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.