Victor A. Rosenberg

Proteomic Biomarkers of Intra-amniotic Inflammation: Relationship with Funisitis and Early-onset Sepsis in the Premature Neonate

Our goal was to determine the relationship between 4 amniotic fluid (AF) proteomic biomarkers (human neutrophil defensins 2 and 1, calgranulins C and A) characteristic of intra-amniotic inflammation, and funisitis and early-onset sepsis in premature neonates. The mass restricted (MR) score was generated from AF obtained from women in preterm labor (n = 123). The MR score ranged from 0–4 (none to all biomarkers present). Funisitis was graded histologically and interpreted in relation to the MR scores. Neonates (n = 97) were evaluated for early-onset sepsis. There was significant correlation between the severity of AF inflammation and the presence (53/123) and grades of funisitis (p < 0.001). Funisitis occurred independently of the amniocentesis-to-delivery interval or status of the membranes and was best predicted by an MR score 3–4 and an earlier gestational age (GA) at delivery. Neonates born to women with an MR score 3–4 had an increased incidence of suspected/confirmed sepsis, even after adjusting for GA at birth. Calgranulin C had the highest association with clinically significant funisitis, while calgranulin A had the strongest association with early-onset sepsis. To conclude, AF proteomic analysis shows that women with MR scores 3–4 are more likely to have histologic funisitis, and deliver neonates with early-onset sepsis.

Multidimensional Proteomics Analysis of Amniotic Fluid to Provide Insight into the Mechanisms of Idiopathic Preterm Birth

Background Though recent advancement in proteomics has provided a novel perspective on several distinct pathogenetic mechanisms leading to preterm birth (inflammation, bleeding), the etiology of most preterm births still remains elusive. We conducted a multidimensional proteomic analysis of the amniotic fluid to identify pathways related to preterm birth in the absence of inflammation or bleeding. Methodology/Principal Findings A proteomic fingerprint was generated from fresh amniotic fluid using surface-enhanced laser desorbtion ionization time of flight (SELDI-TOF) mass spectrometry in a total of 286 consecutive samples retrieved from women who presented with signs or symptoms of preterm labor or preterm premature rupture of the membranes. Inflammation and/or bleeding proteomic patterns were detected in 32% (92/286) of the SELDI tracings. In the remaining tracings, a hierarchical algorithm was applied based on descriptors quantifying similarity/dissimilarity among proteomic fingerprints. This allowed identification of a novel profile (Q-profile) based on the presence of 5 SELDI peaks in the 10–12.5 kDa mass area. Women displaying the Q-profile (mean±SD, gestational age: 25±4 weeks, n = 40) were more likely to deliver preterm despite expectant management in the context of intact membranes and normal amniotic fluid clinical results. Utilizing identification-centered proteomics techniques (fluorescence two-dimensional differential gel electrophoresis, robotic tryptic digestion and mass spectrometry) coupled with Protein ANalysis THrough Evolutionary Relationships (PANTHER) ontological classifications, we determined that in amniotic fluids with Q-profile the differentially expressed proteins are primarily involved in non-inflammatory biological processes such as protein metabolism, signal transduction and transport. Conclusion/Significance Proteomic profiling of amniotic fluid coupled with non-hierarchical bioinformatics algorithms identified a subgroup of patients at risk for preterm birth in the absence of intra-amniotic inflammation or bleeding, suggesting a novel pathogenetic pathway leading to preterm birth. The altered proteins may offer opportunities for therapeutical intervention and future drug development to prevent prematurity.

Using proteomic analysis of the human amniotic fluid to identify histologic chorioamnionitis.

