http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, Early Online: 1–5 ! 2015 Informa UK Ltd. DOI: 10.3109/14767058.2015.1015416
ORIGINAL ARTICLE
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The role of extracellular inducer of matrix metalloproteinases in premature rupture of membranes Gennady T. Sukhikh1, Natalia E. Kan2, Victor L. Tyutyunnik2, Maya V. Sannikova2, Elena A. Dubova3, Konstantin A. Pavlov3, Elrad Y. Amiraslanov2, and Nataliya V. Dolgushina4 1
Directorate, Federal State Budget Institution, ‘‘Research Center for Obstetrics, Gynecology and Perinatology’’ of Ministry of Healthcare of the Russian Federation, Moscow, Russian Federation, 2Obstetric Observational Department, 3Department of Pathology, and 4R&D Department, Federal State Budget Institution, ‘‘Research Center for Obstetrics, Gynecology and Perinatology’’ of Ministry of Healthcare of the Russian Federation, Moscow, Russian Federation Abstract
Keywords
Objective: To investigate the role of matrix metalloproteinases (MMP-2, MMP-9) and their inducer (CD147) in premature rupture of membranes (PROM) at term labor. Methods: In a cross-sectional study, 24 women aged 19–39, with 37–40-week pregnancy, and no clinical and histological signs of chorioamnionitis, were divided into two groups with and without PROM. The histological and immunohistochemical study of the fetal membranes was performed with polyclonal rabbit antibodies to MMP-2/MMP-9 and monoclonal rabbit antibodies to CD147. Results: The analysis of MMP revealed the increase of MMP-9 expression in the amniotic epithelium during premature membrane rupture both in rupture area, and beyond it, and increased MMR-2 expression in the mesodermal cells. We also found high level of CD147 in the amniotic epithelium in PROM group. The above-mentioned changes were found in all areas of fetal membranes, regardless of the rupture localization. Conclusions: The study results demonstrate the increased expression of MMR-2 and MMR-9, which regulate the catabolism of fetal membrane extracellular matrix proteins, in amniotic membranes of women with PROM at term labor. The increased expression of CD147 may be one of the mechanisms triggering PROM in the absence of infection.
CD147, MMP-2, MMP-9, premature rupture of membranes (PROM), spontaneous delivery
Introduction The premature rupture of membranes (PROM) is a complication of 3–15.6% of pregnancies and deliveries [1–4]. The PROM origin remains unknown; the scientific community discusses the role of the degradation or remodeling of extracellular matrix (ECM) components regulated by Zn2+dependent proteolytic enzymes – matrix metalloproteinases (MMP) [5,6]. Among the factors affecting ECM remodeling, a special role is played by MMP-2 and MMP-9 as well as by ECM metalloproteinase inducer – EMMPRIN/CD147 [5]. CD147 is a transmembrane glycoprotein with molecular mass of 44–66 kDa containing two extracellular immunoglobulin domains. CD147 plays an important role in spermatogenesis, implantation, cell growth and adhesion, angiogenesis, endometrial cell decidualization, immune cell activation and MMP induction [5,7–9]. Some previous studies have demonstrated the role of
Address for correspondence: Nataliya V. Dolgushina, R&D Department, Federal State Budget Institution, ‘‘Research Center for Obstetrics, Gynecology and Perinatology’’ of Ministry of Healthcare of the Russian Federation, 4, Oparin street, Moscow 117997, Russian Federation. Tel: +7-903-960-83-42. E-mail: [email protected]
History Received 29 January 2015 Accepted 2 February 2015 Published online 9 March 2015
MMP in the PROM [10–12]. Li et al. [5] showed that CD147 is expressed in the human placenta and fetal membranes. This calls for the research into CD147 role in physiological and pathological processes in fetal membranes during the term and preterm pregnancy and delivery, including PROM. Study objective was to investigate the role of MMP-2 and MMP-9 and their inducer (CD147) in premature membrane rupture at term labor.
Materials and methods The cross-sectional study was performed at Obstetric Observational Department of the Research Center for Obstetrics, Gynecology and Perinatology. In this article, we present the initial data of 24 women, recruited in the study. The inclusion criteria were: age 19–39, non-assisted singleton pregnancy resulted at term, spontaneous delivery ranging from 37 to 40 weeks of gestation. All subjects gave written informed consent for participation in the study. The study was approved by Ethics committee for biomedical research of Federal State Budget institution ‘‘Research Center for Obstetrics, Gynecology and Perinatology’’. Women with clinical or morphological signs of chorioamnionitis and with induced delivery were excluded from the study.
