362

Fortunato et al, MMPITIMP imbalance during PROM

1. Perinat, Med. 27 (1999) 362-3!lJ!...

MMPITIM:P imbalance in amniotic fluid during PROM: an indirect support for endogenous pathway to membrane rupture
Stephen J. Fortunato+', Ramkumar Menon1,2,\ and Salvatore J. Lembardi! (Maternal Fetal Group, 2Perinatal Research Center of The Women's Health Re search and Education Foundation, and 3Aquinas College, Nashville, rn, USA

1 Introduction Preterm premature rupture of the fetal mem branes (PROM) is a major complication of preg nancy associated with over 40 % of preterm labor. Fetal membrane extracellular matrix (ECM), comprising the basement membrane and the un derlying stroma, connects amnion and chorion cells together. Degradation of ECM by excessive matrix metalloprotease (MMP) activity has been described as the cause of rupture [4, 9, 17, 18]. MMPs are members of a family of at least fifteen Zn dependent endopeptidases capable of degrad ing specific components of the ECM. Under physiological conditions, MMP activity is pre cisely controlled at the transcriptional, transla tional and post translational levels [3, 14]. Post translational regulation can be mediated by tissue inhibitors of metalloproteinases (TIMPs). They act as natural inhibitors of MMP activity by forming an inactive 1:1 stoichiometric complex with the MMPs. Gelatinases QvIMP2 and 9) are members of an :MtviIIf~le of specifically degrading Type IV collagen of the basement membrane. Our laboratory has documented the expression pattern of gelatinases and ~s in human fetal membranes [6, 7]. We propose that an imbal ance in the levels of gelatinases and their inhibi tors may result in the increased activity of MMPs resulting in ECM degradation. We have already documented this phenomenon in human fetal membranes in culture in response to bacte-

rial lipopolysaccharide [5]. In this study we are examining the levels of gelatinases and TIMPs in the amniotic fluid of women with pathologi cal complications of pregnancy like PROM, pre term labor (PTL) compared with the levels seen at term. Active, TIMP free forms of the gela tinases in the amniotic fluid are also quantitated in this study. The imbalance in the molar ratio between these enzymes and their inhibitors with respect to their significance in PROM is also discussed. 2 Materials and methods ~

This study has been approved by the nstitutional Review Boardat The Women's Ho ital at Centennial Medical Center, Nashville, , as an exempt protocol.

Amniotic fluid samples from atients who met the entry criteria were selecte from our bank of previously collected and fro en samples. This study included women in e following cate gories. Group 1 (n = 16) onsisted of women with preterm premature rup e of the membranes (pROM group) which wa defined as spontane ous amniorrhexis prior to the onset of labor be fore 37 weeks gestation. embrane rupture was diagnosed using positiv identifiers such as nit razine test, fern test, ooling and oligohydram nios on ultrasound ex ination in the presence of

1999 by Walter de Gruyter GmbH & Co. KG Berlin

New York

364 TIMPs in the 'otic fluid was subjected to Tu key-Kramer ultiple comparison test. A p value < 0.05 wa considered significant. ~ ~ 3 Results

Fortunato et al, MMPITIMP imbalance during PROM

No statistically significant difference was no ticed between term and PTL groups (figure 1). Analysis of TIMP-2 levels documented decreased levels of this protein in the amniotic fluid of women with PROM. The levels were 98 ng/ml (SD 46) in the PROM group compared to 176 ng/ml (SD 68) in term (p < 0.05) and 237 ng/ml (SD 132) in PTL (p < 0.001) groups (figure 1). Specific bioactivity assays were employed to document the TIMP free forms of MMP2 and 9 because the levels mentioned above include pro, active and TIMP bound forms of both gela tinases. The bioactivity measured here documents biologically active, TIMP free MMPs. Zymogra phy followed by densitometric quantitation docu mented a significant increase in the active forms of MMP2 in PROM group (233 pg/ml; SD 167) compared to the term group (135 pg/ ml; SD 97; P < 0.05) and the PTL group (132 pg/ml; SD 86; P < 0.05). No significant change was noticed between PTL and term groups. It is interesting to note that less than 0.01 % of the total IR-MMP2 was active in any of these samples (figure 2).

Amniotic fluid IR-MMP2 levels are increased during PROM (2,125, SD 429 ng/ml) com pared to term (1,455, SD 714 ng/ml; p < 0.01). Although elevated, the levels of IR-MMP2 in the PTL group (1,862, SD 714 ng/ml) was not sig nificantly different compared to the other two groups (p > 0.05) (figure 1). Similarly amniotic fluid levels of IR-MMP9 were also elevated in PROM groups (15.03, SD 14.5 ng/ml) com pared to PTL (3.75, SD 10.09 ng/ml; p < 0.01) and term (0.2, SD 0.8 ng/ml; p < 0.001). A significant difference between the term and PTL groups was not observed (figure 1; see inset). TIMP-l levels were also elevated during PROM as documented by ELISA. In the PROM groups the levels were 3143 ng/ml (SD 1499) where as term and PTL groups showed levels of 1891.6 ng/ml (SD 1012; p < 0.05) and 2406 ng/ml (SD 1461; P > 0.05) respectively.

