Secondary Logo

Journal Logo

The Effect of Progestins on Tumor Necrosis Factor α-Induced Matrix Metalloproteinase-9 Activity and Gene Expression in Human Primary Amnion and Chorion Cells In Vitro

Allen, Terrence K. MBBS, FRCA*; Feng, Liping MD; Nazzal, Matthew BS*; Grotegut, Chad A. MD, MHS; Buhimschi, Irina A. MD‡§; Murtha, Amy P. MD

doi: 10.1213/ANE.0000000000000708
Obstetric Anesthesiology: Research Report
Free

BACKGROUND: Current treatment modalities for preventing preterm premature rupture of membranes are limited, but progestins may play a role. Tumor necrosis factor α (TNFα) enhances matrix metalloproteinase-9 (MMP-9) gene expression and activity in fetal membranes, contributing to membrane weakening and rupture. We previously demonstrated that progestins attenuate TNFα-induced MMP-9 activity in a cytotrophoblast cell line. However, whether they have a similar effect in primary amnion and chorion cells of fetal membranes is unknown. In this study, we evaluated the effect of progestins on basal and TNFα-induced MMP-9 activity and gene expression in primary chorion and amnion cells harvested from the fetal membranes of term nonlaboring patients.

METHODS: Primary amnion and chorion cells were isolated from fetal membranes obtained from term uncomplicated nonlaboring patients following elective cesarean delivery (n = 11). Confluent primary amnion and chorion cell cultures were both pretreated with vehicle (control), progesterone (P4), 17α-hydroxyprogesterone caproate (17P), or medroxyprogesterone acetate (MPA) at 10–6 M concentration for 6 hours followed by stimulation with TNFα at 10 ng/mL for an additional 24 hours. Cell cultures pretreated with the vehicle only served as the unstimulated control and the vehicle stimulated with TNFα served as the stimulated control. Both controls were assigned a value of 100 units. Cell culture medium was harvested for MMP-9 enzymatic activity quantification using gelatin zymography. Total RNA was extracted for quantifying MMP-9 gene expression using real-time quantitative PCR. Basal MMP-9 activity and gene expression data were normalized to the unstimulated control. TNFα-stimulated MMP-9 activity and gene expression were normalized to the stimulated control. The primary outcome was the effect of progestins on TNFα-induced MMP-9 enzymatic activity in term human primary amnion and chorion cells in vitro. Secondary outcomes included the effect of progestin therapy on TNFα-induced MMP-9 gene expression and on basal MMP-9 activity and gene expression in primary amnion and chorion cells in vitro.

RESULTS: Primary cells were harvested from 11 patients. Compared with the unstimulated control, TNFα increased MMP-9 activity (P = 0.005 versus control in primary amnion cells and P < 0.001 versus control in primary chorion cells) and MMP-9 gene expression (P = 0.030 versus control in primary amnion cells, P < 0.001 versus control in primary chorion cells). Compared with the unstimulated controls, MPA, but not P4 or 17P, reduced basal MMP-9 activity [mean difference (95% CI) −49.6 (−81.9, −17.3) units, P = 0.001] and gene expression [mean difference (95% CI) −53.4 (−105.9, −0.9) units, P = 0.045] in primary amnion cells. Compared with the stimulated control, MPA also reduced TNFα-induced MMP-9 activity [mean difference (95% CI) −69.0 (−91.8, −46.3) units, P < 0.001] and gene expression [mean difference (95% CI) −86.0 (−120.7, −51.3) units, P < 0.001] in primary amnion cells. Progestin pretreatment had no significant effect on basal or TNFα-induced MMP-9 activity and gene expression in primary chorion cells.

CONCLUSIONS: The inhibitory effect of MPA on both basal and TNFα-induced MMP-9 activity and gene expression in primary amnion cells demonstrate a possible mechanism by which progestins may prevent fetal membrane weakening leading to preterm premature rupture of membranes.

