- Email: [email protected]
Identification of biomarkers for preterm delivery in mid-trimester amniotic fluid
Placenta 34 (2013) 873e878
Contents lists available at SciVerse ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Identification of biomarkers for preterm delivery in mid-trimester amniotic fluidq,qq A. Kim a, E.S. Lee b, J.C. Shin c, **,1, H.Y. Kim d, *,1 a
Department of Obstetrics and Gynecology, Institute of Wonkwang Medical Science, College of Medicine, Wonkwang University, Iksan, Republic of Korea Department of Obstetrics and Gynecology, College of Medicine, SoonChunHyang University, Seoul, Republic of Korea c Department of Obstetrics and Gynecology, College of Medicine, Seoul, Republic of Korea d Department of Obstetrics and Gynecology, College of Medicine, Kosin University, Busan, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 24 June 2013
Objective: We investigated whether the level of vascular endothelial growth factor (VEGF) and inflammatory markers in mid-trimester amniotic fluid have predictive value for spontaneous preterm birth in singleton pregnancy. Method: Our subjects were 72 pregnant women who were undertaken with amniocentesis from 16 to 19 weeks of gestation. 36 cases were women with preterm delivery, and other 36 cases were matched women with full-term delivery. Stored amniotic fluid was investigated after the delivery. The levels of matrix metalloproteinases-8 (MMP-8), interleukin-6 (IL-6), C-reactive protein (CRP), and VEGF were measured by enzyme-linked immunosorbent assay (ELISA) and Western blot. Results: The levels of MMP-8 and IL-6 in preterm group were significantly higher than control group (5.76 1.53 ng/ml vs 4.89 1.77 ng/ml and 170.54 55.69 pg/ml vs 141.92 57.21 pg/ml, respectively) (p < 0.05). In terms of VEGF, the levels were elevated in preterm group (30.76 4.06 pg/ml vs 22.36 7.03 pg/ml) (p < 0.05). Conclusion: This study suggests that elevated levels of IL-6 and MMP-8 in amniotic fluid at mid-trimester are predictive of preterm delivery, and that VEGF which is representative of angiogenesis can be a new and useful predictor of preterm delivery. Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
Keywords: Preterm birth MMP-8 IL-6 VEGF Amniotic fluid
1. Introduction Preterm birth is an important obstetric problem that causes considerable costs to healthcare systems. Also preterm-birthassociated perinatal morbidities, which are responsible for almost half of all long-term neurological disabilities, and neonatal mortalities reap a huge financial and emotional burden for the affected families. Preterm birth is defined as delivery that occurs before 37th weeks of gestation; it accounts for 10% of all pregnancies [1]. Over the last 25 years, there has been an increase in intensive research in this area because of a rising number of medically-indicated preterm q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. qq This study was supported by a grant from Kosin University College of Medicine (2012). * Corresponding author. Tel.: þ82 51 990 6226; fax: þ82 51 990 3300. ** Corresponding author. Tel.: þ82 2 590 2749; fax: þ82 2 595 1549. E-mail addresses: [email protected] (J.C. Shin), [email protected] (H.Y. Kim). 1 The last two authors equally contributed to this work for correspondence.
births in singletons, and because of increased instances of preterm delivery of multiple pregnancies conceived with assisted reproductive technology (ART) [2]. Preterm delivery is responsible for a significant percentage of neonatal morbidity and mortality. The etiology of preterm birth is unknown and is likely associated with multifactorial causes, including infection stress and demographic factors. Therefore, efforts have been made to identify predictors of preterm birth, especially since some of the causes of preterm delivery can be treated [1]. Biomarkers have been defined as ‘’parameters, which can be measured in a biological sample, and which provide information on potential effects of that exposure” [3]. Based on the known risk factors and pathways of preterm birth, several biomarkers have been tested to see if they can predict spontaneous preterm birth. Among those risk factors evaluated to date, fetal fibronectin in cervicovaginal fluid and ultrasound assessment of cervical length have been most strongly and consistently associated with subsequent spontaneous preterm birth. The test is most accurate in predicting spontaneous preterm birth within 7e10 days of a woman presenting with symptoms [4].
0143-4004/$ e see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.placenta.2013.06.306
874
A. Kim et al. / Placenta 34 (2013) 873e878
An inflammatory response and microbial invasion of the amniotic cavity are generally regarded as a pathologic state associated with preterm labor [2]. The molecular mechanisms by which intrauterine inflammation causes preterm labor are not well understood, although a number of cytokines, chemokines, and inflammatory mediators have been implicated. And inflammatory cells have been known to secret a plethora of proangiogenic factors including VEGF [5]. Until now, no other biomarkers have been consistently found to be useful in clinical settings to predict spontaneous preterm birth in asymptomatic women, especially at mid-trimester [6]. In this reason, the study aimed to identify and determine the accuracy of matrix metalloproteinases-8 (MMP-8), interleukin-6 (IL-6), and C-reactive protein (CRP), for inflammatory markers and vascular endothelial growth factor (VEGF) for an angiogenic marker in mid-trimester amniotic fluid for the prediction of spontaneous preterm birth in women with singleton pregnancies. 2. Materials and methods 2.1. Study design The research was designed to be a prospective study. Collection of amniotic fluid samples from January 2009 to June 2012 and clinical data were approved by the Institutional Review Board of Kosin Medical Center. All patients gave written informed consent, in accordance with the Helsinki criteria. After excluding fetal aneuploidies, anomalies, and cases who experienced pregnancy loss within 30 days of amniocentesis, we enrolled and stored the samples of amniotic fluid for later analysis. Post-delivery patient obstetric data were reviewed, and the clinical outcomes were obtained. Gestational age was determined based on the last menstrual period and the first trimester obstetric ultrasound evaluation (crown rump length at 7e9 weeks). Preterm delivery was defined as birth before 37 weeks of gestation. 2.2. Patients A total of 526 pregnant women with singleton gestations were carried out amniocentesis and their samples of amniotic fluid were stored until delivery. Amniocentesis was performed for proper clinical indications (advanced maternal age, abnormal quad/triple test, family history of chromosomal abnormalities, suspected fetal anomalies or viral infection, and maternal request) at 16e19 weeks of gestation. Among 526 women with available samples of amniotic fluid, the study included 72 women for study objects. Patients were invited to donate amniotic fluid for research purposes. The clinical outcome was obtained by chart review. Inclusion criteria were uneventful pregnancy course before the procedure, absence of congenital fetal malformations, absence of clinical signs of infection, normal volume of amniotic fluid as assessed by ultrasound, and healthy pregnant woman without chronic or medical disease. Any preterm delivery associated with an obstetrical complication, such as hypertensive disorders in pregnancy, obstetrical hemorrhage, fetal growth restriction, or premature rupture of the membrane, was excluded from the amniotic fluid analysis. We retrieved samples from every case known to have resulted in delivery before 37 weeks of gestation (n ¼ 36) and 36 control samples from women who delivered at 37 weeks of gestation. The control samples were matched with the preterm group at a 1:1 ratio from sampling until testing (storage time). Matches were based on maternal age, gestational age (weeks) at the time of amniocentesis, and the indication for the procedure. 2.3. Collection of amniotic fluid and storage Transabdominal amniocentesis was performed with a 21-gauge needle under ultrasound guidance to evaluate the position of the fetus. Amniotic fluid was first taken for further diagnostic testing, depending on the indication of the invasive procedure. Afterward, 5 ml from a total volume of 20 ml of amniotic fluid was collected for research purposes. Samples were transported immediately to the laboratory in a capped sterile syringe; amniotic fluid samples were then centrifuged for 10 min at 400rpm and stored in aliquots at 70 C until analysis at the completion of follow-up. 2.4. Enzyme-linked immunosorbent assay (ELISA) Invitrogen assay kits (Carlsbad, CA, USA) were used for measuring matrix MMP8, IL-6, CRP, and VEGF. These kits are based on the solid phase sandwich enzymelinked immunosorbent assay (ELISA) method. During the first incubation, samples were pipetted into wells coated with antibodies specific for human MMP-8, IL-6, CRP, and VEGF, followed by the addition of a biotinylated second antibody. During
