Gardnerella vaginalis alters cervicovaginal epithelial cell function through microbe-specific immune responses.

BACKGROUND: The cervicovaginal (CV) microbiome is highly associated with vaginal health and disease in both pregnant and nonpregnant individuals. An overabundance of Gardnerella vaginalis (G. vaginalis) in the CV space is commonly associated with adverse reproductive outcomes including bacterial vaginosis (BV), sexually transmitted diseases, and preterm birth, while the presence of Lactobacillus spp. is often associated with reproductive health. While host-microbial interactions are hypothesized to contribute to CV health and disease, the mechanisms by which these interactions regulate CV epithelial function remain largely unknown. RESULTS: Using an in vitro co-culture model, we assessed the effects of Lactobacillus crispatus (L. crispatus) and G. vaginalis on the CV epithelial barrier, the immune mediators that could be contributing to decreased barrier integrity and the immune signaling pathways regulating the immune response. G. vaginalis, but not L. crispatus, significantly increased epithelial cell death and decreased epithelial barrier integrity in an epithelial cell-specific manner. A G. vaginalis-mediated epithelial immune response including NF-κB activation and proinflammatory cytokine release was initiated partially through TLR2-dependent signaling pathways. Additionally, investigation of the cytokine immune profile in human CV fluid showed distinctive clustering of cytokines by Gardnerella spp. abundance and birth outcome. CONCLUSIONS: The results of this study show microbe-specific effects on CV epithelial function. Altered epithelial barrier function through cell death and immune-mediated mechanisms by G. vaginalis, but not L. crispatus, indicates that host epithelial cells respond to bacteria-associated signals, resulting in altered epithelial function and ultimately CV disease. Additionally, distinct immune signatures associated with Gardnerella spp. or birth outcome provide further evidence that host-microbial interactions may contribute significantly to the biological mechanisms regulating reproductive outcomes. Video Abstract.

Intrauterine Inflammation Leads to Select Sex- and Age-Specific Behavior and Molecular Differences in Mice.

Sex-specific differences in behavior have been observed in anxiety and learning in children exposed to prenatal inflammation; however, whether these behaviors manifest differently by age is unknown. This study assesses possible behavioral changes due to in utero inflammation as a function of age in neonatal, juvenile, and adult animals and presents potential molecular targets for observed differences. CD-1 timed pregnant dams were injected in utero with lipopolysaccharide (LPS, 50 µg/animal) or saline at embryonic day 15. No differences in stress responses were measured by neonatal ultrasonic vocalizations between LPS- and saline-exposed groups of either sex. By contrast, prenatal inflammation caused a male-specific increase in anxiety in mature but not juvenile animals. Juvenile LPS-exposed females had decreased movement in open field testing that was not present in adult animals. We additionally observed improved memory retrieval after in utero LPS in the juvenile animals of both sexes, which in males may be related to a perseverative phenotype. However, there was an impairment of long-term memory in only adult LPS-exposed females. Finally, gene expression analyses revealed that LPS induced sex-specific changes in genes involved in hippocampal neurogenesis. In conclusion, intrauterine inflammation has age- and sex-specific effects on anxiety and learning that may correlate to sex-specific disruption of gene expression associated with neurogenesis in the hippocampus.

IFNγ-Producing γ/δ T Cells Accumulate in the Fetal Brain Following Intrauterine Inflammation.

Intrauterine inflammation impacts prenatal neurodevelopment and is linked to adverse neurobehavioral outcomes ranging from cerebral palsy to autism spectrum disorder. However, the mechanism by which a prenatal exposure to intrauterine inflammation contributes to life-long neurobehavioral consequences is unknown. To address this gap in knowledge, this study investigates how inflammation transverses across multiple anatomic compartments from the maternal reproductive tract to the fetal brain and what specific cell types in the fetal brain may cause long-term neuronal injury. Utilizing a well-established mouse model, we found that mid-gestation intrauterine inflammation resulted in a lasting neutrophil influx to the decidua in the absence of maternal systemic inflammation. Fetal immunologic changes were observed at 72-hours post-intrauterine inflammation, including elevated neutrophils and macrophages in the fetal liver, and increased granulocytes and activated microglia in the fetal brain. Through unbiased clustering, a population of Gr-1+ γ/δ T cells was identified as the earliest immune cell shift in the fetal brain of fetuses exposed to intrauterine inflammation and determined to be producing high levels of IFNγ when compared to γ/δ T cells in other compartments. In a case-control study of term infants, IFNγ was found to be elevated in the cord blood of term infants exposed to intrauterine inflammation compared to those without this exposure. Collectively, these data identify a novel cellular immune mechanism for fetal brain injury in the setting of intrauterine inflammation.

