α-Lipoic acid blocks the GMCSF induced protease/protease inhibitor spectrum associated with fetal membrane weakening in-vitro.

INTRODUCTION: We use an in-vitro human fetal membrane (FM) explant-based model to study inflammation-induced FM weakening, a prerequisite for PPROM. In this system, GMCSF is a critical intermediate, both necessary and sufficient for TNFα and thrombin induced FM weakening. α-Lipoic-acid (LA) blocks TNFα and thrombin, as well as GMCSF-induced weakening. Recently, we reported LA concomitantly blocks GMCSF-induction of MMPs 2, 9 and 10 and inhibition of TIMPs 1-3. The aim of this study was to show that LA blocks GMCSF-induced increases in additional proteases and reductions in additional protease inhibitors. METHODS: FM fragments were cultured±LA and then±GMCSF. In other experiments, weak versus strong, fresh FM were cultured without additions. Fragments were strength tested and media analyzed by multiplex protein ELISA for proteases and protease inhibitors. RESULTS: GMCSF induced FM weakening and concomitantly increased several Proteases (Cathepsin-S, Proteinase-3, Elastase-2) and decreased several protease inhibitors (NGAL, Cystatin-C, HE4 and Thrombospondin1). LA inhibited GMCSF-induced FM weakening and all enzymatic changes. Untreated weaker versus stronger regions of fresh FM showed comparable differences in proteases and protease inhibitor patterns to GMCSF-stimulated versus controls. CONCLUSION: LA blocks GMCSF-induced human FM weakening and associated protease increases and inhibitor decreases. The GMCSF-induced spectrum of protease/protease-inhibitor changes is similar to that in the natural weak FM fragments. In concert with previously reported GMCSF-induced changes in MMPs & TIMPs, these other protease and protease-inhibitor changes presumably facilitate FM weakening and rupture. LA blocks these GMCSF effects and therefore may be a useful agent to prevent PPROM.

In vitro selenium supplementation suppresses key mediators involved in myometrial activation and rupture of fetal membranes.

Spontaneous preterm birth, which can affect up to 20% of all pregnancies, is the greatest contributor to perinatal morbidity and mortality. Infection is the leading pathological cause of spontaneous preterm birth. Infection activates the maternal immune system, resulting in the upregulation of pro-inflammatory and pro-labor mediators that activate myometrial contractions and rupture of fetal membranes. Anti-inflammatory agents therefore have the potential for the prevention of spontaneous preterm birth. Selenium, an essential micronutrient, has been shown to be a potent anti-inflammatory regulator. Notably, clinical and epidemiological studies have suggested a link between selenium and preterm birth. Thus, the aim of this study was to assess the effect of selenite (an inorganic form of selenium) on the expression of pro-inflammatory and pro-labor mediators in human gestational tissues. Human fetal membranes and myometrium were pre-incubated with or without selenite before incubation with the bacterial product lipopolysaccharide (LPS) to stimulate inflammation associated with preterm birth. Selenite blocked LPS-induced expression of pro-inflammatory cytokines and chemokines and enzymes involved in remodelling of myometrium and degradation of fetal membranes. Of note, selenite also suppressed myometrial activation induced by inflammation as evidenced by a decrease in LPS-induced prostaglandin signalling and myometrial cell contractility. These effects of selenite were mediated by the MAPK protein ERK as selenite blunted LPS induced activation of ERK. In conclusion, selenite suppresses key mediators involved in inflammation induced activation of mediators involved in active labor in human fetal membranes and myometrium. These findings support recent clinical studies demonstrating selenium supplementation is associated with decreased incidence of spontaneous preterm birth.

Effect of n-3 PUFA-rich fish oil supplementation during late gestation on kidding, uterine involution and resumption of follicular activity in goat.

