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Perinatology Research Branch, Program for Perinatal Research and Obstetrics, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, and Detroit, MIDepartment of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI
Perinatology Research Branch, Program for Perinatal Research and Obstetrics, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, and Detroit, MIDepartment of Obstetrics and Gynecology, University of Michigan Medical School, Ann Arbor, MI, Department of Epidemiology and Biostatistics, Michigan State University College of Human Medicine, East Lansing, MI, and Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI
Perinatology Research Branch, Program for Perinatal Research and Obstetrics, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, and Detroit, MI
Perinatology Research Branch, Program for Perinatal Research and Obstetrics, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, and Detroit, MI
Perinatology Research Branch, Program for Perinatal Research and Obstetrics, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, and Detroit, MI
Perinatology Research Branch, Program for Perinatal Research and Obstetrics, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, and Detroit, MIDepartment of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI
Perinatology Research Branch, Program for Perinatal Research and Obstetrics, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, and Detroit, MIDepartment of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI
Perinatology Research Branch, Program for Perinatal Research and Obstetrics, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, and Detroit, MIDepartment of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MIDepartment of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI
Progestogen (vaginal progesterone or 17-alpha-hydroxyprogesterone caproate [17OHP-C]) administration to patients at risk for preterm delivery is widely used for the prevention of preterm birth (PTB). The mechanisms by which these agents prevent PTB are poorly understood. Progestogens have immunomodulatory functions; therefore, we investigated the local effects of vaginal progesterone and 17OHP-C on adaptive and innate immune cells implicated in the process of parturition.
Study Design
Pregnant C57BL/6 mice received vaginal progesterone (1 mg per 200 μL, n = 10) or Replens (control, 200 μL, n = 10) from 13 to 17 days postcoitum (dpc) or were subcutaneously injected with 17OHP-C (2 mg per 100 μL, n = 10) or castor oil (control, 100 μL, n = 10) on 13, 15, and 17 dpc. Decidual and myometrial leukocytes were isolated prior to term delivery (18.5 dpc) for immunophenotyping by flow cytometry. Cervical tissue samples were collected to determine matrix metalloproteinase (MMP)-9 activity by in situ zymography and visualization of collagen content by Masson’s trichrome staining. Plasma concentrations of progesterone, estradiol, and cytokines (interferon [IFN]γ, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, keratinocyte-activated chemokine/growth-related oncogene, and tumor necrosis factor-α) were quantified by enzyme-linked immunosorbent assays. Pregnant mice pretreated with vaginal progesterone or Replens were injected with 10 μg of an endotoxin on 16.5 dpc (n = 10 each) and monitored via infrared camera until delivery to determine the effect of vaginal progesterone on the rate of PTB.
Results
The following results were found: (1) vaginal progesterone, but not 17OHP-C, increased the proportion of decidual CD4+ regulatory T cells; (2) vaginal progesterone, but not 17OHP-C, decreased the proportion of decidual CD8+CD25+Foxp3+ T cells and macrophages; (3) vaginal progesterone did not result in M1→M2 macrophage polarization but reduced the proportion of myometrial IFNγ+ neutrophils and cervical active MMP-9-positive neutrophils and monocytes; (4) 17OHP-C did not reduce the proportion of myometrial IFNγ+ neutrophils; however, it increased the abundance of cervical active MMP-9-positive neutrophils and monocytes; (5) vaginal progesterone immune effects were associated with reduced systemic concentrations of IL-1β but not with alterations in progesterone or estradiol concentrations; and (6) vaginal progesterone pretreatment protected against endotoxin-induced PTB (effect size 50%, P = 0.011).
Conclusion
Vaginal progesterone, but not 17OHP-C, has local antiinflammatory effects at the maternal-fetal interface and the cervix and protects against endotoxin-induced PTB.
Preterm neonates are at an increased risk for short- and long-term morbidity, and prematurity represents a substantial burden for society and the health care system.
Institute of Medicine Committee on Understanding Premature Birth and Assuring Healthy Outcomes. Societal costs of preterm birth. In: Behrman RE, Butler AS, eds. Preterm birth: causes, consequences, and prevention. Washington (DC); 2007.
Therefore, the prevention of PTB is a health care priority.
Cervical assessment with ultrasound coupled with the administration of vaginal progesterone represents the main strategy to prevent PTB in nulliparous women and in those without a prior history of prematurity.
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.
Effect of progesterone on cervical shortening in women at risk for preterm birth: secondary analysis from a multinational, randomized, double-blind, placebo-controlled trial.
Vaginal progesterone reduces the rate of preterm birth in women with a sonographic short cervix: a multicenter, randomized, double-blind, placebo-controlled trial.
Vaginal progesterone in women with an asymptomatic sonographic short cervix in the midtrimester decreases preterm delivery and neonatal morbidity: a systematic review and metaanalysis of individual patient data.
Vaginal progesterone vs. cervical cerclage for the prevention of preterm birth in women with a sonographic short cervix, previous preterm birth, and singleton gestation: a systematic review and indirect comparison metaanalysis.
Society for Maternal-Fetal Medicine Publications Committee Progesterone and preterm birth prevention: translating clinical trials data into clinical practice.
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.
Effect of progesterone on cervical shortening in women at risk for preterm birth: secondary analysis from a multinational, randomized, double-blind, placebo-controlled trial.
Vaginal progesterone reduces the rate of preterm birth in women with a sonographic short cervix: a multicenter, randomized, double-blind, placebo-controlled trial.
Vaginal progesterone in women with an asymptomatic sonographic short cervix in the midtrimester decreases preterm delivery and neonatal morbidity: a systematic review and metaanalysis of individual patient data.
Vaginal progesterone vs. cervical cerclage for the prevention of preterm birth in women with a sonographic short cervix, previous preterm birth, and singleton gestation: a systematic review and indirect comparison metaanalysis.
Society for Maternal-Fetal Medicine Publications Committee Progesterone and preterm birth prevention: translating clinical trials data into clinical practice.
