Amniotic Fluid Proteins Research Articles

Bioactive proteins in healthy pregnancies and preeclampsia: relevance to hypertension and proteinuria

Background Bioactive proteins, such as cytokines and chemokines, have not been systematically evaluated in healthy and preeclamptic pregnancies. We aimed to investigate the difference of these proteins between healthy and preeclamptic pregnancies in order to help clarify their potential roles in the pathogenesis of hypertension and proteinuria in preeclampsia. Methods Samples of amniotic fluid and maternal/umbilical cord blood were collected from normal pregnancies and women with preeclampsia for examination of bioactive proteins. Fifty-three pregnant women were enrolled in this study. Of them, 30 pregnant women were recruited as healthy controls, and 23 pregnant women were diagnosed with preeclampsia. An antibody array was used to screen for higher levels of cytokines and related proteins in amniotic fluid than in the blood samples, and these proteins were then selected for quantification by immunoassay. Results Interleukin-1 receptor 4, hepatocyte growth factor, and urokinase plasminogen activator receptor were significantly elevated in the blood of preeclampsia patients. In particular, interleukin-1 receptor 4 was 8-fold higher in preeclampsia patients than in the healthy pregnancies. Moreover, in cord blood samples hepatocyte growth factor and interleukin-8 were significantly higher in preeclampsia patients. Conclusions Because of the biologic activities, Interleukin-1 receptor 4, hepatocyte growth factor, urokinase plasminogen activator receptor and interleukin-8 in maternal and/or cord blood could play a role in the pathogenesis of hypertension and proteinuria in preeclampsia.

PL.45 High-Sensitivity C-Reactive Protein (HS-CRP) in Amniotic Fluid Obtained at Caesarean Section: A Feasibility Study

IntroductionThe most important risk factor for post-partum maternal infection is caesarean section (CS). High-sensitivity C-reactive protein (HS-CRP) accurately detects low concentrations of CRP as a predictor of inflammation in blood....

Amniotic fluid protein profiles of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria.

ObjectiveThis study aimed to evaluate the amniotic fluid protein profiles and the intensity of intraamniotic inflammatory response to Ureaplasma spp. and other bacteria, using the multiplex xMAP technology.MethodsA retrospective cohort study was undertaken in the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic. A total of 145 pregnant women with preterm prelabor rupture of membranes between gestational age 24+0 and 36+6 weeks were included in the study. Amniocenteses were performed. The presence of Ureaplasma spp. and other bacteria was evaluated using 16S rRNA gene sequencing. The levels of specific proteins were determined using multiplex xMAP technology.ResultsThe presence of Ureaplasma spp. and other bacteria in the amniotic fluid was associated with increased levels of interleukin (IL)-6, IL-8, IL-10, brain-derived neurotropic factor, granulocyte macrophage colony stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1, and matrix metalloproteinasis-9. Ureaplasma spp. were also associated with increased levels of neurotropin-3 and triggering receptor expressed on myeloid cells-1.ConclusionsThe presence of Ureaplasma spp. in the amniotic fluid is associated with a slightly different protein profile of inflammatory response, but the intensity of inflammatory response to Ureaplasma spp. is comparable with the inflammatory response to other bacteria.

Serum and Amniotic Fluid Eosinophil Cationic Protein Levels in Misoprostol Induced Pregnancies

The aim of this study was to determine the concentration of eosinophil cationic protein (ECP) in maternal serum (MS) and amniotic fluid (AF) at term and assess misoprostol induced changes in their mean concentrations. Methods: A total of 53 pregnant women were included in the study: 24 were in active labor, and a further 29 were not in labor and misoprostol induction was performed. The initial 3 mL of amniotic fluid was collected and processed. In all pregnant subjects, maternal blood samples were obtained from the cubital vein at the time of amniotic fluid sampling. The concentration of ECP in serum and AF was measured by chemiluminescence method with the immulite 2000 analyzer (Immulite2000® ECP, DPC Diagnostic Products Corporation 5700 West 96th Street Los Angeles, CA, USA). Results: Misoprostol induced pregnant showed significantly higher concentrations ECP than the control pregnant women in serum. AF-ECP concentrations were not significantly different between two groups. There was no significant difference between MS and AF-ECP concentrations in control subjects. Conclusion: The association we encountered between labor induction with misoprostol and increased systemic production of ECP should be investigated further.

