Edward K. Chien

A New Model for Inflammation-Induced Preterm Birth: The Role of Platelet-Activating Factor and Toll-Like Receptor-4

Preterm birth is a leading cause of neonatal morbidity and mortality. Despite a growing body of evidence correlating inflammation with preterm birth, the signal transduction pathways responsible for the emptying of the uterus in the setting of intrauterine inflammation has not been elucidated. We now report a unique, reproducible mouse model of localized intrauterine inflammation. This model results in 100% preterm delivery with no maternal mortality. Using our model, we also show that platelet-activating factor is a crucial mediator of both inflammation-induced preterm birth and fetal demise. Using C3H/HeJ mice, we demonstrate that toll-like receptor-4 (TLR-4) plays a role in lipopolysaccharide-induced preterm birth but not in inflammation-induced fetal death. Immunohistochemistry studies demonstrate the presence of the platelet-activating factor receptor in both endometrial glands and smooth muscle in uterine tissues. Molecular studies demonstrate the differential expression of platelet-activating factor receptor and TLR-4 in uterine and cervical tissue throughout gestation. Quantitative polymerase chain reaction revealed an up-regulation of TLR-4 in the fundal region of the uterus in response to intrauterine inflammation. The use of this model will increase our understanding of the significant clinical problem of inflammation-induced preterm birth and will elucidate signal transduction pathways involved in an inflammatory state.

Antenatal Betamethasone for Women at Risk for Late Preterm Delivery

BACKGROUND Infants who are born at 34 to 36 weeks of gestation (late preterm) are at greater risk for adverse respiratory and other outcomes than those born at 37 weeks of gestation or later. It is not known whether betamethasone administered to women at risk for late preterm delivery decreases the risks of neonatal morbidities. METHODS We conducted a multicenter, randomized trial involving women with a singleton pregnancy at 34 weeks 0 days to 36 weeks 5 days of gestation who were at high risk for delivery during the late preterm period (up to 36 weeks 6 days). The participants were assigned to receive two injections of betamethasone or matching placebo 24 hours apart. The primary outcome was a neonatal composite of treatment in the first 72 hours (the use of continuous positive airway pressure or high-flow nasal cannula for at least 2 hours, supplemental oxygen with a fraction of inspired oxygen of at least 0.30 for at least 4 hours, extracorporeal membrane oxygenation, or mechanical ventilation) or stillbirth or neonatal death within 72 hours after delivery. RESULTS The primary outcome occurred in 165 of 1427 infants (11.6%) in the betamethasone group and 202 of 1400 (14.4%) in the placebo group (relative risk in the betamethasone group, 0.80; 95% confidence interval [CI], 0.66 to 0.97; P=0.02). Severe respiratory complications, transient tachypnea of the newborn, surfactant use, and bronchopulmonary dysplasia also occurred significantly less frequently in the betamethasone group. There were no significant between-group differences in the incidence of chorioamnionitis or neonatal sepsis. Neonatal hypoglycemia was more common in the betamethasone group than in the placebo group (24.0% vs. 15.0%; relative risk, 1.60; 95% CI, 1.37 to 1.87; P<0.001). CONCLUSIONS Administration of betamethasone to women at risk for late preterm delivery significantly reduced the rate of neonatal respiratory complications. (Funded by the National Heart, Lung, and Blood Institute and the Eunice Kennedy Shriver National Institute of Child Health and Human Development; ClinicalTrials.gov number, NCT01222247.).

