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Is midtrimester short cervix a sign of intraamniotic inflammation?

      Objective

      We sought to determine the relationship between the degree of cervical shortening and intraamniotic inflammation in patients presenting with a midtrimester short cervix.

      Study Design

      Amniocentesis was performed on singleton pregnancies between 16-24 weeks' gestation with a sonographic cervical length (CL) ≤ 25 mm. The fluid was assayed for 25 cytokines. Spearman correlations were used to determine which cytokines correlate with CL. Stepwise regression identified the most significant cytokine and a receiver operating characteristic curve determined the CL cutoff predictive of intraamniotic inflammation.

      Results

      In all, 109 amniotic fluid samples were analyzed. Most (21 of 25) cytokines were inversely correlated to CL. Monocyte chemotactic protein (MCP)-1 was the most significant by stepwise regression. Using a cutoff of MCP-1 > 1500 pg/mL, CL of 5 mm had an 86% sensitivity, 85% specificity, 58% positive predictive value, and 96% negative predictive value to predict elevated MCP-1 levels. After excluding patients with intraamniotic infection or labor, findings were similar.

      Conclusion

      CL ≤ 5 mm is associated with significant increases in amniotic fluid inflammatory cytokines, even in the absence of infection or labor. In the future, differentiation of those with and without inflammation may aid in choosing therapy directed at the cause of cervical shortening.

      Key words

      The preterm birth rate continues to increase in the United States despite intense research to reduce its prevalence.
      • Ananth C.V.
      • Joseph K.S.
      • Oyelese Y.
      • Demissie K.
      • Vintzileos A.M.
      Trends in preterm birth and perinatal mortality among singletons: United States, 1989 through 2000.
      It has been proposed that preterm birth is the end result of a cascade of events that may begin with infection, inflammation, ischemia, premature activation of the fetal hypothalamic-pituitary axis, maternal-fetal hemorrhage, or uterine overdistension.
      • Peltier M.R.
      Immunology of term and preterm labor.
      • Rust O.A.
      • Atlas R.O.
      • Reed J.
      • van Gaalen J.
      • Balducci J.
      Revisiting the short cervix detected by transvaginal ultrasound in the second trimester: why cerclage therapy may not help.
      • Lockwood C.J.
      • Kuczynski E.
      Markers of risk for preterm delivery.
      A midtrimester short cervix is an early risk factor and strong predictor for spontaneous preterm birth.
      • Iams J.D.
      • Goldenberg R.L.
      • Meis P.J.
      • et al.
      The length of the cervix and the risk of spontaneous premature delivery: National Institute of Child Health and Human Development Maternal Fetal Medicine Unit Network.
      It has been suggested that it is a marker for preterm birth originating from any of the above-listed pathways. Many interventions (ie, cerclage, progesterone therapy) that have been suggested to treat the short cervix have had mixed results in the clinical setting.
      • Fonseca E.B.
      • Celik E.
      • Parra M.
      • Singh M.
      • Nicolaides K.H.
      Progesterone and the risk of preterm birth among women with a short cervix.
      • Berghella V.
      • Odibo A.O.
      • To M.S.
      • Rust O.A.
      • Althuisius S.M.
      Cerclage for short cervix on ultrasonography: meta-analysis of trials using individual patient-level data.
      This may be, in part, a result of our inability to differentiate the multiple origins of the short cervix. In addition, there is a possibility that treatment failures may be dependent on the degree of cervical shortening. Thus, it is important to differentiate the precise cause of the cervical shortening to better aid in tailoring therapy.
      For Editors' Commentary, see Table of Contents
      Some studies have suggested that cervical insufficiency may be the result of an inflammatory process.
      • Lee S.E.
      • Romero R.
      • Park C.W.
      • Jun J.K.
      • Yoon B.H.
      The frequency and significance of intraamniotic inflammation in patients with cervical insufficiency.
      However, no studies have determined the correlation between the degree of cervical shortening and inflammation. Therefore, the purpose of this study was to determine the degree of cervical shortening and amniotic fluid cytokine levels in patients with short cervix in the midtrimester.

