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Vaginal progesterone, but not 17α-hydroxyprogesterone caproate, has antiinflammatory effects at the murine maternal-fetal interface

  • Amy-Eunice Furcron
    Affiliations
    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

    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI
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  • Roberto Romero
    Correspondence
    Roberto Romero, MD, DMedSci.
    Affiliations
    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

    Department 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
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  • Olesya Plazyo
    Affiliations
    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
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  • Ronald Unkel
    Affiliations
    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
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  • Yi Xu
    Affiliations
    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
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  • Sonia S. Hassan
    Affiliations
    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

    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI
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  • Piya Chaemsaithong
    Affiliations
    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

    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI
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  • Arushi Mahajan
    Affiliations
    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI
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  • Nardhy Gomez-Lopez
    Correspondence
    Corresponding author: Nardhy Gomez-Lopez, PhD.
    Affiliations
    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

    Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI

    Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI
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Published:August 08, 2015DOI:https://doi.org/10.1016/j.ajog.2015.08.010

      Objective

      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.

      Key words

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      Preterm labor: one syndrome, many causes.
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      Infection and labor. V. Prevalence, microbiology, and clinical significance of intraamniotic infection in women with preterm labor and intact membranes.
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      Amniotic fluid interleukin 6 in preterm labor. Association with infection.
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      Amniotic fluid white blood cell count: a rapid and simple test to diagnose microbial invasion of the amniotic cavity and predict preterm delivery.
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      The preterm labor syndrome.
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      Amniotic fluid interleukin-6: correlation with upper genital tract microbial colonization and gestational age in women delivered after spontaneous labor versus indicated delivery.
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      Serum C-reactive protein, white blood cell count, and amniotic fluid white blood cell count in women with preterm premature rupture of membranes.
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      Isolation of Ureaplasma urealyticum from the amniotic cavity and adverse outcome in preterm labor.
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      The fetal inflammatory response syndrome.
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      Clinical implications of detection of Ureaplasma urealyticum in the amniotic cavity with the polymerase chain reaction.
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      The role of infection in preterm labour and delivery.
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      Clinical significance of intra-amniotic inflammation in patients with preterm labor and intact membranes.
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      The role of inflammation and infection in preterm birth.
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      The intensity of the fetal inflammatory response in intraamniotic inflammation with and without microbial invasion of the amniotic cavity.
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      Prevalence and clinical significance of sterile intra-amniotic inflammation in patients with preterm labor and intact membranes.
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      • et al.
      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.
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      • et al.
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      • et al.
      Prelabor rupture of membranes between 34 and 37 weeks: the intraamniotic inflammatory response and neonatal outcomes.
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      • et al.
      Maternal intravascular inflammation in preterm premature rupture of membranes.
      • Mittal P.
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      • et al.
