http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, Early Online: 1–6 ! 2014 Informa UK Ltd. DOI: 10.3109/14767058.2014.942627
ORIGINAL ARTICLE
Diagnostic and prognostic value of presepsin in preterm deliveries Karin Malı´cˇkova´1, Michal Koucky´2, Antonı´n Parˇ´ızek2, Kveˇta Pelinkova´1, Helena Brodska´1, Zdeneˇk Ha´jek2, Anna Germanova´2, Oto Mestek3, and Toma´sˇ Zima1 J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by University of Newcastle on 09/25/14 For personal use only.
1
Institute of Medical Biochemistry and Laboratory Diagnostics and 2Department of Gynecology and Obstetrics, General University Hospital and 1st Faculty of Medicine of Charles University in Prague, Prague, Czech Republic, and 3Institute of Chemical Technology Prague, Prague, Czech Republic Abstract
Keywords
Problem: To evaluate the association between serum presepsin (soluble CD14 antigen subtype, sCD14-ST) levels soon after the appearance of signs of preterm delivery and preterm delivery within 48 h, before the 34th and 37th gestational weeks and the possible additional value of concurrently evaluated ultrasound vaginal cervicometry with serum presepsin measurement. Methodology: A total of 60 females were included. Serum presepsin was measured by a chemiluminescent immunoassay. Sonographic evaluation of cervical length in all females was conducted by transvaginal ultrasound. Results: Patients who delivered within 48 h after analysis showed significantly higher presepsin concentrations compared to females with later deliveries. Higher presepsin was proven also for deliveries before/after weeks 34 and 37. A combined finding of cervical length shortening below 18 mm and presepsin level increasing above 623.5 pg/mL could point to the significantly high risk of preterm delivery. Conclusion: Elevated maternal serum concentration of sCD14-ST could be an independent and relevant risk factor for preterm delivery.
Cervical length, inflammation, inflammatory biomarkers, sCD14-ST
Background Preterm delivery is defined as delivery before the 37th gestational week. Prematurity is the leading cause of neonatal mortality and is associated with a wide range of morbidity in survivors. Despite strong efforts to reduce preterm deliveries, the rate has remained relatively stable over the years and is about 7% in the Czech Republic [1]. The etiology of preterm delivery is complex and multifactorial. It is known that both maternal and fetal factors contribute to risk, and inflammation is supposed to be the crucial pathogenetic mechanism of preterm delivery processes [2]. Inflammation is a complex process that reflects the local and systemic responses to different immunological and nonimmunological stimuli. It can be initiated by different ways and is characterized by activation of acute phase response and release of specific markers. The causes of ongoing inflammatory processes in preterm deliveries are not well understood, but it is accepted that continuous feto–maternal conflict results in local and systemic cell activation and triggering of inflammatory cascades [3–6].
Address for correspondence: Karin Malı´cˇkova´, MD, Laboratory of the Clinical Immunology and Allergology, Institute of Medical Biochemistry and Laboratory Diagnostics of the First Faculty of Medicine and General University Hospital, Charles University in Prague, Karlovo na´m. 32, 121 11 Prague 2, Czech Republic. Tel: +420 2496 6469, 6341. E-mail: [email protected]
History Received 20 April 2014 Revised 28 June 2014 Accepted 4 July 2014 Published online 28 July 2014
A number of studies have assessed the relationship between preterm delivery and maternal levels of different biomarkers of systemic and/or local inflammation, such as high-sensitivity C-reactive protein (hs-CRP) [7–9], interleukin-6 (IL-6) [10,11] or fetal fibronectin [12–14]; however, none of them has sufficient sensitivity and specificity to predict preterm delivery independently. For this reason, researchers continue to search for other accessible, costeffective, fast and precise biomarkers that can provide additional information about delivery-associated inflammatory cascades in the maternal immune system. In this work, we have focused on the novel biomarker of neutrophil activation – presepsin. Presepsin (soluble CD14 antigen subtype, sCD14-ST) is the novel serum marker of severe inflammation such as sepsis or systemic inflammatory response syndrome [15–17]. Production of sCD14-ST during the inflammation is probably induced by enzyme degradation during leukocyte activation. Presepsin is normally present in very low concentrations in the serum of healthy individuals and has been shown to be increased in local infections, SIRS, sepsis or severe sepsis [18]. Based on this premise, we have hypothesized that presepsin could take part in the inflammatory cascades during a preterm delivery. To test this hypothesis, we have evaluated the following: (1) the association between serum presepsin levels early after signs of preterm delivery appear
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and preterm delivery within 48 h, before 34th and 37th gestational weeks; (2) the comparison of presepsin and established markers of inflammation severity such as hs-CRP, IL-6 and calprotectin (MRP 8/14); and (3) the possible additional value of concurrently evaluated ultrasound vaginal cervicometry with serum presepsin measurement.
Patients and methods
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Interventions Sonographic evaluation of cervical length in all females from Prenatal Risk Care Unit was conducted by transvaginal ultrasound. Sonographic examinations were performed with standard equipment (Acuson XP128, Oceanside, CA). All examinations were performed by an experienced sonographer. Transvaginal cervical length measurements were obtained using the technique described by Iams [21].
