http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, 2014; 27(10): 1015–1019 ! 2014 Informa UK Ltd. DOI: 10.3109/14767058.2013.847917
ORIGINAL ARTICLE
Short and inflamed cervix predicts spontaneous preterm birth (COLIBRI study)* Evelyne Raiche1, Annie Ouellet1, Maryse Berthiaume2, E´ric Rousseau3, and Jean-Charles Pasquier1 Department of Obstetrics and Gynecology, 2Centre de Recherche Clinique E´tienne-LeBel, and 3Department of Physiology and Biophysics, Universite´ de Sherbrooke, Sherbrooke, Quebec, Canada
1
Abstract
Keywords
Objective: To develop a new strategy of predicting spontaneous preterm birth (sPTB) by combination of transvaginal ultrasound (TVUS) assessment and inflammatory proteins detection in vaginal secretions. Methods: Prospective study of 87 women referred for cervical length assessment with a standardized TVUS combined to vaginal secretions sampling. Samples were analyzed for presence of 10 cytokines. Main outcome was sPTB (537 weeks of gestation). Associations were assessed with the chi-square, Fisher’s exact test (p50.05) and Wald’s logistic regression. Results: sPTB occurred in 25.3% of women at a median gestational age of 35.6 weeks of gestation. Short cervix (525 mm) (n ¼ 24) was associated with sPTB (p50.01) as interleukine (IL)1b, IL-8 and IL-10 in vaginal secretions (p50.05). In multivariate analysis, short cervix and IL-8 in vaginal secretions were independently associated with sPTB (OR 3.58 (95%CI 1.02; 12.61) and 14.55 (95%CI 1.64; 128.83), respectively) as their combination (OR 4.33 (95%CI 1.25; 14.95)). By categorizing cervical length by presence of IL-8, sPTB occurred in 55.6% of women with a short inflamed cervix. Conclusion: COLIBRI study used a novel, single-step method of vaginal secretions sampling during TVUS and demonstrated that combination of short cervix and IL-8 in vaginal secretions is a promising sPTB predictive test.
Cervical length, cytokines, inflammation, pre-term birth, transvaginal ultrasound, vaginal secretions
Introduction Preterm birth is the leading cause of perinatal mortality and morbidity and a major cause of long-term health problems [1]. Spontaneous preterm birth (sPTB), with intact and ruptured membranes, is a heterogeneous pathology arising from different pathways in which inflammation plays a major role [2,3]. Activation of collagenases and prostaglandins, by inflammation and intra-amniotic infection, promote degradation of the cervix and amniotic membranes and enhance uterine contractility and rupture of membranes [4–6]. It is urgent to categorize women with a combination of predictive factors improving the quality of predictive results, but also to propose a personalized therapeutic strategy [7].
*Results were presented at: SMFM 30th Annual Meeting – The Pregnancy MeetingtM, Chicago, IL, February 1–6, 2010; SMFM 31st Annual Meeting – The Pregnancy MeetingtM, San Francisco, CA, February 7–12, 2011. Address for correspondence: Dr Jean-Charles Pasquier, MD, PhD, Department of Obstetrics and Gynecology, Universite´ de Sherbrooke, 3001, 12e avenue Nord, Sherbrooke, QC J1H 3T5, Canada. Tel: (819)346-1110 by 0, pager 4403; (819)-679-2212 (home). Fax: (819)-819 8206434. E-mail: [email protected]
History Received 27 May 2013 Accepted 19 September 2013 Published online 31 October 2013
Two approaches have been described to predict sPTB, ultrasound and biochemical markers. Cervical shortening is an integrated part of the pathophysiological process leading to sPTB and may be associated with other factors such as funnelling and sludge [8]. Transvaginal ultrasound (TVUS) measurement of cervical length, described by Iams in the 1990s [9] is the most reliable method to assess the cervix and represents the best predictive test for sPTB with a positive predictive value ranging from 30% to 55% for sPTB high-risk singleton and twin pregnancies [10,11]. Several pro-inflammatory proteins, identified in vaginal secretions, were associated with sPTB including IL-1b, IL-6, IL-8, MMPs, IL-10 and TIMP-1 [12,13] as well as fetal fibronectine [14]. Some of these, e.g. IL-8 was associated with short cervical length and intra-amniotic infection [15]. Recently, new therapeutic strategies appear in the management of prematurity such as pessary to strengthen the cervix [16]. To deal with these innovations, it is important to select women in order to prevent sPTB by offering adapted treatment to prematurity etiology; antiinflammatory treatment to women with inflammation detected non-invasively and pessary or cerclage to women with mechanical short cervix due to cervix incompetence or cone biopsy.
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We designed a standardized examination allowing sampling of vaginal secretion in the same exam of TVUS with two Dacron swabs set on the ultrasound probe. We used this combined strategy to categorize ultrasound finding by the presence of an inflammatory process. The main objective of our study was to enhance sPTB prediction by a better comprehensive categorization of high-risk patient combining cervical length and inflammatory status of vaginal secretion.
Methods Study population We conducted a prospective cohort study of women referred to maternal and fetal medicine (MFM) clinic for sPTB risk evaluation by TVUS at the Centre Hospitalier Universitaire de Sherbrooke (CHUS), Quebec, Canada, referral center for women living in the surrounding area of Shebrooke. The Ethics Committee of our center approved the study protocol in April 2008 (Comite´ d’e´thique de la recherche en sante´ chez l’humain du CHUS, project 08-020, ClinicalTrials.gov identifier: NCT01007513). Women were recruited consecutively from June 2008 to July 2009. Inclusion criteria: Women were eligible if pregnant with a live singleton or multifetal pregnancy and referred for TVUS between 20 and 34 weeks of gestation. TVUS was performed for clinical indicated reason according to women physician. Women were referred for risk factors of sPTB, either from previous history of sPTB or clinical reason in this pregnancy (multiple pregnancy, soften cervix or contractile activity without active labor). Exclusion criteria: Exclusion criteria were active labor at the time of TVUS or delivery on the TVUS day, fetuses with major congenital anomalies, placenta previa and PPROM. Women with an induced preterm birth for medical reason were excluded from analysis. A research nurse provided information regarding the study protocol and all women gave a written informed consent before participating in the study. Sonographic assessment of the cervix Cervical length was evaluated by the standard method described by Iams and if several values were collected, the shorter length was retained [9,11]. TVUS examinations were performed by seven experienced radiological technologists and controlled by four obstetricians. Examinations were performed either on a GE Voluson 730 Expert with the RIC5-9H (5 MHz) probe (GE Voluson 730 Expert, Little Chalfont, UK) or on a Toshiba Aplia XG with a PVT-661VT (6 MHz) probe without ultrasound gel over the probe (Toshiba Aplio XG, Tustin, CA, USA). All images were stored and read by two independent observers to ensure internal validity of the ultrasound findings. TVUS findings were reported to physician as they were noted in the file. Cervicovaginal fluid analysis We developed an original method with Dacron swabs set on an endovaginal probe (Figure 1). Swabs were left in place during the entire TVUS examination, lasting between 5 and 10 min, and primarily collected vaginal secretions from the posterior fornix. The samples were diluted in 1 mL PBS,
Figure 1. TVUS probe with an affixed Dacron swab as used in the present protocol.