      A molecular signature of an arrest of descent in human parturition.
      • Brennan D.J.
      • McGee S.F.
      • Rexhepaj E.
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      • Robson M.
      • O'Herlihy C.
      Identification of a myometrial molecular profile for dystocic labor.
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      • Romero R.
      • et al.
      Characterization of the myometrial transcriptome in women with an arrest of dilatation during labor.
      Pathological inflammation can result from the activation of innate immunity in response to microbial products
      • Shynlova O.
      • Nedd-Roderique T.
      • Li Y.
      • Dorogin A.
      • Lye S.J.
      Myometrial immune cells contribute to term parturition, preterm labour and post-partum involution in mice.
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      • Kim Y.M.
      • et al.
      Divergent trophoblast responses to bacterial products mediated by TLRs.
      • Kim Y.M.
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      • et al.
      Toll-like receptor-2 and -4 in the chorioamniotic membranes in spontaneous labor at term and in preterm parturition that are associated with chorioamnionitis.
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      • et al.
      Activation of TLR3 in the trophoblast is associated with preterm delivery.
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      • et al.
      Viral infection of the placenta leads to fetal inflammation and sensitization to bacterial products predisposing to preterm labor.
      • Cardenas I.
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      • et al.
      Nod1 activation by bacterial iE-DAP induces maternal-fetal inflammation and preterm labor.
      • Romero R.
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      • et al.
      Damage-associated molecular patterns (DAMPs) in preterm labor with intact membranes and preterm PROM: a study of the alarmin HMGB1.
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      • et al.
      Lipopolysaccharide appears to activate human endometrial endothelial cells through TLR-4-dependent and TLR-4-independent mechanisms.
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      Regulation of apoptosis and innate immune stimuli in inflammation-induced preterm labor.
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      • et al.
      Human fetal membranes generate distinct cytokine profiles in response to bacterial Toll-like receptor and nod-like receptor agonists.
      or activation of the adaptive immune response.
      • Kim J.S.
      • Romero R.
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      • et al.
      Involvement of Hofbauer cells and maternal T cells in villitis of unknown aetiology.
      • Ito M.
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      • et al.
      A role for IL-17 in induction of an inflammation at the fetomaternal interface in preterm labour.
      • Xu Y.
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      • et al.
      Peripheral CD300a+CD8+ T lymphocytes with a distinct cytotoxic molecular signature increase in pregnant women with chronic chorioamnionitis.
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      • et al.
      Fetal intervention increases maternal T cell awareness of the foreign conceptus and can lead to immune-mediated fetal demise.
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      Increased maternal T cell microchimerism in the allogeneic fetus during LPS-induced preterm labor in mice.
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      • Hassan S.S.
      • Kaye E.B.
      • Gomez-Lopez N.
      An imbalance between innate and adaptive immune cells at the maternal-fetal interface occurs prior to endotoxin-induced preterm birth.
      A breakdown of maternal-fetal tolerance is now recognized as a mechanism of disease for spontaneous premature labor/delivery.
      • Romero R.
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      • Fisher S.J.
      Preterm labor: one syndrome, many causes.
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      • et al.
      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.
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      • et al.
      The frequency, clinical significance, and pathological features of chronic chorioamnionitis: a lesion associated with spontaneous preterm birth.
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      • et al.
      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.
      • Lee J.
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      • et al.
      A signature of maternal anti-fetal rejection in spontaneous preterm birth: chronic chorioamnionitis, anti-human leukocyte antigen antibodies, and C4d.
      • Lee J.
      • Romero R.
      • Chaiworapongsa T.
      • et al.
      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.
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      • et al.
      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.
      • Gomez-Lopez N.
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      • Arenas-Hernandez M.