Racial/ethnic standards for fetal growth: The NICHD Fetal Growth Studies

OBJECTIVE Fetal growth is associated with long-term health yet no appropriate standards exist for the early identification of undergrown or overgrown fetuses. We sought to develop contemporary fetal growth standards for 4 self-identified US racial/ethnic groups. STUDY DESIGN We recruited for prospective follow-up 2334 healthy women with low-risk, singleton pregnancies from 12 community and perinatal centers from July 2009 through January 2013. The cohort comprised: 614 (26%) non-Hispanic whites, 611 (26%) non-Hispanic blacks, 649 (28%) Hispanics, and 460 (20%) Asians. Women were screened at 8w0d to 13w6d for maternal health status associated with presumably normal fetal growth (aged 18-40 years; body mass index 19.0-29.9 kg/m(2); healthy lifestyles and living conditions; low-risk medical and obstetrical history); 92% of recruited women completed the protocol. Women were randomized among 4 ultrasonography schedules for longitudinal fetal measurement using the Voluson E8 (GE Healthcare, Milwaukee, WI). In-person interviews and anthropometric assessments were conducted at each visit; medical records were abstracted. The fetuses of 1737 (74%) women continued to be low risk (uncomplicated pregnancy, absent anomalies) at birth, and their measurements were included in the standards. Racial/ethnic-specific fetal growth curves were estimated using linear mixed models with cubic splines. Estimated fetal weight (EFW) and biometric parameter percentiles (5th, 50th, 95th) were determined for each gestational week and comparisons made by race/ethnicity, with and without adjustment for maternal and sociodemographic factors. RESULTS EFW differed significantly by race/ethnicity >20 weeks. Specifically at 39 weeks, the 5th, 50th, and 95th percentiles were 2790, 3505, and 4402 g for white; 2633, 3336, and 4226 g for Hispanic; 2621, 3270, and 4078 g for Asian; and 2622, 3260, and 4053 g for black women (adjusted global P < .001). For individual parameters, racial/ethnic differences by order of detection were: humerus and femur lengths (10 weeks), abdominal circumference (16 weeks), head circumference (21 weeks), and biparietal diameter (27 weeks). The study-derived standard based solely on the white group erroneously classifies as much as 15% of non-white fetuses as growth restricted (EFW <5th percentile). CONCLUSION Significant differences in fetal growth were found among the 4 groups. Racial/ethnic-specific standards improve the precision in evaluating fetal growth.

Mild gestational diabetes mellitus and long-term child health

OBJECTIVE To evaluate whether treatment of mild gestational diabetes mellitus (GDM) confers sustained offspring health benefits, including a lower frequency of obesity. RESEARCH DESIGN AND METHODS Follow-up study of children (ages 5–10) of women enrolled in a multicenter trial of treatment versus no treatment of mild GDM. Height, weight, blood pressure, waist circumference, fasting glucose, fasting insulin, triglycerides, and HDL cholesterol were measured. RESULTS Five hundred of 905 eligible offspring (55%) were enrolled. Maternal baseline characteristics were similar between the follow-up treated and untreated groups. The frequencies of BMI ≥95th (20.8% and 22.9%) and 85th (32.6% and 38.6%) percentiles were not significantly different in treated versus untreated offspring (P = 0.69 and P = 0.26). No associations were observed for BMI z score, log waist circumference, log triglycerides, HDL cholesterol, blood pressure, or log HOMA-estimated insulin resistance (HOMA-IR). The effect of treatment was different by sex for fasting glucose and log HOMA-IR (P for interaction = 0.002 and 0.02, respectively) but not by age-group (5–6 and 7–10 years) for any outcomes. Female offspring of treated women had significantly lower fasting glucose levels. CONCLUSIONS Although treatment for mild GDM has been associated with neonatal benefits, no reduction in childhood obesity or metabolic dysfunction in the offspring of treated women was found. However, only female offspring of women treated for mild GDM had lower fasting glucose.

Placental growth factor is a potent vasodilator of rat and human resistance arteries