From the *Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina; Department of Obstetrics and Gynecology Duke University Medical Center, Durham, North Carolina; Center for Perinatal Research, The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio; and §Departments of Pediatrics and Obstetrics/Gynecology, The Ohio State College of Medicine, Columbus, Ohio.

Accepted for publication February 4, 2015.

Funding: NIH T32 award Grant No. T32 GM008600 and The Society of Obstetric Anesthesia and Perinatology/Gertie Marx Research Grant to Dr. Allen.

The authors declare no conflicts of interest.

This report was previously presented, in part as an Oral Presentation in the Best Paper of the Meeting Session, at the Society for Obstetric Anesthesiology and Perinatology’s Annual Meeting, Toronto, Canada, May 14–18, 2014.

Reprints will not be available from the authors.

Address correspondence to Terrence K. Allen, MBBS, FRCA, Department of Anesthesiology, Duke University Medical Center, Box 3094, Durham, NC 27710. Address e-mail to terrence.allen@dm.duke.edu.

The rate of preterm delivery in the United States is 11.4%,1 which is the highest preterm delivery rate of all industrialized nations. Up to 30% of preterm deliveries result from preterm premature rupture of membranes (PPROM)—rupture of membranes prior to the onset of labor and before 37 weeks of gestation.2 Infants born as a result of PPROM are at a greater risk of developing complications related to prematurity compared with those resulting from spontaneous preterm delivery from other etiologies.3,4 The resulting emotional impact on parents and the significant health care costs incurred make PPROM a major public health problem.4

Mechanisms leading to PPROM are distinctly different from those that result in other causes of preterm delivery.5–7 Inflammation from localized or systemic infections, uterine overdistension or increased genetic susceptibility, stimulate the release of inflammatory cytokines such as tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β), which, in turn, stimulate a localized inflammatory response in fetal membranes.2,6–11 Inflammatory cytokines stimulate the release of matrix metalloproteinases (MMPs) from the amnion and chorion layer of fetal membranes. Matrix metalloproteinases, specifically matrix metalloproteinase-9 (MMP-9), degrade the extracellular matrix proteins, such as types I, III, and IV collagen, fibronectin, and laminin.12 These matrix proteins provide the tensile strength for fetal membranes, particularly in the amnion layer. They also form the basement membrane for cell adhesion. Increased MMP-9 expression and activity leads to matrix protein degradation, resulting in cell detachment from the basement membrane, apoptotic cell death and a reduction in tissue tensile strength.12 The clinical importance of MMP-9 in the pathophysiology of PPROM is highlighted by the following: (1) Increased MMP-9 protein levels in amniotic fluid and enhanced MMP-9 gene expression in fetal membranes have been associated with PPROM compared with other causes of preterm delivery, (2) A polymorphism in the promoter region for the MMP-9 gene in human amnion epithelial cells is associated with an increased risk of PPROM, and (3) TNFα-induced MMP-9 expression results in a significant decrease in the force and work needed to rupture human term fetal membranes in in-vitro studies.6,12,13

Clinically, progestin therapy has been used for the prevention of recurrent preterm birth, but its role in the prevention of PPROM remains unclear.14,15 Progestins such as medroxyprogesterone acetate (MPA) have been shown to inhibit cytokine-induced MMP-1 and MMP-3 expression and activity in term decidua cells in vitro.16 Progestins also inhibit basal and TNFα induced apoptosis in fetal membranes.17 We have also demonstrated that pretreatment with MPA inhibits TNFα-induced MMP-9 activity in a human cytotrophoblast cell line, highlighting a possible mechanism by which progestins may prevent PPROM.18 However, the effect of progestins on cytokine-induced MMP-9 activity and gene expression in human primary chorion and amnion cells has not been described. The underlying hypothesis for this study is that progestins attenuate TNFα-induced MMP-9 activity and gene expression in human primary amnion and chorion cells. This could indicate a possible mechanism by which progestins may prevent inflammation-induced fetal membrane rupture. The primary outcome of this study was to evaluate the effect of progestin pretreatment on TNFα-induced MMP-9 enzymatic activity in term human primary amnion and chorion cells in vitro. Secondary outcomes included the effect of progestin pretreatment on TNFα- induced MMP-9 gene expression, basal MMP-9 enzymatic activity and basal MMP-9 gene expression.

