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Amniotic Fluid Infection, Cytokines, and Adverse Outcome Among Infants at 34 Weeks’ Gestation or Less

From the Departments of Obstetrics and Gynecology and Pediatrics, University of Washington, Seattle, Washington; and Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania.

Address reprint requests to: Jane Hitti, MD, Department of Obstetrics and Gynecology, University of Washington, Box 356460, Seattle, WA 98195; E-mail: jhitti{at}u.washington.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
OBJECTIVE: We examined the hypothesis that amniotic fluid (AF) infection and elevated cytokine concentrations may cause neonatal injury beyond that expected solely from prematurity.

METHODS: The effects of exposure to AF infection and elevated cytokine concentrations were measured in 151 infants born to afebrile women in preterm labor with intact membranes at less than or equal to 34 weeks’ gestation. Amniotic fluid was collected by amniocentesis for culture and determination of tumor necrosis factor- and interleukin-6. Cytokine concentrations, stratified by AF infection, were compared for three gestational age groups. We then examined the associations between a positive AF culture or elevated AF tumor necrosis factor- concentration and adverse neonatal outcomes, adjusted for birth weight.

RESULTS: Amniotic fluid from 45 (30%) of 151 pregnancies had microorganisms, an elevated tumor necrosis factor- concentration, or both. Amniotic fluid cytokine concentrations were significantly higher among women in preterm labor at less than or equal to 30 weeks, compared with 31–34 weeks. Nine of 11 infants who died at less than or equal to 24 hours of age had AF infection or elevated AF tumor necrosis factor-. For the 140 surviving infants, AF infection and/or an elevated AF tumor necrosis factor- was associated with respiratory distress syndrome (adjusted odds ratio [OR] 1.7), grade 3–4 intraventricular hemorrhage (adjusted OR 2.2), necrotizing enterocolitis (adjusted OR 1.8), and multiple organ dysfunction (adjusted OR 3.0).

CONCLUSION: Among infants born at less than or equal to 34 weeks to women who have intact membranes and are initially afebrile, those exposed to AF bacteria or cytokines have more adverse neonatal outcomes than unexposed infants of similar birth weight.

Very low birth weight (VLBW) infants (less than 1500 g) have increased morbidity and mortality.^1,2 Most morbidity among VLBW infants is attributed to immature organ function. However, a substantial proportion of VLBW infants are exposed to amniotic fluid (AF) infection³ and its associated proinflammatory response with production of cytokines such as tumor necrosis factor- and interleukin-6 in AF.^4,5

Recent evidence points to the fetus as a major contributor to the inflammatory response caused by AF infection.^6–9 Elevated AF cortisol levels with AF infection also indicate a fetal response to infection.¹⁰ Although a certain amount of neonatal morbidity results from prematurity, AF infection and the associated fetal and maternal inflammatory responses may further increase neonatal morbidity. Associations are reported between AF infection and respiratory distress syndrome (RDS),¹¹ bronchopulmonary dysplasia,¹² periventricular leukomalacia,^13,14 and cerebral palsy,^14,15 but the effect of AF infection on neonatal morbidity in excess of that expected from prematurity has not been consistently addressed by adjustment for birth weight or gestational age.