The objectives of this study were to determine whether placental growth factor (PlGF) exerts a vasodilatory effect on rat uterine vessels (arcuate arteries and veins) and to examine regional differences in reactivity by comparing these responses to those of comparably sized mesenteric vessels. We also sought to examine and compare its effects on human uterine and subcutaneous vessels. All vessels were studied in vitro, under pressurized (rat) or isometric wire-mounted (human) conditions, and exposed to a range of PlGF concentrations. Inhibitors of nitric oxide and prostaglandin synthesis were included in an effort to understand the causal mechanism(s). In rat uterine arteries, the effects of receptor inhibition and activation using selective ligands for VEGFR-1 (PlGF) vs. VEGFR-2 (VEGF-E) were determined, and real-time RT-PCR was performed to evaluate the effect of pregnancy on relative abundance of VEGFR-1 and VEGFR-2 message in the vascular wall. PlGF was a potent vasodilator of all vessels studied, with greatest sensitivity observed in rat uterine arteries. Pregnancy significantly augmented dilator sensitivity to PlGF, and this effect was associated with selective upregulation of VEGFR-1 message in the pregnant state. The contribution of nitric oxide was appreciable in rat and human uterine arteries, with lesser effects in rat uterine veins and mesenteric arteries, and with no observable effect in human subcutaneous vessels. Based on these results, we conclude that PlGF is a potent vasodilator of several vessel types in both humans and rats. Its potency and mechanism vary with physiological state and vessel location and are mediated solely by the VEGFR-1 receptor subtype. Gestational changes in the uterine circulation suggest that this factor may play a role in modulating uterine vascular remodeling and blood flow during the pregnant state.

Cytomegalovirus-induced mirror syndrome associated with elevated levels of circulating antiangiogenic factors.

BACKGROUND: We describe a case where Mirror syndrome was characterized by altered levels of antiangiogenic proteins (soluble fms–like tyrosine kinase 1 [sFlt1] and soluble endoglin). CASE: We describe a pregnant patient with severe fetal and placental edema induced by congenital cytomegalovirus (CMV) infection that was associated with preeclampsia. Fetal CMV was confirmed histologically, whereas antiangiogenic factors were demonstrated to be elevated in maternal but not fetal blood. The levels of sFlt1 and soluble endoglin in our patient’s serum before delivery were 116.5 ng/mL (normal pregnancy 19.3 ng/mL and preeclampsia 66.0 ng/mL, representing mean values before delivery) and 107.4 ng/mL (normal pregnancy 18.7 ng/mL and preeclampsia 52.6 ng/mL, representing mean values before delivery), respectively. In contrast, the values of sFlt1 and soluble endoglin in the cord blood were relatively low at 2.1 ng/mL and 8.2 ng/mL, respectively. CONCLUSION: If this observation is confirmed, CMV infection may be cited as a cause of Mirror syndrome and preeclampsia phenotypes associated with this disorder may be related to increased circulating antiangiogenic factors.

Nitroglycerin for relaxation to establish a fetal airway (EXIT procedure).

BACKGROUND: The ex utero intrapartum treatment (EXIT) procedure is a technique designed to establish an airway at the time of delivery in fetuses at risk of airway obstruction and requires maintenance of uterine relaxation to continue placental perfusion and prevent placental separation. We describe the use of intravenous nitroglycerin to maintain uterine relaxation during the EXIT procedure. CASE: A 17-year-old primigravida with a fetus known to have an anterior neck mass was admitted for a scheduled operative delivery at 38 weeks of gestation using a modified EXIT procedure. Anesthesia was administered with a combined spinal-epidural technique. Intravenous nitroglycerin was administered as a bolus and then as a continuous infusion to maintain uterine relaxation until evaluation of the neonatal airway was completed. CONCLUSION: Intravenous nitroglycerin is an effective agent for maintenance of uterine relaxation and placental perfusion during the EXIT procedure.

