Background
Progesterone, acting via its nuclear receptors called progesterone receptors, promotes myometrial relaxation during pregnancy, and suspension of this activity triggers labor. We previously found that 20α-hydroxysteroid dehydrogenase causes a local withdrawal of progesterone in the term and preterm myometrium by converting the progesterone into an inactive form before it accesses the progesterone receptors.
Objective
We hypothesized that a selective progesterone receptor modulator called promegestone, which is not metabolized by 20α-hydroxysteroid dehydrogenase, would sustain progesterone receptor signaling and prevent/delay term labor and preterm labor in mice.
Study Design
In the term labor mouse model, promegestone (0.2 mg/dam) or a vehicle were administered subcutaneously in timed-pregnant CD-1 mice at gestational days 15, 16, and 17 (term gestational days, 19.5). In the inflammation preterm labor model, pregnant mice received promegestone or a vehicle on gestational days 15, 16, and 17, which was 24 hours before, immediately before, and 24 hours after systemic bacterial endotoxin (50 μg intraperitoneal; lipopolysaccharide group) or vehicle (saline) administration. The maternal and fetal tissues were collected on gestational day 16 6 hours after lipopolysaccharide±promegestone injection and at term gestational day 18.75. The protein levels of 10 cytokines were measured by multiplex immunoassay in maternal plasma and amniotic fluid. Myometrial, decidual, and placental messenger RNA levels of multiple cytokines and procontractile proteins were evaluated by real-time polymerase chain reaction and confirmed by immunoblotting.
Results
Promegestone prevented term labor and maintained mice pregnancy postterm >24 hours. The litter size and fetal weights were not different from the controls. Promegestone prevented systemic bacterial-endotoxin-induced preterm labor in 100% of the mice, blocked uterine contractions, significantly inhibited all systemic inflammation-induced myometrial cytokines, and partially inhibited decidual and placental inflammation. Promegestone did not prevent bacterial-endotoxin-induced fetal toxicity.
Conclusion
Promegestone a selective progesterone receptor modulator that binds progesterone receptors with high affinity and is not metabolized by 20α-hydroxysteroid dehydrogenase could completely suppress term parturition and systemic bacterial-endotoxin-induced preterm birth in mice. We suggest that such selective progesterone receptor modulators may represent a potential therapeutic approach to the prevention of preterm labor in women at high risk of preterm birth.
Introduction
Preterm birth (PTB)—delivery of the preterm neonate before the full 37 weeks of gestation—is the leading cause of perinatal and infant mortality worldwide. Existing therapies to prevent preterm labor (PTL) focus on suppressing the contractility of the uterine smooth muscle that makes up the the myometrium, but they are generally ineffective and have not reduced the incidence of PTB. Tocolytics (myometrial relaxants), antibiotics, and cervical cerclage have been used to prevent PTB. However, these interventions show efficacy only in a limited groups of patients. , In particular, the efficacy of antibiotic administration in women experiencing PTL with intact membranes was demonstrated by Yoon et al, though such treatments are effective only when used for intraamniotic infection/inflammation. Asymptomatic intrauterine infection is commonly a precursor of preterm premature rupture of membranes (PPROM) . Thus, antibiotic prophylaxis in PPROM has been shown to prolong pregnancy, reduce maternal chorioamnionitis, and reduce neonatal morbidity. Progestin prophylaxis has also been used to prevent PTB, but research on its therapeutic application has produced contradictory data. Out of multiple clinical studies, only one conducted in women with singleton pregnancies and a short cervix (indicative of increased risk for PTB) measured at midgestation resulted in a positive outcome for prophylactic therapy against PTB. This study used a vaginal suppository with micronized progesterone (P4). Data from other studies suggest that P4 has no tocolytic activity and that it does not delay parturition. Despite disappointing clinical trials, the concept of progestin prophylaxis to mitigate PTB risk remains plausible in lieu of the key role of P4 in maintaining pregnancy and blocking labor. The development of effective P4-based therapies to control myometrial contractility is therefore a major priority.
Why was this study conducted?
Progesterone (P4) has anti inflammatory and anticontractile effects on the myometrium but has limited capability to prevent preterm labor in women. We speculate that the local metabolism of P4 in the myometrium limits the efficacy of natural P4. This study assessed the prophylactic potential of a nonmetabolizable selective progesterone receptor modulator (SPRM) called promegestone in preterm birth prevention.
