Objective
The objective of the study was to determine the diagnostic indices and predictive values by bedside assessment of amniotic fluid interleukin-6 (IL-6) concentration in the identification of microbial invasion of the amniotic cavity (MIAC) and/or histological chorioamnionitis (HCA) in patients with preterm prelabor rupture of membranes.
Study Design
One hundred twenty-four women with singleton pregnancies were included in this study. The amniotic fluid was sampled by transabdominal amniocentesis at the time of admission. IL-6 concentrations were assessed with an immunoassay.
Results
The presence of MIAC, HCA, or the coexistence of both was associated with higher amniotic fluid concentrations of IL-6 in both a crude and adjusted analysis. The amniotic fluid concentration of IL-6 of 1000 pg/mL was determined to be the best cutoff value for the prediction of MIAC (sensitivity of 50%, specificity of 95%, positive predictive value of 82%, negative predictive value of 81%, and likelihood ratio of 8.4) or both MIAC and HCA (sensitivity of 60%, specificity of 94%, positive predictive value of 75%, negative predictive value of 88%, and likelihood ratio of 9.4).
Conclusion
The bedside assessment of amniotic fluid IL-6 seems to be an easy, rapid, and inexpensive method for the prediction of MIAC or both MIAC and HCA in pregnancies complicated by preterm prelabor rupture of membranes.
Preterm prelabor rupture of membranes (PPROM), which is defined as the leakage of the amniotic fluid occurring before the onset of regular uterine activity and prior to 37 gestational weeks, is responsible for approximately one third of all preterm deliveries. PPROM represents a serious obstetrical condition that is often complicated by microbial invasions of the amniotic cavity (MIAC), intraamniotic infections/inflammation, and histological chorioamnionitis (HCA). This condition is also related to worse outcome than spontaneous preterm labor with intact membranes.
Our previous work has shown that the presence of both MIAC and HCA is associated with strong intraamniotic and fetal inflammatory response. This indicates that this subgroup constitutes the worst case scenario of PPROM. Neonates from this subgroup of PPROM can be jeopardized by their high risk of infections, such as early-onset sepsis, and thus might benefit from an active management including the induction of labor. Early identification of MIAC and HCA seems to be important for making an optimal clinical decision.
Information about MIAC and HCA are usually not available in time to be useful in making decisions on the management of women with PPROM. Traditional markers from the maternal blood (C-reactive protein, white blood cell count) and the amniotic fluid (white blood cell count, glucose, and Gram stain) are still used for the detection of MIAC and HCA, despite their limited clinical utility.
There is evidence that the evaluation of amniotic fluid interleukin (IL)-6 is superior to that of amniotic fluid white blood cell count, amniotic fluid glucose, or Gram stains for the prediction of MIAC and HCA. Romero et al has reported that the diagnostic indices of amniotic fluid proteomic markers are not superior to that of amniotic fluid concentration of IL-6 in the identification of intraamniotic inflammation and MIAC. Additionally, some investigators have reported that IL-6 determinations can be performed at the bedside.
Despite the fact that bedside IL-6 has been tested on the cervicovaginal fluid of PPROM pregnancies, there is a paucity of information on the diagnostic and prognostic value of this test of amniotic fluid retrieved by transabdominal amniocentesis form PPROM pregnancies. An accurate and rapid IL-6 bedside test would be of great clinical utility in predicting PPROM complications.
The primary aim of this study was to determine the diagnostic indices and predictive values by bedside assessment of amniotic fluid IL-6 concentration in the identification of MIAC and/or HCA in patients with PPROM.
Materials and Methods
Sample collection
Between January 2012 and July 2013, a prospective cohort study was conducted on pregnant women at the gestational ages of 24+0 or 36+6 weeks, who were admitted to the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic. Pregnant women with singleton pregnancies complicated by PPROM and with a maternal age of 18 years or older were invited to participate in the study. Women with signs of fetal growth restriction, the presence of either congenital or chromosomal fetal abnormalities, gestational or pregestational diabetes, gestational hypertension, preeclampsia, signs of fetal hypoxia, or significant vaginal bleeding were excluded from the study.
