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Progesterone inhibits in vitro fetal membrane weakening

      Objective

      Inflammation/infection and abruption are leading causes of preterm premature rupture of the membranes. Recently, we identified granulocyte-macrophage colony-stimulating factor (GM-CSF) as a critical mediator of both tumor necrosis factor-α– (TNF; modeling inflammation) and thrombin-induced (modeling abruption) weakening of the fetal membranes. We found that (1) TNF and thrombin both induced GM-CSF in the choriodecidua, (2) blockade of GM-CSF action with neutralizing antibodies inhibited both TNF- and thrombin-induced fetal membrane weakening, and (3) GM-CSF alone induced fetal membrane weakening. GM-CSF is thus part of an overlap of the inflammation and abruption-induced fetal membrane weakening pathways. The effects of progesterone analogs on the pathways by which fetal membranes are weakened have not been investigated. We examined the effects of progesterone, medroxyprogesterone acetate (MPA) and 17α-hydroxyprogesterone (HP) on TNF- and thrombin-induced fetal membrane weakening.

      Study Design

      Full-thickness fetal membranes from uncomplicated term repeat cesarean deliveries were mounted in Transwell inserts in Minimum Essential Medium alpha and incubated at 37°C in 5% CO2. The choriodecidua side of the fetal membrane fragments were preincubated with progesterone, MPA, HP, or vehicle for 24 hours. Fetal membranes were then exposed to TNF, thrombin, or GM-CSF on the choriodecidua side for an additional 48 hours. The fetal membrane tissues were then strength tested, and medium from the choriodecidua and amnion compartments was assayed for GM-CSF content.

      Results

      TNF and thrombin both weakened fetal membranes and elevated media GM-CSF levels on the choriodecidua side of the fetal membrane. Pretreatment with progesterone, MPA, or HP inhibited both TNF- and thrombin-induced fetal membrane weakening and also inhibited the induced increase in GM-CSF. GM-CSF decreased fetal membrane rupture strength by 68%, which was inhibited by progestogen pretreatment with a potency order: progesterone <MPA <HP.

      Conclusion

      Progestogen pretreatment blocks TNF- and thrombin-induced fetal membrane weakening by inhibiting both the production and action of GM-CSF. These findings are consistent with the administration of progestogens in the prevention of preterm premature rupture of the membranes.