We have shown that dietary supplementation of n-3 polyunsaturated fatty acid (n-3 PUFA)-rich fish oil (FO) around the breeding time improved the utero-ovarian functions in the goat. Here, we investigated the effect of FO supplementation during the periparturient period on serum n-3 PUFA, prostaglandin F2α metabolite (PGFM), placental expulsion, uterine involution, resumption of oestrus and neonatal vigour. Rohilkhandi goat in advanced gestation (n = 16) was divided into two equal groups. One group was supplemented with FO containing 26% n-3 long-chain PUFA at the rate of 156 mg per kg body weight, while the control group was fed isocaloric palm oil (PO) from -3 to +3 week of kidding. Dietary FO increased serum concentration of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) by 7.3- and 6.6-fold, respectively, after 6 weeks of supplementation. Goats in FO group expelled the foetal membranes 99.1 min earlier (p < .01) than those of PO group. Further, dietary FO significantly decreased the serum PGFM on day 7 post-partum. However, no difference was found on uterine involution, which was complete by day 20 post-partum in either group. Resumption of follicular activity by day 5 post-partum was 87.5% in the FO as compared to 25% in the PO group (p < .05). Similarly, occurrence of behavioural oestrus by day 90 post-partum was 57.1% in goats of the FO group while none of does was in the PO group (p < .01) expressed oestrus. It was concluded that feeding FO-rich diet during -3 to +3 weeks of kidding decreased the PGFM till day 7 post-partum, hastened the expulsion of foetal membranes and reduced the time from kidding to first post-partum oestrus in Rohilkhandi does.

Maternal Glucocorticoids Make the Fetal Membrane Thinner: Involvement of Amniotic Macrophages.

Glucocorticoid use during pregnancy is known to increase the risk of preterm birth and preterm premature rupture of membranes (pPROM). Here, we investigated the mechanism of how glucocorticoids weaken the fetal membranes. The amnion mesenchymal layer was significantly thinner in pregnant women treated with prednisolone and in corticosterone-injected mice than in control groups. Matrix metalloproteinase (MMP)-9 mRNA and its activity, COX2 mRNA levels, and prostaglandin E2 synthesis were increased, whereas type 1 collagen (COL1A1) mRNA levels were decreased in the fetal membranes of corticosterone-injected mice. Unexpectedly, the proliferation and migration of macrophages were observed around the corticosterone-injected amnion, and IL-1ß was released from these macrophages. In human amnion mesenchymal cells, cortisol did not change MMP mRNA expression, whereas IL-1ß treatment robustly increased MMP and COX2 mRNA expression. COL1A1 mRNA level was decreased by both cortisol and IL-1ß. These data suggest that the recruitment of amniotic macrophages by glucocorticoids plays a pivotal role in weakening of the fetal membranes, leading to the pathogenesis of pPROM.

Potent anti-inflammatory effects of honokiol in human fetal membranes and myometrium.

BACKGROUND: Preterm birth is the most prominent complication attributing to poor pregnancy and neonatal outcome. Infection is most commonly implicated in preterm birth; it initiates a cascade of inflammatory events that leads to the rupture of fetal membranes and spontaneous uterine contractions. Anti-inflammatory agents may thus be a therapeutic approach to prevent the premature rupture of fetal membranes and block contractions. In non-gestational tissues, the polyphenol honokiol has been shown to possess potent anti-inflammatory properties. PURPOSE: The aim of this study was to investigate the effect of honokiol on pro-inflammatory mediators in human gestational tissues. METHODS: Fetal membranes, myometrium and freshly isolated amnion cells and primary myometrial cells were treated with honokiol in the absence or presence of the products lipopolysaccharide (LPS) and fibroblast-stimulating lipopeptide-1 (fsl-1), the viral dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) or the pro-inflammatory cytokines TNF or IL1B. A luciferase assay was used to determine the effect of honokiol on nuclear factor kappa B (NF-κB) RelA transcriptional activity. RESULTS: Honokiol significantly decreased pro-inflammatory cytokine (IL1A, IL6) and chemokine (CXCL8, CXCL1, CCL2) mRNA expression and secretion from fetal membranes (amnion and choriodecidua) and myometrium stimulated with LPS, fsl-1 or poly(I:C). In amnion cells, honokiol also significantly decreased the expression and secretion of the extracellular matrix degrading enzyme MMP9. Moreover, in myometrium, honokiol significantly suppressed the expression of the contraction associated protein PTGFR, the secretion of the uterotonic prostaglandins PGE2 and PGF2α, and blocked TNF-induced myometrial cell contractility. Finally, honokiol significantly suppressed IL1B- and TNF-induced NF-κB RelA transcriptional activity in primary amnion and myometrial cells. CONCLUSIONS: Honokiol reduced the expression of pro-inflammatory and pro-labour mediators in human amnion, choriodecidua and myometrium and that this may be facilitated through the suppression of NF-κB activation. These results indicate that the polyphenol honokiol may be a potent therapeutic for the prevention of preterm birth.

