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Clinical significance of intra-amniotic inflammation in patients with preterm labor and intact membranes
Clinical significance of intra-amniotic inflammation in patients with preterm labor and intact membranes Bo Hyun Yoon, MD, PhD, Roberto Romero, MD, Jeong Bin Moon, MD, Soon-Sup Shim, MD, Miha Kim, MD, Gilja Kim, MT, and Jong Kwan Jun, MD, PhD Seoul, Korea OBJECTIVE: The purpose of this study was to determine the frequency and clinical significance of intraamniotic inflammation in patients with preterm labor and intact membranes. STUDY DESIGN: Amniocentesis was performed in 206 patients with preterm labor and intact membranes. Amniotic fluid was cultured for aerobic and anaerobic bacteria and mycoplasmas. The diagnosis of intraamniotic inflammation was made in patients with a negative amniotic fluid culture on the basis of amniotic fluid concentrations of interleukin-6 (>2.6 ng/mL, derived from receiver operating characteristic curve analysis). Statistical analysis was conducted with contingency tables and survival techniques. RESULTS: Intra-amniotic inflammation (negative amniotic fluid culture but elevated amniotic fluid interleukin-6) was more common than intra-amniotic infection (positive amniotic fluid culture regardless of amniotic fluid interleukin-6 concentration; 21% [44/206 women] vs 10% [21/206 women]; P < .001). The amniocentesisto-delivery interval was significantly shorter in patients with intra-amniotic inflammation than in patients with a negative culture and without an inflammation (median, 20 hours [range, 0.1-2328 hours] vs median, 701 hours [range, 0.1-3252 hours], respectively; P < .0001). Spontaneous preterm delivery of <37 weeks was more frequent in patients with intra-amniotic inflammation than in those with a negative culture and without inflammation (98% vs 35%; P < .001). Patients with intra-amniotic inflammation had a significantly higher rate of adverse outcome than patients with a negative culture and without intra-amniotic inflammation. Adverse outcomes included clinical and histologic chorioamnionitis, funisitis, early preterm birth, and significant neonatal morbidity. There were no significant differences in the rate of adverse outcomes between patients with a negative culture but with intra-amniotic inflammation and patients with intra-amniotic infection (positive culture regardless of amniotic fluid interleukin-6 concentration). CONCLUSION: Intra-amniotic inflammation/infection complicates one third of the patients with preterm labor (32%; 65/206 women), and its presence is a risk factor for adverse outcome. The outcome of patients with microbiologically proven intra-amniotic infection is similar to that of patients with intra-amniotic inflammation and a negative amniotic fluid culture. We propose that the treatment of patients in preterm labor be based on the operational diagnosis of intra-amniotic inflammation rather than the diagnosis of intra-amniotic infection because the latter diagnosis cannot be undertaken rapidly. (Am J Obstet Gynecol 2001;185:1130-6.)
Key words: Amniotic fluid, infection, inflammation, interleukin-6, preterm labor, prematurity, chorioamnionitis
Microbial invasion of the amniotic cavity in patients with preterm labor and intact membranes is a risk factor for the progression of labor to preterm delivery despite tocolysis, spontaneous rupture of membranes, clinical
From the Department of Obstetrics and Gynecology, Seoul National University College of Medicine; and the Laboratory of Fetal Medicine Research, Clinical Research Institute, Seoul National University Hospital. Supported by grant 2000-N-NL-01-C-078 from the Korea Institute of Science and Technology Evaluation and Planning (KISTEP), Republic of Korea. Presented at the Twenty-first Annual Meeting of the Society for MaternalFetal Medicine, Reno, Nev, February 5-10, 2001. Reprint requests: Bo Hyun Yoon, MD, PhD, Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, Seoul National University College of Medicine, Seoul, 110-744, Korea. Copyright © 2001 by Mosby, Inc. 0002-9378/2001 $35.00 + 0 6/6/117680 doi:10.1067/mob.2001.117680
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chorioamnionitis, and adverse neonatal outcome (ie, sepsis, non-infection-related complications).1-10 The gold standard for the diagnosis of infection in clinical medicine is the isolation and identification of the micro-organism from body fluids and tissues of patients with suspected infection. However, results of microbial culture may take days, and are often not available in time for some clinical decisions. In contrast to the diagnosis of infection, the identification of intrauterine inflammation can be easily and rapidly detected by laboratory tests (such as an amniotic fluid white blood cell count and cytokine determinations). A substantial number of patients with preterm labor and intact membranes or preterm premature rupture of membranes have demonstrable intra-amniotic inflammation with a negative amniotic fluid culture.11-15 The frequency and clinical significance of intra-amniotic
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inflammation without microbial invasion remains to be determined. The purpose of this study was to determine the frequency and maternal/neonatal outcome of patients with intra-amniotic inflammation but with a negative amniotic fluid culture. Material and methods Study population. The study population consisted of consecutive patients admitted to Seoul National University Hospital between January 1993 and July 1998 with the diagnosis of preterm labor and intact membranes (<36 weeks of gestation) and singleton gestation who underwent amniocentesis. Amniocentesis is routinely offered to all patients who are admitted with the diagnosis of preterm labor at our institution. Amniocentesis was performed after written informed consent was obtained. The institutional review board of our institution approved the collection of biologic materials and data from these patients for research purposes. Patients were divided into 3 groups according to the results of amniotic fluid culture and amniotic fluid concentrations of interleukin-6 (IL-6). Group 1 consisted of 141 women with a negative amniotic fluid culture and without an elevated amniotic fluid IL-6 concentration; group 2 consisted of 44 women with a negative amniotic fluid culture but with an elevated amniotic fluid IL-6 concentration (intra-amniotic inflammation); group 3 consisted of 21 women with a positive amniotic fluid culture, regardless of amniotic fluid IL-6 concentration (intraamniotic infection). Amniotic fluid. Amniotic fluid was retrieved by transabdominal amniocentesis and was cultured for aerobic and anaerobic bacteria and for genital mycoplasmas (Ureaplasma urealyticum and Mycoplasma hominis) according to methods described in detail previously.14 Fluid that was not used for diagnostic studies was centrifuged and stored at –70°C until assayed. IL-6 concentrations were measured with a commercially available enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minn). The sensitivity of the test was <1.0 pg/mL. Intraand interassay coefficients of variation were <10%. Diagnosis of chorioamnionitis, funisitis, and neonatal morbidity. Clinical chorioamnionitis was diagnosed when a temperature was elevated to 37.8°C and when ≥2 of the following criteria were present: uterine tenderness, malodorous vaginal discharge, maternal leukocytosis (>15,000 cells/mm3), maternal tachycardia (>100 beats/min), and fetal tachycardia (>160 beats/min). Histologic chorioamnionitis was defined in the presence of acute inflammatory changes on examination of a membrane roll and chorionic plate of the placenta; funisitis was diagnosed in the presence of neutrophil infiltration into the umbilical vessel walls or Wharton’s jelly with the use of criteria published previously.14, 16 Neonatal sepsis, respiratory distress syndrome, pneumonia,
bronchopulmonary dysplasia, intraventricular hemorrhage, and necrotizing enterocolitis were diagnosed according to the definitions that were previously described in detail.14, 16 Statistical analysis. Proportions were compared with the Fisher exact test. The Kruskal-Wallis analysis of variance (ANOVA) test was used for comparison of continuous variables among the groups. Multiple comparisons among groups were performed with Mann-Whitney U tests. The McNemar test was used for comparison of proportions of correlated samples. The generalized Wilcoxon test for survival analysis was used to compare the amniocentesis-to-delivery interval. Patients delivered for maternal or fetal indications were treated as censored observations, with a censoring time equal to the amniocentesis-to-delivery interval. Logistic regression analysis was used to examine the relationship between the presence of intra-amniotic inflammation or positive amniotic fluid culture and outcome of interest (ie, preterm delivery, neonatal complications) after adjusting for the effects of confounding variables (gestational age and the degree of cervical dilatation). A probability value of <.05 was considered significant. Results Characteristics of study population. Table I compares characteristics of the study population. Patients with a negative amniotic fluid culture but with intra-amniotic inflammation (group 2) had a significantly lower mean gestational age at amniocentesis and more advanced cervical dilatation at presentation than patients with a negative culture and without intra-amniotic inflammation (group 1). In contrast, there were no differences in the clinical characteristics between patients in groups 2 and 3. Result of amniotic fluid culture. Amniocentesis was performed in 209 patients with preterm labor and intact membranes during the study period. The prevalence of positive amniotic fluid culture was 10% (21/209 patients). U urealyticum was the most common micro-organism isolated from the amniotic cavity (n = 14 patients). Other isolates included Staphylococcus aureus (n = 2 patients), Enterococcus spp (n = 2 patients), Acinetobacter spp (n = 2 patients), Lactobacillus spp (n = 2 patients), and 1 isolate each of Candida albicans, Porphyromonas asaccharolytica, and coagulase-negative Staphylococcus. Three patients with a negative amniotic fluid culture but without remaining amniotic fluid for IL-6 determinations were excluded from the further analysis because they could not be evaluated with respect to the presence or absence of intra-amniotic inflammation. Diagnosis of intra-amniotic inflammation. The diagnosis of intra-amniotic inflammation was based on amniotic fluid concentration of IL-6. Fig 1 displays a receiver operating characteristic curve that describes the performance of amniotic fluid IL-6 concentration in the identification
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Table I. Characteristics of study population according to the results of amniotic fluid culture and IL-6 concentrations Negative amniotic fluid culture
Characteristic Maternal age§ (y) Nulliparity (n) Gestational age at amniocentesis (wk) Cervical dilation at presentation (cm)
Low IL-6 (group 1; n = 141)
Positive amniotic fluid culture
P*
High IL-6 (group 2; n = 44)
P†
Group 3 (n = 21)
P‡
29.5 ± 4.3 74 (52%) 31.3 ± 3.2
NS < .05 < .001
29.7 ± 3.6 15 (34%) 28.5 ± 4.1
NS NS NS
30.2 ± 5.3 9 (43%) 29.1 ± 3.9
NS NS <.01
0.8 ± 1.1
< .001
3.3 ± 2.7
NS
2.6 ± 2.2
<.001
Values given as mean ± SD. Low IL-6, interleukin-6 <2.6 ng/mL; High IL-6, interleukin-6 >2.6 ng/mL; NS, not significant. *Comparison between groups 1 and 2. †Comparison between groups 2 and 3. ‡Comparison between groups 3 and 1. §P > .1, by Kruskal-Wallis ANOVA test. P < .001, by Kruskal-Wallis ANOVA test.
Fig 1. Receiver operating characteristic curve that describes the performance of amniotic fluid IL-6 concentration in the identification of a positive amniotic fluid culture (area under the curve, 0.84: SE, 0.04; z, 9.26; P < .0001).
of positive amniotic fluid culture (area under the curve, 0.84; SE, 0.04; z, 9.26; P < .0001). A value of 2.6 ng/mL was at the knee of the curve and was used to diagnose the presence of intra-amniotic inflammation. Prevalence of intra-amniotic inflammation. Amniotic fluid concentrations of IL-6 were higher than 2.6 ng/mL in 63 of 206 patients (31%) in the study, in 44 of 185 patients (24%) with a negative amniotic fluid culture (group 2, intra-amniotic inflammation), and in 19 of 21 patients (90%) with a positive amniotic fluid culture (group 3, intra-amniotic infection). Intra-amniotic inflammation (group 2) was more common than intraamniotic infection (group 3; 21% [44/206] vs 10% [21/206]; P < .001, McNemar test).
Fig 2. Frequency of a positive amniotic fluid (AF ) culture and intra-amniotic inflammation (negative AF culture but with an AF IL-6 of higher than 2.6 ng/mL) as a function of gestational age. The lower the gestational age, the higher the frequency of a positive amniotic fluid culture and intra-amniotic inflammation (P < .01 for culture and P < .001 for inflammation, respectively). Hash-marked columns represent intra-amniotic inflammation. Black columns represent positive amniotic fluid culture.