Back to Top | Article Outline

METHODS

Fetal Membrane Collection and Primary Cell Harvesting

This study was approved by the Duke Medicine Institutional Review Board with a waiver of consent for fetal membrane collection. Full thickness fetal membrane samples were collected from term healthy patients at elective cesarean delivery without prior rupture of membranes or labor. To anonymize sample collection, both the identification of suitable patients and the collection of fetal membranes were performed by authorized research personnel who had no further involvement in subsequent experiments. Samples were collected when these research personnel were available between September 2013 and July 2014. Immediately following delivery of the placenta, pieces of fetal membranes (approximately 10 × 10 cm) were cut from the placenta and transported in Dulbecco modified eagle media—Ham’s F12 (DIMEM/F12) (Gibco®, Life Technologies, Carlsbad, CA) culture media with 10% fetal bovine serum (FBS) and antibiotics and antimycotics (streptomycin and penicillin and amphotericin B). Fetal membranes were washed in culture media to remove blood and debris and then cut into 5 × 5 cm squares. The amnion layer was peeled off from the choriodecidua with forceps, and the chorion then bluntly dissected from the decidua with a scalpel blade. Using a previously described protocol, primary amnion and chorion epithelial cells were harvested.19 Briefly, both the amnion and chorion were diced using two scalpel blades and then digested in 0.125% trypsin with 0.2% collagenase for 60 to 90 minutes at 37°C with intermittent shaking to promote tissue digestion. The cells were then filtered through 4 layers of sterile gauze and centrifuged at 2000 rpm for 5 minutes. The resulting pellet was resuspended in serum-free media and layered on a cell separation gradient prepared with an Optiprep™ Density Gradient Medium (Sigma-Aldrich, St. Louis, MO) column (4%:6%:8%:10%:20%:30%:40%) and centrifuged at 2000 rpm for 30 minutes. Harvested cells were washed in DIMEM/F12 media with 10% FBS and antibiotics and antimycotics, cultured in 12-well cell culture plates and grown to 80% to 90% confluence in 21% oxygen, 5% CO2 at 37°C. Immunocytochemistry staining was performed using cytokeratin and vimentin to determine the purity of the cell population cultured.20

Back to Top | Article Outline

Experimental Conditions

The experimental conditions are highlighted in Figure 1. For each fetal membrane sample collected, unpassaged cultured primary amnion and chorion cells were switched to serum-free media for 24 hours. Using our previously described methodology, the 2-cell culture wells were each pretreated with ethanol 0.01% as vehicle (Decon Labs, King of Prussia, PA), progesterone (P4) (Sigma-Aldrich), medroxyprogesterone 17-acetate (MPA) (Sigma-Aldrich), or 17α-hydroxyprogesterone caproate (17P) (Steraloids, Newport, RI) at 10–6 M for 6 hours in serum-containing media.18 The concentrations of progestins used were based on placental levels measured in pregnancy.21 Prior experiments performed in our lab and previously published data have established that concentrations of progestins in the micromolar range have therapeutic effects in vitro.17,18,22,23 The cell cultures were switched to serum-free media supplemented with the vehicle and progestins. One cell culture well for each treatment group was incubated with TNFα 10 ng/mL for an additional 24 hours in serum-free media.18 The vehicle without TNFα served as the unstimulated control for basal MMP-9 activity and gene expression. The vehicle control with TNFα served as the stimulated control for the progestin pretreated groups, stimulated with TNFα. Following 24 hours of TNFα stimulation, cell culture medium for MMP-9 activity quantification by gelatin zymography was harvested, centrifuged at 15000 rpm for 5 minutes and the supernatant was immediately frozen at −80°C. Cell lysates were harvested with TRIzol® reagent (Life Technologies) and frozen at −80°C for RNA extraction and real-time quantitative PCR (RT-qPCR).

