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Gravett MG, Novy MJ, Rosenfeld RG, et al. Diagnosis of Intra-amniotic Infection by Proteomic Profiling and Identification of Novel Biomarkers. JAMA. 2004;292(4):462–469. doi:10.1001/jama.292.4.462
Author Affiliations: Division of Reproductive Sciences, Oregon National Primate Research Center, Beaverton, Ore (Drs Gravett and Novy); Departments of Obstetrics and Gynecology (Dr Gravett) and Pediatrics (Drs Rosenfeld, Roberts, and Nagalla) and Division of Biostatistics, Public Health and Preventive Medicine (Dr Lapidus), Oregon Health and Science University, and ProteoGenix Inc (Drs Reddy, Jacob, and McCormack and Mr Turner), Portland, Ore; Lucile Packard Foundation for Children's Health, Palo Alto, Calif (Dr Rosenfeld); Department of Obstetrics and Gynecology, University of Washington (Drs Hitti and Eschenbach), Seattle.
Context 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
Design, Setting, and Participants 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).
Main Outcome Measures 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
Despite improvements in prenatal care, preterm birth occurs in 11.8%
of births in the United States and remains the major obstetrical problem in
developed countries.1 Recently, intra-amniotic
infection (IAI) has been implicated as a major cause of preterm birth. Intra-amniotic
infections cause more than 50% of very low-birth-weight neonates born before
30 weeks of gestation and account for the highest number of neonatal deaths,
the most serious complications, and a disproportionate share of aggregate
perinatal health care costs (estimated in the United States to be $4 billion
to $6 billion annually).2,3
Current evidence indicates that if a microorganism causes preterm birth,
it usually does so by infection of the chorioamnion and the amniotic fluid
or fetus. Infection of the amniotic fluid or the fetal membranes (ie, chorioamnionitis)
may be acute or chronic depending on specific virulence factors, the microbial
inoculum size, and host defense mechanisms; thus, the timing and severity
of the clinical presentation vary widely. Most women in preterm labor with
intact membranes and a positive amniotic fluid microbial culture are refractory
to standard tocolytic therapy and rapidly deliver compared with women in preterm
labor with sterile amniotic fluid.4 Moreover,
antibiotic therapy has not prevented preterm delivery in most studies, possibly
because the patient subgroup with early IAI that might benefit from antibiotic
treatment is identified too late or not at all.3,5 Improved
diagnostic methods are needed to identify women who may benefit from specific
interventions, such as antibiotics or anti-inflammatory agents.6
Recent advances in proteomics present a new opportunity to examine the
global expression of proteins in tissues and fluids. The proteins or peptides
that are preferentially expressed and identified in a disease or pathologic
state are well suited for the development of convenient, rapid, sensitive,
and specific diagnostic assays. The objective of this study was to discover
novel biomarkers for subclinical or occult IAI by proteomic profiling methods.
We used surface-enhanced laser desorption-ionization/time-of-flight (SELDI-TOF)
mass spectrometry, gel electrophoresis, and tandem mass spectrometry to characterize
amniotic fluid peptides in pregnant rhesus monkeys with experimental IAI.
The proteome profile that was identified in infected monkeys was then tested
for its ability to detect subclinical IAI among women in preterm labor.
The protocol was approved by the Institutional Animal Care and Utilization
Committee of the Oregon National Primate Research Center and followed humane
animal care guidelines. Nineteen pregnant rhesus monkeys (Macaca mulatta) with timed gestations were chronically catheterized
and maintained postoperatively, as previously described.7 Each
animal served as its own control for proteomic analysis by serially sampling
blood and amniotic fluid before and after infection. Experimental IAI was
established between days 126 and 138 of gestation (term is 167 days) by intra-amniotic
inoculation of (1) 106 colony-forming units (CFU) of group B Streptococcus, type III, suspended in saline (n = 7); (2)
107 CFU of Ureaplasma parvum (formerly Ureaplasma urealyticum, serovar 1) suspended in 10B media
(n = 6); or (3) 105 to 107 CFU of Mycoplasma hominis (n = 6) suspended in phosphate-buffered saline with
10% sucrose and 2% endotoxin-free fetal calf serum.