Two year infant outcomes for children exposed to supplemental intravaginal progesterone gel in utero: secondary analysis of a multicenter, randomized, double-blind, placebo-controlled trial.
Increased incidence of gestational diabetes in women receiving prophylactic 17alpha-hydroxyprogesterone caproate for prevention of recurrent preterm delivery.
Obstetrix Collaborative Research N. Failure of 17-hydroxyprogesterone to reduce neonatal morbidity or prolong triplet pregnancy: a double-blind, randomized clinical trial.
Prevention of preterm delivery by 17 alpha-hydroxyprogesterone caproate in asymptomatic twin pregnancies with a short cervix: a randomized controlled trial.
also differ between both compounds. Therefore, these terms should not be used interchangeably.
The mechanisms of action whereby progestogens prevent PTB are unknown. There has been considerable interest in the role of progesterone in the maintenance of myometrial quiescence.
Gap junctions and myometrial steroid hormone receptors in pregnant and postpartum rats: a possible cellular basis for the progesterone withdrawal hypothesis.
The length of the cervix and the risk of spontaneous premature delivery. Eunice Kennedy Shriver National Institute of Child Health and Human Development Maternal Fetal Medicine Unit Network.
Prediction of patient-specific risk of early preterm delivery using maternal history and sonographic measurement of cervical length: a population-based prospective study.
Pharmacologic actions of progestins to inhibit cervical ripening and prevent delivery depend on their properties, the route of administration, and the vehicle.
Medroxyprogesterone acetate modulates remodeling, immune cell census, and nerve fibers in the cervix of a mouse model for inflammation-induced preterm birth.
and a microarray study, which demonstrated that the cervices of mice treated with medroxyprogesterone acetate had a different expression of inflammatory genes compared to the control group.
Morphologic and histochemical evidence for the occurrence of collagenolysis and for the role of neutrophilic polymorphonuclear leukocytes during cervical dilation.
in: Ellwood E. Anderson A. The cervix in pregnancy and labor: clinical and biochemical investigations. Churchill Livingstone,
Edinburgh (United Kingdom)1981
Infection and labor. V. Prevalence, microbiology, and clinical significance of intraamniotic infection in women with preterm labor and intact membranes.
Amniotic fluid interleukin-6: correlation with upper genital tract microbial colonization and gestational age in women delivered after spontaneous labor versus indicated delivery.
A novel molecular microbiologic technique for the rapid diagnosis of microbial invasion of the amniotic cavity and intra-amniotic infection in preterm labor with intact membranes.
Toll-like receptor-2 and -4 in the chorioamniotic membranes in spontaneous labor at term and in preterm parturition that are associated with chorioamnionitis.
Villitis of unknown etiology is associated with a distinct pattern of chemokine up-regulation in the feto-maternal and placental compartments: implications for conjoint maternal allograft rejection and maternal anti-fetal graft-versus-host disease.
Maternal HLA panel-reactive antibodies in early gestation positively correlate with chronic chorioamnionitis: evidence in support of the chronic nature of maternal anti-fetal rejection.
Characterization of the fetal blood transcriptome and proteome in maternal anti-fetal rejection: evidence of a distinct and novel type of human fetal systemic inflammatory response.
Detection of anti-HLA antibodies in maternal blood in the second trimester to identify patients at risk of antibody-mediated maternal anti-fetal rejection and spontaneous preterm delivery.
) during late pregnancy in guinea pigs can increase the release of proinflammatory cytokines by the amniochorion, cervix, and decidual-myometrial tissues.
This hormone can also increase the proportion of CD4+CD25+ regulatory T cells (Tregs), which are key in the control of the adaptive immune response, in the uterine tissues during mid-pregnancy in mice.
The objectives of this study were to determine the effects of vaginal progesterone and 17OHP-C on the following: (1) the proportion of CD4+ Tregs and CD8+CD25+Foxp3+ T cells at the maternal-fetal interface (myometrium and decidua); (2) the proportion and phenotype of macrophages (M1-like or M2-like) at the maternal-fetal interface; (3) the proportion of neutrophils and their cytokine production at the maternal-fetal interface; and (4) matrix metalloproteinase (MMP)-9 activity in the cervix.
Finally, we sought to determine whether pretreatment with vaginal progesterone could prevent endotoxin-induced PTB.
Materials and Methods
Animals
C57BL/6 mice were bred in the animal care facility at the C. S. Mott Center for Human Growth and Development at Wayne State University (Detroit, MI), and housed under a circadian cycle (12 hours of light and 12 hours of dark). Females 8–12 weeks old were mated with male mice of proven fertility. Female mice were examined daily between 8:00 and 9:00 am for the presence of a vaginal plug, which denoted 0.5 days postcoitum (dpc). Upon observation of vaginal plugs, the female mice were then separated from the males and were housed in different cages. A weight gain of >2 g confirmed pregnancy at 12.5 dpc. Procedures were approved by the Institutional Animal Care and Use Committee at Wayne State University (protocol number A09-08-12).
Progestogen administration
Pregnant females received vaginal progesterone (Crinone 8% vaginal gel; Fleet Laboratories Ltd, Watford, Herts, United Kingdom) at a concentration of 1 mg per 200 μL (n = 10) or 200 μL of Replens (Lil’ Drug Store Products, Inc, Cedar Rapids, IA) as a control (n = 10) from 13 to 17 dpc (Figure 1A).
Figure 1Animal model and identification of decidual CD4+ Tregs and CD8+CD25+Foxp3+ T cells
A, Vaginal progesterone administration scheme. B, Gating strategy used to identify CD4+ Tregs (CD4+CD25+Foxp3+ T cells) and CD8+CD25+Foxp3+ T cells in decidual tissue. CD3+ T cells were gated within the total lymphocyte gate (FSC vs SSC). The green histogram represents the autofluorescence control. CD4+ Tregs and CD8+CD25+Foxp3+ T cells were gated within the CD4+ and CD8+ gates, respectively. C, Proportions of decidual CD4+ Tregs in mice treated with vaginal progesterone or Replens (control). D, Proportions of decidual CD8+CD25+Foxp3+ T cells in mice treated with vaginal progesterone or Replens (control). E, Proportions of decidual CD4+ Tregs in mice injected with 17OHP-C or castor oil (control). F, Proportions of decidual CD8+CD25+Foxp3+ T cells in mice injected with 17OHP-C or castor oil (control) (n = 10 each). Data are represented as mean ± SEM.