In an in-vitro model using human fetal membranes, α-lipoic acid inhibits inflammation induced fetal membrane weakening.

INTRODUCTION: We established an in-vitro model for the study of human fetal membrane (FM) weakening leading to pPROM. In this model, granulocyte-macrophage colony-stimulating factor (GM-CSF) is a critical intermediate for both tumor necrosis factor-α (TNF; modeling infection/inflammation) and thrombin (modeling decidual bleeding/abruption)-induced weakening. Thus, inhibitors of FM weakening can be categorized as targeting GM-CSF production, GM-CSF downstream action, or both. Most progestogens inhibit both, except 17-α hydroxyprogesterone caproate which inhibits FM weakening at only one point, GM-CSF production. α-lipoic acid (LA), an over-the-counter dietary supplement, has also been previously shown to inhibit TNF and thrombin induced FM weakening. OBJECTIVE: To determine the point of action of LA inhibition of FM weakening. METHODS: FM fragments were mounted in Transwell inserts and preincubated with/without LA/24 h, then with/without addition of TNF, thrombin or GM-CSF. After 48 h, medium was assayed for GM-CSF, and FM fragments were rupture-strength tested. RESULTS: TNF and thrombin both weakened FM and increased GM-CSF levels. GM-CSF also weakened FM. LA inhibited both TNF and thrombin induced FM weakening and concomitantly inhibited the increase in GM-CSF in a concentration-dependent manner. In addition, LA inhibited GM-CSF induced FM weakening in a concentration dependent manner. CONCLUSIONS: LA blocks TNF and thrombin induced FM weakening at two points, inhibiting both GM-CSF production and downstream action. Thus, we speculate that LA may be a potential standalone therapeutic agent, or supplement to current therapy for prevention of pPROM related spontaneous preterm birth, if preclinical studies to examine feasibility and safety during pregnancy are successfully accomplished.

Progesterone Inhibits Apoptosis in Fetal Membranes by Altering Expression of Both Pro- and Antiapoptotic Proteins.

OBJECTIVE: Progesterone supplementation prevents preterm birth (PTB) in some high-risk women, but its mechanism of action is unknown. One-third of PTB is associated with preterm premature rupture of membranes (PPROMs). We have previously shown that progesterone inhibits basal and Tumor Necrosis Factor (TNF) α-induced apoptosis in an explant model of human fetal membranes. This study investigates the molecular mechanisms responsible for progesterone-mediated inhibition of apoptosis in fetal membranes. METHODS: Human fetal membranes were collected at elective cesarean at term (no labor, no infection; n = 6), washed, and pretreated with/without progesterone (125 ng/mL) for 24 hours. Thereafter, membranes were treated with/without TNFα (50 ng/mL) and/or progesterone for 48 hours, harvested, and homogenized. Apoptosis was determined by evaluating caspase-3, -8, and -9 activities. Expression of pro- BH3 interacting domain death against, Bc1-2 associated X protein (BID, BAX) and antiapoptotic proteins (X-linked inhibitor of apoptosis protein [XIAP], Bcl-2, FLICE inhibitory protein [FLIP]) were measured by Western blot. RESULTS: TNFα increased apoptosis (measured by caspase-3, -8, and -9 activities) in fetal membranes, and this effect was abrogated by progesterone. Under basal conditions, progesterone suppressed expression of the proapoptotic protein, BID, by 0.45 (0.14)-fold, and increased expression of the antiapoptotic proteins, Bcl-2 and XIAP; no change was seen in BAX or FLIP. In contrast, TNFα increased BID expression by 5.15 (2.92)-fold, which was prevented by pretreatment with progesterone. CONCLUSIONS: Progesterone inhibits apoptosis in fetal membranes by suppressing expression of the proapoptotic protein, BID (for both basal and TNFα-induced apoptosis), and upregulating expression of the antiapoptotic proteins, XIAP and Bcl-2 (under basal conditions only). These data provide a mechanism by which progesterone supplementation may prevent PPROM and PTB in some women at high risk.

Inhibition of gingipains prevents Porphyromonas gingivalis-induced preterm birth and fetal death in pregnant mice.

