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Preventing cervical ripening: the primary mechanism by which progestational agents prevent preterm birth?

  • Hua Xu
    Affiliations
    Center for Research in Reproduction and Women’s Health, Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA.
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  • Juan M. Gonzalez
    Affiliations
    Center for Research in Reproduction and Women’s Health, Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA.
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  • Ella Ofori
    Affiliations
    Center for Research in Reproduction and Women’s Health, Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA.
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  • Michal A. Elovitz
    Correspondence
    Reprints: Michal A. Elovitz, MD, Center for Research on Reproduction and Women’s Health, 1354 Biomedical Research Bldg II/III, 421 Curie Blvd, Philadelphia, PA 19104-6142.
    Affiliations
    Center for Research in Reproduction and Women’s Health, Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, PA.
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      Objective

      Recent clinical trials suggest that progestational agents may prevent preterm birth, specifically in women with short cervices. These studies sought to assess novel pathways by which progestational agents (PAs) may modify signal transduction pathways that are involved in cervical ripening.

      Study Design

      A microarray analysis was performed on pregnant mouse cervix that was exposed to a MPA. Appropriate microarray and cluster analyses were performed. Target genes of interest were investigated in both PA– and inflammation-exposed cervices by quantitative polymerase chain reaction and immunohistochemistry.

      Results

      Microarray analysis identified both the previously recognized and novel pathways that are involved in cervical ripening. PAs differentially rejulate expression of claudin-2, hyaluronan synthase 2, and lipocalin 2. Claudin expression is significantly decreased by inflammation, which is prevented by PAs.

      Conclusion

      PAs significantly modulate gene expression in the cervix in the presence and absence of inflammation. The regulation of these pathways, specifically claudin proteins, may be a critical mechanism by which PAs prevent preterm birth, especially in women with premature cervical shortening.

