Antimicrobial peptides in amniotic fluid: defensins,calprotectin and bacterial/permeability-increasingprotein in patients with microbial invasion of theamniotic cavity, intra-amniotic inflammation,preterm labor and premature rupture of membranesJ. Espinoza1, T. Chaiworapongsa1, R. Romero1, S. Edwin1, C. Rathnasabapathy 1, R. Gomez2,E. Bujold3, N. Camacho1, Y. M. Kim1, S. Hassan3, S. Blackwell3, J. Whitty3, S. Berman3,M. Redman3, B. H. Yoon4 and Y. Sorokin3

1Perinatology Research Branch, National Institute of Child Health and Human Development NIH/DHSS, Bethesda,Maryland, USA2CEDIP, Department of Obstetrics and Gynecology, Sotero del Rio Hospital, Puente Alto, Chile3Department of Obstetrics and Gynecology, Wayne State University/Hutzel Hospital, Detroit, Michigan, USA4Department of Obstetrics and Gynecology, Seoul National University, Seoul, Korea

Objective: Neutrophil defensins (HNP 1–3), bactericidal/permeability-increasing protein (BPI)and calprotectin (MRP8/14) are antimicrobial peptides stored in leukocytes that act as effectormolecules of the innate immune response. The purpose of this study was to determine whetherparturition, premature rupture of the membranes (PROM) and microbial invasion of the amnioticcavity (MIAC) are associated with changes in amniotic fluid concentrations of these antimicrobialpeptides.Study design: Amniotic fluid was retrieved by amniocentesis from 333 patients in the followinggroups: group 1, mid-trimester with a subsequent normal pregnancy outcome (n = 84); group 2,preterm labor and intact membranes without MIAC who delivered at term (n = 36), or prematurely(n = 52) and preterm labor with MIAC (n = 26); group 3, preterm PROM with (n = 26) andwithout (n = 26) MIAC; and group 4, term with intact membranes in the absence of MIAC, inlabor (n = 52) and not in labor (n = 31). The concentrations of HNP 1–3, BPI and calprotectinin amniotic fluid were determined by specific and sensitive immunoassays. Placentae of patientsin both preterm labor with intact membranes and preterm PROM groups who delivered within 72 hof amniocentesis were examined. Non-parametric statistics, receiver-operating characteristic(ROC) curves and Cox regression models were used for analysis. A p value of < 0.05 was consideredstatistically significant.Results: Intra-amniotic infection was associated with a significant increase in amniotic fluidconcentrations of immunoreactive HNP 1–3, BPI and calprotectin in both women with pretermlabor and intact membranes, and women with preterm PROM. Preterm PROM was associated witha significant increase in amniotic fluid concentrations of immunoreactive HNP 1–3, BPI andcalprotectin. Preterm parturition was associated with a significant increase in amniotic fluid con-centrations of immunoreactive HNP 1–3, BPI and calprotectin, while parturition at term was asso-ciated with a significant increase in amniotic fluid concentrations of immunoreactive HNP 1–3.Among patients with preterm labor and intact membranes, elevation of amniotic fluid HNP 1–3,BPI and calprotectin concentrations was associated with intra-amniotic inflammation, histologicalchorioamnionitis and a shorter interval to delivery.Conclusion: MIAC, preterm parturition and preterm PROM are associated with increasedamniotic fluid concentrations of immunoreactive HNP 1–3, BPI and calprotectin. Moreover,elevated amniotic fluid concentrations of BPI, immunoreactive HNP 1–3 and calprotectin are asso-ciated with intra-amniotic inflammation, histological chorioamnionitis and shorter amniocentesis-to-delivery interval in patients presenting with preterm labor with intact membranes.

The Journal of Maternal–Fetal and Neonatal Medicine 2003;13:2–21

Correspondence: Dr R. Romero, Perinatology Research Branch, NICHD NIH/DHHS, Wayne State University/Hutzel Hospital, Departm entof Obstetrics and Gynecology, 4707 St. Antoine Boulevard, Detroit, MI 48201, USAã 2003 The Parthenon Publishing Group 2 Received 15–10–02 Accepted 20–10–02

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Key words: AMNIOTIC FLUID; PRETERM LABOR; PARTURITION; INFECTION; HNP 1–3; BPI;MRP8/14; CALPROTECTIN; CALGRANULIN A; CALGRANULIN B; DEFENSIN; ANTIMICROBIALPEPTIDES

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INTRODUCTION

The amniotic cavity is considered to be a sterile compart-ment under normal circumstances. This is presumablyaccomplished by the participation of components of theinnate immune system, which include the cervicalepithelium1, cervical mucus plug2–5, chorioamnioticmembranes6,7, and cellular components of decidua, amnionand chorion, including neutrophils, macrophages, naturalkiller (NK) cells and trophoblast8,9.

Although intact chorioamniotic membranes arethought to protect against microbial invasion of theamniotic cavity (MIAC), this physical barrier cannot byitself be responsible for the sterile nature of amniotic fluidin normal pregnancy because bacteria can cross the intactmembranes10,11. Indeed, patients with intact membranescan have MIAC, as this has been demonstrated in patientsin the mid-trimester of pregnancy12–14 with preterm laborand intact membranes15–17 or even spontaneous labor atterm18. This suggests that, in addition to the physicalbarrier, other mechanisms must be operative to preventbacterial proliferation within the amniotic cavity.

Amniotic fluid is known to have antimicrobial proper-ties, which were reported by Cattaneo in 194919. Sincethen, several investigators have confirmed this findingand attempted to identify the amniotic fluid componentsaccounting for its antimicrobial activity20–25.

Antimicrobial peptides, part of the innate limb of theimmune response, have been identified in plants, insectsand all vertebrates examined to date. Neutrophils containseveral antimicrobial peptides including neutrophil defen-sins (human neutrophil peptides (HNP) 1, 2 and 3)26–29,bactericidal/permeability-increasing protein (BPI)30–35 andcalprotectin (MRP8/14)27,36–38. The purpose of this studywas to determine whether amniotic fluid contains immuno-reactive antimicrobial peptides (HNP 1–3, BPI andMRP8/14) and whether their concentration changes withMIAC, parturition and premature rupture of membranes(PROM).

