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ABSTRACT: Severe preeclampsia is associated with increased neutrophil activation and elevated serum soluble endoglin (sEng) and soluble Flt-1 (sFlt-1) in the maternal circulation. To dissect the contribution of systemic inflammation and anti-angiogenic factors in preeclampsia, we investigated the relationships between the circulating markers of neutrophil activation and anti-angiogenic factors in severe preeclampsia or systemic inflammatory state during pregnancy.
Serum sEng, sFlt-1, placenta growth factor (PlGF), interleukin-6 (IL-6), calprotectin, and plasma α-defensins were measured by ELISA in 88 women of similar gestational age stratified as: severe preeclampsia (sPE, n = 45), maternal systemic inflammatory response (SIR, n = 16) secondary to chorioamnionitis, pyelonephritis or appendicitis, and normotensive controls (CRL, n = 27). Neutrophil activation occurred in sPE and SIR, as α-defensins and calprotectin concentrations were two-fold higher in both groups compared to CRL (P < 0.05 for each). IL-6 concentrations were highest in SIR (P < 0.001) but were higher in sPE than in CRL (P < 0.01). sFlt-1 (P < 0.001) and sEng (P < 0.001) were ≈20-fold higher in sPE compared to CRL, but were not elevated in SIR. In women with sPE, anti-angiogenic factors were not correlated with markers of neutrophil activation (α-defensins and calprotectin) or inflammation (IL-6).
Increased systemic inflammation in sPE and SIR does not correlate with increased anti-angiogenic factors, which were specifically elevated in sPE indicating that excessive systemic inflammation is unlikely to be the main contributor to severe preeclampsia.
Angiogenesis 05/2012; 15(3):341-8. · 6.06 Impact Factor
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ABSTRACT: Severe preeclampsia is associated with increased neutrophil activation and elevated serum soluble endoglin (sEng) and soluble Flt-1 (sFlt-1) in the maternal circulation. To dissect the contribution of systemic inflammation and anti-angiogenic factors in preeclampsia, we investigated the relationships between the circulating markers of neutrophil activation and anti-angiogenic factors in severe preeclampsia or systemic inflammatory state during pregnancy.
Serum sEng, sFlt-1, placenta growth factor, interleukin-6 (IL-6), calprotectin, and plasma α-defensins concentrations were measured by ELISA in 88 women of similar gestational age stratified as: severe preeclampsia (sPE, n = 45), maternal systemic inflammatory response (SIR, n = 16) secondary to chorioamnionitis, pyelonephritis or appendicitis; and normotensive controls (CRL, n = 27). Neutrophil activation occurred in sPE and SIR, as α-defensins and calprotectin concentrations were two-fold higher in both groups compared to CRL (P < 0.05 for each). IL-6 concentrations were highest in SIR (P < 0.001), but were higher in sPE than in CRL (P < 0.01). sFlt-1 (P < 0.001) and sEng (P < 0.001) were ≈20-fold higher in sPE compared to CRL, but were not elevated in SIR. In women with sPE, anti-angiogenic factors were not correlated with markers of neutrophil activation (α-defensins, calprotectin) or inflammation (IL-6).
Increased systemic inflammation in sPE and SIR does not correlate with increased anti-angiogenic factors, which were specifically elevated in sPE indicating that excessive systemic inflammation is unlikely to be the main contributor to severe preeclampsia.
Angiogenesis 03/2012; 15(3):333-40. · 6.06 Impact Factor
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Victor A Rosenberg,
Irina A Buhimschi,
Charles J Lockwood,
Michael J Paidas,
Antonette T Dulay,
Wenda Ramma,
Sonya S Abdel-Razeq, Guomao Zhao,
Shakil Ahmad,
Asif Ahmed,
Catalin S Buhimschi
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ABSTRACT: 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.
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.