Accumulating epidemiological evidence indicates that infection with Porphyromonas gingivalis which is a major periodontal pathogen, causes preterm birth and low birth weight. However, virulence factors of P. gingivalis responsible for preterm birth/low birth weight remain to be elucidated. In this study, using P. gingivalis-infected pregnant mice as an in vivo model, we investigated whether gingipains-cysteine proteinases produced by P. gingivalis-affect preterm birth and low birth weight. We found that intravenous infection of pregnant mice with P. gingivalis induced higher accumulation of the bacterium in the placenta than that in other organs. Compared to infection with P. gingivalis wild-type, infection with a gingipain-deficient P. gingivalis mutant KDP136 led to significant reduction in preterm birth and pregnancy loss. Although repetitive low-level infections of P. gingivalis failed to induce preterm birth and fetal death, it induced suppressive effects on IFN-γ production. Therapeutically, treatment with ginginpain inhibitors prevented fetal death and preterm birth caused by P. gingivalis infection and resulted in recovery of IFN-γ suppression caused by repetitive chronic P. gingivalis infection. These results indicate that gingipains are major virulence factors of P. gingivalis responsible for preterm birth/low birth, and gingipain inhibitors may be useful not only as a therapeutic agent for periodontal diseases, but also as a preventive medicine for preterm birth/low birth weight.

In an in-vitro model using human fetal membranes, 17-α hydroxyprogesterone caproate is not an optimal progestogen for inhibition of fetal membrane weakening.

BACKGROUND: The progestogen 17-α hydroxyprogesterone caproate (17-OHPC) is 1 of only 2 agents recommended for clinical use in the prevention of spontaneous preterm delivery, and studies of its efficacy have been conflicting. We have developed an in-vitro model to study the fetal membrane weakening process that leads to rupture in preterm premature rupture of the fetal membranes (pPROM). Inflammation/infection associated with tumor necrosis factor-α (TNF-α) induction and decidual bleeding/abruption associated thrombin release are leading causes of preterm premature rupture of the fetal membranes. Both agents (TNF-α and thrombin) cause fetal membrane weakening in the model system. Furthermore, granulocyte-macrophage colony-stimulating factor (GM-CSF) is a critical intermediate for both TNF-α and thrombin-induced fetal membrane weakening. In a previous report, we demonstrated that 3 progestogens, progesterone, 17-alpha hydroxyprogesterone (17-OHP), and medroxyprogesterone acetate (MPA), each inhibit both TNF-α- and thrombin-induced fetal membrane weakening at 2 distinct points of the fetal membrane weakening pathway. Each block both the production of and the downstream action of the critical intermediate granulocyte-macrophage colony-stimulating factor. OBJECTIVE: The objective of the study was to characterize the inhibitory effects of 17-OHPC on TNF-α- and thrombin-induced fetal membrane weakening in vitro. STUDY DESIGN: Full-thickness human fetal membrane fragments from uncomplicated term repeat cesarean deliveries were mounted in 2.5 cm Transwell inserts and cultured with/without 17-alpha hydroxyprogesterone caproate (10-9 to 10-7 M). After 24 hours, medium (supernatant) was removed and replaced with/without the addition of tumor necrosis factor-alpha (20 ng/mL) or thrombin (10 U/mL) or granulocyte-macrophage colony-stimulating factor (200 ng/mL). After 48 hours of culture, medium from the maternal side compartment of the model was assayed for granulocyte-macrophage colony-stimulating factor and the fetal membrane fragments were rupture strength tested. RESULTS: Tumor necrosis factor-alpha and thrombin both weakened fetal membranes (43% and 62%, respectively) and increased granulocyte-macrophage colony-stimulating factor levels (3.7- and 5.9-fold, respectively). Pretreatment with 17-alpha hydroxyprogesterone caproate inhibited both tumor necrosis factor-alpha- and thrombin-induced fetal membrane weakening and concomitantly inhibited the induced increase in granulocyte-macrophage colony-stimulating factor in a concentration-dependent manner. However, contrary to our prior reports regarding progesterone and other progestogens, 17-alpha hydroxyprogesterone caproate did not also inhibit granulocyte-macrophage colony-stimulating factor-induced fetal membrane weakening. CONCLUSION: 17-Alpha hydroxyprogesterone caproate blocks tumor necrosis factor-alpha- and thrombin-induced fetal membrane weakening by inhibiting the production of granulocyte-macrophage colony-stimulating factor. However, 17-alpha hydroxyprogesterone caproate did not also inhibit granulocyte-macrophage colony-stimulating factor-induced weakening. We speculate that progestogens other than 17-alpha hydroxyprogesterone caproate may be more efficacious in preventing preterm premature rupture of the fetal membranes-related spontaneous preterm birth.