The tracking of lipopolysaccharide through the feto-maternal compartment and the involvement of maternal TLR4 in inflammation-induced fetal brain injury.

PROBLEM: Exposure to intrauterine inflammation (IUI) has been shown to induce fetal brain injury and increase the risk of acquiring a neurobehavioral disorder. The trafficking of the inflammatory mediator, lipopolysaccharide (LPS), in the pregnant female reproductive tract in the setting of IUI and the precise mechanisms by which inflammation induces fetal brain injury are not fully understood. METHOD OF STUDY: FITC-labeled LPS was utilized to induce IUI on E15, tissues were collected, and fluorescence was visualized via the Spectrum IVIS. Embryo transfer was utilized to create divergent maternal and fetal genotypes. Wild-type (WT) embryos were transferred into TLR4-/- pseudopregnant dams (TLR4-/-mat /WTfet ). On E15, TLR4-/-mat /WTfet dams or their WT controls (WTmat /WTfet ) received an intrauterine injection of LPS or phosphate-buffered saline (PBS). Endotoxin and IL-6 levels were assessed in amniotic fluid, and cytokine expression was measured via QPCR. RESULTS: Lipopolysaccharide trafficked to the uterus, fetal membranes, placenta, and the fetus and was undetectable in other tissues. Endotoxin was present in the amniotic fluid of all animals exposed to LPS. However, the immune response was blunted in TLR4-/-mat /WTfet compared with WT controls. CONCLUSION: Intrauterine administered LPS is capable of accessing the entire feto-placental unit with or without a functional maternal TLR4. Thus, bacteria or bacterial byproducts in the uterus may negatively impact fetal development regardless of the maternal genotype or endotoxin response. Despite the blunted immune response in the TLR4-deficient dams, an inflammatory response is still ignited in the amniotic cavity and may negatively impact the fetus.

HIF-1α Stabilization Increases miR-210 Eliciting First Trimester Extravillous Trophoblast Mitochondrial Dysfunction.

Preeclampsia is associated with first trimester placental dysfunction. miR-210, a small non-coding RNA, is increased in the preeclamptic placenta. The effects of elevated miR-210 on placental function remain unclear. The objectives of this study were to identify targets of miR-210 in first trimester primary extravillous trophoblasts (EVTs) and to investigate functional pathways altered by elevated placental miR-210 during early pregnancy. EVTs isolated from first trimester placentas were exposed to cobalt chloride (CoCl2), a HIF-1α stabilizer and hypoxia mimetic, and miR-210 expression by qPCR, HIF1α protein levels by western blot and cell invasion were assessed. A custom TruSeq RNA array, including all known/predicted miR-210 targets, was run using miR-210 and miR-negative control transfected EVTs. Mitochondrial function was assessed by high resolution respirometry in transfected EVTs. EVTs exposed to CoCl2 showed a dose and time-dependent increase in miR-210 and HIF1α and reductions in cell invasion. The TruSeq array identified 49 altered genes in miR-210 transfected EVTs with 27 genes repressed and 22 enhanced. Three of the top six significantly repressed genes, NDUFA4, SDHD, and ISCU, are associated with mitochondrial function. miR-210 transfected EVTs had decreased maximal, complex II and complex I+II mitochondrial respiration. This study suggests that miR-210 alters first trimester trophoblast function. miR-210 overexpression alters EVT mitochondrial function in early pregnancy. Mitochondrial dysfunction may lead to increased reactive oxygen species, trophoblast cell damage and likely contributes to the pathogenesis of preeclampsia.

Cervicovaginal microbiota and local immune response modulate the risk of spontaneous preterm delivery.