      Key words

      Preterm birth remains the leading contributor to neonatal morbidity and death in the United States; there have been great efforts to investigate effective treatment and/or preventative strategies. In the 1960s-1980s several trials investigated the use of different progestational agents in their ability to prevent preterm birth.
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      Many of these studies had design flaws or were underpowered. A metaanalysis in 1990 suggested that progestational agents indeed could prevent preterm birth.
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      Progestogen administration in pregnancy may prevent preterm delivery.
      In 2003, Meis et al
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      Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate.
      reported the results of a multicenter randomized control trial with 17-alpha hydroxyprogesterone acetate (17OHPC) to women with previous preterm birth. This study demonstrated a significant reduction in recurrent preterm birth in those women who were treated compared with women who received placebo.
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      • Thom E.
      • et al.
      Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate.
      At the same time, another study demonstrated that vaginal progesterone suppositories also significantly decreased the rate of preterm birth.
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      Prophylactic administration of progesterone by vaginal suppository to reduce the incidence of spontaneous preterm birth in women at increased risk: a randomized placebo-controlled double-blind study.
      The 2 populations that were studied had notable differences, as did the progestational agent that was used for each study. In the study of Meis et al, the authors hypothesized that 17OHPC reduced preterm birth through its ability to promote myometrial quiescence.
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      • Thom E.
      • et al.
      Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate.
      A significant amount of in vitro data support the concept that progestational agents can target contractile-associated proteins, through their interaction with target genes such as NF-κB and COX-2,
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      Human labour is associated with nuclear factor-kappaB activity which mediates cyclo-oxygenase-2 expression and is involved with the “functional progesterone withdrawal.”.
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      Progesterone represses interleukin-8 and cyclo-oxygenase-2 in human lower segment fibroblast cells and amnion epithelial cells.
      thus this theory appears biologically plausible. However, patients in the National Institute of Child Health and Human Development Maternal-Fetal Medicine Unit Network randomized control trial were not any less likely to have admission for preterm labor, presumably a surrogate for contractions.
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      • Thom E.
      • et al.
      Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate.
      Furthermore, more recent in vitro studies do not support the ability of progestational agents to suppress myometrial activity.
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      • et al.
      Influence of progesterone on myometrial contractility in pregnant mice treated with lipopolysaccharide.
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      Progesterone enhances the tocolytic effect of ritodrine in isolated pregnant human myometrium.
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      Functional effects of 17alpha-hydroxyprogesterone caproate (17P) on human myometrial contractility in vitro.
      What remains unclear then is how the additional amount of progesterone in these reported trials levels could have a significant biologic effect, considering the already increased level of progesterone in human third-trimester pregnancy. We hypothesize that progestational agents prevent preterm birth by targeting pathways that are involved in cervical ripening.
      Cervical ripening is a natural process of the gradual remodeling of connective tissue in the cervix during pregnancy.
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      Dynamics of cervical remodeling during pregnancy and parturition: mechanisms and current concepts.
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      • Garfield R.E.
      Cervical ripening: biochemical, molecular, and clinical considerations.
      Histologic evidence has demonstrated that this progressive event includes the degradation and rearrangement of collagen and the increased production of matrix metalloproteinases and glycosoaminoglycans.
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      Regulation of hyaluronan expression during cervical ripening.
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      • van Dissel-Emiliani F.M.
      Changes in water content, collagen degradation, collagen content, and concentration in repeated biopsies of the cervix of pregnant cows.
      • Kelly R.W.
      Inflammatory mediators and cervical ripening.
      • Sennstrom M.B.
      • Ekman G.
      • Westergren-Thorsson G.
      • et al.
      Human cervical ripening, an inflammatory process mediated by cytokines.
      Clinical and animal studies suggest that premature cervical shortening and/or cervical ripening contributes to preterm birth. In fact, 2 recent clinical trials suggest that women with a short cervix appear to benefit from the administration of vaginal progesterone with decreased rates of preterm birth, compared with placebo.
      • DeFranco E.A.
      • O’Brien J.M.
      • Adair C.D.
      • et al.
      Vaginal progesterone is associated with a decrease in risk for early preterm birth and improved neonatal outcome in women with a short cervix: a secondary analysis from a randomized, double-blind, placebo-controlled trial.
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      • Celik E.
      • Parra M.
      • Singh M.
      • Nicolaides K.H.
      Progesterone and the risk of preterm birth among women with a short cervix.
      Whether progestational agents target signal transduction pathways in the cervix that promote or contribute to preterm birth has not been investigated.
      Progestational agents are a group of steroid hormones that include natural hormone progesterone and derivations of this hormone such as medroxyprogesterone acetate (MPA) and 17OHP. These agents are known to effect gene transcription, mainly through the activation of the progesterone receptor.
      • Edwards D.P.
      Regulation of signal transduction pathways by estrogen and progesterone.
      However, there are well-established data that MPA and even progesterone can modulate selectively the progesterone receptor and activate the glucocorticoid receptor and have nongenomic actions.
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      • Else T.
      • Bamberger A.M.
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      • Schulte H.M.
      Dissociative glucocorticoid activity of medroxyprogesterone acetate in normal human lymphocytes.
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      Non-genomic effects of progestins: inhibition of cell growth and increased intracellular levels of cyclic nucleotides.
      • Simoncini T.
      • Mannella P.
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      • et al.
      Differential signal transduction of progesterone and medroxyprogesterone acetate in human endothelial cells.
      We previously demonstrated that progesterone, MPA, and 17OHP can all decrease the rate of inflammation-induced preterm birth in a mouse model but that MPA was the most efficacious.
      • Elovitz M.
      • Wang Z.
      Medroxyprogesterone acetate, but not progesterone, protects against inflammation-induced parturition and intrauterine fetal demise.
      • Elovitz M.A.
      • Mrinalini C.
      The use of progestational agents for preterm birth: lessons from a mouse model.
      It is noted that systemic progesterone withdrawal occurs before parturition in rodents, but not in humans. However, there is evidence that a functional progesterone withdrawal is involved in human parturition, which suggests the validity of the use of rodent models to investigate progesterone and its prevention of preterm birth.
      • Zakar T.
      • Hertelendy F.
      Progesterone withdrawal: key to parturition.
      It has been hypothesized that progestational agents may inhibit preterm birth through the modulation of the immune response in both the uterus and the cervix. However, it is noted that, in many of the recent clinical studies, these progestational agents are administered in the second trimester. Whether inflammatory pathways that may be implicated in preterm birth and/or cervical ripening are activated already at this time in gestation is not clear. Therefore, we hypothesize that progestational agents may regulate and, in fact, inhibit preterm cervical ripening. With the use of microarray technology and confirmatory molecular experiments, these studies were performed to determine whether progestational agents in the presence or absence of intrauterine inflammation differentially regulated signal transduction pathways that are implicated in cervical ripening.

      Materials and Methods

      Animals

      CD-1 out-bred, timed-pregnant mice were purchased from Charles-Rivers laboratories (Wilmington, MA). Animals were shipped on day 8 and 12 after mating. Animals were acclimated in our facility for 3 or 7 days before use in these experiments. All the experiments were performed in accordance with the National Institute of Health Guidelines on laboratory animals and with approval from the University of Pennsylvania’s Committee on Animal Use and Care.

