Objective
The objective of the study was to determine the frequency and clinical significance of intraamniotic inflammation in asymptomatic women with a sonographic short cervix (SCX) in the midtrimester.
Study Design
This cohort study included 47 asymptomatic women (14-24 weeks) with an SCX (≤15 mm) who underwent amniocentesis. Women with multiple gestation, cerclage, or cervical dilatation greater than 2 cm were excluded. Intraamniotic inflammation was defined as an elevated amniotic fluid (AF) matrix metalloproteinase-8 concentration (>23 ng/mL).
Results
(1) intraamniotic infection was found in 4.3% of patients; (2) among patients with a negative AF culture, the prevalence of intraamniotic inflammation was 22.2%; and (3) patients with a negative AF culture, but with intraamniotic inflammation, had a higher rate of delivery within 7 days (40% vs 5.7%; P = .016) and a shorter median diagnosis-to-delivery interval than those without intraamniotic inflammation (18 vs 42 days; P = .01).
Conclusion
Twenty-two percent of patients with a midtrimester SCX have intraamniotic inflammation. The risk of preterm delivery within 7 days for these patients is 40%.
A sonographic short cervix (SCX) in the midtrimester of pregnancy is a powerful predictor of a subsequent spontaneous preterm delivery. The shorter the sonographic cervical length, the higher is the risk of spontaneous preterm labor/delivery, and asymptomatic women between 14 and 24 weeks of gestation with a cervical length of 15 mm or less have a nearly 50% rate of spontaneous preterm delivery at 32 weeks or less.
The importance of identifying asymptomatic patients with a sonographic SCX in the midtrimester derives from the evidence that these patients, whether or not they have a history of preterm birth, may be candidates for therapeutic interventions such as vaginal progesterone, cervical cerclage, antibiotics, or indomethacin. However, these therapeutic measures may not be beneficial in all patients. Thus, the clinical management of an asymptomatic pregnant woman with a sonographic SCX in the midtrimester is still an obstetrical challenge.
A plausible explanation for the lack of a single effective treatment is that a sonographic SCX is a syndrome that may be caused by multiple etiologies. Of note, intraamniotic infection has been recently implicated in the pathophysiology of asymptomatic short cervix. Indeed, the subclinical microbial invasion of the amniotic cavity has been detected in 9% of patients with a sonographic cervical length less than 25 mm.
Among patients with preterm labor and intact membranes or with preterm prelabor rupture of membranes (PROM), intraamniotic inflammation, regardless of the microbial status of the amniotic cavity, has been associated with similar adverse pregnancy outcomes as in cases with a proven intraamniotic infection. Recently an association between an SCX (≤5 mm) in the midtrimester and elevated concentrations of inflammatory cytokines in amniotic fluid has been proposed.
Matrix metalloproteinase (MMP)-8, also known as neutrophil collagenase, is a sensitive marker of inflammation. Amniotic fluid MMP-8 concentration has been demonstrated to be a superior index of intraamniotic inflammation than amniotic fluid white blood cell (WBC) count or interleukin (IL)-6.
The aims of this study were to determine the frequency and clinical significance of intraamniotic inflammation (defined by amniotic fluid MMP-8 concentration) in asymptomatic women in the midtrimester of pregnancy with a sonographic SCX and to examine whether there is a relationship between amniotic fluid and maternal plasma MMP-8 concentrations.
Materials and Methods
Study population
This retrospective cohort study consisted of women with a singleton pregnancy admitted to Hutzel Women’s Hospital, between January 2002 and June 2008, with the diagnosis of an asymptomatic SCX. Using a computer-based search of our clinical and sonographic databases, consecutive patients with a cervical length of 15 mm or less diagnosed between 14 and 24 weeks of gestation, as determined by a documented transvaginal ultrasound examination (TVUS) and those who underwent an amniocentesis within 1 week of diagnosis were identified.
Patients with 1 or more of the following conditions were excluded: (1) multifetal pregnancy; (2) preterm labor or preterm PROM at the time of diagnosis; (3) a cervical cerclage (placed before or after the diagnosis of an SCX); (4) cervical dilatation greater than 2 cm by digital examination; (5) placenta previa; and (6) fetuses with chromosomal and/or congenital anomalies.