MATERIALS AND METHODS

A cross-sectional study was constructed by searching ourclinical database and bank of biological samples. This studyincluded women in four groups. Group 1 consisted ofwomen in the mid-trimester (14–18 weeks) of pregnancywho underwent amniocentesis for genetic indicationsand delivered normal infants at term (n = 84). Group 2included women with preterm labor and intact membranes

who were subdivided into the following categories: thosewith preterm labor who delivered at term with a negativeamniotic fluid culture for micro-organisms (n = 36); thosewith preterm labor who delivered preterm (< 37 weeks)with a negative amniotic fluid culture for micro-organisms(n = 52); and those with preterm delivery with MIAC(n = 26). Preterm labor was defined by the presence ofregular uterine contractions occurring at a frequency of atleast two every 10 min and cervical changes before 37 com-pleted weeks of gestation. MIAC was defined as a positiveamniotic fluid culture for micro-organisms. Group 3 con-sisted of women with preterm PROM with (n = 26) andwithout (n = 26) MIAC. PROM was diagnosed asamniorrhexis before the onset of spontaneous labor.Membrane rupture was diagnosed with the use of vaginalpooling, by ferning, or by a positive nitrazine test. The indi-cations for amniocentesis in patients of both groups 2 and 3were for the detection of MIAC and fetal lung maturity.Group 4 included women with term gestations (³ 37 weeksof gestation) with intact membranes and without MIAC.This group was subdivided into two subgroups composed ofthose not in labor (n = 31) and those in labor (n = 52).Women at term underwent amniocentesis for the assess-ment of lung maturity prior to Cesarean section or for thediagnosis of MIAC. Amniotic fluid not required for clinicalpurposes was centrifuged at 4°C for 10 min to remove cellu-lar and particulate matter, and stored at -70°C. A sampleof amniotic fluid was transported to the laboratory forculture of aerobic/anaerobic bacteria and Mycoplasmaspecies. Amniotic fluid white blood cell (WBC) counts andassessment of glucose concentrations were not performedin some cases. The results of these tests were used forsubsequent clinical management.

All women provided informed consent prior to thecollection of amniotic fluid. The collection of amnioticfluid was approved by the Human InvestigationCommittees of the participating institutions, and itsutilization for research purposes was approved by theInstitutional Review Board of the National Institute ofChild Health and Human Development. Many of thesesamples have been used previously in studies of cytokinesand arachidonic acid metabolites in amniotic fluid.

Assays for antimicrobial peptides

Concentrations of BPI, HNP 1–3 and MRP8/14 inamniotic fluid were determined with commercially

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available enzyme-linked immunoassays (Table 1). Theassays are specific for human proteins and were validated inour laboratory for amniotic fluid prior to use. Briefly, BPI,HNP 1–3 and MRP8/14 immunoassays are based on thesandwich format. During the first step of the assays,amniotic fluid specimens are incubated in duplicate wherethe analyte present in the samples or the standard bindsto the microtiter plate precoated with specific antibodies.Following incubation, repeated washings are performed toremove unbound materials. Step two involves incubationwith biotinylated second antibody, which binds to thecaptured antigen in the microtiter plate to form a sand-wich. After removing the excess and unbound materials,streptavidin peroxidase conjugate is added. The detectionof bound peroxidase in each well is determined by the addi-tion of tetramethylbenzidine (TMB) substrate in each ofthese assays. The reaction is stopped by the additionof either 2M citric acid or 1M sulfuric acid, and theresultant color is measured at 450 nm in an automatedmicrotiter plate-based spectrophotometer (Bio-Tek Instru-ments, Winooski, VT, USA). The concentrations ofindividual analytes in amniotic fluid are determined byinterpolation from the respective standard curve. Thecalculated sensitivity, inter- and intra-assay coefficients ofvariation for BPI, HNP 1–3 and MRP8/14 immunoassaysin our laboratory are displayed in Table 1.

Placental pathology

The presence or absence of acute inflammatory lesionsin the extraplacental membranes (histologic chorio-amnionitis) and the umbilical cord (funisitis) was assessedas previously described39 in patients with preterm laborwith intact membranes, as well as those with pretermPROM who delivered within 72 h of amniocentesis. Thisperiod of time was selected to preserve a meaningful tempo-ral relationship between antimicrobial peptide amnioticfluid concentrations and placental pathologic findings.

Statistical analysis

The Shapiro–Wilk and Kolmogorov–Smirnov tests wereused to test for normal distribution of the data. Since anormal distribution was not achieved after logarithmic

transformation, Kruskal–Wallis and Mann–Whitney Utests were used to determine the differences of the medianamong groups. Spearman rank correlation was utilized toassess the correlations, and c2 or Fisher’s exact test wereused for comparisons of proportion variables. Amongpatients with preterm labor and intact membranes,receiver-operating characteristic (ROC) curve analysis wasemployed for the identification of patients who had MIAC,those who had intra-amniotic inflammation, and thosewho delivered within various intervals. The compositemarker was recorded as a score of 1–4. Score 1 was givenwhen none of the studied antimicrobial peptide concentra-tions was above the cut-off values and score 4 when allthree markers were above the cut-off values. Cox regressionanalysis was applied to examine the interval from amnio-centesis to delivery, according to antimicrobial peptideamniotic fluid concentrations, while controlling for otherconfounding factors. A p value of < 0.05 was consideredstatistically significant (SPSS 10.0, SPSS Inc., Chicago, IL,USA).

RESULTS

Immunoreactive BPI was detectable in 61.6% (205/333) ofthe cases, immunoreactive calprotectin in 95.8% (318/332)and immunoreactive HNP 1–3 in all amniotic fluidsamples. The gestational age at amniocentesis and theproportion of patients with detectable BPI and calprotectinin each subgroup are displayed in Table 2. There were nosignificant differences in the median gestational age atamniocentesis among the three subgroups of patients withpreterm labor and intact membranes, or between the twosubgroups of preterm PROM (p > 0.05 for each). BPI wasdetected in only 9.5% (8/84) of mid-trimester samples frompatients with normal pregnancy outcome, whereascalprotectin was detectable in 84.5% (71/84) of the casesand HNP 1–3 in all amniotic fluid samples of this group.

Women at term and not in labor had significantlyhigher median amniotic fluid concentrations of BPI andcalprotectin than women in the mid-trimester whodelivered a normal neonate at term, but not of HNP 1–3(Figure 1). Furthermore, when the analysis includedwomen in the mid-trimester who delivered a normalneonate at term, women with preterm labor and intact

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Analyte Company City, country SensitivityIntra-assay CV

(%)Interassay CV

(%)

BPIHNP 1–3Calprotectin (MRP 8/14)

HyCult BiotechnologyHyCult BiotechnologyBMA Biomedical AG

Uden, The NetherlandsUden, The NetherlandsAugst, Switzerland

71.25 pg/ml19.46 pg/ml0.686 ng/ml

1.741.977.3

4.01.24

11.7

BPI; bactericidal/permeability-increasing protein; HNP 1–3: human neutrophil defensins 1, 2 and 3

Table 1 Sensitivity, interassay and intra-assay coefficients of variation (CV) of study assay kits

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membranes who delivered at term and women at term withintact membranes and not in labor, there was a positivecorrelation between amniotic fluid concentrations of bothBPI and calprotectin with gestational age (Spearman’s rhocoefficient 0.7; p < 0.001 for both). In contrast, there wasno significant correlation between the amniotic fluid con-centration of immunoreactive HNP 1–3 and gestationalage (Spearman’s rho coefficient - 0.002; p = 0.9).