      Mouse model of inflammation-induced preterm birth

      A mouse model of intrauterine inflammation, as previously described,
      • Elovitz M.
      • Wang Z.
      Medroxyprogesterone acetate, but not progesterone, protects against inflammation-induced parturition and intrauterine fetal demise.
      • Elovitz M.A.
      • Wang Z.
      • Chien E.K.
      • Rychlik D.F.
      • Phillippe M.
      A new model for inflammation-induced preterm birth: the role of platelet-activating factor and Toll-like receptor-4.
      • Elovitz M.
      • Mrinalini C.
      Can MPA alter TLR expression in a mouse model of localized intrauterine inflammation?.
      • Elovitz M.A.
      • Mrinalini C.
      • Sammel M.D.
      Elucidating the early signal transduction pathways leading to fetal brain injury in preterm birth.
      was used for these studies. Briefly, on day 15 of gestation, timed-pregnant CD-1 dams were placed under isoflurane anesthesia. A minilaparotomy was performed, and the lower right uterine horn was identified. Lipopolysaccharide (LPS; Sigma, St. Louis, MO), 250 μg in a 100-μL volume or sterile saline solution (100 μL) was infused between 2 gestational sacs, with care not to inject into the amniotic cavity. As previously reported, this model results in a high rate of preterm birth within 24 hours and does not result in maternal death.
      • Elovitz M.
      • Wang Z.
      Medroxyprogesterone acetate, but not progesterone, protects against inflammation-induced parturition and intrauterine fetal demise.
      • Elovitz M.
      • Mrinalini C.
      Preterm labor and 17-P: lessons from a mouse model.
      For these studies, dams were randomized to treatment with saline solution, LPS, LPS + MPA, or MPA alone.

      Cervical tissues

      For all studies, the cervix was identified; adherent adipose, bladder, and rectum were carefully dissected off the cervical tissues before being processed for other studies. Cervical tissues were collected from dams in 3 different experimental groups: (1) E15 dams treated with MPA (1 mg/dam) or vehicle with cervix harvested 48 hours later (microarray and quantitative polymerase chain reaction [QPCR]); (2) E15 dams treated subcutaneously with progesterone (2 mg/dam; Sigma; n = 4), MPA (1 mg/dam; Sigma; n = 4), dexamethasone (0.1 mg/dam; Sigma; n = 4) or an equal volume of vehicle (n = 3) with cervices harvested 24 hours later (QPCR); and (3) E15 dams treated with intrauterine saline solution, intrauterine LPS, intrauterine LPS + MPA, or MPA alone with cervices harvested 6 hours later (immunohistochemistry).

      Microarray analysis

      For microarray experiments, E15 timed-pregnant CD-1 dams were randomized to subcutaneous injection, on the back of neck region, with MPA (1 mg/dam; n = 6) or an equal volume of vehicle (pure ethanol; n = 6). Cervical tissues from each treatment group were harvested 48 hours after injection. Total RNA from each tissue was extracted with trizol (Invitrogen, Carlsbad, CA) and purified with the Qiagen RNeasy kit (Qiagen, Valencia, CA). For all RNA specimens, 260/280 ratios were >1.7. The complementary DNA (cDNA) was amplified with the WT-Ovation Pico RNA Amplification System (NuGEN Technologies, San Carlos, CA). The microarray analysis was performed by the microarray facility at University of Pennsylvania. The gene chip (1 for each sample; total, 12) mouse genome 430V2 (Affymetrix, Santa Clara, CA), which contains >39,000 mouse transcripts, was used to create cell intensity files. The guanine cytosine (GC) robust multi-array average signal values were calculated from the cell intensity files with the use of Stratagene ArrayAssist Lite (version 3.4; Stratagene, La Jolla, CA), which produced the chip (CHP) files with unlogged signal levels and Affymetrix flags. The chip files were imported into Partek genomic suite V6.3B (St Louis, MO), retaining genes flagged as present in at least 4 of 12 samples and with a fold change of ≥1.5. The Log2 transformed GC robust multiarray average signal values were exported into Excel (Microsoft Corporation, Redmond, WA) files and put into SAM V3.02 (Significance Analysis of Microarray; Stanford University, San Francisco, CA) for statistical analysis. The probability value of any given gene was calculated by t-test analysis comparing the 2 treatment groups.