OBJECTVE: To estimate the relationship between histologic chorioamnionitis and four amniotic fluid proteomic biomarkers characteristic of inflammation (defensins 2 and 1, calgranulins C and A). METHODS: One hundred fifty-eight women with singleton pregnancies had a clinically indicated amniocentesis to rule out inflammation and infection in the context of preterm labor or preterm premature rupture of membranes. A proteomic fingerprint (Mass Restricted score) was generated from amniotic fluid using surface-enhanced laser desorption ionization time-of-flight mass spectrometry. The Mass Restricted score ranges from 0 to 4 (none to all four biomarkers present) in direct relationship with severity of intra-amniotic inflammation. Presence or absence of biomarkers was analyzed in relationship to placental pathology. Criteria for severity of histologic chorioamnionitis were 3 stages and 4 grades of inflammation of the amnion, choriodecidua and chorionic plate. RESULTS: The prevalence of histologic chorioamnionitis was 64% (stage I 12%, stage II 16%, and stage III 37%). The Mass Restricted score significantly correlated with stages of histologic chorioamnionitis (r=0.539, P<.001), grades of choriodeciduitis (r=0.465, P<.001), and amnionitis (r=0.536, P<.001). African-American women were overrepresented in the group with severe inflammation (Mass Restricted score 3–4, P=.022). Of the four biomarkers of the Mass Restricted score, calgranulin C had the strongest relationship with presence of stage III chorioamnionitis, independent of race, amniocentesis-to-delivery interval, and gestational age. CONCLUSION: Proteomic analysis of amniotic fluid provides an opportunity for early recognition of histologic chorioamnionitis. This methodology may in the future identify candidates for antenatal therapeutic interventions. LEVEL OF EVIDENCE: II

Ultrasound evaluation of the uterine scar after cesarean delivery: a randomized controlled trial of one- and two-layer closure.

OBJECTIVE: To survey the uterine scar thickness by ultrasonography in women randomly assigned to one- or two-layer hysterotomy closure after primary cesarean delivery. METHODS: This was a randomized, blinded trial of uterine scar closure with ultrasonographic follow-up. Thirty consecutive patients undergoing primary cesarean delivery were enrolled and randomly assigned to one- or two-layer closure of the hysterotomy. Ultrasound surveillance of the uterine scar thickness was performed at baseline (before surgery) and 48 hours, 2 weeks, and 6 weeks post partum. RESULTS: Patient compliance with the postpartum surveillance protocol was 90%, and the uterine scar was visualized in 99% of attempted ultrasonographic examinations. There were no differences between groups at baseline or at any of the follow-up evaluations. An initial 5- to 6-fold increase in uterine scar thickness was observed, followed by a gradual decrease with the 6-week measurements still thicker than baseline. Repeated measures analysis of variance showed significant variation across time points starting either at baseline (P<.001) or at 48 hour postoperatively (P<.001), but this variation did not depend on closure type (P=.79 for all visits and P=.81 beginning with 48-hour postoperative time point). CONCLUSION: The process of uterine scar remodeling can be successfully monitored by ultrasonography. Uterine scar thickness diminishes progressively after both one- or two-layer closure but does not vary with mode of hysterotomy closure. The uterine scar thickness remains increased even at 6 weeks post partum, suggesting that the process of uterine scar remodeling extends beyond the traditional postpartum period. CLINCAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT00224250 LEVEL OF EVIDENCE: I

Heparin Elevates Circulating Soluble fms-Like Tyrosine Kinase-1 Immunoreactivity in Pregnant Women Receiving Anticoagulation Therapy