Preterm parturition, in the absence of MIAC, was asso-ciated with a significantly higher median amniotic fluidconcentration of HNP 1–3, BPI and calprotectin(Figure 2), whereas spontaneous labor at term was asso-ciated with a significantly higher median amniotic fluidconcentration of only HNP 1–3, but not of BPI andcalprotectin (Figure 3).

MIAC was associated with a significantly higher medianamniotic fluid concentration of HNP 1–3, BPI and

calprotectin in women with preterm labor and intact mem-branes, and in those with preterm PROM (Figures 2 and 4,respectively) than in women with sterile amniotic fluid(negative amniotic fluid culture). Similarly, in the absenceof MIAC, women with preterm PROM had a significantlyhigher median amniotic fluid concentration of HNP 1–3,BPI and calprotectin than those with preterm labor andintact membranes who delivered at term (Figure 5).

There were positive correlations among the amnioticfluid concentrations of the three antimicrobial peptidesand the amniotic fluid WBC count, and negative correla-tions among the concentrations of these antimicrobialpeptides and amniotic fluid glucose concentrations, withthe exception of calprotectin (Table 3). Similarly, therewere positive correlations among the amniotic fluidconcentrations of the three antimicrobial peptides, exceptbetween calprotectin and HNP 1–3 (Table 3).

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Gestational age atamniocentesis (weeks) BPI detectable Calprotectin detectable

Median Range % n % n

Mid-trimester and normal outcomesPreterm labor and delivery at termPreterm labor and delivery pretermPreterm labor with MIACPreterm PROM with no MIACPreterm PROM with MIACTerm with no laborTerm and in labor

1628.827.426.830.729.139.339.3

14–1821.2–34.119.3–34.6

19–34.122–33.719–33.438–4237–41.5

9.544.463.596.276.996.296.892.3

8/8416/3633/5225/2620/2625/2630/3148/52

84.597.2

100100100100100100

71/8435/3652/5225/2526/2626/2631/3152/52

Total 28.1 14–42 61.6 205/333 95.8 318/332

BPI, bactericidal/permeability-increasing protein; MIAC, microbial invasion of the amniotic cavity; PROM, premature rupture ofmembranesHuman neutrophil defensins 1–3 were detectable in all samples (333/333)

Table 2 Gestational age at amniocentesis and the proportion of samples that had detectable amniotic fluid antibacterial peptideconcentrations in each group

BPI (ng/ml) HNP 1–3 (ng/ml) Calprotectin (mg/ml)

Amniotic fluid white blood cell count (cells/mm3)

Amniotic fluid glucose (mg/dl)

BPI (ng/ml)

HNP 1–3 (ng/ml)Calprotectin (mg/ml)

0.5p < 0.001*

-0.4p = 0.003*

1

0.5p < 0.001*

-0.6p < 0.001*

0.3p < 0.001*

1

0.3p = 0.04*

NS

0.58p < 0.001*

NS1

*Statistically significant, p < 0.05; NS, statistically non-significantBPI, bactericidal/permeability-increasing protein; HNP, human neutrophil defensins

Table 3 Spearman’s rho correlations of antibacterial peptide concentration, white blood cell count and glucose concentration inamniotic fluid

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Among patients who delivered within 72 h of amnio-centeses, placental pathology was available in 89% (33/37)of those with preterm labor with intact membranes, andin 96% (27/28) of those with preterm PROM. Patientswith evidence of inflammation in the extraplacentalmembranes (histologic chorioamnionitis) and/or umbilicalcord (funisitis) had a significantly higher median amnioticfluid concentration of BPI, HNP 1–3 and MRP8/14than those without inflammation (all p values < 0.05; seeTable 4).

The diagnostic performance of each antimicrobialpeptide concentration in amniotic fluid was assessed among

patients who presented with preterm labor with intactmembranes. Table 5 displays various cut-off values (derivedfrom ROC curves) of amniotic fluid concentrations of thethree antimicrobial peptides for the identification ofpatients who had MIAC, intra-amniotic inflammation(defined as amniotic fluid WBC count either ³ 50 or³ 100 cells/ml), or those who delivered following spon-taneous labor, within 48 h, 72 h and 7 days. The ROCcurves for the identification of MIAC and intra-amnioticinflammation (defined as amniotic fluid WBC count³ 50 cells/ml) are displayed in Figure 6. The diagnosticefficacy of antimicrobial peptide concentrations and other

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Figure 1 Amniotic fluid concentration of bactericidal/permeability-increasing protein (BPI), human neutrophil defensins (HNP) 1–3and calprotectin in women in the mid-trimester who delivered a normal neonate at term and in women at term without labor. (a) Themedian amniotic fluid concentration of BPI in women at term without labor was significantly higher than in women in the mid-trimester(median 1.99 ng/ml, range 0–26.04 vs. median 0 ng/ml, range 0–2.41, respectively;p < 0.001). (b) The median amniotic fluid concentra-tion of HNP 1–3 in women at term without labor was not significantly higher than in women in the mid-trimester (median 5.56 ng/ml,range 1.31–42.46 vs. median 2.8 ng/ml, range 1.21–43.89, respectively; p = 0.2). (c) The median amniotic fluid concentration ofcalprotectin in women at term without labor was significantly higher than in women in the mid-trimester (median 11.9 mg/ml, range1.6–49.9 vs. median 1.45 mg/ml, range 0–14.4, respectively; p < 0.001). Dotted lines indicate detection limits: (a) 0.07 ng/ml;(b) 0.02 ng/ml; (c) 0.007 mg/ml; *, statistically significant

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previously published markers in amniotic fluid (Gramstain, WBC count ³ 50 cells/ml and glucose concentration

£ 14 mg/dl) for the identification of MIAC are displayedin Table 6.

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Figure 2 Amniotic fluid concentration of bactericidal/permeability-increasing protein (BPI), human neutrophil defensins (HNP) 1–3and calprotectin in women with preterm labor and intact membranes. (a) The median amniotic fluid concentration of BPI was signifi-cantly higher in women with preterm labor who delivered preterm than in women who delivered at term (median 0.88 ng/ml, range0–169 vs. median 0 ng/ml, range 0–8.35, respectively; p = 0.005). Similarly, the median amniotic fluid concentration of BPI in womenwith microbial invasion of the amniotic cavity (MIAC) was significantly higher than in women with preterm labor without MIAC whodelivered preterm (median 71.59 ng/ml, range 0–285 vs. median 0.88 ng/ml, range 0–169, respectively; p< 0.001). (b) The medianamniotic fluid concentration of HNP 1–3 was significantly higher in women with preterm labor who delivered preterm than in womenwho delivered at term (median 2.93 ng/ml, range 1.16–342.4 vs. median 1.71 ng/ml, range 1.13–9.28, respectively;p = 0.004). Similarly,the median amniotic fluid concentration of HNP 1–3 in women with MIAC was significantly higher than in women with preterm laborwithout MIAC who delivered preterm (median 195.18 ng/ml, range 1.13–424.7 vs. median 2.93 ng/ml, range 1.16–342.4, respectively;p< 0.001). (c) The median amniotic fluid concentration of calprotectinwas significantly higher in women with preterm labor who deliv-ered preterm than in women who delivered at term (median 15.3 mg/ml, range 1.8–1023.3 vs. median 11.72 mg/ml, range 0–52.5, respec-tively; p = 0.008). Similarly, the median amniotic fluid concentration of calprotectin in women with MIAC was significantly higherthan in women with preterm labor without MIAC who delivered preterm (median 188.1 mg/ml, range 5.05–1013.9 vs. preterm delivery,median 15.3 mg/ml, range 1.8–1023.3, respectively; p < 0.001). Dotted lines indicate detection limits: (a) 0.07 ng/ml; (b) 0.02 ng/ml;(c) 0.007 mg/ml; *, statistically significant

