High MMP-9 and TNF-a expression increase in preterm premature rupture of membranes 33 *Department of Obstetrics and Gynecology, Faculty of Medicine, Universitas Sebelas Maret/ RSUD Dr. Moewardi Surakarta Corresponding author: dr. Sri Sulistyowati, SpOG *Department of Obstetrics and Gynecology, Faculty of Medicine, Universitas Sebelas Maret/ RSUD Dr. Moewardi Surakarta Jl. Ir Sutami 36A Surakarta, Jawa Tengah 57126 Phone: +62812 2968 215 Email: elis_spog@yahoo.co.id Univ Med 2016;35:33-9 DOI: 10.18051/UnivMed.2016.v35.33-39 pISSN: 1907-3062 / eISSN: 2407-2230 This open access article is distributed under a Creative Commons Attribution-Non Commercial-Share Alike 4.0 International License ABSTRACT UNIVERSA MEDICINA January-April, 2016January-April, 2016January-April, 2016January-April, 2016January-April, 2016 Vol.35 - No.1 Vol.35 - No.1 Vol.35 - No.1 Vol.35 - No.1 Vol.35 - No.1 High MMP-9 and TNF-ααααα expression increase in preterm premature rupture of membranes Sri Sulistyowati*, Yuniarsih Zakia*, and Soetrisno* BACKGROUND Preterm delivery is one of the causes of high perinatal morbidity and mortality. Matrix metalloproteinase 9 (MMP-9) is important for extracellular matrix (ECM) remodeling and may cause preterm labor and premature rupture of membranes (PROM). Tumor necrosis factor- á (TNF-α) as a pro-inflammatory cytokine plays a role in stimulating uterine activity and cervical ripening by degrading the ECM of the amniotic membranes through MMP-9. This study aimed to determine differences between MMP-9 and TNF-α expression of the membranes in preterm delivery with premature rupture of membranes (PPROM) and without PROM. METHODS An analytic observational study with cross-sectional approach was conducted in 24 subjects, who were divided into 2 groups, with 12 subjects in the preterm delivery group with PROM and 12 subjects in the preterm delivery group without PROM. The expression of MMP-9 and TNF-α in the amniotic membrane was determined by immunohistochemistry. Data were analyzed using the t test. RESULTS MMP-9 expression in the amniotic membrane of preterm delivery subjects with PROM (8.6 ± 3.1%/field) differed significantly with that of preterm delivery subjects without PROM (5.5 ± 2.3%/ field) (p=0.001). TNF-α expression in the amniotic membrane of preterm delivery subjects with PROM (8.0 ± 3.0%/field) also differed significantly with that of preterm delivery subjects without PROM (3,3 ± 1.5%/field) (p=0.000). CONCLUSION Expression of MMP-9 and TNF-α was higher in the amniotic membrane of preterm delivery subjects with PROM than in preterm delivery subjects without PROM and can thus be used as predictor to avoid PPROM. Keywords: MMP-9, TNF-α, preterm delivery, premature rupture of membranes DOI: http://dx.doi.org/10.18051/UnivMed.2016.v35.33-39 34 Sulistyowati, Zakia, Soetrisno MMP-9 and TNF-α expression in premature rupture INTRODUCTION P r e t e r m p r e m a t u r e r u p t u r e o f t h e membranes (PPROM) is the spontaneous rupture of the fetal membranes during pregnancy before 37 weeks in the absence of regular uterine contractions. Preterm PROM occurs in 3–8% of pregnancies and in one-third of cases results in preterm delivery. PPROM is an important cause of perinatal morbidity, since it is associated with infection and compression of the umbilical c o r d c a u s e d b y l a c k o f a m n i o t i c f l u i d (oligohydramnion).(1) The prevalence rate of preterm delivery differs by country. In developed countries such as Europe, the rate ranges between 5 and 11%.(2) In the USA around one of nine infants is born preterm (11.9%).(3) In Indonesia the preterm prevalence rate is roughly 47.35%.(4) Preterm delivery increases the perinatal and neonatal complications, with a fetal mortality risk of 1–2% and increased perinatal morbidity such as the respiratory distress syndrome and neonatal sepsis.(1) There are many factors that may cause preterm delivery, such as infection, uterine overdistension, uteroplacental ischemia, e n d o c r i n e f a c t o r s , a n d c e r v i c a l a n d immunological disorders that may trigger preterm delivery. (5) One of the causes of infections in pregnancy is premature rupture of the membranes (PROM).(6) The incidence of PROM in Indonesia ranges from 4.5 to 7.6%.