We examined the relationship of AF infection and the concentration of tumor necrosis factor- and interleukin-6 in AF with gestational age among afebrile women in preterm labor with intact membranes. A high concentration of AF cytokines among pregnancies at an early gestational age would indicate that the fetal immune system is capable of a vigorous response to bacterial antigens by midgestation and could explain a portion of the morbidity sustained by preterm neonates when exposed to AF infection. We then examined whether the neonatal outcome was adversely affected by the presence of AF infection or elevated tumor necrosis factor- after adjustment for birth weight. Our hypothesis was that infants exposed to AF bacteria or elevated tumor necrosis factor- have higher rates of neonatal morbidity than do unexposed infants of similar birth weight.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The study population included 151 women in premature labor and their infants born at less than or equal to 34 weeks’ gestation. Women were admitted to the University of Washington Medical Center, Swedish Medical Center, and Virginia Mason Medical Center in Seattle, Washington, between June 25, 1991, and June 30, 1997. All women provided written informed consent for study participation. The Institutional Review Board of each participating hospital approved the study protocol. Participants were at gestational ages of 20–34 completed weeks by obstetric estimate, determined from menstrual dating or from the earliest available ultrasound. The study population is derived from a larger cohort study of infectious causes of prematurity (n = 287), and has previously been described in detail.¹¹ Preterm labor was defined as the spontaneous onset of regular uterine contractions at a frequency of six or more an hour with either documented cervical change or a cervical dilatation of greater than or equal to 1 cm or cervical effacement of greater than or equal to 50%. Women with cervical cerclage, placenta previa, abruption placenta, diabetes, hypertension, and preeclampsia were considered eligible if they met study criteria for preterm labor. Women with twin gestations were included, and infant outcome data for twin A analyzed. Women with cervical dilatation greater than 4 cm, ruptured membranes at admission, and high order multiple gestations were excluded from the study. Women with clinical signs of overt AF infection at admission (fever greater than 38C, significant uterine tenderness, or foul-smelling AF) were excluded.

Participants completed a standardized demographic and reproductive history questionnaire at study entry. Transabdominal amniocentesis was performed under ultrasound guidance for 306 study participants, but 11 subjects (4%) were lost to follow-up, and eight (3%) had a major fetal malformation. Of the remaining 287 evaluable participants, 153 (53%) delivered at less than or equal to 34 weeks. Complete medical records could not be obtained for two infants born at outside hospitals, leaving 151 mother-infant pairs for these analyses.

Amniotic fluid was placed in an anaerobic transport vial (Port-a-Cul, Becton Dickinson Microbiology Systems, Cockeysville, MD) and inoculated into culture media for aerobic and anaerobic bacteria and genital mycoplasmas within 12 hours of collection. Methods for the culture and identification of these organisms were designed to detect low quantities of bacteria through the use of broth enrichment, as previously described.⁵ A Gram stain was also prepared from the AF. If microorganisms were detected on the AF Gram stain or if the culture was positive, these results were relayed to the managing obstetrician and pediatrician.

The remaining AF was stored at -70C until assayed for tumor necrosis factor- and interleukin-6 using commercial enzyme immunoassays specific for each cytokine (Genzyme Diagnostics, Cambridge, MA; Biosource, Camarillo, CA). The mean concentration of two duplicate samples of 25 uL each was calculated. The coefficient of variation between duplicate samples was less than or equal to 20%. Standard positive controls of recombinant tumor necrosis factor- or interleukin-6 in buffered solution were run simultaneously with the study specimens for each cytokine assay. The lower limit of detection was 30 pg/mL for tumor necrosis factor- and 70 pg/mL for interleukin-6. An AF tumor necrosis factor- greater than 30 pg/mL and an AF interleukin-6 greater than 2000 pg/mL were considered elevated⁵ (85th and 80th percentiles for this study population, respectively). Amniotic fluid tumor necrosis factor- assays were completed for 138 (91%) and interleukin-6 for 145 (96%) of 151 participants. Amniotic fluid cytokine results were not available to the clinical staff because they were assayed in batches, usually after the patient had been discharged from the hospital.