centrifuged for 2 min at 5000 rpm and froze within 10 min at 80 C. Samples were analyzed using multiplex antibody array from RayBiotech (Raybiotech, Atlanta, GA, USA). Choice of cytokines was based on immunological pathway and known association of IL-1b, IL-6, IL-8, MMPs, IL-10 and TIMP-1 with sPTB [17–20]. Cytokines were detected by immunoluminescence with a Molecular Imager VersaDoc MP 5000 System (Molecular Imager VersaDoc MP 5000 System from Bio-Rad, Hercules, CA, USA) with the following sensitivities: IL-6, IL-8 and MMP-8 (1 pg/ml); IL-10 and TNF-a (10 pg/ml); IL-1b, GM-CSF and TIMP-1 Definitions and study procedures A research nurse collected information, consent forms, ultrasound reports, images and questionnaires. Gestational age was determined by last menstrual period and confirmed by ultrasound examination, or by ultrasound examination alone if gestational ages differ by more than one week. Demographic data were reviewed from charts and from a questionnaire filled out by the patient and the research nurse at the time of inclusion. The pre-pregnancy body mass index (BMI) was calculated from body weight at the first pregnancy appointment with height reported by the patient. At the time of TVUS visit, questionnaire was filled to assess referral reasons for TVUS, presence of contractions, bleeding, usage of prometrium vaginal suppositories (the only progesterone prophylaxis treatment for sPTB in our center), antibiotics, betamethasone or tocolytic therapy. Smoking and drug usage, work schedule, ethnicity and marital status were reported by the patient and reviewed in charts. Pregnancy and delivery outcomes were reviewed from charts. sPTB was defined as delivery before the 37th week of gestation from premature labor with intact or ruptured membranes. Term birth was defined as delivery after 37 weeks of gestation. Induced preterm births were excluded. Statistical analysis Statistical analyses were performed with NCSS 2007 (NCSS, Kaysville, UT, USA). Pregnancy and delivery data were analysed with Fisher’s exact test and the Chi-square test according to expected frequency. The Mann–Whitney U test was used to analyze the continuous variables not normally distributed and the t-test for variables normally distributed. Results with a p value 50.05 were considered statistically significant. ROC curves were used to determine the cut-off
Short inflamed cervix
DOI: 10.3109/14767058.2013.847917
concentration of cytokines significantly present in vaginal secretions analyzed by Multiplex Membrane from RayBiotech. A sensitivity and specificity of 60% for sPTB prediction was defined as the positive cut-off threshold used to categorize data according either to the presence or absence of cytokines. Categorical data were further analyzed with Fisher’s exact test and the Chi-square test as appropriate. We also stratified our data and analyzed them for singleton and multiple pregnancies. We categorized cervical length by the presence of IL-8. A multivariate analysis by stepwise ascending logistic regression of Wald was performed using all predictive factors showing a trend for sPTB in multivariate analysis (p50.1).
Results During the study period, 123 women were approached to be included in the cohort; six were ineligible and 19 refused to participate. A total of 98 women were recruited and signed the consent form. Two women were excluded at the time of analysis because they delivered in another center without accessible file, nine for induced preterm birth. The final analysis included 87 women. The most frequent referral reason was preterm uterine activity (n ¼ 46) and mean gestational age at TVUS examination was 27.3 (95% CI 26.6–28.1) weeks (Table 1). As expected, several risks factors for sPTB were observed: 27.9% of women had previous sPTB or late spontaneous abortion and 14.9% of pregnancies were multifetal (n ¼ 12 twins and n ¼ 1 triplets). Among 87 women, 25.3% delivered with sPTB (n ¼ 22) and 74.7% with term birth (TB). In the sPTB group, reason for admission in the delivery room was preterm labor in 54.5% (n ¼ 12) and PPROM in 45.5% (n ¼ 10) of cases (Table 2). Median gestational ages at delivery were 35.6 (95% CI 34.1–36.3) and 38.7 (95% CI 38.6–39.1) weeks in women with sPTB and TB, respectively (p50.001; Table 2). Clinical risk factors of sPTB (maternal age, BMI, smoking, obstetrical history of sPTB or late abortion and multifetal pregnancy) did not differ between both groups.