Fig 2 displays the frequency of a positive amniotic fluid culture and intra-amniotic inflammation as a function of gestational age. The lower the gestational age, the higher the frequency of a positive amniotic fluid culture and intraamniotic inflammation (P < .01 and P < .001, respectively). Amniocentesis-to-delivery interval. Fig 3 shows the interval from amniocentesis to delivery. In patients who were delivered because of maternal or fetal indications (n = 34 women), this interval was treated as a censored observation. Patients with a negative amniotic fluid culture but with intra-amniotic inflammation (group 2) had a significantly shorter median amniocentesis-to-delivery interval than women with a negative culture and without intra-amniotic inflammation (group 1; P < .0001). However, there was no difference in the median amniocentesis-to-delivery interval between patients with a negative
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amniotic inflammation (group 2) and 16% of the women (21/132) had a positive amniotic fluid culture (group 3). Thirty-four patients had maternal and/or fetal indication for preterm delivery. Of the remaining 172 women, 57% of the women (98/172 women) had a spontaneous preterm delivery. Forty percent of these patients with a spontaneous preterm delivery (39/98 women) had intraamniotic inflammation (group 2) and only 19% of these women (19/98 women) had a positive amniotic fluid culture (group 3). Comment
Fig 3. Survival analysis of amniocentesis-to-delivery interval (group 1: median, 701 hours [range, 0.1-3252 hours]; group 2, median, 20 hours [range, 0.1-2328 hours]; group 3, median, 11 hours [range, 0.1-983 hours]) according to the results of amniotic fluid (AF) culture and IL-6 concentrations. NS, Not significant.
amniotic fluid culture but with intra-amniotic inflammation (group 2) and patients with a positive culture regardless of amniotic fluid IL-6 concentration (group 3). Multivariate survival analysis demonstrated that the amniocentesis-to-delivery interval in patients with a negative amniotic fluid culture but with intra-amniotic inflammation (group 2) was significantly shorter than that of the group with a negative culture and without intra-amniotic inflammation (group 1) after the adjustment of gestational age and the degree of cervical dilatation at amniocentesis (hazards ratio, 3.1; 95% CI, 2.3-4.1; P < .0001, Cox proportional hazards model analysis). Outcomes of study population. Tables II and III compare pregnancy and neonatal outcomes of study population. Patients with a negative amniotic fluid culture but with intra-amniotic inflammation (group 2) had a significantly higher rate of adverse outcomes than patients with a negative culture and without intra-amniotic inflammation (group 1). Adverse outcomes included higher rates of clinical chorioamnionitis, preterm delivery, histologic chorioamnionitis, funisitis, low Apgar scores, admission to neonatal intensive care unit, neonatal respiratory distress syndrome, pneumonia, sepsis, intraventricular hemorrhage (≥grade II), and bronchopulmonary dysplasia and lower gestational age at birth and birth weight. However, no differences were found between patients with a negative amniotic fluid culture but with intra-amniotic inflammation (group 2) and patients with a positive culture regardless of amniotic fluid IL-6 concentration (group 3; P > .1). One hundred thirty-two patients delivered preterm newborns (<37 weeks); 33% of the women (43/132) had intra-
Our results indicate that intra-amniotic inflammation is more common than microbiologically proven intra-amniotic infection in patients with preterm labor and intact membranes (21% vs 10%; P < .001) and that intra-amniotic inflammation per se is associated with adverse maternal and neonatal outcome. A major finding of this study is that the maternal and neonatal outcome of patients with intraamniotic infection did not differ from that of patients with intra-amniotic inflammation without demonstrable intraamniotic infection. In regard to microbial invasion of the amniotic cavity, the prevalence, the type of microorganism, the inverse relationship with gestational age, and the association with adverse outcome are consistent with our previously reported observations and with reports of other investigators.1-10 Moreover, we observed that the frequency of intra-amniotic inflammation is double that of demonstrable intra-amniotic infection, also in keeping with observations previously reported by us15 and other authors.17 In this study, 40% of all patients with preterm labor who delivered a preterm neonate spontaneously had an elevated amniotic fluid IL-6 but negative amniotic fluid culture. What is the cause of the inflammatory process in cases of negative culture? We have previously reported18 that, with the use of the polymerase chain reaction with specific primers to U urealyticum, many patients with intra-amniotic inflammation but with a negative culture for U urealyticum had microbial footprints for this microorganism in the amniotic fluid. Moreover, these patients had cytologic, pathologic, biochemical, and clinical evidence of inflammation. Therefore, we suspect that some patients with intra-amniotic inflammation of unknown cause will eventually be proved to have infection that cannot be demonstrated today with the use of standard microbiologic techniques. These laboratory procedures demand knowledge on the culture conditions of all potential pathogens, information that clearly is not available. Yet, a subgroup of patients with inflammation in the amniotic cavity may have an extra-amniotic infection, with micro-organisms located primarily in the decidua or the space between chorion and amnion. Previous studies have demonstrated that amniotic fluid IL-6 may be elevated in these patients.15 Of course, we do not dismiss
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Table II. Pregnancy outcome of study population according to the results of amniotic fluid culture and interleukin-6 concentrations
Pregnancy outcome
Negative amniotic fluid culture High IL-6 P* P† (group 2; Unadjusted Adjusted§ n = 44) (Un)adjusted§#
(group 1; n = 141)
Gestational age 36.2 ± 3.8 at delivery (wk; mean ± SD) Amniocentesis to delivery interval (n) ≤48 hr 24 (17%) ≤72 hr 29 (21%) ≤ 7 days 36 (26%) Preterm delivery 68 (48%) (< 37 wk) Spontaneous 40/113 (35%) preterm delivery¶ (<37 wk) Clinical 3 (2%) chorioamnionitis Histologic 18/67 (27%) chorioamnionitis Funisitis 2/67 (3%)
<.001
—
29.0 ± 4.4
NS
Positive amniotic fluid culture Group 3 (n = 21)
P‡ Unadjusted
29.5 ± 4.1
Adjusted§
<.001
—
<.001 <.001 <.001 <.001
<.001 <.001 <.001 <.01
32 (73%) 33 (75%) 40 (91%) 43 (98%)
NS NS NS NS
16 (76%) 16 (76%) 20 (95%) 21 (100%)
<.001 <.001 <.001 <.001
<.01 <.01 <.01 NS
<.001
<.001
39/40 (98%)
NS
19/19 (100%)
<.001
NS
<.05
<.05
5 (11%)
NS
5 (24%)
<.001
<.05
<.001
< .001
30/35 (86%)
NS
15/18 (83%)
<.001
<.01
<.001
<.001
17/35 (49%)
NS
11/18 (61%)
<.001
<.001
Low IL-6, interleukin-6 lower than 2.6 ng/mL; High IL-6, interleukin-6 higher than 2.6 ng/mL; NS, not significant. *Comparison between groups 1 and 2. †Comparison between groups 2 and 3. ‡Comparison between groups 3 and 1. §Adjusted for gestational age and the degree of cervical dilatation at amniocentesis (logistic regression analysis). #The differences between groups 2 and 3 were not significant before and after the adjustment for the gestational age and the degree of cervical dilation at amniocentesis. P < .001, by Kruskal-Wallis ANOVA test. ¶Thirty-four cases that were delivered for maternal or fetal indications were excluded from this analysis.