Figure 1

Figure 1

Back to Top | Article Outline

Gelatin Zymography

Gelatin zymography was used to quantify MMP-9 enzymatic activity in vitro using the manufacturer’s protocol.18 Briefly, harvested cell culture media was incubated in a 1:1 ratio with a Novex® (Life Technologies) tris-glycine sample buffer sodium dodecyl sulfate for 10 minutes at room temperature. Samples were loaded onto a 10% Novex gelatin zymogram gel and electrophoresed for 90 minutes. The gels were run in duplicates and were incubated in Novex renaturing buffer for 30 minutes, followed by Novex developing buffer for a further 30 minutes to allow the enzymes to renature. After incubation with fresh developing buffer for a further 16 to 18 hours, the gels were washed with deionized water and stained with Novex Simplyblue Safestain for 1 hour. The gel was destained by washing with deionized water for 2 hours at room temperature and MMP-9 activity was quantified by analyzing band densities at 88 and 92 kDa using Image J® densitometry software (NIH, Bethesda, MD). The duplicate densitometry readings were averaged for each treatment group.

Back to Top | Article Outline

Real-Time Quantitative Reverse Transcription (RT)-qPCR

Total RNA was extracted from TRIzol lysates using the RNAeasy® minikit (Qiagen, Valencia, CA). RNA concentration was measured using the Nanodrop® spectrophotometer (Thermo Scientific, Wilmington, DE) and 1μg RNA was reverse transcribed into complementary DNA (cDNA) using the Superscript III® first-strand synthesis system (Life Technologies). cDNA (25–50 ng) was used for RT-qPCR with prevalidated Taqman® gene expression probes (Life Technologies) targeted against MMP-9 (assay ID: Hs00234579_m1) in both amnion and chorion samples. The housekeeping gene, glyceraldehyde-3- phosphate dehydrogenase (assay ID:Hs03929097_g1), was used in amnion samples (Life Technologies). For chorion samples, β-actin was used as the housekeeping gene with the SYBR® green detection method (Bio-Rad, Hercules, CA) and primer pairs for β-actin (RealTimePrimers.com accession ID: NM_001101.2). RTqPCR was performed with the iCycler iQ™ Real-Time PCR Detection System (Bio-Rad). Samples were run in duplicates and the mean cycle thresholds (CT) were normalized to average glyceraldehyde-3- phosphate dehydrogenase CT values for amnion samples and β-actin for chorion samples, respectively. A control sample without reverse transcriptase was included with each RT-qPCR run.