Failure to predict and understand the causes of preterm birth, the leading cause of neonatal morbidity and mortality, have limited effective interventions and therapeutics. From a cohort of 2000 pregnant women, we performed a nested case control study on 107 well-phenotyped cases of spontaneous preterm birth (sPTB) and 432 women delivering at term. Using innovative Bayesian modeling of cervicovaginal microbiota, seven bacterial taxa were significantly associated with increased risk of sPTB, with a stronger effect in African American women. However, higher vaginal levels of ß-defensin-2 lowered the risk of sPTB associated with cervicovaginal microbiota in an ethnicity-dependent manner. Surprisingly, even in Lactobacillus spp. dominated cervicovaginal microbiota, low ß-defensin-2 was associated with increased risk of sPTB. These findings hold promise for diagnostics to accurately identify women at risk for sPTB early in pregnancy. Therapeutic strategies could include immune modulators and microbiome-based therapeutics to reduce this significant health burden.

The Absence of TLR4 Prevents Fetal Brain Injury in the Setting of Intrauterine Inflammation.

BACKGROUND: Exposure to intrauterine inflammation during pregnancy is linked to brain injury and neurobehavioral disorders in affected children. Innate immunity, specifically Toll-like receptor (TLR) signaling pathways are present throughout the reproductive tract as well as in the placenta, fetal membranes, and fetus. The TLR pathways are mechanistically involved in host responses to foreign pathogens and may lead to brain injury associated with prenatal inflammation. OBJECTIVE: We aimed to determine whether the activation of the TLR4 signaling pathway, in the mother and fetus, is critical to fetal brain injury in the setting of intrauterine inflammation. METHODS: A mini-laparotomy was performed on time pregnant C57B6 mice and 2 knockout mouse strains lacking the function of the Tlr4 and Myd88 genes on embryonic day 15. Intrauterine injections of Escherichia coli lipopolysaccharide or saline were administered as described previously. Dams were killed 6 hours postsurgery, and placental, amniotic fluid, and fetal brain tissue were collected. To assess brain injury, quantitative polymerase chain reaction (qPCR) analysis was performed on multiple components of the NOTCH signaling pathway, including Hes genes. Interleukin (IL) IL6, IL1ß, and CCL5 expression was assessed using qPCR and enzyme-linked immunosorbent assay. RESULTS: Using an established mouse model of intrauterine inflammation, we demonstrate that the abrogation of TLR4 signaling eliminates the cytokine response in mother and fetus and prevents brain injury associated with increased expression of transcriptional effectors of the NOTCH signaling pathway, Hes1 and Hes5. CONCLUSIONS: These data show that the activation of the TLR4 signaling pathway is necessary for the development of fetal brain injury in response to intrauterine inflammation.

Common Cervicovaginal Microbial Supernatants Alter Cervical Epithelial Function: Mechanisms by Which Lactobacillus crispatus Contributes to Cervical Health.

Cervicovaginal (CV) microbiota is associated with vaginal health and disease in non-pregnant women. Recent studies in pregnant women suggest that specific CV microbes are associated with preterm birth (PTB). While the associations between CV microbiota and adverse outcomes have been demonstrated, the mechanisms regulating the associations remain unclear. As the CV space contains an epithelial barrier, we postulate that CV microbiota can alter the epithelial barrier function. We investigated the biological, molecular, and epigenetic effects of Lactobacillus crispatus, Lactobacillus iners, and Gardnerella vaginalis on the cervical epithelial barrier function and determined whether L. crispatus mitigates the effects of lipopolysaccharide (LPS) and G. vaginalis on the cervical epithelial barrier as a possible mechanism by which CV microbiota mitigates disease risk. Ectocervical and endocervical cells treated with L. crispatus, L. iners, and G. vaginalis bacteria-free supernatants alone or combined were used to measure cell permeability, adherens junction proteins, inflammatory mediators, and miRNAs. Ectocervical and endocervical permeability increased after L. iners and G. vaginalis exposure. Soluble epithelial cadherin increased after exposure to L. iners but not G. vaginalis or L. crispatus. A Luminex cytokine/chemokine panel revealed increased proinflammatory mediators in all three bacteria-free supernatants with L. iners and G. vaginalis having more diverse inflammatory effects. L. iners and G. vaginalis altered the expression of cervical-, microbial-, and inflammatory-associated miRNAs. L. crispatus mitigated the LPS or G. vaginalis-induced disruption of the cervical epithelial barrier and reversed the G. vaginalis-mediated increase in miRNA expression. G. vaginalis colonization of the CV space of a pregnant C57/B6 mouse resulted in 100% PTB. These findings demonstrate that L. iners and G. vaginalis alter the cervical epithelial barrier by regulating adherens junction proteins, cervical immune responses, and miRNA expressions. These results provide evidence that L. crispatus confers protection to the cervical epithelial barrier by mitigating LPS- or G. vaginalis-induced miRNAs associated with cervical remodeling, inflammation, and PTB. This study provides further evidence that the CV microbiota plays a role in cervical function by altering the cervical epithelial barrier and initiating PTB. Thus, targeting the CV microbiota and/or its effects on the cervical epithelium may be a potential therapeutic strategy to prevent PTB.