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G. T. Sukhikh et al.
J Matern Fetal Neonatal Med, Early Online: 1–5
The patients were divided into two groups: group I included 12 patients with deliveries complicated by PROM, and group II included 12 patients without PROM during the delivery. The PROM was determined as a discharge of the amniotic fluid earlier than 2 h prior the regular labor. The amniotic fluid was verified by the level of a2-microglobulin in the posterior vaginal fornix. In group I, the latency period between PROM and labor onset was more than 4 h and composed (6.2 ± 1.4) h. The patients in group II had spontaneous or artificial rupture of membranes after 6-cm cervical dilation. We did not use intravenous oxytocin or other drugs for labor induction in both groups of women. In each group, we made one-time measurement of MMP-2, MMP-9 and CD147 expression level in amniotic membranes. The histological study of fetal membranes was performed following common methods [13]. The fetal membrane tissue samples were taken immediately after delivery. The fetal membranes were studied in the rupture area and beyond the rupture area. The rupture was determined by visual assessment of fetal membranes during the sampling as an area adjacent to the fetal membrane defect. The area included the 10-cm long part of the fetal membrane 1.0 cm from the edge of the defect. The area beyond the rupture was determined visually during the sampling. The histological study of the tissues was performed on paraffin-embedded sections stained with hematoxylin and eosin. The immunohistochemical study of the tissues was performed on 3–4-mm thick paraffin-embedded sections with ready-to-use polyclonal rabbit antibodies to MMP-2 and MMP-9 (Spring Bioscience, Pleasanton, CA) and with monoclonal rabbit antibodies to CD147 (1:100 solution, EPR4052 clone, Epitomics, Burlingame, CA). Automated immunohistochemistry/in situ hybridization (IHC/ISH) slide staining system Ventana Benchmark XT (Roche Group, Basel, Switzerland) with a standard staining protocol was used to perform an immunohistochemical study. MMPs and CD147 expression levels were evaluated in optical density units with Nikon Eclipse 80i microscope and NIS-Element AR 3.2 software (Nikon Corp., Tokyo, Japan) by a single researcher. The data were processed with Statistica 8 (Statsoft Inc., Tulsa, OK) and IBM SPSS Statistics 19 for Windows (IBM Corp., Armonk, NY) software. p50.05 was assumed
statistically significant. Mann–Whitney non-parametric Utest was used to compare the median values of two independent samples for continuous data. The categorical data were presented as percentages and assessed by v2 test.
Results We revealed no statistically significant differences in the rate of pregnancy and delivery complications, the duration of the delivery periods, amount of blood loss, and postpartum complications in two groups of patients. The study participants had comparable baseline characteristics, including the history of infectious and inflammatory diseases (Table 1). The histological analysis of the fetal membranes revealed no statistically significant differences in two groups of patients. It is noteworthy that no leukocyte infiltration was found suggesting the absence of inflammation. The immunohistochemical analysis revealed the increased expression of MMP and CD147 in various components of the fetal membranes. The mesodermal MMP-2 expression was significantly higher in group I, both in the rupture area (p ¼ 0.0004) and beyond it (p ¼ 0.0448) (Table 2). We found no significant differences in MMP-2 expression by amniotic epithelium cells, as well as trophoblast and basal plate decidual cells between the groups (p40.05). The significant increase was Table 1. Clinical characteristics of the study population (n ¼ 24).
Parameter Age, yrs Rate of nulliparity, n (%) Gestational age on inclusion, weeks STD before pregnancy, n (%) Chronic salpingophoritis, n (%) URTI during the pregnancy, n (%) Polyhydramnios, n (%) Oligohydramnios, n (%) Fetal growth retardation, n (%) Chronic fetal hypoxia, n (%) Mild preeclampsia, n (%)
Group I (PROM)
Group II (w/o PROM)
28.3 ± 5.6 1 (8.3) 38.3 ± 0.9 3 (25.0) 2 (16.7) 3 (25.0) 1 (8.3) 0 (0) 0 (0) 1 (8.3) 2 (16.7)
30.3 ± 3.0 0 (0) 39.1 ± 1.0 4 (33.3) 1 (8.3) 2 (16.7) 0 (0) 2 (16.7) 1 (8.3) 2 (16.7) 3 (25.0)
The data are percentages and assessed by v2 test. STD, sexually transmitted diseases; URTI, upper respiratory tract infection. *The results have statistically significant differences.