Gestational ages were established by first-trimester fetal biometry. In the Czech Republic, women with PPROM at less than 34 weeks of gestation are treated with corticosteroids to accelerate the lung maturation (2 doses of 14 mg betamethasone administered intramuscularly 24 hours apart), tocolytics for 48 hours, and antibiotics, whereas no treatment except antibiotics is initiated to delay delivery after 34 weeks.
The management of PPROM in the Czech Republic is active (except <28 gestational weeks); the induction of labor or the termination of pregnancy is initiated no later than 72 hours after the rupture of the membranes, depending on the gestational age of the pregnancy, the fetal status, the maternal serum concentrations of C-reactive protein, and cervicovaginal group B Streptococcus colonization.
PPROM was diagnosed by examination with a sterile speculum to verify the pooling of amniotic fluid in the vagina after confirming the presence of insulin-like growth factor binding proteins (ACTIM PROM test; MedixBiochemica, Kauniainen, Finland) in the vaginal fluid.
Ultrasound-guided transabdominal amniocentesis was performed on admission but before the administration of antibiotics, corticosteroids, and tocolytics, and approximately 2-3 mL of amniotic fluid was aspirated. A total of 100 μL of noncentrifuged amniotic fluid was used for the bedside assessment of IL-6 concentrations. The remaining amniotic fluid was immediately transported to the microbiology laboratory for polymerase chain reaction testing for Ureaplasma spp., Mycoplasma hominis , and Chlamydia trachomatis as well as for aerobic and anaerobic cultivation.
The placentas were collected and fixed in 10% neutral buffered formalin. Tissue samples were obtained from the placenta (at least 2 samples), umbilical cord (typically 1 sample), and placental membranes (at least 2 samples), and the samples were processed and embedded in paraffin. Sections of tissue blocks were stained with hematoxylin and eosin.
The study was approved by the Institutional Review Board Committee (March 19, 2008; no. 200804 SO1P). All women provided their written informed consent and were self-reported to be white.
Detection of Ureaplasma spp , M hominis , and C trachomatis
Deoxyribonucleic acid (DNA) was isolated from the amniotic fluid with the QIAamp DNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions (protocol for the isolation of bacterial DNA from biological fluids). Real-time polymerase chain reaction was performed on a Rotor-Gene 6000 instrument (QIAGEN) with the commercial kit AmpliSens C trachomatis / Ureaplasma / M hominis -FRT (Federal State Institution of Science, Central Research Institute of Epidemiology, Moscow, Russia) to detect the DNA from Ureaplasma spp, M hominis , and C trachomatis in a common polymerase chain reaction tube. For a control, we included a polymerase chain reaction run for beta-actin, a housekeeping gene, to examine the presence of inhibitors of the polymerase chain reaction.
The threshold cycle value of the real-time polymerase chain reaction for Ureaplasma spp was used for the relative quantification of the microbial load of Ureaplasma spp in the amniotic fluid. The threshold cycle value is the intersection between an amplification curve and a threshold line. This measures the concentration of the target DNA in the polymerase chain reaction. Under ideal conditions (most polymerase chain reactions are close to 100% efficient), the amount of the target amplicon present increases at a rate of 1 log 10 every 3.32 cycles, which means that increasing the bacterial load of Ureaplasma spp in the amniotic fluid results in lower threshold cycle values.
Diagnosis of MIAC
MIAC was defined as a positive polymerase chain reaction analysis for Ureaplasma spp and/or M hominis and/or C trachomatis and/or growth of any bacteria in the amniotic fluid, except for coagulase-negative Staphylococcus epidermidis , which was considered a skin contaminant.
