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Intra-amniotic inflammation is thought to result from an ascending infection and is considered to precede intrauterine infection1. In preterm labor with intact membranes, intra-amniotic inflammation has been described as a risk factor for preterm delivery; it has been associated with a shorter latency time to delivery and with a significantly increased rate of neonatal adverse outcomes, even in the absence of demonstrable positive cultures2–6. Patients with intra-amniotic inflammation are more likely to be refractory to tocolysis and to rupture their membranes spontaneously3, 6. It has also been reported that short cervical length is related to intra-amniotic infection7, 8 and inflammation9, and that the earlier the gestational age, the higher the risk of intra-amniotic inflammation2. However, no clear cut-off in cervical length or gestational age, above which the risk of finding intra-amniotic inflammation is low, has been clearly established. This may be relevant, particularly considering the risks of an invasive procedure, the fact that intra-amniotic inflammation in women with preterm labor with intact membranes is found in only 10–30% of cases and that strategies to treat intra-amniotic inflammation are not yet available, making the best clinical approach controversial. Although various inflammatory markers, such as amniotic fluid white blood cell count and glucose, cytokine (interleukin-6; IL-6) and matrix metalloproteinase-8 (MMP-8) concentrations, have been proposed to provide rapid prognostic information10–12, all require an invasive procedure.
Stratification of the risk based on non-invasive markers could substantially reduce the need for invasive procedures, thereby increasing the efficiency of the test. The purpose of the study was to evaluate cervical length and gestational age as non-invasive markers that could be used to select those women with preterm labor and intact membranes at highest risk of intra-amniotic inflammation, in whom amniocentesis would be performed to assess the inflammatory status of the amniotic fluid.
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A prospective cohort study was performed from October 2005 to April 2008 at the Hospital Clínic of Barcelona, Catalonia, Spain. The patients enrolled in this study were pregnant women with clinical symptoms of preterm labor and intact membranes between 22 and 36 weeks of gestation. Multiple pregnancies, patients with clinical signs of chorioamnionitis at admission or those who were receiving antibiotics for any reason were considered ineligible for this study. Gestational age was established according to the first-trimester ultrasound scan. Written informed consent was obtained from all subjects to donate amniotic fluid for research purposes. The Institutional Review Board of the Hospital Clinic approved the collection and use of these samples and information for research purposes.
Transvaginal cervical length was measured on admission and transabdominal amniocentesis was performed within the first 48 h. Cultures for genital mycoplasma, aerobic and anaerobic bacteria, as well as amniotic fluid glucose concentration, white blood cell counts and Gram stains were performed immediately after collection, and their results were available for clinical management. The remaining amniotic fluid was centrifuged at 4000 rpm for 10 min at 4 °C and stored at −70 °C until assayed for IL-6, the results of which were not used for clinical decision making. All maternal and neonatal medical records were reviewed and perinatal outcome was recorded. Three groups with different inflammatory and infectious status of the amniotic fluid were compared: women with low IL-6 and negative culture, women with high IL-6 and negative culture, and women with positive culture.
A complete course of antenatal steroids, comprising betamethasone 12 mg intramuscular injection with two doses given 24 h apart, was administered from 24 to 34 weeks. Tocolysis was considered in all cases in the absence of clinical chorioamnionitis, abruptio placentae and fetal compromise. Maternal and fetal status were closely monitored for signs of chorioamnionitis, labor and/or fetal compromise. Prophylactic parenteral broad-spectrum antibiotics were given in the following situations: advanced cervical dilatation (defined as a cervical dilatation > 4 cm), diagnosis of intra-amniotic infection and development of clinical signs of chorioamnionitis. The antibiotic regimen of choice was parenteral ampicillin (1 g every 6 h) and gentamicin (80 mg every 8 h) given for 5 days. It has been demonstrated in our institution that these antibiotics eradicate most of the microorganisms that could potentially lead to the development of chorioamnionitis. According to our protocol, in the absence of clinical chorioamnionitis, women with a positive amniotic fluid culture were given parenteral antibiotics for 10 days and monitored closely for signs of chorioamnionitis or fetal compromise until 34 weeks when they were allowed to labor. Induction of labor was based on gestational age and maternal or fetal clinical signs of chorioamnionitis.