Patients included in this study were enrolled in a longitudinal protocol whose aim was to identify biochemical markers for the prediction of adverse pregnancy outcomes. As part of this protocol, patients are followed with TVUS every 2-4 weeks beginning as early as 14 weeks of gestation. A fraction of patients included in this study were diagnosed with an SCX during a routine TVUS examination and were sent to labor and delivery for further evaluation. Patients who agreed to participate were enrolled in our protocol and a repeat TVUS examination was performed. Digital assessment of the cervix was performed in all patients. The ultrasound findings were recorded and stored in a dedicated database. After the diagnosis of an SCX was made, the patients were referred to the labor and delivery ward for further evaluation and management. Both the sonographic cervical length and the result of the digital vaginal examination were available to the managing physicians.
In our institution, the majority of patients newly diagnosed with a sonographic cervical length less than 25 mm in the midtrimester undergo an initial evaluation during which patients are assessed for signs and symptoms of preterm labor. If preterm labor is ruled out, tocolysis and prophylactic antibiotics are not administered. In addition, these patients are offered an amniocentesis to determine the microbial status of the amniotic cavity, as previous observations suggested an association between SCX and histologic chorioamnionitis and intraamniotic infection. Patients were counseled by clinicians, and those who agreed to undergo an amniocentesis as part of their clinical management were asked to donate amniotic fluid and allow collection of clinical information for research purposes. Further management of these patients was at the discretion of the attending physician.
All participating women provided written informed consent prior to the collection of amniotic fluid and maternal blood samples. The use of clinical and ultrasound data and the utilization of samples for research purposes was approved by the institutional review boards of Wayne State University and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (National Institutes of Health, Department of Health and Human Services). Some of these samples have been used previously to study microbial invasion of the amniotic cavity and other biological markers of disease.
Definitions and study procedures
Gestational age was determined by the last menstrual period or by ultrasound if the sonographic determination of gestational age was not consistent with the last menstrual period dating by more than 1 week. Gestational age at diagnosis was defined as the earliest gestational age at which a cervical length of 15 mm or less was recorded.
Data regarding pregnancy outcomes were obtained from the clinical and research records. Patients who were lost to follow-up and for whom delivery data were not available were censored from the last available follow-up visit. Spontaneous preterm labor was defined by the presence of regular uterine contractions occurring at a frequency of at least 2 every 10 minutes associated with cervical changes before 37 completed weeks of gestation.
Intraamniotic infection was defined as a positive amniotic fluid culture for microorganisms (aerobic/anaerobic bacteria or genital mycoplasmas). Intraamniotic inflammation was defined as an amniotic fluid MMP-8 concentration greater than 23 ng/mL, as determined by enzyme-linked immunosorbent assay. Based on the results of the amniotic fluid culture and the amniotic fluid MMP-8 concentration, patients were classified as having 1 of the following conditions: (1) intraamniotic infection; (2) intraamniotic inflammation without intraamniotic infection; and (3) no intraamniotic infection or inflammation. Histologic chorioamnionitis was diagnosed based on the presence of inflammatory cells in the chorionic plate and/or chorioamniotic membranes, and acute funisitis was diagnosed by the presence of neutrophils in the wall of the umbilical vessels and/or Wharton’s jelly, using the criteria previously described.
Sonographic assessment of the cervix
Transvaginal ultrasound examination was conducted with commercially available 2-dimensional and 3-dimensional ultrasound systems (Acuson Sequoia; Siemens Medical Systems, Mountain View, CA, and Voluson E8; GE Healthcare, Milwaukee, WI) equipped with endovaginal transducers with frequency ranges of 5–7.5 and 5–9 MHz, respectively. All sonographic examinations of the cervical length were performed by registered diagnostic medical sonographers using a technique previously described and reviewed by an experienced physician.
Sample collection
Amniotic fluid samples were obtained by transabdominal amniocentesis, transported to the laboratory in a sterile capped syringe, and cultured for aerobic/anaerobic bacteria and genital mycoplasmas. WBC count, glucose concentration, and Gram stain were also performed shortly after the amniotic fluid collection. The results of these tests were used for the clinical management of the patients. Amniotic fluid not required for clinical assessment was centrifuged for 10 minutes at 4°C, and the supernatant was aliquoted and stored at –70°C until analysis.