Table 2. The comparison of MMP-2 and MMR-9 expression levels in the fetal membranes and placental cells. MMP-2 Localization Fetal membranes in the rupture area Fetal membranes beyond the rupture area Villi
PROM Amniotic epithelium Mesenchymal cells Basal plate trophoblast Basal plate decidual cells Amniotic epithelium Mesenchymal cells Basal plate trophoblast Basal plate decidual cells Endothelial cells Synticiotrophoblast Mesenchymal cells
21.5 ± 10.6 15.8 ± 4.6 18.1 ± 6.8 18.6 ± 8.2 18.5 ± 11.6 15.4 ± 6.7 17.6 ± 6.2 19.0 ± 6.2 9.3 ± 3.9 12.0 ± 4.8 9.7 ± 3.9
MMR-9
w/o PROM
p value
PROM
17.1 ± 7.8 9.2 ± 1.8 18.3 ± 3.3 17.8 ± 2.0 18.3 ± 8.3 12.2 ± 1.9 20.5 ± 2.1 19.1 ± 4.5 4.6 ± 4.8 7.9 ± 8.0 5.8 ± 5.7
0.1313 0.0004* 0.4446 0.2035 0.3312 0.0448* 0.2223 0.1250 0.0202* 0.2884 0.1250
28.8 ± 3.7 14.2 ± 1.4 23.1 ± 3.9 19.0 ± 4.5 31.6 ± 2.9 15.6 ± 3.3 24.1 ± 4.8 18.2 ± 2.3 12.0 ± 2.7 20.1 ± 4 15.3 ± 2.9
The data are M±s in optical density units and assessed by Mann–Whitney U-test. *The results have statistically significant differences.
w/o PROM
p value
25.2 ± 2.2 14.6 ± 2.2 22.3 ± 2.5 18.5 ± 2.6 26.4 ± 2.7 16.8 ± 3.8 22.5 ± 1.6 19.7 ± 1.4 11.2 ± 1.6 22 ± 2.8 15.8 ± 2.6
0.0196* 0.2470 0.9546 0.9095 0.0007* 0.0976 0.5742 0.0933 0.7732 0.2268 0.9018
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DOI: 10.3109/14767058.2015.1015416
MMPs/CD147 role in PROM
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Figure 1. Box and whisker plot, defining CD147 expression in the fetal membrane amniotic epithelium in the rupture area (A) and beyond the rupture area (B), in the group with PROM (1) and without PROM (2). The median of CD147 expression is significantly higher in women with PROM both in the rupture area (p50.0001) and beyond it (p ¼ 0.0030).
found in MMP-2 expression in the endothelial cells of acroteric villi in group I (p ¼ 0.0202). MMP-9 expression in group I was significantly higher in the amniotic epithelium both in the rupture area (p ¼ 0.0196) and beyond it (p ¼ 0.0007). No significant differences were found in MMP-9 expression by chorionic and amniotic mesoderm, as well as trophoblast and basal plate decidual cells between the groups (p40.05). CD147 expression in group I was significantly higher in the amniotic epithelium both in the rupture area (p50.0001) and beyond it (p ¼ 0.0030) (Figure 1). Moreover, group I demonstrated the decrease of CD147 expression by mesodermal cells, that was not significant in the rupture area (p40.05) and significant beyond it (p ¼ 0.0334) (Figure 2).
Discussion The results of this study confirm those from previous investigations that MMP-9 is predominantly expressed by epithelium while MMP-2 is expressed by stromal elements [14]. In the first trimester, MMP-2 and MMP-9 are mainly distributed in the extravillous trophoblast and villous cytotrophoblast, respectively [9]. The labor onset is accompanied by the significant increase of MMP-2 and MMP-9 activity in the amnion and decidual tissue [11]. MMP-9 plays an important role in the placenta delivery [15]. Our data demonstrate the higher expression of MMP-2 in the mesoderm and MMP-9 in the amniotic epithelium of the fetal membranes together with increased MMP-2 level in the amniotic rather than the chorionic tissues, suggesting different association and localization of the MMPs in question in PROM after term delivery. The analysis of CD147 expression in various fetal membrane structures is especially interesting. We found higher CD147 expression in the amniotic epithelium of fetal membranes both in the rupture area and beyond it, and lower expression in the amniotic mesoderm cells in the patients with deliveries complicated by PROM. Besides that, we found higher CD147 level in the amniotic epithelium of both areas in question compared to mesenchymal cells of fetal