Key findings
Our study shows that promegestone prevents preterm labor in a mouse model by limiting the inflammation of uterine/fetal tissues and inhibiting the expression of procontractile genes/proteins in the myometrium.
What does this add to what is known?
This study reveals that the local metabolism of P4 is a key factor in determining the efficacy of progestins in inhibiting labor contractility. An SPRM that is not a substrate for metabolism by 20α-hydroxysteroid dehydrogenase can maintain P4/progesterone receptor signaling, promote myometrial quiescence, and prevent inflammation-induced preterm birth.
Progesterone, as its name implies, is a progestation hormone that maintains myometrial quiescence for most of pregnancy and blocks labor. It mainly inhibits the expression of genes in myometrial cells, whose products, referred collectively as contraction-associated proteins (CAPs), increase contractility and excitability to produce the labor state. Some important CAPs include connexin43 (encoded by GJA1), the oxytocin receptor (encoded by OXTR ) , cyclooxygenase type 2 (encoded by PTGS2 ), and proinflammatory proteins such as the nuclear factor kappa B (NF-κB), interleukin 1 (IL-1), chemokine (C-X-C motif) ligand 8 (CXCL8/IL8), and chemokine (C-C motif) ligand 2 (CCL2/MCP1). Myometrial activation and the initiation of labor requires the withdrawal of P4, which in most viviparous species examined to date, is achieved by a fall in the peripheral levels of the hormone. This does not occur in women. Instead, circulating P4 levels remain elevated throughout pregnancy, declining only after the delivery of the placenta. Nevertheless, blocking P4 action anytime during human pregnancy, for example by administration of the specific antagonist—RU486, induces PTL. Therefore, it is hypothesized that human parturition is triggered by a functional withdrawal of P4, whereby myometrial cells become refractory to the progestational actions of the hormone. However, the precise molecular mechanism by which this is achieved remains unclear. Progesterone affects cervical, decidual, and fetal membrane cells, and its action is mediated through the progesterone receptors (PRA/B and PRMC1/2). We have recently proposed a model, whereby P4 acting via PRA/B promotes myometrial relaxation during pregnancy by suppressing the expression of CAP genes and the genes within inflammatory pathways , Moreover, we discovered that close to the onset of human labor and despite the high levels of P4 in maternal blood, the intracellular P4 concentrations in myometrial cells decreased because of increased expression of the P4-metabolizing enzyme—20alpha-hydroxysteroid dehydrogenase (20αHSD, encoded by AKR1C1 ), which converts P4 into its PR-inactive metabolite, 20alpha-hydroxyprogesterone (20αOHP). We found that the 20αHSD protein was significantly elevated in the human myometrium during term labor (TL) as compared with term myometrium that was not in labor (NIL). In mice, 20αHSD up-regulation and associated myometrial P4 withdrawal is common to both term and PTL. For instance, in pregnant mice, uterine inflammation caused by the bacterial endotoxin lipopolysaccharide (infection mimetic LPS) initiated PTL and was associated with an increased expression of 20αHSD in the myometrium. We concluded that in mice and humans, the metabolism of P4 in myometrial cells into 20αOHP decreases local P4 levels, which subsequently spares the receptor (PR-A) from binding to the hormone. Our published data indicate that when it is not bound to P4, PR-A switches from a transcriptional repressor to a transcriptional activator of the CAP genes and thus serves as a key trigger for the onset of labor. Based on these findings, we reasoned that administration of a progestin that is not metabolized by 20αHSD would keep the PRs in a ligand-bound state and that this might represent a plausible therapeutic strategy to prevent PTB. To test this hypothesis, we evaluated the ability of promegestone (PMG) (also known as R5020), a selective progesterone receptor modulator (SPRM) that is not metabolized by 20αHSD, to prevent/delay inflammation-induced PTB in a mouse model. Our study provides novel information regarding the potential of nonmetabolized, synthetic P4 agonists as a new class of prophylactic drugs effective for the prevention of PTB.