(7) PROM contributes to 30–40% of preterm deliveries. The prevalence rate of PROM in preterm pregnancies is around 2–3%.(6) PROM is an important problem in obstetrics, as it is associated with preterm delivery.(8) Matrix metalloproteinase-9 (MMP-9) is the main MMP involved in normal delivery and also plays an important role in pathological delivery. Matrix metalloproteinase-9 is the most active MMP in the amnion, and is found to increase significantly in the amniotic membranes after the onset of contractions. Matrix metalloproteinase, also known as matrixin, plays an important role in the breakdown and remodeling of the extracellular matrix (ECM), finally causing both p r e t e r m d e l i v e r y a n d p r e t e r m P R O M . Manipulation of MMP may play a role in preventing spontaneous preterm delivery.(9) Preterm PROM increases MMP-9 concentrations in the amniotic fluid, causes amniotic fluid infection, threatened preterm delivery and poor neonatal outcome.(10) Tumor necrosis factor-α (TNF-α) is a pro- inflammatory cytokine that is mainly produced in monocytes and/or macrophages. Excessive secretion of TNF-α may cause deleterious effects, such as abortion and preterm delivery. Increase or non-increase of the TNF-α pro- inflammatory cytokine presumably plays an important role in preventing preterm delivery.(11) In preterm delivery, TNF-α stimulates uterine activity and the cervical ripening process by producing prostaglandin and cortisol, and degrading the ECM of the membranes through the MMP-2 and MMP-9 pathways.(12) The TNF-α concentration in the amniotic fluid in preterm delivery increases significantly in comparison with term delivery. The TNF-α (and/or IL-6) concentration in amniotic fluid at mid-trimester may identifiy pregnant mothers at risk for chorioamnionitis, which may cause preterm delivery.(13) The purpose of this study was to compare the expression of MMP-9 and TNF-α in the membranes in preterm delivery with PROM and that in preterm delivery without PROM. METHODS Design of the study An analytical-observational study with cross- sectional approach was conducted at the Department of Obstetrics and Gynecology, RSUD Dr. Moewardi Surakarta, from December 2014 until May 2015. Subjects of the study The study subjects were pregnant mothers who had undergone preterm delivery with PROM and without PROM. The sample size was 12 per 35 group, based on the difference between the prevalence of preterm delivery without PROM and the prevalencs of preterm delivery with PROM, the difference being 0.3, with α=1.96 and β= 0.2.(14) There were thus 12 preterm delivery subjects with PROM and 12 preterm delivery subjects without PROM. The inclusion criteria were pregnant mothers delivering at RSUD Dr. Moewardi Surakarta, who had undergone preterm delivery with PROM and preterm delivery without PROM, and whose infants were without major congenital abnormalities. The exclusion criteria were pregnant mothers with chronic diseases such as diabetes mellitus, kidney and heart diseases, and hypertension; pregnant mothers with systemic inflammation and infection, such as pneumonia, typhoid, malaria, hepatitis; pregnant mothers who were smokers, drinkers, and drug abusers, on antibiotic treatment, with trauma in pregnancy, twin pregnancies, intrauterine fetal death, and abnormally shaped uteri. Immunohistochemistry For all subjects, immunohistochemical examination of MMP-9 and TNF-α expression in the membranes was performed at the Pathological Anatomy Laboratory, Universitas Sebelas Maret, Surakarta. Matrix metalloproteinase 9 was determined immunohistochemically by the avidin biotin complex method, using Bioss MMP-9 antibody reagent bs-0397R. MMP-9 expression in the membranes was examined under the microscope at 400X magnification. MMP-9 expression for a strong positive result was shown by a reddish-brown color in the cell nucleus and the cytoplasm, for a moderate positive result by a dark brown color, for a weak positive result by a light brown color, and for a negative result by a bluish color. The histological score was then calculated as percentages per field of view, the data being of continuous scale. Similarly, expression of tumor necrosis factor α was determined immunohistochemically by the avidin biotin complex method, using Bioss TNF-α antibody reagent bs-0078R. Expression of TNF-α in the membranes