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 11/2011; 124(23):2543-53. · 14.74 Impact Factor
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ABSTRACT: Classic IL-6 signaling is conditioned by the transmembrane receptor (IL-6R) and homodimerization of gp130. During trans-signaling, IL-6 binds to soluble IL-6R (sIL-6R), enabling activation of cells expressing solely gp130. Soluble gp130 (sgp130) selectively inhibits IL-6 trans-signaling. To characterize amniotic fluid (AF) IL-6 trans-signaling molecules (IL-6, sIL-6R, sgp130) in normal gestations and pregnancies complicated by intra-amniotic inflammation (IAI), we studied 301 women during second trimester (n = 39), third trimester (n = 40), and preterm labor with intact (n = 131, 85 negative IAI and 46 positive IAI) or preterm premature rupture of membranes (PPROM; n = 91, 61 negative IAI and 30 positive IAI). ELISA, Western blotting, and real-time RT-PCR were used to investigate AF, placenta, and amniochorion for protein and mRNA expression of sIL-6R, sgp130, IL-6R, and gp130. Tissues were immunostained for IL-6R, gp130, CD15(+) (polymorphonuclear), and CD3(+) (T cell) inflammatory cells. The ability of sIL-6R and sgp130 to modulate basal and LPS-stimulated release of amniochorion matrix metalloprotease-9 was tested ex vivo. We showed that in physiologic gestations, AF sgp130 decreases toward term. AF IL-6 and sIL-6R were increased in IAI, whereas sgp130 was decreased in PPROM. Our results suggested that fetal membranes are the probable source of AF sIL-6R and sgp130. Immunohistochemistry and RT-PCR revealed increased IL-6R and decreased gp130 expression in amniochorion of women with IAI. Ex vivo, sIL-6R and LPS augmented amniochorion matrix metalloprotease-9 release, whereas sgp130 opposed this effect. We conclude that IL-6 trans-signaling molecules are physiologic constituents of the AF regulated by gestational age and inflammation. PPROM likely involves functional loss of sgp130.
The Journal of Immunology 03/2011; 186(5):3226-36. · 5.79 Impact Factor
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ABSTRACT: Activation of the receptor for advanced glycation end products (RAGE) mediates cellular injury. Soluble forms of RAGE [soluble RAGE (sRAGE), endogenous secretory (esRAGE)] bind RAGE ligands, thereby preventing downstream signaling and damage.
The objective of the study was to characterize the changes in maternal serum, amniotic fluid, and cord blood soluble receptor for advanced glycation end products (sRAGE) during physiological gestation and to provide insight into mechanisms responsible for RAGE activation in preeclampsia.
This was a cross-sectional study at a tertiary university hospital.
We studied 135 women in the following groups: nonpregnant controls (n = 16), healthy pregnant controls (n = 68), pregnant women with chronic hypertension (n = 13), or pregnant women with severe preeclampsia (sPE; n = 38).
sRAGE and esRAGE levels were evaluated in vivo by ELISA in maternal serum, amniotic fluid, and cord blood and in vitro after stimulation of the amniochorion and placental explants with lipopolysaccharide or xanthine/xanthine oxidase. Placenta and amniochorion were immunostained for RAGE. Real-time quantitative PCR measured RAGE mRNA.
Pregnant women had significantly decreased serum sRAGE compared with nonpregnant subjects (P < 0.001). sPE women had higher serum and amniotic fluid sRAGE and esRAGE relative to those expected for gestational age (P < 0.001). Cord blood sRAGE remained unaffected by sPE. RAGE immunoreactivity and mRNA expression appeared elevated in the amniochorion of sPE women. Xanthine/xanthine oxidase (but not lipopolysaccharide) significantly up-regulated the release of sRAGE (P < 0.001) in the amniochorion explant system.
Fetal membranes are a rich source of sRAGE. Elevated maternal serum and amniotic fluid sRAGE and esRAGE, paralleled by increased RAGE expression in the amniochorion, suggest activation of this system in sPE.
The Journal of clinical endocrinology and metabolism 02/2011; 96(3):689-98. · 6.50 Impact Factor
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Mark J Wehrum,
Irina A Buhimschi,
Carolyn Salafia,
Stephen Thung,
Mert O Bahtiyar,
Erica F Werner,
Katherine H Campbell,
Christine Laky,
Anna K Sfakianaki, Guomao Zhao,
Edmund F Funai,
Catalin S Buhimschi
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ABSTRACT: We sought to characterize serum angiogenic factor profile of women with complete placenta previa and determine if invasive trophoblast differentiation characteristic of accreta, increta, or percreta shares features of epithelial-to-mesenchymal transition.
We analyzed gestational age-matched serum samples from 90 pregnant women with either complete placenta previa (n = 45) or uncomplicated pregnancies (n = 45). Vascular endothelial growth factor (VEGF), placental growth factor, and soluble form of fms-like-tyrosine-kinase-1 were immunoassayed. VEGF and phosphotyrosine immunoreactivity was surveyed in histological specimens relative to expression of vimentin and cytokeratin-7.