FSC, forward scatter; 17OHP-C, 17-alpha-hydroxyprogesterone caproate; SSC, side scatter; Treg, regulatory T cell.
Furcron. Vaginal progesterone, but not 17OHP-C, has antiinflammatory effects. Am J Obstet Gynecol 2015.
A second group of mice was injected subcutaneously with 2 mg per 100 μL of 17OHP-C (n = 10; Compounding Solutions, Shelby Township, MI) or 100 μL of castor oil (European Pharmacia Grade; ACROS Organics, Thermo Fisher Scientific, Waltham, MA) as a control (n = 10) on 13, 15, and 17 dpc.
This source of 17OHP-C is used clinically at the Detroit Medical Center, and previous studies demonstrated that compounded 17OHP-C had adequate potency compared to the U.S. Food and Drug Administration-approved agent.
The administration of vaginal progesterone or Replens (control) was performed starting on 13 dpc to mimic the treatment regimen followed by pregnant women with a short cervix.
Vaginal progesterone administration is generally started between 20–23 weeks of gestation in women with a short cervix,
Vaginal progesterone reduces the rate of preterm birth in women with a sonographic short cervix: a multicenter, randomized, double-blind, placebo-controlled trial.
which is equivalent to approximately 13 dpc in mice during midgestation. Administration of 17OHP-C or the castor oil control began on 13 dpc and continued on alternating days in mice since women receive this synthetic progesterone on a weekly basis.
All mice were euthanized prior to term delivery (18.5 dpc) and decidual, myometrial, and cervical tissues were harvested.
Leukocyte isolation
Immediately after collection, myometrial and decidual tissues were mechanically disaggregated in a cell dissociating reagent (Accutase; Life Technologies, Grand Island, NY) using scissors for approximately 1–2 minutes, as previously described.
Samples were then incubated at 37°C for 35 minutes with gentle shaking (MaxQ 4450 benchtop orbital shaker; Thermo Fisher Scientific, Marietta, OH). The cell suspensions were filtered using a 100 μm cell strainer (Fisher Scientific, Hanover Park, IL) and washed with flow cytometry (FACS) buffer [bovine-serum albumin 0.1% (Sigma-Aldrich, St. Louis, MO), sodium azide 0.05% (Fisher Chemicals, Fair Lawn, NJ), and 1× phosphate-buffered saline (PBS; Fisher Scientific Bioreagents, Fair Lawn, NJ)]. The resulting pellet was resuspended in FACS buffer and used for immunophenotyping.
Immunophenotyping
Cell suspensions were incubated with a monoclonal mouse CD16/CD32 antibody (FcγIII/II receptor; BD Biosciences, San Jose, CA) for 10 minutes at 4°C. The cells were then washed with FACS buffer and incubated for 30 minutes at 4°C with the corresponding extracellular and/or intracellular fluorochrome-conjugated antibodies (Supplemental Table). Tregs were determined in decidual and myometrial tissues using the extracellular markers CD3, CD4, CD8, and CD25 and the transcriptional factor Foxp3. Innate leukocyte populations including macrophages, dendritic cells (DCs), natural killer (NK) cells, and neutrophils were also identified in the decidual and myometrial tissues using the extracellular markers CD45, F4/80, CD11c, CD49b, and Ly6G.
Foxp3 staining was performed using the Foxp3/transcription factor staining buffer set (eBioscience, San Diego, CA). For cytokine staining, the Cytofix/Cytoperm fixation/permeabilization solution kit (BD Biosciences) was used, following the manufacturer’s recommendations. Unstained cells were treated with the same protocol and used as autofluorescence controls. Cell suspensions were acquired and analyzed using the LSRFortessa flow cytometer and BD FACSDiva software, version 8.0 (BD Biosciences), respectively. Figures were prepared using FlowJo Software version 10 (FlowJo, LLC, Ashland, OR).
In situ MMP-9 zymography
To determine the MMP-9 activity in cervical tissue, in situ zymography was performed as described by Hadler-Olsen et al.
Cervical tissue sections were fixed in ethanol and embedded in paraffin; from these blocks, 5-μm-thick sections were cut and mounted on FisherBrand Superfrost microscope slides (Fisher Scientific) and heated to 59°C. Slides were further deparaffinized in xylene and rehydrated in graded alcohol baths. The gelatinase reaction was performed using the EnzChek gelatinase/collagenase assay kit (Life Technologies), and to verify the enzyme specificity, tissue sections were preincubated for 1 hour with 200 μL of 10 mM phenanthroline, a metal chelator and general inhibitor of metalloproteinases.
The remaining slides were preincubated with a reaction buffer, and a substrate was prepared by dissolving 1 mg DQ gelatin (Life Technologies) in 1.0 mL of deionized water and diluted 1:50 with reaction buffer. Substrate solution (200 μL) with or without 10 mM phenanthroline was then added to the tissue sections. All slides were incubated in a dark humidity chamber at 37°C for 2 hours, and the negative control slides were incubated at -20°C for 2 hours.
Following incubation, the sections were rinsed twice with deionized water, fixed in 4% neutral buffered formalin for 10 minutes in the dark, and then rinsed twice with 1× PBS prior to mounting with ProLong Gold Antifade reagent with 4',6-diamidino-2-phenylindole (DAPI; Life Technologies). The slides were scanned using the Pannoramic MIDI digital slide scanner (PerkinElmer, Inc, Waltham, MA), and annotations were made by laboratory personnel who then utilized 3DHISTECH software (3DHISTECH Kft, Budapest, Hungary) to assess the number of positive cells.