Stillbirth was defined as fetal death before delivery. Early neonatal demise was defined as death at less than 24 hours of age. For purposes of analysis, stillbirth and early neonatal demise were combined into one outcome measure (stillbirth/early demise). Neonatal sepsis was defined as a positive blood culture (excluding Staphylococcus epidermidis) within the first 7 days of life. The attending neonatologist determined other neonatal morbidity using published Vermont-Oxford definitions for each outcome of interest.¹⁶ Amniotic fluid cytokine results were not available to attending neonatologists. Respiratory distress syndrome was defined as the requirement for supplemental oxygen to prevent cyanosis, the clinical observation of dyspnea, and the presence of air bronchograms and a reticulogranular pattern on a chest radiograph.¹⁶ Bronchopulmonary dysplasia was defined as a persistent oxygen requirement at 36 weeks’ corrected gestational age, accompanied by a compatible chest radiograph.¹⁷ Intraventricular hemorrhage was detected by cranial ultrasound at 14 days of life for all infants less than 1500 g and for larger infants as clinically indicated, with an earlier or follow-up ultrasound as needed. The severity of intraventricular hemorrhage was graded according to the criteria of Volpe; severe intraventricular hemorrhage was defined as grade 3 (intraventricular hemorrhage with ventricular dilatation) or grade 4 (parenchymal hemorrhage).¹⁸ Patent ductus arteriosus was defined by a compatible heart murmur and/or Doppler evidence of left-to-right ductus shunting, accompanied by at least two of the following signs: bounding peripheral arterial pulses, hyperdynamic precordial pulsation, radiographic evidence of cardiomegaly or pulmonary edema, or inability to decrease ventilator settings after 48 hours of age.¹⁹ Echocardiography was performed only if required to confirm clinical and radiographic findings. Necrotizing enterocolitis was defined either by findings at surgery or postmortem, or by the presence of a bilious gastric aspirate, abdominal distension or blood in stool, accompanied by radiographic findings of pneumatosis intestinalis, hepatobiliary gas, or pneumoperitoneum.¹⁶ Multisystem organ dysfunction was defined by the presence of two or more of the following: RDS, necrotizing enterocolitis, or severe intraventricular hemorrhage. Patent ductus arteriosus and bronchopulmonary dysplasia were excluded from the definition of multisystem organ dysfunction because the clinical definition of these conditions overlapped with RDS.

The statistical methods included comparison of demographic and reproductive characteristics, cytokine concentrations, and pregnancy outcomes for women with a positive or negative AF culture. The group with a negative AF culture was further stratified into those with an elevated AF tumor necrosis factor- (greater than 30 pg/mL) and those with a low AF tumor necrosis factor- (less than or equal to 30 pg/mL). The nine women with a negative AF culture and no tumor necrosis factor- determination were included in the low tumor necrosis factor- group. ² or Fisher’s exact tests were used to examine differences in categorical variables between groups. The Mann-Whitney U test was used to examine differences in distributions of continuous variables between groups. The Bonferroni method was used to correct for multiple comparisons between the positive AF culture, negative AF culture, and elevated tumor necrosis factor- and negative AF culture and low tumor necrosis factor- groups. Logistic regression was used to estimate the associations between a positive AF culture or an elevated concentration of AF tumor necrosis factor- and neonatal outcomes of interest, with adjustment for infant birth weight as a continuous variable. Analyses for RDS also included adjustment for infant sex, maternal race, and mode of delivery as categorical variables, as these factors are associated with RDS. Because infant sex, maternal race, and mode of delivery are not consistently associated with the other neonatal outcomes examined, no adjustment was made for these factors in the analysis of outcomes other than RDS.

The sample size of 300 for the original study population was calculated for its primary objective, to evaluate the associations between AF infection, host inflammatory response, and preterm birth. For the purposes of the present study, our sample size of 36 exposed and 104 unexposed infants surviving greater than 24 hours had 85% power to detect a significant difference (P < .05) in the rate of RDS between groups, assuming the rate of RDS in each group was similar to that previously reported.¹¹

	RESULTS

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Microorganisms were recovered from 30 (20%) of 151 AF specimens. Eight AF cultures grew single or mixed anaerobic organisms, and five cultures grew mixed aerobic and anaerobic flora. Ureaplasmaurealyticumwas isolated from four samples, Escherichiacolifrom three, Group B Streptococcus from two, Mycoplasmahominisfrom two, Candidaalbicansfrom two, and other aerobic isolates from four.

The AF tumor necrosis factor- concentration was greater than 30 pg/mL in 21 (81%) of 26 culture-positive specimens and 15 (13%) of 112 culture-negative specimens tested for tumor necrosis factor- (P < .001). The AF interleukin-6 concentration was greater than 2000 pg/mL in 25 (86%) of 29 culture-positive specimens and 26 (22%) of 115 culture-negative specimens tested for interleukin-6 (P < .001).