Material and Methods
Animal model
The Hsd:ICR (CD-1) outbred mice used for these experiments were purchased from Harlan Laboratories ( http://www.harlan.com/ ). All mice were housed under specific pathogen-free conditions at the Toronto Centre for Phenogenomics (TCP) on a 12L:12D cycle and were administered food and water ad libitum. All animal experiments were approved by the TCP Animal Care Committee (ACC AUP#21-0164H). Female mice were mated overnight with males and the day of vaginal plug detection was designated as the gestational day (GD) 0.5 of pregnancy. The average time of term delivery in our facility was GD 19–20 (GD, 19.5). Preterm delivery was defined as the finding of at least one fetus in the cage within 24 hours of bacterial endotoxin lipopolysaccharide (LPS) administration. Vaginal bleeding alone was not considered as evidence of delivery in the absence of the other signs.
Experimental design
Promegestone and progesterone administration
The PMG used for this study was sourced from Perkin Elmer (Cat # NLP004005MG, Waltham, MA), and P4 from Sigma Aldrich (Cat # P8783, MO). Pregnant CD-1 mice (n=36) were randomly divided, and one-third of animals (n=12) were given a subcutaneous (SC) injection of PMG (0.2 mg/dam, n=12), one-third received an SC injection of P4 (1 mg/dam, n=12), and one-third received an SC injection of ethanol/corn oil (Vehicle, n=12). PMG/P4/vehicle was first administered to pregnant mice on GD 14.5. Twenty-four hours later, on GD 15.5, the mice received the second injection of PMG/P4/vehicle before the LPS or saline injection ( Figure 1 ). On GD 16.5, the animals that did not develop PTL received a third injection of PMG/P4/vehicle. The dose of PMG (0.2 mg/dam in 100 μL of corn oil/ethanol) was established by Kuon et al. A P4 dose (1 mg/dam in 100 μl of corn oil/ethanol) was used as described by Young et al and Hirsch et al. ,
Term labor model
PMG (0.2 mg/dam), P4 (1 mg/dam), or a vehicle was administered subcutaneously in 100 μL corn oil/ethanol at gestation days 14.5, 15.5, 16.5 (n=8; term GD, 19.5). P4-treated and PMG-treated animals that carried pregnancy to full term (GD, 19.1–19.9) were sacrificed during labor. Our criteria of labor was the delivery of at least 1 pup. Mice that did not deliver at term were sacrificed 24 hours postterm (GD, 20.5). The effects of PMG on delivery time, maternal weight gain, litter size, and weight of neonates (for vehicle and P4 groups) and fetal weight (PMG group) were assessed ( Table 1 ). In a replicate group of animals, pregnant PMG-treated and vehicle-treated mice (n=6 per group) were sacrificed before term delivery on GD 18.75 from 6 to 8 PM . We recorded the maternal weight gain, litter size, and the fetal and placental weight ( Table 2 ). Maternal (myometrium, plasma) and fetal tissues were collected for analysis.
| Outcome measures | Vehicle | Progesterone | Promegestone |
|---|---|---|---|
| Term birth (number of dams) | 8/8 | 6/6 | 1/8 |
| Postterm birth (number of dams) | 0 | 0 | 7/8 |
| Delayed parturition (%) | 0 | 0 | 87.5 |
| Pregnancy weight gain (GD 15-labor, g) | 14.8±2 a | 14.7±3.67 a | 13.83±5.24 a |
| Litter size (n) | 12.67±3.2 b | 14.31±2.5 b | 12.88±4.42 b |
| Neonatal/fetal weight (g) | 1.52±0.2 c | 1.76±0.16 c | 1.70±0.21 c |
a Indicates that pregnancy weight gain is not significantly different between groups ( P >.05)
b Indicates that litter size is not significantly different between groups ( P >.05)
c Indicates that neonatal/fetal weight is not significantly different between groups ( P >.05).