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The interval to delivery was calculated to assess therelationship between intra-amniotic inflammation andthe duration of the amniocentesis-to-delivery interval.Overall, the amniocentesis-to-delivery interval was signifi-cantly shorter in patients whose amniotic fluid concentra-tions of antibacterial peptides were above the cut-off valuesderived from ROC curves than those below the cut-offvalues (Table 7). Cox proportional hazard model was usedto examine the relationship between the duration of theamniocentesis-to-delivery interval and various anti-bacterial peptide concentrations in amniotic fluid, whileadjusting for the status of amniotic fluid culture, gestational

age and cervical dilatation at amniocentesis. Spontaneouslabor and delivery was entered in the model as the eventof interest. The cut-off values for BPI, HNP 1–3 andMRP8/14 were derived from the ROC curve analysis for theidentification of patients who had intra-amniotic fluidinflammation (amniotic fluid WBC count ³ 50 cells/ml).Each antibacterial peptide was entered in a separate model,as there was a high correlation in amniotic fluid amongthem. Table 8 displays the hazard ratio and 95% confidenceintervals for the interval from amniocentesis to deliveryaccording to amniotic fluid concentration of BPI, HNP1–3 and MRP8/14 at the cut-off value used to identify

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Figure 3 Amniotic fluid concentration of bactericidal/permeability-increasing protein (BPI), human neutrophil defensins (HNP) 1–3and calprotectin in women at term with and without labor and without microbial invasion of the amniotic cavity (MIAC). (a) Themedian amniotic fluid concentration of BPI was not significantly different in women at term with and without labor (median 2.59 ng/ml,range 0–68.21 vs. median 1.99 ng/ml, range 0–26.04, respectively; p = 0.5). (b) The median amniotic fluid concentration of HNP 1–3was significantly higher in women at term in labor than in women at term without labor (median 12.1 ng/ml, range 1.14–982 vs. median5.56 ng/ml, range 1.31–42.46, respectively; p = 0.02). (c) The median amniotic fluid concentration of calprotectin was not significantlydifferent in women at term with and without labor (median 10.14 mg/ml, range 1.7–135.2 vs. median 11.9 mg/ml, range 1.6–49.9, respec-tively; p = 0.5). Dotted lines indicate detection limits: (a) 0.07 ng/ml; (b) 0.02 ng/ml; (c) 0.007 mg/ml; *, statistically significant

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intra-amniotic inflammation. Survival curves for variousantibacterial peptides are displayed in Figure 7.

Among patients with preterm labor and intact mem-branes who delivered within 72 h and whose placenta hadbeen examined, 70% (23/33) had evidence of placentalinflammation, while 30% (10/33) did not. All except onepatient (95% (19/20)) who had amniotic fluid concentra-tion of BPI ³ 15.5 ng/ml and HNP 1–3 ³ 53.2 ng/ml anddelivered within 72 h had evidence of inflammation in theextraplacental membranes and/or umbilical cord. Con-versely, less than one-fifth (17% (4/23)) of patients who

delivered within 72 h with placental inflammation did nothave elevated amniotic fluid concentrations of BPI andHNP 1–3 above these cut-off levels.

DISCUSSION

Host defense against microbial invasion involves theparticipation of the innate and adaptive immune response.The innate component of the immune system is the firstline of defense and includes a wide range of largely non-specific mechanisms (in contrast to the specific mechanism

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Figure 4 Amniotic fluid concentration of bactericidal/permeability-increasing protein (BPI), human neutrophil defensins (HNP) 1–3and calprotectin in women with preterm premature rupture of membranes (PROM) with and without microbial invasion of the amnioticcavity (MIAC). (a) The median amniotic fluid concentration of BPI was significantly higher in women with preterm PROM and withMIAC than in women with preterm PROM without MIAC (median 32.66 ng/ml, range 0–157 vs. median 2.03 ng/ml, range 0–35.26,respectively; p = 0.008). (b) The median amniotic fluid concentration of HNP 1–3 was significantly higher in women with pretermPROM and MIAC than in women with preterm PROM without MIAC (median 78.78 ng/ml, range 1.65–944.5 vs. median 2.92 ng/ml,range 1.23–157.65, respectively; p < 0.001). (c) The median amniotic fluid concentration of calprotectin was significantly higher inwomen with preterm PROM and MIAC than in women with preterm PROM without MIAC (median 29.13 mg/ml, range 5.4–906.1 vs.median 15.3 mg/ml, range 5.2–149.8, respectively; p = 0.03). Dotted lines indicate detection limits: (a) 0.07 ng/ml; (b) 0.02 ng/ml;(c) 0.007 mg/ml; *, statistically significant

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of the adaptive component), which act rapidly and withoutprevious experience with the pathogen40–45. The threecomponents of innate immunity42 include:

(1) Mechanical barriers such as skin and mucous mem-branes that prevent microbial invasion. The epitheliaand mucous membranes can produce enzymes (e.g.lysozyme) and antimicrobial peptides, which enhancethe effects of the physical barrier. These mechanismspresumably evolved to prevent overgrowth of com-mensal micro-organisms and colonization of certainbody cavities that are normally sterile (such as the

cerebrospinal fluid and amniotic cavity), as well asmicrobial invasion in surfaces normally exposed to bac-teria, such as skin, vagina, gastrointestinal lumen, etc.

(2) Cellular components that include tissue macrophages,which are present at the site of entry and can phago-cytose micro-organisms that penetrate the epithelia, aswell as other cells, such as neutrophils, called to thesite of invasion.

(3) The inflammatory response, which leads to the accu-mulation of plasma proteins, complement component,humoral factors, etc.

Antimicrobial peptides in amniotic fluid Espinoza et al.