Masson’s trichrome staining
Cervical tissue sections were fixed in 4% paraformaldehyde upon harvesting and stored at 4°C in ethanol before being embedded into paraffin blocks. The embedded tissues were then cut into 5-μm-thick sections, placed onto salinized slides, deparaffinized with xylene, and hydrated with ethanol and water. The staining was performed on the Dako AutostainerPlus (Dako, Carpinteria, CA) using Masson’s trichrome stain kit (American MasterTech, Lodi, CA), according to the manufacturer’s protocol. Briefly, the sections were mordanted in Bouin solution overnight at room temperature, rinsed in water, stained with Weigert’s hematoxylin for 3 minutes, rinsed again in water, and stained with Biebrich Scarlet-Acid Fuchsin solution for 15 minutes.
After the second rinse, the slides were incubated with phosphomolybdic/phosphotungstic acid for 15 minutes, stained with Aniline Blue stain for 10 minutes, rinsed, and incubated with 1% acetic acid for 5 minutes. The sections were then dehydrated in a series of alcohol baths, and then a coverslip was placed. The images were taken using the Pannoramic MIDI digital slide scanner (PerkinElmer, Inc).
Decidual protein extracts
Decidual tissue samples were collected from mice treated with vaginal progesterone or Replens (control) at 18.5 dpc and placed in small Petri dishes with sterile 1× PBS (n = 10 each). Tissues were incubated in a 12-well culture plate (Falcon multiwell plates for cell culture; Becton Dickinson Labware, Franklin Lanes, NJ), using a single well per tissue with 1 mL of Gibco Dulbecco’s Modified Eagle Medium (Life Technologies) supplemented with 1% Gibco antibiotic-antimycotic solution (Life Technologies) for 24 hours at 37°C in 5% CO2. Following incubation, tissues were homogenized using a Tissue Tearor (BioSpec Products, Inc, Bartlesville, OK) and centrifuged at 15,000 × g for 30 minutes at 4°C to obtain a cell-free supernatant containing the protein extract.
Enzyme-linked immunosorbent assays (ELISAs)
Blood samples, obtained by cardiac puncture from mice that received vaginal progesterone, Replens, 17OHP-C, or castor oil were placed in tubes containing heparin (Sigma-Aldrich). Plasma samples were then obtained by centrifugation. Plasma progesterone and estradiol concentrations were measured using the PROG-EASIA ELISA kit (GenWay Biotech, Inc, San Diego, CA) and the Calbiotech mouse/rat estradiol ELISA kit (Calbiotech Inc, Spring Valley, CA), respectively, according to the manufacturer’s instructions.
The concentrations of interferon (IFN)γ, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, keratinocyte-activated chemokine/growth-related oncogene (KC/GRO), and tumor necrosis factor (TNF)-α in plasma were measured with sensitive and specific immunoassays (Meso Scale Discovery, Gaithersburg, MD), according to the manufacturer's instructions. IL-10 was also determined in the decidual protein extracts.
The sensitivities of the assays were as follows: 0.022 pg/mL (IFNγ), 0.104 pg/mL (IL-1β), 0.179 pg/mL (IL-2), 0.098 pg/mL (IL-4), 0.066 pg/mL (IL-5), 0.825 pg/mL (IL-6), 0.425 pg/mL (IL-10), 8.578 pg/mL (IL-12p70), 0.218 pg/mL (KC/GRO), and 0.164 pg/mL (TNFα). The interassay and intraassay coefficients of variation were below 15% and 7%, respectively.
Endotoxin-induced preterm birth in animals treated with vaginal progesterone or placebo
Pregnant mice were pretreated with vaginal progesterone or Replens (control) from 13 to 17 dpc as previously described (n = 10 each). On 16.5 dpc, the mice were challenged with an intraperitoneal injection of 10 μg of an endotoxin (lipopolysaccharides from Escherichia coli, O55:B5; Sigma-Aldrich) in 200 μL of 1× PBS.
Video recording provided precise measurements of the gestational age, duration of active labor, and rate of stillbirth. Gestational age at birth was calculated from the identification of the vaginal plug (0.5 dpc) through the delivery of the first pup. Active labor was defined as the time elapsed from the delivery of the first pup through the delivery of the last pup. The rate of stillbirth was defined as the number of pups born dead among the total litter size. PTB was defined as fetal delivery before 18 dpc.
Statistical analysis
Statistical analyses were performed using SPSS, version 21.0 (IBM Corp, Armonk, NY). A Shapiro-Wilk test was performed to determine whether data were normally distributed. Because the data did not have a normal distribution, Mann-Whitney U tests were performed. The Fisher's exact test was used to compare proportions. Graphical data were presented as mean ± SEM. A value of P < .05 was considered statistically significant.
Results
Administration of vaginal progesterone, but not 17OHP-C, increases the proportion of CD4+ Tregs in decidual tissue
We first determined the proportions of CD4+ Tregs (CD4+CD25+Foxp3+ T cells) and CD8+CD25+Foxp3+ T cells in myometrial and decidual tissues following vaginal progesterone or 17OHP-C administration to pregnant mice. Figure 1B shows the gating strategy used to analyze CD4+ Tregs and CD8+CD25+Foxp3+ T cells in myometrial and decidual tissues.
Vaginal progesterone administration increased the proportion of decidual CD4+ Tregs when compared to the group receiving Replens (control) (Figure 1C); however, it decreased the proportion of decidual CD8+CD25+Foxp3+ T cells (Figure 1D).
Administration of 17OHP-C did not have such effects (Figures 1, E and F, P > .05). Moreover, vaginal progesterone administration did not alter the proportion of myometrial CD4+ Tregs or CD8+CD25+Foxp3+ T cells (Figure 2). Therefore, the administration of vaginal progesterone, but not 17OHP-C, increased the proportion of CD4+ Tregs in decidual tissue.