      Materials and Methods

      Under an institutional review board-approved protocol, singleton pregnancies presenting to our hospital perinatal testing center between April 1998 and December 2006 with risk factors for spontaneous preterm birth were screened with serial transvaginal ultrasound beginning at 16 weeks' gestation. Risk factors for preterm birth included history of spontaneous preterm birth, second-trimester pregnancy loss, previous cervical surgery (conization or loop excision), or documented uterine anomaly. In addition, low-risk, asymptomatic singleton pregnancies between 16-24 weeks' gestation were screened for evidence of cervical shortening with transabdominal ultrasound as part of routine anatomic survey. If the cervix appeared short (≤ 25 mm) transabdominally, a transvaginal ultrasound was performed. Transvaginal cervical length (CL) measurement was obtained using the standardized technique described by Rust et al.
      • Rust O.A.
      • Atlas R.O.
      • Jones K.J.
      • Benham B.N.
      • Balducci J.
      A randomized trial of cerclage versus no cerclage among patients with ultrasonographically detected second-trimester preterm dilatation of the internal os.
      Patients with ultrasonographic evidence of a short cervix, defined as a transvaginal distal CL ≤ 25 mm (and exacerbation of these ultrasound findings after transfundal and/or suprapubic pressure), were offered enrollment into randomized controlled trials of cerclage vs either no cerclage or progesterone therapy for treatment of short cervix. All patients with a short cervix underwent an ultrasound-guided transabdominal amniocentesis to exclude intraamniotic infection (low glucose, elevated white blood cell count, and aerobic/anaerobic culture) before study enrollment. A total of 5 mL of unspun amniotic fluid was aliquoted into 15-mL polypropylene tubes and stored at -70°C for future cytokine analysis.
      Patients were excluded for the following conditions: any known fetal chromosomal or structural anomaly, multiple gestation, ruptured membranes, vaginal bleeding, or the need for an obstetrically indicated delivery.
      A comprehensive amniotic fluid cytokine analysis of all patients with a short cervix was undertaken to determine the correlation of each cytokine level with CL. We used the Bio-Plex array system (Bio-Rad Laboratories Inc, Hercules, CA) to simultaneously assay and quantify 25 different cytokine concentrations. The cytokines evaluated were interleukin (IL)-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, eotaxin, granulocyte colony-stimulating factor (G-CSF), interferon-γ, inducible protein-10, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1a, MIP-1b, platelet-derived growth factor (PDGF) bb, tumor necrosis factor-α, vascular endothelial growth factor, and regulated on activation, normal T-cell expressed, and secreted (RANTES) protein.
      The amniotic fluid samples were analyzed by Bio-Plex array system (Bio-Rad Laboratories Inc) according to the manufacturer's protocols. In all, 50 μL of amniotic fluid was placed in a 96-well filtration plate supplied with the assay kit. Premixed beads (50 μL) coated with target capture antibodies were used (5000 beads/well/cytokine). The data were analyzed using software (Bio-Plex Manager, Version 3.0; Bio-Rad Laboratories Inc) with 5-parameter logistic regression curve fitting. For statistical purposes, cytokine levels were determined to be sufficient if the median level was at least as high as the median from healthy control subjects.
      • Chow S.S.
      • Craig M.E.
      • Jones C.A.
      • et al.
      Differences in amniotic fluid and maternal serum cytokine levels in early midtrimester women without evidence of infection.
      Data on maternal demographics, gestational age at study entry, risk factors for spontaneous preterm birth, and CL measurements were recorded from the hospital cerclage database.
      Spearman correlation was used to determine which, if any, cytokines correlated with CL (univariate analysis) and scatterplots were created to depict the relationship between CL and some of the most significant cytokine levels. A stepwise regression analysis was used to determine which cytokine or cytokines were the most predictive of CL. A receiver operating characteristic (ROC) curve was used to determine the best CL cutoff to predict intraamniotic inflammation (namely MCP-1 > 1500 pg/mL). This MCP-1 level was chosen as the dependent variable because it has been shown by previous study,
      • Esplin M.S.
      • Romero R.
      • Chaiworapongsa T.
      • et al.
      Monocyte chemotactic protein-1 is increased in the amniotic fluid of women who deliver preterm in the presence or absence of intra-amniotic infection.
      and a review of our own data,