Placental immune state shifts with gestational age.

PROBLEM: Placental immunologic functions are implicated in both the maintenance of a healthy pregnancy and the pathogenesis of obstetric complications. Immune populations at the maternal-fetal interface are hypothesized to support fetomaternal tolerance, defend the fetus from infection, and contribute to labor initiation. Despite the many potential roles of placental immune cells in normal and abnormal pregnancy, little is known about placental immune population dynamics over gestation, particularly near parturition. METHOD OF STUDY: A daily placental immune cell census was established in a murine model by flow cytometry from mid to late gestation and compared to the maternal systemic immune census. Shifts in the placental immune state were further characterized through cytokine ELISAs. RESULTS: The placental immune census is distinct from the maternal systemic immune census, although the cells are primarily maternal in origin. Near term parturition, the placenta contains fewer CD11c-positive myeloid cells and regulatory T cells, and there is a concurrent decrease in placental IL-9 and IL-35. CONCLUSION: The immune profile of the placenta demonstrates a decrease in both regulatory immune cell types and cytokines late in gestation. Establishing the placental immune population dynamics over a healthy pregnancy will allow future investigation of placental immune cells during abnormal pregnancy.

Colonization of the cervicovaginal space with Gardnerella vaginalis leads to local inflammation and cervical remodeling in pregnant mice.

The role of the cervicovaginal (CV) microbiome in regulating cervical function during pregnancy is poorly understood. Gardnerella vaginalis (G. vaginalis) is the most common bacteria associated with the diagnosis of bacterial vaginosis (BV). While BV has been associated with preterm birth (PTB), clinical trials targeting BV do not decrease PTB rates. It remains unknown if G. vaginalis is capable of triggering molecular, biomechanical and cellular events that could lead to PTB. The objective of this study was to determine if cervicovaginal colonization with G. vaginalis, in pregnant mice, induced cervical remodeling and modified cervical function. CD-1 timed-pregnant mice received a 5X108 CFU/mL intravaginal inoculation of G. vaginalis or control on embryonic day 12 (E12) and E13. On E15, the mice were sacrificed and cervicovaginal fluid (CVF), amniotic fluid (AF), cervix, uterus, placentas and fetal membranes (FM) were collected. Genomic DNA was isolated from the CVF, placenta, uterus and FM and QPCR was performed to confirm colonization. IL-6 was measured in the CVF and AF and soluble e-cadherin (seCAD) was assessed in the CVF by ELISA. RNA was extracted from the cervices to evaluate IL-10, IL-8, IL-1ß, TNF-α, Tff-1, SPINK-5, HAS-1 and LOX expression via QPCR. Mucicarmine and trichrome staining was used to assess cervical mucin and collagen. Biomechanical properties of the cervix were studied using quasi-static tensile load-to-failure biomechanical tests. G. vaginalis successfully colonized the CV space. This colonization induced immune responses (increased IL-6 levels in CVF and AF, increased mRNA expression of cervical cytokines), altered the epithelial barrier (increased seCAD in the CVF), induced cervical remodeling (increased mucin production, altered collagen) and altered cervical biomechanical properties (a decrease in biomechanical modulus and an increase in maximum strain). The ability of G. vaginalis to induce these molecular, immune, cellular and biomechanical changes suggests that this bacterium may play a pathogenic role in premature cervical remodeling leading to PTB.

Exposure to intrauterine inflammation alters metabolomic profiles in the amniotic fluid, fetal and neonatal brain in the mouse.