Diagnosis of HCA
The degree of neutrophilic infiltration was evaluated separately in the free membranes (amnion and chorion-decidua), in the chorionic plate, and in the umbilical cord, according to the criteria provided by Salafia et al. A diagnosis of HCA was made based on the presence of histological grades of chorion-decidua, 3-4; chorionic plate, 3-4; umbilical cord, 1-4; and/or amnion, 1-4. Histological grades of umbilical cord 1-4 were categorized as the presence of funisitis. Histopathological examinations were performed by a single pathologist who was blinded to the clinical status of the patient.
Amniotic fluid IL-6 concentrations
IL-6 concentrations were assessed with a lateral flow immunoassay Milenia QuickLine IL-6 using MileniaPOCScan Reader (Millennia Biotec GmbH, Giessen, Germany). The measurement range was 50-10,000 pg/mL. Intralot and interlot variation was 12.1% and 15.5%, respectively. The equipment was housed in the Labor and Delivery ward and the tests were performed in that location.
Statistical analysis
The demographic characteristics were compared using either an unpaired Student t test or the nonparametric Mann-Whitney U test for continuous variables, and the data are presented as the means ± SD and the medians (range), respectively. Categorical variables were compared using the χ 2 test and are presented as percentages. Spearman’s rank correlation test was used to analyze the correlations among continuous variables.
The normality of the data was tested using the D’Agostino-Pearson omnibus normality test and the Shapiro-Wilk test. Because the amniotic fluid concentrations of IL-6 were not normally distributed, a nonparametric test (Mann-Whitney U test) was used for analyses. For the analysis of the amniotic fluid IL-6 concentrations among the subgroups of women based on the presence and absence of MIAC and/or HCA, a nonparametric Kruskal-Wallis test, followed by a Dunn’s multiple comparisons test, was used.
A Spearman partial correlation was used to adjust the data for the gestational age at sampling. Receiver-operator characteristic curves were constructed to determine the predictive value of IL-6 for the presence of MIAC, HCA, and both MIAC and HCA. All P values were from 2-sided tests, and all statistical analyses were performed using SPSS version 19.0 for Mac OS X (SPSS Inc, Chicago, IL) and with GraphPad Prism version 5.03 for Mac OS X (GraphPad Software, La Jolla, CA).
Results
In total, 124 women with PPROM at gestational ages between 24+0 and 36+6 weeks were recruited. The overall rate of MIAC was 31% (38 of 124), and HCA was found in 71% of women (89 of 124). Funisitis was identified in 43% of women (53 of 124). The presence of both MIAC and HCA was observed in 24% (30 of 124) of women. Demographics and clinical data are shown in Table 1 .
| Characteristic | With MIAC (n = 38) | Without MIAC (n = 84) | P value a | With HCA (n = 89) | Without HCA (n = 35) | P value b | With both MIAC and HCA (n = 30) | Without both MIAC and HCA (n = 94) | P value c |
|---|---|---|---|---|---|---|---|---|---|