We observed greater hospitalization and corticoprophylaxis (p50.05) in women with sPTB than in TB group and there was a trend toward higher antibiotic use, tocolysis and utilization of prometrium vaginal suppositories (p ¼ 0.19, 0.07 and 0.11, respectively). At ultrasound examination, mean cervical length in sPTB group was 25.3 mm (95% CI 19.9–30.6 mm) compared to 32.8 mm (95% CI 30.3–35.3 mm) in the TB group (p50.01). The positive predictive value of a short cervix was 45.8% for sPTB. Funnelling was present in 24 patients, had a trend for sPTB (p ¼ 0.10) and was associated with short cervix (p50.01). In vaginal secretions, IL-1b was detected in 19.5% of women, IL-6 in 5.7%, IL-8 in 72.4%, IL-10 in 4.6%, MMP-8 and MMP-9 in 81.6% and 74.7% and TIMP-1 in 19.5% (Table 2). Association between the presence of cytokines in vaginal secretions and ultrasound factors was limited to TIMP1 associated with short cervical length (p ¼ 0.01). IL-1b, IL-8 and IL-10 were associated with sPTB (p 0.05), while IL-6 showed a trend for association with sPTB (p 0.1). The positive predictive values of IL-1b, IL-8 and IL-10 for sPTB were 44.4%, 31.8% and 75.0% and their negative predictive values were 82.1%, 96.7% and 79.3%, respectively. We categorized data in four groups by cervical length and presence of IL-8: short inflamed cervix; short and cold (non-inflamed) cervix; long inflamed cervix; long and cold cervix. The best predictive value for sPTB was for a short inflamed cervix (PPV 55.6%) and the most protective situation was a long and cold cervix with no occurrence of sPTB in that setting. We did a stratified analysis of our data for singletons and multiple pregnancies. In the singleton group, 17 over 74 patients had sPTB. The median gestational age at delivery was 35.9 (95% CI: 34.3–36.4) for sPTB group and 39.0 (95% CI: 38.7–39.3). Association between short cervical length and sPTB was observed (mean cervical length: 22.6 mm (95% CI: 16.9–26.4 mm) and 32.9 mm (95% CI: 30.1–35.6 mm; p50.01) in the sPTB and TB group). Funnelling was also associated with sPTB (p ¼ 0.04) as the ‘‘U’’ shape funnel
Table 1. Demographic characteristics of the study population. Population characteristics Maternal age (mean, yrs (95% CI)) Pre-pregnancy BMI (median, kg/m2 (95% CI)) Nulliparous Previous preterm delivery or late abortion Tobacco Illicit drugs Single Unemployed Caucasian Singleton Condition at ultrasound examination Referral reason Uterine contractile activity Modified cervix at physical exam Previous preterm delivery or late abortion Multifetal pregnancy Gestational age at ultrasound (mean, wks (95% CI))
1017
sPTB (n ¼ 22) 29.6 23.4 7 8 4
(27.3–31.8) (21.2–28.4) (31.8) (36.4) (19.0) 0 1 (4.5) 0 19 (86.4) 17 (77.3)
TB (n ¼ 65) 28.9 22.3 29 16 10 1 2 11 59 57
(27.9–30.0) (21.0–24.3) (44.6) (25.0) (15.4) (1.6) (3.1) (19.0) (90.8) (87.7)
33 11 15 6 27.3
(50.8) (16.9) (23.1) (9.2) (26.4–28.1)
p Value 0.56a 0.28b 0.29 0.30 0.73* 1.0* 1.0* 0.11* 0.68* 0.30* 0.83
13 2 5 2 27.6
(59.1) (9.1) (22.7) (9.1) (25.9–29.2)
0.72a
Data are n(%) unless indicated otherwise. For statistical analysis, the Chi-square test was used to establish the p value unless the value is identified by an asterisk (*), in which the analysis was performed using the Fisher’s exact test, or by an a or a b, in which T-test for two samples (a) or Mann–Whitney U (b) tests were used according to normality of the distribution.
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Table 2. Pregnancy outcomes. Study population Gestational age at delivery (median, wks (95% CI)) Delay between TVUS and delivery (mean, days (95% CI)) Admission reason in delivery roomz Spontaneous labor Rupture of membranes Labor induction C-section before labor Vaginal delivery Preterm delivery risk management Cerclage Antibiotics Prometrium Tocolysis Corticotherapy Hospitalization during pregnancy Group B Streptococcus Inflammatory factors IL-1b IL-6 IL-8 IL-10 MMP-8 MMP-9 TIMP-1
sPTB (n ¼ 22)
TB (n ¼ 65)
p Value
35.6 (34.1–36.3) 47.4 (36.3–58.5)
38.7 (38.6–39.1) 82.4 (76.4–88.3)
50.001 50.001 50.001z
12 (54.4) 10 (45.5) 0 0 17 (77.3)
21 11 25 8 47
(32.3) (16.9) (38.5) (12.3) (72.3)
0.65
3 6 9 6 16 14 3
(13.6) (27.3) (40.9) (27.3) (72.7) (63.6) (15.0)
6 9 15 6 20 19 12
(9.2) (13.8) (23.1) (9.2) (30.8) (29.2) (18.5)
0.68* 0.19* 0.10 0.07* 50.001 0.004 1.0*
8 3 21 3 20 19 7
(36.4) (13.6) (95.5) (13.6) (90.9) (86.4) (31.8)
9 2 42 1 51 46 10
(13.8) (3.1) (64.6) (1.5) (78.5) (70.8) (15.4)
0.03* 0.10* 0.005* 0.05* 0.34* 0.17* 0.12*
Data are n(%) unless indicated otherwise. For statistical analysis, the Chi-square test was used to establish the p value unless the value is identified by an asterisk (*), in which the analysis was performed using the Fisher’s exact test. z Admission reasons were analyzed between the two groups and were found to be statistically different.
(p ¼ 0.004). Only IL-8 cytokine was associated with sPTB in this subgroup analysis (p ¼ 0.03). In the multiple pregnancy group, five over 15 patients had a sPTB at a median of 34.1 gestational weeks compare to 37.5 (95% CI: 37.2–37.7) for those who had TB. For multiple pregnancies none of the TVUS markers were associated with sPTB, but sPTB was associated with a detection of IL-1b in vaginal secretion (p ¼ 0.03) and had a trend with MMP-8 and 9 presence in vaginal secretion (p ¼ 0.10). In the entire cohort, we also performed a multivariate analysis with a stepwise model of logistic regression in which predictive factors of sPTB were evaluated. We analyzed factors from TVUS and inflammatory study and controlled for classical clinical sPTB risk factors (maternal age, prior sPTB, smoking and multiple pregnancy). Short cervix, funnelling and presence of IL-1b, IL-8 were associated with sPTB in the multivariate analysis (p50.10). In the Wald logistic regression model with those factors, two were independently associated with sPTB: short cervix with an OR of 3.58 (95% CI: 1.02–12.61) and IL-8 with an OR of 14.55 (95% CI: 1.64–128.83). We repeat the model of analysis by replacing short cervix and IL-8 by their combination and found an OR of 4.33 (95% CI: 1.25–14.95).