INTRODUCTION: Exposure to prenatal inflammation is associated with diverse adverse neurobehavioral outcomes in exposed offspring. The mechanism by which inflammation negatively impacts the developing brain is poorly understood. Metabolomic profiling provides an opportunity to identify specific metabolites, and novel pathways, which may reveal mechanisms by which exposure to intrauterine inflammation promotes fetal and neonatal brain injury. Therefore, we investigated whether exposure to intrauterine inflammation altered the metabolome of the amniotic fluid, fetal and neonatal brain. Additionally, we explored whether changes in the metabolomic profile from exposure to prenatal inflammation occurs in a sex-specific manner in the neonatal brain. METHODS: CD-1, timed pregnant mice received an intrauterine injection of lipopolysaccharide (50 µg/dam) or saline on embryonic day 15. Six and 48 hours later mice were sacrificed and amniotic fluid, and fetal brains were collected (n = 8/group). Postnatal brains were collected on day of life 1 (n = 6/group/sex). Global biochemical profiles were determined using ultra performance liquid chromatography/tandem mass spectrometry (Metabolon Inc.). Statistical analyses were performed by comparing samples from lipopolysaccharide and saline treated animals at each time point. For the P1 brains, analyses were stratified by sex. RESULTS/CONCLUSIONS: Exposure to intrauterine inflammation induced unique, temporally regulated changes in the metabolic profiles of amniotic fluid, fetal brain and postnatal brain. Six hours after exposure to intrauterine inflammation, the amniotic fluid and the fetal brain metabolomes were dramatically altered with significant enhancements of amino acid and purine metabolites. The amniotic fluid had enhanced levels of several members of the (hypo) xanthine pathway and this compound was validated as a potential biomarker. By 48 hours, the number of altered biochemicals in both the fetal brain and the amniotic fluid had declined, yet unique profiles existed. Neonatal pups exposed to intrauterine inflammation have significant alterations in their lipid metabolites, in particular, fatty acids. These sex-specific metabolic changes within the newborn brain offer an explanation regarding the sexual dimorphism of certain psychiatric and neurobehavioral disorders associated with exposure to prenatal inflammation.

miR-143 and miR-145 disrupt the cervical epithelial barrier through dysregulation of cell adhesion, apoptosis and proliferation.

Molecular mechanisms regulating preterm birth (PTB)-associated cervical remodeling remain unclear. Prior work demonstrated an altered miRNA profile, with significant increases in miR-143 and miR-145, in cervical cells of women destined to have a PTB. The study objective was to determine the effect of miR-143 and miR-145 on the cervical epithelial barrier and to elucidate the mechanisms by which these miRNAs modify cervical epithelial cell function. Ectocervical and endocervical cells transfected with miR-negative control, miR-143 or miR-145 were used in cell permeability and flow cytometry assays for apoptosis and proliferation. miR-143 and miR-145 target genes associated with cell adhesion, apoptosis and proliferation were measured. Epithelial cell permeability was increased in miR-143 and miR-145 transfected cervical epithelial cells. Cell adhesion genes, JAM-A and FSCN1, were downregulated with overexpression of miR-143 and miR-145. miR-143 and miR-145 transfection decreased cervical cell number by increasing apoptosis and decreasing cell proliferation through initiation of cell cycle arrest. Apoptosis genes, BCL2 and BIRC5, and proliferation genes, CDK1 and CCND2, were repressed by miR-143 and miR-145. These findings suggest that miR-143 and miR-145 play a significant role in cervical epithelial barrier breakdown through diverse mechanisms and could contribute to premature cervical remodeling associated with PTB.

Tensile Mechanical Properties and Dynamic Collagen Fiber Re-Alignment of the Murine Cervix are Dramatically Altered Throughout Pregnancy.

The cervix is a unique organ able to dramatically change its shape and function by serving as a physical barrier for the growing fetus and then undergoing dramatic dilation allowing for delivery of a term infant. As a result, the cervix endures changing mechanical forces from the growing fetus. There is an emerging concept that the cervix may change or remodel "early" in many cases of spontaneous preterm birth (sPTB). However, the mechanical role of the cervix in both normal and preterm birth remains unclear. Therefore, the primary objective of this study was to determine the mechanical and structural responses of murine cervical tissue throughout a normal gestational time course. In this study, both tissue structural and material properties were determined via a quasi-static tensile load-to-failure test, while simultaneously obtaining dynamic collagen fiber re-alignment via cross-polarization imaging. This study demonstrated that the majority of the mechanical properties evaluated decreased at midgestation and not just at term, while collagen fiber re-alignment occurred earlier in the loading curve for cervices at term. This suggests that although structural changes in the cervix occur throughout gestation, the differences in material properties function in combination with collagen fiber re-alignment as mechanical precursors to regulate term gestation. This work lays a foundation for investigating cervical biomechanics and the role of the cervix in preterm birth.

