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)

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--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.Univ Hosp Hradec Kralove, Biomed Res Ctr, Hradec Kralove, Czech RepublicCharles Univ Prague, Fac Med Hradec Kralove, Dept Obstet & Gynecol, Hradec Kralove, Czech RepublicUniv Def, Fac Mil Hlth Sci, Inst Mol Pathol, Hradec Kralove, Czech RepublicHosp Pardubice, Dept Obstet & Gynecol, Pardubice, Czech RepublicOdense Univ Hosp, Dept Obstet & Gynecol, Svendborg, DenmarkUniversidade Federal de São Paulo, Dept Internal Med, São Paulo, BrazilUniv Texas Med Branch, Dept Obstet & Gynecol, Div Maternal Fetal Med & Perinatal Res, Galveston, TX 77755 USAUniversidade Federal de São Paulo, Dept Internal Med, São Paulo, BrazilWeb of Scienc

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

Volcano plots constructed from iTRAQ quantification data.

<p>The volcano plots show how much and how significantly proteins identified in the exploratory phase of the study using representative pooled samples were altered due to the presence of both MIAC and HCA. The top five proteins with the most profound change are highlighted by their abbreviated names (H4 - histone H4; H32 - histone H3.2; H2B1L - histone H2B type 1-L; CAMP – cathelicidine; PERM – myeloperoxidase). Proteins that were found significantly changed (<i>p</i><0.01) in both replicates are coded in red.</p

Demographic and clinical characteristics of women and newborns involved in the exploratory cohort.

<p>Demographic and clinical characteristics of women and newborns involved in the exploratory cohort.</p

Prediction potential of amniotic fluid cathelicidin cut-off level >4.0 ng/ml for the presence of both MIAC and HCA in PPROM pregnancies.

<p>Prediction potential of amniotic fluid cathelicidin cut-off level >4.0 ng/ml for the presence of both MIAC and HCA in PPROM pregnancies.</p

Demographic and clinical characteristics of the women and newborns involved in the replication cohort.

<p>Demographic and clinical characteristics of the women and newborns involved in the replication cohort.</p

Spojení proteomu amniotické tekutiny v polovině trimestru se spontánním předčasným porodem a délkou těhotenství

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/Ostra 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.Dosavadní výzkum Plodová voda je klinicky přístupná prostřednictvím amniocentézy a její proteinové složení může odpovídat načasování porodu. Časné změny v proteomu plodové vody by proto mohly být spojeny s následným vývojem spontánního předčasného porodu. Cíl Hlavním cílem této studie bylo provést objektivní proteomickou analýzu asociace mezi proteomem plodové vody ve středním trimestru a spontánním předčasným porodem, respektive gestační dobou. Sekundárním cílem bylo ověřit a replikovat nálezy pomocí enzymového imunosorbentního testu s použitím druhé nezávislé kohorty. Metody Do této studie, která byla koncipována ve třech analytických fázích, byly zařazeny ženy podstupující genetickou amniocentézu v polovině trimestru ve Fakultní nemocnici Sahlgrenska / Ostra v období od září 2008 do září 2011; 1) nezaujatá fáze proteomického objevu pomocí LC-MS analýzy 22 žen s následným spontánním předčasným porodem (případy) a 37 žen, které porodily v termínu (kontroly), 2) ověřovací fáze sledovaných proteinů identifikovaných ve stadiu 1 a 3 ) replikační fáze proteinů, která prošla validací pomocí druhé nezávislé kohorty skládající se z 20 případů a 40 shodných kontrol. Výsledky Devět proteinů bylo nominálně významně spojeno jak se spontánním předčasným porodem, tak s gestačním trváním po úpravě na gestační věk při odběru vzorků, ale žádný z proteinů nebyl významný po korekci na vícenásobné testování. Některé z těchto proteinů byly dříve popsány jako asociované se spontánní etiologií PTD a šest z nich bylo tedy validováno pomocí enzymového imunosorbentního testu. Dva z proteinů prošly validací; Lipokalin spojený s neutrofilní gelatinázou a inhibitor aktivátoru plazminogenu 1, ale výsledky nemohly být replikovány ve druhé kohortě. Závěry Lipokalin spojený s neutrofilní gelatinázou a inhibitor aktivátoru plazminogenu 1 jsou potenciálními biomarkery spontánního předčasného porodu a gestačního trvání, ale nálezy nelze replikovat. Negativní zjištění podporuje skutečnost, že žádný z devíti proteinů z průzkumné fáze nebyl významný po korekci na vícenásobné testování

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