      QPCR analysis

      Total RNA was extracted from cervical tissues with trizol (Invitrogen) and purified with the Qiagen RNeasy midi kit (Qiagen), and cDNA was generated with high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Primer sets, conjugated to Taqman MGB probes, were used for QPCR (Applied Biosystems). QPCR reactions were carried out with equivalent dilutions of each cDNA sample on the Applied Biosystems Model 7900 sequence detector PCR machine, as previously reported from our laboratory.
      • Elovitz M.
      • Wang Z.
      Medroxyprogesterone acetate, but not progesterone, protects against inflammation-induced parturition and intrauterine fetal demise.
      • Elovitz M.A.
      • Mrinalini C.
      • Sammel M.D.
      Elucidating the early signal transduction pathways leading to fetal brain injury in preterm birth.
      • Elovitz M.A.
      • Mrinalini C.
      Can medroxyprogesterone acetate alter Toll-like receptor expression in a mouse model of intrauterine inflammation?.
      The relative abundance of the target of interest was divided by the relative abundance of 18S in each sample to generate a normalized abundance for the target of interest. All samples were analyzed in triplicate. For QPCR analyses, target messenger RNA (mRNA) expression was compared between the different treatment groups with 1-way analysis of variance for normally distributed data and analysis of variance on ranks for nonparametric data. If significance was reached (P < .05), then pairwise comparison was performed.

      Immunohistochemistry

      With our mouse model of preterm birth, 4 treatment groups of mice were used for these studies: saline solution, LPS, MPA alone, and MPA + LPS. Cervical tissues were harvested 6 hours after intrauterine injection of LPS or saline solution and prepared for histochemical studies. Paraffin sections of cervical tissue from 3 different dams from each treatment group were stained for claudin-1 and claudin-2 with the corresponding rabbit polycolonal antibodies (Lab Vision, Fremont, CA). Negative control experiments with rabbit immunoglobulin G confirmed the absence of nonspecific staining. The paraffin-embedded tissues were deparaffinated in xylene, rehydrated in ethanol, then subjected to endogenous peroxide quenching. The tissues were heated to near boiling in citrate buffer for 20 minutes to retrieve antigen. After 30 minutes of pretreatment with goat normal serum, the primary antibodies were applied (1:200) for 30 minutes. The biotinylated anti-rabbit immunoglobulin G secondary antibody (Vectastain ABC kit; Vector Laboratories Inc, Burlingame, CA) was used and then developed with diaminobenzidine (Sigma) according to the manufacture’s protocol. The tissues were counterstained with hematoxylin and dehydrated before mounting. All the pictures were taken on a white field microscope at ×4, ×10, and ×40 magnification.

