Objective
Clinically, vaginal progesterone (VP) and 17 alpha-hydroxyprogesterone caproate (17P) have been shown to prevent preterm birth (PTB) in high-risk populations. We hypothesize that treatment with these agents may prevent PTB by altering molecular pathways involved in uterine contractility or cervical remodeling.
Study Design
Using a mouse model, on embryonic day (E)14-E17 CD-1 pregnant mice were treated with: (1) 0.1 mL of 25 mg/mL of 17P subcutaneously; (2) 0.1 mL of castor oil subcutaneously; (3) 0.1 mL of 10 mg/mL of progesterone in a long-lasting Replens (Lil’ Drug Store Products, Inc., Cedar Rapids, IA); or (4) 0.1 mL of the same Replens, with 4 dams per treatment group. Mice were sacrificed 6 hours after treatment on E17.5. Cervices and uteri were collected for molecular analysis.
Results
Exposure to VP significantly increased the expression of defensin 1 compared to Replens ( P < .01) on E17.5. Neither VP nor 17P altered the expression of uterine contraction-associated proteins, progesterone-mediated regulators of uterine quiescence, microRNA involved in uterine contractility, or pathways involved in cervical remodeling. In addition, neither agent had an effect on immune cell trafficking or collagen content in the cervix.
Conclusion
Neither VP nor 17P had any effect on the studied pathways known to be involved in uterine contractility or quiescence. In the cervix, neither VP nor 17P altered pathways demonstrated to be involved in cervical remodeling. Administration of VP was noted to increase the expression of the antimicrobial protein defensin 1. Whether this molecular change from VP results in a functional effect and is a key mechanism by which VP prevents PTB requires further study.
Preterm birth (PTB) remains the leading cause of neonatal morbidity and mortality in the United States. In high-risk populations, 17 alpha-hydroxyprogesterone caproate (17P) and vaginal progesterone (VP) have been shown to prevent PTB. However, the molecular mechanisms by which progesterone supplementation prevents PTB remain poorly understood.
While the precise pathways that govern preterm parturition remain to be discovered, an increase in uterine activity and premature cervical remodeling are believed to be critically involved in the pathogenesis of spontaneous PTB. Recent data suggest the use of progesterone may be important in maintaining uterine quiescence in the latter half of pregnancy by limiting the production of stimulatory prostaglandins and inhibiting the expression of contraction-associated protein genes (ion channels, oxytocin and prostaglandin receptors, and gap junctions) within the myometrium. While contraction-associated proteins have long been associated with myometrial activity, recent work has demonstrated the role of microRNA (miRNA) in uterine activity. A conserved family of miRNA, the miR-200 family, has been found to be significantly induced at term in both mice and human beings. Additionally, 2 down-regulated targets zeb 1 and zeb 2 in this pathway act as transcriptional repressors and are down-regulated at term. Zeb 1, which has been shown to be up-regulated by progesterone in vivo, and zeb 2 inhibit the expression of the contraction-associated genes oxytocin receptor and connexin-43. While human and mouse data support the role of these targets in uterine contractility, the effect of progesterone supplementation on these pathways has not been explored as a possible mechanism by which these agents prevent PTB.
In addition to uterine activity, premature cervical remodeling is believed to be an obligatory step in the pathogenesis of PTB. Clinical studies have shown a short cervix is a risk factor for PTB. Cervical remodeling and eventual cervical ripening is a biomechanical process that involves the gradual change of the connective tissue in the cervix throughout pregnancy. Maintenance of cervical integrity and appropriate timing of cervical remodeling is critical to maintain a pregnancy.
While similar pathways must be involved in the final phases of cervical ripening (to allow passage of the fetus), the inciting events that trigger cervical remodeling appear to differ in the preterm compared to term period. Preterm cervical remodeling pathways involve changes in immune cell trafficking, complement activation, and an altered collagen structure. In addition to these pathways, changes in the epithelial barrier have also been implicated in preterm cervical remodeling. Claudins, which in part regulate cell-cell adhesion and the permeability of the epithelial barrier, undergo changes in expression during cervical remodeling. Microarray data have also shown epithelial cell differentiation pathways are up-regulated with cervical remodeling as well.
In other biological systems, such as the gastrointestinal system, there is a greater understanding of the pathways by which the immune system and the epithelial cell barrier interact leading to pathophysiological states. Once the epithelial barrier becomes a target for bacterial pathogens and/or their byproducts, the mucosal immune response becomes essential in preventing disruption of the underlying stroma. In the epithelial barrier, mucosal immunity consists of antimicrobial proteins, along with immune cells and their related cytokines. Disruption of the cervical epithelial barrier from inflammation may lead to premature cervical remodeling and ultimately PTB.
Based on recent clinical studies demonstrating progestational agents can prevent PTB in high-risk populations, we hypothesize these agents are modifying pathways that are critically involved in uterine activity and/or premature cervical remodeling. These studies sought to assess if supplementation with VP or 17P up-regulated crucial aspects of these pathways as potential mechanisms by which they prevent PTB in clinical trials.