Back to Top | Article Outline

Data Analysis

Our sample size was determined by the availability of eligible patients during the enrollment period of the study. Relative MMP-9 gene expression was quantified using the comparative CT method after normalization to the relevant housekeeping genes for primary chorion and amnion cell samples.24,25 To demonstrate that TNFα-induced MMP-9 activity and gene expression, the stimulated control was normalized to the unstimulated and both were compared using the paired t test for both amnion and chorion primary cell cultures. Because of the variability in MMP-9 activity and gene expression among individual patient samples, both MMP-9 activity and gene expression for the progestin-treated groups were also normalized to the unstimulated control for the basal data for each sample. For TNFα-induced MMP-9 activity and gene expression data, progestin-pretreated groups stimulated with TNFα were normalized to the stimulated control. In both cases, the stimulated and unstimulated controls were assigned a value of 100 units. For basal MMP-9 activity and gene expression, the progestin-treated groups were compared with the unstimulated control. For TNFα-induced MMP-9 activity and gene expression, the stimulated control was compared with the groups pretreated with progestins and stimulated with TNFα. Both comparisons were analyzed using two-way ANOVA to compare progestin treatments within cell types with post hoc Dunnett test for multiple comparisons. Post hoc comparisons were limited to those between the unstimulated control and progestin-treated groups for basal data and the stimulated control and progestin-pretreated groups stimulated with TNFα for TNFα-induced MMP-9 activity and gene expression. To determine whether the distribution of the residuals approximated a normal distribution, quantile-quantile plots generated for each two-way ANOVA model were assessed by visual inspection. A family-wise P < 0.05 adjusted for multiple comparisons was considered significant. Data were summarized as mean ± SEM, mean differences, and 95% confidence intervals, and was analyzed using GraphPad prism (version 6.0 for Mac OS X, GraphPad Software, La Jolla, CA, www.graphpad.com) and SAS Enterprise Guide (Version 5.1, SAS Institute Inc., Cary, NC).

Back to Top | Article Outline

RESULTS

Fetal membranes were harvested from 11 subjects. Primary amnion cells were harvested from the fetal membranes of 11 subjects and primary chorion cells were harvested from 10 subjects. In 1 patient, we were unable to quantify MMP-9 gene expression in the primary amnion samples. In 2 patients, the basal MMP-9 activity in the primary amnion cells was too low to allow adequate quantification by zymography. These data were excluded from the subsequent analysis. Immunocytochemistry staining revealed that our harvested primary cells were >90% epithelial cells in both cultures.

Back to Top | Article Outline

The Effect of TNFα on MMP-9 Activity and Gene Expression

Compared with the unstimulated control TNFα increased MMP-9 activity and MMP-9 gene expression in both primary amnion (Fig. 2) and chorion cells (Fig. 3). The predominant band that was observed by zymography was the 92 kD pro MMP-9 band (Fig. 2A and 3A, top panel).

Figure 2

Figure 2

Figure 3

Figure 3

Back to Top | Article Outline

The Effect of Progestins on Basal MMP-9 Activity and Gene Expression

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).

Table 1

Table 1

Table 2

Table 2

Figure 4

Figure 4

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).

Back to Top | Article Outline

The Effect of Progestins on TNFα-Induced MMP-9 Activity and Gene Expression

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).

Table 3

Table 3

Table 4

Table 4

Figure 5

Figure 5

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).

Back to Top | Article Outline

DISCUSSION

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.

Back to Top | Article Outline

DISCLOSURES

Name: Terrence K. Allen, MBBS, FRCA.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Terrence K. Allen has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Liping Feng, MD.

Contribution: This author helped design the study, conduct the study, and write the manuscript.

Attestation: Liping Feng has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Matthew Nazzal, BS.

Contribution: This author helped conduct the study and write the manuscript.

Attestation: Matthew Nazzal approved the final manuscript.

Name: Chad A. Grotegut, MD, MHS.

Contribution: This author helped analyze the data and write the manuscript.

Attestation: Chad A. Grotegut reviewed the analysis of the data and approved the final manuscript.

Name: Irina A. Buhimschi, MD.

Contribution: This author helped design the study and write the manuscript.

Attestation: Irina A. Buhimschi approved the final manuscript.

Name: Amy P. Murtha, MD.

Contribution: This author helped design the study and write the manuscript.

Attestation: Amy P. Murtha approved the final manuscript.

This manuscript was handled by: Cynthia A. Wong, MD.

Back to Top | Article Outline

FOOTNOTE

a Drugs@FDA. Available at: http://www.accessdata.fda.gov/drugsatfda_docs/label/2007/011839s071lbl.pdf. Accessed January 15, 2015.
Cited Here...