membranes. These topographic characteristics of CD147 distribution are apparently the causes of the increased MMPs expression that we found. This suggests independent CD147 activation in PROM that is different from its activation during the delivery. The increased CD147 expression is described in the uterine mucous membranes and embryonic trophoectoderm during blastocyst implantation [16,17]. At 6–9 weeks of gestation as well as in the middle and in the end of the full-term pregnancy, CD147 is predominantly found in trophoblast and sincitiotrophoblast membranes and trophoblast column villi [18]. In the middle and in the end of the full-term pregnancy, CD147 expression was demonstrated in extravillous cytotrophoblast and decidual tissue. The maximal CD147 expression was found at the 6th week of pregnancy with the consequent gradual decrease. During the 2nd and 3rd trimester, CD147 expression was constant with the gradual decrease in extravillous cytotrophoblast and decidual tissue by the end of the pregnancy [18]. Nowadays, the main role in PROM genesis is attributed to the infection. The inflammation caused by the infection results in the production of inflammation mediators – cytokines, which, in turn, activate MMP causing ECM degradation and PROM. The immediate cause of the rupture is the necrosis of the amniotic epithelium and compact substance. In our study, we included the women with PROM and no signs of inflammation in fetal membranes, which were confirmed by the histological analysis. Mercer et al. [19] suggested that in this case, the strength of the fetal membranes decreases during the pregnancy due to amniotic epithelium apoptosis and ECM degradation resulting in the rupture. The mechanical stretching of the membranes may increase MMP production through cytokine cascade [1,4]. Our study has certain limitations since we studied placenta samples after term spontaneous deliveries that may distort the results. CD147 expression increases in placenta and fetal membranes in term spontaneous deliveries. However, Li et al. [5] found no differences in CD147 localization in placental and fetal membrane structures after vaginal and
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Figure 2. Box and whisker plot, defining CD147 expression in the fetal membrane mesodermal cells in the rupture area (A) and beyond the rupture area (B), in the group with PROM (1) and without PROM (2). The median of CD147 expression is significantly lower beyond the rupture area (p ¼ 0.0334) and insignificantly lower in the rupture area (p40.05).
cesarean deliveries. They demonstrated CD147 expression by syncytiotrophoblast, amniotic epithelium, as well as by extravillous trophoblast and decidual cells, but not by amniotic mesenchymal cells. These data differ from our findings. The study findings suggest the probable pathogenic mechanism of PROM, which results from the disruption of fetal membrane ECM homeostasis. In the absence of infection MMP-2, MMP-9 and CD147 are strongly associated with PROM. On the other hand, the study data call for further research into the degree, level and characteristics of CD147 involvement into the fetal membrane ECM remodeling during the delivery as well as in term and preterm membrane rupture.
Declaration of interest This work was supported by Ministry of Education and Science of the Russian Federation (grant # 16.522.12.2009). The authors report no conflicts of interest.
References 1. Berghella V. (ed.). The cervix. In: Preterm birth: prevention and management. Oxford, UK: Wiley-Blackwell; 2010:50–6. 2. Di Renzo GC, Roura LC, Facchinetti F, et al. Guidelines for the management of spontaneous preterm labor: identification of spontaneous preterm labor, diagnosis of preterm premature rupture of membranes, and preventive tools for preterm birth. J Matern Fetal Neonatal Med 2011;24:659–67. 3. Porat S, Amsalem H, Shah PS, Murphy KE. Transabdominal amnioinfusion for preterm premature rupture of membranes: a systematic review and metaanalysis of randomized and observational studies. Am J Obstet Gynecol 2012;207:393.e1–11. 4. The American College of Obstetricians and Gynecologists. ACOG Practice Bulletin No. 80: premature rupture of membranes. Clinical management guidelines for obstetrician-gynecologists. Obstet Gynecol 2007;109:1007–19. 5. Li W, Alfaidy N, Challis JR. Expression of extracellular matrix metalloproteinase inducer in human placenta and fetal membranes at term labor. J Clin Endocrinol Metab 2004;89:2897–904. 6. Weiss A, Goldman S, Shalev E. The matrix metalloproteinases (MMPS) in the decidua and fetal membranes. Front Biosci 2007;1: 649–59.