was examined under the microscope at 400X magnification. TNF-α expression for a strong positive result was shown by a reddish-brown color in the cell nucleus and the cytoplasm, for a moderate positive result by a dark brown color, for a weak positive result by a light brown color, and for a negative result by a bluish color. The histological score was then calculated as percentages per field of view, the data being of continuous scale. Data analysis The data were analyzed with the t-test using SPSS, with p<0.05 being considered significant. Ethical clearance Ethical clearance wa obtained from the Commission for Medical Research Ethics, RSUD Dr. Moewardi/ Faculty of Medicine, Universitas Sebelas Maret, under No. 642/XI/HREC/2014, dated 11 November 2014. RESULTS There were 50% working mothers in the preterm delivery group with PROM and 25% in the preterm delivery group without PROM. The majority of mothers (75%) in the preterm delivery group with PROM as well as in the preterm delivery group without PROM, had a junior high school education. The percentage of primigravida in the preterm delivery group with PROM and the preterm delivery group without PROM was 75% and 50%, respectively (Table 1). The majority of pregnant mothers were 21– 35 years old, accounting for 83% of the preterm delivery group with PROM, and for 75% of the p r e t e r m d e l i v e r y g r o u p w i t h o u t P R O M . Regarding the age of pregnancy of 34–36 weeks, the percentage in both the preterm delivery group with PROM and the preterm delivery group without PROM was identical, namely 58% (Table 1). From Table 2 it may be seen that the mean MMP 9 expression in the membranes was significantly higher in the preterm delivery group with PROM (8.6 ± 3.1%/field), in comparison Univ Med Vol. 35 No.1 36 Sulistyowati, Zakia, Soetrisno MMP-9 and TNF-α expression in premature rupture Table 1. Distribution of characteristics of study subjects Table 2. Mean expression of MMP-9 and TNF-α in the membranes in the preterm delivery group with PROM and the preterm delivery group without PROM *Significant at p<0.05 with the preterm delivery group without PROM (5.5 ± 2.3%/field) (p=0.001). The mean expression of TNF-α in the membranes was significantly higher in the preterm delivery group with PROM (8.0 ± 3.0%/field) than in the preterm delivery group without PROM (3.3 ± 1.5%/field) (p=0.000). Expression of MMP-9 and TNF-α in the membrane of the pretern delivery groups with and without PROM were showed in Figure 1 and Figure 2. DISCUSSION In our present study we found that the mean MMP-9 expression in the membranes was significantly higher in the preterm delivery group with PROM (8.6 ± 3.1%/field) in comparison with the preterm delivery group without PROM (5.5 ± 2.3%/field). PROM is one of the factors for the initiation of delivery and is an important p r o b l e m , s i n c e i t i s a s s o c i a t e d w i t h t h e complications of pregnancy, one of them being preterm delivery, which results in both infant morbidity and mortality.(8) The pathogenesis of preterm delivery is associated with cytokines, matrix metalloproteinases, and prostaglandins. The amniotic membrane is the inner layer of the fetal membranes that line the amniotic cavity. The amnion consists of an epithelial cell layer above the thicker basement membrane and a spongy collagen layer containing mesenchymal cells. The amnion is part of the fetal development and protects the fetus against mechanical injury by surrounding it with amniotic fluid. The amnion receives its strength from collagen in the basement membrane, especially type IV collagen. Collagen in the basement membrane and collagen 37 MMP-9 expression in preterm delivery group with PROM MMP-9 expression in preterm delivery group without PROM Figure 2. Expression of TNF-α in the membranes of the preterm delivery group with and without PROM, determined by immunohistochemistry and evaluated using an Olympus cx21 microscope at 400x magnification. Reddish-brown color in the cell nucleus and the cytoplasm indicates a strongly positive result; dark brown = moderately positive result; light brown = weakly positive result; bluish color = negative result. TNF-α expression was stronger/higher in the preterm delivery group with PROM in comparison with the preterm delivery group without