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 1 A).

Animal 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, 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. The doses of vaginal progesterone and 17OHP-C were similar to those previously reported in studies using the same animal species. 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% CO ₂ . 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.

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 1 B 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 1 C); however, it decreased the proportion of decidual CD8+CD25+Foxp3+ T cells ( Figure 1 D).

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.

Proportions 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 ) 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 3 A.

Immunophenotyping 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 3 B). In contrast, 17OHP-C administration did not alter the proportion of decidual macrophages ( Figure 3 C). 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 4 A.

M1- 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 4 A. Administration of vaginal progesterone tended to reduce the proportion of myometrial macrophages; however, this reduction did not reach statistical significance ( Figure 5 A). Administration of vaginal progesterone ( Figure 5 B), however, decreased the proportion of IFNγ+ neutrophils (CD11b+Ly6G+F4/80– cells) in the myometrium ( Figure 5 C). 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.

Macrophages 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.

MMP-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 6 E).

In contrast, 17OHP-C administration increased the abundance of active MMP-9-positive cells when compared to the control group (castor oil) ( Figure 6 F). Magnification of active MMP-9-positive cells in Replens (control) revealed these cells to be neutrophils and monocytes ( Figure 6 G). 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.

Plasma 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, and the systemic or intraamniotic administration of IL-1β leads to PTB in mice. 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 8 A); however, the administration of 17OHP-C did not alter the concentration of this cytokine ( Figure 8 B).

Plasma 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.

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 1 B 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 1 C); however, it decreased the proportion of decidual CD8+CD25+Foxp3+ T cells ( Figure 1 D).

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.

Proportions 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 ) 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 3 A.

Immunophenotyping 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 3 B). In contrast, 17OHP-C administration did not alter the proportion of decidual macrophages ( Figure 3 C). 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 4 A.

M1- 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 4 A. Administration of vaginal progesterone tended to reduce the proportion of myometrial macrophages; however, this reduction did not reach statistical significance ( Figure 5 A). Administration of vaginal progesterone ( Figure 5 B), however, decreased the proportion of IFNγ+ neutrophils (CD11b+Ly6G+F4/80– cells) in the myometrium ( Figure 5 C). 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.

Macrophages 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.

MMP-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 6 E).

In contrast, 17OHP-C administration increased the abundance of active MMP-9-positive cells when compared to the control group (castor oil) ( Figure 6 F). Magnification of active MMP-9-positive cells in Replens (control) revealed these cells to be neutrophils and monocytes ( Figure 6 G). 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.

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