Michal A. Elovitz

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.

Animal models of preterm birth

Preterm birth continues to pose a significant clinical dilemma and contributes to both acute and long-term neonatal morbidity. Despite efforts, the incidence of preterm birth has not decreased, partly because of our lack of understanding of the mechanisms that trigger parturition. Animal models are essential research tools for investigating the pathways that promote preterm parturition and for testing therapeutic interventions. Growing evidence correlates infection or inflammation with preterm birth. Consequently, many investigators have created animal models that reflect these findings. Current models of preterm parturition include diverse species, varying means of inducing an inflammatory or infectious state, and different routes of administration. Although each of these models can advance our knowledge, it is important to understand their advantages, disadvantages and unique characteristics. An understanding of such models will hopefully promote continued research that will ultimately lead to a decrease in preterm birth and an improvement in neonatal outcome.

Intrauterine inflammation, insufficient to induce parturition, still evokes fetal and neonatal brain injury.

Exposure to prenatal inflammation is a known risk factor for long term neurobehavioral disorders including cerebral palsy, schizophrenia, and autism. Models of systemic inflammation during pregnancy have demonstrated an association with an immune response an adverse neurobehavioral outcomes for the exposed fetus. Yet, the most common route for an inflammatory exposure to a fetus is from intrauterine inflammation as occurs with chorioamnionitis. The aims of this study were to assess the effect of intrauterine inflammation on fetal and neonatal brain development and to determine if the gestational age of exposure altered the maternal or fetal response to inflammation.

Adverse neonatal outcomes: examining the risks between preterm, late preterm, and term infants

OBJECTIVE There is a relative paucity of data regarding neonatal outcomes in the late preterm cohort (34 to 36 6/7 weeks). This study sought to assess differences in adverse outcomes between infants delivering 32 to 33 6/7, 34 to 36 6/7 weeks, and 37 weeks or later. STUDY DESIGN Data were collected as part of a retrospective cohort study of preterm labor patients (2002-2005). Patients delivering 32 weeks or later were included (n = 264). The incidence of adverse outcomes was assessed. Significant associations between outcomes and gestational age at delivery were determined using chi(2) analyses and Poisson regression modeled cumulative incidence and controlled for confounders. RESULTS Late preterm infants have increased risk of adverse outcomes, compared with term infants. Controlling for confounders, there was a 23% decrease in adverse outcomes with each week of advancing gestational age between 32 and 39 completed weeks (relative risk 0.77, P < .001, 95% confidence interval, 0.71-0.84). CONCLUSION Further investigation regarding obstetrical management and long-term outcomes for this cohort is warranted.

Elucidating the Early Signal Transduction Pathways Leading to Fetal Brain Injury in Preterm Birth

Adverse neurologic outcome, including cerebral palsy, is a significant contributor to long-term morbidity in preterm neonates. However, the mechanisms leading to brain injury in the setting of a preterm birth are poorly understood. In the last decade, there has been a growing body of evidence correlating infection or inflammation with preterm birth. The presence of intrauterine inflammation significantly increases the risk for adverse neurologic outcome in the neonate. These studies were performed to elucidate the early signal transduction pathways activated in the fetal brain that may result in long-term neurologic injury. Using our mouse model of localized intrauterine inflammation, the activation of TH1/TH2 pathways in the placenta, fetus corpus, fetal liver, and fetal brain was investigated. Additional studies determined whether activation of TH1/TH2 pathways could promote cell death and alter glial development. Real-time PCR studies demonstrated that a robust TH1/TH2 response occurs rapidly in the fetal brain after exposure to intrauterine inflammation. The cytokine response in the fetus and placenta was not significantly correlated with the response in the fetal brain. Along with an immune response, cell death pathways were activated early in the fetal brain in response to intrauterine LPS. Implicating TH1/TH2 and cell death pathways in permanent brain injury are our findings of an increase in GFAP mRNA and protein as well as a loss of pro-oligodendrocytes. With increased understanding of the mechanisms by which inflammation promotes brain injury in the preterm neonate, identification of potential targets to limit adverse neonatal outcomes becomes possible.

Inflammation-induced preterm birth alters neuronal morphology in the mouse fetal brain

Adverse neurological outcome is a major cause of long‐term morbidity in ex‐preterm children. To investigate the effect of parturition and inflammation on the fetal brain, we utilized two in vivo mouse models of preterm birth. To mimic the most common human scenario of preterm birth, we used a mouse model of intrauterine inflammation by intrauterine infusion of lipopolysaccharide (LPS). To investigate the effect of parturition on the immature fetal brain, in the absence of inflammation, we used a non‐infectious model of preterm birth by administering RU486. Pro‐inflammatory cytokines (IL‐10, IL‐1β, IL‐6 and TNF‐α) in amniotic fluid and inflammatory biomarkers in maternal serum and amniotic fluid were compared between the two models using ELISA. Pro‐inflammatory cytokine expression was evaluated in the whole fetal brains from the two models. Primary neuronal cultures from the fetal cortex were established from the different models and controls in order to compare the neuronal morphology. Only the intrauterine inflammation model resulted in an elevation of inflammatory biomarkers in the maternal serum and amniotic fluid. Exposure to inflammation‐induced preterm birth, but not non‐infectious preterm birth, also resulted in an increase in cytokine mRNA in whole fetal brain and in disrupted fetal neuronal morphology. In particular, Microtubule‐associated protein 2 (MAP2) staining was decreased and the number of dendrites was reduced (P < 0.001, ANOVA between groups). These results suggest that inflammation‐induced preterm birth and not the process of preterm birth may result in neuroinflammation and alter fetal neuronal morphology.