First, we compared characteristics and pregnancy outcomes between the groups with a positive AF culture (n = 30) and a negative AF culture (n = 121); no significant differences were found in demographic, reproductive, or pregnancy characteristics (Table 1). Of interest, fever in labor was present in only 10% of pregnancies with a positive AF culture. However, compared with the AF culture-negative group, pregnancies with a positive AF culture presented and delivered at an earlier gestational age, had a shorter enrollment to delivery interval, and had infants of lower birth weight (Table 1).

View this table: [in this window] [in a new window]   Table 1. Characteristics of the Population That Delivered at 34 Weeks’ Gestation, Stratified by Amniotic Fluid Culture Results and Amniotic Fluid Tumor Necrosis Factor- Concentration

The relationship between gestational age at enrollment and AF cytokine concentrations was examined after stratification by AF culture in Figure 1. For pregnancies with a positive AF culture, the median concentration of interleukin-6 decreased significantly from 26,210 pg/mL at less than or equal to 26 weeks to 2200 pg/mL at 31–34 weeks. The median tumor necrosis factor- concentration decreased from 726 pg/mL at less than or equal to 26 weeks to 59 pg/mL at 31–34 weeks, but this change was not statistically significant. For pregnancies with a negative AF culture, the concentrations of both tumor necrosis factor- and interleukin-6 decreased significantly with increasing gestational age. Amniotic fluid cytokine concentrations were significantly higher in pregnancies with a positive than negative AF culture at all gestational age intervals in Figure 1.

View larger version (23K): [in this window] [in a new window]   Figure 1. Amniotic fluid (AF) interleukin-6 (IL-6) and tumor necrosis factor- (TNF-) distributions by gestational age at enrollment, stratified by AF culture results. Solid lines indicate the median value for each group. The Kruskal-Wallis analysis of variance was used to determine statistical significance for comparison by gestational age categories, and the Mann-Whitney U test was used to compare the positive and negative AF culture strata within gestational age category. Hitti. Infection and Neonatal Outcome. Obstet Gynecol 2001.

Surviving infants in the positive AF culture group had a lower birth weight (P < .01) and gestational age (P < .001), compared with those in the negative AF culture group (Table 2). The following neonatal outcomes occurred significantly more frequently among infants in the positive AF culture group than in the negative AF culture group: RDS (P < 05), grade 3–4 intraventricular hemorrhage (P < .05), patent ductus arteriosus (P < .001), and necrotizing enterocolitis (P < .01). Infants with a positive AF culture also required more days of ventilator support and supplemental oxygen compared with infants with a negative AF culture (P < .01).

View this table: [in this window] [in a new window]   Table 2. Neonatal Outcome After Delivery at 34 Weeks’ Gestation, Restricted to Infants Surviving >24 Hours

Neonatal bacteremia occurred among three infants in the positive AF culture group, with the same isolate (E. coli, Klebsiella sp., or Group B Streptococcus) detected in both AF and neonatal blood. One infant in the negative AF culture/ low tumor necrosis factor- group had E. coli bacteremia. Neonatal bacteremia was more common in the positive AF culture than in the negative AF culture group (P < .05).

The associations between indicators of AF infection and neonatal morbidity were compared using univariate and multivariate analyses in Table 3. The positive AF culture and the negative culture/elevated AF tumor necrosis factor- groups were combined for these analyses. A positive AF culture and/or elevated tumor necrosis factor- was strongly associated with RDS, grade 3–4 intraventricular hemorrhage, patent ductus arteriosus, and necrotizing enterocolitis in the unadjusted analyses.