      Immune cells in term and preterm labor.
      Therefore, the effects of progesterone in the prevention of preterm delivery may be mediated by the innate and/or adaptive immune system.
      In line with this hypothesis, the administration of RU486 (to block progesterone action
      • Frydman R.
      • Lelaidier C.
      • Baton-Saint-Mleux C.
      • Fernandez H.
      • Vial M.
      • Bourget P.
      Labor induction in women at term with mifepristone (RU 486): a double-blind, randomized, placebo-controlled study.
      ) during late pregnancy in guinea pigs can increase the release of proinflammatory cytokines by the amniochorion, cervix, and decidual-myometrial tissues.
      • Gomez-Lopez N.
      • Tong W.C.
      • Arenas-Hernandez M.
      • et al.
      Chemotactic activity of gestational tissues through late pregnancy, term labor, and RU486-induced preterm labor in guinea pigs.
      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.
      • Mao G.
      • Wang J.
      • Kang Y.
      • et al.
      Progesterone increases systemic and local uterine proportions of CD4+CD25+ Treg cells during midterm 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 thumbnail gr1
      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.
      • Chang J.
      • Zhao Y.
      • Zhao W.
      • Venkataramanan R.
      • Caritis S.N.
      Obstetrical-Fetal Pharmacology Research Units Network. Quality assessment of compounded 17-hydroxyprogesterone caproate.
      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,
      • Hassan S.S.
      • Romero R.
      • Vidyadhari D.
      • et al.
      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.
      • Meis P.J.
      • Klebanoff M.
      • Thom E.
      • et al.
      Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate.
      The doses of vaginal progesterone and 17OHP-C were similar to those previously reported in studies using the same animal species.
      • Nold C.
      • Maubert M.
      • Anton L.
      • Yellon S.
      • Elovitz M.A.
      Prevention of preterm birth by progestational agents: what are the molecular mechanisms?.
      • Elovitz M.A.
      • Mrinalini C.
      The use of progestational agents for preterm birth: lessons from a mouse model.
      • Xu H.
      • Gonzalez J.M.
      • Ofori E.
      • Elovitz M.A.
      Preventing cervical ripening: the primary mechanism by which progestational agents prevent preterm birth?.
      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.
      • Arenas-Hernandez M.
      • Sanchez-Rodriguez E.N.
      • Mial N.T.
      • Robertson S.A.
      • Gomez-Lopez N.
      Isolation of leukocytes from the murine tissues at the maternal-fetal interface.
      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.
      • Hadler-Olsen E.
      • Kanapathippillai P.
      • Berg E.
      • Svineng G.
      • Winberg J.O.
      • Uhlin-Hansen L.
      Gelatin in situ zymography on fixed, paraffin-embedded tissue: zinc and ethanol fixation preserve enzyme activity.
      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 thumbnail gr2
      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.
      To explore whether IL-10 (an antiinflammatory cytokine and a differentiation factor of Tregs
      • Murai M.
      • Turovskaya O.
      • Kim G.
      • et al.
      Interleukin 10 acts on regulatory T cells to maintain expression of the transcription factor Foxp3 and suppressive function in mice with colitis.
      ) 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 thumbnail gr3
      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.
      DC, dendritic cell; FSC, forward scatter; NK, natural killer; 17OHP-C, 17-alpha-hydroxyprogesterone caproate; SSC, side scatter.
      Furcron. Vaginal progesterone, but not 17OHP-C, has antiinflammatory effects. Am J Obstet Gynecol 2015.
      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.
      • Mills C.D.
      • Kincaid K.
      • Alt J.M.
      • Heilman M.J.
      • Hill A.M.
      M-1/M-2 macrophages and the Th1/Th2 paradigm.
      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 thumbnail gr4
      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.
      • Thomson A.J.
      • Telfer J.F.
      • Young A.
      • et al.
      Leukocytes infiltrate the myometrium during human parturition: further evidence that labour is an inflammatory process.
      • Shynlova O.
      • Nedd-Roderique T.
      • Li Y.
      • Dorogin A.
      • Lye S.J.
      Myometrial immune cells contribute to term parturition, preterm labour and post-partum involution in mice.
      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 thumbnail gr5
      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 thumbnail gr6
      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.
      DAPI, 4',6-diamidino-2-phenylindole; MMP, matrix metalloproteinase; 17OHP-C, 17-alpha-hydroxyprogesterone caproate.
      Furcron. Vaginal progesterone, but not 17OHP-C, has antiinflammatory effects. Am J Obstet Gynecol 2015.
      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 thumbnail gr7
      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.