458: Identification of differentially expressed proteins in amniotic fluid between donor and recipient of twin fetuses with twin-to-twin transfusion syndrome

Twin-to-twin transfusion syndrome (TTTS) is a serious complication of monochorionic twin pregnancy with disproportionate blood supply caused by vascular anastomosis between the fetuses. Amniotic fluid (AF) is composed of fetal urine, and it is essential for antenatal diagnosis of TTTS, as well as reflects the various fetal conditions. We performed quantitative proteomic analysis of AF to identify differentially expressed proteins between the donor and recipient in twin fetuses with TTTS.

746: Does the neonatal impact of chorioamnionitis in the setting of PPROM vary depending on degree of prematurity?

setting of PPROM vary depending on degree of prematurity? Shireen de Sam Lazaro, Yvonne Cheng, Jonathan Snowden, Leonardo Pereira, Natali Aziz, Aaron Caughey Oregon Health and Science University, Obstetrics and Gynecology, Portland, OR, University of California-San Francisco, Obstetrics, Gynecology and Reproductive Sciences, San Francisco, CA, Stanford, Obstetrics and Gynecology, Palo Alto, CA OBJECTIVE: To examine if neonatal outcomes vary based on the diagnosis of chorioamnionitis in the setting of pregnancies complicated by preterm premature rupture of membranes, (PPROM) and whether this difference varies by gestational age. STUDY DESIGN: A retrospective cohort study was conducted of all pregnancies complicated by PPROM who delivered between 24 to 36 weeks and 6 days gestation in 2006 in California (n 5229). Pregnancies were grouped by gestational age, and patients who delivered with a diagnosis of chorioamnionitis were compared to patients without this diagnosis. Neonatal outcomes were assessed including necrotizing enterocolitis (NEC), intraventricular hemorrhage (IVH), and sepsis. Dichotomous outcomes were compared using Fisher’s exact test, with P 0.05 indicating statistical significance. RESULTS: 5,229 women were diagnosed with PPROM between 24 weeks to 36 weeks 6 days, and 306 (5.9 %) women were diagnosed with chorioamnionitis prior to delivery during the study period. Neonates born to mothers diagnosed with chorioamnionitis were noted to have significantly higher rates of sepsis across each gestational age (Table). Additionally, amongst the most premature infants, an association between chorioamnionitis and IVH was noted, though this did not reach statistical significance. Chorioamnionitis was not noted to affect the rate of necrotizing enterocolitis across the gestational ages. CONCLUSION: Patients who develop chorioamnionitis in the setting of PPROM are at an increased risk of neonatal sepsis. Additionally, the most premature infants delivered in the setting of chorioamnionitis may be an increased risk of intraventricular hemorrhage. No increased risk for necrotizing enterocolitis was noted amongst neonates delivered with chorioamnionitis compared to those without that diagnosis across all gestational ages. 747 Clinical significance of elevated high-sensitivity C-reactive protein in amniotic fluid obtained at emergency caesarean section Zbigniew Marchocki, Maurice O’Donoghue, Kevin Collins, Keelin O’Donoghue Cork University Maternity Hospital, University College Cork, Obstetrics & Gynaecology, Cork, Ireland, Cork University Maternity Hospital, University College Cork, Microbiology, Cork, Ireland OBJECTIVE: Delivery by caesarean section (CS) is the single most important factor associated with post-partum infection. Risk factors include obesity, prolonged operating time, poor surgical technique and emergency delivery. While the majority of infections are not life threatening they have implications on length of hospital stay, costs and re-admission rates. We examined high-sensitivity C-reactive protein (HS-CRP) levels in amniotic fluid (AF) and maternal serum at the time of CS and correlated results with risk factors for infection and postpartum infectious complications. STUDY DESIGN: This was a prospective observational study of women undergoing elective and emergency CS. AF for HS-CRP analysis was obtained at CS by direct needle aspiration from the intact amnion. Maternal serum HS-CRP was measured before and 3 days after CS. Results were correlated with maternal and neonatal outcomes. RESULTS: Seventy-four women had AF samples collected at CS; of these 53% (39/74) underwent emergency CS. There was a significant difference in HS-CRP in AF at emergency CS, with levels on average 10 fold higher (p 0.009). No difference in AF HS-CRP levels was noted in women with BMI 25 or between smokers. In the emergency CS group, AF HS-CRP levels were not affected by length of labour, fetal blood sampling or pyrexia in labour. However there was a significant difference in AF HS-CRP levels in women who had multiple ( 7) vaginal examinations in labour (p 0.003). Overall, 11% of women developed post-partum infectious complications, but there was no difference in AF HS-CRP levels. Six neonates were admitted to NICU with suspected infection; AF or serum HS-CRP levels were not significantly different. CONCLUSION: AF HS-CRP levels were significantly higher after emergency CS. Changes in AF HS-CRP levels were not associated with early post-partum infectious morbidity among mothers and babies, but our findings are limited by small sample size and further study may determine clinical significance.