      Key words

      See related editorial, page 447
      Preterm premature rupture of the fetal membranes (PPROM) and resultant premature birth is a major cause of infant morbidity and death.
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      Infection/inflammation with cytokine production and decidual bleeding/abruption with thrombin production, respectively, increase the risk of PPROM; however, the mechanisms by which these conditions affect PPROM remain obscure.
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      Concomitant with weakening, these agents induce biochemical and histologic tissue changes that mimic those seen in the physiologic weak zone (fetal membrane rupture initiation site) in the fetal membrane region overlying the cervix.
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      Proinflammatory cytokines found in amniotic fluid induce collagen remodeling, apoptosis, and biophysical weakening of cultured human fetal membranes.
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      • Moore J.J.
      Alpha-lipoic acid inhibits tumor necrosis factor-induced remodeling and weakening of human fetal membranes.
      • Moore R.M.
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      Alpha-lipoic acid inhibits thrombin-induced fetal membrane weakening in vitro.
      • El-Khwad M.
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      Pre-labor fetal membranes have a weak zone overlying the lower uterine pole and cervix.
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      Pre-labour fetal membranes overlying the cervix display alterations in inflammation and NF-kappaB signalling pathways.
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      Studies of the pathways of TNF/interleukin-1ß– and thrombin-induced fetal membrane weakening have demonstrated that the initial tissue and cellular targets of these agents are in the choriodecidua component of the fetal membrane rather than the amnion.
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      • Lemerand K.
      • Kumar D.
      • et al.
      Thrombin weakens the amnion extracellular matrix (ECM) directly rather than through protease activated receptors.
      • Kumar D.
      • Moore R.M.
      • Nash A.
      • et al.
      Decidual GM-CSF is a critical common intermediate necessary for thrombin and TNF induced in-vitro fetal membrane weakening.
      This is consistent with the sources of inflammation or bleeding that originate from the maternal side of the fetal membrane. Using an enhanced version of the model system that mimics the directional aspect of the fetal membrane weakening pathways, we recently demonstrated that TNF or thrombin, when applied to only the choriodecidua side of fetal membrane, caused weakening in the same manner as when both sides of the fetal membrane were exposed. We also identified granulocyte-macrophage colony-stimulating factor (GM-CSF) as a critical intermediate in the fetal membrane weakening pathways that are induced by both TNF and thrombin.
      • Kumar D.
      • Moore R.M.
      • Nash A.
      • et al.
      Decidual GM-CSF is a critical common intermediate necessary for thrombin and TNF induced in-vitro fetal membrane weakening.
      As documented in this previous report, GM-CSF is induced in the choriodecidua by TNF and thrombin in a concentration-dependent manner that is concomitant with TNF- and thrombin-induced fetal membrane weakening. Importantly, GM-CSF alone also causes dose-dependent fetal membrane weakening and blockade of GM-CSF with neutralizing antibody prevents fetal membrane weakening by TNF or thrombin.
      • Kumar D.
      • Moore R.M.
      • Nash A.
      • et al.
      Decidual GM-CSF is a critical common intermediate necessary for thrombin and TNF induced in-vitro fetal membrane weakening.
      These observations suggest that GM-CSF mediates fetal membrane weakening that is induced by both inflammation and bleeding.
      • Kumar D.
      • Moore R.M.
      • Nash A.
      • et al.
      Decidual GM-CSF is a critical common intermediate necessary for thrombin and TNF induced in-vitro fetal membrane weakening.
      It is therefore possible to reduce the fetal membrane weakening process into those events that are involved in the generation of GM-CSF and those events caused by the action of GM-CSF. Similarly, inhibitors of the weakening process can now be categorized as acting to prevent GM-CSF generation, acting to inhibit GM-CSF action, or both. This categorization facilitates the identification of the point of action of specific inhibitors and thus provides information that may allow their use in inhibition of PPROM.
      Currently, progestogens are the only agents recommended by the American College of Obstetricians and Gynecologists for the prevention of preterm delivery.
      American College of Obstetricians and Gynecologists Committee on Practice Bulletins–Obstetrics
      Prediction and prevention of preterm birth. ACOG practice bulletin no. 130.
      Weekly intramuscular administration of 17 (OH) progesterone-caproate or daily vaginal administration of progesterone have been recommended for the prevention of recurrent preterm birth or for short cervical length, respectively.
      American College of Obstetricians and Gynecologists Committee on Practice Bulletins–Obstetrics
      Prediction and prevention of preterm birth. ACOG practice bulletin no. 130.
      • Iams J.D.
      Prevention of preterm parturition.
      However, there is no direct evidence that progestogens inhibit fetal membrane weakening. The cellular targets for progesterone action in fetal membrane are thought to be mainly decidual cells that express the nuclear progesterone receptor (nPR) isoforms (PR-A and PR-B).
      • Merlino A.
      • Welsh T.
      • Erdonmez T.
      • et al.
      Nuclear progesterone receptor expression in the human fetal membranes and decidua at term before and after labor.
      In a previous study, we found that the progesterone analog medroxyprogesterone acetate (MPA) inhibited TNF-induced GM-CSF induction in decidual cells.
      • Arcuri F.
      • Toti P.
      • Buchwalder L.
      • et al.
      Mechanisms of leukocyte accumulation and activation in chorioamnionitis: interleukin 1 beta and tumor necrosis factor alpha enhance colony stimulating factor 2 expression in term decidua.
      It thus seemed appropriate to hypothesize that progesterone would inhibit fetal membrane weakening by blocking GM-CSF production. However, GM-CSF is proposed to act, in part, by the activation of resident decidual mononuclear cells, which express membrane progesterone receptors. Thus, it is also possible that progesterone could block fetal membrane weakening by inhibiting GM-CSF action.
      • Srivastava M.D.
      • Anderson D.J.
      Progesterone receptor expression by human leukocyte cell lines: molecular mechanisms of cytokine suppression.
      • Peltier M.R.
      • Tee S.C.
      • Smulian J.C.
      Effect of progesterone on proinflammatory cytokine production by monocytes stimulated with pathogens associated with preterm birth.
      The studies herein were undertaken to determine whether progestogens inhibit TNF- and thrombin-induced fetal membrane weakening.

      Materials and Methods

      Materials

      Humankine TNF, Humankine GM-CSF (both produced in HEK 293 cells), progesterone, MPA, 17α-hydroxyprogesterone (HP), and other miscellaneous reagents, unless otherwise stated, were obtained from Sigma-Aldrich, St. Louis, MO. Thrombin (from bovine plasma, 1500NIH U/mg protein) was obtained from Thermo Fisher Scientific, Pittsburgh, PA. RU486 (mifepristone) was obtained from Cayman Chemical Co, Ann Arbor, MI.