the first incubation, the human antigen bound simultaneously to the immobilized (capture) antibody on one site and to the solution phase-biotinylated antibody on a second site. After washing, streptavidin-peroxidase (enzyme) was added, which binds to the biotinylated antibody to complete the four-member sandwich. After a third incubation and wash to remove all the unbound enzyme, a substrate solution was added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentrations of MMP-8, IL-6, CRP, and VEGF present in the specimen. The coefficients of variation (CV) of intra-assay and inter-assay precision were 8.5e10.2% for MMP-8, 5.0e5.6% for IL-6, 6.0e9.9% for CRP, and 5.1e9.8% for VEGF, respectively. The minimum detectable doses of MMP-8, IL-6, CRP, and VEGF were < 6 pg/ml, 2 pg/ml, 10 pg/ml, and 5 pg/ml, respectively. 2.5. Western blot A total of 1e2 mL of amniotic fluid was prepared by dilution with sodium dodecyl sulfate (SDS) loading buffer (Fermentas, Waltham, MA, USA), followed by boiling and cooling. The AF samples underwent electrophoresis in a 13.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) (Koma Biotech, Seoul, Korea). Thereafter, proteins were electrotransferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) at 30 V for 1 h. Nonspecific binding was blocked for 1 h in noise-cancelling reagents (Millipore). After washing, membranes were incubated for 2 h at room temperature with antibodies. The antibodies used (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were goat anti-human MMP-8 antibody (M-20), mouse anti-human IL-6 antibody (E-4), rabbit anti-human CRP antibody (c-12), and rabbit anti-human VEGF antibody (147). A BCIP/NBT tablet (SigmaeAldrich, St Louis, Missouri) dissolved in distilled water was used as growth substrate. Chemiluminescence analysis was conducted with Luminata Crescendo Western HRP substrate (Millipore) and autoradiography film (Agfa-Gevaert, Mortsel, Belgium), according to the manufacturer’s instructions. The experiment was replicated three times. Bands produced from the Western blot were showed using Gel DocÔ XRþ with Image Lab software (Bio-Rad, Hercules, CA, USA). 2.6. Statistical analyses Results are expressed as mean and standard deviation according to the distribution of data. KolmogoroveSmirnov’s test was used to evaluate the normality of the distribution of the continuous data. Comparisons between two groups were conducted using Student’s t-test in a normal distribution. Univariate analyses were performed using X2 test and Fisher’s exact test to evaluate the association of preterm birth with factors categorized. The receiver operating characteristic (ROC) curve was applied to calculate each factor’s predictive value for preterm delivery. SPSS version 19.0 (SPSS Inc., Chicago, IL, USA) was used for statistical calculations. P < 0.05 was considered statistically significant.
3. Results Thirty-six patients delivered at <37 weeks gestation; all spontaneous preterm labors included an intact membrane. The included cases were classified into the preterm group. The matched control group included 36 healthy pregnancies with full-term delivery. Table 1 shows the clinical and demographic characteristics of the study samples. There were no significant differences between the preterm delivery group and controls with respect to the mother’s
Table 1 Clinical and demographic characterizations of the subjects. Clinical characteristics
Preterm delivery (n ¼ 36)
Maternal age (years) BMI (kg/m2) Gravidity Gestational age at sampling (weeks) Sex (male/female) Indication of amniocentesis Advanced maternal age Abnormal quad/triple test History of fetal anomaly
34.8 21.7 1.08 18.3
4.3 2.5 0.33 0.2
Term delivery (n ¼ 36) 33.2 21.2 0.93 18.0
4.3 0.3 0.46 0.2
p-value 0.855a 0.862a 0.156a 0.673a
19/17
16/20
0.638b
2 29 5
2 30 4
0.938c
Results are expressed as mean SD. BMI, body mass index. a Student’s t-test. b X2 test. c Fisher’s exact test.
A. Kim et al. / Placenta 34 (2013) 873e878
age, the gestational age of the fetus at amniocentesis, body mass index, neonatal sex, indication of amniocentesis, or gravidity. In comparing biomarkers between the preterm and full-term groups, we found the following mean values: MMP-8 levels were 5.76 ng/ml and 4.89 ng/ml, respectively, and IL-6 levels were 170.54 pg/ml and 141.92 pg/ml, respectively. Both concentrations were significantly different between groups. On the other hand, CRP levels were not significantly different between the preterm and full-term groups. Regarding VEGF as an angiogenic factor, the mean values for the preterm and full-term groups were 30.76 pg/ml and 22.36 pg/ml, respectively; this represents a significant difference between the groups (Table 2, Fig. 1). The above values were analyzed for their ability to predict preterm birth using an ROC curve (Table 3, Fig. 2). ROC analysis was also used to determine the optimal amniotic fluid levels for MMP-8, IL-6, and VEGF that would predict preterm delivery. An appropriate cut-off level for MMP-8 in amniotic fluid was 5.14 ng/ml, with a sensitivity of 69.4% and a specificity of 68.3%. For IL-6 in amniotic fluid, the cut-off value was 134.35 pg/ml, with a sensitivity of 77.8% and a specificity of 61.1%. Amniotic VEGF levels had the highest area under the curve with 0.829. Its predictive cut-off value was 24.995 pg/ml, with a sensitivity of 91.7% and a specificity of 75.0%. Fig. 3 shows the representative Western blot results for levels of mid-trimester amniotic fluid for preterm (Pt2*, Pt3*, Pt5*, and Pt7*) and full-term (Pt1, Pt4, Pt 6, and Pt8) deliveries of IL-6 (25 kDa), MMP-8 (85 and 75 kDa, respectively), VEGF (55 and 40 kDa, respectively), and CRP (120 kDa). The samples were randomly selected among the subjectsdfour samples from the preterm delivery group and four samples from the full-term delivery group. Amniotic fluid from the preterm delivery group showed thicker and darker bands of IL-6, MMP-8, and VEGF, but not of CRP.
4. Discussion This study is the first to report on a significant difference in amniotic fluid concentrations of MMP-8, IL-6, and VEGF during the second trimester in women who underwent preterm delivery without any other obstetric complications. No significant difference in CRP levels was found between women who had preterm or fullterm deliveries. Several studies of predictive factors for preterm delivery point to multiple inflammatory biomarkers in mid-trimester amniotic fluid [3,7e18]. Substantial epidemiological, clinical, microbiological, and placental pathology studies support a link between inflammatory states and preterm birth [19]. In this study, we also found support for a proinflammatory response in amniotic fluid that influences preterm labor. To the best of our knowledge, this is the first report showing that VEGF levels in amniotic fluid during the second trimester are predictive of preterm delivery. Elevated concentrations of CRP in peripheral circulation have been associated with the presence of intrauterine infection. Some investigators have noted an elevated amniotic CRP concentration among women with intrauterine infections compared with study
Table 2 Concentrations of MMP-8, IL-6, CRP, and VEGF measured by ELISA in mid-trimester amniotic fluid of preterm and term groups. Preterm delivery (n ¼ 36) MMP-8 (ng/ml) IL-6 (pg/ml) CRP (ng/ml) VEGF (pg/ml)
5.76 170.54 108.431 30.76
1.53 55.69 16.82 4.06
Results are expressed as mean SD. a Student’s t-test.