Women with previa and invasive placentation (accreta, n = 5; increta, n = 6; percreta, n = 2) had lower systemic VEGF (invasive previa: median 0.8 [0.02-3.4] vs control 6.5 [2.7-10.5] pg/mL, P = .02). VEGF and phosphotyrosine immunostaining predominated in the invasive extravillous trophoblasts that coexpressed vimentin and cytokeratin-7, an epithelial-to-mesenchymal transition feature and tumorlike cell phenotype.
Lower systemic free VEGF and a switch of the interstitial extravillous trophoblasts to a metastable cell phenotype characterize placenta previa with excessive myometrial invasion.
American journal of obstetrics and gynecology 02/2011; 204(5):411.e1-411.e11. · 3.28 Impact Factor
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Catalin S Buhimschi,
Vineet Bhandari,
Antonette T Dulay,
Unzila A Nayeri,
Sonya S Abdel-Razeq,
Christian M Pettker,
Stephen Thung, Guomao Zhao,
Yiping W Han,
Matthew Bizzarro,
Irina A Buhimschi
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ABSTRACT: Intra-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.
We 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 1(st)-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 2(nd)-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.
Antenatal 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.
PLoS ONE 01/2011; 6(10):e26111. · 4.09 Impact Factor
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Catalin S Buhimschi,
Vineet Bhandari,
Antonette T Dulay,
Stephen Thung,
Sonya S Abdel- Razeq,
Victor Rosenberg,
Christina S Han,
Unzila A Ali,
Eduardo Zambrano, Guomao Zhao,
Edmund F Funai,
Irina A Buhimschi
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ABSTRACT: 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.
The Journal of clinical endocrinology and metabolism 07/2010; 95(7):3428-36. · 6.50 Impact Factor
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ABSTRACT: There is little known about healing of the uterus after Cesarean delivery (CD). Uterine wound repair was studied by using two strains of mice with different wound healing characteristics: MRL/MpJ(+/+) (MRL: "high-healer" phenotype) and C57Bl/6 ("low-healer" phenotype). First, we examined the morphology and histology of the uterine wall repair. We identified wound granulation tissue 3 days post-CD in both strains, albeit less in the MRL strain. Macroscopically, no scar could be identified either in MRL or C57Bl/6 mice on day 60 post-CD. However, histologically, we found significant differences in wound integration, inflammation, and collagen birefringence between the two strains of mice. Using a histological index, we provided evidence for significant differences in mitotic activity in the initial phases of uterine healing among strains. Functional behavior of the uterine scar also was analyzed by using biomechanical parameters such as slope (measure of stiffness), yield point (measure of elasticity), and break point (measure of strength). There were significant differences in stiffness of the scarred myometrium between the two phenotypes. MRL mice displayed a significantly lower yield point compared with C57Bl/6. The break point was reached faster on days 15 and 60 in both C57Bl/6 and MRL strains compared with day 3 post-CD. Our findings indicate that differences in regenerative ability translate in histological, mitotic, and functional differences in biomechanical properties of the scarred myometrium after CD.
American Journal Of Pathology 07/2010; 177(1):197-207. · 4.89 Impact Factor
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Catalin S Buhimschi,
Margaret A Baumbusch,
Antonette T Dulay,
Emily A Oliver,
Sarah Lee, Guomao Zhao,
Vineet Bhandari,
Richard A Ehrenkranz,
Carl P Weiner,
Joseph A Madri,
Irina A Buhimschi
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ABSTRACT: 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 S100beta protein. We found significantly lower sRAGE in human fetuses that mounted robust inflammatory responses. HMGB1 levels correlated significantly with levels of interleukin-6 and S100beta in fetal circulation. We then evaluated the levels and areas of tissue expression of RAGE, HMGB1, and S100beta 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 S100beta, at sites of tissue damage. Our findings indicate that RAGE and HMGB1 may be important mediators of cellular injury in fetuses delivered in the setting of inflammation-induced preterm birth.
American Journal Of Pathology 09/2009; 175(3):958-75. · 4.89 Impact Factor
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ABSTRACT: 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 07/2009; 182(11):7244-53. · 5.79 Impact Factor
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ABSTRACT: We hypothesized that the genetic makeup has an impact on the functional behavior of the uterine cervix. Therefore, we compared the biomechanical properties of uterine cervix in postpartum in 2 strains of mice that differ in their underlying regenerative collagen remodeling characteristics: MRL/MpJ+/+ (MRL: high regenerative repair) and C57BL/6 (C57: low regenerative high fibrotic repair).