Discussion We developed an original method allowing vaginal secretions sampling during TVUS. We showed that short cervix and IL-8 were independently associated with sPTB and that categorizing cervical length by IL-8 status could be a new classification associating cervical morphological and inflammatory parameters to predict sPTB. We found a strong association between short cervical length (525 mm) and sPTB with a positive predictive value of 45.8% with sPTB rate of 25.3%. This predictive value is
comparable to those reported in the studies of Vendittelli et al. (54%), Owen et al. (55%) and Goldenberg et al. (54%) for women with preterm labor, prior sPTB and twin pregnancy, respectively, where the preterm birth rate was ranging from 26% to 41% [21–23]. In our cohort, IL-8 appears to be the most interesting cytokine since it was the only one independently associated with sPTB in multivariate analysis. IL-8 is a pro-inflammatory cytokine produces in amnion, choriodecidual, placental, myometrial and cervical tissues [24]. This cytokine plays an important role in neutrophil chemotactic, degranulation and activation and activates collagenases and elastase enhancing cervical ripening, membranes degradation and uterine contraction by activating prostaglandins secretion [25–27]. Association between short cervix, IL-8 and intraamniotic infection or chorioamnionitis was found by Rizzo et al. in a cohort of 144 patients with preterm labor [15]. Sakai et al. demonstrated that IL-8 was both associated with diminution of lactobacilli in vaginal flora and augmentation of abnormal pathologic flora and may be used to predict sPTB with OR of 4.0 (95% CI 1.78–14) [28]. Interestingly, they showed that inflammation detected in vaginal secretion could be treated and normalized in 76.8% leading to a decreased rate of sPTB [29]. In that study they compared vaginal treatment with proviodine iodine washing and chloramphenicol vaginal tablet for a week to bed rest or cerclage and they showed a reduction of sPTB and PPROM [29]. In our study, we originally combined Dacron swabs to the TVUS probe allowing vaginal sampling to detect inflammatory markers in the same convenient and noninvasive test. Since the swabs were set on the TVUS probe during the entire examination, the samplings were therefore considered reliable, reflecting the vaginal posterior fornix inflammatory status and can be easily reproduced in any clinical setting.
DOI: 10.3109/14767058.2013.847917
In regard to study limitations, we did not use vaginal sampling to assess the infectious status and therefore cannot conclude if the presence of inflammatory cytokines in vaginal secretions was related to choriodecidual or vaginal infectious and inflammatory process. Heterogeneity of the population study may have decreased the statistical power of our study. However, our study analyzed for the first time the combination of two different predictive tests in a single exam. The interest to combine these tests could not have been found without an heterogeneous population. Our findings support the evidence that sPTB should be considered as a syndrome and the need for a better categorization of high-risk patients [30]. Perspective COLIBRI study contributes to enhance our scientific knowledge by introducing the short inflamed cervix as a new concept to categorize sPTB risk. The categorization of cervical length by presence of IL-8 can certainly be a new way to approach sPTB prevention by choosing the appropriate treatment, cerclage or pessary for short non-inflamed cervix and/or anti-inflammatory treatment for short or even long inflamed cervix. To enhance the clinical utility of our combined test, a bedside test for IL-8 detection in vaginal secretion could be used, as validated by our research team.
Acknowledgements The authors wish to thank Dr Louise Constantineau and Mrs Jose´e Poulin for their expertise and help to develop the standardized TVUS method. They are thankful to Dr Daniel Blouin, Mrs Lise Lavoie, Mrs Ame´lie Le´veille´, Mrs MariePier Labbe´ and the ultrasound technologists at the MFMclinic of the CHUS for their technical support. They are grateful to Mrs Tania Fayad for her precious help in the writing process and to all patients who agreed to participate in the study.
Declaration of interest None of the authors have a conflict of interest. This study was funded by the principal investigator personal grants.
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8. Bujold E, Pasquier JC, Simoneau J, et al. Intra-amniotic sludge, short cervix, and risk of preterm delivery. J Obstet Gynaecol Can 2006;28:198–202. 9. Iams JD, Paraskos J, Landon MB, et al. Cervical sonography in preterm labor. Obstet Gynecol 1994;84:40–6. 10. Berghella V, Baxter JK, Hendrix NW. Cervical assessment by ultrasound for preventing preterm delivery. Cochrane Database Syst Rev 2009;(3):CD007235. doi: 10.1002/14651858.CD007235.pub2. 11. Mella MT, Berghella V. Prediction of preterm birth: cervical sonography. Semin Perinatol 2009;33:317–24. 12. Genc MR, Ford CE. The clinical use of inflammatory markers during pregnancy. Curr Opin Obstet Gynecol 2010;22:116–21. 13. Goldenberg RL, Goepfert AR, Ramsey PS. Biochemical markers for the prediction of preterm birth. Am J Obstet Gynecol 2005;192: S36–46. 14. Berghella V, Hayes E, Visintine J, Baxter JK. Fetal fibronectin testing for reducing the risk of preterm birth. Cochrane Database Syst Rev 2008;(4):CD006843. doi 10.1002/14651858.CD006843 pub2. 15. Rizzo G, Capponi A, Vlachopoulou A, et al. Ultrasonographic assessment of the uterine cervix and interleukin-8 concentrations in cervical secretions predict intrauterine infection in patients with preterm labor and intact membranes. Ultrasound Obstet Gynecol 1998;12:86–92. 16. Goya M, Pratcorona L, Merced C, et al. Cervical pessary in pregnant women with a short cervix (PECEP): an open-label randomised controlled trial. Lancet 2012;379:1800–6. 17. Puchner K, Iavazzo C, Gourgiotis D, et al. Mid-trimester amniotic fluid interleukins (IL-1beta, IL-10 and IL-18) as possible predictors of preterm delivery. In Vivo 2011;25:141–8. 18. Jacobsson B, Mattsby-Baltzer I, Hagberg H. Interleukin-6 and interleukin-8 in cervical and amniotic fluid: relationship to microbial invasion of the chorioamniotic membranes. BJOG 2005;112:719–24. 19. Gotsch F, Romero R, Kusanovic JP, et al. The anti-inflammatory limb of the immune response in preterm labor, intra-amniotic infection/inflammation, and spontaneous parturition at term: a role for interleukin-10. J Matern Fetal Neonatal Med 2008;21: 529–47. 