Term delivery (n ¼ 36) 4.89 141.92 102.04 22.36
1.77 57.21 11.77 7.03
p-Valuea 0.028 0.035 0.066 <0.001
875
controls [8,20]. Other reports, however, have concluded that, though it is a commonly used laboratory parameter, measurement of CRP is nonspecific and therefore unreliable [7]. More specific biomarkers found in mid-trimester amniotic fluiddMMP8 and IL-6dare well established from a meta-analysis [3]. During the course of an inflammatory process, IL-6 and MMP-8 accumulate in body fluids (e.g. crevicular, synovial, and amniotic fluids). We therefore postulate that increased concentrations of these substances in amniotic fluid can be seen as an index of the presence of intra-amniotic inflammation [15,21]. Although this process is often present for weeks before preterm delivery, it is still a reliable marker because inflammation should be considered chronic in nature. Matrix metalloproteinase-8 (MMP-8), a human neutrophil collagenase, is released from the secondary granules of polymorphonuclear cells in response to chemotactic stimulation during inflammation. Inflammation of amniotic fluid is associated with a dramatic increase in MMP-8 in patients with intact or ruptured membranes [21]. Because as many as 42% of early preterm deliveries (<32 weeks) can be associated with an elevated MMP-8 level, intra-amniotic inflammation must be considered both frequent and important [14] The cytokine IL-6 is probably the most investigated and currently the most established biomarker in amniotic fluid with regard to intrauterine infection or sterile inflammatory immune activation [22]. Several studies have shown that levels of an amniotic IL-6 correlate well with chorioamnion colonization [15]. Amniotic IL-6 levels are thus an excellent marker of intrauterine inflammation that can be evaluated prenatally. IL-6 has also been demonstrated to be a very sensitive marker of tocolytic resistance and impending preterm delivery [23]. Nevertheless, there are no values of MMP-8 and IL-6 for which there is a consensus regarding a diagnostic level of intrauterine inflammation associated with preterm birth. Sadowsky et al. demonstrated that infusion of IL-6 into the amniotic fluid in pregnant monkeys did not induce preterm birth [24]. This suggests that preterm labor may be induced not by a single inflammatory or immune substance, but by interactions between multiple substances or pathways. In contrast to acute inflammation, chronic inflammation causes substantial tissue damage, which may lead to procarcinogenic conditions [5]. In this situation, pathological angiogenesis can be promoted through a continuous recruitment of substances, thereby exacerbating inflammation and damage [25]. Furthermore, under hypoxic conditions, recruited inflammatory cells, such as neutrophils, eosinophils, mast cells, NK cells, and macrophages, secrete proangiogenic factors, including VEGF, tumor necrosis factor-alpha, and other cytokines, increasing vascular permeability and facilitating additional recruitment of immune cells [26]. The precise molecular mechanisms by which reproductive tract infections/inflammation induce vascular changes that promote leukocyte trafficking and disruption of vascular integrity that is associated with abruption have yet to be fully elucidated [19] VEGF belongs to a family of heparin-binding growth factors that play an important role in vasculogenesis, angiogenesis, and vascular permeability throughout the body. Although VEGF alone does not activate the endothelium to induce cell-adhesion molecule expression, it sensitizes endothelial cells to proinflammatory cytokines that upregulate E-selectin, a key mediator of leukocyte trafficking [27]. VEGF, which is abundantly expressed by decidual cells, induces vascular fenestrations and a prothrombotic surface on endometrial endothelial cells [28]. Taken together, these data suggest that VEGF signaling at the maternalefetal interface is a critical mediator of inflammation that can be linked to preterm labor. Brou et al. resulted that VEGF concentrations in fetal plasma are significantly higher in cases of preterm birth, and suggested the possible role of VEGF for preterm labor pathway [29].
876
A. Kim et al. / Placenta 34 (2013) 873e878
Fig. 1. Concentrations of MMP-8, IL-6, CRP, and VEGF as measured by ELISA in mid-trimester amniotic fluid in preterm and full-term delivery groups.
This study provides an early demonstration of a possible preterm labor-instigating association or interaction between the inflammatory mediators, MMP-8 and IL-6, and the angiogenic biomarker, VEGF, in amniotic fluid. The concentrations measured through ELISA were confirmed by adjunctive Western blotting, which is a widely accepted analytical technique used to detect specific proteins in this type of tissue homogenate or extract (Fig. 3). VEGF expression in infected placental tissues appears to be localized primarily to decidual stromal cells [19]. Amniotic fluid inflammation is related to chorioamnionitis, and in this condition, decidual cells at the maternalefetal interface can upregulate VEGF production or an associated pathway to alter vascular permeability. As a result, inflammation of amniotic fluid can Table 3 Receiver operating characteristic analysis with the concentrations of MMP-8, IL-6, CRP, and VEGF measured by ELISA in mid-trimester amniotic fluid. Preterm delivery (n ¼ 36) vs. term delivery (n ¼ 36)
MMP-8 (ng/ml) IL-6 (pg/ml) CRP (ng/ml) VEGF (pg/ml)
Area under curve
95% CI
P-value
Cut-off value
0.637 0.650 0.608 0.829
0.509e0.766 0.517e0.782 0.476e0.740 0.722e0.935
0.045 0.029 0.115 <0.001
5.14 134.35 24.995
Fig. 2. Receiver operating characteristic (ROC) curves that compare the values of MMP8, IL-6, CRP, and VEGF for preterm delivery.
A. Kim et al. / Placenta 34 (2013) 873e878
Fig. 3. Representative Western blot for preterm (Pt2*, Pt3*, Pt5*, and Pt7*) and fullterm (Pt1, Pt4, Pt6, and Pt8) groups showing levels of IL-6 (25 kDa), MMP-8 (85 and 75 kDa, respectively), VEGF (55 and 40 kDa, respectively), and CRP (120 kDa) in midtrimester amniotic fluid.
promote extravasation into the decidua of immune cells, leading to ‘‘decidual activation,’’ all of which promote preterm birth. The lack of blinding of patient test results in previous studies is particularly relevant because women with abnormal results might have received more intensive follow-up or therapeutic care than women with normal results, which would have biased an evaluation of the test’s predictive accuracy. One strength of this study is the fact that we did not know or compare the levels of target molecules in the amniotic fluid until the termination of pregnancy. Another strength is the homogeneity of the participant population, since it is a complex phenotype that is related to multiple mechanisms, including infection/inflammation, uteroplacental ischemia or hemorrhage, uterine overdistension, stress, and other immunologically mediated processes [30]. We homogenized the preterm delivery group by including only patients with spontaneous preterm delivery <37 weeks of gestation in singleton pregnancy. A reliable predictor of spontaneous preterm birth is important because it could allow for the identification of women at high risk of preterm birth, in whom a specific intervention could be tested. Being able to predict which women are likely to have a preterm birth is a prerequisite for the effective use of most interventions aimed at preventing preterm birth. Also, studies that identify predictors of spontaneous preterm birth may improve our understanding of the mechanisms of biological and disease pathways that lead to it and possibly lead to better interventions. Furthermore, they can give the clue of predictive markers with noninvasive methods, unlike amniocentesis. Finally, another reason for predicting preterm birth is that being able to identify women with low risk would avoid unnecessary and costly interventions [3]. This strategy may prevent both the initiation of preterm labor and the fetal complications associated with a pathological process that is silent at mid-trimester but leads to preterm delivery. Patients identified through these methods can be followed and monitored with technologies, such as cervical sonography, and treated with interventions to reduce the rate of preterm delivery. We evaluated the cut-off values for useful biomarkers to predict preterm birth in mid-trimester amniotic fluid. The values were 5.14 ng/ml, 134.35 pg/ml, and 24.995 pg/ml for MMP-8, IL-6, and VEGF, respectively. These levels cannot yet be applied in clinical work; further studies are needed to replicate and corroborate these results. It seems to have variability in biomarker levels over time or between individuals, making it difficult to determine the threshold level for risk of spontaneous preterm birth. Indeed, the combined use of markers has been shown to have better predictive accuracy than