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Figure 5 Amniotic fluid concentration of bactericidal permeability-increasingprotein (BPI), human neutrophil defensins (HNP) 1–3and calprotectin in women with preterm labor and term delivery and women with preterm PROM in the absence of microbial invasionof the amniotic cavity (MIAC). (a) The median amniotic fluid concentration of BPI was significantly higher in women with pretermPROM than in women with preterm labor and term delivery (median 2.03 ng/ml, range 0–35.26 vs. median 0 ng/ml, range 0–8.35,respectively; p = 0.001). (b) The median amniotic fluid concentration of HNP 1–3 was significantly higher in women with pretermPROM than in women with preterm labor and term delivery (median 2.93 ng/ml, range 1.23–157.65 vs. median 1.71 ng/ml, range1.13–9.28, respectively;p = 0.005). (c) The median amniotic fluid concentration of calprotectin was significantly higher in women withpreterm PROM than in women with preterm labor and term delivery (median: 15.3 mg/ml, range 5.2–149.8 vs. median 11.72 mg/ml,range 0–52.5, respectively;p = 0.03). Dotted lines indicate detection limits: (a) 0.07 ng/ml; (b) 0.02 ng/ml; (c) 0.007 mg/ml; *statisticallysignificant

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Preterm labor with intact membranes Preterm PROM

Without placentalinflammation

(n = 10)

With placentalinflammation

(n = 23)p Value

(a)

Without placentalinflammation

(n = 8)

With placentalinflammation

(n = 19)p Value

(b)

BPI (ng/ml)HNP 1–3 (ng/ml)Calprotectin (mg/ml)

1.84 (0–22.11)3.41 (1.3–89.55)

17.04 (7.17–120.9)

7.52 (2.8–285)201.4 (2.58–424.7)

212.72 (5.96–1013.9)

< 0.001*< 0.001*< 0.002*

3.53 (0–13.76)3.9 (1.25–18.72)

12.15 (5.86–37.5)

52.65 (0.39–157)81.7 (1.44–944.5)

34.33 (5.44–90.61)

0.01*0.02*0.02*

BPI, bactericidal/permeability-increasing protein; HNP, human neutrophil defensins(a) Compared between pregnant women with preterm labor and intact membranes with and without placental inflammation(b) Compared between pregnant women with preterm PROM with and without placental inflammation*Statistically significant, p < 0.05

Table 4 Amniotic fluid concentration of antibacterial peptides in pregnant women with preterm labor and preterm prematurerupture of membranes (PROM) with and without placental inflammation

Outcomes of interest Prevalence Cut-off valueSensitivity

(%)Specificity

(%)Area(%) p Value

MIACBPI (ng/ml)HNP 1–3 (ng/ml)Calprotectin (mg/ml)Composite markers

22% (25/113)3.77.8

27.9any 2 or 3 markers ³ cut-off

80808080

79797883

87858484

< 0.001*< 0.001*< 0.001*< 0.001*

AF white blood cell count > 50 cells/mlBPI (ng/ml)HNP 1–3 (ng/ml)Calprotectin (mg/ml)Composite markers

21% (24/112)15.553.248.4

all markers ³ cut-off

92929292

96949398

96959494

< 0.001*< 0.001*< 0.001*< 0.001*

AF white blood cell count > 100 cells/mlBPI (ng/ml)HNP 1–3 (ng/ml)Calprotectin (mg/ml)Composite markers

21% (23/112)19.562.148.4

all markers ³ cut-off

91919191

97949298

96959494

< 0.001*< 0.001*< 0.001*< 0.001*

Spontaneous labor and delivery within 48 hBPI (ng/ml)HNP 1–3 (ng/ml)Calprotectin (mg/ml)Composite markers

25% (23/93)2.73.4

18.5any 2 or 3 markers ³ cut-off

83837883

81717382

89878285

< 0.001*< 0.001*< 0.001*< 0.001*

Spontaneous labor and delivery within 72 hBPI (ng/ml)HNP 1–3 (ng/ml)Calprotectin (mg/ml)Composite markers

29% (27/93)1.52.8

18.5any 2 or 3 markers ³ cut-off

85857485

80687477

88858286

< 0.001*< 0.001*< 0.001*< 0.001*

Spontaneous labor and delivery within 7 daysBPI (ng/ml)HNP 1–3 (ng/ml)Calprotectin (mg/ml)Composite markers

38% (35/93)1.22.8

15.2any 2 or 3 markers ³ cut-off

83838083

84746281

88878187

< 0.001*< 0.001*< 0.001*< 0.001*

MIAC, microbial invasion of the amniotic cavity; AF, amniotic fluid; BPI, bactericidal/permeability-increasing protein;HNP, human neutrophil defensins

Table 5 Diagnostic indices of each antibacterial peptide in amniotic fluid of patients with preterm labor and intact membranes

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In most cases, these three mechanisms are believed tosucceed in preventing the establishment of an infection42.

Antimicrobial peptides are ancient members of theinnate immune system and have been described in plantsand animals (invertebrate and vertebrate), and thus arethought to have played a role in the successful evolution ofcomplex multicellular organisms46. Two major classes ofantimicrobial peptides in mammals have been described:defensins and cathelicidins46. An interesting feature ofantimicrobial peptides is their great diversity (for a cata-log see http://www.bbcm.univ.trieste.it/~(tossi/pag1.htm),

thought to reflect the necessary adaptation of differentspecies to the microbial microenvironment, including thatof food required for the survival of many species46. Forexample, antimicrobial peptides of the frog have not beenconserved in humans47.

Antimicrobial peptides are capable of targeting micro-bial membranes, and not plant or animal cells. This selec-tivity appears to relate to the different properties ofmicrobial and non-microbial membranes. Specifically, theouter layer of bacteria is composed of lipids with negativelycharged phospholipid groups, while the same layer of plant

Antimicrobial peptides in amniotic fluid Espinoza et al.

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Figure 6 Receiver-operating characteristic (ROC) curves of bactericidal/permeability-increasing protein (BPI), human neutrophildefensins (HNP) 1–3, calprotectin and the combined markers (composite marker) in women with preterm labor and intact membranes.(a) ROC curve for the identification of positive amniotic fluid culture for micro-organisms. Areas under the curve for amniotic fluidBPI, 0.87; amniotic fluid HNP 1–3, 0.85; amniotic fluid calprotectin, 0.84, and composite marker, 0.84 (p < 0.001 for all). (b) ROCcurve for the identification of intra-amniotic inflammation (defined as amniotic fluid white blood cell count ³ 50 cells/ml). Areas underthe curve for amniotic fluid BPI, 0.96; amniotic fluid HNP 1–3, 0.95; amniotic fluid calprotectin, 0.94; and composite marker, 0.94(p < 0.001 for all)J

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and animal cells is composed of lipids with no net charge.However, selectivity is imperfect, and it is increasinglybeing recognized that some antimicrobial peptides maybe cytotoxic to mammalian cells (e.g. defensins, NK-lysin,BMAP-27 and BMAP-2848). Since some of these cytotoxiceffects are mediated by the induction of apoptosis48, it ispossible that antimicrobial peptides in the amniotic cavitymay participate in the mechanism of membrane ruptureby inducing cytotoxicity of amnion cells.