      Administration of vaginal progesterone, but not 17OHP-C, reduces the systemic concentration of IL-1β

      Preterm labor is associated with a systemic inflammatory response,
      • Gervasi M.T.
      • Chaiworapongsa T.
      • Naccasha N.
      • et al.
      Phenotypic and metabolic characteristics of maternal monocytes and granulocytes in preterm labor with intact membranes.
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      • Chaiworapongsa T.
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      Maternal intravascular inflammation in preterm premature rupture of membranes.
      and the systemic or intraamniotic administration of IL-1β leads to PTB in mice.
      • Romero R.
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      Interleukin-1 beta intra-amniotic infusion induces tumor necrosis factor-alpha, prostaglandin production, and preterm contractions in pregnant rhesus monkeys.
      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).
      Figure thumbnail gr8
      Figure 8Plasma concentration of IL-1β
      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
      Replens (control) plus endotoxinVaginal progesterone plus endotoxinP value
      Number of mice1010
      Preterm birth, %
      The rate of PTB was defined as the percentage of dams delivering at <18.0 dpc among all births.
      ,
      Fisher's exact test.
      90400.011
      Gestational age, d
      Days elapsed from the detection of a vaginal plug (0.5 dpc) through the delivery of the first pup. Data are shown as mean ± SD.
      ,
      Mann-Whitney U test.
      17.7 ± 0.618.4 ± 0.8.038
      Duration of labor, min
      Time elapsed from the delivery of the first pup through the delivery of the last pup. Data are shown as mean ± SD.
      ,
      Mann-Whitney U test.
      37 ± 20.740.5 ± 30.1NS
      dpc, days postcoitum; NS, not significant. PTB, preterm birth.
      Furcron. Vaginal progesterone, but not 17OHP-C, has antiinflammatory effects. Am J Obstet Gynecol 2015.
      a The rate of PTB was defined as the percentage of dams delivering at <18.0 dpc among all births.
      b Fisher's exact test.
      c Days elapsed from the detection of a vaginal plug (0.5 dpc) through the delivery of the first pup. Data are shown as mean ± SD.
      d Mann-Whitney U test.
      e Time elapsed from the delivery of the first pup through the delivery of the last pup. Data are shown as mean ± SD.