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Figure 1. ELISA documents amniotic fluid levels of MMP2, MMP9 (see inset), TIMPI and TIMP2 in PROM, PTL and at term. Mean values are plotted for graphical representation. 1. Perinat. Med. 27 (1999)

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366 expression and release can be induced by labor or infection [6]. This study documented an increase in IR-MMP9 during PROM. Initially we thought that this small amount of MMP9 would be overpow ered by TIMP 1 and thereby not available for ECM degradation. However; the activity assay docu mented that a small percentage of this IR-MMP9 was actually TIMP free and active. The presence of bioactive MMP9 during PROM and its absence in both the term and PTL groups despite the presence oflR-MMP9, is suggestive of a separate final acti vation pathway for IR-MMP9 found only in the pa thologic milieu associated with PROM. Active MMP2 and stromelysinl (MMP3) can activate IR MMP9 [10). We have already documented both stromelysinl increases in the amniotic fluid during PROM [8] along with increased active MMP2 as documented above. A synergistic action of these MMPs may tum on a cascade of MMP activation capable of degrading various components of the ECM. At term and during PTL the absence of active forms of MMP9 and a non-significant increase in the levels of active MMP2 suggests a definitive role for these MMPs during PROM.

Fortunato et ai, MMPrrlMP imbalance during PROM

sins or collagenases in this calculation, which are also elevated during PROM and are inhibited by TIMP 1. TIMP 1 also shows growth promoting activities in various cell types in addition to its inhibitory action on MMPs [11). This may ex plain why this growth promoterlMMP inhibitor is present in high levels in the amniotic fluid throughout gestation. We speculate that during the growth and development of the placenta, TIMPI may playa dual role. It can control colla genolysis during remodeling of the placenta by inhibiting MMPs while as a growth factor it helps placental development by promoting DNA repli cation and cell growth. The dynamic equilibrium between synthesis and degradation of the ECM components during ges tation, engendered by a balanced activity between the MMPs and the TIMPs maintains the structural and functional integrity of the membrane. This rebuilding activity sustains the increased pressure and volume exerted during placental growth. During PROM the excessive levels of gelatinases and TIMPs reflects a condition in which MMP2 and MMP9 overcome their inhibitors of biologi cal activity. The disruption of this balance can create subtle variations in the control of the MMP axis resulting in PROM rather than tissue remod eling. This pathway is not evident during PTL or at term suggesting a separate terminal pathway for each of these conditions. The presence of active forms of both the gelatinases and the stro melysins in addition to reduced availability of the TIMPs supports the theory that PROM very well mayan endogenous autotoxic disease initiated by certain exogenous factors or stimuli such as in fection. Apoptosis of the fetal membrane cells should be considered as one of the signals for MMP activation predisposing to PROM [12]. Further research is warranted to document the signals that adjudge PROM Vs PTL.

4.3 Overabundanceof amniotic

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TIMPI

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Unlike TIMP2, TIMPI is capable of binding to other members of the MMP family. TIMPI is a multi-functional protein and MMP inhibition is just one of its many properties. Large quantities of TIMPI may indicate a host response designed to minimize the biodegradation initiated by a variety of MMPs (stromelysin, gelatinases, and collagenases) that are active during PROM [2, 8]. We calculated the molar ratio between gelatinases and TIMPs (1 + 2) and found no difference be tween term, PTL or PROM groups (0.3 in all cases). We haven not accounted for the stromelyAbstract Objective: We theorize that excessive degradation of the fetal membrane extracellular matrix (ECM) by spe cific matrix metalloproteinases (MMPs) results in pre term premature rupture of the membranes (PROM). Active, inhibitor free MMP2 and 9 (gelatinase A and B respectively) can degrade the anmiochorion basement membrane Type IV collagen to initiate rupture. This study examines the levels of the gelatinases and their

natural inhibitors (tissue inhibitor of matrix metallopro teinases -TIMPs) in the amniotic fluid during PROM, preterm labor (PTL) and at term. Methods: A total of 51 AF samples were collected from the following groups of patients. Group I: Women with PTL and no ROM (n = 16) Group 2: Women with PROM (n = 16) irrespective of labor status Group 3: Women at term with intact mem-

J. Perinat. Med. 27 (1999)

368 tabolism in premature rupture of amniotic mem branes. Obstet Gynecol 75 (1990) 84 [18] Vadillo-Ortega F, A Hernandez, G Gonzalez Avila, L Bermejo, K Iwata, JF Strauss 3,d: Increased matrix metalloproteinase activity and re duced tissue inhibitor of metalloproteinases-l levels in amniotic fluids from pregnancies compli cated by premature rupture of membranes. Am J Obstet Gynecol 174 (1996) 1371 Received June 14, 1999. Accepted July 10, 1999.

Fortunato et al, MMPrrIMP imbalance during PROM

Stephen 1. Fortunato, M. D. Director of Maternal-Fetal Medicine Perinatal Research Center Suite 310 2201 Murphy Ave. Nashville, TN 37203 USA Tel: +615342-3917 Fax: +615340-6722 e-mail: fortunat@edge.net

1. Perina!. Med. 27 (1999)