747: Clinical significance of elevated high-sensitivity C-reactive protein in amniotic fluid obtained at emergency caesarean section

747: Clinical significance of elevated high-sensitivity C-reactive protein in amniotic fluid obtained at emergency caesarean section

Surfactant protein D modulates levels of IL-10 and TNF-α in intrauterine compartments during lipopolysaccharide-induced preterm birth

Prematurity is the main cause of perinatal mortality and morbidity, and preterm birth is often associated with intrauterine inflammation. Surfactant protein D (SP-D) functions in lung homeostasis and has multiple roles in innate immunity. It is present in amniotic fluid and in gestational tissues. We propose that SP-D may regulate intrauterine inflammatory responses related to preterm labor. Our aim was to investigate the role of SP-D in lipopolysaccharide-induced preterm birth in mice that overexpress rat SP-D (rSP-D) under the human SP-C promoter. SP-D protein in amniotic fluid and in gestational tissues was detected by western analysis. TNF-α, IL-10, and IL-6 concentrations in serum and amniotic fluid and mRNA levels in gestational tissues were quantified using cytometric bead array and ribonuclease protection assay, respectively. Increased levels of SP-D protein were detected in the amniotic fluid and gestational tissues of rSP-D mice. Lipopolysaccharide given at 17days post-coitum to rSP-D dams led to preterm birth of live-born offspring within 18h. Preterm birth of live-born pups was induced with a lower dose of lipopolysaccharide compared to wild-type mice. In rSP-D mice, the lipopolysaccharide-induced levels of TNF-α and IL-10 in amniotic fluid and fetal serum and the expression of IL-10 in placenta and fetal membranes were significantly different from wild-type mice. We conclude that SP-D in fetal and gestational tissues modulates the levels of intrauterine inflammatory mediators involved in preterm birth and may contribute to inflammatory processes related to spontaneous preterm labor.

Amniotic Fluid Cathelicidin in PPROM Pregnancies: From Proteomic Discovery to Assessing Its Potential in Inflammatory Complications Diagnosis

BackgroundPreterm prelabor rupture of membranes (PPROM) complicated by microbial invasion of the amniotic cavity (MIAC) leading to histological chorioamnionitis (HCA) significantly impacts perinatal morbidity. Unfortunately, no well-established tool for identifying PPROM patients threatened by these disorders is available.Methodology/Principal FindingsWe performed an unbiased exploratory analysis of amniotic fluid proteome changes due to MIAC and HCA. From among the top five proteins that showed the most profound and significant change, we sought to confirm results concerning cathelicidin (P49913, CAMP_HUMAN), since an ELISA kit was readily available for this protein. In our exploratory proteomic study, cathelicidin showed a ∼6-fold higher concentration in PPROM patients with confirmed MIAC and HCA. We verified significantly higher levels of cathelicidin in exploratory samples (women without both MIAC and HCA: median 1.4 ng/ml; women with both conditions confirmed: median 3.6 ng/ml; p = 0.0003). A prospective replication cohort was used for independent validation and for assessment of cathelicidin potential to stratify women with MIAC leading to HCA from women in whom at least one of these conditions was ruled out. We confirmed the association of higher amniotic fluid cathelicidin levels with MIAC leading to HCA (the presence of both MIAC and HCA: median 3.1 ng/ml; other women: median 1.4 ng/ml; p<0.0001). A cathelicidin concentration of 4.0 ng/ml was found to be the best cut-off point for identifying PPROM women with both MIAC and HCA. When tested on the validation cohort, a sensitivity of 48%, a specificity of 90%, a likelihood ratio of 5.0, and an area under receiver-operating characteristic curve of 71% were achieved for identification of women with MIAC leading to HCA.ConclusionsOur multi-stage study suggests cathelicidin as a candidate marker that should be considered for a panel of amniotic fluid proteins permitting identification of PPROM women with MIAC leading to HCA.