Back to Top | Article Outline

REFERENCES

1. Hamilton B, Martin J, Ventura S Births: Preliminary Data for 2012. National Vital Statistics Report. 2013. 2013 Hyattsville, MD National Center for Health Statistics Available at: http://www.cdc.gov/nchs/data/nvsr/nvsr62/nvsr62_03.pdf. Accessed July 10, 2014
2. Goldenberg RL, Culhane JF, Iams JD, Romero R. Epidemiology and causes of preterm birth. Lancet. 2008;371:75–84
3. Aziz N, Cheng YW, Caughey AB. Neonatal outcomes in the setting of preterm premature rupture of membranes complicated by chorioamnionitis. J Matern Fetal Neonatal Med. 2009;22:780–4
4. Newman DE, Paamoni-Keren O, Press F, Wiznitzer A, Mazor M, Sheiner E. Neonatal outcome in preterm deliveries between 23 and 27 weeks’ gestation with and without preterm premature rupture of membranes. Arch Gynecol Obstet. 2009;280:7–11
5. Fortunato SJ, Menon R, Lombardi SJ. Amniochorion gelatinase-gelatinase inhibitor imbalance in vitro: a possible infectious pathway to rupture. Obstet Gynecol. 2000;95:240–4
6. Fortunato SJ, Menon R. Distinct molecular events suggest different pathways for preterm labor and premature rupture of membranes. Am J Obstet Gynecol. 2001;184:1399–405
7. Fortunato SJ, Menon R, Lombardi SJ. Role of tumor necrosis factor-[alpha] in the premature rupture of membranes and preterm labor pathways. Am J Obstet Gynecol. 2001;184:1399–405
8. Bryant-Greenwood GD. The extracellular matrix of the human fetal membranes: structure and function. Placenta. 1998;19:1–11
9. Fortunato SJ, Menon R. IL-1 beta is a better inducer of apoptosis in human fetal membranes than IL-6. Placenta. 2003;24:922–8
10. Kendal-Wright CE, Hubbard D, Gowin-Brown J, Bryant-Greenwood GD. Stretch and inflammation-induced Pre-B cell colony-enhancing factor (PBEF/Visfatin) and Interleukin-8 in amniotic epithelial cells. Placenta. 2010;31:665–74
11. Parry S, Strauss JF 3rd. Premature rupture of the fetal membranes. N Engl J Med. 1998;338:663–70
12. Ferrand PE, Parry S, Sammel M, Macones GA, Kuivaniemi H, Romero R, Strauss JF 3rd. A polymorphism in the matrix metalloproteinase-9 promoter is associated with increased risk of preterm premature rupture of membranes in African Americans. Mol Hum Reprod. 2002;8:494–501
13. Kumar D, Fung W, Moore RM, Pandey V, Fox J, Stetzer B, Mansour JM, Mercer BM, Redline RW, Moore JJ. Proinflammatory cytokines found in amniotic fluid induce collagen remodeling, apoptosis, and biophysical weakening of cultured human fetal membranes. Biol Reprod. 2006;74:29–34
14. Meis PJ, Klebanoff M, Thom E, Dombrowski MP, Sibai B, Moawad AH, Spong CY, Hauth JC, Miodovnik M, Varner MW, Leveno KJ, Caritis SN, Iams JD, Wapner RJ, Conway D, O’Sullivan MJ, Carpenter M, Mercer B, Ramin SM, Thorp JM, Peaceman AM, Gabbe SNational Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. . Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate. N Engl J Med. 2003;348:2379–85
15. Fonseca EB, Celik E, Parra M, Singh M, Nicolaides KHFetal Medicine Foundation Second Trimester Screening Group. . Progesterone and the risk of preterm birth among women with a short cervix. N Engl J Med. 2007;357:462–9