7. Braundmeier A, Bi J, Mehrotra P, Nowak R. Endogenous EMMPRIN expression by human uterine fibroblast cells regulates metalloproteinase production, proliferation, and decidualization. Biol Reprod 2010;83:13. 8. Mishra B, Kizaki K, Koshi K, et al. Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its related extracellular matrix degrading enzymes in the endometrium during estrous cycle and early gestation in cattle. Reprod Biol Endocrinol 2010;8:60. 9. Mishra B, Kizaki K, Koshi K, et al. Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its expected roles in the bovine endometrium during gestation. Domest Anim Endocrinol 2012;42:63–73. 10. Fortunato SJ, LaFleur B, Menon R. Collagenase-3 (MMP-13) in fetal membranes and amniotic fluid during pregnancy. Am J Reprod Immunol 2003;49:120–5. 11. Maymon E, Romero R, Pacora P, et al. A role for the 72 kDa gelatinase (MMP-2) and its inhibitor (TIMP-2) in human parturition, premature rupture of membranes and intraamniotic infection. J Perinat Med 2001;29:308–16. 12. Romero R, Chaiworapongsa T, Espinoza J, et al. Fetal plasma MMP-9 concentrations are elevated in preterm premature rupture of the membranes. Am J Obstet Gynecol 2002;187:1125–30. 13. Benirschke K, Kaufmann P, Baergen R. Pathology of the human placenta. New York: Springer-Verlag; 2006. 14. Seval Y, Akkoyunlu G, Demir R, Asar M. Distribution patterns of matrix metalloproteinase (MMP)-2 and -9 and other onhibitors (TIMP-1 and TIMP-2) in the human decidua during early pregnancy. Acta Histochem 2004;106:353–62. 15. Demir-Weusten AY, Seval Y, Kaufmann P, et al. Matrix metalloproteinases-2, -3 and -9 in human term placenta. Acta Histochem 2007;109:403–12. 16. Igakura T, Kadomatsu K, Kaname T, et al. A null mutation in basigin, an immunoglobulin superfamily member, indicates its important roles in peri-implantation development and spermatogenesis. Dev Biol 1998;194:152–65. 17. Kuno N, Kadomatsu K, Fan QW, et al. Female sterility in mice lacking the basigin gene, which encodes a transmembrane glycoprotein belonging to the immunoglobulin superfamily. FEBS Lett 1998;425:191–4. 18. Wang YQ, Li J, Shang T, Wang YL. Expression of extracellular matrix metalloproteinase inducer during normal pregnancy. Zhonhua Fu Chan Ke Za Zhi 2005;40:457–9. 19. Mercer BM, Abdelrahim A, Moore RM, et al. The impact of vitamin C supplementation in pregnancy and in vitro upon fetal membrane strength and remodeling. Reprod Sci 2010;17: 685–95.
ORIGINAL ARTICLE
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The role of extracellular inducer of matrix metalloproteinases in premature rupture of membranes Gennady T. Sukhikh1, Natalia E. Kan2, Victor L. Tyutyunnik2, Maya V. Sannikova2, Elena A. Dubova3, Konstantin A. Pavlov3, Elrad Y. Amiraslanov2, and Nataliya V. Dolgushina4 1
Directorate, Federal State Budget Institution, ‘‘Research Center for Obstetrics, Gynecology and Perinatology’’ of Ministry of Healthcare of the Russian Federation, Moscow, Russian Federation, 2Obstetric Observational Department, 3Department of Pathology, and 4R&D Department, Federal State Budget Institution, ‘‘Research Center for Obstetrics, Gynecology and Perinatology’’ of Ministry of Healthcare of the Russian Federation, Moscow, Russian Federation Abstract
Keywords
Objective: To investigate the role of matrix metalloproteinases (MMP-2, MMP-9) and their inducer (CD147) in premature rupture of membranes (PROM) at term labor. Methods: In a cross-sectional study, 24 women aged 19–39, with 37–40-week pregnancy, and no clinical and histological signs of chorioamnionitis, were divided into two groups with and without PROM. The histological and immunohistochemical study of the fetal membranes was performed with polyclonal rabbit antibodies to MMP-2/MMP-9 and monoclonal rabbit antibodies to CD147. Results: The analysis of MMP revealed the increase of MMP-9 expression in the amniotic epithelium during premature membrane rupture both in rupture area, and beyond it, and increased MMR-2 expression in the mesodermal cells. We also found high level of CD147 in the amniotic epithelium in PROM group. The above-mentioned changes were found in all areas of fetal membranes, regardless of the rupture localization. Conclusions: The study results demonstrate the increased expression of MMR-2 and MMR-9, which regulate the catabolism of fetal membrane extracellular matrix proteins, in amniotic membranes of women with PROM at term labor. The increased expression of CD147 may be one of the mechanisms triggering PROM in the absence of infection.