Intra‐amniotic inflammation predicts microbial invasion of the amniotic cavity but not spontaneous preterm delivery in preterm prelabor membrane rupture

To predict microbial invasion of the amniotic cavity (MIAC) and spontaneous preterm delivery within seven days using a panel of selected proteins from amniotic fluid in a Swedish population of preterm prelabor membrane rupture (PPROM). Prospective cohort study. Evaluation of intra-amniotic inflammation in preterm premature rupture of membranes. Sixty-six pregnant women with preterm prelabor membrane rupture at 22(+0) -33(+6) weeks' gestational age. Methods. Twenty-seven amniotic fluid proteins were assayed by a multiple immunoassay. The intra-amniotic inflammatory response was evaluated according to the presence of MIAC and the risk of spontaneous preterm delivery within seven days. A prediction model was constructed using logistic regression. The overall rates of MIAC and spontaneous preterm delivery within seven days were 20 and 50%, respectively. There was a higher inflammatory response in women with MIAC than in those without. Earlier gestational age at delivery and lower birthweight were observed in the presence of microbial invasion of the amniotic cavity. Amniotic fluid interleukin (IL)-6 and IL-10 were the best predictors of MIAC in terms of sensitivity (69%), specificity (81%), positive predictive value (47%), negative predictive value (91%) and a positive likelihood ratio of 3.6. There were no differences in intra-amniotic inflammatory response according to the risk of spontaneous preterm delivery within seven days. Amniotic fluid IL-6 and IL-10 are the best inflammatory biomarkers to predict MIAC in women with PPROM. Intra-amniotic inflammation does not predict the occurrence of spontaneous preterm delivery within seven days of PPROM.

Intraamniotic inflammatory response to bacteria: analysis of multiple amniotic fluid proteins in women with preterm prelabor rupture of membranes

Objective: To analyse whether intraamniotic inflammation in response to bacteria is different below and above gestational age 32 weeks in pregnancies complicated by preterm prelabor rupture of membranes (PPROM). Methods: A prospective study was performed, and 115 women with singleton pregnancies complicated by PPROM at gestational ages between 240/7 and 366/7 weeks were included in the study. Transabdominal amniocenteses were performed. Amniotic fluid was analysed using polymerase chain reactions for genital mycoplasmas and cultured for aerobic and anaerobic bacteria. The concentrations of 26 proteins in the amniotic fluid were determined simultaneously using multiplex technology. Results: Bacteria were found in the amniotic fluid of 43% (49/115) of the women. The women were stratified into two subgroups according to gestational age 32 weeks. The amniotic fluid levels of four (interleukin-6, interleukin-10, CC chemokine ligands 2, and 3) and one specific (CC chemokine ligands 2) proteins were higher in women with the presence of bacteria in the amniotic fluid below and above 32 gestational weeks, respectively. Conclusions: An intraamniotic inflammatory response to bacteria in pregnancies complicated by PPROM seems to be different below and above 32 weeks of gestation.

507: Intraamniotic inflammatory response to bacteria in preterm prelabor rupture of membranes pregnancies: analysis of multiple amniotic fluid proteins