      Fetal membrane collection and preparation

      Full-thickness fetal membrane fragments from term uncomplicated repeat cesarean deliveries were collected after patient consent and approval by MetroHealth Medical Center’s Institutional Review Board (# IRB10-00861; Cleveland, OH). None of the patients had a history of preterm birth. Fetal membrane tissue was taken from areas remote from the weak zone region, washed in 2 × 250–mL changes of phosphate-buffered saline solution (pH 7.2) and mounted, choriodecidua side down, in 24-mm Transwell (Costar, Corning, NY) inserts that were secured with an O-ring to separate the choriodecidua and amnion sides. The inserts were placed in 6-well culture plates and 2 mL MEM+ (Minimum Essential Medium alpha with Earle’s salts, supplemented with 1 mmol/L L-glutamine, 2.24 g/L sodium bicarbonate [Mediatech, Manassas, VA], 10 ml/L Antibiotic-Antimycotic (Sigma Chemical Co, St. Louis, MO), and 50 mg/L gentamicin sulfate) was added to the upper (amnion side) and the lower (choriodecidua side) chambers.
      • Kumar D.
      • Moore R.M.
      • Nash A.
      • et al.
      Decidual GM-CSF is a critical common intermediate necessary for thrombin and TNF induced in-vitro fetal membrane weakening.
      Progestogens (progesterone, HP, MPA [each 10–7mmol]) or vehicle (0.01% ethanol final concentration) were added to the choriodecidua side 24 hours before the addition of weakening agents. Where indicated, RU486 (10–8 mmol) was also added to the choriodecidua side medium 1 hour before the addition of progestogens. Weakening agents (TNF [20 ng/mL], thrombin [10 U/mL], GM-CSF [200 ng/mL]) were then added to the choriodecidua side, and the cultures were incubated at 37°C in 5% CO2 and 100% relative humidity for an additional 48 hours. After culture, medium from each compartment was collected, centrifuged for 15 minutes at 12,000g/10°C, and the supernatants were stored at –70°C.
      Doses for TNF, thrombin, and GM-CSF that were used in these studies were determined from dose-response studies in our previous publication,
      • Kumar D.
      • Moore R.M.
      • Nash A.
      • et al.
      Decidual GM-CSF is a critical common intermediate necessary for thrombin and TNF induced in-vitro fetal membrane weakening.
      and they were added to the choriodecidua side only, because our previous studies have shown that the choriodecidua (rather than the amnion) is the initial target tissue or the tissue of origin of each.
      • Kumar D.
      • Schatz F.
      • Moore R.M.
      • et al.
      The effects of thrombin and cytokines upon the biomechanics and remodeling of isolated amnion membrane, in vitro.
      • Puthiyachirakkal M.
      • Lemerand K.
      • Kumar D.
      • et al.
      Thrombin weakens the amnion extracellular matrix (ECM) directly rather than through protease activated receptors.
      Progestogens were chosen for study with the following rationale: progesterone was selected as the natural progestogen of major focus. MPA was chosen because it is known to be an effective inhibitor of TNF-induced GM-CSF production.
      • Arcuri F.
      • Toti P.
      • Buchwalder L.
      • et al.
      Mechanisms of leukocyte accumulation and activation in chorioamnionitis: interleukin 1 beta and tumor necrosis factor alpha enhance colony stimulating factor 2 expression in term decidua.
      It also metabolizes less rapidly in tissue culture than does progesterone.
      • Arici A.
      • Marshburn P.B.
      • MacDonald P.C.
      • Dombrowski R.A.
      Progesterone metabolism in human endometrial stromal and gland cells in culture.
      HP was selected as a natural progestogen that has a strong affinity for the membranous progesterone receptor (mPR),
      • Blackmore P.F.
      • Beebe S.J.
      • Danforth D.R.
      • Alexander N.
      Progesterone and 17-hyrdroxyprogesterone.
      • Ashley R.L.
      • Clay C.M.
      • Farmerie G.D.
      • et al.
      Cloning and characterization of an ovine intracellular seven transmembrane receptor for progesterone that mediates calcium mobilization.
      • Smith J.L.
      • Kupchak B.R.
      • Garitaonandia I.
      • et al.
      Heterologous expression of human mPR, mPR, and mPR in yeast confirms their ability to function as membrane progesterone receptors.
      • Fernandes M.S.
      • Pierron V.
      • Michalovich D.
      • et al.
      Regulated expression of putative membrane progestin receptor homologues in human endometrium and gestational tissues.
      • Dressings G.E.
      • Goldberg J.E.
      • Charles N.J.
      • et al.
      Membrane progesterone receptor expression in mammalian tissues: a review of regulation and physiological implications.
      but not the nPR. 17-OH progesterone caproate was not used in this study. The concentration of progestogens used (10–7M) was within the range that is seen in pregnant serum at term. Progestogens were added to the choriodecidua side to ensure that they reached the target tissue concomitant with the weakening agents.

      Strength testing of fetal membrane

      Fetal membrane fragments were strength-tested within the Transwell inserts with the use of our rupture testing apparatus, as previously reported.
      • Moore R.M.
      • Mansour J.M.
      • Redline R.W.
      • Mercer B.M.
      • Moore J.J.
      The physiology of fetal membrane rupture: insight gained from the determination of physical properties.
      • El-Khwad M.
      • Stetzer B.
      • Moore R.M.
      • et al.
      Pre-labor fetal membranes have a weak zone overlying the lower uterine pole and cervix.
      • Kumar D.
      • Moore R.M.
      • Nash A.
      • et al.
      Decidual GM-CSF is a critical common intermediate necessary for thrombin and TNF induced in-vitro fetal membrane weakening.
      Briefly, Transwell-mounted fetal membrane were secured in a 2.5-cm diameter fixture between the aligned horizontal plates of the rupture testing equipment. A motor-driven 1-cm diameter spherical-head plunger that was aligned perpendicular to the fetal membrane was then forced against the amnion side. Fetal membrane displacement and concomitant plunger force were recorded continuously until rupture. From these data, force (rupture strength in newtons) and maximum displacement (centimeters) that were needed to cause fetal membrane rupture were determined.

      GM-CSF determination

      GM-CSF levels in thawed supernatants from the fluid compartments adjacent to the choriodecidua and amnion sides of Transwell cultured fetal membrane were determined with the use of the Human GM-CSF Quantikine ELISA Kit (R&D Systems, Minneapolis, MN), according to the manufacturer’s protocol. Intra- and interassay precision of the assay was 2.7% and 5.3% coefficient of variation, respectively, for cell culture supernatants at a sensitivity level of 3 pg/mL with Escherichia coli expressed, recombinant human GM-CSF as standard.

      Statistical analysis

      All experiments were performed at least in triplicate. Data were analyzed by analysis of variance followed by post-hoc pair-wise comparisons (Holm-Sidak method) with the use of Sigmaplot software (Systat Software, Inc, Chicago, IL). Differences were considered significant when at a probability value of < .05.