The group B Streptococcus isolate was originally
recovered from a neonate with meningitis. The U parvum was
from a low-passaged clinical isolate from placenta and membranes of a patient
with chorioamnionitis and whose neonate had sepsis. The M hominis was an endometrial isolate from a patient with postpartum
endomyometritis. Amniotic fluid samples were collected serially from all animals
during the study period for quantitative microbial cultures, white blood cell
analysis, cytokine and prostaglandin measurements (as previously reported7-9), and proteomic analysis.
Uterine activity was expressed as the hourly contraction area in millimeters
of mercury × seconds per hour. After delivery, usually cesarean delivery,
fetal, decidual, placental, and intermembrane bacterial cultures were obtained
to confirm infection. Histopathologic studies were performed to document chorioamnionitis.
The study population was drawn from 309 women admitted in premature
labor at 22 to 34 weeks of gestation with intact fetal membranes to the University
of Washington Medical Center or associated hospitals in Seattle between June
25, 1991, and June 30, 1997, as previously reported.10 These
women represented 27% of eligible women and did not differ from nonparticipants
in maternal age, race, parity, gravidity, prior preterm delivery, or gestational
age at delivery. Informed consent was obtained and the protocol approved by
the respective institutional review boards.
Transabdominal amniocentesis was performed and maternal venous blood
samples were collected by venipuncture in all study participants at the time
of enrollment. Amniotic fluid microbial cultures were performed for facultative
and anaerobic bacteria and for genital mycoplasma (M hominis, U parvum), as previously described.10 Preterm labor was defined as regular uterine contractions
at a frequency of 10 minutes or less with either documented cervical change
or a cervical dilatation of more than 1 cm or effacement greater than 50%.
Subclinical IAI was defined by a positive amniotic fluid microbial culture
and/or an amniotic fluid interleukin 6 (IL-6) concentration greater than 2
ng/mL, histologic evidence for chorioamnionitis (defined as presence of ≥10
polymorphonuclear leukocytes per 400× field in 10 nonadjacent fields11 and absence of uterine tenderness or fever). We have
previously reported that an amniotic fluid IL-6 concentration of more than
2 ng/mL was at or above the 75th percentile for the entire study population
and was associated with the detection of bacteria by polymerase chain reaction.12 Women with cervical dilatation of more than 4 cm
or ruptured membranes at admission were excluded.
A subset of patients was identified from this study population for proteomic
analysis as reported herein. This subset included 3 groups of 11 patients
each. Group 1 included all patients with evidence of subclinical IAI as defined
herein for whom amniotic fluid and maternal serum was available; group 2 was
a randomly selected subset of patients without documented intrauterine infection
but with preterm birth at 35 weeks of gestation or earlier; and group 3 consisted
of randomly selected patients without infection and with preterm labor responsive
to tocolytic therapy who subsequently delivered near term (>35 weeks of gestation).
Samples were stored at −20°C until analysis and not thawed more
than twice. Repeated thawing did not affect SELDI-TOF profiles or protein
A total of 0.5 to 3.0 µg of unfractionated protein from amniotic
fluid was analyzed on 3 different protein chip arrays, a normal-phase NP2
0 (SiO2 surface), a reverse-phase H4 (hydrophobic surface), and
immobilized nickel (IMAC) chips (Ciphergen Biosystems Inc, Fremont, Calif).
Chips were incubated for 1 hour with the sample followed by a 5-µL water
wash. After drying, a saturated solution of sinapinic acid in 50% acetonitrile
(vol/vol) and 0.5% trifluoroacetic acid (vol/vol) was added before reading
the arrays on a SELDI-TOF instrument (Ciphergen Protein Biology System II,
Ciphergen Biosystems).13,14 Spectra
were calibrated using internal standards and analyzed using Protein Chip software,
version 3.0 (Ciphergen Biosystems).