View this table: [in this window] [in a new window]   Table 3. Amniotic Fluid Culture and/or Elevated Amniotic Fluid Tumor Necrosis Factor- and Risk of Neonatal Morbidity: Multivariate Analysis

We then examined the associations of AF infection or elevated tumor necrosis factor- with neonatal multisystem organ dysfunction, defined as at least two of the following conditions: RDS, grade 3–4 intraventricular hemorrhage, and necrotizing enterocolitis. A positive AF culture and/or elevated tumor necrosis factor- was strongly associated with multisystem dysfunction in the unadjusted analysis, and these associations remained significant after adjustment for infant birth weight (adjusted OR 3.0).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Infection of the AF appears to be primarily a fetal, rather than a maternal infection.^6,7 Considerable inflammatory response occurs to AF infection by 26 weeks’ gestation. The median concentration of tumor necrosis factor- (726 pg/mL) at less than or equal to 26 weeks in infected AF is similar to that in the cerebral spinal fluid of children with meningitis (787 pg/mL) and in the abdominal fluid of adults with peritonitis (400 pg/mL).^20,21

Infants in the positive AF culture and the negative AF culture/elevated AF tumor necrosis factor- groups had the highest rate of abnormal outcome. Nine of 11 stillbirths or early neonatal deaths occurred in these two groups. The 36 (26% of 140) infants in the AF infection and elevated AF tumor necrosis factor- groups had 39% of RDS, 67% of grade 3–4 intraventricular hemorrhage, 54% of patent ductus arteriosus, and 53% of necrotizing enterocolitis. Proven neonatal sepsis was associated with a positive AF culture. Pregnancies with a negative AF culture and elevated AF tumor necrosis factor- had a similar birth weight, gestational age, and rate of adverse neonatal outcomes (except necrotizing enterocolitis) as those with AF infection.

Amniotic fluid infection and inflammation are probably in the causal pathway of preterm delivery,⁹ and thus the large unadjusted ORs in Table 3 would more closely reflect the total impact of infection on neonatal outcome than the adjusted ORs. However, to determine whether a preterm infant exposed to AF infection and/or elevated AF tumor necrosis factor- had increased morbidity beyond that expected from prematurity per se, a statistical adjustment was made for birth weight. After adjustment for birth weight, infants exposed to AF infection and/or inflammation still had a 70–120% increase in RDS, intraventricular hemorrhage, and necrotizing enterocolitis compared with those with no AF infection or inflammation.

We previously reported the association of tumor necrosis factor- in AF with RDS independent of birth weight in an analysis with fewer patients.¹¹ Periventricular leukomalacia¹³ and cerebral palsy^14,15 are also increased among pregnancies with a proinflammatory response in the AF. Further, RDS, proven or suspected neonatal sepsis, and a combination of severe neonatal morbidity have been associated with elevated fetal plasma interleukin-6, although adjustment was not made for birth weight or gestational age in these analyses.^7,12 The present data add grade 3–4 intraventricular hemorrhage and necrotizing enterocolitis as neonatal outcomes related to AF infection and inflammation. Data in this report are unique because they relate AF infection and inflammation documented by amniocentesis in afebrile women in preterm labor (without clinical infection) with intact membranes (free of vaginal contaminants) to common and severe adverse neonatal outcomes, even after adjustment for birth weight.

These findings add weight to the concept that a systemic fetal inflammatory response results from AF infection.⁷ This inflammatory response appears to result in widespread fetal organ damage to the brain, lung, and gut. Both a positive AF culture and an elevated tumor necrosis factor- were associated with multisystem dysfunction. We chose to focus on tumor necrosis factor- instead of interleukin-6 because tumor necrosis factor- increases very early in the proinflammatory cytokine cascade,²² and thus may be a more specific response to bacterial infection than interleukin-6. Elevated concentrations of AF interleukin-6 were also strongly associated with adverse neonatal outcome (data not shown). Because of the limited number of patients, we could not determine whether a positive culture or the tumor necrosis factor- or interleukin-6 concentration in AF best correlated with organ damage. Dysfunction after AF infection should also be examined in other neonatal organs such as the liver, kidney, endocrine, and hematopoetic systems.

Bacteria in AF,³ the chorioamnion,²³ and histologic chorioamnionitis^23,24 all have been inversely related to gestational age. In this report, an inverse relationship was present between the gestational age at admission in preterm labor and all of the following: a positive AF culture, the prevalence of an elevated cytokine concentration, and the median concentration of tumor necrosis factor- and interleukin-6 in AF. The high prevalence of elevated proinflammatory cytokine concentrations in the AF of pregnancies in preterm labor at low gestational ages is a new finding consistent with the high prevalence of AF infection in the VLBW group.