| Maternal/neonatal outcome measures | Vehicle | PMG | P value |
|---|---|---|---|
| Number of mice | 6 | 6 | |
| Pregnancy weight gain (GD 15–18.75, g) | 11.37±1.93 | 9.6±2.9 | .28 |
| Litter size (n) | 13.33±1.49 | 14.33±2.75 | .46 |
| Fetal weight (g) | 1.1±0.155 | 1.2±0.09 | .23 |
| Placental weight (g) | 0.1±0.02 | 0.15±0.02 | .001 |
Systemic inflammation model
The LPS used for this study was isolated from E. coli , serotype 055:B5 (Sigma, St Louis, MO). On GD 15.5, half of the mice that were treated with PMG/P4/vehicle received an intraperitoneal (IP) injection of 50 μg of LPS in 100 μl of sterile saline (LPS group, n=6 per subgroup) or IP injections of 100 μl of sterile saline (Control group, n=6 per subgroup). Animals were then observed hourly for signs of labor except during the interval period from midnight to 6 am. Starting from 6 am the following day, the animals were monitored every 45–60 minutes until delivery. The delivery time was recorded for every animal ( Table 3 ). In 2 control groups (control+vehicle and control+PMG), none of the pregnant mice delivered before term. An IP injection of 50 μg LPS/mouse on GD 15.5 causes PTB in 100% of animals in the vehicle+LPS group within 24 hours. PMG-treated animals from the LPS group that did not deliver at term were sacrificed 24 hours later (postterm GD, 20.5).
| Outcome measures | LPS+vehicle | LPS+P4 | LPS+PMG |
|---|---|---|---|
| Preterm birth (number of dams) | 6/6 | 2/5 | 0/6 |
| Term birth (number of dams) | 0 | 3/5 | 1/6 |
| Postterm (number of dams) | 0 | 0 | 5/6 |
| Prevented preterm birth (%) | 0 | 60 | 100 |
Blood and tissue collection
Different experimental groups were used to assess short (6 hours)- and long (24 hours)-term outcomes.
Long-term effect study
We evaluated the long-term effect of PMG treatments vs P4 (n=6 in LPS+Vehicle group, n=5 in LPS+P4 group, and n=6 in LPS+PMG group) ( Figure 1 , B). Mice that delivered preterm (n=6 in LPS group and n=2 in LPS+P4 group) were euthanized by carbon dioxide inhalation during PTL. The animals that carried pregnancy to term were sacrificed on GD 19.5 during TL. Animals that carried pregnancy postterm (5 from 6 mice in the LPS+PMG group) were sacrificed on GD 20.5 (24 hours after the average delivery time). The intact uterus of each female mouse was removed and the total number of fetuses, their vital signs, and fetal and placental weights were taken into account.
In a replicate group of animals, pregnant mice that did not deliver preterm after LPS±PMG injection were sacrificed before term delivery on GD 18.75 from 6 to 8 PM. We recorded the maternal weight gain, litter size, fetal and placental weights and fetal viability ( Table 4 ). The maternal (myometrium, plasma etc.) and fetal tissues (placenta, amniotic fluid) were collected for analysis.
| Maternal/neonatal outcome measures | PMG | LPS+PMG | P value |
|---|---|---|---|
| Number of mice | 6 | 6 | |
| Pregnancy weight gain (GD 15–18.75, g) | 9.6±2.9 | 0.52±0.82 | .001 |
| Litter size (n) | 14.33±2.75 | 14.67±2.36 | .84 |
| Fetal weight (g) | 1.2±0.09 | 0.55±0.11 | .001 |
| Placental weight (g) | 0.15±0.02 | 0.123±0.013 | .02 |
| Fetal viability (%) | 98 | 28.4 | .001 |
Short-term effect study
To evaluate the immediate effect of PMG on cytokine expression, we collected maternal and fetal tissues at 6 hours after the LPS injection, n=5–7 group) ( Figure 1 , C). Maternal blood was obtained by cardiac puncture in a lithium-heparin microtainer (Microvette, Sarstedt, Germany). The animals were killed by CO 2 asphyxiation and blood was collected and plasma isolated by centrifugation for 5 minutes at 2000 g and frozen in liquid nitrogen until an assay was done. The maternal liver was collected. The gravid uterus was placed into ice-cold phosphate-buffered saline, bisected longitudinally, and dissected away from both pups and placentas. The decidua basalis was cut away from the myometrial tissue and pooled from all implantation sites. The myometrium collected from both uterine horns was pooled. The decidua parietalis was carefully removed from the myometrial tissue by mechanical scraping on ice. The amniotic fluid was collected from all gestational sacs and pooled and centrifuged for 10 minutes at 5000 g. Ten placentas were randomly pooled from both the uterine horns. All mouse tissues were flash-frozen in liquid nitrogen and stored at –80°C.
