Impact of anesthetics on rat hippocampus and neocortex: A comprehensive proteomic study based on label-free mass spectrometry.

Anesthesia is regarded as an important milestone in medicine. However, the negative effect on memory and learning has been observed. In addition, the impact of anesthetics on postoperative cognitive functions is still discussed. In this work, in vivo experiment simulating a general anesthesia and ICU sedation was designed to assess the impact of two intravenous (midazolam, dexmedetomidine) and two inhalational (isoflurane, desflurane) agents on neuronal centers for cognition (neocortex), learning, and memory (hippocampus). More than 3600 proteins were quantified across both neocortex and hippocampus. Proteomic study revealed relatively mild effects of anesthetics, nevertheless, protein dysregulation uncovered possible different effect of isoflurane (and midazolam) compared to desflurane (and dexmedetomidine) to neocortical and hippocampal proteins. Isoflurane induced the upregulation of hippocampal NMDAR and other proteins of postsynaptic density and downregulation of GABA signaling, whereas desflurane and dexmedetomidine rather targeted mitochondrial VDAC isoforms and protein regulating apoptotic activity.

Comprehensive proteomic investigation of infectious and inflammatory changes in late preterm prelabour rupture of membranes.

Preterm prelabour rupture of membranes beyond the 34th week of gestation (late PPROM) is frequently associated with the risk of the microbial invasion of the amniotic fluid (MIAC) and histological chorioamnionitis (HCA). Hence, we employed a Tandem Mass Tag-based approach to uncover amniotic fluid proteome response to the presence of MIAC and HCA in late PPROM. Protein dysregulation was associated with only five cases in the group of 15 women with confirmed MIAC and HCA. Altogether, 138 amniotic fluid proteins were changed in these five cases exclusively. These proteins were particularly associated with excessive neutrophil responses to infection, such as neutrophil degranulation and extracellular trap formation. We believe that the quantification of these proteins in amniotic fluid may assist in revealing women with the highest risk of excessive inflammatory response in late PPROM.

Mid-trimester amniotic fluid proteome's association with spontaneous preterm delivery and gestational duration.

BACKGROUND: Amniotic fluid is clinically accessible via amniocentesis and its protein composition may correspond to birth timing. Early changes in the amniotic fluid proteome could therefore be associated with the subsequent development of spontaneous preterm delivery. OBJECTIVE: The main objective of this study was to perform unbiased proteomics analysis of the association between mid-trimester amniotic fluid proteome and spontaneous preterm delivery and gestational duration, respectively. A secondary objective was to validate and replicate the findings by enzyme-linked immunosorbent assay using a second independent cohort. METHODS: Women undergoing a mid-trimester genetic amniocentesis at Sahlgrenska University Hospital/Östra between September 2008 and September 2011 were enrolled in this study, designed in three analytical stages; 1) an unbiased proteomic discovery phase using LC-MS analysis of 22 women with subsequent spontaneous preterm delivery (cases) and 37 women who delivered at term (controls), 2) a validation phase of proteins of interest identified in stage 1, and 3) a replication phase of the proteins that passed validation using a second independent cohort consisting of 20 cases and 40 matched controls. RESULTS: Nine proteins were nominally significantly associated with both spontaneous preterm delivery and gestational duration, after adjustment for gestational age at sampling, but none of the proteins were significant after correction for multiple testing. Several of these proteins have previously been described as being associated with spontaneous PTD etiology and six of them were thus validated using enzyme linked immunosorbent assay. Two of the proteins passed validation; Neutrophil gelatinase-associated lipocalin and plasminogen activator inhibitor 1, but the results could not be replicated in a second cohort. CONCLUSIONS: Neutrophil gelatinase-associated lipocalin and Plasminogen activator inhibitor 1 are potential biomarkers of spontaneous preterm delivery and gestational duration but the findings could not be replicated. The negative findings are supported by the fact that none of the nine proteins from the exploratory phase were significant after correction for multiple testing.

Retraction Note to: cGMP-phosphodiesterase 6, transducin and Wnt5a/Frizzled-2-signaling control cGMP and Ca2+ homeostasis in melanoma cells.