      Results

      Progestogens inhibit thrombin- and TNF-induced fetal membrane weakening

      Thrombin or TNF, with or without progestogens, were each added only to the choriodecidua side of the fetal membrane to mimic the physiologic conditions of pregnancy in which the choriodecidua is exposed to the maternal circulation and the amnion is in contact with the amniotic fluid.
      • Kumar D.
      • Schatz F.
      • Moore R.M.
      • et al.
      The effects of thrombin and cytokines upon the biomechanics and remodeling of isolated amnion membrane, in vitro.
      • Puthiyachirakkal M.
      • Lemerand K.
      • Kumar D.
      • et al.
      Thrombin weakens the amnion extracellular matrix (ECM) directly rather than through protease activated receptors.
      • Kumar D.
      • Moore R.M.
      • Nash A.
      • et al.
      Decidual GM-CSF is a critical common intermediate necessary for thrombin and TNF induced in-vitro fetal membrane weakening.
      Compared with control membrane strength, thrombin or TNF each markedly decreased fetal membrane rupture strength in all studies (both P < .01). Preincubation with progesterone (10–7mmol) inhibited both thrombin- and TNF-induced fetal membrane weakening (both P < .05; Figure 1, A). MPA (10–7mmol) preincubation also inhibited fetal membrane weakening that was induced by thrombin (P = .01) and by TNF (P = .02; Figure 1, B). Finally, HP (10–7mmol) preincubation inhibited fetal membrane weakening that was induced by thrombin and TNF (both P < .01; Figure 1, C). Preincubation with progesterone, MPA, or HP alone did not affect rupture strength (Figure 1).
      Figure thumbnail gr1
      Figure 1Progestogens inhibit thrombin- and TNF α–induced fetal membrane weakening
      Preincubation with A, progesterone (10–7 mmol), B, MPA (10–7 mmol), or C, 17α-hydroxyprogesterone (10–7 mmol) for 24 hours inhibited fetal membrane weakening by either TNF (20 ng/mL) or Thr (10 U/mL) applied for 48 additional hours. In all studies, all agents were applied to only the choriodecidual side of the fetal membranes. Strength testing was done at 72 hours for all fetal membrane fragments. The data shown in each panel are for 1 representative experiment that was performed in triplicate with each condition. Each experiment was repeated 3 times with 3 different placentas (data are presented as mean ± SD). Symbols designate pairs of columns with significant differences (A, the asterisk and plus symbols indicate P < .01, and the circumflex accent and number symbols indicate P < .05; B, the asterisk, plus, and circumflex accent symbols indicate P = .01, and the number symbol indicates P = .02; C, all of the symbols indicate P < .01).
      C, control; HP, 17α-hydroxyprogesterone; MPA, medroxyprogesterone acetate; P, progesterone; Thr, thrombin; TNF, tumor necrosis factor α.
      Kumar. Progesterone analogs inhibit fetal membrane weakening. Am J Obstet Gynecol 2015.

      Effect of progestogens on GM-CSF production

      Because GM-CSF is a critical intermediate in both the TNF- and thrombin-induced fetal membrane weakening pathways,
      • Kumar D.
      • Moore R.M.
      • Nash A.
      • et al.
      Decidual GM-CSF is a critical common intermediate necessary for thrombin and TNF induced in-vitro fetal membrane weakening.
      further studies were done to determine whether progestogens inhibit TNF- or thrombin-induced fetal membrane weakening by blocking GM-CSF production. Consistent with our previous report, TNF and thrombin each increased GM-CSF release on the choriodecidua side concomitant with fetal membrane weakening (Figure 2; P < .01).
      • Kumar D.
      • Moore R.M.
      • Nash A.
      • et al.
      Decidual GM-CSF is a critical common intermediate necessary for thrombin and TNF induced in-vitro fetal membrane weakening.
      MPA markedly inhibited GM-CSF production by both TNF and thrombin to control levels (P < .01). HP markedly inhibited the GM-CSF increase that was produced by TNF (P < .01) but only partially inhibited the GM-CSF increase that was produced by thrombin (P < .05). Finally, progesterone partially inhibited the GM-CSF increase produced by TNF (P < .05). The progesterone effect on thrombin-induced GM-CSF production was not significant. Only small amounts of GM-CSF were detected on the amnion side of the fetal membrane (Figure 2).
      Figure thumbnail gr2
      Figure 2Effect of progestogens on TNF α– and thrombin-induced GM-CSF
      GM-CSF released on the choriodecidua side of the fetal membranes (left) and the amnion side of the fetal membranes (right) in the experiment shown in is shown for A, progesterone, B, MPA, and C, 17α-hydroxyprogesterone. The data shown in each panel are for 1 representative experiment that was performed in triplicate with each condition. Each experiment was repeated 3 times using 3 different placentas (data are presented as mean ± SD). Symbols designate pairs of columns with significant differences (A, the asterisk and number symbols indicate P < .01, and the plus symbol indicates P < .05; B, all of the symbols indicate P < .01; C, the asterisk, number, and plus symbols indicate P < .01, and the circumflex accent symbol indicates P < .05).
      C, control; GM-CSF, granulocyte-macrophage colony-stimulating factor; HP, 17α-hydroxyprogesterone; MPA, medroxyprogesterone acetate; P, progesterone; Thr, thrombin; TNF, tumor necrosis factor α.
      Kumar. Progesterone analogs inhibit fetal membrane weakening. Am J Obstet Gynecol 2015.