Women with preterm birth have a distinct cervicovaginal metabolome.

OBJECTIVE: Metabolomics has the potential to reveal novel pathways involved in the pathogenesis of preterm birth (PTB). The objective of this study was to investigate whether the cervicovaginal (CV) metabolome was different in asymptomatic women destined to have a PTB compared with term birth. STUDY DESIGN: A nested case-control study was performed using CV fluid collected from a larger prospective cohort. The CV fluid was collected between 20-24 weeks (V1) and 24-28 weeks (V2). The metabolome was compared between women with a spontaneous PTB (n = 10) to women who delivered at term (n = 10). Samples were extracted and prepared for analysis using a standard extraction solvent method. Global biochemical profiles were determined using gas chromatography/mass spectrometry and ultra-performance liquid chromatography/tandem mass spectrometry. An ANOVA was used to detect differences in biochemical compounds between the groups. A false discovery rate was estimated to account for multiple comparisons. RESULTS: A total of 313 biochemicals were identified in CV fluid. Eighty-two biochemicals were different in the CV fluid at V1 in those destined to have a PTB compared with term birth, whereas 48 were different at V2. Amino acid, carbohydrate, and peptide metabolites were distinct between women with and without PTB. CONCLUSION: These data suggest that the CV space is metabolically active during pregnancy. Changes in the CV metabolome may be observed weeks, if not months, prior to any clinical symptoms. Understanding the CV metabolome may hold promise for unraveling the pathogenesis of PTB and may provide novel biomarkers to identify women most at risk.

Can microRNA profiling in maternal blood identify women at risk for preterm birth?

OBJECTIVE: MicroRNAs (miRNAs), which are highly conserved single-stranded noncoding RNAs that play a crucial role in gene regulation, have now been identified as important players in many diseases states. MiRNAs have also been demonstrated to be reliable and useful biomarkers to identify those women who are at risk for specific adverse outcomes. The objective of this study was to determine whether miRNA profiles in maternal blood are different in women who are destined to have a preterm, compared with a term, birth. STUDY DESIGN: A nested case-control study was performed with maternal serum that was collected as part of a larger prospective cohort. MiRNA expression profiles in maternal serum were compared between women who ultimately had a preterm birth (n = 40) compared with term birth (n = 40). MiRNA expression profiles were created with the use of the Affymetrix GeneChip miRNA Array. The data were analyzed with the significance of analysis of microarrays and principle components analyses. A false discovery rate of 20% was used to determine the most differentially expressed miRNAs. RESULTS: Of the 5640 miRNAs that were analyzed on the array, 4 miRNAs were significantly different between cases and control subjects. Two of the 4 miRNAs were mature miRNAs. The fold difference in expression was <2 for all 4 miRNAs. CONCLUSION: MiRNA profiles in maternal blood were not significantly different in women who were destined to have a preterm, compared with a term, birth. MiRNAs in maternal blood are unlikely to become clinically useful biomarkers for the prediction of preterm birth.

Differential methylation of genes associated with cell adhesion in preeclamptic placentas.