The Editor-in-Chief has retracted this article [1] due to errors in Figs. 1b, c and 4.

Quantification of cellular protein and redox imbalance using SILAC-iodoTMT methodology.

Under normal conditions, the cellular redox status is maintained in a steady state by reduction and oxidation processes. These redox alterations in the cell are mainly sensed by protein thiol residues of cysteines thus regulating protein function. The imbalance in redox homeostasis may therefore regulate protein turnover either directly by redox modulating of transcription factors or indirectly by the degradation of damaged proteins due to oxidation. A new analytical method capable of simultaneously assessing cellular protein expression and cysteine oxidation would provide a valuable tool for the field of cysteine-targeted biology. Here, we show a workflow based on protein quantification using metabolic labeling and determination of cysteine oxidation using reporter ion quantification. We applied this approach to determine protein and redox changes in cells after 5-min, 60-min and 32-h exposure to H2O2, respectively. Based on the functional analysis of our data, we confirmed a biological relevance of this approach and its applicability for parallel mapping of cellular proteomes and redoxomes under diverse conditions. In addition, we revealed a specific pattern of redox changes in peroxiredoxins in a short time-interval cell exposure to H2O2. Overall, our present study offers an innovative, versatile experimental approach to the multifaceted assessment of cellular proteome and its redox status, with broad implications for biomedical research towards a better understanding of organismal physiology and diverse disease conditions.

Interferon-regulated suprabasin is essential for stress-induced stem-like cell conversion and therapy resistance of human malignancies.

Radiation and chemotherapy represent standard-of-care cancer treatments. However, most patients eventually experience tumour recurrence, treatment failure and metastatic dissemination with fatal consequences. To elucidate the molecular mechanisms of resistance to radio- and chemotherapy, we exposed human cancer cell lines (HeLa, MCF-7 and DU145) to clinically relevant doses of 5-azacytidine or ionizing radiation and compared the transcript profiles of all surviving cell subpopulations, including low-adherent stem-like cells. Stress-mobilized low-adherent cell fractions differed from other survivors in terms of deregulation of hundreds of genes, including those involved in interferon response. Exposure of cancer cells to interferon-gamma but not interferon-beta resulted in the development of a heterogeneous, low-adherent fraction comprising not only apoptotic/necrotic cells but also live cells exhibiting active Notch signalling and expressing stem-cell markers. Chemical inhibition of mitogen-activated protein kinase/ERK kinase (MEK) or siRNA-mediated knockdown of extracellular signal-regulated kinase 1/2 (Erk1/2) and interferon responsible factor 1 (IRF1) prevented mobilization of the surviving low-adherent population, indicating that interferon-gamma-mediated loss of adhesion and anoikis resistance required an active Erk pathway interlinked with interferon signalling by transcription factor IRF1. Notably, a skin-specific protein suprabasin (SBSN), a recently identified oncoprotein, was among the top scoring genes upregulated in surviving low-adherent cancer cells induced by 5-azacytidine or irradiation. SBSN expression required the activity of the MEK/Erk pathway, and siRNA-mediated knockdown of SBSN suppressed the low-adherent fraction in irradiated, interferon-gamma- and 5-azacytidine-treated cells, respectively, implicating SBSN in genotoxic stress-induced phenotypic plasticity and stress resistance. Importantly, SBSN expression was observed in human clinical specimens of colon and ovarian carcinomas, as well as in circulating tumour cells and metastases of the 4T1 mouse model. The association of SBSN expression with progressive stages of cancer development indicates its role in cancer evolution and therapy resistance.

Conventional-Flow Liquid Chromatography-Mass Spectrometry for Exploratory Bottom-Up Proteomic Analyses.