One hundred micrograms of amniotic fluid protein from control and infected
samples was reduced with iodoacetamide and resolved on a 15% sodium dodecyl
sulfate-polyacrilamide gel electrophoresis (SDS-PAGE).15 The
gel was stained with Coomassie blue R-250, and distinct bands from each lane
were cut from the gel, destained, and digested in-gel with trypsin for 24
hours at 37°C using the method of Courchesne and Patterson.16 Peptides
were then extracted with 0.1% trifluoroacetic acid and purified using Zip-Tip
c18 pipette tips from Millipore. After in-gel digestion, samples were analyzed
on a Q-Tof-2 mass spectrometer (Micromass, Manchester, England) coupled to
a CapLC (Waters Inc, Milford, Mass) and/or on an ion trap (LCQ, ThermoFinnigan,
San Jose, Calif) coupled to a 1100 Capillary LC System (Agilent Technologies,
Foster City, Calif). Reverse-phase separation was performed using an Integrafrit
C18 75-µm ID × 15-cm fused silica capillary column (Q-TOF2) and
Zorbax C-18 0.5-mm × 150-mm microbore column using a
10 µL min−1 flow rate and a gradient of 0% to 40% (75% acetonitrile in water)
over 1 hour (LCQ). For Q-Tof-2 analysis, masses of 400 to 1500 Da were scanned
for the mass spectrometry survey and masses of 50 to 1900 Da were scanned
for tandem mass spectrometry. Tandem mass spectrometry spectra were acquired
in an automated mode using standard LCQ software.
Proteomic Analysis. Analysis of mass spectrometry/mass
spectrometry spectra was performed using SEQUEST software, version 3.1 (ThermoFinnigan)
and DTASelect (Scripps Research Institute, La Jolla, Calif) as described.17,18 Searches were run with the default
parameters using a combined, indexed, nonredundant database of protein sequences
obtained from the Protein Information Resource and SwissProt (http://www.expasy.org). S-carboxyamidated cysteine was the only considered modification.
Spectra from the LCQ mass spectrometer were filtered with a cross-correlation
score cutoff of 2.4 for the doubly charged ions. Each spectra and proposed
sequence pair selected by DTASelect were visually inspected and the final
results were input into a Microsoft Access database (Microsoft, Redmond, Wash).
Study Population/Outcomes. The 3 groups of
women were compared using 1-way analysis of variance for continuous data (P values from F statistics are presented) and by the Pearson χ2 or 2-tailed Fisher exact test for categorical data. For each analysis,
overall differences were assessed; no pairwise comparisons were evaluated
because of the small sample size. All analyses were performed using SAS, version
8 (SAS Institute Inc, Cary, NC).
Using the Pearson χ2 statistic with 2 degrees of freedom
and 33 patients allocated equally into the 3 patient groups, the power to
detect an effect size of 0.54 was calculated (NCSS and PASS Number Cruncher
Statistical Systems, Kaysville, Utah). As an example, an effect of this size
corresponds approximately to correctly classifying 9 of 11 in group 1, 7 of
11 in group 2, and 9 of 11 in group 3. Thus, the sample size was adequate
to detect effects that would be observed in evaluating currently available
diagnostic tests that have sensitivity of 80% and a specificity of 80%.
Immunogenic peptides from corresponding proteins were used to generate
rabbit polyclonal antibodies (DSL Laboratories, Webster, Tex). Affinity-purified
antibodies were then used for Western blots. One hundred micrograms of amniotic
fluid protein was resolved on 4% to 20% SDS-PAGE and transferred to polyvinyl
difluoride membranes. From maternal serum, 300 µg of protein was used
for immunoprecipitation using insulinlike growth factor binding protein 1
(IGFBP-1) monoclonal antibody (DSL Laboratories) and products underwent Western
blot analysis. For detection of calgranulin B in maternal serum, 150 µg
of albumin-depleted maternal serum was used for Western blot. The membranes
were blocked with 5% fat-free milk in phosphate-buffered saline-tris for 45
minutes at room temperature and incubated with 1 µg/mL of primary antibody
(IGFBP-1, azurocidin, vitamin D binding protein from DSL Laboratories, and
calgranulin B from Santa Cruz Biotechnology, Santa Cruz, Calif) overnight
at 4°C. After 3 washes with TBST tris-buffered saline containing 0.1%
(vol/vol) Tween-20, the membrane was incubated with IgG-HRP secondary antibody
(Sigma Chemical Co, St Louis, Mo) and visualized with enhanced chemiluminescence.