20. Becher N, Hein M, Danielsen CC, Uldbjerg N. Matrix metalloproteinases in the cervical mucus plug in relation to gestational age, plug compartment, and preterm labor. Reprod Biol Endocrinol 2010;8:113,7827-8-113. 21. Vendittelli F, Mamelle N, Munoz F, Janky E. Transvaginal ultrasonography of the uterine cervix in hospitalized women with preterm labor. Int J Gynaecol Obstet 2001;72:117–25. 22. Owen J, Yost N, Berghella V, et al. Mid-trimester endovaginal sonography in women at high risk for spontaneous preterm birth. JAMA 2001;286:1340–8. 23. Goldenberg RL, Iams JD, Miodovnik M, et al. The preterm prediction study: Risk factors in twin gestations. National Institute of Child Health and Human Development Maternal-fetal Medicine Units Network. Am J Obstet Gynecol 1996;175:1047–53. 24. Laham N, Brennecke SP, Rice GE. Interleukin-8 release from human gestational tissue explants: effects of gestation, labor, and chorioamnionitis. Biol Reprod 1999;61:823–7. 25. Kelly RW, Leask R, Calder AA. Choriodecidual production of interleukin-8 and mechanism of parturition. Lancet 1992;339: 776–7. 26. Maradny EE, Kanayama N, Halim A, et al. Effects of neutrophil chemotactic factors on cervical ripening. Clin Exp Obstet Gynecol 1995;22:76–85. 27. Sennstrom MK, Brauner A, Lu Y, et al. Interleukin-8 is a mediator of the final cervical ripening in humans. Eur J Obstet Gynecol Reprod Biol 1997;74:89–92. 28. Sakai M, Ishiyama A, Tabata M, et al. Relationship between cervical mucus interleukin-8 concentrations and vaginal bacteria in pregnancy. Am J Reprod Immunol 2004;52:106–12. 29. Sakai M, Sasaki Y, Yoneda S, et al. Elevated interleukin-8 in cervical mucus as an indicator for treatment to prevent premature birth and preterm, pre-labor rupture of membranes: a prospective study. Am J Reprod Immunol 2004;51:220–5. 30. Kramer MS, Papageorghiou A, Culhane J, et al. Challenges in defining and classifying the preterm birth syndrome. Am J Obstet Gynecol 2012;206:108–12.
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ORIGINAL ARTICLE
Short and inflamed cervix predicts spontaneous preterm birth (COLIBRI study)* Evelyne Raiche1, Annie Ouellet1, Maryse Berthiaume2, E´ric Rousseau3, and Jean-Charles Pasquier1 Department of Obstetrics and Gynecology, 2Centre de Recherche Clinique E´tienne-LeBel, and 3Department of Physiology and Biophysics, Universite´ de Sherbrooke, Sherbrooke, Quebec, Canada
1
Abstract
Keywords
Objective: To develop a new strategy of predicting spontaneous preterm birth (sPTB) by combination of transvaginal ultrasound (TVUS) assessment and inflammatory proteins detection in vaginal secretions. Methods: Prospective study of 87 women referred for cervical length assessment with a standardized TVUS combined to vaginal secretions sampling. Samples were analyzed for presence of 10 cytokines. Main outcome was sPTB (537 weeks of gestation). Associations were assessed with the chi-square, Fisher’s exact test (p50.05) and Wald’s logistic regression. Results: sPTB occurred in 25.3% of women at a median gestational age of 35.6 weeks of gestation. Short cervix (525 mm) (n ¼ 24) was associated with sPTB (p50.01) as interleukine (IL)1b, IL-8 and IL-10 in vaginal secretions (p50.05). In multivariate analysis, short cervix and IL-8 in vaginal secretions were independently associated with sPTB (OR 3.58 (95%CI 1.02; 12.61) and 14.55 (95%CI 1.64; 128.83), respectively) as their combination (OR 4.33 (95%CI 1.25; 14.95)). By categorizing cervical length by presence of IL-8, sPTB occurred in 55.6% of women with a short inflamed cervix. Conclusion: COLIBRI study used a novel, single-step method of vaginal secretions sampling during TVUS and demonstrated that combination of short cervix and IL-8 in vaginal secretions is a promising sPTB predictive test.
Cervical length, cytokines, inflammation, pre-term birth, transvaginal ultrasound, vaginal secretions
Introduction Preterm birth is the leading cause of perinatal mortality and morbidity and a major cause of long-term health problems [1]. Spontaneous preterm birth (sPTB), with intact and ruptured membranes, is a heterogeneous pathology arising from different pathways in which inflammation plays a major role [2,3]. Activation of collagenases and prostaglandins, by inflammation and intra-amniotic infection, promote degradation of the cervix and amniotic membranes and enhance uterine contractility and rupture of membranes [4–6]. It is urgent to categorize women with a combination of predictive factors improving the quality of predictive results, but also to propose a personalized therapeutic strategy [7].
*Results were presented at: SMFM 30th Annual Meeting – The Pregnancy MeetingtM, Chicago, IL, February 1–6, 2010; SMFM 31st Annual Meeting – The Pregnancy MeetingtM, San Francisco, CA, February 7–12, 2011. Address for correspondence: Dr Jean-Charles Pasquier, MD, PhD, Department of Obstetrics and Gynecology, Universite´ de Sherbrooke, 3001, 12e avenue Nord, Sherbrooke, QC J1H 3T5, Canada. Tel: (819)346-1110 by 0, pager 4403; (819)-679-2212 (home). Fax: (819)-819 8206434. E-mail: [email protected]
History Received 27 May 2013 Accepted 19 September 2013 Published online 31 October 2013
Two approaches have been described to predict sPTB, ultrasound and biochemical markers. Cervical shortening is an integrated part of the pathophysiological process leading to sPTB and may be associated with other factors such as funnelling and sludge [8]. Transvaginal ultrasound (TVUS) measurement of cervical length, described by Iams in the 1990s [9] is the most reliable method to assess the cervix and represents the best predictive test for sPTB with a positive predictive value ranging from 30% to 55% for sPTB high-risk singleton and twin pregnancies [10,11]. Several pro-inflammatory proteins, identified in vaginal secretions, were associated with sPTB including IL-1b, IL-6, IL-8, MMPs, IL-10 and TIMP-1 [12,13] as well as fetal fibronectine [14]. Some of these, e.g. IL-8 was associated with short cervical length and intra-amniotic infection [15]. Recently, new therapeutic strategies appear in the management of prematurity such as pessary to strengthen the cervix [16]. To deal with these innovations, it is important to select women in order to prevent sPTB by offering adapted treatment to prematurity etiology; antiinflammatory treatment to women with inflammation detected non-invasively and pessary or cerclage to women with mechanical short cervix due to cervix incompetence or cone biopsy.