Due to its sensitivity and productivity, bottom-up proteomics based on liquid chromatography-mass spectrometry (LC-MS) has become the core approach in the field. The de facto standard LC-MS platform for proteomics operates at sub-µL/min flow rates, and nanospray is required for efficiently introducing peptides into a mass spectrometer. Although this is almost a "dogma", this view is being reconsidered in light of developments in highly efficient chromatographic columns, and especially with the introduction of exceptionally sensitive MS instruments. Although conventional-flow LC-MS platforms have recently penetrated targeted proteomics successfully, their possibilities in discovery-oriented proteomics have not yet been thoroughly explored. Our objective was to determine what are the extra costs and what optimization and adjustments to a conventional-flow LC-MS system must be undertaken to identify a comparable number of proteins as can be identified on a nanoLC-MS system. We demonstrate that the amount of a complex tryptic digest needed for comparable proteome coverage can be roughly 5-fold greater, providing the column dimensions are properly chosen, extra-column peak dispersion is minimized, column temperature and flow rate are set to levels appropriate for peptide separation, and the composition of mobile phases is fine-tuned. Indeed, we identified 2 835 proteins from 2 µg of HeLa cells tryptic digest separated during a 60 min gradient at 68 µL/min on a 1.0 mm × 250 mm column held at 55 °C and using an aqua-acetonitrile mobile phases containing 0.1% formic acid, 0.4% acetic acid, and 3% dimethyl sulfoxide. Our results document that conventional-flow LC-MS is an attractive alternative for bottom-up exploratory proteomics.

Plasma concentration of fibronectin is decreased in patients with hypertrophic cardiomyopathy.

OBJECTIVES: To confirm the initial iTRAQ-based proteomic findings related to the plasma fibronectin level and hypertrophic cardiomyopathy using an antibody-based assay. METHODS: The iTRAQ technique was used for the discovery proteomic analysis of the pooled plasma samples. The ELISA was used for verification of fibronectin plasma concentration in individual samples. Additional five related plasma proteins were assessed: matrix metalloproteinase 2, matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 1, tissue inhibitor of metalloproteinase 2 and brain natriuretic peptide. RESULTS: The plasma fibronectin level in patients with hypertrophic cardiomyopathy was significantly lower in comparison to the healthy subjects [215.5±47.3µg/ml, n=17 vs. 376.7±134.8µg/ml, n=17; p<0.0001]. CONCLUSION: In this study we present and confirm our initial proteomic findings and we suggest fibronectin as a potential indicator of an extracellular matrix remodeling related to the hypertrophic cardiomyopathy.

Proteomic analysis of cerebrospinal fluid for relapsing-remitting multiple sclerosis and clinically isolated syndrome.

Early diagnosis and treatment of multiple sclerosis (MS) in the initial stages of the disease can significantly retard its progression. The aim of the present study was to identify changes in the cerebrospinal fluid proteome in patients with relapsing-remitting MS and clinically isolated MS syndrome who are at high risk of developing MS (case group) compared to healthy population (control) in order to identify potential new markers, which could ultimately aid in early diagnosis of MS. The protein concentrations of each of the 11 case and 15 control samples were determined using a bicinchoninic acid assay. Nanoscale liquid chromatography coupled with tandem mass spectrometry was used for protein identification. Proteomics data were processed using the Perseus software suite and R. The results were filtered using the Benjamini-Hochberg procedure for the false discovery rate (FDR) correction (FDR<0.05). The results showed that, 26 proteins were significantly dysregulated in case samples compared to the controls. Nine proteins were found to be significantly less abundant in case samples, while the abundance of 17 proteins was significantly increased in case samples compared to controls. Three of the proteins were previously linked to RR MS, including immunoglobulin (Ig) γ-1 chain C region, Ig heavy chain V-III region BRO and Ig κ chain C region. Three proteins that were uniquely expressed in patients with RR MS were identified and these proteins may serve as prognostic biomarkers for identifying patients with a high risk of developing RR MS.

Proteomic Analysis of Early Mid-Trimester Amniotic Fluid Does Not Predict Spontaneous Preterm Delivery.