Preterm labor was defined as the presence of regular uterine contractions, with a frequency of at least two every 10 min, and cervical changes. Clinical chorioamnionitis was defined following Gibbs' criteria as a temperature elevation to 37.8 °C and two or more of the following criteria: uterine tenderness, malodorous vaginal discharge, fetal tachycardia (more than 160 beats per min (bpm)), maternal tachycardia (more than 100 bpm) and maternal leukocytosis (> 15 000/mm3)13. Intra-amniotic infection was defined by the presence of a positive amniotic fluid culture. Intra-amniotic inflammation was defined by the presence of high levels of IL-6 in amniotic fluid. Histological chorioamnionitis was defined as an infiltration of the chorion and/or amnion by polymorphonuclear leukocytes14. Neonatal composite morbidity included the presence of any of the following criteria: intraventricular hemorrhage, respiratory distress syndrome, congenital sepsis and periventricular leukomalacia suspected by ultrasound examination. Puerperal endometritis was considered in patients with a temperature ≥ 38 °C on two occasions 4 h apart (excluding the day of delivery), associated with uterine tenderness, foul-smelling lochia, and no other apparent source of infection13.
Receiver–operating characteristics (ROC) curve analysis was employed to display the relationship between sensitivity and false-positive rate (1—specificity), and to select the best cut-off value for IL-6 for diagnosing intra-amniotic infection. This value was then used as a basis for determining a cut-off of IL-6 for defining intra-amniotic inflammation. Diagnostic indices (sensitivity, specificity) and positive and negative predictive values (PPV and NPV, respectively) in detecting intra-amniotic infection were calculated for IL-6. Linear-by-linear association and ANOVA with polynomial contrast analysis were used to evaluate trends among the three study groups. Two-by-two contingency tables were constructed and Chi-square or Fisher's exact tests were used to identify significant differences among test performances. Continuous data were compared with Student's t-test. Kaplan–Meier probability plots were generated based on the interval between amniocentesis and delivery in the three groups. Differences between the survival functions of the groups were analyzed using the log rank test. Univariate and logistic regression analyses were performed to investigate the relationship between the occurrence of the inflammatory status and various explanatory variables. Sensitivity and PPVs of a combination of gestational age at admission and cervical length in predicting intra-amniotic inflammation were calculated. P < 0.05 was considered statistically significant. The P-values reported do not assume equality of variances. Statistical analyses were performed using SPSS version 14.0 statistical software (SPSS Inc., Chicago, IL, USA).
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Over the study period, from a total of 130 eligible patients with clinical symptoms of preterm labor, 93 patients were not receiving antibiotics at admission and gave signed informed consent. Gestational ages at admission and at delivery of the 37 women not included were comparable to those for the study population.
Thirty-five patients (37.6%) were admitted before 28 weeks of gestation, 24 (25.8%) between 28 and 32 weeks, 21 (22.6%) between 32 + 1 and 34 weeks, and 13 (14%) at > 34 weeks of gestation. Mean ± SD gestational age and cervical length at admission and gestational age at delivery of the entire study population were 29.4 ± 3.4 weeks, 15.7 ± 11.0 mm and 33.6 ± 5.4 weeks, respectively. Cervical length was not obtained in four women because they refused to undergo transvaginal sonography.
ROC curve analysis of the ability of IL-6 to diagnose intra-amniotic infection, showed an area under the curve of 0.86 (95% CI, 0.77–0.92). The best cut-off value for predicting the presence of intra-amniotic infection was IL-6 ≥ 13.4 ng/mL. However, in order to make our findings consistent with a previously reported cut-off described by Romero et al.11, we changed our value to ≥ 11.3 ng/mL when defining intra-amniotic inflammation, which yielded a similar diagnostic performance. The sensitivity, specificity, PPV and NPV of high IL-6 levels (≥ 11.3 ng/mL) in diagnosing intra-amniotic infection were 84.6%, 81.3%, 42.3% and 97%, respectively.
The overall rate of intra-amniotic inflammation in patients with preterm labor was 28% (26/93) whereas proven intra-amniotic infection was detected in 14% of patients (13/93). In all but two cases with a positive amniotic fluid culture, IL-6 concentrations were ≥ 11.3 ng/mL. In one of these, the amniotic fluid was positive for Proteus mirabilis and had an IL-6 level of 1.4 ng/mL; the other was positive for Ureaplasma urealyticum and had an IL-6 level of 4.3 ng/mL. In these two cases, the gestational age at admission was < 28 weeks and cervical length < 15 mm. The most common microorganism isolated from the amniotic cavity of women with intra-amniotic infection was U. urealyticum (n = 4). Others found included: Candida albicans (n = 2), Proteus mirabilis (n = 1), Fusobacterium spp (n = 1), Streptococcus viridans (n = 1), Staphylococcus epidermidis (n = 1), Peptostreptococcus spp (n = 1), Escherichia coli (n = 1) and Bacteroides fragilis (n = 1).
