condition (2%O2 ) or treated with hypoxic mimicking agent cobalt chloride. Our results showed that reduced nephrin and podoplanin expression were associated with downregulation of CuZn-SOD expression in podocytes when cells were cultured under lowered oxygen or hypoxic conditions. Nephrin shed in urinary specimen from preeclamptic women was also determined by immunoprecipitation/immunoblotting. The molecular sizes of nephrin that corresponded to that were lost when cells were cultured under hypoxic conditions. We concluded that increased oxidative stress plays a significant role in inducing podocyte protein shedding in preeclampsia.

Aberrant pro-atrial natriuretic peptide/corin/natriuretic peptide receptor signaling is present in maternal vascular endothelium in preeclampsia.

OBJECTIVE: Corin is a serine protease that converts pro-atrial natriuretic peptide (pro-ANP) to atrial natriuretic peptide (ANP), a cardiac hormone that regulates salt-water balance and blood pressure. ANP is degraded by natriuretic peptide receptor (NPR). This study was to determine if aberrant pro-ANP/corin/NPR signaling is present in maternal vascular system in preeclampsia. STUDY DESIGN: Maternal venous blood was obtained from 197 pregnant women (84 normotensive, 16 complicated with chronic hypertension (CHT), 11 mild and 86 severe preeclampsia). Plasma corin and pro-ANP concentrations were measured by enzyme-linked immunosorbent assay. Maternal subcutaneous fat tissue was obtained from 12 pregnant women with cesarean section delivery (6 normotensive and 6 preeclampsia). Vascular ANP and its receptors NPR-A, NPR-B, and NPR-C expression were examined by immunostaining of paraffin embedded subcutaneous fat tissue sections. RESULTS: Corin concentrations were significantly higher in mild (2.78 ±â€¯0.67 ng/ml, p < .05) and severe (2.53 ±â€¯0.18 ng/ml, p < .01) preeclampsia than in normotensive (1.58 ±â€¯0.08 ng/ml) and CHT (1.55 ±â€¯0.20 ng/ml) groups. Pro-ANP concentrations were significantly higher in CHT (1.59 ±â€¯0.53 ng/ml, p < .05) and severe preeclampsia (1.42 ±â€¯0.24 ng/ml, p < .01) than in normotensive (0.48 ±â€¯0.06 ng/ml) and mild preeclampsia (0.52 ±â€¯0.09 ng/ml) groups. ANP and NPR-B expression was undetectable in maternal vessels from normotensive and preeclamptic pregnancies, but reduced NPR-A expression and increased NPR-C expression was found in maternal vessel endothelium in preeclampsia. CONCLUSIONS: ANP is a vasodilator and NPR-C is a clearance receptor for ANP. The finding of upregulation of NPR-C expression suggests that circulating ANP clearance or degradation is increased in preeclampsia. These results also suggest that pro-ANP/corin/NPR signaling is dominant in the vascular system in preeclampsia.

Reduced CD200 expression is associated with altered Th1/Th2 cytokine production in placental trophoblasts from preeclampsia.

PROBLEM: To determine if altered trophoblast CD200 and CD200R expressions promote inflammatory cytokine production in preeclamptic placentas. METHODS OF STUDY: Placental tissue CD200 and CD200R expressions were determined by immunostaining. Tissue sections from first-, second-, and third-trimester, normal term, and preeclamptic placentas were used. CD200 and CD200R expressions and cytokine production of TNFα, sTNFR1, INFγ, IL-4, IL-6, IL-8, and IL-10 were determined in trophoblasts from normal and preeclamptic placentas and in normal trophoblasts transfected with CD200 siRNA. RESULTS: CD200, but not CD200R, expression was significantly reduced in trophoblasts from preeclamptic compared to normal placentas. Trophoblast from preeclamptic placentas and trophoblast transfected with CD200 siRNA produced significantly more TNFα, sTNFR1, IL-6, and IL-8, but significantly less IL-10, than trophoblasts from normal control placentas. CONCLUSION: Downregulation of CD200 expression resulted in an imbalance of increased Th1 cytokine and decreased Th2 cytokine production in placental trophoblasts in preeclampsia.