OBJECTIVE: The aim of this study was to identify early proteomic biomarkers of spontaneous preterm delivery (PTD) in mid-trimester amniotic fluid from asymptomatic women. METHODS: This is a case-cohort study. Amniotic fluid from mid-trimester genetic amniocentesis (14-19 weeks of gestation) was collected from 2008 to 2011. The analysis was conducted in 24 healthy women with subsequent spontaneous PTD (cases) and 40 randomly selected healthy women delivering at term (controls). An exploratory phase with proteomics analysis of pooled samples was followed by a verification phase with ELISA of individual case and control samples. RESULTS: The median (interquartile range (IQR: 25th; 75th percentiles) gestational age at delivery was 35+5 (33+6-36+6) weeks in women with spontaneous PTD and 40+0 (39+1-40+5) weeks in women who delivered at term. In the exploratory phase, the most pronounced differences were found in C-reactive protein (CRP) levels, that were approximately two-fold higher in the pooled case samples than in the pooled control samples. However, we could not verify these differences with ELISA. The median (25th; 75th IQR) CRP level was 95.2 ng/mL (64.3; 163.5) in women with spontaneous PTD and 86.0 ng/mL (51.2; 145.8) in women delivering at term (p = 0.37; t-test). CONCLUSIONS: Proteomic analysis with mass spectrometry of mid-trimester amniotic fluid suggests CRP as a potential marker of spontaneous preterm delivery, but this prognostic potential was not verified with ELISA.

Microbial invasion and histological chorioamnionitis upregulate neutrophil-gelatinase associated lipocalin in preterm prelabor rupture of membranes.

Our recent exploratory proteomic study suggested increased levels of neutrophil-gelatinase associated lipocalin (P80188, NGAL_HUMAN) due to microbial invasion of the amniotic cavity (MIAC) and histological chorioamnionitis (HCA) in women with preterm prelabor rupture of the membranes. In this study, we verified the proteomics findings by assessing the amniotic fluid NGAL by ELISA in the original exploratory cohort. The NGAL level was significantly higher in women positive for both MIAC and HCA compared to women with both conditions ruled out (median 75.1 ng/ml versus 27.9 ng/ml; p < 0.0001). For independent validation and to assess NGALs potential to stratify women positive for both MIAC and HCA from women in whom at least one of these conditions was absent, we subsequently designed a retrospective replication cohort. Significantly higher NGAL levels were found in women positive for both MIAC and HCA (median 65.9 ng/ml versus 34.2 ng/ml; p = 0.0061). Significantly higher levels of NGAL were confirmed only in strata below 32 weeks of gestation. Based on the observed likelihood ratio, the best predictive cutoff level (47.1 ng/ml) was evaluated in both cohorts. Data from the verification cohort implied that NGAL is a valuable clinical marker for revealing MIAC leading to HCA; however, this potential was not replicated in the replication cohort.

Proteomic investigation of embryonic rat heart-derived H9c2 cell line sheds new light on the molecular phenotype of the popular cell model.

Due to their cardiac origin, H9c2 cells rank among the most popular cell lines in current cardiovascular research, yet molecular phenotype remains elusive. Hence, in this study we used proteomic approach to describe molecular phenotype of H9c2 cells in their undifferentiated (i.e., most frequently used) state, and its functional response to cardiotoxic drug doxorubicin. Of 1671 proteins identified by iTRAQ IEF/LC-MSMS analysis, only 12 proteins were characteristic for striated muscle cells and none was cardiac phenotype-specific. Targeted LC-SRM and western blot analyses confirmed that undifferentiated H9c2 cells are phenotypically considerably different to both primary neonatal cardiomyocytes and adult myocardium. These cells lack proteins essential for formation of striated muscle myofibrils or they express only minor amounts thereof. They also fail to express many proteins important for metabolism of muscle cells. The challenge with clinically relevant concentrations of doxorubicin did not induce a proteomic signature that has been previously noted in primary cardiomyocytes or adult hearts. Instead, several alterations previously described in other cells of mesodermal origin, such as fibroblasts, were observed (e.g., severe down-regulation of collagen synthesis pathway). In conclusion, the molecular phenotype of H9c2 cells resembles very immature myogenic cells with skeletal muscle commitment upon differentiation and thus, translatability of findings obtained in these cells deserves caution.