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Subjects Participant enrollment was undertaken from August 2012 to August 2013 at the Department of Gynecology and Obstetrics of General University Hospital and 1st Faculty of Medicine of Charles University in Prague. All patients with a singleton pregnancy presenting to the Prenatal Risk Care Unit were between the 24th and 32nd week of pregnancy. A total of 60 females were included. The following clinical outcomes were recorded in all subjects: (1) delivery within 48 h after the examination; (2) delivery before pregnancy week 34; and (3) delivery before week 37. Control groups: a total of 61 pregnant females were included, 25 of them at 10th week of early physiological pregnancy, 15 women with pregnancy between weeks 20 and 23 and 12 females between weeks 37 and 41 of physiological pregnancy terminated thereafter with the term delivery. None of the examined groups have shown any symptoms of apparent systemic infection. Ethical aspects The study was approved by the Ethics Committee of 1st Faculty of Medicine of Charles University in Prague (No. 1162/12S-IV). All women included have read, dated and signed the informed consent form and were fully informed of the nature, significance and implications of this study. Laboratory examinations A peripheral blood sample was drawn from the cubital vein to the tube without an additive. Clotted blood samples were centrifuged for 10 min at ambient temperature and 1300g, and separated serum aliquots were frozen at 80 C and placed in the serum biobank. The frozen serum samples were thawed once on ice before analysis. Presepsin was measured by the chemiluminescent immunoassay Pathfast Presepsin (Mitsubishi Chemical, Tokyo, Japan). As described by Spanuth et al. [19], excellent comparability between the different sample matrices (such as serum, EDTA plasma, heparin plasma and citrate plasma) across the full measurement range was proven, and the suitability of serum samples for presepsin determination was established. Therefore, normal presepsin values in healthy individuals 5200 pg/mL in plasma [20] can be related to serum samples as well. Hs-CRP was measured by nephelometry with CardioPhase hs-CRP (Siemens, Mu¨nchen, Germany). Serum IL-6 was measured by h-IL-6 Platinum ELISA (eBioScience, San Diego, CA). Serum calprotectin was measured by enzyme-linked immunosorbent assay (MRP8/14, Bu¨hlmann, Scho¨nenbuch, Switzerland).
Statistical analysis For sample size determination, the freeware Epi InfoTM 6.04 from the Centers for Disease Control and Prevention (USA) was used. The cutoff point of serum presepsin was determined using the web application Cutoff Finder [22]. Subsequent statistical analysis was performed using the software Statistica CZ 10.0 (StatSoft Inc., Tulsa, OK). Standard descriptive statistical analyses were performed, including frequency distributions for categorical data and calculations of medians and interquartile ranges for continuous variables. Different groups of patients were compared using the Wilcoxon nonparametric test. Spearman’s rank correlation coefficient was used as a measure of linear relationship between two sets of data. A p value smaller than 0.05 was considered significant.
Results Based on the fact that the annual birthrate in the Czech Republic is about 100 000 newborns and preterm delivery rate fluctuates annually around 7% [23], a necessary sample size of 53 was determined to achieve a confidence level of 90%. Therefore, our cohort size of n ¼ 60 was evaluated as sufficient for the purpose of this study. The baseline descriptive characteristics of patients with risk pregnancies are listed in Table 1. Three quarters of examined females from the Prenatal Risk Care Unit delivered prematurely. There were no age differences between cohorts with and without preterm delivery. The obtained values for presepsin measurements in all cohorts are included in Table 2. Presepsin and pregnancy outcome Risk patients who delivered within 48 h after the analyses showed significantly higher presepsin concentrations compared to females with later deliveries. Higher presepsin was also proven for deliveries before week 34 compared to post-week 34 as well as for before and after week 37. Table 1. Descriptive characteristics of examined Prenatal Risk Care Unit cohort, n ¼ 60. Age, years, median (IQR) 34 Pregnancy week at the examination, median (IQR) 28 Abnormal contractions, n (%) 23 Vaginal bleeding, n (%) 9 Complete funneling, n (%) 20 Delivery within 48 h after the examination, n (%) 9 Delivery 448 h and before week 34, n (%) 21 Delivery 4week 34 and 5week 37, n (%) 15 Term delivery 4week 37, n (%) 15 Newborn birth weight (g), median (IQR) 2496 IQR, interquartile range.
(30.5–35.3) (25–30) (38.3%) (15.0%) (33.3%) (15.0%) (35.0%) (25.0%) (25.0%) (1850–3200)
Presepsin in preterm deliveries
DOI: 10.3109/14767058.2014.942627
Other inflammatory biomarkers assessed have shown significant differences too, but none of them as significant as presepsin (see Figure 1). The odds ratio (OR) represents the odds that a preterm birth will occur given a particular test result, compared to the odds of the preterm birth occurring in the absence of this test result. Table 3 shows the ORs for assessed inflammatory biomarkers and for cervicometry, where medians were used as cut-off values.
Based on the online Cutoff Finder [22], the optimal cutoff point for preterm delivery is represented by the concentration of presepsin 623.5 pg/mL (calculated median value for whole cohort was 454 pg/mL). The receiver operating characteristics analysis of presepsin for preterm delivery revealed an area under the curve (AUC) of greater than 0.5 on all three pregnancy outcomes: delivery within 48 h (AUC ¼ 0.863; p ¼ 0.002), delivery before week 34 (AUC ¼ 0.791; p ¼ 0.042) and delivery before week 37 (AUC ¼ 0.788; p ¼ 0.044) (Figure 2).
Table 2. Non-parametric univariate statistics for presepsin measurements in all cohorts.
Correlation between biomarkers
sCD14-ST (pg/mL) J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by University of Newcastle on 09/25/14 For personal use only.
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Week 10, physiological pregnancy, n ¼ 25 Weeks 20–22, physiological pregnancy, n ¼ 15 Weeks 37–41, physiological pregnancy, n ¼ 12 Weeks 24–32, Prenatal Risk Care Unit, n ¼ 60 sCD14-ST, presepsin.
Median
Interquartile range
0.273
0.00–3.230
95.35
25.50–128.25
143.50
106.75–175.80
454.00
262.00–569.50
As presepsin serum concentrations rise, the other biomarkers tends to increase too. Presepsin levels were in significant positive correlation with hs-CRP (r ¼ 0.599, p50.001) and MRP8/14 (r ¼ 0.625, p50.001). With IL-6, only a tendency to the positive correlation was observed (r ¼ 0.317, p ¼ 0.061). Simultaneous cervical length measurement and serum presepsin detection For a binary outcome that measures the presence or absence of preterm delivery, a survey logistic regression model was used to analyze the relationship between the binary dependent
Figure 1. Presepsin and different pregnancy outcomes; comparison to hs-CRP, IL-6 and MRP8/14. Horizontal lines represent medians. sCD14-ST, presepsin; hs-CRP, high-sensitivity C-reactive protein; IL-6, interleukin 6; and MRP 8/14, calprotectin.
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Table 3. OR is an estimate with confidence interval for the relationship of preterm delivery with measured parameters in binary (‘‘yes or no’’) variables.