Background— Alterations in circulating levels of pro- and antiangiogenic factors have been associated with adverse pregnancy outcomes. Heparin is routinely administered to pregnant women, but without clear knowledge of its impact on these factors. Methods and Results— We conducted a longitudinal study of 42 pregnant women. Twenty-one women received prophylactic heparin anticoagulation, and 21 healthy pregnant women served as controls. Compared with gestational age-matched controls, heparin treatment was associated with increased circulating levels of soluble fms-like tyrosine kinase-1 (sFlt-1) in the third trimester (P<0.05), in the absence of preeclampsia, placental abruption, or fetal growth restriction. Heparin had no effect on circulating levels of vascular endothelial growth factor, placenta growth factor, or soluble endoglin as assessed by ELISA. In vitro, low-molecular weight and unfractionated heparins stimulated sFlt-1 release from placental villous explants, in a dose- and time-dependent manner. This effect was not due to placental apoptosis, necrosis, alteration in protein secretion, or increased transcription. Western blot analysis demonstrated that heparin induced shedding of the N-terminus of Flt-1 both in vivo and in vitro as indicated by a predominant band of 100–112 kDa. By using an in vitro angiogenesis assay, we demonstrated that serum of heparin-treated cases inhibited both basal and vascular endothelial growth factor-induced capillary-like tube formation. Conclusions— Heparin likely increases the maternal sFlt-1 through shedding of the extracellular domain of Flt-1 receptor. Our results imply that upregulation of circulating sFlt-1 immunoreactivity in pregnancy is not always associated with adverse outcomes, and that heparins protective effects, if any, cannot be explained by promotion of angiogenesis.

Nucleated red blood cells are a direct response to mediators of inflammation in newborns with early-onset neonatal sepsis.

OBJECTIVE The objective of the study was to test the hypothesis that inflammation modulates fetal erythroblastosis and/or the release of nucleated red blood cells (NRBCs) independent of hypoxia or fetal stress. We sought to determine whether fetal inflammation is associated with an elevation in neonatal NRBC count in the setting of inflammation-associated preterm birth. STUDY DESIGN The relationships between peripheral NRBC count, histological chorioamnionitis, umbilical cord interleukin (IL)-6, erythropoietin (EPO), cortisol, and acid-base status were analyzed in 68 preterm singletons, born to mothers who had an amniocentesis to rule out infection. Proteomic profiling of amniotic fluid identified presence of intraamniotic inflammation according to established parameters. NRBC counts were assessed within 1 hour of birth. Early-onset neonatal sepsis (EONS) was established based on hematological and microbiological indices. IL-6, EPO, and cortisol levels were measured by immunoassays. Fetal acid-base status was determined within 10 minutes of delivery. Parametric or nonparametric statistics were used. RESULTS Fetuses with EONS (n = 19) were delivered at earlier gestational ages (mean +/- SD: 27.1 +/- 2.8 weeks, P = .001) and more often by mothers with intraamniotic inflammation (P = .022) and histological chorioamnionitis (P < .001). Neonates with EONS had higher absolute NRBC counts (P = .011). NRBC counts were directly correlated with cord blood IL-6 levels (P < .001) but not with EPO, cortisol or parameters of acid-base status levels regardless of EONS status. These relationships remained following correction for gestational age, diabetes, intrauterine growth restriction, and steroid exposure. CONCLUSION In the setting of inflammation-associated preterm birth and in the absence of hypoxia, elevations in NRBCs in the early neonatal period may be a direct response of exposure to inflammatory mediators in utero.

Multidimensional system biology: genetic markers and proteomic biomarkers of adverse pregnancy outcome in preterm birth.

Premature birth before 37 weeks of gestation is a significant public health problem. Each year, 4.5 million premature infants are born worldwide. Despite extensive research and a variety of interventions, the rate of preterm birth has steadily increased over the past 20 years and reached a high of 12.8% in 2006. The etiology of most preterm births remains elusive and is likely multifactorial, with many pathophysiological pathways involved, such as excessive stretching, oxidative stress, decidual hemorrhage, and infection. Genomics and proteomics have emerged to provide a better comprehension of the pathophysiological conditions leading to preterm birth, thereby providing a perspective for improving neonatal outcome.

Stillbirth at Term in Women of Advanced Maternal Age in the United States: When Could the Antenatal Testing Be Initiated?

