Monochorionic/diamniotic twin gestation appears to be a risk factor for CHDs. Conditions that lead to abnormal placentation may also contribute to abnormal heart development, especially in MC/DA twin pregnancies complicated with TTTS. Fetal echocardiography may be considered for all MC/DA twin gestations because ventricular septal defects and pulmonary stenosis are the most common defects.

BackgroundIntra-amniotic infection and/or inflammation (IAI) are important causes of preterm birth and early-onset neonatal sepsis (EONS). A prompt and accurate diagnosis of EONS is critical for improved neonatal outcomes. We sought to explore the cord blood proteome and identify biomarkers and functional protein networks characterizing EONS in preterm newborns.Methodology/Principal FindingsWe studied a prospective cohort of 180 premature newborns delivered May 2004-September 2009. A proteomics discovery phase employing two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry identified 19 differentially-expressed proteins in cord blood of newborns with culture-confirmed EONS (n = 3) versus GA-matched controls (n = 3). Ontological classifications of the proteins included transfer/carrier, immunity/defense, protease/extracellular matrix. The 1st-level external validation conducted in the remaining 174 samples confirmed elevated haptoglobin and haptoglobin-related protein immunoreactivity (Hp&HpRP) in newborns with EONS (presumed and culture-confirmed) independent of GA at birth and birthweight (P<0.001). Western blot concurred in determining that EONS babies had conspicuous Hp&HpRP bands in cord blood (“switch-on pattern”) as opposed to non-EONS newborns who had near-absent “switch-off pattern” (P<0.001). Fetal Hp phenotype independently impacted Hp&HpRP. A Bayesian latent-class analysis (LCA) was further used for unbiased classification of all 180 cases based on probability of “antenatal IAI exposure” as latent variable. This was then subjected to 2nd-level validation against indicators of adverse short-term neonatal outcome. The optimal LCA algorithm combined Hp&HpRP switch pattern (most input), interleukin-6 and neonatal hematological indices yielding two non-overlapping newborn clusters with low (≤20%) versus high (≥70%) probability of IAI exposure. This approach reclassified ∼30% of clinical EONS diagnoses lowering the number needed to harm and increasing the odds ratios for several adverse outcomes including intra-ventricular hemorrhage.Conclusions/SignificanceAntenatal exposure to IAI results in precocious switch-on of Hp&HpRP expression. As EONS biomarker, cord blood Hp&HpRP has potential to improve the selection of newborns for prompt and targeted treatment at birth.

TLRs are pattern recognition transmembrane receptors that play key roles in innate immunity. A recently discovered soluble truncated form of TLR2 (sTLR2) acts as a decoy receptor, down-regulating the host inflammatory response to bacteria. To identify the presence and functional role of sTLR2 in modulating the intraamniotic inflammatory response to infection, we studied 109 amniotic fluid samples of women with normal pregnancy outcomes (n ‫؍‬ 28) and women with (n ‫؍‬ 39) and without (n ‫؍‬ 42) intraamniotic infection. We sought to demonstrate a functional role of the amniotic fluid sTLR2 in modulating the TLR2 inflammatory signaling in vitro by using a villous explant system. Two sTLR2 forms were identified, and specificity was confirmed with neutralizing peptides. We showed that sTLR2 is present constitutively in amniotic fluid, its levels are gestational age dependent, and we determined that the sTLR2 quantity and functional engagement modulates the intensity of the intraamniotic inflammation elicited by Gram-positive bacteria. In vitro, we demonstrated that challenging placental villous explants with a specific TLR2 agonist (Pam3Cys) induced a significant cytokine response. Notably, preincubation of the preterm, but not near-term, amniotic fluid with Pam3Cys significantly inhibited the ability of this TLR2 agonist to elicit a cytokine reaction. Moreover, depletion of sTLR2 from preterm amniotic fluid removed its neutralizing property. Monensin significantly diminished sTLR2 immunoreactivity, indicating that sTLR2 is the result of intracellular posttranslational processing of TLR2. We conclude that sTLR2 is part of the amniotic fluid innate immune system and participates in regulating the inflammatory response to microbial pathogens. The Journal of Immunology, 2009, 182: 7244 -7253.T he innate immune system is an archaic defense mechanism, phylogenetically preserved to be at the forefront of resistance to microbial infections (1). The human TLRs are essential for triggering an inflammatory innate immune response (2). To date, 13 mammalian TLRs have been identified and 10 of these are present in humans (3). At the maternal-fetal interface, TLRs are expressed not only in immune cells, but also in the trophoblast and decidual cells (4, 5). Moreover, their expression pattern varies according to the stage of pregnancy (5). Such findings provide evidence that during pregnancy, placental TLRs may play a key role in modulating the inflammatory response triggered by infection.TLRs are transmembrane receptors that mediate host defense through the engagement of pathogen-associated molecular patterns (PAMPs) 3 , which are ubiquitous constituents of the bacterial wall (6). TLR2 was the first of 10 human TLRs, proven to be precisely involved in recognition of PAMPs (lipoproteins, peptidoglycan, glycolipids, nucleic acids), representing broad groups of microbial species such as Gram-positive bacteria, Mycobacteria, spirochetes, and Mycoplasmataceae (7,8,9). Traditionally, it has been thought that the extracellular recepto...