      Keeler S, Kiefer D, Rust O, et al. Comprehensive amniotic fluid cytokine profile evaluation in women with a short cervix: which cytokine(s) correlate(s) best with outcome? Poster presented at: the 29th Annual Meeting of the Society for Maternal–Fetal Medicine; Jan. 26-31, 2009; San Diego, CA.

      to correlate with adverse pregnancy outcome and short interval to delivery. The patients were then divided into 2 groups, based on the CL derived from the ROC curve. As the data were not normally distributed, the cytokine levels of the 2 groups were compared using the Wilcoxon rank sum test. Results were considered statistically significant at P < .05, with Bonferroni correction for those statistics involving all 25 cytokines (P < .002). All calculations were performed using software (SAS 9.1 for Windows; SAS Institute, Cary, NC).
      Intraamniotic infection or labor are known to affect cytokine expression. To eliminate these potential confounders, the same analyses were repeated after excluding patients with evidence of either infection (defined as a positive aerobic/anaerobic culture, or low glucose [≤14 mg/dL]) or labor (defined as delivery within 72 hours of amniocentesis).

      Results

      In all, 109 amniotic fluid samples from singleton pregnancies were analyzed. The mean maternal age was 26.6 ± 3.6 years and the mean gestational age at study entry was 20.4 ± 2.0 weeks. Whites composed 48.6% (n = 53) of the population; 32 (29.4%) were Hispanic; 19 (17.4%) were African American; and 5 (4.6%) were classified as “other.” Regarding obstetric history, 45.9% (n = 50) were nulliparous and 43 (39.4%) had a previous preterm birth. The median CL was 12 mm (range, 0-25 mm). Two (1.9%) patients had a positive amniotic fluid culture and 5 (4.8%) had an amniotic fluid glucose ≤ 14 mg/dL.
      Table 1 displays the median cytokine level along with the Spearman correlation between the cytokine levels and CL (univariate analysis). Although many cytokines were inversely correlated with CL, not all were detected in significant quantities. Only IL-6, IL-8, G-CSF, MCP-1, MIP-1b, and RANTES protein were both significantly correlated with CL and detected in substantial quantities. Scatterplots depicting the relationship between CL and 4 of the cytokines are shown in Figure 1. Levels of most cytokines appear to become elevated at a CL of ≤ 5 mm (eg, MCP-1, IL-6, IL-8). For most cytokines, the significant correlations were driven largely by high cytokine levels at very short CL measurements. This finding also accounts for how some cytokines (eg, IL-1β) were not detected in large quantities, yet still managed to obtain extremely significant correlations.
      TABLE 1Correlation of cytokine levels with cervical length
      Cytokine nameMedian level (pg/mL)Spearman correlationP value
      Bonferroni correction; P < .002 required for significance;
      IL-1β0.9-0.43869< .0001
      IL-1ra4502.0-0.00778.9360
      IL-20.20-0.22650.0179
      IL-40.5-0.25052.0086
      IL-50.32-0.19775.0393
      IL-6
      cytokines that were detected in sufficient quantities and achieved a statistically significant correlation.
      388.5-0.31995.0011
      IL-73.3-0.06093.5291
      IL-8
      cytokines that were detected in sufficient quantities and achieved a statistically significant correlation.
      319.3-0.30374.0016
      IL-915.7-0.21031.0282
      IL-100.9-0.37005< .0001
      IL-120.6-0.36840< .0001
      IL-130.8-0.26862.0047
      IL-156.9-0.26232.0059
      IL-170-0.51118< .0001
      Eotaxin12.8-0.19573.0414
      G-CSF
      cytokines that were detected in sufficient quantities and achieved a statistically significant correlation.
      212.0-0.38694< .0001
      IFN-γ83.2-0.26399.0055
      IP-1014927.1-0.02206.8199
      MCP-1
      cytokines that were detected in sufficient quantities and achieved a statistically significant correlation.
      452.9-0.41757< .0001
      MIP-1a0-0.45683< .0001
      MIP-1b
      cytokines that were detected in sufficient quantities and achieved a statistically significant correlation.
      47.5-0.33004.0005
      PDGF bb272.0-0.05873.5441
      TNF-α0-0.31933.0007
      RANTES protein