Inhibition of PIM1 kinase attenuates inflammation-induced pro-labour mediators in human foetal membranes in vitro.

STUDY QUESTION: Does proviral integration site for Moloney murine leukaemic virus (PIM)1 kinase play a role in regulating the inflammatory processes of human labour and delivery? SUMMARY ANSWER: PIM1 kinase plays a critical role in foetal membranes in regulating pro-inflammatory and pro-labour mediators. WHAT IS KNOWN ALREADY: Infection and inflammation have strong causal links to preterm delivery by stimulating pro-inflammatory cytokines and collagen degrading enzymes, which can lead to rupture of membranes. PIM1 has been shown to have a role in immune regulation and inflammation in non-gestational tissues; however, its role has not been explored in the field of human labour. STUDY DESIGN, SIZE, DURATION: PIM1 expression was analysed in myometrium and/or foetal membranes obtained at term and preterm (n = 8-9 patients per group). Foetal membranes, freshly isolated amnion cells and primary myometrial cells were used to investigate the effect of PIM1 inhibition on pro-labour mediators (n = 5 patients per treatment group). PARTICIPANTS/MATERIALS, SETTING AND METHODS: Foetal membranes, from term and preterm, were obtained from non-labouring and labouring women, and from preterm pre-labour rupture of membranes (PPROM) (n = 9 per group). Amnion was collected from women with and without preterm chorioamnionitis (n = 8 per group). Expression of PIM1 kinase was determined by qRT-PCR and western blotting. To determine the effect of PIM1 kinase inhibition on the expression of pro-inflammatory and pro-labour mediators induced by bacterial products lipopolysaccharide (LPS) (10 µg/ml) and flagellin (1 µg/ml) and pro-inflammatory cytokine tumour necrosis factor (TNF) (10 ng/ml), chemical inhibitors SMI-4a (20 µM) and AZD1208 (50 µM) were used in foetal membrane explants and siRNA against PIM1 was used in primary amnion cells. Statistical significance was set at P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: PIM1 expression was significantly increased in foetal membranes after spontaneous term labour compared to no labour at term and in amnion with preterm chorioamnionitis compared to preterm with no chorioamnionitis. There was no change in PIM1 expression with preterm labour or PPROM compared to preterm with no labour or PPROM. In human foetal membranes, PIM1 inhibitors SMI-4a and AZD1208 significantly decreased the expression of pro-inflammatory cytokine interleukin-6 (IL6) and chemokines CXCL8 and CCL2 mRNA and release, prostaglandin prostaglandin F2α (PGF2α) release, adhesion molecule intercellular adhesion molecule 1 mRNA expression and release, and oxidative stress marker 8-isoprostane release after stimulation with either LPS or flagellin. Primary amnion cells transfected with PIM1 siRNA also showed decreased expression of IL6, CXCL8 and CCL2, PTGS2 mRNA and PGF2α release, and matrix metalloproteinase-9 (MMP9) expression, when stimulated with TNF. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: The conclusions were drawn from in vitro experiments using foetal membrane explants and primary cells isolated from amnion. Animal models are necessary to determine whether PIM1 kinase inhibitors can prevent spontaneous preterm birth in vivo. WIDER IMPLICATIONS OF THE FINDINGS: PIM1 kinase inhibitors may provide a novel therapeutic approach for preventing spontaneous preterm birth. STUDY FUNDING/COMPETING INTEREST(S): Associate Professor Martha Lappas is supported by a Career Development Fellowship from the National Health and Medical Research Council (NHMRC; grant no. 1047025). Funding for this study was provided by the NHMRC (grant no. 1058786), Norman Beischer Medical Research Foundation and the Mercy Research Foundation. The authors have no conflict of interest.

Restored in vivo-like membrane lipidomics positively influence in vitro features of cultured mesenchymal stromal/stem cells derived from human placenta.