      Results

      Microarray and pathway analysis

      The complete data profiles from the microarray analyses have been deposited in Gene Expression Omnibus of the National Center for Biotechnology Information. Of all the genes that were present on the chip, a total of 357 genes were identified as being present in at least 4 of 12 samples and having a minimal fold change of ≥1.5. These 357 genes were retained for analysis using significance analysis method (SAM). With a more stringent probability value (P < .01), 33 genes were significantly different between MPA and vehicle-treated cervices (Table 1).
      TABLE 1Microarray analysis results
      Probe IDGene nameFold changeP valueQ value (%)Description
      Up-regulated genes
      1419195_atWfdc15b2.642.20E-050WAP four-disulfide core domain 15B
      1416125_atFkbp51.81.00E-040FK506 binding protein 5
      1423844_s_atCbs2.286.50E-040Cystathionine beta-synthase
      1448995_atCxcl41.616.60E-040Chemokine (C-X-C motif) ligand 4
      1424067_atIcam11.531.90E-0310.47Intercellular adhesion molecule
      1417936_atCcl91.942.00E-0310.47Chemokine (C-C motif) ligand 9
      1418204_s_atAif11.912.10E-0310.47Allograft inflammatory factor 1
      1445349_at2.072.20E-0310.470 day neonate lung cDNA, RIKEN full-length enriched library, clone: E030049A20 pr
      1454935_atD930001I22Rik1.512.30E-0310.47RIKEN cDNA D930001I22 gene
      1416051_atC21.772.60E-0310.47Complement component 2 (within H-2S)
      1415897_a_atMgst11.542.80E-0313.96Microsomal glutathione S-transferase 1
      1441603_atSstr33.412.90E-0313.96Somatostatin receptor 3
      1417231_atCldn25.793.10E-0313.96Claudin 2
      1419015_atWisp22.273.30E-0313.96WNT1 inducible signaling pathway protein 2
      1426706_s_stXylb1.694.00E-0317.45Xylulokinase homolog (H. influenzae)
      1455000_atGpr681.594.30E-0317.45G protein-coupled receptor 68
      1417836_atGpx71.994.70E-0317.45Glutathione peroxidase 7
      1460431_atGcnt11.675.80E-0321.82Glucosaminyl (N-acetyl) transferase 1, core 2
      1450792_atTyrobp1.627.30E-0321.82TYRO protein tyrosine kinase binding protein
      1435879_atAkt31.737.30E-0321.82Thymoma viral proto-oncogene 3
      1433467_atSlc7a61.587.40E-0321.82Solution carrier family 7 (cationic amino acid transporter, y+ system), member 6
      1433977_atHs3st3b12.389.10E-0326.18Heparan sulfate (glucosamine) 3-O-sulfotransferase 3B1
      1446345_at2.789.40E-0326.18
      1444242_at1.739.70E-0326.18
      1443870_atAbcc41.539.80E-0326.18ATP-binding cassette, sub-family C (CFTR/MRP), member 4
      Down-regulated genes
      1418678_atHas2-4.411.00E-040Hyaluronan synthase 2
      1417266_atCcl6-1.783.90E-0422.44Chemokine (C-C motif) ligand 6
      1434211_atSh3bgrl2-1.586.00E-0422.44SH3 domain binding glutamic acid-rich protein like 2
      1452106_atNpnt-1.891.80E-0339.09Nephronectin
      Genes that have a fold change of 1.5 or greater and false discovery rate less than 40% are listed.
      Xu. Preventing cervical ripening: the primary mechanism by which progestational agents prevent preterm birth? Am J Obstet Gynecol 2008.
      To identify MPA-mediated biologic and molecular activities on the cervix, microarray results were analyzed by the use of the functional clustering tool in the Database for Annotation Visualization, and Integrated Discovery (DAVID) (http://david.abcc.ncifcrf.gov/tools.jsp). For the inputted gene list, 226 genes were used that had a false discovery rate <40% and a fold change of ≥1.5. The background gene list includes all genes on the Mouse Affymetrix Chip. The functional annotations were clustered with medium classification stringency. The use of this functional annotation clustering tool revealed that MPA alters and/or regulates a number of biologic and molecular processes that include extracellular region, immune and defense response, and glycoprotein and chemokine activity (Table 2). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that, compared with vehicle, MPA significantly altered a number of cellular pathways in the pregnant cervix (Figure 1).
      TABLE 2Functional annotation clustering
      Enichment scoreAnnotation clustersBenjamini (corrected p value)
      11.17Extracellular region3.20E-16
      Extracellular space2.90E-16
      Glycoprotein1.10E-06
      5.21Immunoglobulin, immunoglobin-like5.20E-03
      4.12Defense response3.20E-04
      Immune response4.80E-03
      2.58Chemokine activity6.70E-03
      Chemokine receptor binding6.70E-03
      G-protein-coupled receptor binding9.70E-03
      2.29Epidermal growth factor (EGF)6.10E-03
      Xu. Preventing cervical ripening: the primary mechanism by which progestational agents prevent preterm birth? Am J Obstet Gynecol 2008.
      Figure thumbnail gr1
      FIGURE 1KEGG pathway analysis of MPA-modulated genes by DAVID
      Of 226 selected modulated genes, 79 genes were classified. The number of classified genes in each pathway is indicated next to the pathway. Some genes may be represented in >1 functional category.
      Xu. Preventing cervical ripening: the primary mechanism by which progestational agents prevent preterm birth? Am J Obstet Gynecol 2008.