The Role of Inflammation and Infection in Preterm Birth

Much emphasis in recent decades has been devoted to inflammation and infection as a premier causal mechanism of preterm birth. This article explores the epidemiologic, clinical, and animal data that exist to support this conceptual paradigm as well as proposed mechanisms through which to potentially mitigate the adversity of prematurity. Truly successful interventions are not likely to occur until the pathogenesis of preterm birth and the role of inflammation in causing not only parturition but also fetal and neonatal injury is fully elucidated.

Rethinking IUGR in preeclampsia: dependent or independent of maternal hypertension?

Objective:Chronic hypertension (CHTN) is a risk factor for both intrauterine growth restriction (IUGR) as well as preeclampsia. This study was performed to: (1) describe the prevalence of IUGR in women with preeclampsia (with and without CHTN) compared with controls, (2) investigate the relationship between preeclampsia and maternal CHTN with IUGR, and (3) investigate the relationship between IUGR and severity of preeclampsia.Study Design:A case–control study was performed. Cases were patients identified with preeclampsia. Controls were patients presenting for delivery at term (⩾37 weeks). IUGR prevalence by case–control status, or severity of disease was evaluated using Pearson χ2 tests. Multivariable logistic regression was used to control for confounders.Result:In all, 430 cases and 568 controls were studied. Preeclamptic women have a 2.7 (CI (1.94 to 3.86)) and 4.3 (CI (2.58 to 7.17)) times increased odds of having a fetus with IUGR at <10 and <5% compared with controls in adjusted analyses. There was a significant interaction between CHTN and IUGR. Therefore, in women without CHTN, women with PEC had increased odds of IUGR, whereas in women with CHTN, there was no difference in odds of IUGR in women with or without preeclampsia. Within the cases, severe preeclampsia was associated with IUGR<10% (AOR=1.82 (1.11 to 2.97)) but not IUGR<5% (AOR=1.6 (0.85 to 2.86)).Conclusion:Preeclampsia is independently associated with the development of IUGR. As suggested earlier, women with CHTN do not have the highest prevalence of IUGR, suggesting disparate pathways by which IUGR develops in women with superimposed preeclampsia compared with preeclampsia alone.

miR-210 Inhibits Trophoblast Invasion and Is a Serum Biomarker for Preeclampsia

Preeclampsia is characterized by hypertension and proteinuria in pregnant women. Its exact cause is unknown. Preeclampsia increases the risk of maternal and fetal morbidity and mortality. Although delivery, often premature, is the only known cure, early targeted interventions may improve maternal and fetal outcomes. Successful intervention requires a better understanding of the molecular etiology of preeclampsia and the development of accurate methods to predict women at risk. To this end, we tested the role of miR-210, a miRNA up-regulated in preeclamptic placentas, in first-trimester extravillous trophoblasts. miR-210 overexpression reduced trophoblast invasion, a process necessary for uteroplacental perfusion, in an extracellular signal-regulated kinase/mitogen-activated protein kinase-dependent manner. Conversely, miR-210 inhibition promoted invasion. Furthermore, given that the placenta secretes miRNAs into the maternal circulation, we tested if serum expression of miR-210 was associated with the disease. We measured miR-210 expression in two clinical studies: a case-control study and a prospective cohort study. Serum miR-210 expression was significantly associated with a diagnosis of preeclampsia (P = 0.007, area under the receiver operator curves = 0.81) and was predictive of the disease, even months before clinical diagnosis (P < 0.0001, area under the receiver operator curve = 0.89). Hence, we conclude that aberrant expression of miR-210 may contribute to trophoblast function and that miR-210 is a novel predictive serum biomarker for preeclampsia that can help in identifying at-risk women for monitoring and treatment.

Preventing cervical ripening: the primary mechanism by which progestational agents prevent preterm birth?

OBJECTIVE Recent clinical trials suggest that progestational agents may prevent preterm birth, specifically in women with short cervices. These studies sought to assess novel pathways by which progestational agents (PAs) may modify signal transduction pathways that are involved in cervical ripening. STUDY DESIGN A microarray analysis was performed on pregnant mouse cervix that was exposed to a MPA. Appropriate microarray and cluster analyses were performed. Target genes of interest were investigated in both PA- and inflammation-exposed cervices by quantitative polymerase chain reaction and immunohistochemistry. RESULTS Microarray analysis identified both the previously recognized and novel pathways that are involved in cervical ripening. PAs differentially regulate expression of claudin-2, hyaluronan synthase 2, and lipocalin 2. Claudin expression is significantly decreased by inflammation, which is prevented by PAs. CONCLUSION PAs significantly modulate gene expression in the cervix in the presence and absence of inflammation. The regulation of these pathways, specifically claudin proteins, may be a critical mechanism by which PAs prevent preterm birth, especially in women with premature cervical shortening.