Table III. Neonatal outcome of study population according to the results of amniotic fluid culture and interleukin-6 concentrations Negative amniotic fluid culture
Neonatal outcome
Low IL-6 (group 1; n = 141)
Birth weight 2683 ± 785 (g; mean ± SD) 1-min Apgar score <7 (n) 32 (23%) 5-min Apgar score <7 (n) 18 (13%) Admission to neonatal 39/135 (29%) intensive care unit¶ (n) Neonatal complications (n) Congenital sepsis¶ 0/135 (0%) Respiratory distress¶ 8/135 (6%) Pneumonia¶ 2/135 (1%) Intraventricular hemorrhage 13/135 (10%) (≥ grade II)¶ Bronchopulmonary 4/135 (3%) dysplasia¶ Necrotizing enterocolitis¶ 1/135 (1%)
P*
Positive amniotic fluid culture P†
P‡
Unadjusted
Adjusted§
High IL-6 (group 2; n = 44)
<.001
—
1449 ± 691
NS
1497 ± 719
<.001
—
<.001 <.001 <.001
<.001 <.01 <.001
32 (73%) 27 (61%) 31/36 (86%)
NS NS NS
12 (57%) 10 (48%) 18/18 (100%)
<.01 <.001 <.001
<.05 NS <.01
<.01 <.001 <.01 <.001
<.01 <.05 <.05 <.05
4/36 (11%) 11/36 (31%) 5/36 (14%) 13/36 (36%)
NS NS NS NS
1/18 (6%) 6/18 (33%) 2/18 (11%) 8/18 (44%)
NS <.01 .07 <.001
NS <.05 NS <.05
<.001
<.01
9/36 (25%)
NS
4/18 (22%)
<.01
NS
<.01
NS
4/36 (11%)
NS
0/18 (0%)
NS
NS
Group 3 (Un)adjusted§# (n = 21)
Unadjusted Adjusted§
Low IL-6, interleukin-6 lower than 2.6 ng/mL; High IL-6, interleukin-6 higher than 2.6 ng/mL; NS, not significant. *Comparison between groups 1 and 2. †Comparison between groups 2 and 3. ‡Comparison between groups 3 and 1. §Adjusted for gestational age and the degree of cervical dilatation at amniocentesis (logistic regression analysis). #The differences between groups 2 and 3 were not significant before and after the adjustment for the gestational age and the degree of cervical dilation at amniocentesis. P < .001, by Kruskal-Wallis ANOVA test. ¶Seventeen infants were excluded from the analysis because they died in utero (n = 6 infants), were not actively resuscitated at birth, or died in the delivery room despite intensive resuscitative efforts as a result of extreme prematurity (n = 10 infants) or congenital anomaly (n = 1 infant) and thus could not be evaluated with respect to the presence or absence of complications.
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the possibility that inflammation may have a noninfectious cause. Two patients with microbial invasion of the amniotic cavity did not have an elevated IL-6 above the cutoff selected in our study. This may represent earlier stages of the infection process in which the host did not have time to elicit a full cytokine response or, alternatively, these cases may represent microbial invasion of the amniotic cavity in patients who are hyporesponders. Functional polymorphism in several cytokines (including IL-6) have been reported.19-22 Hyporesponder mothers may not mount enough of a proinflammatory cytokine response in the decidua for labor to be initiated. Consequently, micro-organisms would proliferate in the decidua, cross intact membranes, and stimulate fetal cells to produce cytokines into the amniotic cavity. If the fetal genotype determines hyporesponsiveness, microbial invasion of the amniotic cavity would be associated with low concentrations of cytokines in amniotic fluid. Another explanation for our findings is that microorganisms differ in their capacity to stimulate cytokine production by the host. Indeed, not all endotoxins are equally potent in stimulating cytokine production.23, 24 The major finding of our study was that the outcome of patients with intra-amniotic inflammation but with a negative amniotic fluid culture (group 2) was similar to that of patients with demonstrable infection (group 3) and much worse than that of patients without inflammation and with a negative amniotic fluid culture (group 1). This was the case for both maternal and fetal complications. Therefore, we propose that patients with evidence of intra-amniotic inflammation, regardless of whether there is demonstrable infection, be considered a separate group from patients without inflammation in clinical studies and randomized trials in preterm labor. The identification of patients belonging to this inflammatory cluster and who are at risk for adverse outcomes would have substantial clinical implications. For example, it would be ideal if tocolytic trials could identify patients with inflammation and either exclude or stratify these patients, given the substantial likelihood of preterm delivery within 24, 48, and 72 hours (Table II). Otherwise, there is a risk of considering therapies to be ineffective, which may benefit the subset of patients without inflammation. This is critical, given the high frequency of intra-amniotic inflammation observed in this study. The diagnosis of intra-amniotic inflammation can be readily made by either a cytokine assay or an amniotic fluid white blood cell count. Moreover, we have recently proposed that screening can be conducted by assaying cytokines in cervical fluid in preterm premature rupture of membranes.25 Therefore, we propose that the operational definition of preterm labor with intraamniotic inflammation be used to classify patients with preterm labor in studies of therapeutic interventions and to evaluate short- and long-term neonatal outcome. The pathophysiologic and clinical implications of the presence of intra-amniotic inflammation in so many pa-
tients with preterm labor appear not to have been fully understood in clinical obstetric practice. Antimicrobial treatment of patients with infection often leads to the release of microbial products (eg, endotoxin), which may exacerbate the cytokine response and lead to clinical worsening.26 This issue is a major concern in the treatment of septic shock and often considered a cause of death.27 A similar scenario may occur in patients with microbial invasion of the decidua and/or the amniotic cavity. Antimicrobial agents may lead to an initial worsening of the inflammatory response, which may accelerate the process of parturition and/or fetal damage. Basic and clinical research is urgently required to understand the value of agents that modulate the inflammatory response in obstetrics. It is possible that transient down-regulation of the effects of the inflammatory response would permit the time that is required to eradicate the infectious process, without injury to the fetus. Under these circumstances, prolongation of pregnancy may be safe and desirable.