Maternal blood samples were collected into Vacutainer (Becton Dickinson, Franklin Lakes, NJ) tubes immediately before or after the amniocentesis. The samples were then centrifuged at 1300 × g for 10 minutes at 4°C and the obtained plasma was stored at –70°C until assayed.
Following delivery, the placenta, umbilical cord, and chorioamniotic membranes were collected and the presence or absence of histologic chorioamnionitis and/or funisitis was assessed.
Determination of MMP-8 concentration
Amniotic fluid and maternal plasma concentrations of MMP-8 were determined by sensitive enzyme-linked immunoassays (GE Healthcare/Amersham Pharmacia Biotech Inc, Piscataway, NJ). Immunoassays were carried out according to the manufacturer’s recommendations. The calculated inter- and intraassay coefficients of variation for MMP-8 immunoassays in our laboratory were 1.7% and 3.9%, respectively, and the sensitivity was 0.101 ng/mL.
Statistical analysis
Shapiro-Wilk and Kolmogorov-Smirnov tests were used to test for normal distribution of the data. As amniotic fluid and maternal plasma MMP-8 concentrations were not normally distributed, Mann-Whitney U test was used for comparisons of continuous variables. Comparisons of proportions among groups were performed using Fisher’s exact test for categorical variables.
Correlations between amniotic fluid MMP-8 concentrations and cervical length at diagnosis, gestational age at amniocentesis, diagnosis-to-delivery interval, and maternal plasma MMP-8 concentrations were examined using Spearman’s rank correlation test. Multivariate logistic regression analysis was performed to determine the relationship between the amniotic fluid MMP-8 concentrations (as a categorical variable of ≤23 ng/mL and >23 ng/mL), gestational age at diagnosis, and cervical length in the prediction of preterm delivery within 7 days from diagnosis.
A Kaplan-Meier survival analysis was performed to assess the diagnosis-to-delivery interval according to the presence or absence of intraamniotic inflammation. A P < .05 was considered statistically significant. The statistical package used was SPSS version 12.0 (SPSS Inc, Chicago, IL).
Results
Demographic and clinical characteristics of the study population
During the study period, 53 asymptomatic patients who met the inclusion criteria were identified. Amniotic fluid samples for retrospective analysis of MMP-8 concentration were not available for 6 patients; thus, 47 women (88.7%) were included in the final analysis.
The overall rate of intraamniotic inflammation was 23.4% (11/47). Intraamniotic infection was identified in 2 of 47 patients (4.3%). Amniotic fluid WBC count of 100 cells/mm 3 or greater was found in 3 of 47 patients (6.4%). Among patients with a negative amniotic fluid culture for microorganisms, the rate of intraamniotic inflammation was 22.2% (10/45), whereas only 4.4% (2/45) had an amniotic fluid WBC count of 100 cells/mm 3 or greater.
Of the 2 patients with intraamniotic infection, 1 had a positive amniotic fluid culture for Fusobacterium nucleatum. This patient developed clinical chorioamnionitis shortly after admission and labor was induced. The second patient had a positive amniotic fluid culture for ureaplasma urealyticum (without intraamniotic inflammation) and received intravenous azithromycin for 7 days with a subsequent term delivery.
Of the 45 patients without intraamniotic infection, 1 patient was lost to follow-up from 32 weeks of gestation, 8 patients (18.2%) delivered at term, and 36 (81.8%) had a spontaneous preterm delivery before 37 completed weeks of gestation. Spontaneous preterm delivery followed a spontaneous preterm labor in 24 patients (67%) and preterm PROM in 12 patients (33%) (2 patients were induced following preterm PROM because of clinical chorioamnionitis).
Table 1 displays the demographic and clinical characteristics of patients with a negative amniotic fluid culture, with (n = 10) and without (n = 35) intraamniotic inflammation. The gestational age range of patients included in this study was 16 weeks and 3 days to 24 weeks of gestation. Patients with intraamniotic inflammation had a shorter gestational age at diagnosis and a lower cervical length than those without intraamniotic inflammation.