Figure 2Proportions of myometrial CD4+ Tregs and CD8+CD25+Foxp3+ T cells
Proportions of myometrial CD4+ Tregs (CD4+CD25+Foxp3+ T cells) and CD8+CD25+Foxp3+ T cells in mice treated with vaginal progesterone or Replens (control) (n = 10 each). Data are represented as mean ± SEM.
Treg, regulatory T cell.
Furcron. Vaginal progesterone, but not 17OHP-C, has antiinflammatory effects. Am J Obstet Gynecol 2015.
) mediated the increase in CD4+ Tregs, we determined the concentration of this cytokine in decidual tissue. No differences were observed in the concentration of IL-10 between the decidual protein extracts upon vaginal progesterone or Replens (control) administration (Supplemental Figure). These results do not support a role for IL-10 in the increase of decidual CD4+ Tregs upon administration of vaginal progesterone.
Administration of vaginal progesterone, but not 17OHP-C, decreases the proportion of macrophages in decidual tissue
To further characterize the decidual microenvironment following vaginal progesterone or 17OHP-C administration, the proportion of innate immune cells was determined. The gating strategy used to analyze NK cells (CD45+CD49b+ cells), DCs (CD45+CD11c+ cells), neutrophils (CD45+Ly6G+ cells), and macrophages (CD45+F4/80+ cells) in decidual tissue is shown in Figure 3A.
Figure 3Immunophenotyping of innate immune cells in decidual tissue
A, Gating strategy used to identify NK cells (CD45+CD49b+ cells), DCs (CD45+CD11c+ cells), neutrophils (CD45+Ly6G+ cells), and macrophages (CD45+F4/80+ cells) in decidual tissue. B, Proportions of decidual macrophages in mice treated with vaginal progesterone or Replens (control). C, Proportions of decidual macrophages in mice injected with 17OHP-C or castor oil (control) (n = 10 each). Data are represented as mean ± SEM.
Vaginal progesterone administration reduced the proportion of macrophages in decidual tissue when compared to Replens (control) (Figure 3B). In contrast, 17OHP-C administration did not alter the proportion of decidual macrophages (Figure 3C). No differences were found in the proportions of decidual neutrophils, NK cells, or DCs between the 2 groups of mice (data not shown).
To characterize the phenotype of decidual macrophages upon vaginal progesterone administration, we determined the expression of M1-like and M2-like markers including inducible NO synthase (iNOS), IFNγ, Arg1, and IL-4.
The gating strategy used to determine M1-like (CD11b+Ly6G-F4/80+iNOS+ or IFNγ+ cells) and M2-like (CD11b+Ly6G-F4/80+Arg1+ or IL4+ cells) macrophages in decidual tissue is shown in Figure 4A.
Figure 4M1- and M2-like macrophages in decidual tissue
A, Gating strategy used to identify M1-like (CD11b+ Ly6G-F4/80+ IFNγ+ or iNOS+ cells) and M2-like (CD11b+ Ly6G-F4/80+ IL4+ or Arg1+ cells) macrophages. The green histogram represents the autofluorescence control. B and C, Proportions of M1-like (CD11b+Ly6G-F4/80+ IFNγ+ or iNOS+ cells) macrophages in decidual tissue from mice treated with vaginal progesterone or Replens (control). D and E, Proportions of M2-like (CD11b+Ly6G-F4/80+ IL4+ or Arg1+ cells) macrophages in decidual tissue from mice treated with vaginal progesterone or Replens (control) (n = 10 each). Data are represented as mean ± SEM.
IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase.
Furcron. Vaginal progesterone, but not 17OHP-C, has antiinflammatory effects. Am J Obstet Gynecol 2015.
We hypothesized that vaginal progesterone administration would reduce the proportion of M1-like macrophages and/or would cause an M1→M2 macrophage polarization. Administration of vaginal progesterone did not change the proportion of M1-like (Figure 4, B and C) or M2-like (Figure 4, D and E) macrophages. Therefore, vaginal progesterone reduced the proportion of decidual macrophages, yet did not reduce M1-like macrophages or cause an M1→M2 macrophage polarization.
Administration of vaginal progesterone, but not 17OHP-C, reduces the proportion of IFNγ+ neutrophils in the myometrium
Uterine/myometrial macrophages and neutrophils have been implicated in the onset of term and preterm labor.
We therefore sought to determine whether vaginal progesterone or 17OHP-C administration alters the proportion of these innate immune cells in myometrial tissue.
The gating strategy used to determine macrophages, neutrophils, and their expression of IFNγ or IL-4 was similar to the strategy used in Figure 4A. Administration of vaginal progesterone tended to reduce the proportion of myometrial macrophages; however, this reduction did not reach statistical significance (Figure 5A). Administration of vaginal progesterone (Figure 5B), however, decreased the proportion of IFNγ+ neutrophils (CD11b+Ly6G+F4/80– cells) in the myometrium (Figure 5C). 17OHP-C administration did not reduce the proportion of IFNγ+ neutrophils in the myometrium (data not shown). These results demonstrate that vaginal progesterone administration reduced the proportion of pro-inflammatory neutrophils in the myometrium.
Figure 5Macrophages and neutrophils in the myometrium
A, Proportions of myometrial macrophages in mice treated with vaginal progesterone or Replens (control). B, Proportions of myometrial neutrophils in mice treated with vaginal progesterone or Replens (control). C, Proportions of myometrial IFNγ+ neutrophils (CD11b+Ly6G+F4/80− cells) in mice treated with vaginal progesterone or Replens (control) (n = 10 each). Data are represented as mean ± SEM.
IFN, interferon.
Furcron. Vaginal progesterone, but not 17OHP-C, has antiinflammatory effects. Am J Obstet Gynecol 2015.
Administration of vaginal progesterone, but not 17OHP-C, reduces the abundance of active MMP-9-positive cells in the cervix
We further investigated whether vaginal progesterone and 17OHP-C had effects on MMP-9 activity and collagen content in cervical tissue. Administration of vaginal progesterone or 17OHP-C increased MMP-9 activity (green staining) (Figure 6, A and B) and altered collagen integrity (blue staining; Figure 6, C and D) in cervical tissue.