Intracellular Signaling and Phasic Myometrial Contractions

This article reviews recently reported observations regarding the intracellular signal transduction mechanisms involved in the generation of phasic contractions occurring in myometrial tissue. The presence of cell surface receptors for classic uterotonic agonists (including oxytocin, norepinephrine, vasopressin, acetylcholine, and prostaglandins [PGs]) has been well described; all are seven-membrane-spanning, G protein-coupled receptors. Occupancy of these receptors, coupled through members of the Gq and/or Gi families of heterotrimetric G proteins, results in stimulation of the phospholipase C-β (PCL-β) isoforms. Nonclassic uterotonic agonists, such as growth factors and cytokines, also activate the phosphatidylinositol (PI)-signaling pathway, in this case through tyrosine kinase receptor-mediated activation of the phospholipase C-γ (PCL-γ) isoforms. Several recent reports have demonstrated that activation of the PI-signaling pathway in uterine myocytes results in the development of cytosolic calcium oscillation-like phenomena. These cytosolic calcium oscillations appear to arise from receptive cycles of emptying and refill of the endoplasmic reticulum calcium stores along with the influx of extracellular calcium. Calcium release from the endoplasmic reticulum calcium stores appears to be mediated by the inositol trisphosphate-sensitive and the ryanodine-sensitive receptor/channels; isoforms for both of these receptor/channels have been shown to be expressed in myometrial tissue. In summary, receptor-mediated activation of the PI-signaling pathway and the generation of cytosolic calcium oscillations appear to produce intermitten calcium transients that result in the development and maintenance of phasic myometrial contractions.

Prostaglandin E2-regulated cervical ripening: analysis of proteoglycan expression in the rat cervix.

OBJECTIVE Prostaglandins reduce cervical resistance by reorganizing collagen fibrils. Proteoglycans are involved in collagen fibril organization and structure. We evaluated the changes in proteoglycan composition induced by prostaglandin E(2) (PGE(2)). STUDY DESIGN Prostaglandins were administered intravaginally to induce cervical ripening in timed pregnant Sprague-Dawley rats. Changes in proteoglycan messenger ribonucleic acid (mRNA) expression were measured using reverse transcription (RT-PCR) for core protein. Fluorophore assisted carbohydrate gel electrophoresis (FACE) was used to evaluate proteoglycan glycosaminoglycan composition along with size exclusion high-performance liquid chromatography (HPLC). RESULTS No change in core protein mRNA expression was detected after PGE(2) treatment. Total glycosaminoglycan (GAG) decreased more than 20% after PGE(2) (P = .02). FACE demonstrated a shift in disaccharide subunit composition after PGE(2), with a decrease in 4-sulfated disaccharides (P = .02). HPLC confirmed a decrease in total GAG (P = .04). CONCLUSION Although there was no change in core protein mRNA expression, alterations in GAG composition was detected after PGE(2). The decrease in sulfated GAG could decrease electrostatic interactions that would weaken interfibrillar interactions. These findings would be consistent with a decline in cervical resistance.

The mechanisms underlying Bay K 8644-stimulated phasic myometrial contractions.

Objective: Phasic myometrical contractions appear to be produced by calcium transients resulting from the activation of the phosphatidylinositol-signaling pathway. Bay K 8644, an L-type calcium channel activator, produces an increase in frequency and intensity of phasic myometrial contractions. These studies were performed to test the hypothesis that Bay K 8644-stimulated contractions were mediated through mechanisms involving phosphoinositide-specific phospholipase C activation and cytosolic calcium oscillation-like mechanisms. Methods: In vitro contraction studies and intracellular calcium imaging were performed on longitudinal strips of uterine tissue obtained from mature virgin Sprague-Dawley rats. Isometric contraction data were computer digitized, analyzed for contraction area, and nomalized for cross-sectional area. Dose-response studies were performed using previously reported inhibitors of cytosolic calcium oscillation mechanisms. In addition, qualitative inositol-phosphate production studies were performed after prelabeling uterine tissue in vitro with 3H-inositol. Subsequently, the labeled inositol phosphates were separated and recovered using anion exchange chromatography. Results: Bay K 8644 produced periodic calcium transients or oscillations along with a dose-related increase in contractile activity and a signfificant increase in inositol-phosphate production. In contrast, neomycin (an inhibitor of phospholipase C), adenine (an inhibitor of calcium-induced calcium release), nifedipine (an L-type calcium channel blocker), and EGTA (a calcium chelator) significantly inhibited Bay K 8644-stimulated contractile activity. Conclusions: These results are consistent with the hypothesis that Bay K 8644, through its facilitation of increased intracellular calcium, results in the activation of the phosphatidylinsitol-signaling pathway and cytosolic calcium oscillation-like phenomena, thereby resulting in the generation of phasic myometrial contractions.