877
individual markers alone because there are several heterogeneous pathways that lead to spontaneous preterm birth, including inflammatory and angiogenic responses. Further prospective, large-scale, longitudinal studies are needed to determine the novel biomarkers that have more predictive accuracy, alone or combination. We excluded the women with complications, which can cause preterm labor, such as pre-eclampsia, obstetrical hemorrhage, or premature preterm rupture of membrane. In general population, many maternal or fetal conditions can lead to preterm birth. The analysis of MMP-8, IL-6, and VEGF concentrations in amniotic fluid of women with those conditions may give us more information for preterm labor with or without obstetric complications. It must be preceded as further study for clinical application to predict preterm birth in general pregnant women. In conclusion, the diagnostic value of CRP in mid-trimester amniotic fluid for spontaneous preterm birth is questionable. However, despite still unrevealed mechanisms, elevated inflammatory mediators, MMP-8 and IL-6, are associated with preterm birth among women with an intact membrane. Elevated concentration of VEGF as angiogenic parameter is also related to preterm birth. This analogizes the interaction between inflammation and angiogenic pathways in amniotic fluid and the maternalefetal interface to promote spontaneous preterm labor. Conflicts of interest The authors declare that there are no conflicts of interest. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.placenta.2013.06.306. References [1] Krupa FG, Faltin D, Cecatti JG, Surita FG, Souza JP. Predictors of preterm birth. Int J Gynaecol Obstet 2006;94(1):5e11. [2] Goldenberg RL, Culhane JF, Iams JD, Romero R. Epidemiology and causes of preterm birth. Lancet 2008;371(9606):75e84. [3] Conde Agudelo A, Papageorghiou AT, Kennedy SH, Villar J. Novel biomarkers for the prediction of the spontaneous preterm birth phenotype: a systematic review and meta-analysis. BJOG 2011;118(9):1042e54. [4] Defranco EA, Lewis DF, Odibo AO. Improving the screening accuracy for preterm labor: is the combination of fetal fibronectin and cervical length in symptomatic patients a useful predictor of preterm birth? A systematic review. Am J Obstet Gynecol 2013;208(3):233.e1e6. [5] Kim Y, West XZ, Byzova TV. Inflammation and oxidative stress in angiogenesis and vascular disease. J Mol Med 2013;91(3):323e8. [6] Gervasi M, Romero R, Bracalente G, Erez O, Dong Z, Hassan S, et al. Midtrimester amniotic fluid concentrations of interleukin-6 and interferongamma-inducible protein-10: evidence for heterogeneity of intra-amniotic inflammation and associations with spontaneous early (<32 weeks) and late (>32 weeks) preterm delivery. J Perinat Med 2012;40(4):329e43. [7] Borna S, Abdollahi A, Mirzaei F. Predictive value of mid-trimester amniotic fluid high-sensitive C-reactive protein, ferritin, and lactate dehydrogenase for fetal growth restriction. Indian J Pathol Microbiol 2009;52(4):498e500. [8] Ghezzi F, Franchi M, Raio L, Di Naro E, Bossi G, D’Eril GV, et al. Elevated amniotic fluid C-reactive protein at the time of genetic amniocentesis is a marker for preterm delivery. Am J Obstet Gynecol 2002;186(2):268e73. [9] Spong CY, Sherer DM, Ghidini A, Jenkins CB, Seydel FD, Eglinton GS. Secondtrimester amniotic fluid or maternal serum interleukin-10 levels and small for gestational age neonates. Obstet Gynecol 1996;88(1):24e8. [10] Spong CY, Scherer DM, Ghidini A, Pezzullo JC, Salafia CM, Eglinton GS. Midtrimester amniotic fluid tumor necrosis factor-alpha does not predict smallfor-gestational-age infants. Am J Reprod Immunol 1997;37(3):236e9. [11] Ghidini A, Jenkins CB, Spong CY, Pezzullo JC, Salafia CM, Eglinton GS. Elevated amniotic fluid interleukin-6 levels during the early second trimester are associated with greater risk of subsequent preterm delivery. Am J Reprod Immunol 1997;37(3):227e31. [12] Figueroa R, Garry D, Elimian A, Patel K, Sehgal PB, Tejani N. Evaluation of amniotic fluid cytokines in preterm labor and intact membranes. J Matern Fetal Neonatal Med 2005;18(4):241e7.
878
A. Kim et al. / Placenta 34 (2013) 873e878
[13] Apuzzio J, Chan Y, Al Khan A, Illsley N, Kim P, Vonhaggen S. Second-trimester amniotic fluid interleukin-10 concentration predicts preterm delivery. J Matern Fetal Neonatal Med 2004;15(5):313e7. [14] Yoon BH, Oh SY, Romero R, Shim SS, Han SY, Park JS, et al. An elevated amniotic fluid matrix metalloproteinase-8 level at the time of mid-trimester genetic amniocentesis is a risk factor for spontaneous preterm delivery. Am J Obstet Gynecol 2001;185(5):1162e7. [15] Wenstrom KD, Andrews WW, Hauth JC, Goldenberg RL, DuBard MB, Cliver SP. Elevated second-trimester amniotic fluid interleukin-6 levels predict preterm delivery. Am J Obstet Gynecol 1998;178(3):546e50. [16] Pressman EK, Thornburg L, Glantz JC, Earhart A, Wall PD, Ashraf M, et al. Inflammatory cytokines and antioxidants in midtrimester amniotic fluid: correlation with pregnancy outcome. Am J Obstet Gynecol 2011;204(2):155.e1e7. [17] Bamberg C, Fotopoulou C, Thiem D, Roehr C, Dudenhausen JW, Kalache KD. Correlation of midtrimester amniotic fluid cytokine concentrations with adverse pregnancy outcome in terms of spontaneous abortion, preterm birth, and preeclampsia. J Matern Fetal Neonatal Med 2012;25(6):812e7. [18] Bamberg C, Fotopoulou C, Linder M, Roehr CC, Dudenhausen JW, Henrich W, et al. Mid-trimester amniotic fluid concentrations of the proinflammatory cytokines IL-6, IL-8, TNF-Î, and lipopolysaccharide binding protein in normal pregnancies: a prospective evaluation according to parity, gestational age, and fetal gender. J Perinat Med 2011;39(4):403e9. [19] Snegovskikh V, Schatz F, Arcuri F, Toti P, Kayisli UA, Murk W, et al. Intraamniotic infection upregulates decidual cell vascular endothelial growth factor (VEGF) and neuropilin-1 and -2 expression: implications for infectionrelated preterm birth. Reprod Sci 2009;16(8):767e80. [20] Ozer KT, Kavak ZN, GÃkaslan H, Elter K, Pekin T. Predictive power of maternal serum and amniotic fluid CRP and PAPP-A concentrations at the time of genetic amniocentesis for the preterm delivery. Eur J Obstet Gynecol Reprod Biol 2005;122(2):187e90.
[21] Maymon E, Romero R, Pacora P, Gomez R, Athayde N, Edwin S, et al. Human neutrophil collagenase (matrix metalloproteinase 8) in parturition, premature rupture of the membranes, and intrauterine infection. Am J Obstet Gynecol 2000;183(1):94e9. [22] Weissenbacher T, Laubender RP, Witkin S, Gingelmaier A, Schiessl B, Kainer F, et al. Diagnostic biomarkers of pro-inflammatory immune-mediated preterm birth. Arch Gynecol Obstet 2013;287(4):673e85. [23] Hillier SL, Witkin SS, Krohn MA, Watts DH, Kiviat NB, Eschenbach DA. The relationship of amniotic fluid cytokines and preterm delivery, amniotic fluid infection, histologic chorioamnionitis, and chorioamnion infection. Obstet Gynecol 1993;81(6):941e8. [24] Sadowsky DW, Adams KM, Gravett MG, Witkin S, Novy MJ. Preterm labor is induced by intraamniotic infusions of interleukin-1beta and tumor necrosis factor-alpha but not by interleukin-6 or interleukin-8 in a nonhuman primate model. Am J Obstet Gynecol 2006;195(6):1578e89. [25] Costa C, Incio J, Soares R. Angiogenesis and chronic inflammation: cause or consequence? Angiogenesis 2007;10:149e66. [26] Kundu JK, Surh Y. Emerging avenues linking inflammation and cancer. Free Radic Biol Med 2012;52(3):2013e37. [27] Stannard AK, Khurana R, Evans IM, Sofra V, Holmes DI, Zachary I. Vascular endothelial growth factor synergistically enhances induction of E-selectin by tumor necrosis factor-alpha. Arterioscler Thromb Vasc Biol 2007;27(3):494e502. [28] Krikun G, Huang SJ, Schatz F, Salafia C, Stocco C, Lockwood CJ. Thrombin activation of endometrial endothelial cells: a possible role in intrauterine growth restriction. Thromb Haemost 2007;97(2):245e53. [29] Brou L, Almli LM, Pearce BD, Bhat G, Drobek CO, Fortunato S, et al. Dysregulated biomarkers induce distinct pathways in preterm birth. BJOG 2012;119(4):458e73. [30] Romero R, Espinoza J, Kusanovic JP, Gotsch F, Hassan S, Erez O, et al. The preterm parturition syndrome. BJOG 2006;113(Suppl. 3):17e42.