BACKGROUND: The study of lipid metabolism in stem cell physiology has recently raised great interest. The role of lipids goes beyond the mere structural involvement in assembling extra- and intra-cellular compartments. Nevertheless, we are still far from understanding the impact of membrane lipidomics in stemness maintenance and differentiation patterns. In the last years, it has been reported how in vitro cell culturing can modify membrane lipidomics. The aim of the present work was to study the membrane fatty acid profile of mesenchymal stromal cells (MSCs) derived from human fetal membranes (hFM-MSCs) and to correlate this to specific biological properties by using chemically defined tailored lipid supplements (Refeed®). METHODS: Freshly isolated hFM-MSCs were characterized for their membrane fatty acid composition. hFM-MSCs were cultivated in vitro following a classical protocol and their membrane fatty acid profile at different passages was compared to the profile in vivo. A tailored Refeed® lipid supplement was developed with the aim of reducing the differences created by the in vitro cultivation and was tested on cultured hFM-MSCs. Cell morphology, viability, proliferation, angiogenic differentiation, and immunomodulatory properties after in vitro exposure to the tailored Refeed® lipid supplement were investigated. RESULTS: A significant modification of hFM-MSC membrane fatty acid composition occurred during in vitro culture. Using a tailored lipid supplement, the fatty acid composition of cultured cells remained more similar to their in vivo counterparts, being characterized by a higher polyunsaturated and omega-6 fatty acid content. These changes in membrane composition had no effect on cell morphology and viability, but were linked with increased cell proliferation rate, angiogenic differentiation, and immunomodulatory properties. In particular, Refeed®-supplemented hFM-MSCs showed greater ability to express fully functional cell membrane molecules. CONCLUSIONS: Culturing hFM-MSCs alters their fatty acid composition. A tailored lipid supplement is able to improve in vitro hFM-MSC functional properties by recreating a membrane environment more similar to the physiological counterpart. This approach should be considered in cell therapy applications in order to maintain a higher cell quality during in vitro passaging and to influence the outcome of cell-based therapeutic approaches when cells are administered to patients.

All-trans retinoic acid promotes wound healing of primary amniocytes through the induction of LOXL4, a member of the lysyl oxidase family.

Thirty percent of preterm births directly result from preterm premature rupture of fetal membranes (PPROM). Clinical management currently proposes using a collagen plug to mechanically stop loss of amniotic fluid. Vitamin A and its active metabolite (retinoic acid) have well-known pro-healing properties and could thus make good candidates as a proposable adjuvant to this mechanical approach. Here we investigate the molecular mechanisms involved in the pro-healing properties of all-trans retinoic acid (atRA) in fetal membranes via an approach using an in vitro primary amniocyte wound model and transcriptomics. The results demonstrate that atRA promotes migration in primary amniocytes, improving wound healing in vitro by up to 90%. This effect is mediated by the induction of LOXL4, which plays a crucial role in the dynamics of the extracellular matrix by regulating collagen reticulation. This new insight into how atRA exerts its pro-healing properties prompts us to propose using atRA as a candidate strategy to help prevent future PPROM.

High Mobility Group-Box 1 (HMGB1) levels are increased in amniotic fluid of women with intra-amniotic inflammation-determined preterm birth, and the source may be the damaged fetal membranes.

BACKGROUND: High Mobility Group Box-1 (HMGB1) is considered a prototype alarmin molecule. Upon its extracellular release, HMGB1 engages pattern recognition receptors and the Receptor for Advanced Glycation End-products (RAGE) followed by an outpouring of inflammatory cytokines, including interleukin (IL)-6. METHODS: We assayed the amniotic fluid (AF) levels of HMGB1 and IL-6 in 255 women that either had a normal pregnancy outcome or delivered preterm. Immunohistochemistry on fetal membranes was used for cellular localization and validation of immunoassay findings. HMGB1 also was analyzed in amniochorion tissue explants subjected to endotoxin. RESULTS: AF HMGB1 levels are not gestational age regulated but are increased in women with intra-amniotic inflammation and preterm birth. The likely source is the damaged amniochorion, as demonstrated by immunohistochemistry and explant experiments. CONCLUSIONS: Our research supports a role for HMGB1 in the inflammatory response leading to preterm birth. As a delayed phase cytokine, in utero exposure to elevated AF HMGB1 levels may have an impact on the newborn beyond the time of birth.

Progesterone, Inflammatory Cytokine (TNF-α), and Oxidative Stress (H2O2) Regulate Progesterone Receptor Membrane Component 1 Expression in Fetal Membrane Cells.