Infection was rapidly established following intra-amniotic inoculation.
Uterine contractility, which increased from basal levels of 100 mm Hg ×
s/h to greater than 10 000 mm Hg × s/h, occurred at a mean (SD)
of 33 (9) hours after inoculation with group B Streptococcus; a mean (SD) of 43 (14) hours after inoculation with U parvum; and a mean (SD) of 62 (14) hours after inoculation with M hominis. Uterine contractions led to progressive cervical
dilatation and effacement in all instances. Increases in uterine contractility
were preceded by significant elevations in the proinflammatory cytokines tumor
necrosis factor α, IL-1β, IL-6, and IL-8 and prostaglandins E2 and F2α, as previously reported.7-9 No
animal demonstrated clinical signs of IAI prior to onset of labor. Chorioamnionitis
was confirmed histologically in all cases.
Demographic and delivery data for the patients are summarized in Table 1. There were no differences in maternal
age, race/ethnicity, or parity among the 3 groups. Patients with subclinical
IAI had a somewhat earlier gestational age at enrollment and delivered at
a significantly earlier gestational age than patients with preterm delivery
without infection or those with term delivery. Ninety-one percent of patients
with occult IAI delivered within 7 days of enrollment in this study.
Four of 11 patients with occult IAI had microorganisms recovered (2
with Escherichia coli, 1 with Candida albicans, and 1 with mixed anaerobes). Neither U parvum nor M hominis was isolated. The other
7 patients with infection were identified on the basis of markedly elevated
amniotic fluid IL-6 concentrations (>2 ng/mL). The mean (SD) amniotic fluid
concentration of IL-6 was 27.7 (7.8) ng/mL among these patients compared with
0.68 (0.20) ng/mL among those with preterm delivery without infection and
0.25 (0.13) ng/mL among those with preterm contractions but term delivery
Whole-spectrum SELDI-TOF mass spectrometry analyses collected at 235
laser intensity of amniotic fluid extracts bound to chemically defined normal-phase
chip arrays revealed several peak intensity differences in 3- to 5-kDa and
11- to 12-kDa regions between infected and noninfected primate and human amniotic
fluid (Figure 1). The 11-kDa cluster
was differentially expressed between control and infected in all cases (P = .004). Longitudinal sampling following group B Streptococcus inoculation revealed that the 11-kDa peak
was first noted as early as 12 hours after inoculation and preceded increases
in hourly contraction area in infected animals.
As shown in Figure 2, the
11-kDa peak was also detected in experimental IAI induced by the other 2 microbes.
In the human cohort, all 11 patients with confirmed occult IAI and none of
the 11 patients who subsequently delivered at term had the SELDI-TOF mass
spectrometry profile consistent with IAI and characterized by the presence
of the 11-kDa peak. This profile was also present in 2 of 11 patients in whom
infection was not confirmed but who delivered prematurely (Table 1). These differences in the proteome profile could not be
attributed to differences in gestational age.
Global analysis of primate and human infected and noninfected amniotic
fluid using 1-dimensional gel electrophoresis separation and in-gel digestion
of specific bands identified expression of various immunoregulatory proteins
not described previously in IAI (Table 2). Liquid chromatography–tandem mass spectrometry analysis
of the approximately 11-kDa band from 1-dimensional gel identified several
polypeptides derived from calgranulins and a unique fragment of IGFBP-1.
To validate the differential expression of proteins identified in IAI,
we selected 2 markers identified from the 11-kDa SELDI-TOF mass spectrometry
profile (calgranulin B and IGFBP-1), 1 immunoregulatory molecule (azurocidin),
and an unregulated protein (vitamin D binding protein) identified in global
protein expression analysis. As shown in Figure 3, Western blot analysis confirmed all 3 biomarkers showing
differential expression, consistent with the protein identification experiments
performed on IAI amniotic fluid.