Table 5. Serum levels of biomarkers and clinical signs of imminent preterm delivery. Present
Delivery Presepsin Hs-CRP IL-6
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MRP 8/14 Cervical length
Within 48 h Before week Before week Within 48 h Before week Before week Within 48 h Before week Before week Within 48 h Before week Before week Within 48 h Before week Before week
34 37 34 37 34 37 34 37 34 37
28.7 13.5 4.0 1.30 2.88 2.25 3.25 3.29 4.11 4.26 4.00 2.98 5.85 5.71 4.00
OR (CI)
p value
(9.3–57.8) (3.3–54.7) (1.37–11.7) (0.31–5.40) (0.95–8.71) (0.8–6.32) (0.56–12.51) (1.0–11.54) (1.37–11.74) (0.81–22.5) (1.27–12.6) (1.04–8.52) (2.7–10.2) (1.7–18.9) (1.4–11.7)
50.001 50.001 0.011 0.718 0.062 0.124 0.102 0.611 0.045 0.088 0.017 0.045 0.002 0.004 0.011
Within 48 h, n ¼ 9; before week 34, n ¼ 21; and before week 37, n ¼ 15. sCD14-ST, presepsin; hs-CRP, high-sensitivity C-reactive protein; IL-6, interleukin 6; and MRP 8/14, calprotectin. As cut-off values, medians were used.
Abnormal uterine contractions sCD14-ST, pg/mL 578.50 (361–852) hs-CRP, mg/L 3.58 (1.83–5.81) IL-6, ng/L 18.9 (7.54–26.2) MRP8/14, mg/mL 8.71 (3.65–14.25) Vaginal bleeding sCD14-ST, pg/mL hs-CRP, mg/L IL-6, ng/L MRP8/14, mg/mL
371.5 5.12 22.47 6.60
Absent
339 3.22 11.4 5.2
p value
(263–470) (1.61–6.27) (5.95–15.10 (1.35–7.35)
(315–500) 405 (263–633) (3.57–6.42) 3.99 (2.05–5.31 (18.5–25.79) 15.96 (13.63–19.22) (0.73–11.87) 5.21 (2.99–13.78)
NS NS NS NS NS NS NS NS
Complete funneling sCD14-ST, pg/mL 783 (471–991) 339 (259–438) 0.035 hs-CRP, mg/L 3.08 (2.22–5.78) 3.87 (2.99–6.12) NS IL-6, ng/L 14.98 (11.65–18.87) 17.04 (14.04–20.31) NS MRP8/14, mg/mL 11.45 (3.32–13.88) 5.2 (2.35–6.97) 0.033 Medians (interquartile ranges, IQR) of measured parameters in females with and without assessed clinical signs of preterm delivery. sCD14-ST, presepsin; hs-CRP, high-sensitivity C-reactive protein; IL-6, interleukin 6; and MRP 8/14, calprotectin.
Comparison of examined biomarkers in association with clinical signs of imminent preterm delivery Abnormal uterine contractions, unexplained vaginal bleeding and ultrasound-proven funneled cervix were assessed. In patients with complete funneling, presepsin serum levels were significantly higher compared to patients with cervix competence (p ¼ 0.035) (Table 5). Similarly, MRP8/4 levels were higher in females with complete funneling phenomena (p ¼ 0.033). Neither of the other clinical signs was associated with significantly elevated sCD14-ST levels.
Discussion Figure 2. Comparison of presepsin levels in three pregnancy outcomes: delivery within 48 h, delivery before week 34 and delivery before week 37. The ROC curves of different outcomes are plotted in different shapes. Table 4. OR as an estimate with confidence interval for the relationship of preterm delivery with combined simultaneous cervical length and serum presepsin measurements.
Simultaneous Presepsin + cervical length Measurement
Delivery
OR (CI)
p values
Within 48 h Before week 34
50.4 (5.1–571) 105 (8.5–1290)
50.001 50.001
Before week 37
33.0 (3.4–324)
0.003
Medians were used as cut-off values.
variable and a combination of two analyses: cervicometry and presepsin measurement (Table 4). Because the probability of the assessed outcome (preterm delivery) is less than 10% [1], the OR and relative risk can be used interchangeably [24]. Therefore, the combined findings of cervical length shortening below 18 mm and presepsin level increasing above 623.5 pg/mL could point to a significantly high risk of preterm delivery.
To our knowledge, this is the first study that alerts to an increased presepsin level in women with preterm deliveries. High presepsin serum concentrations could provide a warning of delivery within 48 h after the examination, as well as of preterm delivery before the 34th or 37th pregnancy week. The odds that a preterm birth will occur given an increased presepsin level seems to be significantly higher compared to other inflammatory markers such as hs-CRP, IL-6 or MRP 8/14. Moreover, a simultaneous cervical length and presepsin serum concentrations measurement significantly outweighs the odds for each individual examination. Presepsin (sCD14-ST) is the novel serum marker of severe inflammation, such as sepsis or systemic inflammatory response syndrome. The subject CD14 molecule is a glycoprotein expressed mainly on the surface of monocytes/ macrophages and serves as a receptor for an endotoxin (lipopolysaccharide) [25]. The CD14 molecule includes soluble (sCD14) as well as membrane-bound CD14 (mCD14). Several soluble CD14 subtypes having different molecular weights are present in blood. Among them, a soluble low-molecular-weight CD14 subtype of about 13 kDa (sCD14-ST) was confirmed as a diagnostic marker of systemic inflammation.