      QPCR

      To verify the results that were seen in microarray, selected targets were examined with QPCR (Table 3). Consistent with the microarray results, pretreatment of MPA resulted in a significant upregulation in mRNA expression of Claudin-2, hyaluronan syntheses 2 (Has-2) and lipocalin 2 (Lcn2) by QPCR analysis at 48 hours of treatment (Table 3). Other tested targets demonstrated consistent fold changes as observed in microarray, but these did not reach statistical significance (Table 3).
      TABLE 3Validation of microarray findings by QPCR
      GeneMicroarrayQPCR (48 hours)
      Fold changeFold changeP value
      P value calculated by T-test or Mann Whitney Rank Sum
      Claudin25.792.010.04
      Hyaluronan synthase 2-4.41-3.30.04
      Lipocalin 23.012.510.04
      Intercellular adhesion molecule 1 (Icam 1)1.531.550.35
      Chemokine ligand 4 (Cxcl 4)1.611.240.57
      Retinoic acid induced 14 (Rai14)-1.45-1.330.41
      FK506 binding protein 5 (Fkbp5)1.81.460.31
      Epoxide hydrolase 1 (Ephx1)1.41.30.91
      Syncollin8.333.710.7
      Xu. Preventing cervical ripening: the primary mechanism by which progestational agents prevent preterm birth? Am J Obstet Gynecol 2008.
      a P value calculated by T-test or Mann Whitney Rank Sum
      To determine whether the effects of MPA on cervical tissues differed by exposure time, mRNA expression changes of those selected targets were assessed by QPCR with mouse cervical tissues that were treated with MPA, progesterone, or dexamethasone for 24 hours (Table 4). Consistent with upregulation that was observed at 48 hours, claudin-2 mRNA was significantly elevated 6.8 fold (P = .003) by MPA at 24 hours. Progesterone, but not dexamethasone, also demonstrated a significant upregulation of claudin-2 expression at 24 hours (P = .04). Has-2 and Lcn2 mRNA were modulated by MPA at a 24-hour time point that was similar to the observed fold change in microarray, but this did not reach statistical significance (Table 4). MPA significantly increased Fkbp5 mRNA expression at 24 hours.
      TABLE 4Effect of progestational agents on gene expression at 24 hours
      GeneMultiple Comparison (4 treatment groups)
      4 treatment groups: MPA, DEX, Progesterone and Vehicle exposed cervices. Multiple comparisons by One-way ANOVA or ANOVA on Ranks. Pair-wise comparisons were performed only is statistical significance was reached (P < 0.05).
      MPAProgesteroneDEX
      Fold changeP valueFold changeP valueFold changeP value
      Claudin2P = 0.0017.80.0034.90.041.50.73
      Hyaluronan synthase 2P = 0.06-1.9NP1.6NP2NP
      Lipocalin 2P = 0.171.7NP-2.6NP-2.3NP
      Intercellular adhesion molecule 1 (Icam 1)P = 0.033.1NS1.2NS1NS
      Chemokine (C-X-C motif) ligand 4 (Cxcl 4)P = 0.071.5NP1.2NP-1.1NP
      Retinoic acid induced 14P = 0.071.1NP-1.1NP-1.3NP
      FK506 binding protein 5P = 0.0052.30.0021.40.271.10.87
      Epoxide hydrolase 1P = 0.61.4NP1.1NP1NP
      SyncollinP = 0.641.9NP2.3NP1.2NP
      MPA = medroxyprogesterone acetate; DEX = dexamethasone; NP = not performed; NS = not significant
      Xu. Preventing cervical ripening: the primary mechanism by which progestational agents prevent preterm birth? Am J Obstet Gynecol 2008.
      a 4 treatment groups: MPA, DEX, Progesterone and Vehicle exposed cervices. Multiple comparisons by One-way ANOVA or ANOVA on Ranks. Pair-wise comparisons were performed only is statistical significance was reached (P < 0.05).

      Immunohistochemistry

      Given that the claudin-2 mRNA expression was significantly modulated by MPA (TABLE 3, TABLE 4), immunohistochemical detection of claudin-2 and its family member claudin-1 was performed on cervical tissues. We explored claudin protein expression in our mouse model of preterm birth in the various treatment groups. On E15, there is a significant expression of claudin-1 and claudin-2 in the cervical epithelial and stromal regions (Figures 2, A and 3, A). Intrauterine LPS reduced expression of claudin-1 and claudin-2 in the stromal regions and led to disruption of the epithelial layer with a loss of claudin expression (Figures 2, B and 3, B). Pretreatment of dams with MPA before LPS (which inhibits preterm birth as previously published
      • Elovitz M.
      • Wang Z.
      Medroxyprogesterone acetate, but not progesterone, protects against inflammation-induced parturition and intrauterine fetal demise.
      • Elovitz M.A.
      • Mrinalini C.
      The use of progestational agents for preterm birth: lessons from a mouse model.
      ) prevented the LPS-induced decrease of claudin-1 expression in the epithelium and preserved the integrity of the epithelial layer (Figure 2, C). However, staining for claudin-1 remained decreased in the cervical stroma of MPA + LPS–treated dams, compared with saline solution–dams. With respect to claudin-2, pretreatment with MPA before intrauterine LPS prevented some of the loss of claudin-2 expression in the epithelial layer, and stromal staining mimicked that of saline solution–treated cervices (Figure 3, D). MPA treatment alone at 6 hours appeared to increase the expression of claudin-1 in the epithelial layer (Figure 2, D). MPA treatment alone for 6 hours did not appear to have a profound effect on claudin-2 expression (Figure 3, D).
      Figure thumbnail gr2
      FIGURE 2Immunohistochemical representation of claudin 1 in cervix
      Sections are imaged at ×40 magnification. Four groups (n = 3 for each group) were stained for claudin 1 expression. A, Saline solution; B, LPS; C, MPA + LPS; D, MPA alone. One representative image from each group is shown.
      Xu. Preventing cervical ripening: the primary mechanism by which progestational agents prevent preterm birth? Am J Obstet Gynecol 2008.
      Figure thumbnail gr3
      FIGURE 3Immunohistochemical representation of claudin 2 in cervix
      Sections are imaged at ×40 magnification. Four groups (n = 3 for each group) were stained for claudin 2 expression. A, Saline solution; B, LPS; C, MPA + LPS; D, MPA alone. One representative image from each group is shown.
      Xu. Preventing cervical ripening: the primary mechanism by which progestational agents prevent preterm birth? Am J Obstet Gynecol 2008.

