Pretreatment with MPA significantly reduced basal MMP-9 activity in primary amnion cells compared with the unstimulated control [mean difference (95% CI) = −49.6 (−81.9, −17.3) units, P = 0.001] (Fig. 4A, Table 1). Pretreatment with MPA also significantly reduced basal MMP-9 gene expression in primary amnion cells compared with the unstimulated control [mean difference (95% CI) = −53.4 (−105.9, −0.9) units, P = 0.045] (Fig. 4B, Table 2). Pretreatment with P4 and 17P did not significantly reduce basal MMP-9 activity and gene expression when compared with the unstimulated control in primary amnion cells (Fig. 4, A and B, Tables 1 and 2).
In chorion cells, no significant differences in basal MMP-9 activity between the unstimulated control and the progestin treated groups were observed (Fig. 4A, Table 1). Similarly no differences in basal MMP-9 gene expression were observed between the unstimulated control and the progestin-treated groups (Fig. 4B, Table 2).
Pretreatment with MPA significantly reduced TNFα-induced MMP-9 activity in primary amnion cells compared with the stimulated control [mean difference (95% CI) = −69.0 (−91.8, −46.3) units, P < 0.001] (Fig. 5A, Table 3). Pretreatment with MPA also significantly reduced TNFα-induced MMP-9 gene expression in primary amnion cells compared with the stimulated control [mean difference (95% CI) = −86.0 (−120.7, −51.3) units, P < 0.001] (Fig. 5B, Table 4). Pretreatment with P4 and 17P did not significantly reduce TNFα-induced MMP-9 activity and gene expression compared with the stimulated control in primary amnion cells (Fig. 5, A and B, Tables 3 and 4).
No significant differences in TNFα-induced MMP-9 activity were observed between the progestin-treated groups stimulated with TNFα and the stimulated control in primary chorion cells (Fig. 5A, Table 3). Similarly, no significant differences in TNFα-induced MMP-9 gene expression were observed between the progestin-treated groups stimulated with TNFα and the stimulated control in primary chorion cells (Fig. 5B, Table 4).
Our results demonstrate that MPA pretreatment significantly inhibited both basal and TNFα-induced MMP-9 activity and gene expression in primary amnion cells. Interestingly, P4 and 17P did not inhibit basal and TNFα-induced MMP-9 activity and gene expression in primary amnion cell cultures. Progestin pretreatment also did not significantly inhibit basal and TNFα-induced MMP 9 activity and gene expression in primary chorion cells.
Our findings suggest that the amnion layer maybe a site of action for progestin therapy in preventing inflammation-induced fetal membrane tissue remodeling. The amnion layer is responsible for most of the tensile strength of fetal membranes and ultimately has to rupture for PPROM to occur.26 In vitro studies have demonstrated that the sequence of events that leads to fetal membrane rupture includes fetal membrane distension, separation of the amnion and choriodecidua, choriodecidual rupture, further nonelastic amnion distension followed ultimately by rupture of the amnion.26 MMP-9 significantly contributes to the initiation of membrane rupture by degrading collagen fibers in the spongy layer of the amnion, leading to dissociation of the amnion and chorion.27 This is in addition to degrading collagen and other extracellular matrix proteins in the compact and fibroblast layer along with the basement membrane of the amnion, which also contributes to the reduction in tensile strength. A reduction in fetal membrane tensile strength is directly correlated with MMP-9 protein expression in fetal membranes in term laboring patients.28 TNFα-induced MMP-9 protein expression and the resulting collagen remodeling have also been correlated with fetal membrane weakness in vitro.13 Additionally, inhibition of TNFα-induced fetal membrane weakening by alpha-lipoic acid has been associated with a concomitant reduction in TNFα-induced MMP-9 expression in full thickness fetal membranes and primary amnion epithelial cells.29 We hypothesize, based on the profound suppression by MPA of MMP-9 activity and gene expression in primary unstimulated and TNFα-stimulated amnion cells, that this is a mechanism by which progestins may act to maintain fetal membrane integrity in the setting of inflammation and other idiopathic causes of PPROM. However, tissue biomechanical studies are now needed to confirm this hypothesis.
Our findings suggest that the reductions in basal and TNFα-induced MMP-9 activity in primary amnion cells by MPA are mainly a result of effects on gene expression. In most tissues,MMP-9 gene expression is low but can be induced by inflammatory cytokines. Transcriptional activation of MMP-9 gene expression by inflammatory cytokines occurs primarily through the transcription factors nuclear factor κB and activator protein 1 pathways in fetal membranes.30,31 Additionally, there are transcription factors that negatively regulate MMP-9 gene expression, such as kisspetin and metastasis associated gene 1.32–34 These repressors inhibit MMP-9 gene expression either by inhibiting nuclear factor-κB activation or by forming a repressor complex that binds to the MMP-9 promoter site and maintaining it in a silenced state.32,33 Inhibition of positive regulators, or induction of negative regulators of MMP-9 gene expression, maybe mechanisms by which MPA inhibits both basal and TNFα-induced MMP-9 mRNA expression in primary amnion cells.