Presepsin in preterm deliveries
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DOI: 10.3109/14767058.2014.942627
CD14 molecule exists in two forms: the form anchored to the cell membrane by a glycosylphosphatidylinositol tail (membranous mCD14) and a soluble form (sCD14) [26]. Soluble CD14 either appears after shedding of mCD14 or is directly secreted from intracellular vesicles. CD14 is expressed mainly by macrophages and at significantly lesser extent by neutrophils and dendritic cells. sCD14 is secreted by the liver and blood monocytes. Lipopolysaccharide is considered as the main CD14 ligand; however, it also recognizes other pathogen-associated molecular patterns [27]. Macrophages play an important role at the human maternal–fetal interface during normal placental development, and the misregulation of these cells can result in pregnancy complications. As demonstrated by Houser [28], dominance of macrophages over dendritic cells suggests that macrophages could be the most important antigen presenting cells at the maternal–fetal interface. They could be able to initiate a T cell response to invading pathogens and simultaneously avoid the risk of prematurely priming T cells toward fetal antigens. Aberrant activity of uterine macrophages is linked to the pathogenesis of preterm delivery [29–32]. It is known that preterm delivery can be accompanied by excessive maternal inflammatory response to pregnancy [2]. During pregnancy, the female carries a fetus partly foreign to her immune system because of the expression of paternal antigens. Despite this, the fetus is normally tolerated and not rejected due to systemic and local physiological changes in the maternal immune system. As suggested by Wegmann et al. [33], the immune system during physiological pregnancy is associated with weakened cell-mediated immunity and increased humoral immunity, at least directed toward the fetus and placenta (so-called Th2 shift). In contrast to normal pregnancy, there are indications of increased inflammatory responses in pathological pregnancies [34]. Roberts et al. [35] suggested that proinflammatory mediators released from the placenta are responsible for the endothelial damage, with a subsequent dysfunctional cascade of coagulation, vasoconstriction and excessive maternal inflammatory response triggering the results in preterm deliveries. These inflammatory cascades contribute significantly to the pathogenesis of preterm labor, likely by the release of proinflammatory cytokines that stimulate uterogenic agents such as prostaglandin E2 and F2 biosynthesis by the decidua or amnion, and thus initiate labor [36]. In preterm deliveries, several studies have shown increased concentrations of proinflammatory cytokines in maternal peripheral blood [37], in amniotic fluid [38,39] or in cord blood [40]. Generally, any acute inflammatory response is characterized by the arrival of neutrophils and the subsequent recruitment of monocytes that differentiate into macrophages and dendritic cells on site. Both neutrophils and macrophages phagocytose harmful particles (such as microbes); they also have an important role in orchestrating the immune response through the production of pro-inflammatory cytokines such as IL-1, IL-6 and tumor necrosis factor alpha. Therefore, it could be hypothesized that the significant elevation of serum sCD14-ST levels could be at least an epiphenomenon of monocyte/macrophage activation during inflammatory processes, leading to preterm delivery.
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The principal finding of this study is that elevated maternal serum concentrations of sCD14-ST probably outweigh ‘‘classical’’ systemic inflammatory markers such as IL-6 and hs-CRP in assessed association with preterm births. Several prior studies have explored the association between maternal serum concentrations of analytes and preterm delivery and/or neonatal morbidities; however, most of them have explored the relationship between IL-6 [4] or CRP [9] and preterm delivery or infectious and non-infectious neonatal outcomes [3] focused on pregnancies with either preterm labor [8] or preterm rupture of membranes [40]. Our findings for IL-6, hs-CRP and MRP8/14 are in accordance with mentioned studies, so that sCD14-ST figures are at least encouraging for further investigation. Our study has some limitations. Because infection is a major cause of spontaneous preterm delivery in the late second and early third trimester [2], high maternal serum concentration of such a systemic inflammatory marker as sCD14-ST in women at increased risk for spontaneous early preterm delivery could be in a way understandable. However, the maternal compartment differs from the fetal compartment, and the fetal inflammatory compartment is not necessarily reflected in maternal serum. Our study does not contain any information about funisitis or fetal inflammatory response syndrome in offspring. Moreover, our hypothesis of sCD14ST as an epiphenomenon of monocyte/macrophage activation is not supported by other biomarkers of leukocyte activation, such as intracellular cytokine production in peripheral blood monocytes or flow cytometric detection of membrane scavenger receptors. However, we believe that our main result remains valid because elevated levels of serum sCD14-ST were significantly associated with preterm deliveries. Clearly, only further experiments can demonstrate the real pathogenetic mechanisms of monocyte/macrophage participation on preterm delivery mechanisms. In conclusion, the development of strategies for risk stratification and prediction of morbidity in preterm deliveries includes the identification of simple, rapid and safe markers of inflammation in women that are at increased risk for preterm birth. This study provided new information, suggesting that elevated maternal serum concentrations of sCD14-ST could be one such independent and relevant risk factor for preterm delivery.
Declaration of interest This project was supported by the research projects PRVOUK P25/LF1/2 and UNCE No. 204024 of Charles University in Prague as well as by RVO VFN 64165/2012 of General University Hospital in Prague.
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compared to procalcitonin. 20th IFCC-EFLM European Congress of Clinical Chemistry and Laboratory Medicine, Milan (Italy), 2013. M129, p: S150. Available from: http://euromedlab.expomeeting.it/wp-content/uploads/2013/03/POSTERS.pdf [last accessed 10 Jul 2014]. Iams JD. Prediction and early detection of preterm labor. Obstet Gynecol 2003;101:402–12. Budczies J, Klauschen F, Sinn BV, et al. Cutoff Finder: a comprehensive and straightforward web application enabling rapid biomarker cutoff optimization. Plos One 2012;7:e51862. Ritschelova I, ed. Statistical Yearbook of the Czech Republic. Prague (CZ): Czech Statistical Office 2013;828p. Available from: http://www.czso.cz/ [last accessed 10 Jul 2014]. Tripepi G, Torino C, D’Arrigo G, et al. An overview of standard statistical methods for assessing exposure-outcome link in survival analysis (part I): basic concepts. Aging Clin Exp Res 2012;24: 109–12. Dupuy AM, Philippart F, Pe´an Y, et al.; Maurice Rapin Institute Biomarkers Group. Role of biomarkers in the management of antibiotic therapy: an expert panel review: I – currently available biomarkers for clinical use in acute infections. Ann Intensive Care 2013;3:22 doi: 10.1186/2110-5820-3-22. Kirkland TN, Viriyakosol S. Structure-function analysis of soluble and membrane-bound CD14. Prog Clin Biol Res 1998;397:79–87. Simmons DL, Tan S, Tenen DG, et al. Antigen CD14 is a phospholipid anchored membrane protein. Blood 1997;73:284–9. Houser BL. Decidual macrophages and their roles at the maternal– fetal interface. Yale J Biol Med 2012;85:105–18. Tamblyn JA, Lissauer DM, Powell R, et al. The immunological basis of villitis of unknown etiology – review. Placenta 2013;34: 846–55. Nagamatsu T, Schust DJ. The immunomodulatory roles of macrophages at the maternal-fetal interface. Reprod Sci 2010;17:209–18. Yellon SM, Mackler AM, Kirby MA. The role of leukocyte traffic and activation in parturition. J Soc Gynecol Investig 2003;10: 323–38. Winkler M, Rath W. The role of cytokines in the induction of labor, cervical ripening and rupture of the fetal membranes. Z Geburtshilfe Neonatol 1996;200:1–12. Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bidirectional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a TH2 phenomenon? Immunol Today 1993;14: 353–6. Picklesimer AH, Jared HL, Moss K, et al. Racial differences in C-reactive protein levels during normal pregnancy. Am J Obstet Gynecol 2008;199:523.e1-6. doi: 10.1016/j.ajog.2008.04.017. Roberts RM, Chen Y, Ezashi T, Walker AM. Interferons and the maternal-conceptus dialog in mammals. Semin Cell Dev Biol 2008; 19:170–7. Keelan JA, Blumenstein M, Helliwell RJ, et al. Cytokines, prostaglandins and parturition – a review. Placenta 2003;24: S33–46. Yoon BH, Yang SH, Jun JK, et al. Maternal blood C-reactive protein, white blood cell count, and temperature in preterm labor: a comparison with amniotic fluid white blood cell count. Obstet Gynecol 1996;87:231–7. Buhimschi CS, Buhimschi IA, Abdel-Razeq S, et al. Proteomic biomarkers of intra-amniotic inflammation: relationship with funisitis and early-onset sepsis in the premature neonate. Pediatr Res 2007;61:318–24. Lyon D, Cheng CY, Howland L, et al. Integrated review of cytokines in maternal, cord, and newborn blood: part I – associations with preterm birth. Biol Res Nurs 2010;11:371–6. Kurki T, Teramo K, Ylikorkala O, Paavonen J. C-reactive protein in preterm premature rupture of the membranes. Arch Gynecol Obstet 1990;247:31–7.
ORIGINAL ARTICLE
Diagnostic and prognostic value of presepsin in preterm deliveries Karin Malı´cˇkova´1, Michal Koucky´2, Antonı´n Parˇ´ızek2, Kveˇta Pelinkova´1, Helena Brodska´1, Zdeneˇk Ha´jek2, Anna Germanova´2, Oto Mestek3, and Toma´sˇ Zima1 J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by University of Newcastle on 09/25/14 For personal use only.
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Institute of Medical Biochemistry and Laboratory Diagnostics and 2Department of Gynecology and Obstetrics, General University Hospital and 1st Faculty of Medicine of Charles University in Prague, Prague, Czech Republic, and 3Institute of Chemical Technology Prague, Prague, Czech Republic Abstract
Keywords
Problem: To evaluate the association between serum presepsin (soluble CD14 antigen subtype, sCD14-ST) levels soon after the appearance of signs of preterm delivery and preterm delivery within 48 h, before the 34th and 37th gestational weeks and the possible additional value of concurrently evaluated ultrasound vaginal cervicometry with serum presepsin measurement. Methodology: A total of 60 females were included. Serum presepsin was measured by a chemiluminescent immunoassay. Sonographic evaluation of cervical length in all females was conducted by transvaginal ultrasound. Results: Patients who delivered within 48 h after analysis showed significantly higher presepsin concentrations compared to females with later deliveries. Higher presepsin was proven also for deliveries before/after weeks 34 and 37. A combined finding of cervical length shortening below 18 mm and presepsin level increasing above 623.5 pg/mL could point to the significantly high risk of preterm delivery. Conclusion: Elevated maternal serum concentration of sCD14-ST could be an independent and relevant risk factor for preterm delivery.
Cervical length, inflammation, inflammatory biomarkers, sCD14-ST
Background Preterm delivery is defined as delivery before the 37th gestational week. Prematurity is the leading cause of neonatal mortality and is associated with a wide range of morbidity in survivors. Despite strong efforts to reduce preterm deliveries, the rate has remained relatively stable over the years and is about 7% in the Czech Republic [1]. The etiology of preterm delivery is complex and multifactorial. It is known that both maternal and fetal factors contribute to risk, and inflammation is supposed to be the crucial pathogenetic mechanism of preterm delivery processes [2]. Inflammation is a complex process that reflects the local and systemic responses to different immunological and nonimmunological stimuli. It can be initiated by different ways and is characterized by activation of acute phase response and release of specific markers. The causes of ongoing inflammatory processes in preterm deliveries are not well understood, but it is accepted that continuous feto–maternal conflict results in local and systemic cell activation and triggering of inflammatory cascades [3–6].
Address for correspondence: Karin Malı´cˇkova´, MD, Laboratory of the Clinical Immunology and Allergology, Institute of Medical Biochemistry and Laboratory Diagnostics of the First Faculty of Medicine and General University Hospital, Charles University in Prague, Karlovo na´m. 32, 121 11 Prague 2, Czech Republic. Tel: +420 2496 6469, 6341. E-mail: [email protected]
History Received 20 April 2014 Revised 28 June 2014 Accepted 4 July 2014 Published online 28 July 2014
A number of studies have assessed the relationship between preterm delivery and maternal levels of different biomarkers of systemic and/or local inflammation, such as high-sensitivity C-reactive protein (hs-CRP) [7–9], interleukin-6 (IL-6) [10,11] or fetal fibronectin [12–14]; however, none of them has sufficient sensitivity and specificity to predict preterm delivery independently. For this reason, researchers continue to search for other accessible, costeffective, fast and precise biomarkers that can provide additional information about delivery-associated inflammatory cascades in the maternal immune system. In this work, we have focused on the novel biomarker of neutrophil activation – presepsin. Presepsin (soluble CD14 antigen subtype, sCD14-ST) is the novel serum marker of severe inflammation such as sepsis or systemic inflammatory response syndrome [15–17]. Production of sCD14-ST during the inflammation is probably induced by enzyme degradation during leukocyte activation. Presepsin is normally present in very low concentrations in the serum of healthy individuals and has been shown to be increased in local infections, SIRS, sepsis or severe sepsis [18]. Based on this premise, we have hypothesized that presepsin could take part in the inflammatory cascades during a preterm delivery. To test this hypothesis, we have evaluated the following: (1) the association between serum presepsin levels early after signs of preterm delivery appear
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and preterm delivery within 48 h, before 34th and 37th gestational weeks; (2) the comparison of presepsin and established markers of inflammation severity such as hs-CRP, IL-6 and calprotectin (MRP 8/14); and (3) the possible additional value of concurrently evaluated ultrasound vaginal cervicometry with serum presepsin measurement.