      Comment

      Although the American College of Obstetrics and Gynecology supports the use of 17OHP in high-risk patients for the prevention of preterm birth, the mechanisms by which 17OHP or other progestational agents prevent preterm birth remain unclear. Most recent clinical trials suggest that progesterone may have a significant reduction on preterm birth in those women with short cervices. The animal and molecular studies presented here demonstrate that both previously recognized and novel pathways are differentially modulated by progestational agents in the cervix. These findings suggest a biological mechanism by which women with a short cervix benefit from the administration of progestational agents. Furthermore, because these effects were noted by progesterone and MPA, not dexamethasone, these studies suggest that modulated pathways result from activation of progesterone, not glucocorticoid receptor. Although these studies are unable to determine whether progestational agents prevent preterm birth by inhibition of these specific pathways, these findings do provide novel insight into possible molecular mechanisms by which progestational agents target and/or modulate cervical ripening.
      Because the women in the trial of Meis et al
      • Meis P.J.
      • Klebanoff M.
      • Thom E.
      • et al.
      Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate.
      were assigned randomly to 17OHP early in pregnancy, presumably before the onset of contractions or cervical ripening, we believe that the administration of MPA, at this point in mouse gestation, is a strength to our study. A microarray with MPA, instead of 17OHP or progesterone, was performed because of our previous work that demonstrated that, in mice, MPA has greater efficacy in the prevention of inflammation-induced preterm birth than 17OHP or progesterone.
      • Elovitz M.
      • Wang Z.
      Medroxyprogesterone acetate, but not progesterone, protects against inflammation-induced parturition and intrauterine fetal demise.
      • Elovitz M.
      • Mrinalini C.
      Preterm labor and 17-P: lessons from a mouse model.
      It is a recognized limitation that MPA is not used in the recent human clinical trials and that MPA and progesterone are known to activate similar and divergent signal transduction pathways.
      • Bamberger C.M.
      • Else T.
      • Bamberger A.M.
      • Beil F.U.
      • Schulte H.M.
      Dissociative glucocorticoid activity of medroxyprogesterone acetate in normal human lymphocytes.
      • Simoncini T.
      • Mannella P.
      • Fornari L.
      • et al.
      Differential signal transduction of progesterone and medroxyprogesterone acetate in human endothelial cells.
      The use of microarray technology provided the ability to discover novel pathways that might be involved in progestational-mediated effects on the cervix. A strength of these reported studies is that we confirmed our microarray data, not just in the same cervical samples but also in separate time-course experiments and in this described model of preterm birth. Furthermore, we performed separate techniques (QPCR and immunohistochemistry) to confirm our findings.
      The claudin family contains approximately 24 family members.
      • Krause G.
      • Winkler L.
      • Mueller S.L.
      • Haseloff R.F.
      • Piontek J.
      • Blasig I.E.
      Structure and function of claudins.
      • Moriwaki K.
      • Tsukita S.
      • Furuse M.
      Tight junctions containing claudin 4 and 6 are essential for blastocyst formation in preimplantation mouse embryos.
      The primary molecular role of claudins is to determine the permeability and the charge selectivity of the paracellular pathway to small ions by assembling into multimers to form paracellular pores.
      • Krause G.
      • Winkler L.
      • Mueller S.L.
      • Haseloff R.F.
      • Piontek J.
      • Blasig I.E.
      Structure and function of claudins.
      The observed upregulation of claudin by progestational agents in our study suggests that these agents can modify epithelial and endothelial tight junctions in cervix. Tight junctions are an important mode of cell-cell adhesion in epithelial and endothelial cellular layers.
      • Moriwaki K.
      • Tsukita S.
      • Furuse M.
      Tight junctions containing claudin 4 and 6 are essential for blastocyst formation in preimplantation mouse embryos.
      They create a boundary between the apical and the basolateral plasma membrane and recruit various signaling molecules at their cytoplasmic surface.
      • Moriwaki K.
      • Tsukita S.
      • Furuse M.
      Tight junctions containing claudin 4 and 6 are essential for blastocyst formation in preimplantation mouse embryos.
      It is possible that, through regulation of claudins, progestational agents modulate a wide range of cellular activities in epithelial and endothelial layers, which include cell-cell communication and adhesion and morphologic construction. Currently, there is a paucity of data that demonstrate how inflammation disrupts the cervical epithelia. However, alterations of claudin proteins have been associated with proinflammatory challenges.
      • Gao W.-M.
      • Chadha M.S.
      • Kline A.E.
      • et al.