      Progestogens inhibit GM-CSF–induced fetal membrane weakening

      Studies were then performed to determine whether progestogens inhibit TNF- and thrombin-induced fetal membrane weakening by inhibiting the action of GM-CSF. Consistent with our previous report,
      • Kumar D.
      • Moore R.M.
      • Nash A.
      • et al.
      Decidual GM-CSF is a critical common intermediate necessary for thrombin and TNF induced in-vitro fetal membrane weakening.
      GM-CSF incubation on the choriodecidua side of fetal membrane induced significant (P < .01) fetal membrane weakening (Figure 3). This GM-CSF–induced fetal membrane weakening was almost completely inhibited by preincubation with either MPA (10–7 mmol) or HP (10–7 mmol; both P < .01). Although preincubation with progesterone (10–7 mmol) also blunted GM-CSF–induced fetal membrane weakening, its effect was not statistically significant. Rupture strength after incubation with progesterone, HP, or MPA alone was not different from control membranes (Figure 3).
      Figure thumbnail gr3
      Figure 3Progestogens inhibit GM-CSF–induced fetal membrane weakening
      Preincubation with progesterone, MPA, or 17α-hydroxyprogesterone (all 10–7 mmol) for 24 hours inhibited fetal membrane weakening by GM-CSF (200 ng/mL) applied for 48 additional hours. In all studies, all agents were applied to only the choriodecidual side of the fetal membranes. Strength testing was done at 72 hours for all fetal membrane fragments. The data shown are for 1 representative experiment that was performed in triplicate with each condition. The experiment was repeated 3 times with 3 different placentas (data are presented as mean ± SD). Symbols designate pairs of columns with significant differences (all symbols indicate P < .01).
      C, control; G, GM-CSF; GM-CSF, granulocyte-macrophage colony-stimulating factor; HP, 17α-hydroxyprogesterone; MPA, medroxyprogesterone acetate; P, progesterone.
      Kumar. Progesterone analogs inhibit fetal membrane weakening. Am J Obstet Gynecol 2015.

      RU486 blocks HP inhibition of GM-CSF–induced fetal membrane weakening

      In the experiment with RU486, consistent with the previously presented experiments, GM-CSF significantly decreased fetal membrane rupture strength, and preincubation with HP inhibited the GM-CSF–induced fetal membrane weakening. RU486 (10–8 mmol) that was applied 1 hour before HP blocked HP inhibition of GM-CSF–induced weakening (P < .01; Figure 4).
      Figure thumbnail gr4
      Figure 4RU486 blocks 17α-hydroxyprogesterone inhibition of GM-CSF–induced fetal membrane weakening
      Preincubation initially with RU486 (10–8 mmol) for 1 hour and then with 17α-hydroxyprogesterone (10–7 mmol) for 24 hours blocked the inhibition of GM-CSF (200 ng/mL applied for 48 additional hours) induced fetal membrane weakening by 17α-hydroxyprogesterone. In all studies, all agents were applied only to the choriodecidual side of the fetal membrane. Strength testing was done at 72 hours for all fetal membrane fragments. The data shown are for 1 representative experiment that was performed in triplicate with each condition. The experiment was repeated 3 times with 3 different placentas (data are presented as mean ± SD). Symbols designate pairs of columns with significant differences (all symbols indicate P < .01).
      C, control; G, GM-CSF; GM-CSF, granulocyte-macrophage colony-stimulating factor; HP, 17α-hydroxyprogesterone; R, RU486 (mifepristone).
      Kumar. Progesterone analogs inhibit fetal membrane weakening. Am J Obstet Gynecol 2015.

