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Amniotic Fluid Matrix Metalloproteinase-9 Levels in Women With Preterm Labor and Suspected Intra-amniotic Infection

From the Department of Obstetrics and Gynecology, University of Florida College of Medicine, Gainesville, Florida.

Address reprint requests to: Gregory J. Locksmith, MD, Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Boulevard, Route 0587, Galveston, TX 77555-0587, E-mail: gjlocksm{at}utmb.edu

	Abstract

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Objective: To determine the accuracy of amniotic fluid (AF) matrix metalloproteinase-9 measurements for diagnosing intra-amniotic infection in women with preterm labor.

Methods: We performed amniocenteses in 44 women between 22 and 35 weeks’ gestation who presented to our center with preterm labor and clinical suspicion of intra-amniotic infection. Each sample was analyzed by glucose measurement, Gram stain, and culture for aerobes, anaerobes, and mycoplasmas. We tested the AF for matrix metalloproteinase-9 using gelatin zymography and a commercial enzyme-linked immunosorbent assay (ELISA) system. We calculated accuracy and confidence intervals (CIs) for AF matrix metalloproteinase-9, glucose, and Gram stain for diagnosing intra-amniotic infection, using culture as the criterion standard.

Results: All patients who had matrix metalloproteinase-9 detectable by ELISA also demonstrated matrix metalloproteinase-9 by zymography. Six cases of intra-amniotic infection were confirmed by culture (prevalence 14%). The performance statistics of AF matrix metalloproteinase-9 for diagnosing intra-amniotic infection were: sensitivity 83% (95% CI 53, 99), specificity 95% (95% CI 88, 99), positive predictive value 71% (95% CI 37, 99), and negative predictive value 97% (95% CI 92, 99). Two women had false-positive results; one had gram-negative rods on the AF Gram stain and developed clinical signs and symptoms of chorioamnionitis several hours after amniocentesis and the other had a purulent vaginal discharge and an AF glucose level less than 15 mg/dL. Both delivered within 24 hours of amniocentesis.

Conclusion: Measuring matrix metalloproteinase-9 in the AF appeared to be reliable for diagnosing intra-amniotic infection. An elevated matrix metalloproteinase-9 concentration in the AF at a preterm gestational age may portend imminent delivery regardless of microbiologic confirmation of intra-amniotic infection.

The relation between spontaneous preterm delivery and intrauterine infection has been clearly demonstrated over the past decade^1,2; however, the management of pregnant women with subclinical intra-amniotic infection before term is unclear. It is not certain whether perinatal outcome is optimized by attempting to prolong pregnancy with tocolytic and antimicrobial agents or by delivery. Despite this controversy, the development of rapid tests for identification of microbial invasion of the amniotic cavity has been an important research pursuit.

The standard for diagnosing intrauterine infection is microbiologic culture of amniotic fluid (AF) obtained by transabdominal amniocentesis. The main disadvantages of AF studies are slow culture times and inability to identify intrauterine extra-amniotic pathogens. Other diagnostic tests commonly used in practice include AF Gram stain, white blood cell count, glucose concentration, and interleukin-6. Of these tests, interleukin-6 is probably the most accurate predictor of positive AF culture,³ but it is limited by a low positive predictive value, relatively high expense, and limited availability. Amniotic fluid glucose analysis may be less sensitive than interleukin-6 in detecting intra-amniotic infection but is less expensive, can be done rapidly, and is more readily available.