      Comment

      Principal findings of the study

      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
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      ; 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.
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      and this suppressive function is largely due to their expression of the transcription factor Foxp3.
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      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.
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      which might be due to the diminished suppressive function of CD4+ Tregs in preterm labor.
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      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.
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      Pregnancy-associated diseases are characterized by the composition of the systemic regulatory T cell (Treg) pool with distinct subsets of Tregs.
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      T regulatory cells: regulating both term and preterm labor?.
      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.
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      An imbalance between innate and adaptive immune cells at the maternal-fetal interface occurs prior to endotoxin-induced preterm birth.
      Because progesterone plays a central role in pregnancy maintenance
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      and increases CD4+ Tregs with a suppressive function during midgestation,
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      Progesterone increases systemic and local uterine proportions of CD4+CD25+ Treg cells during midterm pregnancy in mice.
      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.
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      Depletion of CD8+ cells abolishes the pregnancy protective effect of progesterone substitution with dydrogesterone in mice by altering the Th1/Th2 cytokine profile.
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      Progesterone and HMOX-1 promote fetal growth by CD8+ T cell modulation.
      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.
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      Human CD8+CD25+ thymocytes share phenotypic and functional features with CD4+CD25+ regulatory thymocytes.
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      IL-6 positively regulates Foxp3+CD8+ T cells in vivo.
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      IL-6 positively regulates Foxp3+CD8+ T cells in vivo.
      During midgestation, CD8+Foxp3+ T cells expressing CD103 are found in the spleen in which they suppress immune responses via ICOS-B7h.
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      B7h (ICOS-L) maintains tolerance at the fetomaternal interface.
      Recently we reported that splenic CD8+CD25+Foxp3+ T cells, which produce IL-10, increased in endotoxin-induced PTB.
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      An imbalance between innate and adaptive immune cells at the maternal-fetal interface occurs prior to endotoxin-induced preterm birth.
      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.
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      Interleukin-6 controls uterine Th9 cells and CD8+ T regulatory cells to accelerate parturition in 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.
      • Osman I.
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      • et al.
      Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes, decidua, cervix and myometrium before and during labour at term.
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      Leukocytes infiltrate the myometrium during human parturition: further evidence that labour is an inflammatory process.
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      • Robson S.C.
      Macrophages and not granulocytes are involved in cervical ripening.
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      • Mahendroo M.S.
      Timing of neutrophil activation and expression of proinflammatory markers do not support a role for neutrophils in cervical ripening in the mouse.
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      • Dorogin A.
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      Monocyte chemoattractant protein-1 (CCL-2) integrates mechanical and endocrine signals that mediate term and preterm labor.
      • Gonzalez J.M.
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      • Yang F.
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      Complement activation triggers metalloproteinases release inducing cervical remodeling and preterm birth in mice.
      • Gonzalez J.M.
      • Dong Z.
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      • Girardi G.
      Cervical remodeling/ripening at term and preterm delivery: the same mechanism initiated by different mediators and different effector cells.
      • Payne K.J.
      • Clyde L.A.
      • Weldon A.J.
      • Milford T.A.