The precise mechanism of antimicrobial activity isincompletely understood. Some antimicrobial peptidesfollow the Shai–Matsuzaki–Huang model, which proposesbinding of the bacterial membrane’s outer leaflet with theantimicrobial peptide, integration of the peptide intothe membranes, formation of pores, and sometimes trans-port of the lipids and peptides into the inner leaflet46,49–51.Antimicrobial peptides operating through this model areeffective at micromolar concentrations, while those usingother models achieve bacterial killing at nanomolar con-centration (e.g. nisin)52. The proposed mechanisms bywhich microbial killing is achieved include the creation ofpores allowing leakage of microbial content, activationof enzymes that degrade the bacterial cell wall, disruptionof the architecture and function of the microbial wall, andfatal depolarization of the membrane46,49–51.

Over the past few decades, considerable focus has beenplaced on mechanisms responsible for microbial killing byhost cells53. Activated neutrophils increase oxygen con-

sumption during the course of inflammatory responses –‘the respiratory burst’ – and this results in the transfer ofelectrons to molecular oxygen and the generation of re-active oxygen species, which participate in microbialkilling. However, an increasing body of evidence indicatesthat oxygen-independent killing of micro-organisms is alsopossible. Indeed, neutrophils deprived of oxygen in vitrohave microbicidal activity54 and acid extracts of neutro-phils can kill bacteria53, as can cationic proteins andpeptides isolated from such extracts55.

Neutrophil antimicrobial peptides can be released out-side the cells (e.g. into amniotic fluid or cervical mucus) orinside the cells into the phagolysosome, where micro-organisms would be exposed to extremely high concentra-tions of these agents35. In addition to their role in innateimmunity, antimicrobial peptides can also participate inthe initiation of the adaptive immune response by anumber of mechanisms including chemotaxis of immaturedendritic cells56, CD4/CD45RA+ (naive memory) andCD8+ T cells by alpha defensins57, and chemotaxis ofCD45RO+ (memory T cells) by human beta defensin-258.

The current investigation was undertaken as part of along-term research program to determine why some womendevelop intrauterine infection during or before pregnancywhile others do not. Work by several groups including ourshas focused on the potential role of antimicrobial peptidesin the lower genital tract (cervix, vaginal fluid, amnion,chorion, endometrium, etc.)1,2,4–7,9,59,60. Amniotic fluid has

13

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Sensitivity SpecificityOdds ratio with

95% CIMarkers % n % n

Gram stain positiveAmniotic fluid WBC count ³ 50 cells/mlAmniotic fluid glucose £ 14 mg/dlGram stain positive plus amniotic fluid WBC count ³ 50 cells/mlGram stain positive plus amniotic fluid glucose £ 14 mg/dlAmniotic fluid WBC count ³ 50 cells/ml plus glucose £ 14 mg/dlGram stain positive plus amniotic fluid WBC count ³ 50 cells/ml

plus amniotic fluid glucose £ 14 mg/dlBPI ³ 3.7 ng/mlHNP 1–3 ³ 7.8 ng/mlCalprotectin ³ 27.9 mg/mlComposite markers (any 2 or 3 antibacterial peptides with

concentration ³ cut-off)Gram stain positive plus amniotic fluid WBC count ³ 50 cells/ml

plus composite markers

44684480607680

80808080

88

11/2517/2511/2520/2515/2519/2520/25

20/2520/2520/2520/25

22/25

99927992797373

79797883

81

85/8679/8668/8679/8668/8663/8663/86

68/8668/8667/8671/86

70/86

66.7 (7.9–558.4)23.9 (7.6–75.1)

2.9 (1.2–7.6)45.1 (12.9–157)

5.7 (2.2–14.7)8.6 (3.0–24.4)

10.9 (3.7–32.6)

15.1 (4.9–45.8)15.1 (4.9–45.8)14.1 (4.7–42.6)18.9 (6.1–58.4)

32.1 (8.5–120.5)

BPI; bactericidal/permeability-increasing protein; HNP, human neutrophil defensins; CI, confidence interval

Table 6 Diagnostic indices of various amniotic fluid antibacterial peptide concentrations and other previously published markers(amniotic fluid Gram stain, white blood cell (WBC) count ³ 50 cells/ml and glucose concentration £ 14 mg/dl) for the identification ofmicrobial invasion of the amniotic cavity (n = 25) in patients with preterm labor and intact membranes who had the results for everymarker (n = 111)

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been known to have antimicrobial activity19–25, but itsmolecular basis is unknown. The current study focused onthree naturally occurring antimicrobial agents: HNP 1–3,BPI and calprotectin. Previous studies by our group haveexamined amniotic fluid lactoferrin61, phospholipase A2

62

as well as other enzymes implicated in host defense such as

neutrophil elastase63, neutrophil collagenase (MMP-8)64–66

and matrilysin (MMP-7)67.Defensins are small cationic antimicrobial peptides that

have been classified according to their size and patternof disulfide bonding into alpha, beta and theta defensins68.Alpha defensins are abundant in neutrophils, some

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Interval from amniocentesis to delivery (h)

Cut-off values n Median Range p Value

BPI (ng/ml)

HNP 1–3 (ng/ml)

Calprotectin (mg/ml)

Composite markers

³ 15.5< 15.5³ 53.2< 53.2³ 48.4< 48.4

all markers above the cut-offnot all markers above the cut-off

2787288628852490

14636

19675

24624

13588

2–6511.5–2904

2–6511.5–2904

2–8401.5–2904

2–6511.5–2904

< 0.001*

< 0.001*

< 0.001*

< 0.001*

*Statistically significant, p < 0.05BPI; bactericidal/permeability-increasing protein; HNP, human neutrophil defensins

Table 7 Comparisons of interval from amniocentesis to delivery in patients who had amniotic fluid antibacterial peptideconcentration above the cut-off values for the identification of intra-amniotic inflammation (white blood cell count ³ 50 cells/ml)and those who did not

Beta Wald Hazard ratio 95% CI

BPI (ng/ml)BPI ³ 15.5 ng/mlMIACGestational age (weeks)Cervical dilatation (cm)

1.50.70.10.3

8.42.39.2

13.5

4.32.01.11.4

1.6–11.50.8–5.0

1.04–1.181.2–1.7

HNP 1–3 (ng/ml)HNP 1–3 ³ 53.2 ng/mlMIACGestational age (weeks)Cervical dilatation (cm)

1.60.60.10.3

13.12.0

10.314.6

4.91.91.11.4

2.1–11.90.8–4.4

1.04–1.191.2–1.7

Calprotectin (mg/ml)Calprotectin ³ 48.4 mg/mlMIACGestational age (weeks)Cervical dilatation (cm)

0.81.20.080.3

5.411.9

6.812.2

2.23.41.11.4

1.1–4.21.7–6.9

1.02–1.161.2–1.6

Composite markersAll markers ³ cut-offMIACGestational age (weeks)Cervical dilatation (cm)

1.40.80.10.3

7.62.98.5

13.9

3.92.21.11.4

1.5–10.10.8–5.4

1.03–1.181.2–1.7

BPI; bactericidal/permeability-increasing protein; MIAC, microbial invasion of the amniotic cavity; HNP, human neutrophil defensins;CI, confidence interval

