Diagnosis of intra-amniotic infection by proteomic profiling and identification of novel biomarkers.
BACKGROUND
Intra-amniotic infection (IAI) is commonly associated with preterm birth and adverse neonatal sequelae. Early diagnosis of IAI, however, has been hindered by insensitive or nonspecific tests.


OBJECTIVE
To identify unique protein signatures in rhesus monkeys with experimental IAI, a proteomics-based analysis of amniotic fluid was used to develop diagnostic biomarkers for subclinical IAI in amniotic fluid and blood of women with preterm labor.


METHODS
Surface-enhanced laser desorption-ionization/time-of-flight mass spectrometry, gel electrophoresis, and tandem mass spectrometry were used to characterize amniotic fluid peptides in 19 chronically instrumented pregnant rhesus monkeys before and after experimental IAI. Candidate biomarkers were determined by liquid chromatography-tandem mass spectrometry. Polyclonal antibodies were generated from synthetic peptides for validation of biomarkers of IAI. Amniotic fluid peptide profiles identified in experimental IAI were subsequently tested in a cohort of 33 women admitted to Seattle, Wash, hospitals between June 25, 1991, and June 30, 1997, with preterm delivery at 35 weeks or earlier associated with subclinical IAI (n = 11), preterm delivery at 35 weeks or earlier without IAI (n = 11), and preterm contractions with subsequent term delivery at later than 35 weeks (n = 11).


METHODS
Identification of peptide biomarkers for occult IAI.


RESULTS
Protein expression profiles in amniotic fluid showed unique signatures of overexpression of polypeptides in the 3- to 5-kDa and 10- to 12-kDa molecular weight ranges in all animals after infection and in no animal prior to infection. In women, the 10- to 12-kDa signature was identified in all 11 patients with subclinical IAI, in 2 of 11 with preterm delivery without IAI, and in 0 of 11 with preterm labor and term delivery without infection (P<.001). Peptide fragment analysis of the diagnostic peak in amniotic fluid identified calgranulin B and a unique fragment of insulinlike growth factor binding protein 1, which were also expressed in maternal serum. Mapping of other amniotic fluid proteins differentially expressed in IAI identified several immunoregulators not previously described in amniotic fluid.


CONCLUSIONS
This proteomics-based characterization of the differential expression of amniotic fluid proteins in IAI identified a distinct proteomic profile in an experimental primate chorioamnionitis model that detected subclinical IAI in a human cohort with preterm labor. These diagnostic protein expression signatures, complemented by immunodetection of specific biomarkers in amniotic fluid and in maternal serum, might have application in the early detection of IAI.
Division of Reproductive Sciences, Oregon National Primate Research Center, Beaverton, Ore, USA. gravettm@oshu.edu
Michael G
MG
Gravett
Miles J
MJ
Novy
Ron G
RG
Rosenfeld
Ashok P
AP
Reddy
Thomas
T
Jacob
Mark
M
Turner
Ashley
A
McCormack
Jodi A
JA
Lapidus
Jane
J
Hitti
David A
DA
Eschenbach
Charles T
CT
Roberts
Srinivasa R
SR
Nagalla
D000328:Adult; D000653:Amniotic Fluid / Q000737:chemistry* / Q000382:microbiology; D000818:Animals; D015415:Biomarkers / Q000032:analysis*; D015153:Blotting, Western; D040502:Calgranulin B / Q000378:metabolism; D002821:Chorioamnionitis / Q000378:metabolism* / Q000382:microbiology; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007239:Infections / Q000378:metabolism; D018970:Insulin-Like Growth Factor Binding Protein 1; D018969:Insulin-Like Growth Factor Binding Proteins / Q000378:metabolism; D008253:Macaca mulatta; D007752:Obstetric Labor, Premature / Q000378:metabolism*; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000378:metabolism; D020543:Proteome / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D015415:Biomarkers; D040502:Calgranulin B; C032231:IGFBP1 protein, human; D018970:Insulin-Like Growth Factor Binding Protein 1; D018969:Insulin-Like Growth Factor Binding Proteins; D011257:Pregnancy Proteins; D020543:Proteome