Maternal characteristics are summarized in Table 1. Regardless of the results of the microbial culture, gestational age at admission and cervical length were significantly lower among the group of women with intra-amniotic inflammation.
Table 1. Maternal and amniotic fluid characteristics of the study population
|Variable||Negative culture||Positive culture (n = 13)||P*|
|Low IL-6 (n = 65)||High IL-6 (n = 15)|
|Maternal age (years)||28.2 ± 6.3||30.4 ± 6.5||33.1 ± 8.0||0.012|
|Nulliparous||38 (58.5)||8 (53.3)||6 (46.2)||0.701|
|Previous preterm delivery||7 (10.8)||2 (13.3)||3 (23.1)||0.250|
|GA at admission (weeks)||30.6 ± 3.3||26.9 ± 3.9||26.2 ± 4.0||< 0.001|
| < 28 weeks||15 (23.1)||10 (66.7)||10 (76.9)||< 0.001|
| < 32 weeks||35 (53.8)||13 (86.7)||11 (84.6)||0.008|
|Bishop score||3.5 ± 2.5||6.6 ± 3.4||7.0 ± 3.1||< 0.001|
|Cervical length (mm)||18.7 ± 10.5||10.5 ± 11.0||7.4 ± 6.1||< 0.001|
| < 15 mm||21 (33.9)||11 (73.3)||10 (83.3)||< 0.001|
| < 25 mm||41 (66.1)||12 (80.0)||12 (100)||0.013|
|Amniotic fluid glucose (mg/mL)||50.3 ± 23.2||27.9 ± 20.1||7.3 ± 9.6||< 0.001|
|Amniotic fluid IL-6 (ng/mL)||1.02 (0.44–1.79)||86.2 (47.3–152.5)||75.6 (16.2–133.5)||< 0.001|
Perinatal outcomes are summarized in Table 2. Regardless of the intra-amniotic microbial status, gestational age at delivery was significantly lower among the group of women with intra-amniotic inflammation. In this group, a higher percentage of the women delivered before 28 and 32 weeks of gestation, and the interval between amniocentesis and delivery was shorter. Mean birth weight was significantly lower and time spent in the neonatal intensive care unit was significantly longer among pregnancies complicated by intra-amniotic inflammation. Mortality and the neonatal composite morbidity rates were significantly higher in neonates whose mothers developed intra-amniotic inflammation than in those who did not.
Table 2. Perinatal outcomes of the study population
|Variable||Negative culture||Positive culture (n = 13)||P*|
|Low IL-6 (n = 65)||High IL-6 (n = 15)|
|GA at delivery (weeks)||35.8 ± 4.0||29.5 ± 4.8||26.9 ± 3.9||< 0.001|
| < 28 weeks†||4/15 (26.7)||7/10 (70)||9/10 (90)||0.001|
| < 32 weeks‡||10/35 (28.6)||10/13 (76.9)||11/11 (100)||< 0.001|
| < 37 weeks||31/65 (47.7)||13/15 (86.7)||13/13 (100)||< 0.001|
|Amniocentesis to delivery (days)||34.7 ± 29.1||18.0 ± 33.5||4.9 ± 5.0||< 0.001|
| < 14 days||20/65 (30.8)||12/15 (80)||13/13 (100)||< 0.001|
|Clinical chorioamnionitis||4/65 (6.2)||2/15 (13.3)||5/13 (38.5)||0.002|
|Birth weight (g)||2661 ± 901||1463 ± 778||1077 ± 548||< 0.001|
|5-min Apgar score < 7||1/54 (1.9)||1/13 (7.7)||4/13 (30.8)||0.004|
|UA pH < 7.20||7/48 (14.6)||5/14 (35.7)||0 (0)||0.948|
|Admission to NICU||26/65 (40)||11/15 (73.3)||10/13 (76.9)||0.003|
|Duration of stay in NICU (days)||8.0 ± 17.3||35.9 ± 33.8||37.6 ± 34.3||< 0.001|
|Composite neonatal morbidity||8/65 (12.3)||7/15 (46.7)||9/13 (69.2)||< 0.001|
|Neonatal mortality||1/65 (1.5)||3/15 (20)||5/13 (38.5)||< 0.001|
Remarkably, maternal characteristics and perinatal outcomes were similar between the intra-amniotic inflammation with negative amniotic fluid culture and the intra-amniotic infection groups (Tables 1 and 2).