Cervical tensile proprieties were assessed on day 3, 15, and 60 postpartum in MRL (n = 14) and C57 (n = 13) mice (4-5 animals at each time point). Stress-strain curves were generated using Shimadzu EZ-test instrumentation. Cervical tissue was stretched by 0.42 mm/min until rupture. Parameters of viscoelasticity including slope (a measure of stiffness), yield point (YP; moment when tissue changes its proprieties from elastic to plastic), and break point (BP; measure of tissue strength) were recorded and analyzed blindly between strains. Data were normalized to the weight of the tissue and analyzed by 2-way analysis of variance. Histological and collagen birefringence evaluation of the uterine cervix (MRL: n = 4; C57: n = 4) was performed 5 days after delivery.
At 3 and 15 days postpartum, cervices of MRL mice were significantly more compliant than those of C57 (P < .001). MRL mice displayed a significant increase in stiffness from day 3 to day 60 (slope, median +/- SEM: day 3: 3.1 +/- 0.5 vs day 15: 20.3 +/- 4.9 vs day 60: 33.1 +/- 3.5 N/mm per gram; P < .001). In contrast, the stiffness of C57 cervices reached maximum on day 15 (slope day 3: 14.1 +/- 4.3 vs day 15: 40.0 +/- 6.5 N/mm per gram; P = .02) and rested at a similar level on day 60 (day 60: 26.1 +/- 7.0 N/mm per gram; day 60 vs day 15: P = .937). More force was required to reach YP in C57 on day 3 (C57: 72.5 +/- 14.7 vs MRL: 19.9 +/- 1.6 N/g; P < .001) but not on either day 15 (C57: 156.1 +/- 27.5 vs MRL: 109.2 +/- 26.0 N/g; P = .120) or on day 60 (C57: 143.4 +/- 26.5 vs MRL: 164.5 +/- 18.7 N/g; P = .412). There was a significant decrease in BP in both strains on both day 15 and day 60 compared with day 3 postpartum (P = .856 for strain, P = .008 for day). MRL mice displayed significantly less cervical collagen birefringence compared with C57 control (P < .001) but increased proteoglycan staining and increased water content.
We provide evidence that genetic makeup may have an impact on cervical tissue remodeling and function. There are significant differences in postpartum cervical stiffness and compliance that vary with the regenerative collagen remodeling phenotype.
American journal of obstetrics and gynecology 02/2009; 200(4):434.e1-7. · 3.28 Impact Factor
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ABSTRACT: The cause of preeclampsia remains unknown and the diagnosis can be uncertain. We used proteomic-based analysis of urine to improve disease classification and extend the pathophysiologic understanding of preeclampsia.
Urine samples from 284 women were analyzed by surface-enhanced laser desorption/ionization. In the exploratory phase, 59 samples were used to extract the proteomic fingerprint characteristic of severe preeclampsia requiring mandated delivery and to develop a diagnostic algorithm. In the challenge phase, we sought to prospectively validate the algorithm in 225 women screened for a variety of high- and low-risk conditions, including preeclampsia. Of these, 19 women were followed longitudinally throughout pregnancy. The presence of biomarkers was interpreted relative to clinical classification, need for delivery, and other urine laboratory measures (ratios of protein to creatinine and soluble fms-like tyrosine kinase-1 to placental growth factor). In the translational phase, biomarker identification by tandem mass spectrometry and validation experiments in urine, serum, and placenta were used to identify, quantify, and localize the biomarkers or related proteins.
We report that women with preeclampsia appear to present a unique urine proteomic fingerprint that predicts preeclampsia in need of mandated delivery with highest accuracy. This characteristic proteomic profile also has the ability to distinguish preeclampsia from other hypertensive or proteinuric disorders in pregnancy. Pregnant women followed longitudinally who developed preeclampsia displayed abnormal urinary profiles more than 10 weeks before clinical manifestation. Tandem mass spectrometry and de novo sequencing identified the biomarkers as nonrandom cleavage products of SERPINA1 and albumin. Of these, the 21 amino acid C-terminus fragment of SERPINA1 was highly associated with severe forms of preeclampsia requiring early delivery. In preeclampsia, increased and aberrant SERPINA1 immunoreactivity was found in urine, serum, and placenta, in which it localized predominantly to placental villi and placental vascular spaces adherent to the endothelium. In addition, significant perivascular deposits of misfolded SERPINA1 aggregates were exclusively identified in preeclamptic placentae.