Contents lists available at SciVerse ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Identification of biomarkers for preterm delivery in mid-trimester amniotic fluidq,qq A. Kim a, E.S. Lee b, J.C. Shin c, **,1, H.Y. Kim d, *,1 a
Department of Obstetrics and Gynecology, Institute of Wonkwang Medical Science, College of Medicine, Wonkwang University, Iksan, Republic of Korea Department of Obstetrics and Gynecology, College of Medicine, SoonChunHyang University, Seoul, Republic of Korea c Department of Obstetrics and Gynecology, College of Medicine, Seoul, Republic of Korea d Department of Obstetrics and Gynecology, College of Medicine, Kosin University, Busan, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 24 June 2013
Objective: We investigated whether the level of vascular endothelial growth factor (VEGF) and inflammatory markers in mid-trimester amniotic fluid have predictive value for spontaneous preterm birth in singleton pregnancy. Method: Our subjects were 72 pregnant women who were undertaken with amniocentesis from 16 to 19 weeks of gestation. 36 cases were women with preterm delivery, and other 36 cases were matched women with full-term delivery. Stored amniotic fluid was investigated after the delivery. The levels of matrix metalloproteinases-8 (MMP-8), interleukin-6 (IL-6), C-reactive protein (CRP), and VEGF were measured by enzyme-linked immunosorbent assay (ELISA) and Western blot. Results: The levels of MMP-8 and IL-6 in preterm group were significantly higher than control group (5.76 1.53 ng/ml vs 4.89 1.77 ng/ml and 170.54 55.69 pg/ml vs 141.92 57.21 pg/ml, respectively) (p < 0.05). In terms of VEGF, the levels were elevated in preterm group (30.76 4.06 pg/ml vs 22.36 7.03 pg/ml) (p < 0.05). Conclusion: This study suggests that elevated levels of IL-6 and MMP-8 in amniotic fluid at mid-trimester are predictive of preterm delivery, and that VEGF which is representative of angiogenesis can be a new and useful predictor of preterm delivery. Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
Keywords: Preterm birth MMP-8 IL-6 VEGF Amniotic fluid
1. Introduction Preterm birth is an important obstetric problem that causes considerable costs to healthcare systems. Also preterm-birthassociated perinatal morbidities, which are responsible for almost half of all long-term neurological disabilities, and neonatal mortalities reap a huge financial and emotional burden for the affected families. Preterm birth is defined as delivery that occurs before 37th weeks of gestation; it accounts for 10% of all pregnancies [1]. Over the last 25 years, there has been an increase in intensive research in this area because of a rising number of medically-indicated preterm q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. qq This study was supported by a grant from Kosin University College of Medicine (2012). * Corresponding author. Tel.: þ82 51 990 6226; fax: þ82 51 990 3300. ** Corresponding author. Tel.: þ82 2 590 2749; fax: þ82 2 595 1549. E-mail addresses: [email protected] (J.C. Shin), [email protected] (H.Y. Kim). 1 The last two authors equally contributed to this work for correspondence.
births in singletons, and because of increased instances of preterm delivery of multiple pregnancies conceived with assisted reproductive technology (ART) [2]. Preterm delivery is responsible for a significant percentage of neonatal morbidity and mortality. The etiology of preterm birth is unknown and is likely associated with multifactorial causes, including infection stress and demographic factors. Therefore, efforts have been made to identify predictors of preterm birth, especially since some of the causes of preterm delivery can be treated [1]. Biomarkers have been defined as ‘’parameters, which can be measured in a biological sample, and which provide information on potential effects of that exposure” [3]. Based on the known risk factors and pathways of preterm birth, several biomarkers have been tested to see if they can predict spontaneous preterm birth. Among those risk factors evaluated to date, fetal fibronectin in cervicovaginal fluid and ultrasound assessment of cervical length have been most strongly and consistently associated with subsequent spontaneous preterm birth. The test is most accurate in predicting spontaneous preterm birth within 7e10 days of a woman presenting with symptoms [4].
0143-4004/$ e see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.placenta.2013.06.306
874
A. Kim et al. / Placenta 34 (2013) 873e878
An inflammatory response and microbial invasion of the amniotic cavity are generally regarded as a pathologic state associated with preterm labor [2]. The molecular mechanisms by which intrauterine inflammation causes preterm labor are not well understood, although a number of cytokines, chemokines, and inflammatory mediators have been implicated. And inflammatory cells have been known to secret a plethora of proangiogenic factors including VEGF [5]. Until now, no other biomarkers have been consistently found to be useful in clinical settings to predict spontaneous preterm birth in asymptomatic women, especially at mid-trimester [6]. In this reason, the study aimed to identify and determine the accuracy of matrix metalloproteinases-8 (MMP-8), interleukin-6 (IL-6), and C-reactive protein (CRP), for inflammatory markers and vascular endothelial growth factor (VEGF) for an angiogenic marker in mid-trimester amniotic fluid for the prediction of spontaneous preterm birth in women with singleton pregnancies. 2. Materials and methods 2.1. Study design The research was designed to be a prospective study. Collection of amniotic fluid samples from January 2009 to June 2012 and clinical data were approved by the Institutional Review Board of Kosin Medical Center. All patients gave written informed consent, in accordance with the Helsinki criteria. After excluding fetal aneuploidies, anomalies, and cases who experienced pregnancy loss within 30 days of amniocentesis, we enrolled and stored the samples of amniotic fluid for later analysis. Post-delivery patient obstetric data were reviewed, and the clinical outcomes were obtained. Gestational age was determined based on the last menstrual period and the first trimester obstetric ultrasound evaluation (crown rump length at 7e9 weeks). Preterm delivery was defined as birth before 37 weeks of gestation. 2.2. Patients A total of 526 pregnant women with singleton gestations were carried out amniocentesis and their samples of amniotic fluid were stored until delivery. Amniocentesis was performed for proper clinical indications (advanced maternal age, abnormal quad/triple test, family history of chromosomal abnormalities, suspected fetal anomalies or viral infection, and maternal request) at 16e19 weeks of gestation. Among 526 women with available samples of amniotic fluid, the study included 72 women for study objects. Patients were invited to donate amniotic fluid for research purposes. The clinical outcome was obtained by chart review. Inclusion criteria were uneventful pregnancy course before the procedure, absence of congenital fetal malformations, absence of clinical signs of infection, normal volume of amniotic fluid as assessed by ultrasound, and healthy pregnant woman without chronic or medical disease. Any preterm delivery associated with an obstetrical complication, such as hypertensive disorders in pregnancy, obstetrical hemorrhage, fetal growth restriction, or premature rupture of the membrane, was excluded from the amniotic fluid analysis. We retrieved samples from every case known to have resulted in delivery before 37 weeks of gestation (n ¼ 36) and 36 control samples from women who delivered at 37 weeks of gestation. The control samples were matched with the preterm group at a 1:1 ratio from sampling until testing (storage time). Matches were based on maternal age, gestational age (weeks) at the time of amniocentesis, and the indication for the procedure. 2.3. Collection of amniotic fluid and storage Transabdominal amniocentesis was performed with a 21-gauge needle under ultrasound guidance to evaluate the position of the fetus. Amniotic fluid was first taken for further diagnostic testing, depending on the indication of the invasive procedure. Afterward, 5 ml from a total volume of 20 ml of amniotic fluid was collected for research purposes. Samples were transported immediately to the laboratory in a capped sterile syringe; amniotic fluid samples were then centrifuged for 10 min at 400rpm and stored in aliquots at 70 C until analysis at the completion of follow-up. 2.4. Enzyme-linked immunosorbent assay (ELISA) Invitrogen assay kits (Carlsbad, CA, USA) were used for measuring matrix MMP8, IL-6, CRP, and VEGF. These kits are based on the solid phase sandwich enzymelinked immunosorbent assay (ELISA) method. During the first incubation, samples were pipetted into wells coated with antibodies specific for human MMP-8, IL-6, CRP, and VEGF, followed by the addition of a biotinylated second antibody. During