Patients and methods
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Interventions Sonographic evaluation of cervical length in all females from Prenatal Risk Care Unit was conducted by transvaginal ultrasound. Sonographic examinations were performed with standard equipment (Acuson XP128, Oceanside, CA). All examinations were performed by an experienced sonographer. Transvaginal cervical length measurements were obtained using the technique described by Iams [21].
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Subjects Participant enrollment was undertaken from August 2012 to August 2013 at the Department of Gynecology and Obstetrics of General University Hospital and 1st Faculty of Medicine of Charles University in Prague. All patients with a singleton pregnancy presenting to the Prenatal Risk Care Unit were between the 24th and 32nd week of pregnancy. A total of 60 females were included. The following clinical outcomes were recorded in all subjects: (1) delivery within 48 h after the examination; (2) delivery before pregnancy week 34; and (3) delivery before week 37. Control groups: a total of 61 pregnant females were included, 25 of them at 10th week of early physiological pregnancy, 15 women with pregnancy between weeks 20 and 23 and 12 females between weeks 37 and 41 of physiological pregnancy terminated thereafter with the term delivery. None of the examined groups have shown any symptoms of apparent systemic infection. Ethical aspects The study was approved by the Ethics Committee of 1st Faculty of Medicine of Charles University in Prague (No. 1162/12S-IV). All women included have read, dated and signed the informed consent form and were fully informed of the nature, significance and implications of this study. Laboratory examinations A peripheral blood sample was drawn from the cubital vein to the tube without an additive. Clotted blood samples were centrifuged for 10 min at ambient temperature and 1300g, and separated serum aliquots were frozen at 80 C and placed in the serum biobank. The frozen serum samples were thawed once on ice before analysis. Presepsin was measured by the chemiluminescent immunoassay Pathfast Presepsin (Mitsubishi Chemical, Tokyo, Japan). As described by Spanuth et al. [19], excellent comparability between the different sample matrices (such as serum, EDTA plasma, heparin plasma and citrate plasma) across the full measurement range was proven, and the suitability of serum samples for presepsin determination was established. Therefore, normal presepsin values in healthy individuals 5200 pg/mL in plasma [20] can be related to serum samples as well. Hs-CRP was measured by nephelometry with CardioPhase hs-CRP (Siemens, Mu¨nchen, Germany). Serum IL-6 was measured by h-IL-6 Platinum ELISA (eBioScience, San Diego, CA). Serum calprotectin was measured by enzyme-linked immunosorbent assay (MRP8/14, Bu¨hlmann, Scho¨nenbuch, Switzerland).
Statistical analysis For sample size determination, the freeware Epi InfoTM 6.04 from the Centers for Disease Control and Prevention (USA) was used. The cutoff point of serum presepsin was determined using the web application Cutoff Finder [22]. Subsequent statistical analysis was performed using the software Statistica CZ 10.0 (StatSoft Inc., Tulsa, OK). Standard descriptive statistical analyses were performed, including frequency distributions for categorical data and calculations of medians and interquartile ranges for continuous variables. Different groups of patients were compared using the Wilcoxon nonparametric test. Spearman’s rank correlation coefficient was used as a measure of linear relationship between two sets of data. A p value smaller than 0.05 was considered significant.
Results Based on the fact that the annual birthrate in the Czech Republic is about 100 000 newborns and preterm delivery rate fluctuates annually around 7% [23], a necessary sample size of 53 was determined to achieve a confidence level of 90%. Therefore, our cohort size of n ¼ 60 was evaluated as sufficient for the purpose of this study. The baseline descriptive characteristics of patients with risk pregnancies are listed in Table 1. Three quarters of examined females from the Prenatal Risk Care Unit delivered prematurely. There were no age differences between cohorts with and without preterm delivery. The obtained values for presepsin measurements in all cohorts are included in Table 2. Presepsin and pregnancy outcome Risk patients who delivered within 48 h after the analyses showed significantly higher presepsin concentrations compared to females with later deliveries. Higher presepsin was also proven for deliveries before week 34 compared to post-week 34 as well as for before and after week 37. Table 1. Descriptive characteristics of examined Prenatal Risk Care Unit cohort, n ¼ 60. Age, years, median (IQR) 34 Pregnancy week at the examination, median (IQR) 28 Abnormal contractions, n (%) 23 Vaginal bleeding, n (%) 9 Complete funneling, n (%) 20 Delivery within 48 h after the examination, n (%) 9 Delivery 448 h and before week 34, n (%) 21 Delivery 4week 34 and 5week 37, n (%) 15 Term delivery 4week 37, n (%) 15 Newborn birth weight (g), median (IQR) 2496 IQR, interquartile range.
(30.5–35.3) (25–30) (38.3%) (15.0%) (33.3%) (15.0%) (35.0%) (25.0%) (25.0%) (1850–3200)
Presepsin in preterm deliveries
DOI: 10.3109/14767058.2014.942627
Other inflammatory biomarkers assessed have shown significant differences too, but none of them as significant as presepsin (see Figure 1). The odds ratio (OR) represents the odds that a preterm birth will occur given a particular test result, compared to the odds of the preterm birth occurring in the absence of this test result. Table 3 shows the ORs for assessed inflammatory biomarkers and for cervicometry, where medians were used as cut-off values.
Based on the online Cutoff Finder [22], the optimal cutoff point for preterm delivery is represented by the concentration of presepsin 623.5 pg/mL (calculated median value for whole cohort was 454 pg/mL). The receiver operating characteristics analysis of presepsin for preterm delivery revealed an area under the curve (AUC) of greater than 0.5 on all three pregnancy outcomes: delivery within 48 h (AUC ¼ 0.863; p ¼ 0.002), delivery before week 34 (AUC ¼ 0.791; p ¼ 0.042) and delivery before week 37 (AUC ¼ 0.788; p ¼ 0.044) (Figure 2).