Progesterone receptor membrane component 1 (PGRMC1) is an important novel mediator of progesterone (P4) function in fetal membrane cells. We demonstrated previously that PGRMC1 is differentially expressed in fetal membranes among pregnancy subjects and diminished in preterm premature rupture of membrane subjects. In the current study, we aim to elucidate whether PGRMC1 expression is regulated by P4, tumor necrosis factor α (TNF-α), and H2O2 in fetal membrane cells. Primary cultured membrane cells were serum starved for 24 hours followed by treatments of P4, 17 hydroxyprogesterone caproate, and medroxyprogesterone 17 acetate (MPA) at 10(-7) mol/L with ethanol as vehicle control; TNF-α at 10, 20, and 50 ng/mL with phosphate-buffered saline (PBS) as control; and H2O2 at 10 and 100 µmol/L with culture media as control for 24, 48, and 72 hours. The messenger RNA (mRNA) and protein expression of PGRMC1 was quantified using polymerase chain reaction and Western blotting, respectively. We found that PGRMC1 protein expression was regulated by MPA, TNF-α, and H2O2 in a dose-dependent manner. This regulation is also specific to the type of cell (amnion, chorion, or decidua). The upregulation of PGRMC1 by MPA might be mediated through glucocorticoid receptor (GR) demonstrated using amnion and chorion cells model with GR knockdown by specific small interfering RNA transfection. The mRNA expression of PGRMC1 was decreased by H2O2 (100 µmol/L) treatment in amnion cells, which might ultimately result in downregulation of PGRMC1 protein as our data demonstrated. None of other treatments changed PGRMC1 mRNA level in these cells. We conclude that these stimuli act as regulatory factors of PGRMC1 in a cell-specific manner.

Exposure of chick embryos to cadmium changes the extra-embryonic vascular branching pattern and alters expression of VEGF-A and VEGF-R2.

Cadmium (Cd) has several industrial applications, and is found in tobacco products, a notable source of human exposure. Vascular endothelial cells are key targets of Cd toxicity. Here, we aim to quantify the alteration to vascular branching pattern following Cd exposure in the chick extra-embryonic membrane (EEM) using fractal analysis, and explore molecular cues to angiogenesis such as VEGF-A and VEGF-R2 expression following Cd treatment. Chicken embryos were incubated for 60 h to Hamburger-Hamilton developmental stage 16-17, then explanted and treated with 50 µL of 50 µmol cadmium acetate (CdAc) or an equivalent volume of equimolar sodium acetate (NaAc). Images of embryos and their area vasculosa (AV) were captured and analyzed at 4 different time points (4, 8, 24 and 48 h) following treatment. Vascular branching in the AV was quantified using its fractal dimension (Df), estimated using a box counting method. Gallinaceous VEGF ELISA was used to measure the VEGF-A concentration in the EEM following treatment, with determination of the relative expression of VEGF-A and VEGF-R2 using quantitative real-time RT-PCR. Vascular branching increased monotonically in the control group at all time points. The anti-angiogenic effect of Cd exposure on the AV was reflected by a significant reduction in Df when compared with controls. Df was more markedly reduced in cultures with abnormal embryos. The expression of VEGF-A protein, and VEGF-A and VEGF-R2 mRNA were reduced in Cd-exposed EEMs. Both molecules contribute to growth, vessel sprouting and branching processes, which supports our findings using fractal analysis.

Effect of oleic acid supplementation on prostaglandin production in maternal endometrial and fetal allantochorion cells isolated from late gestation ewes.

INTRODUCTION: Elevated circulating non-esterified fatty acids including oleic acid (OA) are associated with many pregnancy related complications. Prostaglandins (PGs) play crucial roles during parturition. We investigated the effect of OA supplementation on PG production using an in vitro model of ovine placenta. METHODS: Maternal endometrium (ME) and fetal allantochorion (FC) were collected in late pregnancy (day 135). Confluent cells were cultured in serum-free medium supplemented with 0, 20 or 100 µM OA and challenged with control medium, oxytocin (OT, 250 nM), lipopolysaccharide (LPS, 0.1 µg/ml) or dexamethasone (DEX, 5 µM). Spent medium was harvested at 2 and 24 h after challenge for quantifying PGs. RESULTS: In ME cells OA increased PGE2 production moderately but attenuated PGF2α production leading to a doubling of the PGE2:PGF2α ratio (E:F) (P < 0.01). Without OA, both OT and LPS stimulated PG production for about 3-fold (P < 0.01) without changing the E:F ratio. In the ME cells challenged with OT, OA decreased both PGE2 and PGF2α production by up to 70% (P < 0.01) whereas in LPS treated cells OA increased the E:F ratio. In FC cells PGE2 production at 2 h was stimulated by 100 µM OA (P < 0.05). In these cells LPS caused a 3-fold increase in PGE2 (P < 0.01), an effect which was completely inhibited by DEX. DISCUSSION: OA supplementation favours basal PGE2 production in both ME and FC. In ME OA increased E:F ratios and antagonized the stimulatory effect of OT on PG production. This suggests that raised circulating OA may affect both the initiation and progression of parturition.