Table 8 Hazard ratio of amniotic fluid concentration of antibacterial peptides above the cut-off value used for the identificationof intra-amniotic inflammation (³ 50 cells/ml) in relation to interval from amniocentesis to delivery (h)

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macrophage populations, and Paneth cells of the smallintestine69–71. Some alpha defensins are produced byepithelial cells such as alpha defensin 51. Beta defensinsare present mostly in the epithelium of several organs,including the bronchial tree and epidermis72–74. Thetadefensins (also known as circular mini-defensins) havebeen identified only in primate phagocytes to date75. Theantimicrobial spectrum of defensins is broad and includesbacteria, fungi and some enveloped viruses in vitro71,76,77.Alpha and beta defensins have limited antimicrobialactivity in fluids with physiological concentrations of salt(e.g. 150 mmol/l of NaCl)71,76, and thus their antimicrobialactivity is more likely to take place inside neutrophilsand macrophages, as well as on the surface of mucousmembranes71,76. Neutrophil defensins include HNP-1, 2and 3, and comprise 30–50% of total protein in azurophilgranules of human neutrophils. HNP 1–3 are also chemo-tactic to monocytes, immature dendritic cells and T cells26.In the presence of microbial invasion, the concentrationsof defensins increase in relevant biological fluids such as

peripheral blood78, amniotic fluid79, pleural fluid, broncho-alveolar lavage, urine and cerebrospinal fluid80.

BPI is found in the membrane, lumen of the azurophilicgranules of the neutrophil33,81 and eosinophil32. BPI haseffects not only in micro-organisms, but also in host cells(for example, it modulates the inflammatory response tolipopolysaccharide (LPS)). The cationic BPI would bindto the negatively charged microbial surface34,82. The anti-microbial effects against Gram-negative bacteria aremediated by: (1) damage to the outer and inner lipid mem-branes of microorganisms34,35,83,84; (2) enhancement ofphagocytosis by neutrophils85,86; and (3) binding andneutralizing of the lipid A moiety of LPS30,87,88, probably byincreasing the size of the LPS aggregates and preventingthe delivery of LPS monomers to the CD14 receptor, thuspreventing cell activation and production of pro-inflammatory cytokines35. Interestingly, Levy reported thatneutrophils of newborns at term contain the same amountsof HNP 1–3, but at least three- to four-fold less BPI per cellthan adult neutrophils89. There is substantial evidence

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Figure 7 Amniocentesis-to-delivery interval according to the amniotic fluid concentrations of bactericidal permeability-increasingprotein (BPI), human neutrophil defensins (HNP) 1–3, calprotectin and the composite marker. Patients with amniotic fluidconcentrations of BPI ³ 15.5 ng/ml, HNP 1–3 ³ 53.2 ng/ml and calprotectin ³ 48.4 mg/ml had a significantly shorter amniocentesis-to-delivery interval than patients below those cut-off values

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that Gram-negative bacteria and LPS increase the plasmaconcentration of BPI90. Experimental endotoxemia inhealthy human volunteers or the administration of recom-binant tumor necrosis factor (TNF)-a increased theconcentration of neutrophil surface-bound BPI, as wellas the plasma concentration of BPI91,92. Furthermore,simultaneous administration of LPS of Escherichia coli andrecombinant BPI (rBPI23) to human volunteers reducedthe circulating endotoxin concentrations and resulted inthe reduction in the plasma concentration of TNF, solubleTNF receptors p55 and p75, interleukin (IL)-6, IL-8 andIL-10, most likely by preventing the interaction of LPSwith the CD14 receptor on the macrophage surface93.Similar results were reported in experimental E. colibacteremia in baboons94. Thus, BPI is thought todown-regulate the pro-inflammatory response elicited byLPS.

The existence of antimicrobial activity in the cytoplasmof neutrophils has been known for many years. Indeed,neutrophil death with release of their cellular content wasconsidered a mechanism of defense against Candida albicansinfections95. This antimicrobial activity of the neutrophilcytoplasm (and not of its granules) was attributed to anabundant calcium-binding protein96, subsequently identi-fied as calprotectin or calcium-binding leukocyte L1protein97,98, the cystic fibrosis antigen99, MRP 8 andMRP 14100 and calgranulins A and B101. Calprotectin(MRP8/14) is a protein that belongs to the S100 familyof calcium-binding proteins. The calprotectin complex iscomposed of 8- and 14-kDa subunits and represents about50% of the total cytosolic protein of neutrophils36,although it is also present in macrophages and epidermis102.The antimicrobial activities of calprotectin against bacteriaand fungi appear to be mediated by zinc chelation103.Calprotectin is elevated in plasma of patients with activepulmonary tuberculosis, and the plasma level depends onthe volume of lung tissue involved104. In vitro experimentshave demonstrated an inverse correlation between neutro-phil viability and calprotectin release, indicating thathuman calprotectin is not secreted extracellularly, butreleased upon neutrophil disruption37,38. Therefore, therelease of calprotectin into the amniotic fluid is likely toreflect neutrophil disruption and/or cell death.

Our results demonstrate that MIAC, preterm parturitionand prelabor rupture of membranes at preterm gestation areassociated with high amniotic fluid concentrations of HNP1–3, BPI and calprotectin. Spontaneous labor at term isassociated with a high amniotic fluid concentration ofdefensins, but not of BPI or calprotectin. Moreover,elevated amniotic fluid concentrations of BPI, HNP 1–3and calprotectin are associated with intra-amniotic inflam-mation, histological chorioamnionitis and shorter amnio-centesis-to-delivery interval in patients presenting withpreterm labor with intact membranes.

In our study, immunoreactive BPI was detectable inapproximately 10% of amniotic fluid samples obtainedduring the mid-trimester of pregnancy. In contrast,immunoreactive HNP 1–3 and calprotectin were detect-able in virtually all samples. These observations suggestthat BPI is not constitutively present in mid-trimesteramniotic fluid. The amniotic fluid concentration of BPIand calprotectin increased with advancing gestational age(see Figure 1). It is tempting to postulate that this may bebeneficial to the host in preparation for parturition at term,since the likelihood of MIAC is greater during labor thanin the absence of parturition. These findings may explain,at least in part, the increased amniotic fluid inhibitoryactivity against micro-organisms in the last trimester aspreviously reported25.

Our findings that MIAC in patients with preterm laborwith intact membranes and preterm PROM was associatedwith a significant increase in amniotic fluid concentrationof neutrophil defensins are consistent with the study ofHeine and co-workers79. They reported a high amnioticfluid concentration of neutrophil defensins in patients whohad a positive amniotic fluid culture for micro-organismsand/or histological chorioamnionitis79. To our knowledge,this is the first report of increased amniotic fluid concentra-tions of BPI and calprotectin in patients with MIAC.