Figure 6MMP-9 activity and collagen content in cervical tissue
A, MMP-9 activity (green staining) in mice treated with vaginal progesterone or Replens (control). B, MMP-9 activity (green staining) in mice injected with 17OHP-C or castor oil (control). Nuclei were stained with DAPI. White arrows represent active MMP-9-positive cells. Scale bars in ×10 and ×40: 200 μm and 50 μm, respectively. C, Masson’s trichrome staining of cervical tissue from mice treated with vaginal progesterone or Replens (control). Scale bars in ×10 and ×40: 200 μm and 50 μm, respectively. D, Masson’s trichrome staining of cervical tissue from mice injected with 17OHP-C or castor oil (control). Collagen fibers are stained in blue. Scale bars in ×10 and ×40: 200 μm and 50 μm, respectively. E, Semiquantification of active MMP-9-positive cells in the cervices from mice treated with vaginal progesterone or Replens (control). F, Semiquantification of active MMP-9-positive cells in cervices from mice injected with 17OHP-C or castor oil (control) (n = 5 each). Data are represented as mean ± SEM. G, Magnified image of active MMP-9-positive neutrophils and monocytes in cervical tissue from control mice. Scale bars: 20 μm.
While analyzing the images, we observed that the cervices in the control group (Replens) had an abundant number of active MMP-9-positive cells (white arrows). Semiquantification of these cells revealed that vaginal progesterone administration reduced the abundance of active MMP-9-positive cells when compared to Replens (control) (Figure 6E).
In contrast, 17OHP-C administration increased the abundance of active MMP-9-positive cells when compared to the control group (castor oil) (Figure 6F). Magnification of active MMP-9-positive cells in Replens (control) revealed these cells to be neutrophils and monocytes (Figure 6G). Therefore, vaginal progesterone and 17OHP-C increased MMP-9 activity and altered collagen integrity in the cervix. However, only vaginal progesterone reduced the infiltration of active MMP-9-positive neutrophils and monocytes.
Administration of vaginal progesterone or 17OHP-C is not associated with changes in the systemic concentrations of progesterone or estradiol
To investigate whether the immune effects of vaginal progesterone or 17OHP-C were associated with a change in the systemic levels of sex steroids, we quantified the concentrations of progesterone and estradiol in the plasma. Administration of vaginal progesterone or 17OHP-C did not change the systemic concentrations of progesterone or estradiol (Figure 7, A and B). These results demonstrate that the local immunomodulatory effects of vaginal progesterone in decidual, myometrial, and cervical tissues were not associated with systemic changes in sex steroids.
Figure 7Plasma concentrations of progesterone and estradiol
A, Progesterone and estradiol concentrations in mice treated with vaginal progesterone or Replens (control). B, Progesterone and estradiol concentrations in mice injected with 17OHP-C or castor oil (control). Plasma samples were collected at 18.5 dpc (n = 10 each). Data are represented as mean ± SEM.
dpc, days postcoitum; 17OHP-C, 17-alpha-hydroxyprogesterone caproate.
Furcron. Vaginal progesterone, but not 17OHP-C, has antiinflammatory effects. Am J Obstet Gynecol 2015.
Therefore, we evaluated whether the administration of vaginal progesterone or 17OHP-C had an effect on the systemic concentration of IL-1β. Vaginal progesterone reduced by 20% the plasma concentrations of IL-1β (Figure 8A); however, the administration of 17OHP-C did not alter the concentration of this cytokine (Figure 8B).
A, IL-1β concentrations in mice treated with vaginal progesterone or Replens (control). B, IL-1β concentration in mice injected with 17OHP-C or castor oil (control). Plasma samples were collected at 18.5 dpc (n = 10 each). Data are represented as mean ± SEM.
dpc, days postcoitum; IL, interleukin; 17OHP-C, 17-alpha-hydroxyprogesterone caproate.
Furcron. Vaginal progesterone, but not 17OHP-C, has antiinflammatory effects. Am J Obstet Gynecol 2015.
Pretreatment with vaginal progesterone conferred partial protection (50%) against endotoxin-induced preterm birth
Finally, we evaluated the efficacy of vaginal progesterone in preventing endotoxin-induced preterm birth. Mice pretreated with vaginal progesterone had lower rates of endotoxin-induced preterm birth than mice pretreated with Replens (control) (40% vs 90%, P = 0.011; Table). These results demonstrate that vaginal progesterone administration may be an effective treatment for reducing inflammation-associated preterm labor.
TableVaginal progesterone administration decreases the rate of endotoxin-induced preterm birth
The principal findings of the study include the following: (1) the administration of vaginal progesterone, but not 17OHP-C, increased the proportion of decidual CD4+ Tregs and decreased the proportions of CD8+CD25+Foxp3+ T cells and macrophages in decidual tissue; (2) administration of vaginal progesterone did not cause M1→M2 macrophage polarization; however, it reduced the proportion of IFNγ+ neutrophils in the myometrium and active MMP-9-positive neutrophils and monocytes in the cervix; (3) in contrast, the administration of 17OHP-C increased the abundance of active MMP-9-positive neutrophils and monocytes in the cervix; (4) the immune effects of vaginal progesterone were associated with reduced systemic concentrations of IL-1β but not with alterations in progesterone or estradiol concentrations; and (5) pretreatment with vaginal progesterone was associated with a 50% reduction in endotoxin-induced PTB.
Vaginal progesterone increases the proportion of decidual CD4+ Tregs
Lymphocytes with immunoregulatory properties were described more than 4 decades ago
; however, the lack of specific markers for these cells precluded their characterization using immunophenotypic techniques. CD4+ Tregs are an important subset of T cells, which express CD25 and Foxp3.
Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance.
Evidence that the T cell repertoire of normal rats contains cells with the potential to cause diabetes. Characterization of the CD4+ T cell subset that inhibits this autoimmune potential.