Preeclampsia (PE), a hypertensive disorder of pregnancy, is hypothesized to be associated with, if not mechanistically related to abnormal placental function. However, the exact mechanisms regulating the pathogenesis of PE remain unclear. While many studies have investigated changes in gene expression in the PE placenta, the role of epigenetics in PE associated placental dysfunction remains unclear. Using the genome-wide Illumina Infinium Methylation 450 BeadChip array, we analyzed gene-specific alterations in DNA methylation in placental biopsies collected from normal pregnant women delivering at term (n = 14), with term PE (≥37 weeks; n = 19) or with preterm PE (<37 weeks, n = 12). Of the 485,582 gene loci on the array, compared to controls, 229 loci were differentially methylated in PE placentas and 3411 loci were differentially methylated in preterm PE (step up p-value <0.05 and >5% methylation difference). Functional annotation of the differentially methylated genes in preterm PE placentas revealed a 32 gene cluster in the cadherin and cell adhesion functional groups (Benjamini p<0.00001). Hypermethylation of CDH11 (p = 0.0143), COL5A1 (p = 0.0127) and TNF (p = 0.0098) and hypomethylation of NCAM1 (p = 0.0158) was associated with altered mRNA expression in preterm PE placentas. Demethylation of first trimester extravillous trophoblast cells resulted in altered CDH11 (p = 0.0087), COL5A1 (p = 0.0043), NCAM1 (p = 0.0260) and TNF (p = 0.0022) mRNA expression. These studies demonstrate aberrant methylation, correlating with disease severity, in PE placentas. Furthermore, we provide evidence that disruption of gene-specific methylation in preterm PE placentas and first trimester trophoblasts is significantly associated with altered gene expression demonstrating that epigenetic modifications early in pregnancy can have effects on trophoblast function contributing to PE.

Distinct cervical microRNA profiles are present in women destined to have a preterm birth.

OBJECTIVE: Although premature cervical remodeling is involved in preterm birth (PTB), the molecular pathways that are involved have not been elucidated fully. MicroRNAs (miRNAs) that are highly conserved single-stranded noncoding RNAs that play a crucial role in gene regulation have now been identified as important players in disease states. The objective of this study was to determine whether miRNA profiles in cervical cells are different in women who are destined to have a PTB compared with a term birth. STUDY DESIGN: A nested case-control study was performed. With the use of a noninvasive method, cervical cells were obtained at 2 time points in pregnancy. The cervical cell miRNA expression profiles were compared between women who ultimately had a PTB (n = 10) compared with a term birth (n = 10). MiRNA expression profiles were created with the Affymetrix GeneChip miRNA Array. The data were analyzed with the Significance of Analysis of Microarrays and Principle Components Analyses. A false-discovery rate of 20% was used to determine the most differentially expressed miRNAs. Validation was performed with quantitative polymerase chain reaction. In vitro studies were performed to confirm expression and regulation of select miRNAs. RESULTS: With a false-discovery rate of 20% of the 5640 miRNAs that were analyzed on the array, 99 miRNAs differed between those with a PTB vs a term birth. Qualitative polymerase chain reaction validated the array findings. In vitro studies confirmed expression of select miRNAs in cervical cells. CONCLUSION: MiRNA profiles in cervical cells may distinguish women who are at risk for PTB months before the outcome. With the large downstream effects of miRNAs on gene expression, these studies provide a new understanding of the processes that are involved in premature cervical remodeling and allow for the discovery of new therapeutic targets.

The soluble receptor for advanced glycation end products can prospectively identify patients at greatest risk for preterm birth.

OBJECTIVE: Our primary objective was to determine whether there was an association between levels of antenatal maternal serum soluble RAGE (sRAGE), drawn at the time of presentation with preterm labor (PTL), and subsequent preterm birth (PTB). Secondary objectives were to determine whether levels of sRAGE - analyzed from both antenatal maternal serum (MS) and postpartum umbilical cord serum (CS) - were associated with neonatal sepsis. METHODS: Nested case-control analyses were performed within a prospective cohort of patients at risk for PTB. MS was obtained at enrollment and CS at delivery. The sRAGE levels were analyzed. Non-parametric calculations and receiver-operator analyses were performed. RESULTS: Overall, 39.8% of patients delivered < 37 weeks (n=498) and 15% had neonatal sepsis (n=193). In comparing cases and controls, sRAGE was significantly lower in those with than those without an adverse event (PTB: median MS-sRAGE 771.79 versus 948.485 pg/mL, p=0.004; neonatal sepsis: 25-centile CS-sRAGE 1220.49 versus 2244.41 pg/mL, p=0.0013). Adding MS-sRAGE to models of clinical variables significantly enhanced the ability of the model to predict both PTB (area under the curve [AUC] 0.71 versus 0.79, p=0.004) and neonatal sepsis (AUC 0.65 versus 0.75, p=0.04). The negative predictive value of CS-sRAGE for neonatal sepsis was very strong (NPV=0.91). CONCLUSIONS: The sRAGE can be used to help predict adverse perinatal outcomes. Patients with higher levels of sRAGE - who therefore may have an enhanced capability to regulate their immune response - appear less likely to experience PTB and neonatal sepsis.