Using specific antibodies developed against IGFBP-1 and calgranulin
B, we investigated whether differentially expressed proteins in amniotic fluid
could be identified in pooled maternal serum from a limited number of patients
(n = 5) for whom serum samples were available. In this limited analysis, the
11-kDa proteolytic fragment of IGFBP-1 and of calgranulin B, corresponding
to the 11-kDa peak that was differentially present in infected vs control
amniotic fluid, was also detected in maternal serum in response to IAI (Figure 3B) and in nonhuman primates following
experimental IAI (data not shown). Azurocidin was not detected in serum.
Intra-amniotic infection is an important and potentially preventable
cause of premature births, neonatal sepsis, periventricular leukomalacia/cerebral
palsy, and maternal febrile morbidity.2,3,19-21 Overt
or subclinical IAI is present in at least 50% of extremely premature births;
an inverse relationship has been demonstrated between gestational age at birth
and both the frequency of microorganisms recovered from the chorioamnion and
Despite growing evidence that IAI is responsible for a substantial proportion
of premature births in the United States, antibiotic treatment has had limited
success in treating patients with preterm labor and intact fetal membranes
or in preventing preterm delivery.3,5 This
dilemma may be attributable in part to misclassification and patient selection
bias, whereby the subgroup of women with occult IAI and intact fetal membranes
that might benefit from antibiotics is not reliably distinguished from women
without infection. Accurate and early diagnosis of IAI would facilitate early
and appropriate interventions as well as enhance the design of therapeutic
trials. Early diagnosis of IAI is problematic, however, because clinical signs
and symptoms (including preterm labor) tend to be late manifestations of this
condition. Furthermore, the available noninvasive diagnostic tests have limited
predictive value, or, as in the case of measurement of IL-6, polymerase chain
reaction tests, or microbial cultures, the results are often delayed and amniocentesis
We previously demonstrated in a nonhuman primate model the causal relationship
between IAI with group B Streptococcus, U parvum, or M hominis and preterm labor,
which resembles the clinical progression of events that is observed in women
with IAI.7-9 These
microorganisms are also frequently isolated from the amniotic fluid and chorioamnion
in women with preterm labor and delivery.11,23,24 In
the rhesus monkey, as in women, histologic appearance of the fetal membranes
is consistent with subacute chorioamnionitis.
Therefore, we used this experimental model to search for novel amniotic
fluid protein and peptide biomarkers for IAI with the expectation that they
could similarly be found in human amniotic fluid and would provide the basis
for the subsequent development of sensitive and specific assays to detect
subclinical IAI. We identified a unique amniotic fluid peptide profile in
IAI by SELDI-TOF that appeared within 12 hours after intra-amniotic inoculation
of microorganisms and was reliably present before the onset of labor or other
clinical signs or symptoms of infection. We validated our observations in
a cohort of women in preterm labor by microbiologic, biochemical, or histologic
evidence of chorioamnionitis and in a control group without evidence of infection.
We used 2 distinct proteomic approaches: a low-resolution rapid protein
fingerprinting approach (SELDI-TOF mass spectrometry) that generates distinct
expression profiles and is amenable to developing rapid screening assays,
together with a high-throughput protein identification approach (LC–tandem
mass spectrometry) that provides the identity of the biomarkers suitable for
identification by conventional immunoassays.
The detection of a differentially expressed 11-kDa peak in SELDI-TOF
mass spectrometry in the setting of IAI in the experimental primate model,
which used clinically relevant microorganisms and in women infected with different
microorganisms, confirms this signature for IAI caused by a broad range of
pathogens. Peptides within the differentially expressed peak may represent
a basic intrauterine immune response to infection since one set of proteins
identified in this unique cluster, ie, the calgranulins, are members of the
S-100 calcium-binding protein family, expressed by macrophages and by epithelial
cells in acutely inflamed tissues.25,26 The
second candidate from this cluster, a specific proteolytic fragment of IGFBP-1,
indicates a potential protease-related mechanism in response to infection.