      cytokines that were detected in sufficient quantities and achieved a statistically significant correlation.
      21.9-0.38140< .0001
      VEGF3.5-0.08328.3893
      G-CSF, granulocyte colony-stimulating factor; IFN, interferon; IL, interleukin; IP, inducible protein; MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein; PDGF, platelet-derived growth factor; RANTES, regulated on activation, normal T-cell expressed, and secreted; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.
      Kiefer. Is midtrimester short cervix a sign of intraamniotic inflammation? Am J Obstet Gynecol 2009.
      a Bonferroni correction; P < .002 required for significance;
      b cytokines that were detected in sufficient quantities and achieved a statistically significant correlation.
      Figure thumbnail gr1
      FIGURE 1Scatterplots of selected cytokine levels vs cervical length
      Kiefer. Is midtrimester short cervix a sign of intraamniotic inflammation? Am J Obstet Gynecol 2009.
      Of the cytokines that were detected in significant quantities, stepwise regression showed that MCP-1 was most predictive of a shortened CL. Once MCP-1 entered the model, no other cytokines were significant. The ROC curve showing the ability of CL to predict an MCP-1 level > 1500 pg/mL is shown in Figure 2. The area under the curve was 0.88. Based on the ROC curve, a CL cutoff of 5 mm had an 86% sensitivity, 85% specificity, 58% positive predictive value, and 96% negative predictive value to predict elevated MCP-1 levels (>1500 pg/mL).
      Figure thumbnail gr2
      FIGURE 2ROC curve
      Receiver operating characteristic curve (ROC) for cervical length predictive of monocyte chemotactic protein-1 > 1500 pg/mL.
      Kiefer. Is midtrimester short cervix a sign of intraamniotic inflammation? Am J Obstet Gynecol 2009.
      Table 2 compares the median cytokine levels when patients were divided into 2 groups based on CL (≤ 5 mm and 6-25 mm). In all, 29 patients had a CL ≤ 5 mm; 80 had a CL of 6-25 mm. When compared with those with a CL between 6-25 mm, all the cytokine levels shown in Table 2 were higher in the patients with CL ≤ 5 mm with MCP-1, IL-6, and G-CSF being the most significant. The mean gestational age at enrollment was 20.4 weeks for patients with a CL ≤ 5 mm compared with 20.3 weeks for patient with a CL between 6-25 mm (P = .80).
      TABLE 2Comparison of selected median cytokine levels, based on cervical length
      Cytokine (pg/mL)Cervical length ≤ 5 mm (n = 29)Cervical length 6-25 mm (n = 80)P value
      Wilcoxon rank-sum, Bonferroni correction.
      IL-1ra5727.74210.3.035
      IL-61530.0344.8< .0001
      IL-81531.9271.3.0002
      Eotaxin41.39.2.0002
      G-CSF1402.0166.1< .0001
      IFN-γ258.374.4.001
      MCP-12093.0346.9< .0001
      PDGF bb314.8250.5.044
      RANTES protein57.016.9.001
      G-CSF, granulocyte colony-stimulating factor; IFN, interferon; IL, interleukin; MCP, monocyte chemotactic protein; PDGF, platelet-derived growth factor; RANTES, regulated on activation, normal T-cell expressed, and secreted.
      Kiefer. Is midtrimester short cervix a sign of intraamniotic inflammation? Am J Obstet Gynecol 2009.
      a Wilcoxon rank-sum, Bonferroni correction.
      We also examined the Spearman correlations among patients with a CL between 6-25 mm. None of the cytokine levels were significantly correlated with CL, except IL-1ra. This antiinflammatory cytokine was directly correlated with CL (correlation coefficient, 0.33; P = .003) when the CL was 6-25 mm.
      Nineteen patients had evidence of either labor or infection as defined above. When the analysis was performed on the remaining 90 patients, similar results were found when compared with the entire cohort. Among the 90 patients, 70 had a CL between 6-25 mm and 20 had a CL ≤ 5 mm. The ROC curve analysis suggests the CL cutoff most predictive of an elevated MCP-1 level was < 5 mm, similar to the previous analysis. Although the cytokine levels were, in general, higher in the patients with an extremely short cervix (≤ 5 mm), 2 from those listed in Table 2 were no longer statistically significant, IL-1ra (P = .17) and PDGF (P = .06); the rest remained significant.