507 Intraamniotic inflammatory response to bacteria in preterm prelabor rupture of membranes pregnancies: analysis of multiple amniotic fluid proteins Marian Kacerovsky, Kristin Skogstrand, David Hougaard, Ali Khatibi, Bo Jacobsson University Hospital Hradec Kralove, Department of Obstetrics and Gynecology, Hradec Kralove, Czech Republic, Statens Serum Institut, 3Department of Clinical Biochemistry and Immunology, Copenhagen, Denmark, Statens Serum Institut, Department of Clinical Biochemistry and Immunology, Copenhagen, Denmark, Sahlgrenska University Hospital, Department of Obstetrics and Gynecology, Goteborg, Sweden, Sahlgrenska University Hospital, Department of Obstetrics and Gynecology, Gothenburg, Sweden OBJECTIVE: To analyze whether intraamniotic inflammatory response to bacteria is dependent on gestational age in preterm prelabor rupture membrane (PPROM) pregnancies with using a panel specific proteins. To determine whether is the difference in intraamniotic inflammatory response to genital mycoplasmas and other bacteria. STUDY DESIGN: A prospective follow-up study was performed and 115 women with singleton pregnancies complicated by preterm prelabor rupture of membranes at gestational age between 24 0 and 36 6 weeks were recruited. Amniotic fluid retrieved by transabdominal amniocentesis was analyzed with polymerase chain reaction for genital mycoplasmas, and cultured for aerobic and anaerobic bacteria. The concentrations of 26 proteins in the amniotic fluid were determined simultaneously using multiplex technology. RESULTS: The presence of bacteria in amniotic fluid was found in 43% (49/115) women. The women were stratified into three subgroups according to gestational age. The differences of amniotic fluid proteins between cohorts with and without bacteria were found in the groups with gestational age 24 0 32 0 weeks and 32 1 34 0 for six specific proteins (interleukin 6, 8, 10, granulocyte macrophage colony stimulating factor, monocyte chemotactic protein-1, and macrophage inflammatory protein-1 ) and two proteins (adiponectin, leptin), respectively. No differences were found between women with the presence of genital mycoplasmas and other bacteria in amniotic fluid. CONCLUSION: The intraamniotic inflammatory response to bacteria in amniotic fluid is dependent on gestational age. There is no difference in the intensity of intraamniotic inflammatory response to genital mycoplasmas and the other bacteria. 508 Working conditions and preterm birth in multiple pregnancies Marjo van Melick Dutch Consortium for Studies in Women’s Health and Reproductivity, Department of Obstetrics & Gynaecology, Maastricht, Netherlands OBJECTIVE: Multiple pregnancies are at high risk for preterm birth. Unfortunately, strategies for preventing preterm birth have been largely unsuccessful. Specific physical activities or working conditions are suspected for having an increased effect on preterm birth. The aim of this study was to perform an explorative analysis of the effect of specific work strain in the first 20 weeks of pregnancy on the occurrence of preterm birth in multiple pregnancies before 34 weeks of gestation. STUDY DESIGN: A prospective cohort study was conducted in the Netherlands alongside the multicentre randomized controlled trial ‘Pessaries in multiple pregnancies as a prevention of preterm birth (ProTWIN trial)’. Participants in the ProTWIN trial received questionnaires concerning their working conditions and physical work strain. From these questionnaires specific working conditions of women in the first 20 weeks of pregnancy could be assessed. We assessed whether these conditions were associated with preterm birth before 34 weeks of gestation. RESULTS: Of the 81 participants, 70 (86%) had a paid employment with a mean of 31 working hours per week (range 8-48 hours). The average gestational age at birth in women with and without paid employment was 34 weeks (p 0.79). There were 21 women that delivered before 34 weeks, 17 (81%) of them had a paid employment. Work containing repetitive movements was associated with preterm birth (OR 3.0, 95% CI 1.3 to 6.6). Walking, standing, sitting down, bending over and reaching as well as strenuous work, the need for physical power and an uncomfortable posture were not associated with preterm birth. CONCLUSION: Women with multiple pregnancies and paid employment with heavy physical work strain appear not to be more at risk for preterm birth before 34 weeks of gestation. However, work containing repetitive movements is associated with preterm birth in multiple pregnancies.

10: Fetuin-mediated aggregation of amniotic fluid proteins into calcifying nanoparticles (CNP) and preterm premature rupture of membranes (PPROM)

10: Fetuin-mediated aggregation of amniotic fluid proteins into calcifying nanoparticles (CNP) and preterm premature rupture of membranes (PPROM)

New Approaches to Molecular Diagnostics of Prenatal Pathology

Analysis of the spectrum of amniotic fluid proteins in physiological and abnormal pregnancy using proteomic analysis allowed detection of a number of difference proteins, that are absent or, alternatively, appear in gestosis. Among absent proteins, there were NADPH-dependent carbonyl reductase, epidermal fatty acid-binding protein, haptoglobin, calgranulins A and B. In contrast to proteomic spectrum of amniotic fluid in physiological pregnancy, 7 new proteins appear during gestosis, 3 of them were identified: C area of immunoglobulin K-chain, breast cancer metastasis suppressor-1, and protein-1 containing AIG2-like domain. Possible effects of revealed differences in proteomic spectrum on development of main disturbances during gestosis are discussed. Difference proteins detected in amniotic fluid may serve as gestosis markers.