Immune activation represents an adaptive reaction triggered by both noxious exogenous (microbes) and endogenous [high mobility group box-1 protein (HMGB1), S100 calcium binding proteins] inducers of inflammation. Cell stress or necrosis lead the release of HMGB1 and S100 proteins in the extracellular compartment where they act as damage-associated molecular pattern molecules (or alarmins) by engaging the receptor for advanced glycation end-products (RAGE). Although the biology of RAGE is dictated by the accumulation of damage-associated molecular pattern molecules at sites of tissue injury, the role of RAGE in mediating antenatal fetal injury remains unknown. First, we studied the relationships at birth between the intensity of human fetal inflammation and sRAGE (an endogenous RAGE antagonist), HMGB1, and S100␤ protein. We found significantly lower sRAGE in human fetuses that mounted robust inflammatory responses. HMGB1 levels correlated significantly with levels of interleukin-6 and S100␤ in fetal circulation. We then evaluated the levels and areas of tissue expression of RAGE, HMGB1, and S100␤ in specific organs of mouse fetuses on E16. Using an animal model of endotoxin-induced fetal damage and preterm birth, we determined that inflammation induces a significant change in expression of RAGE and HMGB1, but not S100␤, at sites of tissue damage. Our findings indicate that RAGE and Conventional wisdom holds that the primary causes of the high neonatal morbidity and mortality attendant preterm birth are complications of immature organ systems.1-4 However, a growing body of investigation suggests that the poor outcome observed in many preterm children is not entirely dependent on their gestational age at birth. 2,5,6 After correcting for gestational age, several risk factors remain significantly associated with an increased risk of cerebral palsy, such as intra-amniotic infection, histological chorioamnionitis, prolonged rupture of the membranes, and hypoxemic fetal growth re-

Objective To determine the relationship between presence of amniotic fluid (AF) biomarkers characteristic of inflammation (defensins 2 and 1, calgranulins C and A) and fetal inflammatory status at birth. Design Prospective observational cohort. Setting Tertiary referral University hospital Population 132 consecutive mothers (gestational age, median [interquartile range]: 29.6 [24.1-33.6] weeks), who had a clinically indicated amniocentesis to rule-out infection and their newborns. Methods Intra-amniotic inflammation was diagnosed by mass spectrometry SELDI-TOF. The AF proteomic fingerprint [Mass Restricted (MR) score] ranges from 0-4 (none to all biomarkers present). The intensity of intra-amniotic inflammation was graded based on the number of proteomic biomarkers: MR score 0: “no” inflammation; MR score 1-2: “minimal” inflammation; MR score 3-4: “severe” inflammation. At birth, cord blood was obtained for all cases. Severity of histological chorioamnionitis (HCA) and early onset neonatal sepsis (EONS) was based on established histological and hematological criteria. Interleukin-6 (IL-6) levels were measured by sensitive immunoassays. The cord blood-to-AF IL-6 ratio was used as an indicator of the differential inflammatory response in the fetal versus the AF compartment. Main Outcome Measures to relate proteomic biomarkers of intra-amniotic infection to cord blood IL-6 and to use the latter as the primary marker of fetal inflammatory response. Results Women with intra-amniotic inflammation delivered at an earlier gestational age (ANOVA, P<0.001) and had higher AF IL-6 levels (P<0.001). At birth, neonates of women with “severe” intra-amniotic inflammation had higher cord blood IL-6 levels (P=0.002) and a higher frequency of EONS (P=0.002). EONS was characterized by significantly elevated cord blood IL-6 levels (P<0.001). Out of the 39 neonates delivered by mothers with “minimal” intra-amniotic inflammation, 15 (39%) had umbilical cord blood IL-6 levels above the mean for the group, and 2 neonates had confirmed sepsis. The severity of the neutrophilic infiltrate in the chorionic plate (P<0.001), choriodecidua (P=0.002), umbilical cord (P<0.001), but not amnion (P>0.05) was an independent predictor of the cord blood-to-AF IL-6 ratio. Relationships were maintained following correction for gestational age, birthweight, amniocentesis-to delivery interval, cesarean delivery, status of the membranes, race, MR score, antibiotics and steroid exposure. Conclusion We provide evidence that presence in the AF of proteomic biomarkers characteristic of inflammation is associated with an increased inflammatory status of the fetus at birth. Neonates mount an increased inflammatory status and have positive blood cultures even in the context of “minimal” intra-amniotic inflammation.