Lipopolysaccharide induces cytokine production and decreases extravillous trophoblast invasion through a mitogen-activated protein kinase-mediated pathway: possible mechanisms of first trimester placental dysfunction.

BACKGROUND: Defects in extravillous trophoblast (EVT) function could contribute to placental dysfunction resulting in adverse obstetrical outcomes. Adverse obstetrical outcomes have been highly correlated with intrauterine infection; however, the mechanisms linking infection to placental dysfunction remain unclear. We investigated the effects of inflammation on EVT cytokine production and invasion early in pregnancy and determined the cell signaling pathways mediating this response. METHODS AND RESULTS: In our model of inflammation, EVT cells, isolated following first trimester pregnancy terminations (n= 6) were stimulated with lipopolysaccharide (LPS). LPS induced a dose-dependent increase in interleukin (IL)-8 and IL-6 protein production (P < 0.01) and decreased EVT invasion (P = 0.01) versus control. The LPS-mediated changes in cytokine production (P < 0.001) and invasion (P < 0.001) were reversed by dexamethasone (DEX). Exposure to LPS resulted in an increase in mitogen-activated protein kinase (MAPK) signaling pathway phosphorylation, including p44/42 MAPK (P < 0.01), p38 MAPK (P < 0.05), MAPK extracellular signal-regulated kinase 1/2 (MEK1/2) (P< 0.01) and stress-activated protein kinase/c-Jun N-terminal kinase (JNK; P < 0.001), which was reversed by DEX (P < 0.05) for all MAPKs except p38. MAPK-specific inhibitors to MEK1/2 (U0126), p38 MAPK (SB 202190) and JNK (SP 600125) significantly reversed the LPS-mediated increase in IL-6 (P < 0.001) and IL-8 (P < 0.001) production. While U0126 reversed the LPS-induced decrease in EVT invasion (P < 0.001), SB 202190 (P < 0.001) and SP 600125 (P< 0.001) decreased EVT invasion, further indicating that MEK1/2 phosphorylation may be inflammation dependent while p38 MAPK and JNK phosphorylation occurs independently of an inflammatory stimulus. CONCLUSIONS: LPS increased IL-8 and IL-6 and decreased EVT invasion through activation of MAPK signaling. MEK1/2 activation may contribute to placental dysfunction, in the setting of inflammation-associated adverse obstetrical outcomes.

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. CD-1 timed pregnant mice on embryonic day 15 (E15) and E18.5 were utilized for this study. Dams were randomized to receive intrauterine infusion of lipopolysaccharide (LPS, 50 µg/dam) or normal saline. Different experimental groups were used to assess both acute and long-term outcomes. For each gestational age and each treatment group, fetal brains, amniotic fluid, maternal serum and placentas were collected 6h after intrauterine infusion. Rates of preterm birth, maternal morbidity and litter size were assessed. IL6 levels were assayed in maternal serum and amniotic fluid. An immune response was determined in the fetal brains and placentas by QPCR. Cortical cultures were performed to assess for fetal neuronal injury. Gene expression changes in postnatal day 7 brains from exposed and unexposed pups were determined. In the preterm period, low dose LPS resulted in a 30% preterm birth rate. Litter sizes were not different between the groups at either gestational age. IL6 levels were not significantly increased in maternal serum at either gestational time period. Low dose LPS increased IL6 levels in the amniotic fluid from exposed dams in the term but not preterm period. Regardless of gestational age of exposure, low dose intrauterine LPS activated an immune response in the placenta and fetal brain. Exposure to intrauterine LPS significantly decreased dendritic counts in cortical cultures from both the preterm and term period. Exposure to intrauterine inflammation altered gene expression patterns in the postnatal brain; this effect was dependent on gestational age of exposure. In conclusion, intrauterine inflammation, even in the absence of preterm parturition, can evoke fetal brain injury as evidence by alterations in cytokine expression and neuronal injury. Despite an absent or limited maternal immune response in low dose intrauterine inflammation, the immune system in the placenta is activated which is likely sufficient to induce a fetal immune response and subsequent brain injury. Changes in the fetal brain lead to changes in gene expression patterns into the neonatal period. Subclinical intrauterine inflammation can lead to fetal brain injury and is likely to be mechanistically associated with long term adverse outcomes for exposed offspring.