The matrix metalloproteinases are a family of enzymes that use zinc-dependent catalytic mechanisms to degrade extracellular matrix components. Interstitial collagenase (matrix metalloproteinase-1) cleaves collagen types I, II, and III. The gelatinases (matrix metalloproteinase-2 and -9) further degrade the denatured collagen fragments generated by interstitial collagenase. The gelatinases also cleave various basement membrane components and proteoglycans.^4,5 Previous work has demonstrated that matrix metalloproteinases and their natural inhibitors are produced by amnion, chorion, and decidua and are important in the maintenance and breakdown of the extracellular matrix of the amniochorion and cervix.⁶ Some matrix metalloproteinases (matrix metalloproteinase-1 and matrix metalloproteinase-2) are produced in relatively stable amounts throughout pregnancy, but the production of others (matrix metalloproteinase-3 and matrix metalloproteinase-9) increases during labor.⁷ Chorioamnionitis induces matrix metalloproteinase-9 expression and release from the membranes.⁸

Our primary objective was to assess the accuracy of AF matrix metalloproteinase-9 analysis for detecting intra-amniotic infection in women with preterm labor, intact membranes, and signs and symptoms consistent with subclinical chorioamnionitis. We used AF Gram stain and glucose analysis for comparison. We also sought to determine the importance of elevated quantities of matrix metalloproteinase-9 in the AF by analyzing the pregnancy outcomes of our subjects.

	Materials and Methods

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From July 1, 1996 through May 31, 1998, we performed amniocenteses in 44 women between 22 and 35 weeks’ gestation who presented to the Labor and Delivery Department at Shands Hospital at the University of Florida with preterm labor and clinical suspicion of intra-amniotic infection. Our patient population is 90% indigent and mainly rural. Preterm labor was defined as the presence of regular uterine contractions, at least four per hour, plus documented cervical change, cervical dilatation of at least 2 cm at presentation, or effacement of at least 80% at presentation. Suspicion of intra-amniotic infection was based on maternal fever, fetal tachycardia, or poor response to tocolytic agents. Eligible subjects who consented to amniocentesis were included in the study. We excluded women with ruptured membranes, antibiotic use within 7 days of presentation, and cervical dilatation greater than 5 cm.

Our purpose in performing amniocentesis was to evaluate intra-amniotic infection. In addition to giving informed consent for the procedures, all subjects signed a separate Institutional Review Board consent form to allow analysis of their AF in this study. After collection, all samples were transported to the laboratory for glucose concentration analysis, Gram stain, and culture for aerobes, anaerobes, and mycoplasmas. We saved 3–5 mL of fluid, centrifuged it at 3000 rpm for 5 minutes, and stored the supernatant at -70C to be used for matrix metalloproteinase-9 measurement. Results of the glucose, culture, and Gram stain analyses were used to make management decisions for our subjects, but metalloproteinase levels were not.

Trained personnel in the microbiology laboratory used standard reagents to perform Gram stain on the AF. Fluid aliquots for microbiologic analysis were placed immediately into a 5-mL capped syringe that was devoid of air. These specimens were inoculated within 2 hours of collection and incubated at 37C in a 5% CO₂ environment. Aerobic specimens were inoculated into brain-heart infusion broth and onto blood agar plates. Anaerobic specimens were inoculated into thioglycolate broth and anaerobic blood agar plates and were kept in closed anaerobic systems. Mycoplasma specimens were inoculated into arginine broth, 10B broth, or A₃B broth. We measured the AF glucose concentration by the hexokinase ultraviolet method using the Hitachi model 747 automated analyzer (Boehringer Mannheim Corporation, Indianapolis, IN) after centrifuging the sample for 10 minutes at 3000 rpm.

Gelatin zymography provided a qualitative assessment of the presence of matrix metalloproteinase-9 along with its companion gelatinase, matrix metalloproteinase-2. We used precast zymogram gels (Novel Experimental Technology, San Diego, CA). The methods have been reported previously.⁹ Gelatinases appeared as clear bands against a dark blue background where the protease digested the substrate.