CD147, MMP-2, MMP-9, premature rupture of membranes (PROM), spontaneous delivery
Introduction The premature rupture of membranes (PROM) is a complication of 3–15.6% of pregnancies and deliveries [1–4]. The PROM origin remains unknown; the scientific community discusses the role of the degradation or remodeling of extracellular matrix (ECM) components regulated by Zn2+dependent proteolytic enzymes – matrix metalloproteinases (MMP) [5,6]. Among the factors affecting ECM remodeling, a special role is played by MMP-2 and MMP-9 as well as by ECM metalloproteinase inducer – EMMPRIN/CD147 [5]. CD147 is a transmembrane glycoprotein with molecular mass of 44–66 kDa containing two extracellular immunoglobulin domains. CD147 plays an important role in spermatogenesis, implantation, cell growth and adhesion, angiogenesis, endometrial cell decidualization, immune cell activation and MMP induction [5,7–9]. Some previous studies have demonstrated the role of
Address for correspondence: Nataliya V. Dolgushina, R&D Department, Federal State Budget Institution, ‘‘Research Center for Obstetrics, Gynecology and Perinatology’’ of Ministry of Healthcare of the Russian Federation, 4, Oparin street, Moscow 117997, Russian Federation. Tel: +7-903-960-83-42. E-mail: [email protected]
History Received 29 January 2015 Accepted 2 February 2015 Published online 9 March 2015
MMP in the PROM [10–12]. Li et al. [5] showed that CD147 is expressed in the human placenta and fetal membranes. This calls for the research into CD147 role in physiological and pathological processes in fetal membranes during the term and preterm pregnancy and delivery, including PROM. Study objective was to investigate the role of MMP-2 and MMP-9 and their inducer (CD147) in premature membrane rupture at term labor.
Materials and methods The cross-sectional study was performed at Obstetric Observational Department of the Research Center for Obstetrics, Gynecology and Perinatology. In this article, we present the initial data of 24 women, recruited in the study. The inclusion criteria were: age 19–39, non-assisted singleton pregnancy resulted at term, spontaneous delivery ranging from 37 to 40 weeks of gestation. All subjects gave written informed consent for participation in the study. The study was approved by Ethics committee for biomedical research of Federal State Budget institution ‘‘Research Center for Obstetrics, Gynecology and Perinatology’’. Women with clinical or morphological signs of chorioamnionitis and with induced delivery were excluded from the study.
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The patients were divided into two groups: group I included 12 patients with deliveries complicated by PROM, and group II included 12 patients without PROM during the delivery. The PROM was determined as a discharge of the amniotic fluid earlier than 2 h prior the regular labor. The amniotic fluid was verified by the level of a2-microglobulin in the posterior vaginal fornix. In group I, the latency period between PROM and labor onset was more than 4 h and composed (6.2 ± 1.4) h. The patients in group II had spontaneous or artificial rupture of membranes after 6-cm cervical dilation. We did not use intravenous oxytocin or other drugs for labor induction in both groups of women. In each group, we made one-time measurement of MMP-2, MMP-9 and CD147 expression level in amniotic membranes. The histological study of fetal membranes was performed following common methods [13]. The fetal membrane tissue samples were taken immediately after delivery. The fetal membranes were studied in the rupture area and beyond the rupture area. The rupture was determined by visual assessment of fetal membranes during the sampling as an area adjacent to the fetal membrane defect. The area included the 10-cm long part of the fetal membrane 1.0 cm from the edge of the defect. The area beyond the rupture was determined visually during the sampling. The histological study of the tissues was performed on paraffin-embedded sections stained with hematoxylin and eosin. The immunohistochemical study of the tissues was performed on 3–4-mm thick paraffin-embedded sections with ready-to-use polyclonal rabbit antibodies to MMP-2 and MMP-9 (Spring Bioscience, Pleasanton, CA) and with monoclonal rabbit antibodies to CD147 (1:100 solution, EPR4052 clone, Epitomics, Burlingame, CA). Automated immunohistochemistry/in situ hybridization (IHC/ISH) slide staining system Ventana Benchmark XT (Roche Group, Basel, Switzerland) with a standard staining protocol was used to perform an immunohistochemical study. MMPs and CD147 expression levels were evaluated in optical density units with Nikon Eclipse 80i microscope and NIS-Element AR 3.2 software (Nikon Corp., Tokyo, Japan) by a single researcher. The data were processed with Statistica 8 (Statsoft Inc., Tulsa, OK) and IBM SPSS Statistics 19 for Windows (IBM Corp., Armonk, NY) software. p50.05 was assumed
statistically significant. Mann–Whitney non-parametric Utest was used to compare the median values of two independent samples for continuous data. The categorical data were presented as percentages and assessed by v2 test.