Background The arsenal of maternal and amniotic fluid (AF) immune response to local or systemic infection includes among others the acute-phase reactants IL-6, C-reactive protein (CRP) and procalcitonin (PCT). If these molecules can be used as non-invasive biomarkers of intra-amniotic infection (IAI) in the subclinical phase of the disease remains incompletely known. Methods We used time-matched maternal serum, urine and AF from 100 pregnant women who had an amniocentesis to rule out IAI in the setting of preterm labor, PPROM or systemic inflammatory response (SIR: pyelonephritis, appendicitis, pneumonia) to infection. Cord blood was analyzed in a subgroup of cases. We used sensitive immunoassays to quantify the levels of inflammatory markers in the maternal blood, urine and AF compartment. Microbiological testing and placental pathology was used to establish infection and histological chorioamnionits. Results PCT was not a useful biomarker of IAI in any of the studied compartments. Maternal blood IL-6 and CRP levels were elevated in women with subclinical IAI. Compared to clinically manifest chorioamnionitis group, women with SIR have higher maternal blood IL-6 levels rendering some marginal diagnostic benefit for this condition. Urine was not a useful biological sample for assessment of IAI using either of these three inflammatory biomarkers. Conclusions In women with subclincal IAI, the large overlapping confidence intervals and different cut-offs for the maternal blood levels of IL-6, CRP and PCT likely make interpretation of their absolute values difficult for clinical decision-making.

PURPOSE OF THE REVIEW: Particularities of the fetal immune response to infection cause a heightened inflammatory state that acts synergistically with microbial insult to induce damage. Proteomics offers the opportunity for detecting fetuses at risk of sepsis and neurological injury. RECENT FINDINGS: Molecular tools (16S-rRNA) demonstrate the diversity of microbial agents of intra-amniotic infection exceeds what is suspected clinically or is documented by cultures. The resulting inflammatory process has the potential to damage the fetus in utero. Stepwise algorithms [mass restricted (MR) score] have been developed to extract proteomic profiles characteristic of amniotic fluid (AF) inflammation. The MR score includes 4 proteomic biomarkers: defensin-2, defensin-1, S100A12 and S100A8 proteins. Other AF biomarkers relevant for preterm birth are S100A9 and insulin-like-growth-factor-binding protein 1 (IGFBP-1). S100A12, ligand for the receptor of advanced glycation end-products (RAGE), has the strongest association with histological chorioamnionitis and funisitis. Presence of S100A12 and S100A8 in AF is predictive of early-onset neonatal sepsis and poor neuro-developmental outcome. SUMMARY: Presence of AF proteomic biomarkers of inflammation is associated with increased inflammatory status of the fetus at birth. Future challenges are finding biomarkers that provide insight into molecular mechanisms of chronic fetal and neonatal cellular damage and identify candidates for early neuro-protection strategies.

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 (PϽ0.05), 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 heparin's protective effects, if any, cannot be explained by promotion of angiogenesis. (Circulation. 2011;124:2543-2553.)Key Words: anticoagulation Ⅲ angiogenesis Ⅲ sFlt-1 Ⅲ heparin Ⅲ pregnancy A successful pregnancy outcome requires the proper functioning of the molecular mechanisms responsible for regulating vascular endothelial homeostasis at both the systemic and local decidual/placental levels. 1 Derangements of these mechanisms can lead to major pregnancy-related complications. Recent studies have focused on how alterations in placental trophoblast production of angiogenic proteins may help to explain the pathogenesis of preeclampsia, 2,3 placental abruption, 4 intrauterine growth restriction, and stillbirth. 5 A unifying theory is that excessive release of antiangiogenic factors, such as soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) by hypoxic trophoblasts, antagonize proangiogenic factors such as vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). 3 This phenomenon causes aberrant placental angiogenesis and vasculogenesis. 6 The end result is an increasingly dysfunctional placenta with local and systemic vascular endothelial damage, activation of inflammatory pathways, and enhanced platelet turnover, which result in the...