We sought to determine if advanced maternal age (AMA) is a risk factor for intrauterine fetal demise (IUFD). We used a U.S. Centers for Disease Control and Prevention database and analyzed outcomes in women 15 to 44 years of age with term singleton gestations. Cox proportional hazards models and Cochran-Mantel-Haenszel tests were used. Results were controlled for maternal race and smoking. After excluding congenital anomalies and medical complications, 6,239,399 singleton term deliveries were identified. When compared with women 25 to 29 years of age, the risk of IUFD increased with advancing age: 30 to 34 years, odds ratio [OR] = 1.24 (95% confidence interval [CI], 1.13 to 1.36); 35 to 39 years, OR = 1.45 (95% CI, 1.21 to 1.74), and 40 to 44 years, OR = 3.04 (95% CI, 1.58 to 5.86). The risk of IUFD for women 40 to 44 years of age at 39 weeks is comparable with that of 42 weeks in those 25 to 29 years of age. We concluded that AMA is an independent predictor of IUFD, and a strategy of antenatal testing in those > or = 40 years of age beginning at 38 weeks may be considered.

Amniotic Fluid Angiopoietin-1, Angiopoietin-2, and Soluble Receptor Tunica Interna Endothelial Cell Kinase-2 Levels and Regulation in Normal Pregnancy and Intraamniotic Inflammation-Induced Preterm Birth

BACKGROUND Angiopoietin-1 (Ang-1) and Ang-2 act selectively on endothelial cells by engaging the Tunica interna endothelial cell kinase-2 (Tie2) receptor. A soluble form of Tie2 (sTie2) blocks angiopoietin bioactivity. OBJECTIVE The aim of the study was to characterize changes and expression patterns of Ang-1, Ang-2, and sTie2 in amniotic fluid (AF) and placenta during human pregnancy and intraamniotic inflammation (IAI)-induced preterm birth. DESIGN AND SETTING We conducted a cross-sectional study at a tertiary university hospital. PATIENTS AF levels of Ang-1, Ang-2, and sTie2 were evaluated in 176 women during second trimester (n = 40), third trimester (n = 37), and preterm labor (positive IAI, n = 50; negative IAI, n = 49). Placenta and cord blood of select women were analyzed. MAIN OUTCOME MEASURES Ang-1, Ang-2, sTie2, and IL-6 were evaluated by ELISA. Real-time PCR measured Ang-1, Ang-2, and Tie2 placental mRNA levels. Placenta was immunostained for Ang-1 and Ang-2. Placental explant cultures were stimulated with lipopolysaccharide, Pam3Cys, and modulators of protein synthesis/secretion (cycloheximide, monensin, and brefeldin A). RESULTS In normal pregnancy, the levels and ratios of AF Ang-1, Ang-2, and sTie2 varied with gestational age (GA) (P < 0.001). PCR revealed corresponding changes in placental Ang-1 and Ang-2, but not Tie2, mRNA. IAI raised AF Ang-1, Ang-2, and sTie2 above the expected level for GA without affecting their placental mRNA. Ang-2 immunoreactivity appeared enhanced in areas of villous edema. AF Ang-2/Ang-1 ratio was an important determinant of cord blood IL-6 (P < 0.001). Ex-vivo, sTie2 release was increased by Golgi disrupting but not bacterial mimic agents. CONCLUSIONS Ang-1, Ang-2, and sTie2 are physiological constituents of AF that are GA and IAI regulated. Ang-2/Ang-1 ratio may play a role in modulating the fetal inflammatory response to IAI. Placental sTie2 shedding likely involves a Golgi-mediated mechanism.

MODULATION OF AMNIOTIC FLUID ACTIVIN-A AND INHIBIN-A IN WOMEN WITH PRETERM PREMATURE RUPTURE OF THE MEMBRANES AND INFECTION-INDUCED PRETERM BIRTH

Citation Rosenberg VA, Buhimschi IA, Dulay AT, Abdel‐Razeq SS, Oliver EA, Duzyj CM, Lipkind H, Pettker CM, Buhimschi CS. Modulation of amniotic fluid activin‐A and inhibin‐A in women with preterm premature rupture of the membranes and infection‐induced preterm birth. Am J Reprod Immunol 2012; 67: 122–131