      Comment

      The results presented here demonstrate for the first time that the natural progestogens (progesterone and HP) and the synthetic progestogen (MPA) inhibit both TNF- and thrombin-induced weakening of human fetal membrane in vitro. These observations suggest that progesterone blocks both inflammation- and bleeding-initiated fetal membrane weakening. Previously, we found that GM-CSF is a critical intermediate in TNF- and thrombin-induced weakening of human fetal membrane. In these experiments, HP and MPA each inhibited both GM-CSF production by choriodecidua- and GM-CSF-induced fetal membrane weakening. Although progesterone also decreased both GM-CSF production and GM-CSF–induced weakening, its inhibitory effect did not attain statistical significance. Taken together, these data suggest that progestogens act at multiple points in the pathway to fetal membrane weakening, evidenced by their effect on both GM-CSF production and action.
      GM-CSF is a pro-inflammatory mediator that we have previously shown to be produced by choriodecidua in response to either TNF or thrombin and mediates the fetal membrane weakening action of both agents.
      • Kumar D.
      • Moore R.M.
      • Nash A.
      • et al.
      Decidual GM-CSF is a critical common intermediate necessary for thrombin and TNF induced in-vitro fetal membrane weakening.
      • Arcuri F.
      • Toti P.
      • Buchwalder L.
      • et al.
      Mechanisms of leukocyte accumulation and activation in chorioamnionitis: interleukin 1 beta and tumor necrosis factor alpha enhance colony stimulating factor 2 expression in term decidua.
      Decidual stromal cells have been shown to produce GM-CSF in response to TNF stimulation that is inhibited by MPA.
      • Arcuri F.
      • Toti P.
      • Buchwalder L.
      • et al.
      Mechanisms of leukocyte accumulation and activation in chorioamnionitis: interleukin 1 beta and tumor necrosis factor alpha enhance colony stimulating factor 2 expression in term decidua.
      Our present findings confirm progestogen inhibition of TNF-induced GM-CSF production in choriodecidua by MPA. They also demonstrate inhibition of TNF-induced GM-CSF production by progesterone, and HP and inhibition of thrombin-induced GM-CSF production by HP and MPA (Figure 2).
      GM-CSF is postulated to recruit mononuclear cells, to stimulate their conversion to macrophages, and then to activate macrophages resulting in increased protease production and fetal membrane weakening (Figure 5). Nuclear progesterone receptors have not been found in the amnion or chorion or in the mononuclear cells of the decidua. In fetal membranes, they have been described only in decidual cell nuclei.
      • Merlino A.
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      • et al.
      Nuclear progesterone receptor expression in the human fetal membranes and decidua at term before and after labor.
      Nuclear progesterone receptors have also not been detected in murine macrophages or normal human mononuclear cells.
      • Srivastava M.D.
      • Anderson D.J.
      Progesterone receptor expression by human leukocyte cell lines: molecular mechanisms of cytokine suppression.
      • Lu J.
      • Reese J.
      • Zhou Y.
      • Hirsh E.
      Progesterone-induced activation of membrane-bound progesterone receptors in murine macrophages.
      Decidual monocytes/macrophages and amnion and chorion cells do express mPRs that make progestogen modulation of monocytic cell activation possible.
      • Fernandes M.S.
      • Pierron V.
      • Michalovich D.
      • et al.
      Regulated expression of putative membrane progestin receptor homologues in human endometrium and gestational tissues.
      • Dressings G.E.
      • Goldberg J.E.
      • Charles N.J.
      • et al.
      Membrane progesterone receptor expression in mammalian tissues: a review of regulation and physiological implications.
      • Lu J.
      • Reese J.
      • Zhou Y.
      • Hirsh E.
      Progesterone-induced activation of membrane-bound progesterone receptors in murine macrophages.
      Peripheral blood mononuclear cells that were obtained from pregnant women who had been exposed to sustained progesterone supplementation demonstrate decreased immunoreactivity in vitro to gram-positive and gram-negative bacterial infections.
      • Foglia L.M.
      • Ippolito D.L.
      • Stallings J.D.
      • et al.
      Intramuscular 17α-hydroxyprogesterone caproate administration attenuates immunoresponsiveness of maternal peripheral blood mononuclear cells.
      Progesterone also inhibits proinflammatory cytokines in a fetoplacental artery explant model, inhibits TNF- and interleukin-1–induced MMP-1 and MMP3 in decidua, and inhibits TNF-induced apoptosis in fetal membranes.
      • Sheilds A.D.
      • Wright J.
      • Paonessa D.J.
      • et al.
      Progesterone modulation of inflammatory cytokine production in a fetoplacental artery explant model.
      • Oner C.
      • Schatz F.
      • Kizilay G.
      • et al.
      Progestin-inflammatory cytokine interactions affect matrix metalloproteinase-1 and -3 expression in term decidual cells: implications for treatment of chorioamnionitis-induced preterm delivery.
      • Luo G.
      • Abrahams V.M.
      • Tadesse S.
      • et al.
      Progesterone inhibits basal and TNF alpha- induced apoptosis in fetal membranes: a novel mechanism to explain progesterone-mediated prevention of preterm birth.
      Our results demonstrate that progestogens inhibit GM-CSF–induced fetal membrane weakening (Figure 3).
      Figure thumbnail gr5
      Figure 5Proposed mechanism for fetal membrane weakening
      TNF α (modeling inflammation) and thrombin (modeling decidual bleeding/abruption) act on decidua cells in the choriodecidua to produce GM-CSF. Blocking GM-CSF with neutralizing antibody blocks both thrombin- and TNF α–induced weakening.
      • Kumar D.
      • Moore R.M.
      • Nash A.
      • et al.
      Decidual GM-CSF is a critical common intermediate necessary for thrombin and TNF induced in-vitro fetal membrane weakening.
      GM-CSF is postulated to recruit mononuclear cells, stimulate their conversion to macrophages, and then activate macrophages, which results in protease production and fetal membrane weakening. Progestogens act on both GM-CSF production and action.
      GM-CSF, granulocyte-macrophage colony-stimulating factor; MMP, matrix metaloproteinase; TNF, tumor necrosis factor α.
      Kumar. Progesterone analogs inhibit fetal membrane weakening. Am J Obstet Gynecol 2015.
      In most viviparous species, the process of parturition is initiated by progesterone withdrawal.
      • Young I.R.
      • Renfree M.B.
      • Mesiano S.
      • Shaw G.
      • Jenkin G.
      • Smith R.