PROM. TNF-α expression in preterm delivery with PROM TNF-α expression in preterm delivery without PROM Figure 1. Expression of MMP-9 in the membranes of the preterm delivery groups with and without PROM, determined by immunohistochemistry and evaluated using an Olympus cx21 microscope at 400x magnification. Reddish-brown color in the cell nucleus and the cytoplasm indicates a strongly positive result; dark brown = moderately positive result; light brown = weakly positive result; bluish color = negative result. MMP-9 expression was stronger/higher in the preterm delivery group with PROM in comparison with the preterm delivery group without PROM Univ Med Vol. 35 No.1 38 Sulistyowati, Zakia, Soetrisno MMP-9 and TNF-α expression in premature rupture degradation in the chorioamnion is controlled by matrix metalloproteinases (MMPs). Matrix metalloproteinase-1 (MMP-1) degrades type I, II and III collagens, while MMP-2 and MMP-9 (gelatinase B) degrade type IV collagen. In human chorionic cells, tumor necrotic factor alpha (TNF-α) has been found to promote the production of MMP and prostaglandin E2 (PGE2), and to repress tissue inhibitors of metalloproteinase [TIMPs]). Therefore, TNF-α tends to cause the membranes to weaken and rupture through collagen degradation in the ECM.(15) Increased concentrations of interleukin-6 (IL-6), C-reactive protein (CRP), and matrix metalloproteinase 9 (MMP-9) in the fetal and neonatal compartments have been shown to be associated with increased risk of preterm delivery.(16) MMP-9 expression, apoptosis index (IA) of cervical cells, and MMP-9 gene polymorphism (C-1562T) in pregnancies of 21– 36 weeks without and with PROM have been demonstrated by Sabarudin et al.,(17) who found no significant differences in MMP-9 expression and cervical cell IA between preterm delivery with PROM and preterm delivery without PROM. In the present study, mean TNF-α in the membranes was found to be higher in the preterm delivery group with PROM than in the preterm delivery group without PROM. In preterm d e l i v e r y c a s e s , T N F - α a n d o t h e r p r o - inflammatory cytokines play a role in stimulating uterine activity and cervical ripening by p r o d u c i n g p r o s t a g l a n d i n s , c o r t i s o l , a n d degrading the ECM of the amniotic membranes through the MMP-2 and MMP-9 pathways.(12) TNF-α is a predictor of PROM, since increased TNF-α levels in the patient’s serum may trigger MMP-9 expression, thus causing increased degradation of type IV collagen in the membranes of pregnant women and possibly causing PROM.(8) Tu m o r n e c r o s i s f a c t o r, c a c h e x i n o r cachectin, previously known as tumor necrosis factor-α, is a pleiotropic inflammatory cytokine that is mainly produced in monocytes and/or macrophages. Excessive secretion of the pro- inflmmatory cytokines TNF or IL-1, such as occur in infections of the amnion, has been known to be detrimental to intra-uterine tissues such as in abortion and preterm delivery.(11) Cytokines can increase the production of prostaglandins in the amniotic membranes and decidua. Various factors may be involved in the release of TNF-α and the activation of MMP-9, such as psychological stress, which causes the release of corticotropin releasing hormone (CRH), infection, and inflammation, that directly stimulates the release of inflammatory mediators, such as TNF-α. The concentration of bioactive TNF, the availability of TNF receptors (TNFR1 and TNFR2), and the affinity of TNF for its receptors, and also its signaling pathway, may display different kinerja of TNF-α. TNF-α in most frequently involved in the activation of MMP-9.(13) MMP-9 increases significantly upon exposure of the amnion to TNF-α or IL-1 beta, whereas its secreted concentration in the chorion is not altered. Each of the inflammatory cytokines (IL-1, IL-6, and TNF-α) have been found in higher concentrations in the amniotic fluid of women with PROM. The cytokine that influences the MMP family in pregnancy is TNF-α, which is the most characterized cytokine. This member of the pro- inflammatory cytokines has been shown to be able to trigger MMP expression in various tissues, thus causing the release of active MMP-9 from the amniotic membranes and also triggering apoptosis in the amniotic membranes and activation of MMP-9, which finally causes PROM.