16. Oner C, Schatz F, Kizilay G, Murk W, Buchwalder LF, Kayisli UA, Arici A, Lockwood CJ. Progestin-inflammatory cytokine interactions affect matrix metalloproteinase-1 and -3 expression in term decidual cells: implications for treatment of chorioamnionitis-induced preterm delivery. J Clin Endocrinol Metab. 2008;93:252–9
17. Luo G, Abrahams VM, Tadesse S, Funai EF, Hodgson EJ, Gao J, Norwitz ER. Progesterone inhibits basal and TNF-alpha-induced apoptosis in fetal membranes: a novel mechanism to explain progesterone-mediated prevention of preterm birth. Reprod Sci. 2010;17:532–9
18. Allen TK, Feng L, Grotegut CA, Murtha AP. Progesterone receptor membrane component 1 as the mediator of the inhibitory effect of progestins on cytokine-induced matrix metalloproteinase 9 activity in vitro. Reprod Sci. 2014;21:260–8
19. Murtha AP, Feng L, Yonish B, Leppert PC, Schomberg DW. Progesterone protects fetal chorion and maternal decidua cells from calcium-induced death. Am J Obstet Gynecol. 2007;196:257.e1–5
20. Mills AA, Yonish B, Feng L, Schomberg DW, Heine RP, Murtha AP. Characterization of progesterone receptor isoform expression in fetal membranes. Am J Obstet Gynecol. 2006;195:998–1003
21. Dawood MY, Helmkamp F. Human umbilical arterial and venous progesterone concentrations: effect of fetal sex, weight, and mode of delivery. Obstet Gynecol. 1977;50:450–3
22. Goldman S, Weiss A, Shalev E. The effect of progesterone on gelatinase expression in the decidua and fetal membranes before and after contractions. Am J Obstet Gynecol. 2007;197:521.e1–7
23. Loudon JA, Elliott CL, Hills F, Bennett PR. Progesterone represses interleukin-8 and cyclo-oxygenase-2 in human lower segment fibroblast cells and amnion epithelial cells. Biol Reprod. 2003;69:331–7
24. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3:1101–8
25. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25:402–8
26. Moore RM, Mansour JM, Redline RW, Mercer BM, Moore JJ. The physiology of fetal membrane rupture: insight gained from the determination of physical properties. Placenta. 2006;27:1037–51
27. Poljak M, Lim R, Barker G, Lappas M. Class I to III histone deacetylases differentially regulate inflammation-induced matrix metalloproteinase 9 expression in primary amnion cells. Reprod Sci. 2014;21:804–13
28. Uchide K, Ueno H, Inoue M, Sakai A, Fujimoto N, Okada Y. Matrix metalloproteinase-9 and tensile strength of fetal membranes in uncomplicated labor. Obstet Gynecol. 2000;95:851–5
29. Moore RM, Novak JB, Kumar D, Mansour JM, Mercer BM, Moore JJ. Alpha-lipoic acid inhibits tumor necrosis factor-induced remodeling and weakening of human fetal membranes. Biol Reprod. 2009;80:781–7
30. Lappas M, Permezel M, Rice GE. N-Acetyl-cysteine inhibits phospholipid metabolism, proinflammatory cytokine release, protease activity, and nuclear factor-kappaB deoxyribonucleic acid-binding activity in human fetal membranes in vitro. J Clin Endocrinol Metab. 2003;88:1723–9
31. Li W, Li H, Bocking AD, Challis JR. Tumor necrosis factor stimulates matrix metalloproteinase 9 secretion from cultured human chorionic trophoblast cells through TNF receptor 1 signaling to IKBKB-NFKB and MAPK1/3 pathway. Biol Reprod. 2010;83:481–7
32. Das A, Fernandez-Zapico ME, Cao S, Yao J, Fiorucci S, Hebbel RP, Urrutia R, Shah VH. Disruption of an SP2/KLF6 repression complex by SHP is required for farnesoid X receptor-induced endothelial cell migration. J Biol Chem. 2006;281:39105–13