The survival function displayed in Figure 1 shows that the interval between amniocentesis and delivery was longer in the non-inflammatory group (median, 31 (95% CI, 22.4–39.6) days) than in the group with inflammation but negative culture (median 4 (95% CI, 1.2–6.8) days). Indeed, in the latter, the interval between amniocentesis and delivery was similar to that of the intra-amniotic infection group (median, 3 (95% CI, 0–7.4) days). The differences between the three groups were statistically significant (log rank = 23.4, P < 0.001).
The sensitivity, specificity, PPV and NPV of gestational age at admission < 28 weeks and < 32 weeks, and cervical length < 15 mm and < 25 mm, in predicting intra-amniotic inflammation are shown in Table 3. Logistic regression analysis (Table 4) showed that gestational age < 28 weeks at admission and cervical length < 15 mm were independently associated with the occurrence of intra-amniotic inflammation (IL-6 ≥ 11.3 ng/mL). However, only gestational age at admission < 28 weeks remained significant (odds ratio, 7.3 (95% CI, 2.5–21.2); P < 0.001) when 25 mm was used as a cut-off point for cervical length (odds ratio, 3.0 (95% CI, 0.7–12.3); P = 0.133). Sensitivity and PPVs for predicting intra-amniotic inflammation when gestational age at admission and cervical length were combined are shown in Figure 2.
Figure 2. Sensitivity and positive predictive value of different cut-offs of gestational age and cervical length (in combination) at admission for predicting intra-amniotic inflammation (interleukin-6 ≥ 11.3 ng/mL). CL, cervical length at admission (not measured in four cases); FPR, false-positive rate; GA, gestational age at admission; PPV, positive predictive value; Sens, sensitivity.
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Table 3. Diagnostic indices of different variables for predicting intra-amniotic inflammation (interleukin-6 ≥ 11.3 ng/mL)
|Variable||Sensitivity (%)||Specificity (%)||PPV (%)||NPV (%)|
|GA at admission|
| < 28 weeks||69.2||74.6||51.4||86.2|
| < 32 weeks||84.6||44.8||37.3||88.2|
| < 15 mm||76.0||64.1||45.2||87.2|
| < 25 mm||88.0||32.8||33.8||87.5|
Table 4. Relationship between various explanatory variables and intra-amniotic inflammation (interleukin-6 ≥ 11.3 ng/mL), analyzed by logistic regression analysis
|Explanatory variable||Odds ratio (95% CI)||P|
|Previous preterm delivery||2.8 (0.6–12.6)||0.194|
|GA at admission < 28 weeks||6.2 (2.1–18.7)||0.001|
|Cervical length at admission < 15 mm||4.6 (1.5–14.6)||0.009|
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Our data show overall rates of intra-amniotic inflammation and intra-amniotic infection in patients with preterm labor and intact membranes of 28% and 14%, respectively. They also show that women with pregnancy complicated with preterm labor and intact membranes in which an intra-amniotic inflammatory process is diagnosed have adverse outcomes that are similar to those of patients with intra-amniotic microbial invasion and clearly worse than those without an inflammatory component, confirming previous studies1, 2, 4. In addition, we show that the inflammatory process is particularly common in pregnancies complicated with preterm labor and intact membranes at early gestational ages and with short cervical length (PPV 63.6%), whereas it is rare above 32 weeks (PPV 11.8%).
The major finding of our study is that cervical length and gestational age at admission are independently related to intra-amniotic inflammation. Although short cervical length has been associated with a higher risk of microbial invasion of the amniotic cavity7–9, the ability of cervical length to predict intra-amniotic inflammation rather than infection has only been tested in one study9. Furthermore, it has already been reported that the earlier the gestational age, the higher the risk of intra-amniotic inflammation2. However, no particular gestational age above which an invasive procedure may not be worthwhile has been established. As we have shown that a short cervical length is independently associated with intra-amniotic inflammation, these data can also be used in combination with gestational age at admission as a non-invasive approach to the evaluation of the risk of an underlying inflammatory process. As gestational age < 28 weeks presents a better false-positive rate than does cervical length < 15 mm (25.4% vs. 35.9%, respectively), it seems reasonable to use gestational age as a first step in order to avoid unnecessary procedures.