Materials and Methods
Mouse model
All procedures were performed with Institutional Animal Care and Use Committee approval from the Perelman School of Medicine at the University of Pennsylvania. In this study, CD-1 outbred, timed pregnant mice were used (Charles River Laboratories, Wilmington, MA). 17P (Boothwyn Pharmacy, Boothwyn, PA) injections were prepared by diluting 0.1 mL of 250 mg/mL in 0.9 mL of castor oil (Sigma Chemical Co, St. Louis, MO). VP was prepared by diluting 40 mg of progesterone (Sigma Chemical Co) in 4 mL of a long-lasting Replens (Lil’ Drug Store Products, Inc., Cedar Rapids, IA). On embryonic day (E)14-E17, pregnant CD-1 mice were treated daily with: (1) 0.1 mL of 25 mg/mL of 17P subcutaneously; (2) 0.1 mL of castor oil subcutaneously; (3) 0.1 mL of 10 mg/mL of progesterone in the Replens; or (4) 0.1 mL of the same Replens. After administration of either VP or Replens, the mice were returned to their cages and none of the progesterone solution was noted to be leaking from the vagina. Prior work from our laboratory has demonstrated that administration of castor oil to mice does not induce PTB. Hence, an additional control group of mice exposed to castor oil was deemed unnecessary for these studies. The dose of VP and 17P represent a 25- and 17-fold increase compared to human dosing basing on 70 kg of body weight, respectively. Four dams per treatment group were used. A murine gestation lasts 19-21 days, and treatment on E14-E17 would mimic late second- and third-trimester supplementation in human beings. The same experiment was repeated twice with 4 dams per group for each set of experiments. The first set of experiments provided tissue for molecular analysis of uterine and cervical tissues. The second set provided cervical tissues for histological assessments as described below. On E17.5, 6 hours after the last treatment, dams were euthanized. By harvesting tissue on E17.5, the uterine and cervical tissue could be analyzed prior to the progesterone withdrawal that occurs with term parturition in the murine model. Whole blood was collected from the aorta using a 25-gauge syringe. The blood was allowed to clot at room temperature for 2 hours and then spun down for 20 minutes at 2000 g . The serum was collected and flash frozen in liquid nitrogen. Uterine tissue was harvested. Gestational sacs were removed and the decidua was left in situ. Cervical tissues were carefully dissected from the surrounding organs (bladder and bowel) and adjacent adipose tissue was removed. Tissues were rinsed in sterile saline solution and placed immediately in liquid nitrogen. Samples were stored at −80°C until processed for experiments described below.
Quantitative polymerase chain reaction
Total RNA was extracted from the cervix and uterus with TRIzol (Invitrogen, Grand Island, NY) according to product protocol. Complementary DNA (cDNA) was generated from 2 μg of RNA/sample using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). To assess expression, quantitative polymerase chain reaction (qPCR) was performed using equivalent dilutions of each sample on the Applied Biosystems model 9700 sequence detector polymerase chain reaction (PCR) machine. Primer sets, conjugated to TaqMan MGB probes, were used for qPCR (Applied Biosystems). Uterine targets analyzed were contraction-associated proteins: connexin-43, oxytocin receptor, and cyclooxygenase-2, and progesterone-mediated regulators of uterine quiescence: stat 5b, zeb 1, zeb 2, and 20-alpha-hydroxysteroid dehydrogenase. Cervical targets analyzed included interleukin (IL)-17, IL-22, IL-22R, defensin 1, defensin 3, defensin 4, and SLP1. 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. 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 duplicate. Statistical analyses were performed comparing means or medians depending on whether the data were parametric (Student t test) or nonparametric (Mann-Whitney). Analysis was performed by SigmaStat 3.5 (Aspire Software International, Ashburn, VA).
miRNA qPCR
miRNA was isolated from mouse uterus using TRIzol and the miRNeasy isolation kit (Qiagen, Valencia, CA). cDNA was generated from the isolated miRNA using the miScript reverse transcription II kit (Qiagen) and qPCR was performed on the 7900HT real-time PCR system (Applied Biosystems) using the miScript SYBR green PCR kit (Qiagen) according to the manufacturers’ protocols. The ΔΔCT method was used for relative expression quantification using RQ manager software v2.4 (Applied Biosystems). The endogenous reference gene RNU6B was used for miRNA quantification. All primer sets were purchased from Qiagen: miR-200a (MS00001813), 429 (MS00002366), and RNU6B (MS0001400). Statistical analyses were performed comparing means or medians depending on whether the data were parametric (Student t test) or nonparametric (Mann-Whitney). Analysis was performed by SigmaStat 3.5 (Aspire Software International).