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14. Yoon BH, Romero R, Kim CJ, Jun JK, Gomez R, Choi JH, et al. Amniotic fluid interleukin-6: a sensitive test for antenatal diagnosis of acute inflammatory lesions of preterm placenta and prediction of perinatal morbidity. Am J Obstet Gynecol 1995;172:960-70. 15. Andrews WW, Hauth JC, Goldenberg RL, Gomez R, Romero R, Cassell GH. Amniotic fluid interleukin-6: correlation with upper genital tract microbial colonization and gestational age in women delivered after spontaneous labor versus indicated delivery. Am J Obstet Gynecol 1995;173:606-12. 16. Yoon BH, Romero R, Park JS, Kim CJ, Kim SH, Choi JH, et al. Fetal exposure to an intra-amniotic inflammation and the development of cerebral palsy at the age of three years. Am J Obstet Gynecol 2000;182:675-81. 17. Cassell G, Andrews W, Hauth J, Cutter G, Hamrick W, Baldus K, et al. Isolation of microorganisms from the chorioamnion is twice that from amniotic fluid at cesarean delivery in women with intact membranes [abstract]. Am J Obstet Gynecol 1993;168:424. 18. Yoon BH, Romero R, Kim M, Kim EC, Kim T, Park JS, et al. Clinical implications of detection of Ureaplasma urealyticum in the amniotic cavity with the polymerase chain reaction. Am J Obstet Gynecol 2000;183:1130-7. 19. Stuber F, Petersen M, Bokelmann F, Schade U. A genomic polymorphism within the tumor necrosis factor locus influences plasma tumor necrosis factor-alpha concentrations and outcome of patients with severe sepsis. Crit Care Med 1996;24:381-4.
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Key words: Amniotic fluid, infection, inflammation, interleukin-6, preterm labor, prematurity, chorioamnionitis
Microbial invasion of the amniotic cavity in patients with preterm labor and intact membranes is a risk factor for the progression of labor to preterm delivery despite tocolysis, spontaneous rupture of membranes, clinical
From the Department of Obstetrics and Gynecology, Seoul National University College of Medicine; and the Laboratory of Fetal Medicine Research, Clinical Research Institute, Seoul National University Hospital. Supported by grant 2000-N-NL-01-C-078 from the Korea Institute of Science and Technology Evaluation and Planning (KISTEP), Republic of Korea. Presented at the Twenty-first Annual Meeting of the Society for MaternalFetal Medicine, Reno, Nev, February 5-10, 2001. Reprint requests: Bo Hyun Yoon, MD, PhD, Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, Seoul National University College of Medicine, Seoul, 110-744, Korea. Copyright © 2001 by Mosby, Inc. 0002-9378/2001 $35.00 + 0 6/6/117680 doi:10.1067/mob.2001.117680
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chorioamnionitis, and adverse neonatal outcome (ie, sepsis, non-infection-related complications).1-10 The gold standard for the diagnosis of infection in clinical medicine is the isolation and identification of the micro-organism from body fluids and tissues of patients with suspected infection. However, results of microbial culture may take days, and are often not available in time for some clinical decisions. In contrast to the diagnosis of infection, the identification of intrauterine inflammation can be easily and rapidly detected by laboratory tests (such as an amniotic fluid white blood cell count and cytokine determinations). A substantial number of patients with preterm labor and intact membranes or preterm premature rupture of membranes have demonstrable intra-amniotic inflammation with a negative amniotic fluid culture.11-15 The frequency and clinical significance of intra-amniotic
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inflammation without microbial invasion remains to be determined. The purpose of this study was to determine the frequency and maternal/neonatal outcome of patients with intra-amniotic inflammation but with a negative amniotic fluid culture. Material and methods Study population. The study population consisted of consecutive patients admitted to Seoul National University Hospital between January 1993 and July 1998 with the diagnosis of preterm labor and intact membranes (<36 weeks of gestation) and singleton gestation who underwent amniocentesis. Amniocentesis is routinely offered to all patients who are admitted with the diagnosis of preterm labor at our institution. Amniocentesis was performed after written informed consent was obtained. The institutional review board of our institution approved the collection of biologic materials and data from these patients for research purposes. Patients were divided into 3 groups according to the results of amniotic fluid culture and amniotic fluid concentrations of interleukin-6 (IL-6). Group 1 consisted of 141 women with a negative amniotic fluid culture and without an elevated amniotic fluid IL-6 concentration; group 2 consisted of 44 women with a negative amniotic fluid culture but with an elevated amniotic fluid IL-6 concentration (intra-amniotic inflammation); group 3 consisted of 21 women with a positive amniotic fluid culture, regardless of amniotic fluid IL-6 concentration (intraamniotic infection). Amniotic fluid. Amniotic fluid was retrieved by transabdominal amniocentesis and was cultured for aerobic and anaerobic bacteria and for genital mycoplasmas (Ureaplasma urealyticum and Mycoplasma hominis) according to methods described in detail previously.14 Fluid that was not used for diagnostic studies was centrifuged and stored at –70°C until assayed. IL-6 concentrations were measured with a commercially available enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minn). The sensitivity of the test was <1.0 pg/mL. Intraand interassay coefficients of variation were <10%. Diagnosis of chorioamnionitis, funisitis, and neonatal morbidity. Clinical chorioamnionitis was diagnosed when a temperature was elevated to 37.8°C and when ≥2 of the following criteria were present: uterine tenderness, malodorous vaginal discharge, maternal leukocytosis (>15,000 cells/mm3), maternal tachycardia (>100 beats/min), and fetal tachycardia (>160 beats/min). Histologic chorioamnionitis was defined in the presence of acute inflammatory changes on examination of a membrane roll and chorionic plate of the placenta; funisitis was diagnosed in the presence of neutrophil infiltration into the umbilical vessel walls or Wharton’s jelly with the use of criteria published previously.14, 16 Neonatal sepsis, respiratory distress syndrome, pneumonia,