      The comparative physiology of parturition in mammals.
      Human parturition, however, occurs without systemic progesterone withdrawal. Maternal, fetal, and amniotic fluid concentrations of progesterone and HP remain high throughout pregnancy and labor and delivery.
      • Challis J.R.G.
      Sharp decrease increases in free circulating oestrogens immediately before parturition in sheep.
      • Walsh S.W.
      • Stanczyk F.Z.
      • Novy M.J.
      Daily hormonal changes in the maternal, fetal, and amniotic fluid compartments before parturition in a primate species.
      • Boroditsky R.S.
      • Reyes F.I.
      • Winter J.S.
      • Faiman C.
      Maternal serum estrogen and progesterone concentrations preceding normal labor.
      Term and preterm labor onset in humans is thus postulated to result from a functional progesterone withdrawal, whereby the gestation tissue becomes refractory to progesterone signaling. Several mechanisms for functional progesterone withdrawal have been proposed. One mechanism likely active in the choriodecidua decidual cells is mediated via changes in the relative levels of the nPR isoforms, PR-A and PR-B, coupled with changes in progesterone receptor coregulator levels, which leads to decreased progesterone receptor transcriptional activity.
      • Patel B.
      • Elguero S.
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      • Dahoud W.
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      • Mesiano S.
      Role of nuclear progesterone receptor isoforms in uterine pathophysiology.
      • Byrns M.C.
      Regulation of progesterone signaling during pregnancy: implications for the use of progestins for the prevention of preterm birth.
      Total progesterone receptor expression in human decidual cells is decreased with the onset of term and preterm labor.
      • Lockwood C.J.
      • Stocco C.
      • Murk W.
      • Kayisli U.A.
      • Funai E.F.
      • Schatz F.
      Human labor is associated with reduced decidual cell expression of progesterone, but not glucocorticoid, receptors.
      In a recent study, decidual cell TNF immunostaining, and protein levels, the PR-A/PR-B ratio and type II cyclooxygenase were increased significantly in association with the onset of labor.
      • Jiang Z.Y.
      • Guo Y.Y.
      • Ren H.B.
      • et al.
      Tumor necrosis factor (TNF)-α upregulates progesterone receptor-A by activating the NF-κB signaling pathway in human decidua after labor onset.
      Interestingly, in quiescent (ie, not in labor) decidual cells, TNF induced a concentration-dependent increase in NF-κB activity was associated with increased levels of PR-A and type II cyclooxygenase, which suggests a role for TNF in regulating functional progesterone withdrawal.
      • Jiang Z.Y.
      • Guo Y.Y.
      • Ren H.B.
      • et al.
      Tumor necrosis factor (TNF)-α upregulates progesterone receptor-A by activating the NF-κB signaling pathway in human decidua after labor onset.
      Similarly, abruption-associated preterm delivery is associated with thrombin-mediated functional progesterone withdrawal that involves PR-A and PR-B in decidual cells.
      • Lockwood C.J.
      • Kayisli U.A.
      • Stocco C.
      • et al.
      Abruption-induced preterm delivery is associated with thrombin-mediated functional progesterone withdrawal in decidual cells.
      There is no information on how mPRs change at the end of gestation.
      Whatever the mechanism, progestogens should not be effective in mediating any response in term tissue once functional progesterone withdrawal has occurred. The experiments presented here were performed on term (37-39 weeks of gestation) fetal membranes that were obtained after elective cesarean deliveries in women who were not in clinical labor. We must assume that the robust protective effects of progesterone (and other progestogens) that has been demonstrated in these data were mediated either through the nPRs in cells in the choriodecidua and that the progesterone receptor isoform ratio in the cells was conducive to mediating progestational effects of progesterone or they were mediated through mPR for which changes around labor remain unclear. Because it is not clear at what point in gestation or how suddenly the postulated changes in nPR subtypes occur, further studies are needed to determine whether the protective effects of progestogens remain in fetal membranes that are obtained from women in active labor, in whom a functional progesterone withdrawal mechanism likely would be activated.
      The reported effectiveness of exogenous progesterone in the prevention of preterm birth suggests a persistence of tissue responsiveness to progesterone that is consistent with the results presented here. Clinical trials suggest that progestogen prophylaxis reduces the risk of preterm birth in at-risk women.
      • Iams J.D.
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      • Lockwood C.J.
      • Stocco C.
      • Murk W.
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      • Funai E.F.
      • Schatz F.
      Human labor is associated with reduced decidual cell expression of progesterone, but not glucocorticoid, receptors.
      • Jiang Z.Y.
      • Guo Y.Y.
      • Ren H.B.
      • et al.
      Tumor necrosis factor (TNF)-α upregulates progesterone receptor-A by activating the NF-κB signaling pathway in human decidua after labor onset.
      • Lockwood C.J.
      • Kayisli U.A.
      • Stocco C.
      • et al.
      Abruption-induced preterm delivery is associated with thrombin-mediated functional progesterone withdrawal in decidual cells.
      • Keirse M.J.
      Progestogen administration in pregnancy may prevent preterm delivery.
      • 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.
      • Meis P.J.
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      • Thom E.
      • et al.
      Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate.
      • O’Brien J.M.
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      • et al.
      Progesterone vaginal gel for the reduction of recurrent preterm birth: primary results from a randomized, double-blind, placebo-controlled trial.
      However, observations regarding the capacity for progestogen therapy to prevent PPROM specifically are lacking.
      • Iams J.D.
      Prevention of preterm parturition.
      • Keirse M.J.
      Progestogen administration in pregnancy may prevent preterm delivery.
      • Da Fonseca E.B.
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      • Meis P.J.
      • Klebanoff M.
      • Thom E.
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      Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate.
      • O’Brien J.M.
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      • Lewis D.F.