Proteomics-based characterization of urine in preeclampsia identified a proteomic fingerprint composed of SERPINA1 and albumin fragments, which can accurately diagnose preeclampsia and shows promise to discriminate it from other hypertensive proteinuric diseases. These findings provide insight into a novel pathophysiological mechanism of preeclampsia related to SERPINA1 misfolding, which may offer new therapeutic opportunities in the future.
American journal of obstetrics and gynecology 12/2008; 199(5):551.e1-16. · 3.28 Impact Factor
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ABSTRACT: Intrapartum penicillin G prophylaxis aims to prevent early-onset group B streptococci (GBS) sepsis by interrupting vertical transmission. We examined the relationship between duration of prophylaxis and fetal serum penicillin G levels among fetuses exposed to fewer than 4 hours of prophylaxis compared with longer durations.
In this prospective cohort study, 98 laboring GBS-positive women carrying singleton gestations at 37 weeks or greater were administered 5 million units of intravenous penicillin G followed by 2.5 million units every 4 hours until delivery. Umbilical cord blood samples were collected at delivery, and penicillin G levels were measured by high-performance liquid chromatography. Intraassay and interassay coefficients of variation were less than 3%.
Fetuses exposed to fewer than 4 hours of prophylaxis had higher penicillin G levels than those exposed to greater than 4 hours (P=.003). In multivariable linear regression analysis, fetal penicillin G levels were determined by duration of exposure, time since last dose, dosage, and number of doses, but not maternal body mass index. Penicillin G levels increased linearly until 1 hour (R(2)=.40) and then decreased rapidly according to a power-decay model (R(2)=.67). All subgroups analyzed were above the minimal inhibitory concentration (MIC) for GBS (0.1 micrograms/mL)(P<.002). Individual samples were 10-179-fold above the MIC. In patients receiving maintenance dosing, penicillin G did not accumulate in the cord blood and returned to baseline after each 4-hour interval.
Short durations of prophylaxis achieved levels significantly above the MIC, suggesting a benefit even in precipitous labors. The designation of infants exposed to fewer than 4 hours of prophylaxis as particularly at risk for GBS sepsis may be pharmacokinetically inaccurate.
Obstetrics and Gynecology 09/2008; 112(2 Pt 1):265-70. · 4.73 Impact Factor
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ABSTRACT: Proinflammatory cytokines of placental or systemic origin are thought to play a central role in the pathophysiology of preeclampsia. We sought to estimate the fractional excretion of tumor necrosis factor (TNF)-alpha in relationship to proteinuria in women with severe preeclampsia.
In a cross-sectional study, we evaluated the serum and urine levels of TNF-alpha in 45 women diagnosed with severe preeclampsia (mean+/-standard error of the mean, gestational age 29.1+/-0.5 weeks). Forty-five healthy pregnant women matched for parity, maternal age, and gestational age at recruitment (30.1+/-0.4 weeks) made up the control group. Urinary concentrations were normalized to creatinine. The fractional excretion of TNF-alpha was interpreted in relationship to that of total proteins and soluble fms-like tyrosine kinase-1 (sFlt-1).
We found that the women with preeclampsia had significantly higher serum TNF-alpha concentrations compared with the women in the control group (mean+/-standard error of the mean, preeclampsia: 1.39+/-0.09 versus control: 0.93+/-0.07 pg/mL, P<.001). In contrast, urinary levels of TNF-alpha were significantly decreased in the women with preeclampsia compared with the healthy women (median [interquartile range], preeclampsia: 0.26 [0.10-0.91] versus control: 0.58 [0.21-1.29] pg/mg creatinine, P=.003), even though the hypertensive women had higher levels of proteinuria. In contrast to sFlt-1, urinary TNF-alpha did not correlate with the degree of proteinuria. Additionally, in preeclampsia, the fractional excretion of TNF-alpha was significantly lower (preeclampsia: 1.92% [0.46-4.20] versus control: 7.2% [2.44-12.07], P<.001).
The fractional excretion of TNF-alpha is significantly reduced in women with severe preeclampsia despite proteinuria. The decreased clearance and altered renal excretion of this cytokine may contribute to the exaggerated inflammatory response observed in preeclampsia.
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Obstetrics and Gynecology 07/2008; 112(1):93-100. · 4.73 Impact Factor
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Antonette T Dulay,
Irina A Buhimschi, Guomao Zhao,
Guoyang Luo,
Sonya Abdel-Razeq,
Michael Cackovic,
Victor A Rosenberg,
Christian M Pettker,
Stephen F Thung,
Mert O Bahtiyar,
Vineet Bhandari,
Catalin S Buhimschi
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ABSTRACT: 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.