bronchopulmonary dysplasia, intraventricular hemorrhage, and necrotizing enterocolitis were diagnosed according to the definitions that were previously described in detail.14, 16 Statistical analysis. Proportions were compared with the Fisher exact test. The Kruskal-Wallis analysis of variance (ANOVA) test was used for comparison of continuous variables among the groups. Multiple comparisons among groups were performed with Mann-Whitney U tests. The McNemar test was used for comparison of proportions of correlated samples. The generalized Wilcoxon test for survival analysis was used to compare the amniocentesis-to-delivery interval. Patients delivered for maternal or fetal indications were treated as censored observations, with a censoring time equal to the amniocentesis-to-delivery interval. Logistic regression analysis was used to examine the relationship between the presence of intra-amniotic inflammation or positive amniotic fluid culture and outcome of interest (ie, preterm delivery, neonatal complications) after adjusting for the effects of confounding variables (gestational age and the degree of cervical dilatation). A probability value of <.05 was considered significant. Results Characteristics of study population. Table I compares characteristics of the study population. Patients with a negative amniotic fluid culture but with intra-amniotic inflammation (group 2) had a significantly lower mean gestational age at amniocentesis and more advanced cervical dilatation at presentation than patients with a negative culture and without intra-amniotic inflammation (group 1). In contrast, there were no differences in the clinical characteristics between patients in groups 2 and 3. Result of amniotic fluid culture. Amniocentesis was performed in 209 patients with preterm labor and intact membranes during the study period. The prevalence of positive amniotic fluid culture was 10% (21/209 patients). U urealyticum was the most common micro-organism isolated from the amniotic cavity (n = 14 patients). Other isolates included Staphylococcus aureus (n = 2 patients), Enterococcus spp (n = 2 patients), Acinetobacter spp (n = 2 patients), Lactobacillus spp (n = 2 patients), and 1 isolate each of Candida albicans, Porphyromonas asaccharolytica, and coagulase-negative Staphylococcus. Three patients with a negative amniotic fluid culture but without remaining amniotic fluid for IL-6 determinations were excluded from the further analysis because they could not be evaluated with respect to the presence or absence of intra-amniotic inflammation. Diagnosis of intra-amniotic inflammation. The diagnosis of intra-amniotic inflammation was based on amniotic fluid concentration of IL-6. Fig 1 displays a receiver operating characteristic curve that describes the performance of amniotic fluid IL-6 concentration in the identification
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Table I. Characteristics of study population according to the results of amniotic fluid culture and IL-6 concentrations Negative amniotic fluid culture
Characteristic Maternal age§ (y) Nulliparity (n) Gestational age at amniocentesis (wk) Cervical dilation at presentation (cm)
Low IL-6 (group 1; n = 141)
Positive amniotic fluid culture
P*
High IL-6 (group 2; n = 44)
P†
Group 3 (n = 21)
P‡
29.5 ± 4.3 74 (52%) 31.3 ± 3.2
NS < .05 < .001
29.7 ± 3.6 15 (34%) 28.5 ± 4.1
NS NS NS
30.2 ± 5.3 9 (43%) 29.1 ± 3.9
NS NS <.01
0.8 ± 1.1
< .001
3.3 ± 2.7
NS
2.6 ± 2.2
<.001
Values given as mean ± SD. Low IL-6, interleukin-6 <2.6 ng/mL; High IL-6, interleukin-6 >2.6 ng/mL; NS, not significant. *Comparison between groups 1 and 2. †Comparison between groups 2 and 3. ‡Comparison between groups 3 and 1. §P > .1, by Kruskal-Wallis ANOVA test. P < .001, by Kruskal-Wallis ANOVA test.
Fig 1. Receiver operating characteristic curve that describes the performance of amniotic fluid IL-6 concentration in the identification of a positive amniotic fluid culture (area under the curve, 0.84: SE, 0.04; z, 9.26; P < .0001).
of positive amniotic fluid culture (area under the curve, 0.84; SE, 0.04; z, 9.26; P < .0001). A value of 2.6 ng/mL was at the knee of the curve and was used to diagnose the presence of intra-amniotic inflammation. Prevalence of intra-amniotic inflammation. Amniotic fluid concentrations of IL-6 were higher than 2.6 ng/mL in 63 of 206 patients (31%) in the study, in 44 of 185 patients (24%) with a negative amniotic fluid culture (group 2, intra-amniotic inflammation), and in 19 of 21 patients (90%) with a positive amniotic fluid culture (group 3, intra-amniotic infection). Intra-amniotic inflammation (group 2) was more common than intraamniotic infection (group 3; 21% [44/206] vs 10% [21/206]; P < .001, McNemar test).
Fig 2. Frequency of a positive amniotic fluid (AF ) culture and intra-amniotic inflammation (negative AF culture but with an AF IL-6 of higher than 2.6 ng/mL) as a function of gestational age. The lower the gestational age, the higher the frequency of a positive amniotic fluid culture and intra-amniotic inflammation (P < .01 for culture and P < .001 for inflammation, respectively). Hash-marked columns represent intra-amniotic inflammation. Black columns represent positive amniotic fluid culture.