      • et al.
      Progesterone vaginal gel for the reduction of recurrent preterm birth: primary results from a randomized, double-blind, placebo-controlled trial.
      In most studies, either PPROM rates were not reported or patients with PPROM were excluded from analysis.
      • 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.
      • Meis P.J.
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      • Thom E.
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      Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate.
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      Our results suggest that progestogen therapy may be effective in the prevention of PPROM, provided that it is initiated before the onset of functional progesterone withdrawal in the choriodecidua. Studies in myometrial cells suggest that progesterone, via PR-B, exerts antiinflammatory actions by desensitizing the cells to proinflammatory stimuli.
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      Progesterone receptor plays a major antiinflammatory role in human myometrial cells by antagonism of nuclear factor-kappa B activation of cyclooxygenase 2 expression.
      Thus, if progesterone exerts similar effects on decidual cells, consistent with this scenario, progestogen supplementation may enhance the capacity of endogenous progesterone to exert antiinflammatory actions and thus prevent inflammation-induced fetal membrane weakening.
      There are data that suggest the effectiveness of progestogens in the prevention of preterm delivery might be a function of the dose and the specific progestogen that is administered.
      • Caritis S.N.
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      • Thom E.
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      Relationship between 17-alpha hydroxyprogesterone caproate concentration and spontaneous preterm birth.
      Studies with primary human chorion cells, and in a mouse inflammatory-mediated parturition model, suggest that MPA may be more promising than 17-OH progesterone caproate for prevention of preterm birth.
      • Allen T.K.
      • Feng L.
      • Grotegut C.A.
      • Murtha A.P.
      Progesterone receptor membrane component 1 as the mediator of the inhibitory effect of progestins on cytokine-induced matrix metalloproteinase 9 activity in vitro.
      • Bamberger C.M.
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      Molecular mechanisms of dissociative glucocorticoid activity.
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      Medroxyprogesterone acetate, but not progesterone, protects against inflammation-induced parturition and intrauterine fetal demise.
      • Elovitz M.A.
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      Can medroxyprogesterone acetate alter Toll-like receptor expression in a mouse model of intrauterine inflammation?.
      However, the only randomized clinical trial to use MPA to prevent prematurity in at-risk pregnancies failed to demonstrate benefit.
      • Hobel C.J.
      • Ross M.G.
      • Bemis R.L.
      • et al.
      The West Los Angeles preterm birth prevention project: I, program impact on high-risk women.
      Although progesterone, MPA, and HP each inhibited both TNF- and thrombin-induced fetal membrane weakening, their effects on GM-CSF production and action were different. This may be the result of several factors. Progesterone has been reported to undergo rapid metabolism in cell and tissue studies.
      • Arici A.
      • Marshburn P.B.
      • MacDonald P.C.
      • Dombrowski R.A.
      Progesterone metabolism in human endometrial stromal and gland cells in culture.
      MPA acts at the glucocorticoid receptor in addition to acting on both nPRs and mPRs. HP has much less efficacy at the nPR than the mPR.
      • Blackmore P.F.
      • Beebe S.J.
      • Danforth D.R.
      • Alexander N.
      Progesterone and 17-hyrdroxyprogesterone.
      • Ashley R.L.
      • Clay C.M.
      • Farmerie G.D.
      • et al.
      Cloning and characterization of an ovine intracellular seven transmembrane receptor for progesterone that mediates calcium mobilization.
      • Smith J.L.
      • Kupchak B.R.
      • Garitaonandia I.
      • et al.
      Heterologous expression of human mPR, mPR, and mPR in yeast confirms their ability to function as membrane progesterone receptors.
      • Fernandes M.S.
      • Pierron V.
      • Michalovich D.
      • et al.
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      • Dressings G.E.
      • Goldberg J.E.
      • Charles N.J.
      • et al.
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      The blockade of HP effect by RU486 may not be informative because RU486 can act as an antagonist at mPR and at nPR.
      • Smith J.L.
      • Kupchak B.R.
      • Garitaonandia I.
      • et al.
      Heterologous expression of human mPR, mPR, and mPR in yeast confirms their ability to function as membrane progesterone receptors.
      Additional studies will be necessary to determine whether the differences in inhibition of fetal membrane weakening that is seen with different progestogens are the result of differences in their activity at the progesterone nuclear or membranous receptors, differences in activity at other receptors, or differences in metabolism.
      In summary, our data show that progestogens inhibit TNF- (modeling inflammation) and thrombin- (modeling bleeding) induced fetal membrane weakening. Progestogens also inhibit fetal membrane weakening that is induced by GM-CSF, which is a cytokine that has been shown previously to be a critical intermediate in the pathways of both TNF- and thrombin-induced fetal membrane weakening. Progestogens also inhibit TNF- and thrombin-induced GM-CSF production by choriodecidua. Interestingly, HP, a progestogen with much greater mPR than nPR activity, markedly inhibited both TNF- and thrombin-induced fetal membrane weakening. Based on these findings, we propose that progesterone inhibits inflammation- and bleeding-induced fetal membrane weakening through (1) interaction with nPRs in decidual cells that leads to the inhibition of GM-CSF induction and (2) interaction with mPRs in inhibition of GM-CSF-mediated fetal membrane weakening action. In the context of our in vitro model system, the data also clearly indicate that progestogens are effective on late gestation fetal membrane tissue, which may have implications for current therapeutic regimens for progestogen use in the prevention of PPROM that is associated preterm birth.

      Acknowledgments

      The investigators thank the medical and nursing staffs of MetroHealth Medical Center, Cleveland, OH and Hillcrest Hospital, Mayfield Heights, OH.

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