Table 2. Non-parametric univariate statistics for presepsin measurements in all cohorts.
Correlation between biomarkers
sCD14-ST (pg/mL) J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by University of Newcastle on 09/25/14 For personal use only.
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Week 10, physiological pregnancy, n ¼ 25 Weeks 20–22, physiological pregnancy, n ¼ 15 Weeks 37–41, physiological pregnancy, n ¼ 12 Weeks 24–32, Prenatal Risk Care Unit, n ¼ 60 sCD14-ST, presepsin.
Median
Interquartile range
0.273
0.00–3.230
95.35
25.50–128.25
143.50
106.75–175.80
454.00
262.00–569.50
As presepsin serum concentrations rise, the other biomarkers tends to increase too. Presepsin levels were in significant positive correlation with hs-CRP (r ¼ 0.599, p50.001) and MRP8/14 (r ¼ 0.625, p50.001). With IL-6, only a tendency to the positive correlation was observed (r ¼ 0.317, p ¼ 0.061). Simultaneous cervical length measurement and serum presepsin detection For a binary outcome that measures the presence or absence of preterm delivery, a survey logistic regression model was used to analyze the relationship between the binary dependent
Figure 1. Presepsin and different pregnancy outcomes; comparison to hs-CRP, IL-6 and MRP8/14. Horizontal lines represent medians. sCD14-ST, presepsin; hs-CRP, high-sensitivity C-reactive protein; IL-6, interleukin 6; and MRP 8/14, calprotectin.
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Table 3. OR is an estimate with confidence interval for the relationship of preterm delivery with measured parameters in binary (‘‘yes or no’’) variables.
Table 5. Serum levels of biomarkers and clinical signs of imminent preterm delivery. Present
Delivery Presepsin Hs-CRP IL-6
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MRP 8/14 Cervical length
Within 48 h Before week Before week Within 48 h Before week Before week Within 48 h Before week Before week Within 48 h Before week Before week Within 48 h Before week Before week
34 37 34 37 34 37 34 37 34 37
28.7 13.5 4.0 1.30 2.88 2.25 3.25 3.29 4.11 4.26 4.00 2.98 5.85 5.71 4.00
OR (CI)
p value
(9.3–57.8) (3.3–54.7) (1.37–11.7) (0.31–5.40) (0.95–8.71) (0.8–6.32) (0.56–12.51) (1.0–11.54) (1.37–11.74) (0.81–22.5) (1.27–12.6) (1.04–8.52) (2.7–10.2) (1.7–18.9) (1.4–11.7)
50.001 50.001 0.011 0.718 0.062 0.124 0.102 0.611 0.045 0.088 0.017 0.045 0.002 0.004 0.011
Within 48 h, n ¼ 9; before week 34, n ¼ 21; and before week 37, n ¼ 15. sCD14-ST, presepsin; hs-CRP, high-sensitivity C-reactive protein; IL-6, interleukin 6; and MRP 8/14, calprotectin. As cut-off values, medians were used.
Abnormal uterine contractions sCD14-ST, pg/mL 578.50 (361–852) hs-CRP, mg/L 3.58 (1.83–5.81) IL-6, ng/L 18.9 (7.54–26.2) MRP8/14, mg/mL 8.71 (3.65–14.25) Vaginal bleeding sCD14-ST, pg/mL hs-CRP, mg/L IL-6, ng/L MRP8/14, mg/mL
371.5 5.12 22.47 6.60
Absent
339 3.22 11.4 5.2
p value
(263–470) (1.61–6.27) (5.95–15.10 (1.35–7.35)
(315–500) 405 (263–633) (3.57–6.42) 3.99 (2.05–5.31 (18.5–25.79) 15.96 (13.63–19.22) (0.73–11.87) 5.21 (2.99–13.78)
NS NS NS NS NS NS NS NS
Complete funneling sCD14-ST, pg/mL 783 (471–991) 339 (259–438) 0.035 hs-CRP, mg/L 3.08 (2.22–5.78) 3.87 (2.99–6.12) NS IL-6, ng/L 14.98 (11.65–18.87) 17.04 (14.04–20.31) NS MRP8/14, mg/mL 11.45 (3.32–13.88) 5.2 (2.35–6.97) 0.033 Medians (interquartile ranges, IQR) of measured parameters in females with and without assessed clinical signs of preterm delivery. sCD14-ST, presepsin; hs-CRP, high-sensitivity C-reactive protein; IL-6, interleukin 6; and MRP 8/14, calprotectin.
Comparison of examined biomarkers in association with clinical signs of imminent preterm delivery Abnormal uterine contractions, unexplained vaginal bleeding and ultrasound-proven funneled cervix were assessed. In patients with complete funneling, presepsin serum levels were significantly higher compared to patients with cervix competence (p ¼ 0.035) (Table 5). Similarly, MRP8/4 levels were higher in females with complete funneling phenomena (p ¼ 0.033). Neither of the other clinical signs was associated with significantly elevated sCD14-ST levels.
Discussion Figure 2. Comparison of presepsin levels in three pregnancy outcomes: delivery within 48 h, delivery before week 34 and delivery before week 37. The ROC curves of different outcomes are plotted in different shapes. Table 4. OR as an estimate with confidence interval for the relationship of preterm delivery with combined simultaneous cervical length and serum presepsin measurements.
Simultaneous Presepsin + cervical length Measurement
Delivery
OR (CI)
p values
Within 48 h Before week 34
50.4 (5.1–571) 105 (8.5–1290)
50.001 50.001
Before week 37
33.0 (3.4–324)
0.003
Medians were used as cut-off values.
variable and a combination of two analyses: cervicometry and presepsin measurement (Table 4). Because the probability of the assessed outcome (preterm delivery) is less than 10% [1], the OR and relative risk can be used interchangeably [24]. Therefore, the combined findings of cervical length shortening below 18 mm and presepsin level increasing above 623.5 pg/mL could point to a significantly high risk of preterm delivery.