      • Yellon S.M.
      Residency and activation of myeloid cells during remodeling of the prepartum murine cervix.
      • Hamilton S.
      • Oomomian Y.
      • Stephen G.
      • et al.
      Macrophages infiltrate the human and rat decidua during term and preterm labor: evidence that decidual inflammation precedes labor.
      • Gomez-Lopez N.
      • Bijland M.T.
      • David M.O.
      • Robertson S.A.
      Maternal monocyte-derived cell depletion promotes preterm delivery in mice.
      Macrophage/monocyte neutralization, using an anti-F4/80 antibody, prevents endotoxin-induced PTB,
      • Gonzalez J.M.
      • Franzke C.W.
      • Yang F.
      • Romero R.
      • Girardi G.
      Complement activation triggers metalloproteinases release inducing cervical remodeling and preterm birth in mice.
      which demonstrates that macrophages/monocytes participate in the process of microbial-induced preterm labor. Macrophages/monocytes express progesterone receptors
      • Werb Z.
      • Foley R.
      • Munck A.
      Interaction of glucocorticoids with macrophages. Identification of glucocorticoid receptors in monocytes and macrophages.
      • Lu J.
      • Reese J.
      • Zhou Y.
      • Hirsch E.
      Progesterone-induced activation of membrane-bound progesterone receptors in murine macrophage cells.
      ; 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.
      • Yellon S.M.
      • Burns A.E.
      • See J.L.
      • Lechuga T.J.
      • Kirby M.A.
      Progesterone withdrawal promotes remodeling processes in the nonpregnant mouse cervix.
      • Yellon S.M.
      • Ebner C.A.
      • Elovitz M.A.
      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.
      • Junqueira L.C.
      • Zugaib M.
      • Montes G.S.
      • Toledo O.M.
      • Krisztan R.M.
      • Shigihara K.M.
      Morphologic and histochemical evidence for the occurrence of collagenolysis and for the role of neutrophilic polymorphonuclear leukocytes during cervical dilation.
      • Osman I.
      • Young A.
      • Ledingham M.A.
      • et al.
      Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes, decidua, cervix and myometrium before and during labour at term.
      • Thomson A.J.
      • Telfer J.F.
      • Young A.
      • et al.
      Leukocytes infiltrate the myometrium during human parturition: further evidence that labour is an inflammatory process.
      • Romero R.
      • Ceska M.
      • Avila C.
      • Mazor M.
      • Behnke E.
      • Lindley I.
      Neutrophil attractant/activating peptide-1/interleukin-8 in term and preterm parturition.
      • Winkler M.
      • Fischer D.C.
      • Ruck P.
      • et al.
      Parturition at term: parallel increases in interleukin-8 and proteinase concentrations and neutrophil count in the lower uterine segment.
      • Helmig B.R.
      • Romero R.
      • Espinoza J.
      • et al.
      Neutrophil elastase and secretory leukocyte protease inhibitor in prelabor rupture of membranes, parturition and intra-amniotic infection.
      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.
      • Osman I.
      • Young A.
      • Ledingham M.A.
      • et al.
      Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes, decidua, cervix and myometrium before and during labour at term.
      • Bollapragada S.
      • Youssef R.
      • Jordan F.
      • Greer I.
      • Norman J.
      • Nelson S.
      Term labor is associated with a core inflammatory response in human fetal membranes, myometrium, and cervix.
      Recently, we were able to support this hypothesis by demonstrating that the percentage and total number of myometrial neutrophils increase in endotoxin-induced PTB.
      • Arenas-Hernandez M.
      • Romero R.
      • St Louis D.
      • Hassan S.S.
      • Kaye E.B.
      • Gomez-Lopez N.
      An imbalance between innate and adaptive immune cells at the maternal-fetal interface occurs prior to endotoxin-induced preterm birth.
      Indeed, myometrial neutrophils express inflammatory cytokines such as IL-6, IL-8, TNFα, IFNγ, and IL-4,
      • Young A.
      • Thomson A.J.
      • Ledingham M.
      • Jordan F.
      • Greer I.A.
      • Norman J.E.
      Immunolocalization of proinflammatory cytokines in myometrium, cervix, and fetal membranes during human parturition at term.
      • Arenas-Hernandez M.
      • Romero R.
      • St Louis D.
      • Hassan S.S.
      • Kaye E.B.
      • Gomez-Lopez N.
      An imbalance between innate and adaptive immune cells at the maternal-fetal interface occurs prior to endotoxin-induced preterm birth.
      • Yeaman G.R.
      • Collins J.E.
      • Currie J.K.
      • Guyre P.M.
      • Wira C.R.
      • Fanger M.W.
      IFN-gamma is produced by polymorphonuclear neutrophils in human uterine endometrium and by cultured peripheral blood polymorphonuclear neutrophils.
      which is a characteristic phenotype of activated neutrophils.
      • Ellis T.N.
      • Beaman B.L.
      Murine polymorphonuclear neutrophils produce interferon-gamma in response to pulmonary infection with Nocardia asteroides.