| Maternal age, y | 30.3 ± 7.2 | 30.4 ± 5.0 | .92 | 30.7 ± 5.6 | 29.6 ± 6.3 | .33 | 30.3 ± 7.4 | 30.1 ± 5.2 | .94 |
| Primiparous | 12 (32%) | 46 (55%) | .03 d | 38 (43%) | 20 (57%) | .17 | 9 (30%) | 49 (52%) | .04 d |
| Prepregnancy body mass index | 22.3 (16.5–28.5) | 22.6 (17.5–36.2) | .73 | 22.5 (16.5–36.2) | 22.2 (17.9–32.9) | .18 | 21.6 (16.5–36.2) | 22.6 (17.9–32.9) | .70 |
| Smoking | 10 (26%) | 12 (14%) | .10 | 15 (17%) | 7 (20%) | .79 | 9 (30%) | 13 (14%) | .06 |
| Gestational age at admission, wks plus d | 31+6 (24+2 to 36+6) | 32+3 (24+5 to 36+4) | .37 | 32+0 (24+2 to 36+6) | 33+5 (29+4 to 36+4) | .06 | 31+5 (24+2 to 36+6) | 32+3 (24+5 to 36+4) | .03 d |
| Gestational age at delivery, wks plus d | 32+1 (24+5 to 36+6) | 33+2 (25+2 to 36+4) | .39 | 32+3 (24+5 to 36+6 | 33+6 (30+5 to 36+4) | .09 | 31+6 (24+5 to 36+6) | 33+2 (25+2 to 36+6) | .04 d |
| Latency from PPROM to amniocentesis, h | 5 (1–97) | 5 (1–180) | .98 | 6 (1–180) | 4 (1–72) | .18 | 5 (1–180) | 5 (1–72) | .82 |
| Latency from PPROM to delivery, h | 33 (5–211) | 51 (4–196) | .07 | 49 (5–211) | 27 (4–196) | .12 | 36 (5–211) | 30 (4–196) | .16 |
| Maternal CRP levels at admission, mg/L | 6.5 (1.1–68.5) | 6.1 (0.5–36.1) | .57 | 6.0 (0.5–68.5) | 6.2 (0.5–21.7) | .55 | 8.25 (1.1–68.5) | 6.1 (0.5–36.1) | .14 |
| Microbial invasion of the amniotic cavity | — | — | — | 30 (48%) | 8 (23%) | .28 | 30 (100%) | 8 (17%) | < .0001 d |
| Administration of corticosteroids | 30 (78%) | 60 (71%) | 1.00 | 72 (81%) | 24 (69%) | .16 | 25 (83%) | 71 (76%) | .49 |
| Administration of antibiotics | 38 (100%) | 83 (99%) | 1.00 | 89 (100%) | 34 (97%) | .29 | 30 (100%) | 93 (99%) | 1.00 |
| Induction of labor | 12 (32%) | 18 (21%) | .26 | 24 (27%) | 6 (17%) | .35 | 10 (33%) | 20 (21%) | .22 |
| Cesarean section | 12 (32%) | 35 (41%) | .42 | 36 (40%) | 11 (31%) | .41 | 10 (33%) | 37 (39%) | .67 |
| Birthweight, g | 1836 ± 702 | 2039 ± 511 | .11 | 1839 ± 606 | 2189 ± 536 | .01 d | 1715 ± 708 | 2061 ± 539 | .005 d |
| HCA | 30 (79%) | 59 (70%) | .28 | — | — | — | 30 (100%) | 59 (62%) | < .0001 d |
| Funisitis | 22 (59%) | 31 (37%) | .03 d | — | — | — | 22 (73%) | 31 (33%) | < .0001 d |
| Apgar score <7 at 5 min | 2 (5%) | 4 (5%) | 1.00 | 5 (6%) | 1 (3%) | 1.00 | 2 (7%) | 4 (4%) | .63 |
| Apgar score <7 at 10 min | 1 (3%) | 2 (2%) | 1.00 | 2 (2%) | 1 (3%) | 1.00 | 1 (3%) | 2 (2%) | .56 |
a P value is the comparison between groups with and without MIAC
b P value is the comparison between groups with and without HCA
c P value is the comparison between groups with and without the presence of both MIAC and HCA
All of the microorganisms detected in the amniotic fluid are shown in Table 2 . The most common bacteria was Ureaplasma spp and was identified in 21% of women (26 of 124). Polymicrobial findings were found in 4% of women (5 of 124) ( Ureaplasma spp plus M hominis ).
| The bacterial species | Number of cases |
|---|---|
| Ureaplasma spp | 26 |
| Mycoplasma hominis | 9 |
| Chlamydia trachomatis | 6 |
| Enterococcus faecim | 1 |
| Haemophylus influenzae | 1 |
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