We used a commercial, two-site enzyme-linked immunosorbent sandwich assay (ELISA) (Amersham Life Science, Arlington Heights, IL) to quantitate total matrix metalloproteinase-9 levels in the AF samples. This assay measures concentrations most reliably in the 1–64-ng/mL range. We expected a broad range of matrix metalloproteinase-9 concentrations in our subjects, so all samples were diluted at 1:10 for preliminary analysis. The lower limit of detection for this assay, given this ten-fold dilution, was 6 ng/mL. Samples that contained greater than 640 ng/mL were diluted at 1:100 for final analysis. The intra-assay coefficient of variation for this assay was 5.5% and the interassay coefficient of variation was 9.8%. Each sample was analyzed in duplicate. Matrix metalloproteinase-9 concentrations were determined by interpolation from a curve constructed from known standards.

A required sample size of 45 was estimated assuming that a valuable test predicts the presence or absence of infection for 80% of measurements, compared with 50% accuracy for chance alone. The confidence coefficient was 95% and the desired power level was 80%. We constructed receiver operating characteristic (ROC) curves for matrix metalloproteinase-9 and for glucose concentration. The ROC curve for matrix metalloproteinase-9 was tested for significance against those of the null hypothesis and glucose analysis by comparing the areas under the curves using a modification of the paired t test¹⁰ with the Bonferroni correction (Epistat Services, Richardson, TX). We also compared the accuracy (number of subjects correctly identified as infected or uninfected divided by the total number of subjects) of AF matrix metalloproteinase-9 assessment versus that of chance alone using the z test comparison of proportions with Yates correction for continuity.

We used the Yates-corrected z test for the analysis of demographic characteristics. The Mann-Whitney U test was used to compare matrix metalloproteinase-9 concentrations between infected and uninfected subjects. Sensitivity, specificity, positive predictive value, and negative predictive value of AF Gram stain, glucose (15 mg/dL or less), and matrix metalloproteinase-9 for diagnosing intra-amniotic infection were calculated with 95% confidence intervals (CIs), using culture as the criterion standard.

	Results

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Table 1 presents the demographic characteristics of culture-positive and culture-negative subjects. Women with intra-amniotic infections were more likely to be black and to present at a very early gestational age compared with those with negative cultures.

View larger version (46K): [in this window] [in a new window]   Figure 1. A gelatin zymogram depicting gelatinases (matrix metalloproteinase-2 and -9) in amniotic fluid (AF) samples. Lane 1 contains molecular-weight standards. Lanes 2, 3, and 6 contain AF samples that were culture-positive and matrix metalloproteinase-9–positive by enzyme-linked immunosorbent assay (ELISA). Lane 4 contains a sample that was culture-negative but matrix metalloproteinase-9–positive by ELISA. Lanes 5 and 7–9 contain AF samples that were culture-negative and matrix metalloproteinase-9–negative by ELISA. Lane 10 contains a matrix metalloproteinase-9 standard (92 kd). Note that matrix metalloproteinase-2 (72 kd) appears to be present in all AF samples but matrix metalloproteinase-9 is not.

Figure 2 shows the ROC curves for AF matrix metalloproteinase-9 and AF glucose. Using ROC analysis, AF matrix metalloproteinase-9 was significantly better than chance alone (P < .001) at diagnosing intra-amniotic infection. Analysis of AF matrix metalloproteinase-9 was not superior to AF glucose assessment for diagnosing intra-amniotic infection (P > .99).

View larger version (15K): [in this window] [in a new window]   Figure 2. Receiver operating characteristic curves for amniotic fluid (AF) matrix metalloproteinase-9 (closed circles) and AF glucose (open circles) for diagnosing intra-amniotic infection in women with preterm labor.

	Discussion

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The performance statistics of AF matrix metalloproteinase-9 analysis were similar to those of AF glucose concentration^3,11 and AF interleukin-6 concentration³ from previous studies. Although we found a higher specificity and positive predictive value for AF matrix metalloproteinase-9 than for AF glucose, their ROC curves did not differ significantly. The one subject with a false-negative result for matrix metalloproteinase-9 delivered at term, without any treatment, 4 weeks after amniocentesis. The organism cultured from this woman, Xanthomonas maltophilia, is a ubiquitous organism commonly found on the skin, in hospital water conduits, in contaminated cell cultures, and in deionized water.¹² We believe the organism was a contaminant rather than a true pathogen.