Results We revealed no statistically significant differences in the rate of pregnancy and delivery complications, the duration of the delivery periods, amount of blood loss, and postpartum complications in two groups of patients. The study participants had comparable baseline characteristics, including the history of infectious and inflammatory diseases (Table 1). The histological analysis of the fetal membranes revealed no statistically significant differences in two groups of patients. It is noteworthy that no leukocyte infiltration was found suggesting the absence of inflammation. The immunohistochemical analysis revealed the increased expression of MMP and CD147 in various components of the fetal membranes. The mesodermal MMP-2 expression was significantly higher in group I, both in the rupture area (p ¼ 0.0004) and beyond it (p ¼ 0.0448) (Table 2). We found no significant differences in MMP-2 expression by amniotic epithelium cells, as well as trophoblast and basal plate decidual cells between the groups (p40.05). The significant increase was Table 1. Clinical characteristics of the study population (n ¼ 24).
Parameter Age, yrs Rate of nulliparity, n (%) Gestational age on inclusion, weeks STD before pregnancy, n (%) Chronic salpingophoritis, n (%) URTI during the pregnancy, n (%) Polyhydramnios, n (%) Oligohydramnios, n (%) Fetal growth retardation, n (%) Chronic fetal hypoxia, n (%) Mild preeclampsia, n (%)
Group I (PROM)
Group II (w/o PROM)
28.3 ± 5.6 1 (8.3) 38.3 ± 0.9 3 (25.0) 2 (16.7) 3 (25.0) 1 (8.3) 0 (0) 0 (0) 1 (8.3) 2 (16.7)
30.3 ± 3.0 0 (0) 39.1 ± 1.0 4 (33.3) 1 (8.3) 2 (16.7) 0 (0) 2 (16.7) 1 (8.3) 2 (16.7) 3 (25.0)
The data are percentages and assessed by v2 test. STD, sexually transmitted diseases; URTI, upper respiratory tract infection. *The results have statistically significant differences.
Table 2. The comparison of MMP-2 and MMR-9 expression levels in the fetal membranes and placental cells. MMP-2 Localization Fetal membranes in the rupture area Fetal membranes beyond the rupture area Villi
PROM Amniotic epithelium Mesenchymal cells Basal plate trophoblast Basal plate decidual cells Amniotic epithelium Mesenchymal cells Basal plate trophoblast Basal plate decidual cells Endothelial cells Synticiotrophoblast Mesenchymal cells
21.5 ± 10.6 15.8 ± 4.6 18.1 ± 6.8 18.6 ± 8.2 18.5 ± 11.6 15.4 ± 6.7 17.6 ± 6.2 19.0 ± 6.2 9.3 ± 3.9 12.0 ± 4.8 9.7 ± 3.9
MMR-9
w/o PROM
p value
PROM
17.1 ± 7.8 9.2 ± 1.8 18.3 ± 3.3 17.8 ± 2.0 18.3 ± 8.3 12.2 ± 1.9 20.5 ± 2.1 19.1 ± 4.5 4.6 ± 4.8 7.9 ± 8.0 5.8 ± 5.7
0.1313 0.0004* 0.4446 0.2035 0.3312 0.0448* 0.2223 0.1250 0.0202* 0.2884 0.1250
28.8 ± 3.7 14.2 ± 1.4 23.1 ± 3.9 19.0 ± 4.5 31.6 ± 2.9 15.6 ± 3.3 24.1 ± 4.8 18.2 ± 2.3 12.0 ± 2.7 20.1 ± 4 15.3 ± 2.9
The data are M±s in optical density units and assessed by Mann–Whitney U-test. *The results have statistically significant differences.
w/o PROM
p value
25.2 ± 2.2 14.6 ± 2.2 22.3 ± 2.5 18.5 ± 2.6 26.4 ± 2.7 16.8 ± 3.8 22.5 ± 1.6 19.7 ± 1.4 11.2 ± 1.6 22 ± 2.8 15.8 ± 2.6
0.0196* 0.2470 0.9546 0.9095 0.0007* 0.0976 0.5742 0.0933 0.7732 0.2268 0.9018
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DOI: 10.3109/14767058.2015.1015416
MMPs/CD147 role in PROM
3
Figure 1. Box and whisker plot, defining CD147 expression in the fetal membrane amniotic epithelium in the rupture area (A) and beyond the rupture area (B), in the group with PROM (1) and without PROM (2). The median of CD147 expression is significantly higher in women with PROM both in the rupture area (p50.0001) and beyond it (p ¼ 0.0030).
found in MMP-2 expression in the endothelial cells of acroteric villi in group I (p ¼ 0.0202). MMP-9 expression in group I was significantly higher in the amniotic epithelium both in the rupture area (p ¼ 0.0196) and beyond it (p ¼ 0.0007). No significant differences were found in MMP-9 expression by chorionic and amniotic mesoderm, as well as trophoblast and basal plate decidual cells between the groups (p40.05). CD147 expression in group I was significantly higher in the amniotic epithelium both in the rupture area (p50.0001) and beyond it (p ¼ 0.0030) (Figure 1). Moreover, group I demonstrated the decrease of CD147 expression by mesodermal cells, that was not significant in the rupture area (p40.05) and significant beyond it (p ¼ 0.0334) (Figure 2).