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.
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.
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.
American journal of obstetrics and gynecology 04/2008; 198(4):426.e1-9. · 3.28 Impact Factor
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Catalin S Buhimschi,
Ozhan M Turan,
Edmund F Funai,
Humberto Azpurua,
Mert-Ozan Bahtiyar,
Sifa Turan, Guomao Zhao,
Antonette Dulay,
Vineet Bhandari,
Joshua A Copel,
Irina A Buhimschi
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ABSTRACT: Fetal adaptation to stress is regulated in part by the pituitary-adrenocortical system. The stress hormones dehydroepiandrosterone sulfate (DHEAS) and cortisol have opposing effects: cortisol suppresses while DHEAS enhances immune functions. We sought to estimate the impact of intraamniotic inflammation on fetal adrenal gland volume and cortisol-to-dehydroepiandrosterone sulfate ratio (fetal stress ratio) in pregnancies complicated by preterm birth.
Fifty-one consecutive singleton fetuses of mothers who had an indicated amniocentesis to rule out infection were analyzed. Intraamniotic inflammation was assessed by proteomic profiling of amniotic fluid for the biomarkers of the Mass Restricted score. The Mass Restricted score ranges from 0 (biomarkers absent) to 4 (all biomarkers present), with Mass Restricted scores of 3 or 4 indicating severe intraamniotic inflammation. Fetal adrenal gland volume was assessed by three-dimensional ultrasonography and corrected for estimated fetal weight. Interleukin-6 (IL-6), cortisol, and DHEAS were measured by immunoassay.
Women with intraamniotic inflammation delivered earlier (27.8+/-3.4 weeks, n=16, compared with 32.3+/-3.0 weeks, n=35, P<.001), and their fetuses had higher cord blood IL-6 (P=.011) and higher corrected adrenal gland volumes (P=.027). Cord blood IL-6 levels were in direct relationship with corrected adrenal volume (r=0.372, P=.019), fetal cortisol (r=0.428, P=.010), and DHEAS (r=0.521, P<.001). However, fetuses exposed to intraamniotic inflammation had an overall lower fetal stress ratio (P=.034). These results maintained after adjusting for gestational age, uterine contractions, and steroid exposure.
Fetuses exposed to intraamniotic inflammation have higher adrenal gland volumes and lower cortisol-to-DHEAS ratios, suggesting that the fetal adrenocortical axis plays a role in the intrauterine adaptation to inflammation.
Obstetrics and Gynecology 03/2008; 111(3):715-22. · 4.73 Impact Factor
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ABSTRACT: 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.
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.
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.
PLoS ONE 02/2008; 3(4):e2049. · 4.09 Impact Factor
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ABSTRACT: To test the hypothesis that differences in the matrix arrangement of the lower uterine segment (above and below the reflection of the bladder flap) translate to differences in biomechanical or structural properties of the uterine wall.
We enrolled prospectively 40 women at term randomized to a bladder flap (n=21) versus no bladder flap (n=19) at the time of a cesarean delivery for dystocia. Tensile properties of tissue biopsies from the upper and lower edges of the hysterotomy incision were quantitated using a stretching regimen designed to mimic labor. Parameters such as slope, yield point, and break point were analyzed in relationship to biochemical (total collagen, sulfated glycosaminoglycans, pyridinoline-deoxypyridinoline) and histological (collagen birefringence) properties of the tissue.
The lower edge of the hysterotomy had a higher collagen content than the upper, independently of whether the hysterotomy site was above or below the reflection of the bladder (P<.001 for edge level, P=.8 for bladder flap). The pyridinoline-to-collagen ratio (a reflection of collagen cross-linking) was significantly decreased in the lower edge of the hysterotomy, independently of a bladder flap (P<.001). Neither collagen birefringence nor the amount of sulfated glycosaminoglycans differed among sites. There were no significant differences in biomechanical properties between the upper and lower edges of the hysterotomy, whether or not a bladder flap was created.
The lower uterine segment of women with dystocia maintains its overall biomechanical properties despite significant quantitative and qualitative differences in fibrillar collagen between the upper and lower edge of the hysterotomy incision.
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Obstetrics and Gynecology 03/2007; 109(3):691-700. · 4.73 Impact Factor
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ABSTRACT: 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.
Pediatric Research 03/2007; 61(3):318-24. · 2.70 Impact Factor