Staining of cervical tissue
For histological assessments of the cervix, after euthanizing the dam, the cervix was excised, removing all adjacent tissues and adipose, and fixed in 4% paraformaldehyde for 24 hours. Samples were then immersed and stored in 20% sucrose at 4°C. Cervices were rehydrated in phosphate-buffered saline and a graded series of alcohols, then embedded in paraffin and sectioned at 10 μm. As previously shown, longitudinal sections of cervix from each mouse were stained with picrosirius red to assess collagen content and cross-linked structure. Photomicrographs of birefringence of transmitted polarized light in sections were taken (Zeiss AxioImager A1, ×20 objective). Multiple defined fields of view were analyzed for optical density using National Institutes of Health Image J software (gray-scale threshold determined with uncalibrated Rodbard standard curve). The optical density of birefringent polarized transmitted light is inversely related to the density of collagen content and structure. Thus, low optical density is indicative of areas of bright red birefringence with high collagen content and complex dense cross-linking due to high transmittance of light. By contrast, areas with low birefringence and low collagen content and diffuse structure were dark and had proportionally high optical density values. Other sections were processed by immunohistochemistry to stain macrophages and counterstained with methyl green to identify cell nuclei. The BM-8 (F4/80) antibody was specific for macrophages because reports of cross-reactivity with other immune cells are not a consideration due to their absence or rare presence in the murine cervix. Brightfield scans of sections were taken (Aperio ImageScope). In a snapshot of a calibrated fields of view for each section (approximately 1.251 × 10 6 μm ), cell nuclei and macrophages were counted. Data for collagen and immune cell counts were normalized to cell nuclei density for each individual to account for hypertrophy and hyperplasia of tissue. Data were evaluated by Levene test for homogeneity of variance and determined to be normally distributed ( P > .05). An unpaired Student t test was then used to assess statistical significance between control and treated mice.
Detection of serum progesterone
Progesterone levels were assessed with an enzyme-linked immunosorbent assay (ELISA) kit (USCN Life Science Inc, Houston TX). Four dams per treatment group were used. The detection range was 1.23-100 ng/mL, and the minimal detectable dose of progesterone was <0.48 ng/mL. Due to unknown levels of serum progesterone after VP and 17P administration, initial ELISAs were performed to determine optimal performance. Based on the detection range of the ELISAs and these initial experiments, maternal serum was diluted (1:10). We chose to test serum levels to determine if the administration of either VP or 17P had a measurable increase on circulating levels of progesterone indicating an increase in bioavailability at the cervix and uterus.
Results
Effect of progestational agents on uterine quiescence
Contraction-associated proteins
Neither VP nor systemically administered 17P significantly altered the signal transduction pathways regulating the contraction-associated proteins connexin-43, oxytocin receptor, or cyclooxygenase-2 ( Table ).
| Gene name | Replens | Vaginal progesterone | P value | Castor oil | 17P | P value |
|---|---|---|---|---|---|---|
| Contraction-associated proteins | ||||||
| Connexin-43 | 1.58 ± 0.57 | 1.54 ± 0.32 | .95 | 1.18 ± 0.20 | 1.28 ± 0.05 | .62 |
| Oxytocin receptor | 0.06 ± 0.02 | 0.05 ± 0.01 | .77 | 0.06 ± 0.03 | 0.03 ± 0.01 | .39 |
| Cyclooxygenase-2 | 1.27 ± 0.49 | 1.32 ± 0.40 | .94 | 0.71 ± 0.12 | 1.30 ± 0.29 | .11 |
| Progesterone-mediated regulators of uterine quiescence | ||||||
| Stat 5b | 1.33 ± 0.26 | 1.15 ± 0.18 | .58 | 1.05 ± 0.05 | 0.94 ± 0.08 | .28 |
| Zeb 1 | 1.53 ± 0.34 | 1.05 ± 0.09 | .22 | 1.07 ± 0.10 | 0.77 ± 0.05 | .06 |
| Zeb 2 | 1.58 ± 0.37 | 1.13 ± 0.13 | .30 | 1.15 ± 0.28 | 1.00 ± 0.11 | .63 |
| 20-alpha-hydroxysteroid dehydrogenase | 1.45 ± 0.35 | 0.97 ± 0.24 | .30 | 1.07 ± 0.07 | 1.00 ± 0.29 | .81 |
| Micro-RNA | ||||||
| miR-200a | 0.96 ± 0.21 | 0.99 ± 0.25 | .97 | 1.16 ± 0.20 | 0.94 ± 0.06 | .33 |
| miR-429 | 1.22 ± 0.30 | 1.29 ± 0.23 | .87 | 1.25 ± 0.13 | 1.12 ± 0.23 | .63 |
Progesterone-mediated regulators of uterine quiescence
Neither treatment effected expression of recently discovered mediators of uterine quiescence: stat 5b, zeb 1, zeb 2, and 20-alpha-hydroxysteroid dehydrogenase ( Table ).
miRNA
Neither VP nor 17P effected the production of the miRNAs miR-200a or miR-429 ( Table ).
Effect of progestational agents on cervical remodeling
Mucosal immunity
Exposure to VP significantly increased the expression of defensin 1 compared to Replens ( P < .01) ( Figure 1 ). Treatment with either 17P or VP did not have a statistically significant effect on the production of IL-22R or SLP1 (data not shown). Baseline messenger RNA values of IL-17, IL-22, defensin 3, and defensin 4 were very low and expression was not altered by either progesterone treatment.