To our knowledge, this is the first study that alerts to an increased presepsin level in women with preterm deliveries. High presepsin serum concentrations could provide a warning of delivery within 48 h after the examination, as well as of preterm delivery before the 34th or 37th pregnancy week. The odds that a preterm birth will occur given an increased presepsin level seems to be significantly higher compared to other inflammatory markers such as hs-CRP, IL-6 or MRP 8/14. Moreover, a simultaneous cervical length and presepsin serum concentrations measurement significantly outweighs the odds for each individual examination. Presepsin (sCD14-ST) is the novel serum marker of severe inflammation, such as sepsis or systemic inflammatory response syndrome. The subject CD14 molecule is a glycoprotein expressed mainly on the surface of monocytes/ macrophages and serves as a receptor for an endotoxin (lipopolysaccharide) [25]. The CD14 molecule includes soluble (sCD14) as well as membrane-bound CD14 (mCD14). Several soluble CD14 subtypes having different molecular weights are present in blood. Among them, a soluble low-molecular-weight CD14 subtype of about 13 kDa (sCD14-ST) was confirmed as a diagnostic marker of systemic inflammation.
Presepsin in preterm deliveries
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DOI: 10.3109/14767058.2014.942627
CD14 molecule exists in two forms: the form anchored to the cell membrane by a glycosylphosphatidylinositol tail (membranous mCD14) and a soluble form (sCD14) [26]. Soluble CD14 either appears after shedding of mCD14 or is directly secreted from intracellular vesicles. CD14 is expressed mainly by macrophages and at significantly lesser extent by neutrophils and dendritic cells. sCD14 is secreted by the liver and blood monocytes. Lipopolysaccharide is considered as the main CD14 ligand; however, it also recognizes other pathogen-associated molecular patterns [27]. Macrophages play an important role at the human maternal–fetal interface during normal placental development, and the misregulation of these cells can result in pregnancy complications. As demonstrated by Houser [28], dominance of macrophages over dendritic cells suggests that macrophages could be the most important antigen presenting cells at the maternal–fetal interface. They could be able to initiate a T cell response to invading pathogens and simultaneously avoid the risk of prematurely priming T cells toward fetal antigens. Aberrant activity of uterine macrophages is linked to the pathogenesis of preterm delivery [29–32]. It is known that preterm delivery can be accompanied by excessive maternal inflammatory response to pregnancy [2]. During pregnancy, the female carries a fetus partly foreign to her immune system because of the expression of paternal antigens. Despite this, the fetus is normally tolerated and not rejected due to systemic and local physiological changes in the maternal immune system. As suggested by Wegmann et al. [33], the immune system during physiological pregnancy is associated with weakened cell-mediated immunity and increased humoral immunity, at least directed toward the fetus and placenta (so-called Th2 shift). In contrast to normal pregnancy, there are indications of increased inflammatory responses in pathological pregnancies [34]. Roberts et al. [35] suggested that proinflammatory mediators released from the placenta are responsible for the endothelial damage, with a subsequent dysfunctional cascade of coagulation, vasoconstriction and excessive maternal inflammatory response triggering the results in preterm deliveries. These inflammatory cascades contribute significantly to the pathogenesis of preterm labor, likely by the release of proinflammatory cytokines that stimulate uterogenic agents such as prostaglandin E2 and F2 biosynthesis by the decidua or amnion, and thus initiate labor [36]. In preterm deliveries, several studies have shown increased concentrations of proinflammatory cytokines in maternal peripheral blood [37], in amniotic fluid [38,39] or in cord blood [40]. Generally, any acute inflammatory response is characterized by the arrival of neutrophils and the subsequent recruitment of monocytes that differentiate into macrophages and dendritic cells on site. Both neutrophils and macrophages phagocytose harmful particles (such as microbes); they also have an important role in orchestrating the immune response through the production of pro-inflammatory cytokines such as IL-1, IL-6 and tumor necrosis factor alpha. Therefore, it could be hypothesized that the significant elevation of serum sCD14-ST levels could be at least an epiphenomenon of monocyte/macrophage activation during inflammatory processes, leading to preterm delivery.
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The principal finding of this study is that elevated maternal serum concentrations of sCD14-ST probably outweigh ‘‘classical’’ systemic inflammatory markers such as IL-6 and hs-CRP in assessed association with preterm births. Several prior studies have explored the association between maternal serum concentrations of analytes and preterm delivery and/or neonatal morbidities; however, most of them have explored the relationship between IL-6 [4] or CRP [9] and preterm delivery or infectious and non-infectious neonatal outcomes [3] focused on pregnancies with either preterm labor [8] or preterm rupture of membranes [40]. Our findings for IL-6, hs-CRP and MRP8/14 are in accordance with mentioned studies, so that sCD14-ST figures are at least encouraging for further investigation. Our study has some limitations. Because infection is a major cause of spontaneous preterm delivery in the late second and early third trimester [2], high maternal serum concentration of such a systemic inflammatory marker as sCD14-ST in women at increased risk for spontaneous early preterm delivery could be in a way understandable. However, the maternal compartment differs from the fetal compartment, and the fetal inflammatory compartment is not necessarily reflected in maternal serum. Our study does not contain any information about funisitis or fetal inflammatory response syndrome in offspring. Moreover, our hypothesis of sCD14ST as an epiphenomenon of monocyte/macrophage activation is not supported by other biomarkers of leukocyte activation, such as intracellular cytokine production in peripheral blood monocytes or flow cytometric detection of membrane scavenger receptors. However, we believe that our main result remains valid because elevated levels of serum sCD14-ST were significantly associated with preterm deliveries. Clearly, only further experiments can demonstrate the real pathogenetic mechanisms of monocyte/macrophage participation on preterm delivery mechanisms. In conclusion, the development of strategies for risk stratification and prediction of morbidity in preterm deliveries includes the identification of simple, rapid and safe markers of inflammation in women that are at increased risk for preterm birth. This study provided new information, suggesting that elevated maternal serum concentrations of sCD14-ST could be one such independent and relevant risk factor for preterm delivery.
Declaration of interest This project was supported by the research projects PRVOUK P25/LF1/2 and UNCE No. 204024 of Charles University in Prague as well as by RVO VFN 64165/2012 of General University Hospital in Prague.
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