the first incubation, the human antigen bound simultaneously to the immobilized (capture) antibody on one site and to the solution phase-biotinylated antibody on a second site. After washing, streptavidin-peroxidase (enzyme) was added, which binds to the biotinylated antibody to complete the four-member sandwich. After a third incubation and wash to remove all the unbound enzyme, a substrate solution was added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentrations of MMP-8, IL-6, CRP, and VEGF present in the specimen. The coefficients of variation (CV) of intra-assay and inter-assay precision were 8.5e10.2% for MMP-8, 5.0e5.6% for IL-6, 6.0e9.9% for CRP, and 5.1e9.8% for VEGF, respectively. The minimum detectable doses of MMP-8, IL-6, CRP, and VEGF were < 6 pg/ml, 2 pg/ml, 10 pg/ml, and 5 pg/ml, respectively. 2.5. Western blot A total of 1e2 mL of amniotic fluid was prepared by dilution with sodium dodecyl sulfate (SDS) loading buffer (Fermentas, Waltham, MA, USA), followed by boiling and cooling. The AF samples underwent electrophoresis in a 13.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) (Koma Biotech, Seoul, Korea). Thereafter, proteins were electrotransferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) at 30 V for 1 h. Nonspecific binding was blocked for 1 h in noise-cancelling reagents (Millipore). After washing, membranes were incubated for 2 h at room temperature with antibodies. The antibodies used (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were goat anti-human MMP-8 antibody (M-20), mouse anti-human IL-6 antibody (E-4), rabbit anti-human CRP antibody (c-12), and rabbit anti-human VEGF antibody (147). A BCIP/NBT tablet (SigmaeAldrich, St Louis, Missouri) dissolved in distilled water was used as growth substrate. Chemiluminescence analysis was conducted with Luminata Crescendo Western HRP substrate (Millipore) and autoradiography film (Agfa-Gevaert, Mortsel, Belgium), according to the manufacturer’s instructions. The experiment was replicated three times. Bands produced from the Western blot were showed using Gel DocÔ XRþ with Image Lab software (Bio-Rad, Hercules, CA, USA). 2.6. Statistical analyses Results are expressed as mean and standard deviation according to the distribution of data. KolmogoroveSmirnov’s test was used to evaluate the normality of the distribution of the continuous data. Comparisons between two groups were conducted using Student’s t-test in a normal distribution. Univariate analyses were performed using X2 test and Fisher’s exact test to evaluate the association of preterm birth with factors categorized. The receiver operating characteristic (ROC) curve was applied to calculate each factor’s predictive value for preterm delivery. SPSS version 19.0 (SPSS Inc., Chicago, IL, USA) was used for statistical calculations. P < 0.05 was considered statistically significant.
3. Results Thirty-six patients delivered at <37 weeks gestation; all spontaneous preterm labors included an intact membrane. The included cases were classified into the preterm group. The matched control group included 36 healthy pregnancies with full-term delivery. Table 1 shows the clinical and demographic characteristics of the study samples. There were no significant differences between the preterm delivery group and controls with respect to the mother’s
Table 1 Clinical and demographic characterizations of the subjects. Clinical characteristics
Preterm delivery (n ¼ 36)
Maternal age (years) BMI (kg/m2) Gravidity Gestational age at sampling (weeks) Sex (male/female) Indication of amniocentesis Advanced maternal age Abnormal quad/triple test History of fetal anomaly
34.8 21.7 1.08 18.3
4.3 2.5 0.33 0.2
Term delivery (n ¼ 36) 33.2 21.2 0.93 18.0
4.3 0.3 0.46 0.2
p-value 0.855a 0.862a 0.156a 0.673a
19/17
16/20
0.638b
2 29 5
2 30 4
0.938c
Results are expressed as mean SD. BMI, body mass index. a Student’s t-test. b X2 test. c Fisher’s exact test.
A. Kim et al. / Placenta 34 (2013) 873e878
age, the gestational age of the fetus at amniocentesis, body mass index, neonatal sex, indication of amniocentesis, or gravidity. In comparing biomarkers between the preterm and full-term groups, we found the following mean values: MMP-8 levels were 5.76 ng/ml and 4.89 ng/ml, respectively, and IL-6 levels were 170.54 pg/ml and 141.92 pg/ml, respectively. Both concentrations were significantly different between groups. On the other hand, CRP levels were not significantly different between the preterm and full-term groups. Regarding VEGF as an angiogenic factor, the mean values for the preterm and full-term groups were 30.76 pg/ml and 22.36 pg/ml, respectively; this represents a significant difference between the groups (Table 2, Fig. 1). The above values were analyzed for their ability to predict preterm birth using an ROC curve (Table 3, Fig. 2). ROC analysis was also used to determine the optimal amniotic fluid levels for MMP-8, IL-6, and VEGF that would predict preterm delivery. An appropriate cut-off level for MMP-8 in amniotic fluid was 5.14 ng/ml, with a sensitivity of 69.4% and a specificity of 68.3%. For IL-6 in amniotic fluid, the cut-off value was 134.35 pg/ml, with a sensitivity of 77.8% and a specificity of 61.1%. Amniotic VEGF levels had the highest area under the curve with 0.829. Its predictive cut-off value was 24.995 pg/ml, with a sensitivity of 91.7% and a specificity of 75.0%. Fig. 3 shows the representative Western blot results for levels of mid-trimester amniotic fluid for preterm (Pt2*, Pt3*, Pt5*, and Pt7*) and full-term (Pt1, Pt4, Pt 6, and Pt8) deliveries of IL-6 (25 kDa), MMP-8 (85 and 75 kDa, respectively), VEGF (55 and 40 kDa, respectively), and CRP (120 kDa). The samples were randomly selected among the subjectsdfour samples from the preterm delivery group and four samples from the full-term delivery group. Amniotic fluid from the preterm delivery group showed thicker and darker bands of IL-6, MMP-8, and VEGF, but not of CRP.
4. Discussion This study is the first to report on a significant difference in amniotic fluid concentrations of MMP-8, IL-6, and VEGF during the second trimester in women who underwent preterm delivery without any other obstetric complications. No significant difference in CRP levels was found between women who had preterm or fullterm deliveries. Several studies of predictive factors for preterm delivery point to multiple inflammatory biomarkers in mid-trimester amniotic fluid [3,7e18]. Substantial epidemiological, clinical, microbiological, and placental pathology studies support a link between inflammatory states and preterm birth [19]. In this study, we also found support for a proinflammatory response in amniotic fluid that influences preterm labor. To the best of our knowledge, this is the first report showing that VEGF levels in amniotic fluid during the second trimester are predictive of preterm delivery. Elevated concentrations of CRP in peripheral circulation have been associated with the presence of intrauterine infection. Some investigators have noted an elevated amniotic CRP concentration among women with intrauterine infections compared with study
Table 2 Concentrations of MMP-8, IL-6, CRP, and VEGF measured by ELISA in mid-trimester amniotic fluid of preterm and term groups. Preterm delivery (n ¼ 36) MMP-8 (ng/ml) IL-6 (pg/ml) CRP (ng/ml) VEGF (pg/ml)
5.76 170.54 108.431 30.76
1.53 55.69 16.82 4.06
Results are expressed as mean SD. a Student’s t-test.