Verification of a biomarker discovery approach for detection of Down syndrome in amniotic fluid via multiplex selected reaction monitoring (SRM) assay

Prenatal screening test for Down syndrome (DS) can be improved by discovery of novel biomarkers. A multiplex selected reaction monitoring (SRM) assay was developed to test previously identified thirteen candidate proteins in amniotic fluid (AF). One unique peptide was selected for each protein based on discovery data, while three MS/MS transitions were selected based on intelligent SRM results. For one of the candidates, matrix metalloproteinase-2 (MMP2), ELISA was also performed to validate SRM results in AF and to test serum samples. Comparison of AF samples from DS versus controls via SRM assay revealed five proteins that were differentially expressed. Bile salt-activated lipase, mucin-13, carboxypeptidase A1, and dipeptidyl peptidase 4 showed a decrease in DS-affected AF, and MMP2 showed an increase, in comparison to controls (P<0.05). Discovery-based spectral counting ratios and SRM ratios showed a strong correlation, and MMP2 ELISA further confirmed the validity of the SRM data. Potential implications of differentially expressed proteins during fetal development are proposed. Our data also shows that SRM can provide a high-throughput and accurate platform for biomarker verification.

Chronic chorioamnionitis displays distinct alterations of the amniotic fluid proteome

Acute chorioamnionitis of infectious origin and chronic chorioamnionitis of immunological origin are two major placental lesions of spontaneous preterm birth with elevated amniotic fluid interleukin-6 and CXCL10 concentrations, respectively. The changes in the amniotic fluid proteome associated with intra-amniotic infection and acute chorioamnionitis are well defined, yet alterations unique to chronic chorioamnionitis remain to be elucidated. This study was conducted to determine those amniotic fluid proteins changing specifically in the presence of chronic chorioamnionitis. Amniotic fluid obtained from acute chorioamnionitis, chronic chorioamnionitis and gestational age-matched controls were analysed by two-dimensional (2D) difference in gel electrophoresis and MALDI-TOF analyses. The type of histological inflammation was used to define each condition in preterm labour cases (n = 125) and term not in labour cases (n = 22), and the amniotic fluid concentrations of interleukin-6, CXCL8, CXCL10 and prostaglandin F(2α) were also measured by specific immunoassays. Among preterm labour cases, 31 differentially expressed proteins were identified in chronic chorioamnionitis cases as compared to both acute chorioamnionitis and control cases. Importantly, glycodelin-A, which maintains maternal tolerance against an allogeneic fetus, was decreased in chronic chorioamnionitis, while haptoglobin was increased. We report the amniotic fluid proteome of chronic chorioamnionitis for the first time, and the findings herein strongly suggest that there is a pathophysiological association between the changes of immunomodulatory proteins in the amniotic fluid and chronic chorioamnionitis, a histological manifestation of maternal anti-fetal allograft rejection.

Different protein profile in amniotic fluid with nervous system malformations by surface‐enhanced laser desorption‐ionization/time‐of‐flight mass spectrometry (SELDI‐TOF‐MS) technology

To detect the distinct proteins in amniotic fluid (AF) between nervous system malformations fetuses and normal fetuses. Surface-enhanced laser desorption-ionization/time-of-flight mass spectrometry was used to characterize AF peptides in AF between nervous system malformations fetuses and normal fetuses. WCX2 protein chips were used to characterize AF peptides in AF. Protein chips were examined in a PBSIIC protein reader, the protein profiling was collected by ProteinChip software version 3.1 (Ciphergen Biosystems, Fremont, CA, USA) and analyzed by Biomarker Wizard software (Ciphergen Biosystems). Nine distinct proteins were identified in AF between nervous system malformations fetuses and normal fetuses. Compared with the control group, three proteins with m/z 4967.5 Da, 5258.0 Da, and 11,717.0 Da were down-regulated, and six proteins with m/z 2540.4 Da, 3107.1 Da, 3396.8 Da, 4590.965 Da, 5589.2 Da and 6429.4 Da up-regulated in nervous system malformations fetuses. The results suggest that there are distinct proteins in protein profiling of AF between nervous system malformations fetuses and normal fetuses.