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We designed a standardized examination allowing sampling of vaginal secretion in the same exam of TVUS with two Dacron swabs set on the ultrasound probe. We used this combined strategy to categorize ultrasound finding by the presence of an inflammatory process. The main objective of our study was to enhance sPTB prediction by a better comprehensive categorization of high-risk patient combining cervical length and inflammatory status of vaginal secretion.
Methods Study population We conducted a prospective cohort study of women referred to maternal and fetal medicine (MFM) clinic for sPTB risk evaluation by TVUS at the Centre Hospitalier Universitaire de Sherbrooke (CHUS), Quebec, Canada, referral center for women living in the surrounding area of Shebrooke. The Ethics Committee of our center approved the study protocol in April 2008 (Comite´ d’e´thique de la recherche en sante´ chez l’humain du CHUS, project 08-020, ClinicalTrials.gov identifier: NCT01007513). Women were recruited consecutively from June 2008 to July 2009. Inclusion criteria: Women were eligible if pregnant with a live singleton or multifetal pregnancy and referred for TVUS between 20 and 34 weeks of gestation. TVUS was performed for clinical indicated reason according to women physician. Women were referred for risk factors of sPTB, either from previous history of sPTB or clinical reason in this pregnancy (multiple pregnancy, soften cervix or contractile activity without active labor). Exclusion criteria: Exclusion criteria were active labor at the time of TVUS or delivery on the TVUS day, fetuses with major congenital anomalies, placenta previa and PPROM. Women with an induced preterm birth for medical reason were excluded from analysis. A research nurse provided information regarding the study protocol and all women gave a written informed consent before participating in the study. Sonographic assessment of the cervix Cervical length was evaluated by the standard method described by Iams and if several values were collected, the shorter length was retained [9,11]. TVUS examinations were performed by seven experienced radiological technologists and controlled by four obstetricians. Examinations were performed either on a GE Voluson 730 Expert with the RIC5-9H (5 MHz) probe (GE Voluson 730 Expert, Little Chalfont, UK) or on a Toshiba Aplia XG with a PVT-661VT (6 MHz) probe without ultrasound gel over the probe (Toshiba Aplio XG, Tustin, CA, USA). All images were stored and read by two independent observers to ensure internal validity of the ultrasound findings. TVUS findings were reported to physician as they were noted in the file. Cervicovaginal fluid analysis We developed an original method with Dacron swabs set on an endovaginal probe (Figure 1). Swabs were left in place during the entire TVUS examination, lasting between 5 and 10 min, and primarily collected vaginal secretions from the posterior fornix. The samples were diluted in 1 mL PBS,
Figure 1. TVUS probe with an affixed Dacron swab as used in the present protocol.
centrifuged for 2 min at 5000 rpm and froze within 10 min at 80 C. Samples were analyzed using multiplex antibody array from RayBiotech (Raybiotech, Atlanta, GA, USA). Choice of cytokines was based on immunological pathway and known association of IL-1b, IL-6, IL-8, MMPs, IL-10 and TIMP-1 with sPTB [17–20]. Cytokines were detected by immunoluminescence with a Molecular Imager VersaDoc MP 5000 System (Molecular Imager VersaDoc MP 5000 System from Bio-Rad, Hercules, CA, USA) with the following sensitivities: IL-6, IL-8 and MMP-8 (1 pg/ml); IL-10 and TNF-a (10 pg/ml); IL-1b, GM-CSF and TIMP-1 Definitions and study procedures A research nurse collected information, consent forms, ultrasound reports, images and questionnaires. Gestational age was determined by last menstrual period and confirmed by ultrasound examination, or by ultrasound examination alone if gestational ages differ by more than one week. Demographic data were reviewed from charts and from a questionnaire filled out by the patient and the research nurse at the time of inclusion. The pre-pregnancy body mass index (BMI) was calculated from body weight at the first pregnancy appointment with height reported by the patient. At the time of TVUS visit, questionnaire was filled to assess referral reasons for TVUS, presence of contractions, bleeding, usage of prometrium vaginal suppositories (the only progesterone prophylaxis treatment for sPTB in our center), antibiotics, betamethasone or tocolytic therapy. Smoking and drug usage, work schedule, ethnicity and marital status were reported by the patient and reviewed in charts. Pregnancy and delivery outcomes were reviewed from charts. sPTB was defined as delivery before the 37th week of gestation from premature labor with intact or ruptured membranes. Term birth was defined as delivery after 37 weeks of gestation. Induced preterm births were excluded. Statistical analysis Statistical analyses were performed with NCSS 2007 (NCSS, Kaysville, UT, USA). Pregnancy and delivery data were analysed with Fisher’s exact test and the Chi-square test according to expected frequency. The Mann–Whitney U test was used to analyze the continuous variables not normally distributed and the t-test for variables normally distributed. Results with a p value 50.05 were considered statistically significant. ROC curves were used to determine the cut-off
Short inflamed cervix
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concentration of cytokines significantly present in vaginal secretions analyzed by Multiplex Membrane from RayBiotech. A sensitivity and specificity of 60% for sPTB prediction was defined as the positive cut-off threshold used to categorize data according either to the presence or absence of cytokines. Categorical data were further analyzed with Fisher’s exact test and the Chi-square test as appropriate. We also stratified our data and analyzed them for singleton and multiple pregnancies. We categorized cervical length by the presence of IL-8. A multivariate analysis by stepwise ascending logistic regression of Wald was performed using all predictive factors showing a trend for sPTB in multivariate analysis (p50.1).