Term delivery (n ¼ 36) 4.89 141.92 102.04 22.36
1.77 57.21 11.77 7.03
p-Valuea 0.028 0.035 0.066 <0.001
875
controls [8,20]. Other reports, however, have concluded that, though it is a commonly used laboratory parameter, measurement of CRP is nonspecific and therefore unreliable [7]. More specific biomarkers found in mid-trimester amniotic fluiddMMP8 and IL-6dare well established from a meta-analysis [3]. During the course of an inflammatory process, IL-6 and MMP-8 accumulate in body fluids (e.g. crevicular, synovial, and amniotic fluids). We therefore postulate that increased concentrations of these substances in amniotic fluid can be seen as an index of the presence of intra-amniotic inflammation [15,21]. Although this process is often present for weeks before preterm delivery, it is still a reliable marker because inflammation should be considered chronic in nature. Matrix metalloproteinase-8 (MMP-8), a human neutrophil collagenase, is released from the secondary granules of polymorphonuclear cells in response to chemotactic stimulation during inflammation. Inflammation of amniotic fluid is associated with a dramatic increase in MMP-8 in patients with intact or ruptured membranes [21]. Because as many as 42% of early preterm deliveries (<32 weeks) can be associated with an elevated MMP-8 level, intra-amniotic inflammation must be considered both frequent and important [14] The cytokine IL-6 is probably the most investigated and currently the most established biomarker in amniotic fluid with regard to intrauterine infection or sterile inflammatory immune activation [22]. Several studies have shown that levels of an amniotic IL-6 correlate well with chorioamnion colonization [15]. Amniotic IL-6 levels are thus an excellent marker of intrauterine inflammation that can be evaluated prenatally. IL-6 has also been demonstrated to be a very sensitive marker of tocolytic resistance and impending preterm delivery [23]. Nevertheless, there are no values of MMP-8 and IL-6 for which there is a consensus regarding a diagnostic level of intrauterine inflammation associated with preterm birth. Sadowsky et al. demonstrated that infusion of IL-6 into the amniotic fluid in pregnant monkeys did not induce preterm birth [24]. This suggests that preterm labor may be induced not by a single inflammatory or immune substance, but by interactions between multiple substances or pathways. In contrast to acute inflammation, chronic inflammation causes substantial tissue damage, which may lead to procarcinogenic conditions [5]. In this situation, pathological angiogenesis can be promoted through a continuous recruitment of substances, thereby exacerbating inflammation and damage [25]. Furthermore, under hypoxic conditions, recruited inflammatory cells, such as neutrophils, eosinophils, mast cells, NK cells, and macrophages, secrete proangiogenic factors, including VEGF, tumor necrosis factor-alpha, and other cytokines, increasing vascular permeability and facilitating additional recruitment of immune cells [26]. The precise molecular mechanisms by which reproductive tract infections/inflammation induce vascular changes that promote leukocyte trafficking and disruption of vascular integrity that is associated with abruption have yet to be fully elucidated [19] VEGF belongs to a family of heparin-binding growth factors that play an important role in vasculogenesis, angiogenesis, and vascular permeability throughout the body. Although VEGF alone does not activate the endothelium to induce cell-adhesion molecule expression, it sensitizes endothelial cells to proinflammatory cytokines that upregulate E-selectin, a key mediator of leukocyte trafficking [27]. VEGF, which is abundantly expressed by decidual cells, induces vascular fenestrations and a prothrombotic surface on endometrial endothelial cells [28]. Taken together, these data suggest that VEGF signaling at the maternalefetal interface is a critical mediator of inflammation that can be linked to preterm labor. Brou et al. resulted that VEGF concentrations in fetal plasma are significantly higher in cases of preterm birth, and suggested the possible role of VEGF for preterm labor pathway [29].
876
A. Kim et al. / Placenta 34 (2013) 873e878
Fig. 1. Concentrations of MMP-8, IL-6, CRP, and VEGF as measured by ELISA in mid-trimester amniotic fluid in preterm and full-term delivery groups.
This study provides an early demonstration of a possible preterm labor-instigating association or interaction between the inflammatory mediators, MMP-8 and IL-6, and the angiogenic biomarker, VEGF, in amniotic fluid. The concentrations measured through ELISA were confirmed by adjunctive Western blotting, which is a widely accepted analytical technique used to detect specific proteins in this type of tissue homogenate or extract (Fig. 3). VEGF expression in infected placental tissues appears to be localized primarily to decidual stromal cells [19]. Amniotic fluid inflammation is related to chorioamnionitis, and in this condition, decidual cells at the maternalefetal interface can upregulate VEGF production or an associated pathway to alter vascular permeability. As a result, inflammation of amniotic fluid can Table 3 Receiver operating characteristic analysis with the concentrations of MMP-8, IL-6, CRP, and VEGF measured by ELISA in mid-trimester amniotic fluid. Preterm delivery (n ¼ 36) vs. term delivery (n ¼ 36)
MMP-8 (ng/ml) IL-6 (pg/ml) CRP (ng/ml) VEGF (pg/ml)
Area under curve
95% CI
P-value
Cut-off value
0.637 0.650 0.608 0.829
0.509e0.766 0.517e0.782 0.476e0.740 0.722e0.935
0.045 0.029 0.115 <0.001
5.14 134.35 24.995
Fig. 2. Receiver operating characteristic (ROC) curves that compare the values of MMP8, IL-6, CRP, and VEGF for preterm delivery.
A. Kim et al. / Placenta 34 (2013) 873e878
Fig. 3. Representative Western blot for preterm (Pt2*, Pt3*, Pt5*, and Pt7*) and fullterm (Pt1, Pt4, Pt6, and Pt8) groups showing levels of IL-6 (25 kDa), MMP-8 (85 and 75 kDa, respectively), VEGF (55 and 40 kDa, respectively), and CRP (120 kDa) in midtrimester amniotic fluid.
promote extravasation into the decidua of immune cells, leading to ‘‘decidual activation,’’ all of which promote preterm birth. The lack of blinding of patient test results in previous studies is particularly relevant because women with abnormal results might have received more intensive follow-up or therapeutic care than women with normal results, which would have biased an evaluation of the test’s predictive accuracy. One strength of this study is the fact that we did not know or compare the levels of target molecules in the amniotic fluid until the termination of pregnancy. Another strength is the homogeneity of the participant population, since it is a complex phenotype that is related to multiple mechanisms, including infection/inflammation, uteroplacental ischemia or hemorrhage, uterine overdistension, stress, and other immunologically mediated processes [30]. We homogenized the preterm delivery group by including only patients with spontaneous preterm delivery <37 weeks of gestation in singleton pregnancy. A reliable predictor of spontaneous preterm birth is important because it could allow for the identification of women at high risk of preterm birth, in whom a specific intervention could be tested. Being able to predict which women are likely to have a preterm birth is a prerequisite for the effective use of most interventions aimed at preventing preterm birth. Also, studies that identify predictors of spontaneous preterm birth may improve our understanding of the mechanisms of biological and disease pathways that lead to it and possibly lead to better interventions. Furthermore, they can give the clue of predictive markers with noninvasive methods, unlike amniocentesis. Finally, another reason for predicting preterm birth is that being able to identify women with low risk would avoid unnecessary and costly interventions [3]. This strategy may prevent both the initiation of preterm labor and the fetal complications associated with a pathological process that is silent at mid-trimester but leads to preterm delivery. Patients identified through these methods can be followed and monitored with technologies, such as cervical sonography, and treated with interventions to reduce the rate of preterm delivery. We evaluated the cut-off values for useful biomarkers to predict preterm birth in mid-trimester amniotic fluid. The values were 5.14 ng/ml, 134.35 pg/ml, and 24.995 pg/ml for MMP-8, IL-6, and VEGF, respectively. These levels cannot yet be applied in clinical work; further studies are needed to replicate and corroborate these results. It seems to have variability in biomarker levels over time or between individuals, making it difficult to determine the threshold level for risk of spontaneous preterm birth. Indeed, the combined use of markers has been shown to have better predictive accuracy than