During pregnancy, there is an expansion of antigen-specific CD4+ Tregs that exhibit suppressive functions. This is thought to promote maternal-fetal tolerance and pregnancy maintenance.
A distinct subset of HLA-DR+-regulatory T cells is involved in the induction of preterm labor during pregnancy and in the induction of organ rejection after transplantation.
Indeed, we recently presented evidence that the administration of endotoxin, which causes PTB in mice, leads to a reduction of CD4+ Tregs at the maternal-fetal interface.
Gap junctions and myometrial steroid hormone receptors in pregnant and postpartum rats: a possible cellular basis for the progesterone withdrawal hypothesis.
we hypothesized that administration of vaginal progesterone and 17OHP-C from midgestation to late gestation would lead to an expansion of CD4+ Tregs at the maternal-fetal interface. In this study, administration of vaginal progesterone, but not 17OHP-C, increased the proportion of decidual CD4+ Tregs.
Altogether, these findings suggest that vaginal progesterone administration during late gestation fosters local maternal-fetal tolerance by increasing the proportion of decidual CD4+ Tregs.
Vaginal progesterone reduces the proportion of decidual CD8+CD25+Foxp3+ T cells
In addition to increasing the proportion of CD4+ Tregs, vaginal progesterone administration to pregnant mice reduced the proportion of CD8+CD25+Foxp3+ T cells in decidual tissue. This finding is consistent with previous reports demonstrating that progesterone regulates CD8+ T cell cytokine release and cytotoxicity during pregnancy.
Depletion of CD8+ cells abolishes the pregnancy protective effect of progesterone substitution with dydrogesterone in mice by altering the Th1/Th2 cytokine profile.
CD8+CD25+ T cells expressing Foxp3 seem to share phenotypic, functional, and mechanistic actions with the classical CD4+ Tregs; therefore, they were named CD8+ Tregs.
In addition, CD8+CD25+Foxp3+ T cells are present in both decidual tissues and maternal circulation during term pregnancy and that their proportions are increased by exogenous administration of IL-6, which restores parturition on time in Il6–/– mice.
This supports a role for these cells in the proinflammatory milieu associated with the process of labor.
As a whole, these data suggest that CD8+CD25+Foxp3+ T cells have a proinflammatory phenotype rather than a suppressive phenotype and that vaginal progesterone administration reduces the proportion of these cells in the decidua, thereby having an antiinflammatory role.
Vaginal progesterone decreases the proportion of decidual macrophages
Macrophages/monocytes play central roles in the maintenance of pregnancy and term and preterm parturition, including uterine contractility, cervical ripening, and rupture of the membranes as well as in uterine involution during the postpartum period.
which demonstrates that macrophages/monocytes participate in the process of microbial-induced preterm labor. Macrophages/monocytes express progesterone receptors
; therefore, it is possible that the infiltration and/or function of these cells are regulated by progesterone.
In the study herein, we found that administration of vaginal progesterone decreased the proportion of macrophages in decidual tissues. These data are consistent with previous reports demonstrating that the administration of progesterone reduces the infiltration and migration of macrophages/monocytes into reproductive tissues.
Medroxyprogesterone acetate modulates remodeling, immune cell census, and nerve fibers in the cervix of a mouse model for inflammation-induced preterm birth.
Altogether, these data suggest that vaginal progesterone regulates the infiltration of macrophages/monocytes into decidual tissue, which fosters an antiinflammatory microenvironment at the maternal-fetal interface.
Vaginal progesterone reduces the proportion of IFNγ+ neutrophils in the myometrium
Neutrophils play an important role during term and preterm parturition because they release proinflammatory mediators that are associated with the onset of labor.
Morphologic and histochemical evidence for the occurrence of collagenolysis and for the role of neutrophilic polymorphonuclear leukocytes during cervical dilation.
In the myometrium, the mRNA expression of CXCL8, a neutrophil chemokine, is higher in women who underwent labor than in those who did not undergo labor at term, suggesting a role for neutrophils in myometrial contractions.
Recently, we were able to support this hypothesis by demonstrating that the percentage and total number of myometrial neutrophils increase in endotoxin-induced PTB.
Herein, we found that vaginal progesterone administration reduces the proportion of IFNγ+ neutrophils in the myometrium. This is in line with previous in vitro studies demonstrating that incubation with progesterone reduces the release of CXCL8 in human myometrial biopsies or rabbit uterine cervical fibroblasts.
Progesterone control of interleukin-8 production in endometrium and chorio-decidual cells underlines the role of the neutrophil in menstruation and parturition.
Collectively, the data suggest that vaginal progesterone administration to pregnant mice reduces the infiltration of activated neutrophils into the myometrial tissue, which may be mediated by CXCL8.
Vaginal progesterone, but not 17OHP-C, reduces active MMP-9-positive neutrophils and monocytes in the cervix
We evaluated whether vaginal progesterone or 17OHP-C administration had an effect on MMP-9 activity and collagen integrity. MMPs are a superfamily of zinc enzymes participating in the degradation of the extracellular matrix.
During pregnancy, MMP-9 is expressed by resident cells and infiltrating leukocytes at the maternal-fetal interface and has been associated with the process of labor.
Increased matrix metalloproteinase activity and reduced tissue inhibitor of metalloproteinases-1 levels in amniotic fluids from pregnancies complicated by premature rupture of membranes.
Herein, the in vivo administration of vaginal progesterone increased MMP-9 activity and altered collagen integrity in the cervical stroma. Vaginal progesterone also reduced the infiltration of active MMP-9-positive neutrophils and monocytes. In contrast, 17OHP-C administration increased MMP-9 activity in the cervical stroma, reduced collagen content, and increased infiltration of active MMP-9-positive neutrophils and monocytes.
Collectively, the data demonstrate that administration of vaginal progesterone or 17OHP-C increases MMP-9 activity in the cervical stroma and alters collagen integrity, yet administration of natural progesterone reduces the infiltration of neutrophils and monocytes expressing active MMP-9. Infiltration may be the key element in determining changes in the biomechanical properties of the cervix, which favor parturition.