Real-time polymerase chain reaction analysis
The total RNA was extracted from frozen mouse liver, myometrium, decidua, and placenta using TRIzol (Gibco BRL, Burlington, Ontario, Canada) according to the manufacturer’s instructions (n=5–7/group). The RNA samples were column-purified using the RNeasy Mini Kit (Qiagen, Mississauga, Ontario, Canada) and treated with DNase I (Qiagen) to remove genomic DNA contamination . The process was quality-controlled by measuring yield (μg) and by A 260/280 ratio (Nanodrop ND-1000; Thermo Scientific, Waltham, MA). cDNA synthesis was performed as per the manufacturer’s protocol (iScript cDNA synthesis kit, Bio-Rad, Mississauga, Ontario, Canada). Quantitative real-time polymerase chain reaction (PCR) was performed using LuminoCt SYBR Green QPCR READYMIX (Sigma, St. Luis, MO), CFX-384 Real Time System C1000 Thermal Cycler (Bio-Rad), and specific pairs of primers ( Supplemental Table 1 ). Aliquots (5 ng) of cDNA were used for each PCR reaction run in triplicates. A cycle threshold (Ct) value was recorded for each sample. Each gene was normalized to the expression of 3 housekeeping genes ( Gapdh , Tbp , Hprt ); the expression levels were calculated by the CFX Manager software (version 2.1), and the relative gene expression for LPS-induced and PMG/P4/vehicle-treated animals was presented as the average fold change relative to the control-vehicle sample.
Luminex assay
To evaluate the effect of PMG on proinflammatory cytokines and chemokines, the levels of the proinflammatory cytokines IL1β, IL6, IL12, tumor necrosis factor (TNF)α, interferon (IFN)γ, and CSF2/GM-CSF, anti-inflammatory cytokine IL10, and 3 major chemokines CXCL1/KC, CCL2/MCP1, and CCL4/ Mib1b were measured by the Luminex multiplex assay in the plasma and amniotic fluid using the Bio-Plex Pro Mouse Cytokine 10-Plex Array kit (Bio-Rad, Hercules, CA) ( Supplemental Table 2 ). The magnetic bead kit was run on the Luminex 200 system and Bioplex HTF (Bio-Rad) in accordance with the manufacturer`s instructions. The standards and each of the samples were analyzed in duplicate. Data analysis was performed using the Bio-Plex Manager version 6.0 (Bio-Rad) and presented as concentrations (pg/mL). The Bio-Plex “Assay Working Range” and the sensitivity of each cytokine assay ( www.biorad.com ) is shown in Supplemental Table 2 .
Protein extraction
The myometrial tissues were crushed on dry ice, and the total protein was extracted from the myometrial samples using bead-based homogenization. This was done in precooled SafeSeal Micro tubes (Sarstedt, Numbrecht, Germany) containing the appropriate volume of lysis buffer (0.08 M Tris/HCl, pH 6.8, 2% SDS, 10% Glycerol) supplemented with Halt protease & phosphatase inhibitors (Thermo Fisher Scientific/Pierce Inc, Rockford, IL). The tissues were immediately homogenized by mechanical disruption using Tissuelyser II (Qiagen, MD) for 3 minutes and then incubated on ice for 10 minutes followed by centrifugation at 8000× g for 5 minutes at 4°C. The supernatants were then sonicated on ice for 10 seconds, cooled on ice for 5 minutes, and centrifuged at 18,000× g for 15 minutes at 4°C. The supernatant was collected and stored at −80°C. The protein concentration was determined by bicinchoninic acid protein assay (Thermo Fisher Scientific Inc, Toronto, Ontario, Canada) using bovine serum albumin (Sigma, St. Louise, MO) as the reference standard.
Western blotting
Protein samples (35 μg each) were denatured in NuPAGE lithium dodecyl sulfate loading buffer (Thermo Fisher Scientific/Novex, NP0007, Carlsbad, CA) at 95°C for 10 minutes, subjected to polyacrylamide (Tris-glycine) gel electrophoresis, and transferred onto a polyvinylidene difluoride (PVDF) membrane (Trans-blot Turbo Midi PVDF, BioRad). The membranes were blocked with 5% w/v fat-free milk in 0.05 Tween-tris-buffered saline (TBS-T) solution for 1 hour at room temperature and then incubated with anti-Cx43 primary antibody (1:1000, Millipore, AB1728, Billerica, MA) overnight at 4°C. The membranes were washed 3 times in the TBS-T buffer before incubation with the anti-rabbit- horseradish-peroxidase-conjugated secondary antibody (1:5000, Santa Cruz Biotechnology, SC-2004, Dallas, TX) at a dilution for 1 hour at room temperature. The membranes were stripped and reprobed with a housekeeping protein using anti-ERK-2 antibody (1:2000, Santa Cruz Biotechnology, SC-154). The signals were detected using the Luminata horseradish peroxidase-substrate (Millipore), and imaging was performed with the VersaDoc imaging system (BioRad).