Intact IGFBP-1 is the major IGFBP found in amniotic fluid and is synthesized
by both fetal membranes and maternal decidua.27 Although
the proteolytic cleavage of IGFBPs is a well-characterized phenomenon,28 the particular fragment described in the current
study has not been previously described.
In the second approach, characterization of proteins expressed in amniotic
fluid in control and IAI using LC–tandem mass spectrometry identified
a significant number of infection and immune response–related molecules
in IAI for the first time. Macrophage-capping protein is a Ca+2-sensitive
protein that modulates actin filaments and is involved in inflammatory processes.29 Leukocyte elastase and neutrophil gelatinase-associated
lipocalcin are involved in bacteriostatic and baceterolysis mechanisms.30
In addition to the above immunomodulators, the detection of antibacterial
proteins Fall-39 and azurocidin in amniotic fluid in response to infection
provides new insights into intrauterine immune responses. Antibacterial protein
Fall-39 (LL-37) binds to bacterial lipopolysaccharides, is a potent chemotactic
factor for mast cells, and serves as a first-line defense to prevent local
infection and systemic invasion of microbes.31 Azurocidin
(CAP37) is a cationic antimicrobial protein isolated from human neutrophils
with important implications in host defense and inflammation.32 Glycoprotein-340
variant protein is a scavenger receptor previously identified in lung that
binds to bacteria.33 Identification of these
proteins complements the sensitive proteomic approaches used to identify biomarkers
for IAI. Since our experimental objectives were focused on the discovery of
novel biomarkers unique to IAI, we did not look for the peptide profiles of
cytokines such as IL-6.
Currently available laboratory tests used to confirm the diagnosis of
IAI include measurement of maternal C-reactive protein, direct examination
of amniotic fluid for leukocytes or bacteria on Gram stain, amniotic fluid
microbial culture or polymerase chain reaction, measurement of amniotic fluid
glucose and IL-6 concentrations, and detection of amniotic fluid leukocyte
esterase. Only microbial cultures and broad-spectrum bacterial recombinant
DNA polymerase chain reaction of amniotic fluid appears to be sensitive and
specific for IAI.12,34 However,
microbial cultures for genital mycoplasmas or polymerase chain reaction–based
tests are not widely available and amniocentesis is required to obtain amniotic
One advantage of a proteomics-based approach is that candidate peptide
biomarkers lend themselves to the development of rapid point-of-service and
cost-effective diagnostic immunoassays that can be completed within hours.
By analogy with other amniotic fluid and serum screening tests (eg, aα-fetoprotein
for neural tube defects), we predicted that peptides identified within infected
amniotic fluid could also be detected in maternal serum and might lead to
the development of noninvasive diagnostic tests.35 Our
preliminary results using pooled maternal serum samples suggest the feasibility
of this approach, in that IGFBP-1–restrictive fragment and calgranulin
B are 2 such candidate biomarkers that appear in both amniotic fluid and maternal
blood during IAI but not in the absence of infection (Figure 3B). In contrast, elevated IL-6 concentrations have not been
reported in maternal serum in association with intra-amniotic infection.
In summary, we used a proteomics-based analysis of amniotic fluid to
identify biomarkers for IAI in both an experimental nonhuman primate model
and a cohort of women with preterm labor and occult IAI. Diagnostic protein
expression profiles were identified by SELDI-TOF mass spectrometry profiling
that was both sensitive and specific in detecting IAI. High-throughput analysis
of expressed proteins in amniotic fluid identified expression of several immunoregulatory
molecules for the first time. Immunodetection of proteins detected in the
diagnostic 11-kDa peak (IGFBP-1 and calgranulin B) confirmed the presence
and differential expression of these biomarkers in amniotic fluid and in maternal
blood during IAI.
Our findings hold promise for the future development of rapid and noninvasive
assays to detect occult IAI during pregnancy. This would be an important breakthrough
since it would allow clinicians and investigators to target a particular subgroup
of women at high risk of premature delivery for specific interventions and
in therapeutic trials. Finally, these studies demonstrate the potential utility
of proteomics-based approaches to identify specific biomarkers and diagnostic
profiles for infectious and inflammatory processes and in pathophysiologic
conditions of pregnancy.