Modulation of amniotic fluid activin-a and inhibin-a in women with preterm premature rupture of the membranes and infection-induced preterm birth.

PROBLEM: Activins and inhibins are important modulators of inflammatory processes. We explored activation of amniotic fluid (AF) activin-A and inhibin-A system in women with intra-amniotic infection and preterm premature rupture of the membranes (PPROM). METHOD OF STUDY: We analyzed 78 AF samples: '2nd trimester-control' (n=12), '3rd trimester-control' (n=14), preterm labor with intact membranes [positive-AF-cultures (n=13), negative-AF-cultures (n=13)], and PPROM [positive-AF-cultures (n=13), negative-AF-cultures (n=13)]. Activin-A levels were evaluated ex-vivo following incubation of amniochorion and placental villous explants with Gram-negative lipopolysaccharide (LPS) or Gram-positive (Pam3Cys) bacterial mimics. Ability of recombinant activin-A and inhibin-A to modulate inflammatory reactions in fetal membranes was explored through explants' IL-8 release. RESULTS: Activin-A and inhibin-A were present in human AF and were gestational age-regulated. Activin-A was significantly upregulated by infection. Lower inhibin-A levels were seen in PPROM. LPS elicited release of activin-A from amniochorion, but not from villous explants. Recombinant activin-A stimulated IL-8 release from amniochorion, an effect that was not reversed by inhibin-A. CONCLUSION: Human AF activin-A and inhibin-A are involved in biological processes linked to intra-amniotic infection/inflammation-induced preterm birth.

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 heparin's protective effects, if any, cannot be explained by promotion of angiogenesis.

Interpretation of amniotic fluid white blood cell count in "bloody tap" amniocenteses in women with symptoms of preterm labor.

OBJECTIVE: To estimate whether blood-contaminated amniotic fluid affects the performance of white blood cell (WBC) count in diagnosing intraamniotic inflammation and infection. METHODS: Three hundred fifty-seven consecutive women pregnant with singletons undergoing amniocentesis to rule out infection were enrolled prospectively. A "bloody tap" was defined as a red blood cell (RBC) count of 1,000 cells/mm or more. Proteomics analysis of amniotic fluid was used in this study as the standard for diagnosing inflammation. Infection was confirmed by positive amniotic fluid culture. An amniotic fluid WBC count correction formula was computed using maternal WBC count, hematocrit, and mean corpuscular volume. RESULTS: The prevalence of a bloody tap amniocentesis was 22% (77 of 357). In the absence of inflammation, the amniotic fluid WBC count was significantly higher in bloody tap (median [interquartile range] 18 [9-58] cells/mm) compared with non-bloody tap specimens (4 [1-10] cells/mm; P<.001). The correction formula reversed this difference to a nonsignificant level (bloody tap 0 [0-17] compared with non-bloody tap 3 [1-10] cells/mm; P=.273). In the setting of inflammation, the observed WBC count of bloody tap samples (778 [197-2,062 cells/mm]) was significantly elevated compared with that of the non-bloody tap specimens (616 [105-1,730] cells/mm; P=.023). Correction of the WBC count in bloody tap amniocenteses improved the test accuracy and positive likelihood ratios for inflammation and infection. A correction algorithm was not useful in amniotic fluid specimens with less than 1,000/RBCs/mm or WBC counts more than 1,100 cells/mm. Given the nonlinear relationship between amniotic fluid WBC and RBC, for a rapid correction of WBC count, the number of neutrophils that need to be subtracted from the observed WBC count is variable. CONCLUSION: In the setting of an amniotic fluid sample contaminated with 1,000 RBCs/mm or more, WBC count is a less accurate indicator of inflammation and infection. In such samples, correction of WBC count enhances diagnostic performance for inflammation and infection. LEVEL OF EVIDENCE: II.

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.

Prenatal diagnosis of ectopic intrathoracic kidney in a fetus with a left diaphragmatic hernia.

Intrathoracic renal ectopia as a result of a congenital diaphragmatic hernia (CDH) is a rare congenital anomaly. We present a case in which the prenatal diagnosis of an ectopic intrathoracic kidney was made on routine anatomical survey at 28 weeks' gestation. Color doppler sonography imaging revealed the renal artery coursing into the infant's thorax and was consistent with CDH, but fetal MRI suggested an intact diaphragm. However, neonatal evaluation confirmed the diagnosis of intrathoracic kidney with posterior CDH, which was repaired without complication. In contrast to diaphragmatic hernia with liver or bowel herniation, infants with intrathoracic ectopic kidneys generally do well.

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.

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.

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.

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.

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

OBJECTIVE: 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

Thromboembolism in pregnancy.

Venous thromboembolism in pregnancy is a clinical emergency that has been associated with significant risk for maternal and fetal morbidity and mortality. The adaptation of the maternal hemostatic system to pregnancy predisposes women to an increased risk of thromboembolism. A timely diagnosis of deep venous thrombosis is crucial because up to 24% of patients with untreated deep venous thrombosis develop a pulmonary embolism. Recent clinical guidelines identify compression venous ultrasound as the best way to diagnose deep venous thrombosis in pregnancy and CT pulmonary angiography as the best way to diagnose pulmonary embolism in pregnancy. Therapy involves supportive care and anticoagulation with unfractionated or low molecular weight heparin, depending on the clinical scenario.

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.

Intrapartum maternal glycemic control in women with insulin requiring diabetes: a randomized clinical trial of rotating fluids versus insulin drip.

OBJECTIVE: The purpose of this study was to determine whether continuous insulin infusion provides a greater degree of intrapartum maternal glycemic control than rotating between glucose and non-glucose containing intravenous fluids. STUDY DESIGN: Laboring patients with pregestational or gestational diabetes were recruited and randomized to an "insulin drip" or "rotating fluids" protocol. The primary outcome measure was mean maternal capillary blood glucose (CBG) levels (mg/dL). Power analysis indicated that 16 patients were needed in each arm to find a difference of 10 mg/dL. RESULTS: Fifteen patients were randomized to the rotating fluids protocol and 21 patients to an insulin drip. There was no difference in mean intrapartum maternal CBG levels (103.9 +/- 8.7 mg/dL and 103.2 +/- 17.9 mg/dL in the rotating fluids and insulin drip group, respectively, P = .89). Neonatal outcomes were also similar between the 2 treatment groups. CONCLUSION: In patients with insulin requiring gestational diabetes, intrapartum glycemic control may be comparable with a standard adjusted insulin drip or a rotation of intravenous fluids between glucose and non-glucose containing fluids.