Geeta Bhat

Dysregulated biomarkers induce distinct pathways in preterm birth

Please cite this paper as: Brou L, Almli L, Pearce B, Bhat G, Drobek C, Fortunato S, Menon R. Dysregulated biomarkers induce distinct pathways in preterm birth. BJOG 2012;119:458–473.

Bacterial modulation of human fetal membrane Toll-like receptor expression.

Preterm premature rupture of fetal membranes (pPROM) occurs in 30–40% of spontaneous preterm births (PTB) and is associated with intra‐amniotic infection and inflammation. The membranes may sense and respond to microbes via Toll‐like receptors (TLRs); however, little is known about their expression and regulation in this tissue. The objective of this study was to evaluate the expression of TLRs 1–10 in fetal membranes after exposure to pathogens associated with intra‐amniotic infection and PTB.

Amniotic fluid and maternal race influence responsiveness of fetal membranes to bacteria

Spontaneous preterm birth (PTB) and preterm prelabor rupture of membranes (pPROM) occur more frequently in African-American women than in other racial groups. This may be due to an enhanced inflammatory response to pathogens associated with the condition. It is also possible that amniotic fluid (AF) has different immunomodulatory properties in African-American women that increase their risk of PTB and pPROM. To test this, we cultured fetal membranes from European-American and African-American women with sterile medium (control), Escherichia coli, Gardnerella vaginalis, Group B streptococci (GBS), Polyporphorans gingivalis, Mycoplasma hominis, Ureaplasma urealyticum or Ureaplasma parvum in the presence and absence of 50% autologous AF. Cytokine concentrations were quantified in the conditioned medium. All bacterial species increased IL-8 production. IL-1β and TNF-α production were stimulated by LPS, E. coli, and G. vaginalis compared with control, but responses to Group B streptococci and P. gingivalis were limited to IL-1β and TNF-α respectively. Genital mycoplasmas stimulated TNF-α and IL-10 but had no effect on IL-1β production. African-Americans had twice the IL-1β response to E. coli as European-Americans (P=0.031). Conversely, European-Americans produced more IL-8 in response to LPS than African-Americans (P=0.026). AF had both pro- and anti-inflammatory properties that varied between races and pathogens. These results suggest that the host response to fetal membrane infections is complex and not generalizable. Interventions to prevent PTB and pPROM may need to be customized based on a patients race, type of bacterial infection and factors in her AF.

Long-term alterations in maternal plasma proteome after sFlt1-induced preeclampsia in mice.

OBJECTIVE Preeclampsia is associated with long-term adverse maternal health, such as cardiovascular and metabolic diseases. The objective of this study was to determine whether preeclampsia in a well-characterized animal model that was induced by overexpression of soluble fms-like tyrosine kinase-1 (sFlt1) results in alterations in the maternal circulating proteome that persist long after delivery. STUDY DESIGN CD-1 mice at day 8 of gestation were injected with adenovirus that carried sFlt1 or the murine immunoglobulin G2α Fc fragment as control. Depleted maternal plasma was analyzed 6 months after delivery by label-free liquid chromatography-mass spectrometry assay. The tandem mass spectrometry data were searched against a mouse database, and the resultant intensity data were used to compare abundance of proteins across disease/control plasma pool. Results were analyzed with ingenuity pathways analysis. Right-tailed Fisher exact test was used to calculate a probability value. RESULTS Of 150 proteins that are common for both groups, ingenuity pathways analysis determined 105 proteins that were ready for analysis. Diseases and disorders analysis showed significant enrichment of proteins that are associated with cardiovascular disease. Within this cluster, the most abundant proteins were associated with vascular disease, atherosclerosis, and atherosclerotic lesions. Other top disease clusters were inflammatory response, organismal injury and abnormalities, and hematologic and metabolic disease. CONCLUSION Exposure to sFlt1-induced preeclampsia alters multiple biologic functions in mothers that persist later in life. Our results suggest that some of the long-term adverse outcomes that are associated with preeclampsia actually may be a consequence rather than a mere unmasking of an underlying predisposition. If similar results are found in humans, the development of preventive strategies for preeclampsia should also improve long-term maternal health.

Multivariate adaptive regression splines analysis to predict biomarkers of spontaneous preterm birth

To develop classification models of demographic/clinical factors and biomarker data from spontaneous preterm birth in African Americans and Caucasians.

Proteomics and bioinformatics analysis reveal underlying pathways of infection associated histologic chorioamnionitis in pPROM.