      • Ethuin F.
      • Gerard B.
      • Benna J.E.
      • et al.
      Human neutrophils produce interferon gamma upon stimulation by interleukin-12.
      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.
      • Ito A.
      • Imada K.
      • Sato T.
      • Kubo T.
      • Matsushima K.
      • Mori Y.
      Suppression of interleukin 8 production by progesterone in rabbit uterine cervix.
      • Kelly R.W.
      • Illingworth P.
      • Baldie G.
      • Leask R.
      • Brouwer S.
      • Calder A.A.
      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.
      • Gross J.
      • Lapiere C.M.
      Collagenolytic activity in amphibian tissues: a tissue culture assay.
      • Woessner Jr., J.F.
      Matrix metalloproteinases and their inhibitors in connective tissue remodeling.
      • Vu T.H.
      • Werb Z.
      Matrix metalloproteinases: effectors of development and normal physiology.
      MMP-9 (also known as gelatinase B) was discovered in polymorphonuclear leukocytes and monocytes.
      • Sopata I.
      • Wize J.
      A latent gelatin specific proteinase of human leucocytes and its activation.
      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.
      • Osmers R.G.
      • Blaser J.
      • Kuhn W.
      • Tschesche H.
      Interleukin-8 synthesis and the onset of labor.
      • Gomez-Lopez N.
      • Vadillo-Perez L.
      • Hernandez-Carbajal A.
      • Godines-Enriquez M.
      • Olson D.M.
      • Vadillo-Ortega F.
      Specific inflammatory microenvironments in the zones of the fetal membranes at term delivery.
      • Gomez-Lopez N.
      • Vega-Sanchez R.
      • Castillo-Castrejon M.
      • Romero R.
      • Cubeiro-Arreola K.
      • Vadillo-Ortega F.
      Evidence for a role for the adaptive immune response in human term parturition.
      • Vadillo-Ortega F.
      • Gonzalez-Avila G.
      • Furth E.E.
      • et al.
      92-kd type IV collagenase (matrix metalloproteinase-9) activity in human amniochorion increases with labor.
      • Vadillo-Ortega F.
      • Hernandez A.
      • Gonzalez-Avila G.
      • Bermejo L.
      • Iwata K.
      • Strauss 3rd, J.F.
      Increased matrix metalloproteinase activity and reduced tissue inhibitor of metalloproteinases-1 levels in amniotic fluids from pregnancies complicated by premature rupture of membranes.
      • Athayde N.
      • Edwin S.S.
      • Romero R.
      • et al.
      A role for matrix metalloproteinase-9 in spontaneous rupture of the fetal membranes.
      • Xu P.
      • Alfaidy N.
      • Challis J.R.
      Expression of matrix metalloproteinase (MMP)-2 and MMP-9 in human placenta and fetal membranes in relation to preterm and term labor.
      • Castillo-Castrejon M.
      • Meraz-Cruz N.
      • Gomez-Lopez N.
      • et al.
      Choriodecidual cells from term human pregnancies show distinctive functional properties related to the induction of labor.
      The expression of active MMP-9 is increased in term pregnancies of humans and rabbits,
      • Imada K.
      • Ito A.
      • Sato T.
      • Namiki M.
      • Nagase H.
      • Mori Y.
      Hormonal regulation of matrix metalloproteinase 9/gelatinase B gene expression in rabbit uterine cervical fibroblasts.
      • Stygar D.
      • Wang H.
      • Vladic Y.S.
      • Ekman G.
      • Eriksson H.
      • Sahlin L.
      Increased level of matrix metalloproteinases 2 and 9 in the ripening process of the human cervix.
      and it was localized in human infiltrating leukocytes and murine columnar epithelial cells and fibroblasts.
      • Gonzalez J.M.
      • Dong Z.
      • Romero R.
      • Girardi G.
      Cervical remodeling/ripening at term and preterm delivery: the same mechanism initiated by different mediators and different effector cells.
      • Stygar D.
      • Wang H.
      • Vladic Y.S.
      • Ekman G.
      • Eriksson H.
      • Sahlin L.
      Increased level of matrix metalloproteinases 2 and 9 in the ripening process of the human cervix.
      In vitro experimentation has demonstrated that the incubation of columnar epithelial cells or fibroblasts with progesterone inhibits MMP-9 activity.
      • Imada K.
      • Ito A.
      • Sato T.
      • Namiki M.
      • Nagase H.
      • Mori Y.
      Hormonal regulation of matrix metalloproteinase 9/gelatinase B gene expression in rabbit uterine cervical fibroblasts.
      • Gonzalez J.M.
      • Dong Z.
      • Romero R.
      • Girardi G.
      Cervical remodeling/ripening at term and preterm delivery: the same mechanism initiated by different mediators and different effector cells.
      However, in vitro incubation of human myometrial muscle cells with progesterone is not able to reduce IL-1β or TNFα-induced MMP-9 activity.
      • Roh C.R.
      • Oh W.J.
      • Yoon B.K.
      • Lee J.H.
      Up-regulation of matrix metalloproteinase-9 in human myometrium during labour: a cytokine-mediated process in uterine smooth muscle cells.
      Alternatively, in vitro incubation with progesterone reduces collagen synthesis in a 3-dimensional culture system with human cervical fibroblasts.