Potential Peripartum Markers of Infectious-Inflammatory Complications in Spontaneous Preterm Birth.

Spontaneous preterm birth significantly contributes to the overall neonatal morbidity associated with preterm deliveries. Nearly 50% of cases are associated with microbial invasion of the amniotic cavity followed by an inflammatory response. Robust diagnostic tools for neonates jeopardized by infection and inflammation may thus decrease the overall neonatal morbidity substantially. Amniotic fluid retrieved during labor retains fetal and pregnancy-related protein fingerprint and its sampling does not place any unwanted stress on women. Using exploratory and targeted methods we analyzed proteomes of amniotic fluid sampled at the end of spontaneous preterm labor prior to delivery from women with and without infection and inflammation. Exploratory data indicated several amniotic fluid proteins to be associated with infectious-inflammatory complications in spontaneous preterm birth. LC-SRM analysis subsequently verified statistically significant changes in lipocalin-1 (P = 0.047 and AUC = 0.67, P = 0.046), glycodelin (P = 0.013 and AUC = 0.73, P = 0.013), and nicotinamide phosphoribosyltransferase (P = 0.018 and AUC = 0.71, P = 0.01).

Changes in proteome of the Δhfq strain derived from Francisella tularensis LVS correspond with its attenuated phenotype.

The posttranscriptional regulatory protein Hfq was shown to be an important determinant of the stress resistance and full virulence in the dangerous human pathogen Francisella tularensis. Transcriptomics brought rather limited clues to the precise contribution of Hfq in virulence. To reveal the molecular basis of the attenuation caused by hfq inactivation, we employed iTRAQ in the present study and compared proteomes of the parent and isogenic Δhfq strains. We show that Hfq modulates the level of 76 proteins. Most of them show decreased abundance in the ∆hfq mutant, thereby indicating that Hfq widely acts rather as a positive regulator of Francisella gene expression. Several key Francisella virulence factors including those encoded within the Francisella pathogenicity island were found among the downregulated proteins, which is in a good agreement with the attenuated phenotype of the Δhfq strain. To further validate the iTRAQ exploratory findings, we subsequently performed targeted LC-SRM analysis of selected proteins. This accurate quantification method corroborated the trends found in the iTRAQ data.

Proteomic biomarkers for spontaneous preterm birth: a systematic review of the literature.

This review aimed to identify, synthesize, and analyze the findings of studies on proteomic biomarkers for spontaneous preterm birth (PTB). Three electronic databases (Medline, Embase, and Scopus) were searched for studies in any language reporting the use of proteomic biomarkers for PTB published between January 1994 and December 2012. Retrieved citations were screened, and relevant studies were selected for full-text reading, in triplicate. The search yielded 529 citations, 51 were selected for full-text reading and 8 studies were included in the review. A total of 64 dysregulated proteins were reported. Only 14-3-3 protein sigma, annexin A5, protein S100-A8, protein S100-A12, and inter-α-trypsin inhibitor heavy chain H4 were reported in more than 1 study, but results could not be combined due to heterogeneity in type of sample and analytical platform. In conclusion, according to the existing literature, there are no specific proteomic biomarkers capable of accurately predicting PTB.

Preterm prelabor rupture of membranes (PPROM) is not associated with presence of viral genomes in the amniotic fluid.

BACKGROUND: The role of viral infections in preterm prelabor rupture of the membranes (PPROM) is not established. Studies on the presence of viral genomes in the amniotic fluid (AF) collected in pregnancies complicated by PPROM show contradictory outcomes. OBJECTIVES: To investigate AF samples of PPROM pregnancies for the presence of viral genomes. STUDY DESIGN: AF samples from patients with PPROM were collected during a 4-year (2008-2012) observational study. 174 women were included with selection criteria of singleton pregnancy, PPROM, and maternal age of 18 years and above. PCR was used for detection of human cytomegalovirus (HCMV), herpes simplex virus (HSV), parvovirus B19, human adenoviruses (HAdV), enteroviruses (EV) and human parechovirus (HPeV). The selection of these viral targets was based on literature regarding screening of AF for presence of viral genomes. RESULTS: Only a single sample was positive out of the 174 tested AFs, HCMV DNA was detected. CONCLUSIONS: PPROM is not associated with active viral infections.