INTRODUCTION The presence of microbial invasion of the amniotic cavity (MIAC) and histological chorioamnionitis (HCA) is associated with adverse neonatal outcomes in pregnancies complicated by preterm prelabor rupture of membranes (pPROM). Therefore, there is an urgent need to identify new biomarkers revealing these conditions. The objective of this study is to identify possible biomarkers and their underlying biofunctions in pPROM pregnancies with and without MIAC and HCA. METHODS A total of 72 women with pPROM were recruited. Only women with both MIAC and HCA (n = 19) and all women without these complications (n = 19) having the same range of gestational ages at sampling were included in the study. Samples of amniotic fluid were obtained by transabdominal amniocentesis, processed and analyzed using quantitative shotgun proteomics. Ingenuity pathway analysis was used to identify molecular networks that involve altered proteins. RESULTS Network interaction identified by ingenuity pathway analysis revealed immunological disease and the inflammatory response as the top functions and disease associated with pPROM in the presence of MIAC and HCA. The proteins involved in these pathways were significantly altered between the groups with and without the presence of both MIAC and HCA. Proteins involved included histones H3, H4, H2B, cathelicidin antimicrobial peptide, myeloperoxidase, neutrophil gelatinase-associated lipocalin, matrix metalloproteinase-9, peptidoglycan recognition protein-1 and neutrophil defensin 1, all of which were found to be up-regulated in the presence of MIAC and HCA. CONCLUSION Bioinformatic analysis of proteomics data allowed us to project likely biomolecular pathology resulting in pPROM complicated by MIAC and HCA. As inflammation is not a homogeneous phenomenon, we provide evidence for oxidative-stress-associated DNA damage and biomarkers of reactive oxygen species generation as factors associated with inflammation and proteolysis.

Biomarker Interactions Are Better Predictors of Spontaneous Preterm Birth

Objective: The objective of this study was to assess the role of biomarker interactions as predictors of spontaneous preterm birth (PTB) using multifactor dimensionality reduction (MDR) analysis. With MDR, a nonparametric, unsupervised, model-free approach, we tested for biomarker interactions within maternal–fetal compartments in 2 racial groups: African Americans (AA) and Caucasians (C). Study Design: A total of 36 biomarkers from maternal plasma (MP), cord plasma (CP), and amniotic fluid (AF) were analyzed from 191 patients. The MDR combined attribute selection, construction, and classification to detect biomarker interactions that were assessed for generality and significance using 10× cross-validation and permutation testing. Selected significant interactive models were replicated with additional samples. Results: The interactive model containing interleukin (IL)-2, angiopoietin 2 (ANGPT-2), and IL-6 receptor was significant in AA MP. In AA CP, the IL-8 and tumor necrosis factor (TNF) receptor 1 model was significant. In AA AF, the ANGPT-2 and macrophage inflammatory protein 1 alpha model was significant. Replication of the AA MP model using 54 additional AA MP samples confirmed predictability of these biomarkers. In C AF, interaction was observed between ANGPT-2, monocyte chemotactic protein 3, and TNF-α, but no other interactions were significant in C. Conclusions: Using MDR, we identified biomarker interactions that are predictors of PTB even in the absence of a main effect with a single biomarker.

Fetal membrane biomarker network diversity and disease functions induced by intra-amniotic pathogens.

Intra‐amniotic pathogens and by‐products activate innate immune responses encompassing multitudes of signaling molecules and pathways that can result in spontaneous preterm birth (PTB). This study investigates fetal membrane response to bacterial stimulation using a bioinformatics approach.

Group B Streptococcus colonization and higher maternal IL-1β concentrations are associated with early term births.

Association between maternal Group B Streptococcus (GBS) colonization diagnosed between 35 and 37 weeks of gestation and early term birth (between 37 and 39 weeks) and maternal-fetal inflammatory response associated with this condition were tested. In this cohort study of women delivering at term at Centennial Women’s Hospital in Nashville, TN, GBS status and other clinical and demographic data were obtained from medical records. Exposed women were those testing positive for GBS (GBS positive [n = 490]) and the unexposed tested negative for GBS (GBS negative [n = 1,127]). To determine the inflammatory response associated with GBS, a cross sectional study, maternal and fetal plasma biomarkers (IL-1β, IL-2, IL-6, IL-8, and TNF-α) were measured in the same cohort. T-tests and logistic regression determined association between GBS status, biomarker concentrations and early term birth. Gestational age was reduced to 271.1 (95% CI 270.4, 271.1) for cases compared to 274.7 (95% CI 274.4, 275.1) days for controls (p < 0.0001). The odds of early term birth was increased by threefold in cases (OR 3.28; 95% CI 2.60–4.15; p < 0.0001). The mean birth weight in cases (3285.3 g) (95% CI 3242.6, 3327.9) was lower than the controls, 3373.8 g (95% CI 3348.9, 3398.7) (p = 0.0004). Maternal IL-1β was greater in cases (22.8 ng/ml; range 5.2–157.7 ng/ml) compared to controls (5.7; range 2.4–69.5 ng/ml; p < 0.0001). IL-1β was higher in fetal plasma in cases vs. controls (20.33 vs. 8.18 ng/ml; p = 0.01). A 10 ng/ml increase in maternal IL-1β was associated with increased risk for GBS infection (OR: 1.628, CI: 1.163–2.278; p = 0.0045). GBS colonization shortened gestational age at term and IL-1β concentration in maternal plasma is an indicator of GBS status.