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ARTICLE IN PRESS

JRI-2252; No. of Pages 8

Journal of Reproductive Immunology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jreprimm

Low levels of circulating T-regulatory lymphocytes and short cervical length are associated with preterm labor Michal Koucky´ a,∗ , Karin Malíˇcková b , Tereza Cindrová-Davies c , Anna Germanová a,b , Antonín Paˇrízek a , Marta Kalousová b , Zdenˇek Hájek a , Tomáˇs Zima b a Department of Gynecology and Obstetrics of the First Faculty of Medicine and General Teaching Hospital, Charles University in Prague, Czech Republic b Institute of Medical Biochemistry and Laboratory Diagnostics of the First Faculty of Medicine and General Teaching Hospital, Charles University in Prague, Czech Republic c Department of Physiology, Development and Neuroscience, University of Cambridge, United Kingdom

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Article history: Received 28 January 2014 Received in revised form 24 March 2014 Accepted 2 April 2014

Keywords: Preterm labor Ultrasound cervical length Cervical incompetence T-regulatory lymphocytes

a b s t r a c t Recent discoveries suggest that T-regulatory lymphocytes (Treg) might play an important role in the pathophysiology of preterm labor. The aim of this study was to assess the relationship among the levels of maternal circulating Treg cells, uterine cervical length, and the risk of preterm labor. Sixty women with regular contractions and/or cervical incompetence at 24–32 weeks’ gestation were recruited into a prospective study. Each patient underwent transvaginal ultrasound examination of the cervical length, and regulatory T cells were quantified in peripheral blood samples by flow cytometry. Patients with cervical incompetence were prescribed vaginal progesterone until birth. Measurements of Treg levels and cervical length correlated with the timing of labor. The risk of preterm labor happening within 48 h of testing was demonstrated to be almost 35 times higher (OR = 35.21, CI 13.3; 214, p < 0.001) in the group with simultaneously low Treg values (37 weeks). The study was approved by the Ethics Committee of the 1st Faculty of Medicine, Charles University, Prague (no. 1162/12 S-IV). All women included in the cohort read, dated, and signed the Informed Consent Form and were fully informed of the nature, significance, and implications of the study. 2.2. Cervical assessment The ultrasound examination of the cervical length was conducted by transvaginal ultrasound, using standard

´ M., et al., Low levels of circulating T-regulatory lymphocytes and short cervical Please cite this article in press as: Koucky, length are associated with preterm labor. J. Reprod. Immunol. (2014), http://dx.doi.org/10.1016/j.jri.2014.04.001

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equipment (Acuson XP128, Mountain View, CA, USA). All examinations were performed by an experienced and certified sonographer (certification of the Fetal Medicine Center, London, UK). Transvaginal cervical length measurements were obtained using the technique described by Iams et al. (1996). 2.3. Quantification of T-regulatory lymphocytes Peripheral blood samples (one per patient) were collected from the cubital vein into heparinized tubes straight after vaginal ultrasound cervical examination, and analyzed within 4 h. Regulatory T cells were quantified by flow cytometry using BD PharmingenTM Human Regulatory T Cell Cocktail (BD Biosciences, USA). This three-color cytometric analysis of the CD4+ CD25int/highCD127low live natural Treg cell populations includes anti-human CD4 FITC (clone SK3), anti-human CD25 PE-CyTM 7 (clone 2A3), and anti-human CD127 Alexa Fluor® 647 (clone hIL-7R-M21) antibodies. Data were collected on a FACSCanto cytometer and results were analyzed using FACSDiva software (BD Biosciences). Lymphocytes were identified by light scatter properties and then gated on CD4 vs. CD25. The percentage of Treg cells (CD127−) was identified for each of the CD25 populations (Liu et al., 2006). 2.4. Statistical analysis The sample size was determined using Epi InfoTM 6.04 freeware (Center of Disease Control and Prevention). Subsequent statistical analyses, including frequency distributions for categorical data and calculations of medians and interquartile ranges for continuous variables, were performed using the Statistica CZ 10.0 software (StatSoft Inc, USA). Different groups of patients were compared using the Wilcoxon nonparametric test. Spearman’s rank correlation coefficient was used as a measure of the linear relationship between two sets of data. Results were considered significant at p < 0.05. 3. Results 3.1. Patient characteristics Based on the annual birth rate in the Czech Republic (∼100,000 newborns), and the rate of preterm delivery (∼5–7% per annum), we calculated a minimum sample size of 53 patients (to achieve a 90% confidence level). Our cohort size of n = 60 was thus deemed sufficient for the purposes of this study. Patient details are summarized in Table 1. Three quarters of patients delivered prematurely. There were no

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Table 1 Descriptive characteristics of patients in the cohort, n = 60. Age, years, median (IQR) Gestational age when examined, median (IQR) Abnormal contractions, n (%) Complete funneling, n (%) Vaginal progesterone administration, n (%) Delivery within 48 h of examination, n (%) Delivery before 34 weeks’ gestation, n (%) Delivery between 34 and 37 weeks, n (%) Term delivery (>37 weeks), n (%) Newborn birth weight, g, median (IQR)

32.5 (27.5; 36) 27 (25; 30) 23 (38.3%) 20 (33.3%) 35 (58.3%) 9 (15.0%) 21 (35.0%) 15 (25.0%) 15 (25.0%) 2795 (1862; 3200)

IQR, interquartile range.

differences in maternal age between patients who delivered preterm and those who delivered at term. 3.2. Levels of Treg cells, cervical length, and the timing of labor The Treg cell counts and cervical length were measured in all individuals (Table 2). The relationship among the cervical length, Treg cell count, and the onset of labor is depicted in Fig. 1. The Treg count and cervical length increased with increasing gestational age at delivery. Women with term deliveries had a significantly higher Treg cell count than those who delivered preterm (Fig. 1). Cervical measurements on ultrasound revealed similar differences between the two groups of patients (Fig. 1). Using the ROC curve analysis of Treg cell counts, we calculated the area under curve (AUC) for all preterm delivery groups. The area was greater than 0.7 in all three groups (Fig. 2) and the best correlation was found between low Treg count and delivery within 48 h. In all three preterm groups, the cervical length was as good a predictor of premature birth as the Treg cell count. Unadjusted odds ratios (OR) for preterm delivery were also calculated using pooled Treg cell values and cervical length data from all patients. ORs represent the odds that preterm birth will occur given particular test results, compared with the odds of preterm birth occurring in the absence of these test results. Table 3 shows ORs for Treg cell count, cervical length, and the combination of these two tests. Medians from pooled Treg cell counts (0.031 × 109 /L) and cervical length (17.5 mm) data were used as cut-off values. The data confirm that the odds that preterm delivery will occur given a short cervix (