Fig 2 displays the frequency of a positive amniotic fluid culture and intra-amniotic inflammation as a function of gestational age. The lower the gestational age, the higher the frequency of a positive amniotic fluid culture and intraamniotic inflammation (P < .01 and P < .001, respectively). Amniocentesis-to-delivery interval. Fig 3 shows the interval from amniocentesis to delivery. In patients who were delivered because of maternal or fetal indications (n = 34 women), this interval was treated as a censored observation. Patients with a negative amniotic fluid culture but with intra-amniotic inflammation (group 2) had a significantly shorter median amniocentesis-to-delivery interval than women with a negative culture and without intra-amniotic inflammation (group 1; P < .0001). However, there was no difference in the median amniocentesis-to-delivery interval between patients with a negative
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amniotic inflammation (group 2) and 16% of the women (21/132) had a positive amniotic fluid culture (group 3). Thirty-four patients had maternal and/or fetal indication for preterm delivery. Of the remaining 172 women, 57% of the women (98/172 women) had a spontaneous preterm delivery. Forty percent of these patients with a spontaneous preterm delivery (39/98 women) had intraamniotic inflammation (group 2) and only 19% of these women (19/98 women) had a positive amniotic fluid culture (group 3). Comment
Fig 3. Survival analysis of amniocentesis-to-delivery interval (group 1: median, 701 hours [range, 0.1-3252 hours]; group 2, median, 20 hours [range, 0.1-2328 hours]; group 3, median, 11 hours [range, 0.1-983 hours]) according to the results of amniotic fluid (AF) culture and IL-6 concentrations. NS, Not significant.
amniotic fluid culture but with intra-amniotic inflammation (group 2) and patients with a positive culture regardless of amniotic fluid IL-6 concentration (group 3). Multivariate survival analysis demonstrated that the amniocentesis-to-delivery interval in patients with a negative amniotic fluid culture but with intra-amniotic inflammation (group 2) was significantly shorter than that of the group with a negative culture and without intra-amniotic inflammation (group 1) after the adjustment of gestational age and the degree of cervical dilatation at amniocentesis (hazards ratio, 3.1; 95% CI, 2.3-4.1; P < .0001, Cox proportional hazards model analysis). Outcomes of study population. Tables II and III compare pregnancy and neonatal outcomes of study population. Patients with a negative amniotic fluid culture but with intra-amniotic inflammation (group 2) had a significantly higher rate of adverse outcomes than patients with a negative culture and without intra-amniotic inflammation (group 1). Adverse outcomes included higher rates of clinical chorioamnionitis, preterm delivery, histologic chorioamnionitis, funisitis, low Apgar scores, admission to neonatal intensive care unit, neonatal respiratory distress syndrome, pneumonia, sepsis, intraventricular hemorrhage (≥grade II), and bronchopulmonary dysplasia and lower gestational age at birth and birth weight. However, no differences were found between patients with a negative amniotic fluid culture but with intra-amniotic inflammation (group 2) and patients with a positive culture regardless of amniotic fluid IL-6 concentration (group 3; P > .1). One hundred thirty-two patients delivered preterm newborns (<37 weeks); 33% of the women (43/132) had intra-
Our results indicate that intra-amniotic inflammation is more common than microbiologically proven intra-amniotic infection in patients with preterm labor and intact membranes (21% vs 10%; P < .001) and that intra-amniotic inflammation per se is associated with adverse maternal and neonatal outcome. A major finding of this study is that the maternal and neonatal outcome of patients with intraamniotic infection did not differ from that of patients with intra-amniotic inflammation without demonstrable intraamniotic infection. In regard to microbial invasion of the amniotic cavity, the prevalence, the type of microorganism, the inverse relationship with gestational age, and the association with adverse outcome are consistent with our previously reported observations and with reports of other investigators.1-10 Moreover, we observed that the frequency of intra-amniotic inflammation is double that of demonstrable intra-amniotic infection, also in keeping with observations previously reported by us15 and other authors.17 In this study, 40% of all patients with preterm labor who delivered a preterm neonate spontaneously had an elevated amniotic fluid IL-6 but negative amniotic fluid culture. What is the cause of the inflammatory process in cases of negative culture? We have previously reported18 that, with the use of the polymerase chain reaction with specific primers to U urealyticum, many patients with intra-amniotic inflammation but with a negative culture for U urealyticum had microbial footprints for this microorganism in the amniotic fluid. Moreover, these patients had cytologic, pathologic, biochemical, and clinical evidence of inflammation. Therefore, we suspect that some patients with intra-amniotic inflammation of unknown cause will eventually be proved to have infection that cannot be demonstrated today with the use of standard microbiologic techniques. These laboratory procedures demand knowledge on the culture conditions of all potential pathogens, information that clearly is not available. Yet, a subgroup of patients with inflammation in the amniotic cavity may have an extra-amniotic infection, with micro-organisms located primarily in the decidua or the space between chorion and amnion. Previous studies have demonstrated that amniotic fluid IL-6 may be elevated in these patients.15 Of course, we do not dismiss
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Table II. Pregnancy outcome of study population according to the results of amniotic fluid culture and interleukin-6 concentrations
Pregnancy outcome
Negative amniotic fluid culture High IL-6 P* P† (group 2; Unadjusted Adjusted§ n = 44) (Un)adjusted§#
(group 1; n = 141)
Gestational age 36.2 ± 3.8 at delivery (wk; mean ± SD) Amniocentesis to delivery interval (n) ≤48 hr 24 (17%) ≤72 hr 29 (21%) ≤ 7 days 36 (26%) Preterm delivery 68 (48%) (< 37 wk) Spontaneous 40/113 (35%) preterm delivery¶ (<37 wk) Clinical 3 (2%) chorioamnionitis Histologic 18/67 (27%) chorioamnionitis Funisitis 2/67 (3%)
<.001
—
29.0 ± 4.4
NS
Positive amniotic fluid culture Group 3 (n = 21)
P‡ Unadjusted
29.5 ± 4.1
Adjusted§
<.001
—
<.001 <.001 <.001 <.001
<.001 <.001 <.001 <.01
32 (73%) 33 (75%) 40 (91%) 43 (98%)
NS NS NS NS
16 (76%) 16 (76%) 20 (95%) 21 (100%)
<.001 <.001 <.001 <.001
<.01 <.01 <.01 NS
<.001
<.001
39/40 (98%)
NS
19/19 (100%)
<.001
NS
<.05
<.05
5 (11%)
NS
5 (24%)
<.001
<.05
<.001
< .001
30/35 (86%)
NS
15/18 (83%)
<.001
<.01
<.001
<.001
17/35 (49%)
NS
11/18 (61%)
<.001
<.001
Low IL-6, interleukin-6 lower than 2.6 ng/mL; High IL-6, interleukin-6 higher than 2.6 ng/mL; NS, not significant. *Comparison between groups 1 and 2. †Comparison between groups 2 and 3. ‡Comparison between groups 3 and 1. §Adjusted for gestational age and the degree of cervical dilatation at amniocentesis (logistic regression analysis). #The differences between groups 2 and 3 were not significant before and after the adjustment for the gestational age and the degree of cervical dilation at amniocentesis. P < .001, by Kruskal-Wallis ANOVA test. ¶Thirty-four cases that were delivered for maternal or fetal indications were excluded from this analysis.