      • House M.
      • Tadesse-Telila S.
      • Norwitz E.R.
      • Socrate S.
      • Kaplan D.L.
      Inhibitory effect of progesterone on cervical tissue formation in a three-dimensional culture system with human cervical fibroblasts.
      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.
      • Fonseca E.B.
      • Celik E.
      • Parra M.
      • Singh M.
      • Nicolaides K.H.
      Fetal Medicine Foundation Second Trimester Screening Group. Progesterone and the risk of preterm birth among women with a short cervix.
      • DeFranco E.A.
      • O'Brien J.M.
      • Adair C.D.
      • et al.
      Vaginal progesterone is associated with a decrease in risk for early preterm birth and improved neonatal outcome in women with a short cervix: a secondary analysis from a randomized, double-blind, placebo-controlled trial.
      • Hassan S.S.
      • Romero R.
      • Vidyadhari D.
      • et al.
      Vaginal progesterone reduces the rate of preterm birth in women with a sonographic short cervix: a multicenter, randomized, double-blind, placebo-controlled trial.
      • da Fonseca E.B.
      • Bittar R.E.
      • Carvalho M.H.
      • Zugaib M.
      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.
      • Elovitz M.
      • Wang Z.
      Medroxyprogesterone acetate, but not progesterone, protects against inflammation-induced parturition and intrauterine fetal demise.
      • Elovitz M.A.
      • Mrinalini C.
      The use of progestational agents for preterm birth: lessons from a mouse model.
      It is interesting that systemic administration of IL-1β induces PTB in mice, and pretreatment with the IL-1 receptor antagonist abrogrates this effect.
      • Romero R.
      • Tartakovsky B.
      The natural interleukin-1 receptor antagonist prevents interleukin-1-induced preterm delivery in mice.
      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%,
      • Elovitz M.
      • Wang Z.
      Medroxyprogesterone acetate, but not progesterone, protects against inflammation-induced parturition and intrauterine fetal demise.
      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%.
      • Hassan S.S.
      • Romero R.
      • Vidyadhari D.
      • et al.
      Vaginal progesterone reduces the rate of preterm birth in women with a sonographic short cervix: a multicenter, randomized, double-blind, placebo-controlled trial.
      The mechanisms responsible for the protection against preterm birth in some animals and women, and not in others, remain to be determined.
      Administration of 17OHP-C to women with multiple gestations has been reported to increase the rates of midtrimester fetal loss
      • Combs C.A.
      • Garite T.
      • Maurel K.
      • Das A.
      • Porto M.
      Obstetrix Collaborative Research N. Failure of 17-hydroxyprogesterone to reduce neonatal morbidity or prolong triplet pregnancy: a double-blind, randomized clinical trial.
      and PTB before 32 weeks.
      • Senat M.V.
      • Porcher R.
      • Winer N.
      • et al.
      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.
      • Elovitz M.A.
      • Mrinalini C.
      The use of progestational agents for preterm birth: lessons from a mouse model.
      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.
      • Attardi B.J.
      • Zeleznik A.
      • Simhan H.
      • Chiao J.P.
      • Mattison D.R.
      • Caritis S.N.
      Comparison of progesterone and glucocorticoid receptor binding and stimulation of gene expression by progesterone, 17-alpha hydroxyprogesterone caproate, and related progestins.
      Progesterone and 17OHP-C also induced similar stimulation of endogenous alkaline phosphatase activity.
      • Attardi B.J.
      • Zeleznik A.
      • Simhan H.
      • Chiao J.P.
      • Mattison D.R.
      • Caritis S.N.
      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.
      • Tiboni G.M.
      • Del Corso A.
      • Marotta F.
      Progestational agents prevent preterm birth induced by a nitric oxide synthesis inhibitor in the mouse.
      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

      Figure thumbnail fx1
      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.
      Supplemental TableAntibodies used for immunophenotyping
      Cell MarkerFluorochromeCloneManufacturer
      ARG1FITCPolyclonalR&D Systems
      CD11bPECF594M1/70BD Biosciences
      CD11cAF488N418eBioscience
      CD25PE-Cy7PC61BD Biosciences
      CD3eAPC-Cy7 or PE-Cy5145-2C11BD Biosciences
      CD4APCRM4-5BD Biosciences
      CD45V45030-F11BD Biosciences
      CD49bAPCDX5BD Biosciences
      CD8PE53-6.7BD Biosciences
      F4/80APC-eFluor 780 or PEBM8eBioscience
      Foxp3AF488MF23BD Biosciences
      IFNγV450XMG1.2BD Biosciences
      IL4PE-Cy711B11BD Biosciences
      iNOSPECXNFTeBioscience
      Ly6GAPC-Cy7 or APC1A8BD Biosciences
      Furcron. Vaginal progesterone, but not 17OHP-C, has antiinflammatory effects. Am J Obstet Gynecol 2015.

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