In the clinical setting, a prudent cut-off for gestational age and cervical length should be considered, taking into account that the diagnosis of intra-amniotic inflammation requires the use of an invasive procedure and that the overall risk of intra-amniotic inflammation in preterm labor with intact membranes is, in fact, quite low (10–30%)2, 10, 15. The fact that the combination of both parameters may discriminate the risk of intra-amniotic inflammation is, therefore, of clinical relevance. The sensitivities and PPVs shown in Figure 2 may help to predict the risk for a specific patient, allowing amniocentesis to be reserved for those at highest risk. Women who present with preterm labor at < 28 weeks or who present at between ≥ 28 and < 32 weeks with a cervical length < 15 mm represent a high-risk group for intra-amniotic inflammation. This combination of parameters provides improved diagnostic indices compared to either variable considered alone, with a sensitivity of 84.0% and a PPV of 48.8%.
The rates of intra-amniotic inflammation and infection in our study are similar to those reported by other authors2, 5, 6, 10, 15. In addition, the sensitivity, specificity, PPV and NPV of IL-6 in diagnosing intra-amniotic infection in the present study were 84.6%, 81.3%, 42.3% and 97%, respectively, consistent with those reported in the literature with sensitivities and specificities in the range 80.9–100% and 75–82.5% depending on the risk group (i.e. preterm labor with intact membranes or premature rupture of membranes)2, 10, 11.
One of the main factors that makes our results interesting is that few studies have specifically evaluated the value of markers of intra-amniotic inflammation such as IL-62, 3, 5, 6, 10, 11. Indeed, only three reports indicated specifically that women with high IL-6 levels but a negative amniotic culture had a similar adverse outcome to those with a positive amniotic culture2, 3, 5. IL-6 determination is already a standardized technique and is readily available for clinical use in most laboratories. This is in contrast with other markers of inflammation such as proteomic determinations which need much more sophisticated facilities. Determination of MMP-8 for the same purpose may also play a role4, 12.
The weakness of our study lies in the relatively small sample size and the non-consecutive recruitment of patients. At the time of the study, amniocentesis was not included routinely in the clinical management of patients with preterm labor and intact membranes in our institution and so some patients were not studied, leading to a potential bias. In fact, cervical length and gestational age at admission and gestational age at delivery show, in our study, a population at high risk for preterm delivery in which an intra-amniotic inflammatory process may be somewhat overestimated. However, Romero et al.11 and Yoon et al.2 reported similar rates of infection and inflammation. Nonetheless, our study differs from previous reports in that we performed amniocentesis to study the microbiological status of the amniotic cavity in patients without any suspicion of infection, whereas most authors have studied a mixed population including women with chorioamnionitis16, 17, or women with a diagnosis of cervical insufficiency4.
The identification of intra-amniotic inflammation rather than bacterial infection may be clinically more important because inflammation occurs at an earlier stage of microbial invasion1. The increase in IL-6 levels in amniotic fluid even with a negative culture may identify patients in whom there is intrauterine infection, although such a process is extra-amniotic in nature. In this subgroup of patients, early treatment with antibiotics and/or anti-inflammatory agents may prevent or postpone preterm delivery and improve neonatal complications. However, an effective therapy to treat this situation is not yet available, making amniocentesis controversial as a universal approach. Most clinical guidelines for the management of preterm labor with intact membranes do not include amniocentesis to identify an inflammatory or infectious underlying process. Above 32 weeks, the finding of intra-amniotic inflammation is relatively rare (11.8%) and it would be difficult to justify this procedure, whereas more than half (51.4%) of patients with a gestational age < 28 weeks at admission could have an inflammatory component. This is consistent with the findings of Yoon et al.2, suggesting that the inflammatory pathway may explain preterm labor, particularly at early gestational ages. Stratification of the risk of intra-amniotic inflammation based on non-invasive markers, such as cervical length or gestational age at admission, could select the group at highest risk in which amniocentesis would be recommended to identify the inflammatory status of the amniotic cavity.
Whether this inflammation can be treated or not to improve perinatal outcomes remains unknown. Treatment with antibiotics for preterm labor has failed to demonstrate an improvement in outcomes18, 19 but this may be because the target population was not adequately selected and most women who received treatment probably did not have an underlying inflammatory mechanism. Whether this approach may be useful in women presenting with early preterm labor and a short cervix is unknown. Further clinical trials are necessary to address this hypothesis.