Table III. Neonatal outcome of study population according to the results of amniotic fluid culture and interleukin-6 concentrations Negative amniotic fluid culture
Neonatal outcome
Low IL-6 (group 1; n = 141)
Birth weight 2683 ± 785 (g; mean ± SD) 1-min Apgar score <7 (n) 32 (23%) 5-min Apgar score <7 (n) 18 (13%) Admission to neonatal 39/135 (29%) intensive care unit¶ (n) Neonatal complications (n) Congenital sepsis¶ 0/135 (0%) Respiratory distress¶ 8/135 (6%) Pneumonia¶ 2/135 (1%) Intraventricular hemorrhage 13/135 (10%) (≥ grade II)¶ Bronchopulmonary 4/135 (3%) dysplasia¶ Necrotizing enterocolitis¶ 1/135 (1%)
P*
Positive amniotic fluid culture P†
P‡
Unadjusted
Adjusted§
High IL-6 (group 2; n = 44)
<.001
—
1449 ± 691
NS
1497 ± 719
<.001
—
<.001 <.001 <.001
<.001 <.01 <.001
32 (73%) 27 (61%) 31/36 (86%)
NS NS NS
12 (57%) 10 (48%) 18/18 (100%)
<.01 <.001 <.001
<.05 NS <.01
<.01 <.001 <.01 <.001
<.01 <.05 <.05 <.05
4/36 (11%) 11/36 (31%) 5/36 (14%) 13/36 (36%)
NS NS NS NS
1/18 (6%) 6/18 (33%) 2/18 (11%) 8/18 (44%)
NS <.01 .07 <.001
NS <.05 NS <.05
<.001
<.01
9/36 (25%)
NS
4/18 (22%)
<.01
NS
<.01
NS
4/36 (11%)
NS
0/18 (0%)
NS
NS
Group 3 (Un)adjusted§# (n = 21)
Unadjusted Adjusted§
Low IL-6, interleukin-6 lower than 2.6 ng/mL; High IL-6, interleukin-6 higher than 2.6 ng/mL; NS, not significant. *Comparison between groups 1 and 2. †Comparison between groups 2 and 3. ‡Comparison between groups 3 and 1. §Adjusted for gestational age and the degree of cervical dilatation at amniocentesis (logistic regression analysis). #The differences between groups 2 and 3 were not significant before and after the adjustment for the gestational age and the degree of cervical dilation at amniocentesis. P < .001, by Kruskal-Wallis ANOVA test. ¶Seventeen infants were excluded from the analysis because they died in utero (n = 6 infants), were not actively resuscitated at birth, or died in the delivery room despite intensive resuscitative efforts as a result of extreme prematurity (n = 10 infants) or congenital anomaly (n = 1 infant) and thus could not be evaluated with respect to the presence or absence of complications.
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the possibility that inflammation may have a noninfectious cause. Two patients with microbial invasion of the amniotic cavity did not have an elevated IL-6 above the cutoff selected in our study. This may represent earlier stages of the infection process in which the host did not have time to elicit a full cytokine response or, alternatively, these cases may represent microbial invasion of the amniotic cavity in patients who are hyporesponders. Functional polymorphism in several cytokines (including IL-6) have been reported.19-22 Hyporesponder mothers may not mount enough of a proinflammatory cytokine response in the decidua for labor to be initiated. Consequently, micro-organisms would proliferate in the decidua, cross intact membranes, and stimulate fetal cells to produce cytokines into the amniotic cavity. If the fetal genotype determines hyporesponsiveness, microbial invasion of the amniotic cavity would be associated with low concentrations of cytokines in amniotic fluid. Another explanation for our findings is that microorganisms differ in their capacity to stimulate cytokine production by the host. Indeed, not all endotoxins are equally potent in stimulating cytokine production.23, 24 The major finding of our study was that the outcome of patients with intra-amniotic inflammation but with a negative amniotic fluid culture (group 2) was similar to that of patients with demonstrable infection (group 3) and much worse than that of patients without inflammation and with a negative amniotic fluid culture (group 1). This was the case for both maternal and fetal complications. Therefore, we propose that patients with evidence of intra-amniotic inflammation, regardless of whether there is demonstrable infection, be considered a separate group from patients without inflammation in clinical studies and randomized trials in preterm labor. The identification of patients belonging to this inflammatory cluster and who are at risk for adverse outcomes would have substantial clinical implications. For example, it would be ideal if tocolytic trials could identify patients with inflammation and either exclude or stratify these patients, given the substantial likelihood of preterm delivery within 24, 48, and 72 hours (Table II). Otherwise, there is a risk of considering therapies to be ineffective, which may benefit the subset of patients without inflammation. This is critical, given the high frequency of intra-amniotic inflammation observed in this study. The diagnosis of intra-amniotic inflammation can be readily made by either a cytokine assay or an amniotic fluid white blood cell count. Moreover, we have recently proposed that screening can be conducted by assaying cytokines in cervical fluid in preterm premature rupture of membranes.25 Therefore, we propose that the operational definition of preterm labor with intraamniotic inflammation be used to classify patients with preterm labor in studies of therapeutic interventions and to evaluate short- and long-term neonatal outcome. The pathophysiologic and clinical implications of the presence of intra-amniotic inflammation in so many pa-
tients with preterm labor appear not to have been fully understood in clinical obstetric practice. Antimicrobial treatment of patients with infection often leads to the release of microbial products (eg, endotoxin), which may exacerbate the cytokine response and lead to clinical worsening.26 This issue is a major concern in the treatment of septic shock and often considered a cause of death.27 A similar scenario may occur in patients with microbial invasion of the decidua and/or the amniotic cavity. Antimicrobial agents may lead to an initial worsening of the inflammatory response, which may accelerate the process of parturition and/or fetal damage. Basic and clinical research is urgently required to understand the value of agents that modulate the inflammatory response in obstetrics. It is possible that transient down-regulation of the effects of the inflammatory response would permit the time that is required to eradicate the infectious process, without injury to the fetus. Under these circumstances, prolongation of pregnancy may be safe and desirable.
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