      Immunohistochemical analysis of histone H3 acetylation and methylation: evidence for altered epigenetic signaling following traumatic brain injury in immature rats.
      • Mazzon E.
      • Cuzzocrea S.
      Role of TNF-alpha in lung tight junction alteration in mouse model of acute lung inflammation.
      Although other mechanisms may exist, our studies support this finding and demonstrate that, early in inflammation-induced preterm birth, claudin expression is decreased in cervical epithelia. Furthermore, our studies suggest that progestational agents can prevent this inflammatory effect. Recently, it was demonstrated that, in mouse gestation, the expression of claudin proteins decrease near term, which is consistent with cervical ripening.
      • Timmons B.C.
      • Mitchell S.M.
      • Gilpin C.
      • Mahendroo M.S.
      Dynamic changes in the cervical epithelial tight junction complex and differentiation occur during cervical ripening and parturition.
      These recent studies along with our report suggest that claudins may play a critical role in both preterm and term cervical ripening. Our finding that progestational agents modulate this biological response is a novel and potentially clinically important finding.
      Several reports demonstrate the involvement of hyaluronan in cervical ripening.
      • El Maradny E.
      • Kanayama N.
      • Kobayashi H.
      • et al.
      The role of hyaluronic acid as a mediator and regulator of cervical ripening.
      Hyaluronan levels in human cervix are very low during pregnancy but increase rapidly at the onset of labor.
      • El Maradny E.
      • Kanayama N.
      • Kobayashi H.
      • et al.
      The role of hyaluronic acid as a mediator and regulator of cervical ripening.
      Within the cervix, hyaluronan is expressed in the fibroblasts and can be stimulated by prostaglandins, interleukin-1, and LPS.
      • Tanaka K.
      • Nakamura T.
      • Ikeya H.
      • et al.
      Hyaluronate depolymerization activity induced by progesterone in cultured fibroblasts derived from human uterine cervix.
      • Rath W.
      • Osmers R.
      • Adelmann-Grill B.C.
      • Stuhlsatz H.W.
      • Szevereny M.
      • Kuhn W.
      Biochemical changes in human cervical connective tissue after intracervical application of prostaglandin E2.
      • Chao C.C.
      • Molitor T.W.
      • Hu S.
      Neuroprotective role of IL-4 against activated microglia.
      • Laurent T.C.
      • Fraser J.R.
      The properties and turnover of hyaluronan.
      Cervical Has-2 mRNA expression increases during gestation and declines after birth.
      • Straach K.J.
      • Shelton J.M.
      • Richardson J.A.
      • Hascall V.C.
      • Mahendroo M.S.
      Regulation of hyaluronan expression during cervical ripening.
      Our findings that progestational agents significantly decrease baseline expression of HAS-2 in the cervix during murine pregnancy suggests a possible pathway by which progestational agents may inhibit cervical ripening before the normal initiation of these pathways in gestation.
      Lcn2 is a member of lipocalin family that is expressed in multiple tissues that include the epithelium and exerts a bacteriostatic effect through reduction of iron access to the bacteria.
      • Marques F.
      • Rodrigues A.J.
      • Sousa J.C.
      • et al.
      Lipocalin 2 is a choroid plexus acute-phase protein.
      Lcn2 mRNA expression was significantly upregulated by MPA in our studies. Interestingly, Lcn2 expression was also significantly elevated in the cervix of the 5-alpha reductase knock-out mouse that has a parturition defect that results from failed cervical ripening.
      • Timmons B.C.
      • Mitchell S.M.
      • Gilpin C.
      • Mahendroo M.S.
      Dynamic changes in the cervical epithelial tight junction complex and differentiation occur during cervical ripening and parturition.
      Thus, these studies provide another possible mechanism by which progestational agents may prevent preterm birth. MPA-induced increase in Lcn2 may contribute to immune modulation in a subset of patient, thereby preventing cervical ripening and preterm birth.
      In summary, these studies suggest novel biological and molecular pathways in the cervix that are modulated by progestational agents. The regulation of these specific pathways, both in the presence and absence of inflammation, may provide some of the answers as to how progestational agents prevent preterm birth. Because preterm birth is a complex disorder, it is unlikely that all patients with preterm birth can be treated by 1 agent. Further understanding of the pathways that are reported here may serve to identify subsets of patients with preterm birth who would most benefit from treatment with progestational agents. Allelic alterations in genes in these pathways may be expressed clinically as cervical ripening defects and predispose women to preterm birth. Alternatively, women who are more susceptible to inflammatory responses may have premature cervical ripening through early activation of these pathways. These theories require active investigation if we are to make a significant impact on reducing the preterm birth rate.

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