S-nitrosoglutathione covalently modifies cysteine residues of human carbonyl reductase 1 and affects its activity.

Carbonyl reductase 1 (CBR1 or SDR21C1) is a ubiquitously-expressed, cytosolic, monomeric, and NADPH-dependent enzyme. CBR1 participates in apoptosis, carcinogenesis and drug resistance, and has a protective role in oxidative stress, cancer and neurodegeneration. S-Nitrosoglutathione (GSNO) represents the newest addition to its diverse substrate spectrum, which includes a wide range of xenobiotics and endogenous substances. GSNO has also been shown to covalently modify and inhibit CBR1. The aim of the present study was to quantify and characterize the resulting modifications. Of five candidate cysteines for modification by 2 mM GSNO (positions 26, 122, 150, 226, 227), the last four were analyzed using MALDI-TOF/TOF mass spectrometry and then quantified using the Selected Reaction Monitoring Approach on hyphenated HPLC with a triple quadrupole mass spectrometer. The analysis confirmed GSNO concentration-dependent S-glutathionylation of cysteines at positions 122, 150, 226, 227 which was 2-700 times higher compared to wild-type CBR1 (WT-CBR1). Moreover, a disulfide bond between neighboring Cys-226 and Cys-227 was detected. We suggest a role of these two cysteines as a redox-sensitive cysteine pair. The catalytic properties of wild-type and enzyme modified with 2 mM GSNO were also investigated by steady state kinetic experiments with various substrates. GSNO treatment of CBR1 resulted in a 2-5-fold decrease in kcat with menadione, 4-benzoylpyridine, 2,3-hexanedione, daunorubicin and 1,4-naphthoquinone. In contrast, the same treatment increased kcat for substrates containing a 1,2-diketo group in a ring structure (1,2-naphthoquinone, 9,10-phenanthrenequinone, isatin). Except for 9,10-phenanthrenequinone, all changes in kcat were at least in part compensated for by a similar change in Km, overall yielding no drastic changes in catalytic efficiency. The findings indicate that GSNO-induced covalent modification of cysteine residues affects the kinetic mechanism of CBR1 both in terms of substrate binding and turnover rate, probably by covalent modification of Cys-226 and/or Cys-227.

Amniotic fluid myeloperoxidase in pregnancies complicated by preterm prelabor rupture of membranes.

OBJECTIVE: To determine amniotic fluid myeloperoxidase concentration in women with preterm prelabor rupture of the membranes with microbial invasion of the amniotic cavity and histological chorioamnionitis. METHODS: One hundred eighty-one women with singleton pregnancies with a gestational age between 24+0 and 36+6 weeks were included in this study. Amniocenteses were performed, and myeloperoxidase concentration in the amniotic fluid was determined using ELISA. RESULT: Women with microbial invasion of the amniotic cavity had higher median myeloperoxidase concentration than women without this condition (149.2 ng/mL vs. 54.6 ng/mL; p = 0.0006). Women with the presence of histological chorioamnionitis had higher median myeloperoxidase concentration than women without histological chorioamnionitis (103.7 ng/mL vs. 50.0 ng/mL; p = 0.0001). The presence of both microbial invasion of the amniotic cavity and histological chorioamnionitis was associated with higher median myeloperoxidase concentration (456.0 ng/mL vs. 52.9 ng/mL; p < 0.0001). The results remained significant after adjusting for gestational age. CONCLUSIONS: Increased amniotic fluid myeloperoxidase in microbial invasion of the amniotic cavity and histological chorioamnionitis confirm a role of myeloperoxidase in preterm prelabor rupture of the membranes pathophysiology.

Amniotic fluid cathelicidin in PPROM pregnancies: from proteomic discovery to assessing its potential in inflammatory complications diagnosis.

BACKGROUND: Preterm 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 FINDINGS: We 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. CONCLUSIONS: Our 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.