Results During the study period, 123 women were approached to be included in the cohort; six were ineligible and 19 refused to participate. A total of 98 women were recruited and signed the consent form. Two women were excluded at the time of analysis because they delivered in another center without accessible file, nine for induced preterm birth. The final analysis included 87 women. The most frequent referral reason was preterm uterine activity (n ¼ 46) and mean gestational age at TVUS examination was 27.3 (95% CI 26.6–28.1) weeks (Table 1). As expected, several risks factors for sPTB were observed: 27.9% of women had previous sPTB or late spontaneous abortion and 14.9% of pregnancies were multifetal (n ¼ 12 twins and n ¼ 1 triplets). Among 87 women, 25.3% delivered with sPTB (n ¼ 22) and 74.7% with term birth (TB). In the sPTB group, reason for admission in the delivery room was preterm labor in 54.5% (n ¼ 12) and PPROM in 45.5% (n ¼ 10) of cases (Table 2). Median gestational ages at delivery were 35.6 (95% CI 34.1–36.3) and 38.7 (95% CI 38.6–39.1) weeks in women with sPTB and TB, respectively (p50.001; Table 2). Clinical risk factors of sPTB (maternal age, BMI, smoking, obstetrical history of sPTB or late abortion and multifetal pregnancy) did not differ between both groups.
We observed greater hospitalization and corticoprophylaxis (p50.05) in women with sPTB than in TB group and there was a trend toward higher antibiotic use, tocolysis and utilization of prometrium vaginal suppositories (p ¼ 0.19, 0.07 and 0.11, respectively). At ultrasound examination, mean cervical length in sPTB group was 25.3 mm (95% CI 19.9–30.6 mm) compared to 32.8 mm (95% CI 30.3–35.3 mm) in the TB group (p50.01). The positive predictive value of a short cervix was 45.8% for sPTB. Funnelling was present in 24 patients, had a trend for sPTB (p ¼ 0.10) and was associated with short cervix (p50.01). In vaginal secretions, IL-1b was detected in 19.5% of women, IL-6 in 5.7%, IL-8 in 72.4%, IL-10 in 4.6%, MMP-8 and MMP-9 in 81.6% and 74.7% and TIMP-1 in 19.5% (Table 2). Association between the presence of cytokines in vaginal secretions and ultrasound factors was limited to TIMP1 associated with short cervical length (p ¼ 0.01). IL-1b, IL-8 and IL-10 were associated with sPTB (p 0.05), while IL-6 showed a trend for association with sPTB (p 0.1). The positive predictive values of IL-1b, IL-8 and IL-10 for sPTB were 44.4%, 31.8% and 75.0% and their negative predictive values were 82.1%, 96.7% and 79.3%, respectively. We categorized data in four groups by cervical length and presence of IL-8: short inflamed cervix; short and cold (non-inflamed) cervix; long inflamed cervix; long and cold cervix. The best predictive value for sPTB was for a short inflamed cervix (PPV 55.6%) and the most protective situation was a long and cold cervix with no occurrence of sPTB in that setting. We did a stratified analysis of our data for singletons and multiple pregnancies. In the singleton group, 17 over 74 patients had sPTB. The median gestational age at delivery was 35.9 (95% CI: 34.3–36.4) for sPTB group and 39.0 (95% CI: 38.7–39.3). Association between short cervical length and sPTB was observed (mean cervical length: 22.6 mm (95% CI: 16.9–26.4 mm) and 32.9 mm (95% CI: 30.1–35.6 mm; p50.01) in the sPTB and TB group). Funnelling was also associated with sPTB (p ¼ 0.04) as the ‘‘U’’ shape funnel
Table 1. Demographic characteristics of the study population. Population characteristics Maternal age (mean, yrs (95% CI)) Pre-pregnancy BMI (median, kg/m2 (95% CI)) Nulliparous Previous preterm delivery or late abortion Tobacco Illicit drugs Single Unemployed Caucasian Singleton Condition at ultrasound examination Referral reason Uterine contractile activity Modified cervix at physical exam Previous preterm delivery or late abortion Multifetal pregnancy Gestational age at ultrasound (mean, wks (95% CI))
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sPTB (n ¼ 22) 29.6 23.4 7 8 4
(27.3–31.8) (21.2–28.4) (31.8) (36.4) (19.0) 0 1 (4.5) 0 19 (86.4) 17 (77.3)
TB (n ¼ 65) 28.9 22.3 29 16 10 1 2 11 59 57
(27.9–30.0) (21.0–24.3) (44.6) (25.0) (15.4) (1.6) (3.1) (19.0) (90.8) (87.7)
33 11 15 6 27.3
(50.8) (16.9) (23.1) (9.2) (26.4–28.1)
p Value 0.56a 0.28b 0.29 0.30 0.73* 1.0* 1.0* 0.11* 0.68* 0.30* 0.83
13 2 5 2 27.6
(59.1) (9.1) (22.7) (9.1) (25.9–29.2)
0.72a
Data are n(%) unless indicated otherwise. For statistical analysis, the Chi-square test was used to establish the p value unless the value is identified by an asterisk (*), in which the analysis was performed using the Fisher’s exact test, or by an a or a b, in which T-test for two samples (a) or Mann–Whitney U (b) tests were used according to normality of the distribution.