In the context of infection and/or inflammation,amniotic fluid WBCs may be the source of these anti-bacterial peptides. The significant positive correlationbetween amniotic fluid WBC count and the amniotic fluidconcentration of each antimicrobial peptide examined inthis study supports this concept. Previous studies havesuggested that amniotic fluid WBCs are of fetal origin105.Thus, it is possible that antimicrobial peptides in theamniotic fluid are, at least in part, of fetal origin. Otherpotential sources of antimicrobial peptides may be theleukocytes in the fetal lung106,107, chorioamniotic mem-branes, placenta decidua, etc. Several years ago, our groupdemonstrated that human placenta, amnion and chorionexpressed mRNA for hematopoietic defensins, as demon-strated by the polymerase chain reaction (PCR), Southernblot analysis and partial sequencing of products9. There isno report, to date, regarding the expression of BPI andcalprotectin in chorioamniotic membrane, placenta ordecidua.

Elevated amniotic concentrations of BPI, HNP 1–3 andcalprotectin are associated not only with MIAC, but alsointra-amniotic inflammation, histological chorioamnio-nitis and shorter interval from amniocentesis to deliveryin patients with preterm labor and intact membranes.Previous studies have examined the value of several cyto-kines or enzymes released in the identification of patientswith MIAC or intra-amniotic inflammation during thecourse of inflammation64,67,108–116. We also examinedthe potential value of the antimicrobial peptides in this

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study. For this purpose, the study was restricted to patientswith preterm labor and intact membranes. The best diag-nostic performance of amniotic fluid antimicrobial peptideconcentration was in the identification of intra-amnioticinflammation (defined as an amniotic fluid WBC countabove 50 cells/ml). Antimicrobial peptides had a sensi-tivity of 92% and a specificity of 93–98%. Similarly,amniotic fluid antimicrobial peptide concentration wasrelated to the detection of histological evidence of chorio-amnionitis in patients who delivered within 72 h ofamniotic fluid retrieval. Ninety-five per cent (19/20) ofpatients who had elevated HNP 1–3 (³ 53.2 ng/ml) or BPI(³ 15.5 ng/ml) and delivered within 72 h also had histo-logical chorioamnionitis and/or funisitis. There were fourpatients who had evidence of inflammation in theplacenta, but did not have elevated amniotic fluid concen-tration of the antimicrobial peptides. This observation mayrepresent extra-amniotic infection in the chorio-amnioticspace, which had not reached the amniotic cavity. Indeed,three out of the four patients had negative amniotic fluidculture for micro-organisms. Another possibility is thatmicro-organisms invaded the amniotic cavity after amnio-centesis. It is noteworthy that nine out of ten patients whodelivered within 72 h and showed no evidence of inflam-mation in the extraplacental membranes or umbilical cordhad amniotic concentration of BPI or HNP 1–3 below thecut-off level used in this study for the identification ofintra-amniotic inflammation.

Among patients with preterm labor with intact mem-branes, those with amniotic HNP 1–3 ³ 53.2 ng/ml or BPI³ 15.5 ng/ml had the highest risk for early spontaneouspreterm delivery. Indeed, the risk conferred by an elevationof antimicrobial peptides was higher than that of otherfactors, such as amniotic fluid culture positive for micro-organisms, gestational age and even cervical dilatation atthe time of amniocentesis (see Table 8). This observation isconsistent with those of other studies66,117,118 that reporteda strong association between intra-amniotic inflammationand a shorter interval to delivery in patients with pretermlabor and intact membranes.

The abnormally high concentrations of all threeantimicrobial peptides examined in this study were notassociated with an increased likelihood of early deliverycompared to an elevation of either HNP 1–3 or BPI alone.However, elevation of the three was better than a singleelevation of calprotectin (MRP8/14 ³ 48.4 mg/ml) in pre-dicting interval to delivery (see Table 8).

Comparisons of previously published markers for theidentification of MIAC (Gram stain, WBC count andglucose concentration in amniotic fluid) and the amnioticfluid concentration of the three antimicrobial peptidesrevealed that an amniotic fluid concentration of HNP 1–3of ³ 3.7 ng/ml, BPI of ³ 7.8 ng/ml or calprotectin of³ 27.9 mg/ml, individually, had a sensitivity of 80% for the

identification of MIAC (see Table 5). This high sensitivityis comparable to previous reports of amniotic fluid matrixmetallaproteinase (MMP)-8 concentration (82%)66 andthe amniotic fluid WBC count of ³ 50 cells/ml accompa-nied by Gram stain in this study (80%) (see Table 6).However, the 78–79% specificity reported herein is lowerthan that provided by the Gram stain and amniotic fluidWBC count (92%), but similar to that of MMP-8 (78%).Interestingly, the combined use of the composite anti-microbial peptide marker with Gram stain and amnioticfluid WBC count increased the sensitivity to 88%, with aspecificity of 81% for the identification of MIAC.

In the absence of MIAC, both preterm parturition andpreterm PROM were associated with increased amnioticfluid concentrations of defensins, BPI and calprotectin.Amniotic fluid culture has limitations in the detection ofMIAC, which we have discussed elsewhere119. Modernmicrobiological techniques have the potential to increasethe detection of infection when compared to that ofstandard microbiological techniques119–121. It is possiblethat many patients with intra-amniotic inflammation haveinfections that standard microbiological techniques cannot detect.

Our finding that, in the absence of intrauterine infec-tion, pregnant women in labor at term had a two-foldincrease in amniotic fluid concentration of defensins com-pared to women at term and not in labor is consistent withthe study of Heine and associates79, although in their reportthe difference did not reach statistical significance. Laborat term has been likened to an inflammatory response109.Indeed, there is an association between spontaneous laborat term and increased mRNA expression and concentra-tion of IL-1b, IL6, IL-8 and TNF-a in amnion, chorion anddecidua122–125. It is possible that this low-grade inflamma-tory process accounts for the modest elevation of amnioticfluid defensins observed in spontaneous labor at term.

In conclusion, antimicrobial peptides can be detectedin the amniotic fluid of patients with MIAC andintra-amniotic inflammation, and their concentration inamniotic fluid relates to the risk for impending pretermdelivery and histological chorioamnionitis. Elevated con-centrations of HNP 1–3, BPI and calprotectin have similardiagnostic indices in the identification of intra-amnioticinfection and inflammation to those of other cytokines andenzymes released during the course of inflammation.

ACKNOWLEDGEMENTS

The authors wish to acknowledge the contributions of thenursing staff of the Detroit Medical Center: Ms. SandyField, Ms. Vicki Ineson, Ms. Mabubeh Mahoudieh, Ms.Milagros Kitchen, Ms. Cozette Kelly-Roberts, Ms. DeAndrea Duncan-Tolbert, Ms. Cindy Urbanik, Ms. Audrey

17

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Milliken, Ms. Leandra Ga-Pinlac, and Ms. Kathy Firchau;and the contributions of Ms. Lisa Palmer for expert andtimely data entry and verification of accuracy.

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