The mechanism for the very constant inverse relationship between AF infection/inflammation and gestational age at delivery is unexplained. The first possible explanation is that microbes present in the endometrium earlier in pregnancy may go unrecognized until the fetal immune system begins to respond to antigens at midgestation.²⁵ The recent association of bacterial vaginosis with early pregnancy loss^26,27 suggests that bacteria present in the endometrium in early pregnancy may cause pregnancy loss. Women who do not miscarry early in pregnancy would be at risk to have endometrial infection recognized at midgestation, a time when both the fetal immune system begins to recognize antigens and an increase occurs in infection-related preterm delivery. A second, less likely explanation is a short cervix. A short cervix at midgestation is associated with preterm delivery,²⁸ which could increase the bacterial exposure to the chorioamnion. However, the cervix continues to shorten as pregnancy progresses,²⁹ which ostensibly would increase AF infection as pregnancy proceeds, and we observed the opposite effect.

One potential bias to this study is that detection of bacteria on AF Gram stain or culture was routinely reported to the managing clinician. However, obstetricians at our institution were not likely to deliver a patient solely because of a Gram stain result, but would consider this information in its clinical context. A positive AF culture might be more likely to affect timing of delivery, but these results were not usually positive until at least 48–72 hours after the amniocentesis, and most women with a positive AF culture delivered within 24–48 hours. Even if knowledge of AF Gram stain or culture findings resulted in an earlier delivery, this bias would not explain the increased neonatal morbidity among infants exposed to AF bacteria or cytokines when compared with unexposed infants with adjustment for birth weight. A second potential bias is that the study population excludes infants at gestational ages greater than 34 weeks. We chose to restrict the analysis to infants born at less than or equal to 34 weeks because most neonatal morbidity occurs before 34 weeks, and also because the rate of AF infection is higher early in pregnancy.³ There were two infants in the larger data set who had a positive AF culture and delivered after 34 weeks. The first presented at 33 weeks, had Clostridium isolated from the AF, and delivered at 39 weeks. In this case, the Clostridium may have been a skin contaminant. The second case presented at 33 weeks, had Candida albicans isolated from the AF, and delivered at 36 weeks. A third case presented at 32 weeks, had a negative AF culture, but elevated tumor necrosis factor- and interleukin-6, and delivered at 35 weeks. Thus, of 287 mother-infant pairs with preterm labor at less than or equal to 34 weeks’ gestation, intact membranes, and no clinical evidence of infection, only three (1%) had a positive AF culture or high AF tumor necrosis factor- and delivered after 34 weeks. Therefore, it is unlikely that the decision to limit the study population to infants delivered at less than or equal to 34 weeks would have influenced our findings.

The pregnant women in this report had no clinical evidence of infection at enrollment, and only 10% of those with a positive AF culture became febrile before delivery. Despite a high prevalence of AF infection that can exceed 50% at 26 weeks’ gestation,³ the clinical management of preterm labor does not usually include amniocentesis to detect AF infection or inflammation among afebrile women. Women with preterm labor are treated uniformly with tocolytics to inhibit labor, corticosteroids to mature the fetal lungs, and antibiotics aimed only at prevention of neonatal Group B streptococcal infection. If AF infection or inflammation is associated with adverse neonatal outcome beyond that expected from prematurity, early detection would allow the opportunity to provide treatment to reduce infection or inflammation, or even early delivery, if necessary. Clearly, before cogent treatment strategies can be derived, additional studies are needed to determine which compartments (endometrium, AF, chorioamnion, fetus) are infected, which compartment produces the most inflammation, and whether fetal endothelial activation occurs. Moreover, it is apparent from these data that new treatment strategies that downregulate AF and/or the fetal inflammatory response may be needed.

	Footnotes

 
This work was supported by National Institutes of Health Grant AI-31071.

PII S0029-7844(01)01567-8

Received February 27, 2001. Received in revised form July 6, 2001. Accepted July 19, 2001.

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