(9) Advanced understanding of the biochemical causes of fetal membrane weakening and rupture indicates the possibility of inhibiting the process and ultimately preventing PPROM.(18) In this study, we did not discuss in detail the causes of PROM in the study groups, e.g. we did not examine the occurrence of urinary tract infection (UTI). We also did not differentiate the duration of PROM or the degree of infection. Another limitation of this study was that we did not equalize the leukocyte concentrations in the 39 preterm delivery groups, despite the fact that this can influence the MMP-9 and TNF-α concentrations in preterm deliveries with PROM. Further studies are required that control for confounding variables such as the causes of PROM and the degree of infection. It is expected that pregnant mothers with PROM may be detected earlier so that they can be better managed to prevent preterm delivery resulting in morbidity and mortality of the mother and her fetus. CONCLUSIONS The expression of MMP-9 and TNF-α were higher in the membranes of preterm deliveries with PROM, in comparison with preterm deliveries without PROM. MMP-9 and TNF-α may be strong predictors of PROM, particularly in preterm deliveries. CONFLICT OF INTEREST The authors declare that there is no conflict of interest associated with this study, the authors, and / or the publication of this article. REFERENCES 1. Okeke TC, Enwereji JO, Okoro OS, et al. The incidence and management outcome of preterm premature rupture of membranes (PPROM) in a tertiary hospital in Nigeria. Am J Clin Med Res 2014;2:14–7. 2. Martin JA, Hamilton BE, Sutton PD, et al. Births: final data for 2005. Natl Vital Stat Rep 2007;5;1-103. 3. Spong CY. Prediction and prevention of recurrent spontaneous preterm birth. Obstet Gynecol 2007; 110 2 Pt 1:405–15. 4. Sudarmi. Hubungan ketuban pecah dini > 12 jam dengan gawat janin di ruang bersalin RSUP NTB tahun 2012. Media Bina Ilmiah 2013;7:31–4. 5. Razzak MSA, Al-Sa’adi MAK, Hussainy TAIA. The role of tumor necrosis factor-alpha (TNF- α) in the induction of preterm labor. Karbala J Med 2010;3:779–83. 6. Veleminsky M, Sak P. Management of pregnancy with premature rupture of membranes (PROM). J Health Sci Management Public Health 2006; 192–7. 7. Wiradharma, Kardana I, Wyn DAI. Risiko asfiksia pada ketuban pecah dini di RSUP Sanglah. Sari Pediatri 2013;14:316-9. 8. Wibowo AP, Sulistyowati S, Respati SH. Difference of serum MMP-9 and TNF-α level in preterm and term premature rupture of membranes. IJOG 2015;3:15–8. 9. Weiss A, Goldman S, Shalev E. The matrix metalloproteinases (MMPS) in the decidua and fetal membranes. Front Biosci 2007;1:649–59. 10. Yoon BH, Oh SY, Romero R, 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: 1162-7. 11. Haider SM, Knofler. Human tumour necrosis factor: physiological and pathological roles in placenta and endometrium. Placenta 2009; 30:111–23. 12. Calleja-Agius J, Muttukrishna S, Jauniaux E. The role of tumor necrosis factor-receptors in pregnancy with normal and adverse outcome. Int J Interferon, Cytokine Mediator Res 2012;4: 1–15. 13. Thomakos N, Daskalakis G, Papapanagiotou A, et al. Amniotic fluid interleukin-6 and tumor necrosis factor-a at mid-trimester genetic amniocentesis: relationship to intra-amniotic microbial invasion and preterm delivery. Eur J Obstet Gynecol Reprod Biol 2010;148:147–51. 14. Hulley SB, Cummings SR, Browner WS, et al. Designing clinical research. 4th ed. New York: Lippincot Williams & Wilkins, Wolters Kluwer; 2013. 15. Izumi-Yoned N, Toda A, Okabe M, et al. Alpha 1 antitrypsin activity is decreased in human amnion in premature rupture of the fetal membranes. Mol Hum Reprod 2009;15:49-57. 16. Sorokin Y, Romero R, Mele L, et al. Maternal serum interleukin-6, C-reactive protein, and matrix metalloproteinase-9 concentrations as risk factors for preterm birth <32 weeks and adverse neonatal outcomes. Am J Perinatol 2010; 27:631–40. 17. Sabarudin U, Mose JC, Krisnadi SR. Polimorfisme gen MMP-9, ekspresi MMP-9, dan indeks apoptosis sel serviks pada kehamilan 21 – 36 minggu. MKB 2011;43:199–206. 18. Kumar D, Schatz F, Moore RM, et al. The effects of thrombin and cytokines upon the biomechanics and remodeling of isolated amnion. In Vitro Placenta 2011;32:206-13. Univ Med Vol. 35 No.1