Discussion The results of this study confirm those from previous investigations that MMP-9 is predominantly expressed by epithelium while MMP-2 is expressed by stromal elements [14]. In the first trimester, MMP-2 and MMP-9 are mainly distributed in the extravillous trophoblast and villous cytotrophoblast, respectively [9]. The labor onset is accompanied by the significant increase of MMP-2 and MMP-9 activity in the amnion and decidual tissue [11]. MMP-9 plays an important role in the placenta delivery [15]. Our data demonstrate the higher expression of MMP-2 in the mesoderm and MMP-9 in the amniotic epithelium of the fetal membranes together with increased MMP-2 level in the amniotic rather than the chorionic tissues, suggesting different association and localization of the MMPs in question in PROM after term delivery. The analysis of CD147 expression in various fetal membrane structures is especially interesting. We found higher CD147 expression in the amniotic epithelium of fetal membranes both in the rupture area and beyond it, and lower expression in the amniotic mesoderm cells in the patients with deliveries complicated by PROM. Besides that, we found higher CD147 level in the amniotic epithelium of both areas in question compared to mesenchymal cells of fetal
membranes. These topographic characteristics of CD147 distribution are apparently the causes of the increased MMPs expression that we found. This suggests independent CD147 activation in PROM that is different from its activation during the delivery. The increased CD147 expression is described in the uterine mucous membranes and embryonic trophoectoderm during blastocyst implantation [16,17]. At 6–9 weeks of gestation as well as in the middle and in the end of the full-term pregnancy, CD147 is predominantly found in trophoblast and sincitiotrophoblast membranes and trophoblast column villi [18]. In the middle and in the end of the full-term pregnancy, CD147 expression was demonstrated in extravillous cytotrophoblast and decidual tissue. The maximal CD147 expression was found at the 6th week of pregnancy with the consequent gradual decrease. During the 2nd and 3rd trimester, CD147 expression was constant with the gradual decrease in extravillous cytotrophoblast and decidual tissue by the end of the pregnancy [18]. Nowadays, the main role in PROM genesis is attributed to the infection. The inflammation caused by the infection results in the production of inflammation mediators – cytokines, which, in turn, activate MMP causing ECM degradation and PROM. The immediate cause of the rupture is the necrosis of the amniotic epithelium and compact substance. In our study, we included the women with PROM and no signs of inflammation in fetal membranes, which were confirmed by the histological analysis. Mercer et al. [19] suggested that in this case, the strength of the fetal membranes decreases during the pregnancy due to amniotic epithelium apoptosis and ECM degradation resulting in the rupture. The mechanical stretching of the membranes may increase MMP production through cytokine cascade [1,4]. Our study has certain limitations since we studied placenta samples after term spontaneous deliveries that may distort the results. CD147 expression increases in placenta and fetal membranes in term spontaneous deliveries. However, Li et al. [5] found no differences in CD147 localization in placental and fetal membrane structures after vaginal and
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Figure 2. Box and whisker plot, defining CD147 expression in the fetal membrane mesodermal cells in the rupture area (A) and beyond the rupture area (B), in the group with PROM (1) and without PROM (2). The median of CD147 expression is significantly lower beyond the rupture area (p ¼ 0.0334) and insignificantly lower in the rupture area (p40.05).
cesarean deliveries. They demonstrated CD147 expression by syncytiotrophoblast, amniotic epithelium, as well as by extravillous trophoblast and decidual cells, but not by amniotic mesenchymal cells. These data differ from our findings. The study findings suggest the probable pathogenic mechanism of PROM, which results from the disruption of fetal membrane ECM homeostasis. In the absence of infection MMP-2, MMP-9 and CD147 are strongly associated with PROM. On the other hand, the study data call for further research into the degree, level and characteristics of CD147 involvement into the fetal membrane ECM remodeling during the delivery as well as in term and preterm membrane rupture.
Declaration of interest This work was supported by Ministry of Education and Science of the Russian Federation (grant # 16.522.12.2009). The authors report no conflicts of interest.
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