33. Labrie M, St-Pierre Y. Epigenetic regulation of MMP-9 gene expression. Cell Mol Life Sci. 2013;70:3109–24
34. Yan L, Borregaard N, Kjeldsen L, Moses MA. The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL. J Biol Chem. 2001;276:37258–65
35. Merlino A, Welsh T, Erdonmez T, Madsen G, Zakar T, Smith R, Mercer B, Mesiano S. Nuclear progesterone receptor expression in the human fetal membranes and decidua at term before and after labor. Reprod Sci. 2009;16:357–63
36. Feng L, Antczak BC, Lan L, Grotegut CA, Thompson JL, Allen TK, Murtha AP. Progesterone receptor membrane component 1 (PGRMC1) expression in fetal membranes among women with preterm premature rupture of the membranes (PPROM). Placenta. 2014;35:331–3
37. Lei K, Chen L, Geogiou EX, Sooroanna SR, Khanjani S, Brosens JJ, Bennett PR, Johnson MR. Progesterone acts via the nuclear glucocoticoid receptor to suppress IL-1 beta induced COX-2 expression in human term myometrial cells. PloS One. 2012;7:e50167
38. Kumar D, Schatz F, Moore RM, Mercer BM, Rangaswamy N, Mansour JM, Lockwood CJ, Moore JJ. The effects of thrombin and cytokines upon the biomechanics and remodeling of isolated amnion membrane, in vitro. Placenta. 2011;32:206–13
39. Manuck TA, Lai Y, Meis PJ, Dombrowski MP, Sibai B, Spong CY, Rouse DJ, Durnwald CP, Caritis SN, Wapner RJ, Mercer BM, Ramin SM. Progesterone receptor polymorphisms and clinical response to 17-alpha-hydroxyprogesterone caproate. Am J Obstet Gynecol. 2011;205:135.e1–9
40. Manuck TA, Watkins WS, Moore B, Esplin MS, Varner MW, Jackson GM, Yandell M, Jorde L. Pharmacogenomics of 17-alpha hydroxyprogesterone caproate for recurrent preterm birth prevention. Am J Obstet Gynecol. 2014;210:321.e1–e21
41. Ruddock NK, Shi SQ, Jain S, Moore G, Hankins GD, Romero R, Garfield RE. Progesterone, but not 17-alpha-hydroxyprogesterone caproate, inhibits human myometrial contractions. Am J Obstet Gynecol. 2008;199:391.e1–7
42. Canzoneri BJ, Feng L, Grotegut CA, Bentley RC, Heine RP, Murtha AP. The chorion layer of fetal membranes is prematurely destroyed in women with preterm premature rupture of the membranes. Reprod Sci. 2013;20:1246–54
43. Fortner KB, Grotegut CA, Ransom CE, Bentley RC, Feng L, Lan L, Heine RP, Seed PC, Murtha AP. Bacteria localization and chorion thinning among preterm premature rupture of membranes. PLoS One. 2014;9:e83338
44. Pressman EK, Cavanaugh JL, Woods JR. Physical properties of the chorioamnion throughout gestation. Am J Obstet Gynecol. 2002;187:672–5
45. Strohl A, Kumar D, Novince R, Shaniuk P, Smith J, Bryant K, Moore RM, Novak J, Stetzer B, Mercer BM, Mansour JM, Moore JJ. Decreased adherence and spontaneous separation of fetal membrane layers–amnion and choriodecidua–a possible part of the normal weakening process. Placenta. 2010;31:18–24
46. Arici A, Marshburn PB, MacDonald PC, Dombrowski RA. Progesterone metabolism in human endometrial stromal and gland cells in culture. Steroids. 1999;64:530–4
47. Lee RH, Stanczyk FZ, Stolz A, Ji Q, Yang G, Goodwin TM. AKR1C1 and SRD5A1 messenger RNA expression at term in the human myometrium and chorioamniotic membranes. Am J Perinatol. 2008;25:577–82
© 2015 International Anesthesia Research Society