      Comment

      The midtrimester short cervix poses a significant dilemma for clinicians. Many therapies have been directed at treating the short cervix since it has been shown to be a risk factor for spontaneous preterm birth. Lee et al
      • Lee S.E.
      • Romero R.
      • Park C.W.
      • Jun J.K.
      • Yoon B.H.
      The frequency and significance of intraamniotic inflammation in patients with cervical insufficiency.
      have shown that patients with acute cervical insufficiency (based on visual examination) have evidence of intraamniotic inflammation, even in the absence of detectable infection or labor. Our study demonstrates a correlation between intraamniotic inflammation and the degree of cervical shortening as detected by transvaginal sonography, which has been shown to be superior to bimanual examination.
      • Sonek J.D.
      • Iams J.D.
      • Blumenfeld M.
      • Johnson F.
      • Landon M.
      • Gabbe S.
      Measurement of cervical length in pregnancy: comparison between vaginal ultrasonography and digital examination.
      • Berghella V.
      • Ness A.
      • Bega G.
      • Berghella M.
      Cervical sonography in women with symptoms of preterm labor.
      Similar to Lee et al,
      • Lee S.E.
      • Romero R.
      • Park C.W.
      • Jun J.K.
      • Yoon B.H.
      The frequency and significance of intraamniotic inflammation in patients with cervical insufficiency.
      we found these correlations remained after excluding patient with infection or labor.
      Our criteria for intraamniotic infection were: (1) positive aerobic/anaerobic culture; (2) glucose ≤ 14 mg/dL; or (3) delivery within 72 hours of amniocentesis. We included delivery within 72 hours given the limitations of aerobic/anaerobic cultures or low glucose (81.8% sensitivity)
      • Romero R.
      • Yoon B.H.
      • Mazor M.
      • et al.
      The diagnostic and prognostic value of amniotic fluid white blood cell count, glucose, interleukin-6, and gram stain in patients with preterm labor and intact membranes.
      to detect infection. We believed this criterion would capture those patients with infection not detected by culture or low glucose. In addition, our amniotic fluid samples were not cultured for Mycoplasma and Ureaplasma, which have been shown to be prevalent in patients with preterm labor. This partly explains the lower positive culture rate (1.9%) in our patients compared with previous study in patients with short cervix (9% culture positive; 80% of which were Ureaplasma).
      • Hassan S.
      • Romero R.
      • Hendler I.
      • et al.
      A sonographic short cervix as the only clinical manifestation of intra-amniotic infection.
      Nonculture-based approaches to identify microorganisms in the amniotic cavity can lead to the identification of bacteria that have not traditionally being identified by culture-based approaches.
      • DiGiulio D.B.
      • Romero R.
      • Amogan H.P.
      • et al.
      Microbial prevalence, diversity and abundance in amniotic fluid during preterm labor: a molecular and culture-based investigation.
      We are planning to use these techniques to assay the amniotic fluid for many bacteria, and pathogens associated with preterm labor (eg, Mycoplasma and Ureaplasma). These data will allow us to better determine the relationship between microbial invasion and infection, CL, and the inflammatory response.
      Although other studies have demonstrated increased inflammatory cytokines in patients with preterm labor or preterm rupture of membranes, none have evaluated intraamniotic cytokine levels in patients with a sonographically detected short cervix. Another unique aspect of our study is that we simultaneously evaluated the amniotic fluid for 25 cytokines, rather than focusing on just a few. We selected this panel of cytokines, developed by Bio-Rad Laboratories Inc, because: (1) it includes several key proinflammatory and antiinflammatory cytokines, chemokines, and growth factors that were shown to play key roles in placental immune regulation; and (2) the same panel of amniotic fluid cytokines used in our study have been studied by previous investigators in early midtrimester normal pregnancy and were detected in amniotic fluid.
      • Chow S.S.
      • Craig M.E.
      • Jones C.A.
      • et al.
      Differences in amniotic fluid and maternal serum cytokine levels in early midtrimester women without evidence of infection.
      From this analysis, MCP-1 was selected from the stepwise regression as the cytokine most highly correlated with CL. MCP-1 plays a role in regulating inflammatory processes by recruiting monocytes into foci of active inflammation.
      • Muller W.A.
      New mechanisms and pathways for monocyte recruitment.
      It has been shown to be elevated in cervical secretions and amniotic fluid of patient with preterm labor and preterm premature rupture of membranes.
      • Jacobsson B.
      • Holst R.M.
      • Wennerholm U.B.
      • Andersson B.
      • Lilja H.
      • Hagberg H.
      Monocyte chemotactic protein-1 in cervical and amniotic fluid: relationship to microbial invasion of the amniotic cavity, intra-amniotic inflammation, and preterm delivery.
      In addition, elevated levels of MCP-1 > 1500 pg/mL have been shown in other studies,
      • Esplin M.S.
      • Romero R.
      • Chaiworapongsa T.
      • et al.
      Monocyte chemotactic protein-1 is increased in the amniotic fluid of women who deliver preterm in the presence or absence of intra-amniotic infection.
      corroborated by an analysis of our own data,