The receptors by which MPA initiates these effects in the amnion are unclear. Amnion epithelial cells do not express the PR-A and PR-B subtypes of the progesterone receptor, which is thought to mediate many of the biological effects of progestins.35 Our previous work has demonstrated the presence of a novel progesterone receptor, which may play a role in mediating the effects of progestins in fetal membranes.18,36 This receptor, progesterone membrane component 1, is expressed in all layers of the fetal membranes and in the HTR8/SVneo cytotrophoblast cell line. In the HTR8/SVneo cell line, we demonstrated that progesterone membrane component 1 mediate the inhibitory effect of MPA on TNFα-induced MMP-9 activity.18 Recently, the glucocorticoid receptor has also been implicated as possible receptor mediating the suppression of IL-1β induced increases in COX-2 expression by MPA and P4 in human myometrial cells.37 Both receptor pathways are being investigated in our laboratory.
Progestin therapy did not attenuate basal or TNFα-induced MMP-9 mRNA expression or activity in primary chorion cells. Although the chorion layer is thicker than the amnion, it does not possess its tensile strength. The role of the chorion in inflammation-induced membrane rupture is still unclear, but it does appear to contribute indirectly to the inflammatory process through its production of soluble factors that may weaken the amnion.38 The inflammatory response of the chorion layer and its response to progestin therapy may also be modulated by both the decidua and amnion layer through paracrine effects. This may lead to different responses in cell cultures experiments when compared with tissue culture and in vivo treatment conditions.22 We were also unable to demonstrate a response to P4 or 17P on basal or TNFα induced MMP-9 activity and expression in primary amnion cells. Genetic variations in receptor types and the metabolic enzymes involved in progestin-mediated mechanisms and metabolic pathways may partially explain these results.39,40 For example, we know that the clinical response to 17P therapy is quite variable; up to two-thirds of patients have recurrent preterm births while on treatment.39,40 A lack of response to P4 may also be related to the concentrations of P4 used in our experiments. Myometrial studies have established that higher concentrations of progesterone (10–3 to 10–5 M) suppress both spontaneous myometrial contractility and cytokine-induced cyclooxygenase 2 expression.37,41 However nonspecific steroid effects due to changes in cell membrane fluidity can occur with doses of progesterone in excess of micromolar concentrations.37,41 Another possibility for the lack of response in primary amnion cells is that both P4 and 17P may exert their effects on mechanisms that prevent PPROM independent of MMP-9 activity or gene expression in fetal membranes.
One limitation of our study is that experiments were performed using cells isolated from the fetal membranes of term pregnant patients, and not preterm or PPROM patients. We opted not to use fetal membranes from PPROM patients due to their increased bacterial colonization and because the fetal membranes of these patients have already undergone the pathological changes, such as MMP activation and apoptosis, that result in membrane rupture.42,43 There is also limited data on the biomechanical properties of preterm fetal membranes. But preterm fetal membranes may have greater tensile strength and less regional variation in biomechanical and histological properties than term fetal membranes harvested from patients who did not labor.44,45 Whether primary amnion and chorion cells isolated from preterm fetal membranes would respond to progestin treatment and cytokine stimulation in a similar way to those harvested from term fetal membranes is debatable. Another limitation is that MPA was the only progestin which attenuated both basal and TNFα-induced MMP-9 activity and gene expression in primary amnion cells. MPA bears a category X classification from the Food and Drug Administration and is contraindicated in pregnancy.a Additionally, it is not in clinical use for the prevention of preterm delivery. However, MPA is commonly used in in vitro cell and tissue culture experiments due to its increased stability when compared with other progestins.46 Fetal membranes also express enzymes that metabolize progesterone, and although systematic studies comparing the metabolism of natural and synthetic progestins by the amniochorion have not as yet been conducted, it is possible that P4 may be more effectively metabolized when compared with MPA or 17P in vitro.47
In summary, we demonstrated that MPA inhibits both basal and TNFα-induced MMP-9 activity and gene transcription in primary amnion cells harvested from term pregnant women. Our data highlight one of the mechanisms by which progestins may prevent inflammation-induced fetal membrane weakening that may lead to PPROM. The cell signaling pathways by which MPA decreases MMP-9 activity and gene expression require further investigation in light of the absence of the classic nuclear progesterone receptor in primary amnion cells.
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