Pretreatment with vaginal progesterone reduces the rate of endotoxin-induced preterm birth
Vaginal progesterone administered to women with a sonographic short cervix reduces the rate of PTB.
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.
Vaginal progesterone reduces the rate of preterm birth in women with a sonographic short cervix: a multicenter, randomized, double-blind, placebo-controlled trial.
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.
In addition, pretreatment by injection of natural or medroxyprogesterone acetate prevents endotoxin-induced PTB in mice, which is associated with the down-regulation of the mRNA expression of the inflammatory cytokines Il1β and Tnf.
It is interesting that systemic administration of IL-1β induces PTB in mice, and pretreatment with the IL-1 receptor antagonist abrogrates this effect.
In the current study, pretreatment with vaginal progesterone reduced the frequency of endotoxin-induced PTB by 50% and reduced the systemic concentration of IL-1β. Altogether, these data suggest that pretreatment with vaginal progesterone fosters a local and systemic antiinflammatory response, preventing endotoxin-induced preterm birth.
Although previous studies reported that systemic administration of progesterone reduces the rate of endotoxin-induced preterm birth by 28%,
the current study is the first to demonstrate that vaginal progesterone has this effect. The fact that vaginal progesterone does not prevent endotoxin-induced preterm delivery in all cases is not unexpected, given that even in women with a short cervix, the administration of vaginal progesterone reduced the rate of preterm delivery by only 45%.
Vaginal progesterone reduces the rate of preterm birth in women with a sonographic short cervix: a multicenter, randomized, double-blind, placebo-controlled trial.
Obstetrix Collaborative Research N. Failure of 17-hydroxyprogesterone to reduce neonatal morbidity or prolong triplet pregnancy: a double-blind, randomized clinical trial.
Prevention of preterm delivery by 17 alpha-hydroxyprogesterone caproate in asymptomatic twin pregnancies with a short cervix: a randomized controlled trial.
Similarly, pretreatment with 17OHP-C before endotoxin exposure has adverse effects on pregnant mice, including behavioral changes (lethargy or piloerection) and maternal death.
For these reasons, we did not study the effect of 17OHP-C on endotoxin-induced preterm birth. However, in contrast to previous reports with 17OHP-C, vaginal progesterone followed by endotoxin did not result in demonstrable maternal morbidity or death.
A previous study demonstrated that progesterone binds with more avidity to progesterone receptors than 17OHP-C; however, both progestogens are comparable in eliciting the transactivation of reporter genes as assessed by luciferase activity in the T47D-2963.1 and T47Dco carcinoma cell lines.
Comparison of progesterone and glucocorticoid receptor binding and stimulation of gene expression by progesterone, 17-alpha hydroxyprogesterone caproate, and related progestins.
Comparison of progesterone and glucocorticoid receptor binding and stimulation of gene expression by progesterone, 17-alpha hydroxyprogesterone caproate, and related progestins.
The equivalent biological effect per unit mass of 17OHP-C and progesterone in preventing preterm delivery induced by an inhibitor of nitric oxide synthase has also been shown in CD-1 mice.
These findings suggest that the progestational activity of 17OHP-C and progesterone as measured by these assays are similar. However, this does not seem to translate into changes in the immune cell composition at the maternal-fetal interface. Specifically, the total exposure to 17OHP-C was greater than the total exposure to vaginal progesterone. Yet we observed antiinflammatory effects only with vaginal progesterone.
Conclusion
Our results demonstrate that the administration of vaginal progesterone fosters an antiinflammatory microenvironment at the maternal-fetal interface by increasing CD4+ Tregs and reducing CD8+CD25+Foxp3+ T cells, macrophages, and IFNγ+ neutrophils. In addition, the administration of vaginal progesterone decreases the infiltration of active MMP-9-positive neutrophils and monocytes in the cervix, reduces the plasma concentration of IL-1β, and reduces the frequency of endotoxin-induced preterm birth. Administration of 17OHP-C did not have the same effects as vaginal progesterone. These results provide insight into the mechanisms whereby vaginal progesterone prevents preterm birth.
Acknowledgment
We gratefully acknowledge Akshata Naik, Elly Sanchez-Rodriguez, Dr Eleazar Soto, Marcia Arenas-Hernandez, Nakisha Rutledge, Tamara Roumayah, Yang Jiang, Amapola Balancio, Stella Dewar, Dr Zhong Dong, Lorri McLuckie, Rona Wang, and Sunjay Modi for their contributions to the execution of this study and to Maureen McGerty for her critical readings of the manuscript.
Appendix
Supplemental FigureIL-10 concentrations in decidual protein extracts
IL-10 concentrations in the decidual protein extracts of mice treated with vaginal progesterone or Replens (control) (n = 10 each). Data are represented as mean ± SEM.
IL, interleukin.
Furcron. Vaginal progesterone, but not 17OHP-C, has antiinflammatory effects. Am J Obstet Gynecol 2015.
Institute of Medicine Committee on Understanding Premature Birth and Assuring Healthy Outcomes. Societal costs of preterm birth. In: Behrman RE, Butler AS, eds. Preterm birth: causes, consequences, and prevention. Washington (DC); 2007.
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.
Effect of progesterone on cervical shortening in women at risk for preterm birth: secondary analysis from a multinational, randomized, double-blind, placebo-controlled trial.
Vaginal progesterone reduces the rate of preterm birth in women with a sonographic short cervix: a multicenter, randomized, double-blind, placebo-controlled trial.
Vaginal progesterone in women with an asymptomatic sonographic short cervix in the midtrimester decreases preterm delivery and neonatal morbidity: a systematic review and metaanalysis of individual patient data.
Vaginal progesterone vs. cervical cerclage for the prevention of preterm birth in women with a sonographic short cervix, previous preterm birth, and singleton gestation: a systematic review and indirect comparison metaanalysis.
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