      Keeler S, Kiefer D, Rust O, et al. Comprehensive amniotic fluid cytokine profile evaluation in women with a short cervix: which cytokine(s) correlate(s) best with outcome? Poster presented at: the 29th Annual Meeting of the Society for Maternal–Fetal Medicine; Jan. 26-31, 2009; San Diego, CA.

      to be associated with adverse pregnancy outcome. In our study, CL was able to predict which patients were most likely to have higher MCP-1 levels.
      Although we were able to show significant correlations between cytokine levels and the degree of cervical shortening, our study cohort does not include patients with a normal CL. Thus, these data cannot be used to determine the relationship of intraamniotic inflammation and CL across all CLs. In fact, the significant inverse correlations between CL and cytokine levels did not exist when the CL was 6-25 mm.
      Although this suggests that inflammatory cytokines may play less of a role in those patients with a milder degree of cervical shortening, we should not conclude that there is no inflammation in patients with a CL between 6-25 mm. Chow et al
      • Chow S.S.
      • Craig M.E.
      • Jones C.A.
      • et al.
      Differences in amniotic fluid and maternal serum cytokine levels in early midtrimester women without evidence of infection.
      recently published an expanded amniotic fluid cytokine profile (also using the Bio-Plex system [Bio-Rad Laboratories Inc]) from asymptomatic, noninfected women undergoing genetic amniocentesis in the midtrimester. Although CL was not reported, we can assume that the large majority of these patients did not have a short cervix because 99 (99%) of 100 delivered at term. Median intraamniotic levels of IL-6, IL-8, G-CSF, interferon-γ, MCP-1, PDGF bb, and RANTES protein were all lower in these patients when compared with patients in our study with CL between 6-25 mm. These data suggest that there may be other mechanisms or pathways involved, in addition to mild inflammation, when the CL is between 6-25 mm. These findings provide a biological explanation for the variable success observed in clinical research in which all patients with a short cervix are treated in a similar manner. Some patients may be so far along the inflammation cascade that leads to preterm labor that our current therapies (eg, cerclage, tocolytics, progesterone therapy) may not be effective.
      These data are important because it enables the clinician to use a readily available noninvasive tool, transvaginal sonography, to predict patients at high risk for intraamniotic inflammation based on CL. As pathway-specific therapies to treat cervical shortening and preterm labor evolve, these data may aid in choosing the most appropriate therapy directed at the underlying cause of cervical shortening.

      Acknowledgment

      We wish to thank Martin Feuerman for his assistance with the statistical analysis.

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