IL‐6 Stimulates but is not essential for stratum corneum formation and permeability barrier development during gestation

The regulation of epidermal ontogenesis is a complex process. Previous studies have shown that cytokines (IL-1, TNFalpha and IL-6) regulate permeability barrier homeostasis in adult mice. Recently, we reported that IL-1 and TNFalpha accelerate stratum corneum (SC) formation and permeability barrier development in foetal rodents. Here, we determined whether IL-6 also regulates SC formation and permeability barrier development during late gestation. Using a rat skin explant model, we demonstrated that IL-6 accelerates permeability barrier formation in a time- and dose-dependent fashion. This acceleration of barrier formation is attributable to (a) accelerated lamellar membrane maturation, (b) formation of a multi-layer SC and (c) enhanced expression of epidermal differentiation markers. When comparing epidermis of IL-6-deficient (knockout mice) and wild-type foetal mice at days 16-18, we could not detect any abnormalities in either SC formation or the expression of differentiation markers in knockout (KO) mice. In parallel, the basal expression levels of IL-6 mRNA in epidermis and IL-6 protein in amniotic fluid were very low, with only a minimal change in IL-6 receptor mRNA levels in epidermis of days 16-22 foetal mice. These low IL-6 levels may account, at least in part, for the absence of epidermal abnormalities in IL-6 KO mice. In conclusion, exogenous IL-6 accelerates epidermal ontogenesis, but it is not essential for normal epidermal maturation.

Noninvasive diagnosis of intraamniotic infection: proteomic biomarkers in vaginal fluid

We analyzed the vaginal fluid proteome to identify biomarkers of intraamniotic infection among women in preterm labor. Proteome analysis was performed on vaginal fluid specimens from women with preterm labor, using multidimensional liquid chromatography, tandem mass spectrometry, and label-free quantification. Enzyme immunoassays were used to quantify candidate proteins. Classification accuracy for intraamniotic infection (positive amniotic fluid bacterial culture and/or interleukin-6 >2 ng/mL) was evaluated using receiver-operator characteristic curves obtained by logistic regression. Of 170 subjects, 30 (18%) had intraamniotic infection. Vaginal fluid proteome analysis revealed 338 unique proteins. Label-free quantification identified 15 proteins differentially expressed in intraamniotic infection, including acute-phase reactants, immune modulators, high-abundance amniotic fluid proteins and extracellular matrix-signaling factors; these findings were confirmed by enzyme immunoassay. A multi-analyte algorithm showed accurate classification of intraamniotic infection. Vaginal fluid proteome analyses identified proteins capable of discriminating between patients with and without intraamniotic infection.

Effects of pretreatment protocols on human amniotic fluid protein profiling with SELDI-TOF MS using protein chips and magnetic beads

There is increasing interest in the use of human amniotic fluid (AF) proteomics with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) for diagnosing pregnancy-associated abnormalities. A critical parameter of diagnostic biomarkers is the accuracy and reproducibility of protein patterns. We evaluated the effects of common pretreatment protocols on protein patterns generated using SELDI mass spectrometry with two different protein capture strategies (including functional protein chips and functionalized magnetic beads prior to MS analysis) in AF. Various extrinsic factors involved in processing and storing amniotic fluid, including matrix composition, sample storage time, temperature and freeze-thaw cycles, were analyzed regarding their impact on AF protein patterns using SELDI mass spectrometry with 2 different protein capture strategies. Three extrinsic factors (sample storage for 3days at either room temperature or 4 degrees C or freeze-thawing the sample 5 times) significantly decreased the number or intensities of protein peaks detected in AF. Matrix dilutions also changed the spectra of AF, with more peaks and higher intensities observed with 50% alpha-cyano-4-hydroxycinnamic acid (CHCA). Moreover, protein chips captured more proteins or peptides than magnetic beads on SELDI-TOF MS profiling of AF. These results suggest that extrinsic factors must be taken into account for valid data interpretation to ensure good reproducibility of AF profiling by SELDI mass spectrometry.