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Table 2. Pregnancy outcomes. Study population Gestational age at delivery (median, wks (95% CI)) Delay between TVUS and delivery (mean, days (95% CI)) Admission reason in delivery roomz Spontaneous labor Rupture of membranes Labor induction C-section before labor Vaginal delivery Preterm delivery risk management Cerclage Antibiotics Prometrium Tocolysis Corticotherapy Hospitalization during pregnancy Group B Streptococcus Inflammatory factors IL-1b IL-6 IL-8 IL-10 MMP-8 MMP-9 TIMP-1
sPTB (n ¼ 22)
TB (n ¼ 65)
p Value
35.6 (34.1–36.3) 47.4 (36.3–58.5)
38.7 (38.6–39.1) 82.4 (76.4–88.3)
50.001 50.001 50.001z
12 (54.4) 10 (45.5) 0 0 17 (77.3)
21 11 25 8 47
(32.3) (16.9) (38.5) (12.3) (72.3)
0.65
3 6 9 6 16 14 3
(13.6) (27.3) (40.9) (27.3) (72.7) (63.6) (15.0)
6 9 15 6 20 19 12
(9.2) (13.8) (23.1) (9.2) (30.8) (29.2) (18.5)
0.68* 0.19* 0.10 0.07* 50.001 0.004 1.0*
8 3 21 3 20 19 7
(36.4) (13.6) (95.5) (13.6) (90.9) (86.4) (31.8)
9 2 42 1 51 46 10
(13.8) (3.1) (64.6) (1.5) (78.5) (70.8) (15.4)
0.03* 0.10* 0.005* 0.05* 0.34* 0.17* 0.12*
Data are n(%) unless indicated otherwise. For statistical analysis, the Chi-square test was used to establish the p value unless the value is identified by an asterisk (*), in which the analysis was performed using the Fisher’s exact test. z Admission reasons were analyzed between the two groups and were found to be statistically different.
(p ¼ 0.004). Only IL-8 cytokine was associated with sPTB in this subgroup analysis (p ¼ 0.03). In the multiple pregnancy group, five over 15 patients had a sPTB at a median of 34.1 gestational weeks compare to 37.5 (95% CI: 37.2–37.7) for those who had TB. For multiple pregnancies none of the TVUS markers were associated with sPTB, but sPTB was associated with a detection of IL-1b in vaginal secretion (p ¼ 0.03) and had a trend with MMP-8 and 9 presence in vaginal secretion (p ¼ 0.10). In the entire cohort, we also performed a multivariate analysis with a stepwise model of logistic regression in which predictive factors of sPTB were evaluated. We analyzed factors from TVUS and inflammatory study and controlled for classical clinical sPTB risk factors (maternal age, prior sPTB, smoking and multiple pregnancy). Short cervix, funnelling and presence of IL-1b, IL-8 were associated with sPTB in the multivariate analysis (p50.10). In the Wald logistic regression model with those factors, two were independently associated with sPTB: short cervix with an OR of 3.58 (95% CI: 1.02–12.61) and IL-8 with an OR of 14.55 (95% CI: 1.64–128.83). We repeat the model of analysis by replacing short cervix and IL-8 by their combination and found an OR of 4.33 (95% CI: 1.25–14.95).
Discussion We developed an original method allowing vaginal secretions sampling during TVUS. We showed that short cervix and IL-8 were independently associated with sPTB and that categorizing cervical length by IL-8 status could be a new classification associating cervical morphological and inflammatory parameters to predict sPTB. We found a strong association between short cervical length (525 mm) and sPTB with a positive predictive value of 45.8% with sPTB rate of 25.3%. This predictive value is
comparable to those reported in the studies of Vendittelli et al. (54%), Owen et al. (55%) and Goldenberg et al. (54%) for women with preterm labor, prior sPTB and twin pregnancy, respectively, where the preterm birth rate was ranging from 26% to 41% [21–23]. In our cohort, IL-8 appears to be the most interesting cytokine since it was the only one independently associated with sPTB in multivariate analysis. IL-8 is a pro-inflammatory cytokine produces in amnion, choriodecidual, placental, myometrial and cervical tissues [24]. This cytokine plays an important role in neutrophil chemotactic, degranulation and activation and activates collagenases and elastase enhancing cervical ripening, membranes degradation and uterine contraction by activating prostaglandins secretion [25–27]. Association between short cervix, IL-8 and intraamniotic infection or chorioamnionitis was found by Rizzo et al. in a cohort of 144 patients with preterm labor [15]. Sakai et al. demonstrated that IL-8 was both associated with diminution of lactobacilli in vaginal flora and augmentation of abnormal pathologic flora and may be used to predict sPTB with OR of 4.0 (95% CI 1.78–14) [28]. Interestingly, they showed that inflammation detected in vaginal secretion could be treated and normalized in 76.8% leading to a decreased rate of sPTB [29]. In that study they compared vaginal treatment with proviodine iodine washing and chloramphenicol vaginal tablet for a week to bed rest or cerclage and they showed a reduction of sPTB and PPROM [29]. In our study, we originally combined Dacron swabs to the TVUS probe allowing vaginal sampling to detect inflammatory markers in the same convenient and noninvasive test. Since the swabs were set on the TVUS probe during the entire examination, the samplings were therefore considered reliable, reflecting the vaginal posterior fornix inflammatory status and can be easily reproduced in any clinical setting.
DOI: 10.3109/14767058.2013.847917
In regard to study limitations, we did not use vaginal sampling to assess the infectious status and therefore cannot conclude if the presence of inflammatory cytokines in vaginal secretions was related to choriodecidual or vaginal infectious and inflammatory process. Heterogeneity of the population study may have decreased the statistical power of our study. However, our study analyzed for the first time the combination of two different predictive tests in a single exam. The interest to combine these tests could not have been found without an heterogeneous population. Our findings support the evidence that sPTB should be considered as a syndrome and the need for a better categorization of high-risk patients [30]. Perspective COLIBRI study contributes to enhance our scientific knowledge by introducing the short inflamed cervix as a new concept to categorize sPTB risk. The categorization of cervical length by presence of IL-8 can certainly be a new way to approach sPTB prevention by choosing the appropriate treatment, cerclage or pessary for short non-inflamed cervix and/or anti-inflammatory treatment for short or even long inflamed cervix. To enhance the clinical utility of our combined test, a bedside test for IL-8 detection in vaginal secretion could be used, as validated by our research team.
Acknowledgements The authors wish to thank Dr Louise Constantineau and Mrs Jose´e Poulin for their expertise and help to develop the standardized TVUS method. They are thankful to Dr Daniel Blouin, Mrs Lise Lavoie, Mrs Ame´lie Le´veille´, Mrs MariePier Labbe´ and the ultrasound technologists at the MFMclinic of the CHUS for their technical support. They are grateful to Mrs Tania Fayad for her precious help in the writing process and to all patients who agreed to participate in the study.
Declaration of interest None of the authors have a conflict of interest. This study was funded by the principal investigator personal grants.
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