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

Activation of AMPK in human fetal membranes alleviates infection-induced expression of pro-inflammatory and pro-labour mediators.

INTRODUCTION: In non-gestational tissues, the activation of adenosine monophosphate (AMP)-activated kinase (AMPK) is associated with potent anti-inflammatory actions. Infection and/or inflammation, by stimulating pro-inflammatory cytokines and matrix metalloproteinase (MMP)-9, play a central role in the rupture of fetal membranes. However, no studies have examined the role of AMPK in human labour. METHODS: Fetal membranes, from term and preterm, were obtained from non-labouring and labouring women, and after preterm pre-labour rupture of membranes (PPROM). AMPK activity was assessed by Western blotting of phosphorylated AMPK expression. To determine the effect of AMPK activators on pro-inflammatory cytokines, fetal membranes were pre-treated with AMPK activators then stimulated with bacterial products LPS and flagellin or viral dsDNA analogue poly(I:C). Primary amnion cells were used to determine the effect of AMPK activators on IL-1ß-stimulated MMP-9 expression. RESULTS: AMPK activity was decreased with term labour. There was no effect of preterm labour. AMPK activity was also decreased in preterm fetal membranes, in the absence of labour, with PROM compared to intact membranes. AMPK activators AICAR, phenformin and A769662 significantly decreased IL-6 and IL-8 stimulated by LPS, flagellin and poly(I:C). Primary amnion cells treated with AMPK activators significantly decreased IL-1ß-induced MMP-9 expression. DISCUSSION: The decrease in AMPK activity in fetal membranes after spontaneous term labour and PPROM indicates an anti-inflammatory role for AMPK in human labour and delivery. The use of AMPK activators as possible therapeutics for threatened preterm labour would be an exciting future avenue of research.

Evidence of an immunosuppressive effect of progesterone upon in vitro secretion of proinflammatory and prodegradative factors in a model of choriodecidual infection.

OBJECTIVE: To evaluate whether progesterone (P4) is able to modulate the secretion of tumour necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), IL-6, IL-8, IL-10 and matrix metalloproteinase-9 (MMP-9) after choriodecidual stimulation with lipopolysaccharide (LPS). DESIGN: Chorioamnionitis-elicited preterm delivery is associated with an uncontrolled secretion of proinflammatory cytokines that may induce MMPs, which modify the fine immunological and structural equilibrium at the fetal-maternal interface. SETTING: Instituto Nacional de Perinatología 'Isidro Espinosa de los Reyes', Mexico City. SAMPLE: Twelve human fetal membranes at term from healthy patients were placed in a two-chamber culture system. METHODS: Choriodecidual and amniotic regions were preincubated with 1.0, 0.1, or 0.01 µmol/l P4 for 24 hours; after which the choriodecidual region was costimulated with 1000 ng/ml of LPS for 24 hours. MAIN OUTCOME MEASURES: Descriptive statistics were obtained for each variable. Data distribution was tested for normality using Kolmogorov-Smirnoff and Shapiro-Wilk tests. When distribution was normal, Student's t test was used to analyse for differences among groups. Mann-Whitney's U test was used when data were not normally distributed. RESULTS: Pretreatment with 1.0 µmol/l P4 significantly blunted the secretion of TNF-α, IL-1ß, IL-6, IL-8 and IL-10. MMP-9 was inhibited with 0.1 µmol/l P4. Mifepristone (RU486) blocked the immunosuppressive effect of P4, suggesting a P4 effect mediated by its receptor. CONCLUSION: These results offer evidence to support the concept that P4 can protect the fetal-placental unit through a compensatory mechanism that partially limits the secretion of proinflammatory and prodegradative modulators.