877
individual markers alone because there are several heterogeneous pathways that lead to spontaneous preterm birth, including inflammatory and angiogenic responses. Further prospective, large-scale, longitudinal studies are needed to determine the novel biomarkers that have more predictive accuracy, alone or combination. We excluded the women with complications, which can cause preterm labor, such as pre-eclampsia, obstetrical hemorrhage, or premature preterm rupture of membrane. In general population, many maternal or fetal conditions can lead to preterm birth. The analysis of MMP-8, IL-6, and VEGF concentrations in amniotic fluid of women with those conditions may give us more information for preterm labor with or without obstetric complications. It must be preceded as further study for clinical application to predict preterm birth in general pregnant women. In conclusion, the diagnostic value of CRP in mid-trimester amniotic fluid for spontaneous preterm birth is questionable. However, despite still unrevealed mechanisms, elevated inflammatory mediators, MMP-8 and IL-6, are associated with preterm birth among women with an intact membrane. Elevated concentration of VEGF as angiogenic parameter is also related to preterm birth. This analogizes the interaction between inflammation and angiogenic pathways in amniotic fluid and the maternalefetal interface to promote spontaneous preterm labor. Conflicts of interest The authors declare that there are no conflicts of interest. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.placenta.2013.06.306. References [1] Krupa FG, Faltin D, Cecatti JG, Surita FG, Souza JP. Predictors of preterm birth. Int J Gynaecol Obstet 2006;94(1):5e11. [2] Goldenberg RL, Culhane JF, Iams JD, Romero R. Epidemiology and causes of preterm birth. Lancet 2008;371(9606):75e84. [3] Conde Agudelo A, Papageorghiou AT, Kennedy SH, Villar J. Novel biomarkers for the prediction of the spontaneous preterm birth phenotype: a systematic review and meta-analysis. BJOG 2011;118(9):1042e54. [4] Defranco EA, Lewis DF, Odibo AO. Improving the screening accuracy for preterm labor: is the combination of fetal fibronectin and cervical length in symptomatic patients a useful predictor of preterm birth? A systematic review. Am J Obstet Gynecol 2013;208(3):233.e1e6. [5] Kim Y, West XZ, Byzova TV. Inflammation and oxidative stress in angiogenesis and vascular disease. J Mol Med 2013;91(3):323e8. [6] Gervasi M, Romero R, Bracalente G, Erez O, Dong Z, Hassan S, et al. Midtrimester amniotic fluid concentrations of interleukin-6 and interferongamma-inducible protein-10: evidence for heterogeneity of intra-amniotic inflammation and associations with spontaneous early (<32 weeks) and late (>32 weeks) preterm delivery. J Perinat Med 2012;40(4):329e43. [7] Borna S, Abdollahi A, Mirzaei F. Predictive value of mid-trimester amniotic fluid high-sensitive C-reactive protein, ferritin, and lactate dehydrogenase for fetal growth restriction. Indian J Pathol Microbiol 2009;52(4):498e500. [8] Ghezzi F, Franchi M, Raio L, Di Naro E, Bossi G, D’Eril GV, et al. Elevated amniotic fluid C-reactive protein at the time of genetic amniocentesis is a marker for preterm delivery. Am J Obstet Gynecol 2002;186(2):268e73. [9] Spong CY, Sherer DM, Ghidini A, Jenkins CB, Seydel FD, Eglinton GS. Secondtrimester amniotic fluid or maternal serum interleukin-10 levels and small for gestational age neonates. Obstet Gynecol 1996;88(1):24e8. [10] Spong CY, Scherer DM, Ghidini A, Pezzullo JC, Salafia CM, Eglinton GS. Midtrimester amniotic fluid tumor necrosis factor-alpha does not predict smallfor-gestational-age infants. Am J Reprod Immunol 1997;37(3):236e9. [11] Ghidini A, Jenkins CB, Spong CY, Pezzullo JC, Salafia CM, Eglinton GS. Elevated amniotic fluid interleukin-6 levels during the early second trimester are associated with greater risk of subsequent preterm delivery. Am J Reprod Immunol 1997;37(3):227e31. [12] Figueroa R, Garry D, Elimian A, Patel K, Sehgal PB, Tejani N. Evaluation of amniotic fluid cytokines in preterm labor and intact membranes. J Matern Fetal Neonatal Med 2005;18(4):241e7.
878
A. Kim et al. / Placenta 34 (2013) 873e878
[13] Apuzzio J, Chan Y, Al Khan A, Illsley N, Kim P, Vonhaggen S. Second-trimester amniotic fluid interleukin-10 concentration predicts preterm delivery. J Matern Fetal Neonatal Med 2004;15(5):313e7. [14] Yoon BH, Oh SY, Romero R, Shim SS, Han SY, Park JS, et al. An elevated amniotic fluid matrix metalloproteinase-8 level at the time of mid-trimester genetic amniocentesis is a risk factor for spontaneous preterm delivery. Am J Obstet Gynecol 2001;185(5):1162e7. [15] Wenstrom KD, Andrews WW, Hauth JC, Goldenberg RL, DuBard MB, Cliver SP. Elevated second-trimester amniotic fluid interleukin-6 levels predict preterm delivery. Am J Obstet Gynecol 1998;178(3):546e50. [16] Pressman EK, Thornburg L, Glantz JC, Earhart A, Wall PD, Ashraf M, et al. Inflammatory cytokines and antioxidants in midtrimester amniotic fluid: correlation with pregnancy outcome. Am J Obstet Gynecol 2011;204(2):155.e1e7. [17] Bamberg C, Fotopoulou C, Thiem D, Roehr C, Dudenhausen JW, Kalache KD. Correlation of midtrimester amniotic fluid cytokine concentrations with adverse pregnancy outcome in terms of spontaneous abortion, preterm birth, and preeclampsia. J Matern Fetal Neonatal Med 2012;25(6):812e7. [18] Bamberg C, Fotopoulou C, Linder M, Roehr CC, Dudenhausen JW, Henrich W, et al. Mid-trimester amniotic fluid concentrations of the proinflammatory cytokines IL-6, IL-8, TNF-Î, and lipopolysaccharide binding protein in normal pregnancies: a prospective evaluation according to parity, gestational age, and fetal gender. J Perinat Med 2011;39(4):403e9. [19] Snegovskikh V, Schatz F, Arcuri F, Toti P, Kayisli UA, Murk W, et al. Intraamniotic infection upregulates decidual cell vascular endothelial growth factor (VEGF) and neuropilin-1 and -2 expression: implications for infectionrelated preterm birth. Reprod Sci 2009;16(8):767e80. [20] Ozer KT, Kavak ZN, GÃkaslan H, Elter K, Pekin T. Predictive power of maternal serum and amniotic fluid CRP and PAPP-A concentrations at the time of genetic amniocentesis for the preterm delivery. Eur J Obstet Gynecol Reprod Biol 2005;122(2):187e90.
[21] Maymon E, Romero R, Pacora P, Gomez R, Athayde N, Edwin S, et al. Human neutrophil collagenase (matrix metalloproteinase 8) in parturition, premature rupture of the membranes, and intrauterine infection. Am J Obstet Gynecol 2000;183(1):94e9. [22] Weissenbacher T, Laubender RP, Witkin S, Gingelmaier A, Schiessl B, Kainer F, et al. Diagnostic biomarkers of pro-inflammatory immune-mediated preterm birth. Arch Gynecol Obstet 2013;287(4):673e85. [23] Hillier SL, Witkin SS, Krohn MA, Watts DH, Kiviat NB, Eschenbach DA. The relationship of amniotic fluid cytokines and preterm delivery, amniotic fluid infection, histologic chorioamnionitis, and chorioamnion infection. Obstet Gynecol 1993;81(6):941e8. [24] Sadowsky DW, Adams KM, Gravett MG, Witkin S, Novy MJ. Preterm labor is induced by intraamniotic infusions of interleukin-1beta and tumor necrosis factor-alpha but not by interleukin-6 or interleukin-8 in a nonhuman primate model. Am J Obstet Gynecol 2006;195(6):1578e89. [25] Costa C, Incio J, Soares R. Angiogenesis and chronic inflammation: cause or consequence? Angiogenesis 2007;10:149e66. [26] Kundu JK, Surh Y. Emerging avenues linking inflammation and cancer. Free Radic Biol Med 2012;52(3):2013e37. [27] Stannard AK, Khurana R, Evans IM, Sofra V, Holmes DI, Zachary I. Vascular endothelial growth factor synergistically enhances induction of E-selectin by tumor necrosis factor-alpha. Arterioscler Thromb Vasc Biol 2007;27(3):494e502. [28] Krikun G, Huang SJ, Schatz F, Salafia C, Stocco C, Lockwood CJ. Thrombin activation of endometrial endothelial cells: a possible role in intrauterine growth restriction. Thromb Haemost 2007;97(2):245e53. [29] Brou L, Almli LM, Pearce BD, Bhat G, Drobek CO, Fortunato S, et al. Dysregulated biomarkers induce distinct pathways in preterm birth. BJOG 2012;119(4):458e73. [30] Romero R, Espinoza J, Kusanovic JP, Gotsch F, Hassan S, Erez O, et al. The preterm parturition syndrome. BJOG 2006;113(Suppl. 3):17e42.