Statistical analysis
The Grubbs’ outlier test was utilized to identify and exclude outliers from all data sets. The student t test was carried out for the number of pups, fetal weight, and plasma P4 concentration between the control and PMG groups. To determine the differences between cytokine gene and protein expression, 4 study groups were subjected to a 2-way analysis of variance (ANOVA) followed by Sidak’s or Tukey’s multiple comparison posttest. To compare Cx43 protein expression in the mouse myometrium, t test, 1-way, and 2-way ANOVA were used. The normality equal variance tests were performed with the statistical program GraphPad Prism (version 8.3.1). The level of significance was set at P <.05.
Results
Promegestone blocks term labor in mice
Using pregnant CD-1 mice as a model of normal gestation and TL, we evaluated the effects of PMG and P4 on the pregnancy length. Pregnant dams received daily injections of a vehicle, P4, or PMG for 3 days, starting from GD 15 (75% of mouse gestational length) through to GD 17. PMG prevented term parturition and prolonged the length of gestation. 7/8 mice treated with PMG remained pregnant for at least 24 hours postterm (88%, Table 1 , Figure 1 , A). In contrast, term parturition occurred normally at GD 19 in vehicle- and P4-treated mice. Pregnancy weight gain, litter size, and pup weights were not affected by PMG or P4 ( Table 1 ). At GD 18.75 (term NIL), the placental weights in PMG-treated mice were increased than the vehicle-treated mice, whereas the number of pups and fetal weight were not different ( P <.05, Table 2 ).
Promegestone blocks lipopolysaccharide-induced preterm birth and delays the onset of labor
An intraperitoneal injection of LPS on GD 15.5 (50 μg LPS/mouse) caused PTB within 24 hours in 6 of 6 mice treated with a vehicle and in 2 of 5 mice treated with P4 ( P <.05 compared with vehicle); 3 of 5 mice (60%) treated with P4 delivered at term ( Table 3 , Figure 1 , B). There was no effect of PMG or P4 on maternal mortality or morbidity. The pretreatment of pregnant mice with PMG (0.2 mg/dam/day) 24 hours before LPS injection completely blocked PTB (100%). Moreover, 5 of 6 dams induced with LPS and treated with PMG for 3 days (GD, 15–17, maintained pregnancy at least 24 hours postterm when the experiment was terminated ( Figure 1 , B). The mice in which LPS-induced parturition was prevented by PMG had a litter size similar to the group of mice treated with PMG only. However, the mean pregnancy weight gain between GD 15 and 18.75 and the mean fetal and placental weights measured at GD 18.75 in the mice treated with LPS+PMG were significantly lower than those in the mice treated with PMG alone ( Table 4 , Figure 1 , C). Moreover, though pretreatment with PMG prevented LPS-induced PTB, fetal viability was not preserved. In the mice receiving PMG only, the number of live pups at GD 18.75 was significantly higher than animals receiving LPS+PMG (98% vs 28.4%; P <.001, t test) ( Table 4 ). Similarly, P4 administration decreased the incidence of PTB but did not preserve fetal viability. Three mice injected with LPS that delivered at term after pretreatment with P4 (1 mg/dam) contained mostly dead fetuses in their uterine horns.
Promegestone attenuates lipopolysaccharide-mediated effects on maternal and fetal tissues
PMG significantly inhibited the myometrial expression of the procontractile genes Gja1 and Oxtr in LPS-treated mice 6 hours postinjection ( P <.05) ( Figure 2 , A). The effect of PMG was sustained until term. The abundance of the Gja1 and Oxtr transcripts in the myometrial samples collected at GD 18.75 (term NIL) was significantly lower in the PMG-treated mice than the vehicle-treated mice ( Figure 2 , B).