The 71% positive predictive value of AF matrix metalloproteinase-9 may be explained by failure of our culture methods to diagnose intrauterine infection or by the production of protease under other conditions that are independent of infection. Of the two subjects with negative AF cultures and elevated matrix metalloproteinase-9 levels, both had AF glucose levels less than 15 mg/dL and were delivered within 24 hours. One woman at 34 weeks had spontaneous labor and the other at 26 weeks developed a fever and had labor induced because of a clinical impression of chorioamnionitis.

Of the two known gelatinases, matrix metalloproteinase-9 is associated most specifically with intra-amniotic infection. Fortunato et al⁸ found matrix metalloproteinase-2 in the membranes of nonlaboring women and in women with intra-amniotic infections, but found matrix metalloproteinase-9 only in the membranes of women with intra-amniotic infection. They also showed that matrix metalloproteinase-9 gene expression could be induced in cultured membranes by exposure to lipopolysaccharide or peptidoglycan polysaccharide. Production of this enzyme also appears to be related to other physiologic and pathologic events during pregnancy. Investigators have found that matrix metalloproteinase-9 gene expression⁷ and enzyme levels in the membranes¹³ and AF¹⁴ increase markedly in term pregnancies during active labor. Others^14,15 have found elevated AF concentrations in women with preterm premature rupture of membranes. Tu et al¹⁶ discovered that plasma matrix metalloproteinase-9 concentrations were increased threefold in women who presented with spontaneous rupture of membranes or labor, but were not elevated significantly within 1 week of delivery. These findings suggest that an increase in matrix metalloproteinase-9 may be useful for predicting inevitable preterm delivery or membrane rupture in women with signs and symptoms of preterm labor, regardless of AF culture results. A negative result, however, does not appear to be of much value for ruling out inevitable delivery.

We did not analyze preterm delivery as an outcome variable because many of the women who delivered before term had labor induced or had cesarean delivery based on a clinical impression that the benefits of delivery outweighed those of pregnancy prolongation. Nevertheless, all seven subjects with elevated AF matrix metalloproteinase-9 delivered before term and within 2 weeks of amniocentesis. Six of these women delivered within 48 hours of amniocentesis and five at extremely preterm gestational ages (22–26 weeks). Of 33 women with undetectable matrix metalloproteinase-9 for whom delivery information was available, 19 (58%) delivered preterm, 13 (39%) delivered within 2 weeks, and four (12%) delivered within 2 days of amniocentesis. Two women were excluded from the analysis because of antibiotic exposure. Both had negative AF cultures but markedly elevated matrix metalloproteinase-9 levels and were delivered at 24 weeks’ gestation, within 24 hours of amniocentesis. One woman had labor induced and the other delivered spontaneously.

Amnion, chorion, and decidua are probable sources of matrix metalloproteinases in the AF. In cases of intra-amniotic infection, other cells also may produce these proteases. Activated neutrophils release and activate a wide variety of matrix metalloproteinases.^17,18 McGregor et al¹⁹ have shown that various commensal and pathogenic bacteria produce gelatinases, collagenases, and elastinases in vitro. Immunocytochemical localization studies of infected membranes should improve our understanding of the sources of matrix metalloproteinases in chorioamnionitis.

In this small study, the analysis of AF matrix metalloproteinase-9 concentration was an accurate method for diagnosing and ruling out culture-proved intra-amniotic infection. Analysis of matrix metalloproteinase-9 may also provide a high positive predictive value for diagnosing inevitable preterm delivery. Larger studies are needed to confirm these preliminary findings.

	Footnotes

 
Supported by a grant from the Southern Medical Association.

PII S0029-7844(99)00011-3

Received September 24, 1998. Received in revised form December 1, 1998. Accepted December 30, 1998.

	References

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