Expression data from primary culture human myometrial cells

Inflammation plays a central role in many human diseases. Human parturition also resembles an inflammatory reaction, where progesterone (P4) and progesterone receptors (PRs) have already been demonstrated to suppress contraction-associated gene expression. In our previous studies, we have found that the progesterone actions, including progesterone-induced gene expression and progesterone's anti-inflammatory effect, are mediated by PR, GR or both. In this study, we used microarrays (GSE68171) to find P4 and IL-1β responsive genes and IL-1β responsive genes which were repressed by P4. These data may provide a broader view of gene networks and cellular functions regulated by P4 and IL-1β in human myometrial cells. These data will also help us understand the role of PR and GR in human parturition

Curettage after uterine artery embolization combined with methotrexate treatment for caesarean scar pregnancy.

In the present study, we evaluated the diagnosis and management modalities of caesarean scar pregnancy (CSP). Thirty patients diagnosed with CSP were retrospectively studied between February, 2010 and February, 2012. Twenty-five patients were offered prophylactic uterine artery embolization (UAE) and methotrexate (MTX) prior to uterine suction curettage. Five cases were referred from other hospitals where the initial management with uterine suction curettage had resulted in uncontrollable massive haemorrhage, 4 of the cases had UAE and one proceeded immediately to hysterectomy. In the 25 patients treated with prophylactic UAE and MTX, 12 had laparoscopy-guided curettage and 13 had ultrasound-guided curettage without complication. The results showed that the 25 patients with CSP, who received prophylactic UAE and MTX prior to uterine curettage, recovered without complications. Five patients referred from other hospitals, where uterine curettage was the primary procedure, had severe complications including uncontrolled vaginal bleeding and uterine rupture. Four of the five patients were treated successfully with emergency UAE and the remaining patient underwent emergency hysterectomy as ultrasound examination detected significant haemorrhage between the uterus and the bladder. Of the 25 patients who received prophylactic UAE combined with MTX, there were no reports of irregular menstruation or serious adverse effects. Notably, the decrease in serum human chorionic gonadotropin (HCG) levels 3 days post-surgery was greater with ultrasound-guided curettage (84.3±5.5%) than with laparoscopy-guided curettage (76.3±10.2%). In summary, the data suggested that prophylactic UAE with MTX followed by ultrasound-guided curettage is the most effective therapeutic approach in CSP

Cyclic AMP increases COX-2 expression via mitogen-activated kinase in human myometrial cells

Cyclic AMP (cAMP) is the archetypal smooth muscle relaxant, mediating the effects of many hormones and drugs. However, recently PGI2, acting via cAMP/PKA, was found to increase contraction-associated protein expression in myometrial cells and to promote oxytocin-driven myometrial contractility. Cyclo-oxygenase-2 (COX-2) is the rate-limiting enzyme in prostaglandin synthesis, which is critical to the onset and progression of human labour. We have investigated the impact of cAMP on myometrial COX-2 expression, synthesis and activity. Three cAMP agonists (8-bromo-cAMP, forskolin and rolipram) increased COX-2 mRNA expression and further studies confirmed that this was associated with COX-2 protein synthesis and activity (increased PGE2 and PGI2 in culture supernatant) in primary cultures of human myometrial cells. These effects were neither reproduced by specific agonists nor inhibited by specific inhibitors of known cAMP-effectors (PKA, EPAC and AMPK). We then used shRNA to knockdown the same effectors and another recently described cAMP-effector PDZ-GEF1-2, without changing the response to cAMP. We found that MAPK activation mediated the cAMP effects on COX-2 expression and that PGE2 acts through EP-2 to activate MAPK and increase COX-2. These data provide further evidence in support of a dual role for cAMP in the regulation of myometrial function

The study of progesterone action in human myometrial explants

Study hypothesis: Myometrial explants represent a superior model compared with cell culture models for the study of human myometrial progesterone (P4) signalling in parturition. Study finding: Gene expression analysis showed myometrial explants closely resemble the in vivo condition and the anti-inflammatory action of P4 is not lost with labour onset. What is known already: Circulating P4 levels decline before the onset of parturition in most animals, but not in humans. This has led to the suggestion that there is a functional withdrawal of P4 action at the myometrial level prior to labour onset. However, to date, no evidence of a loss of P4 function has been provided, with studies hampered by a lack of a physiologically relevant model. Study design, samples/materials, methods: Myometrial biopsies obtained at Caesarean section were dissected into explants after a portion was immediately snap frozen (t = 0). Microarray analysis was used to compare gene expression of t = 0 with paired (i) explants, (ii) passage 4 myometrial cell cultures or (iii) the hTERT myometrial cell line. Western blotting and chemokine/cytokine assays were used to study P4 signalling in myometrial explants. Main results and the role of chance: Gene expression comparison of t = 0 to the three models demonstrated that explants more closely resemble the in vivo status. At the protein level, explants maintain both P4 receptor (PR) and glucocorticoid receptor (GR) levels versus t = 0 whereas cells only maintain GR levels. Additionally, treatment with 1 μM P4 led to a reduction in interleukin-1 (IL-1) β-driven cyclooxygenase-2 in explants but not in cells. P4 signalling in explants was PR-mediated and associated with a repression of p65 and c-Jun phosphorylation. Furthermore, the anti-inflammatory action of P4 was maintained after labour onset. Limitations/reasons for caution: There is evidence of basal inflammation in the myometrial explant model. Wider implications of the findings: Myometrial explants constitute a novel model to study P4 signalling in the myometrium and can be used to further elucidate the mechanisms of P4 action in human labour. Large scale data: Data deposited at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=gvmpggkurbgxfqf&acc=GSE77830

Chemical profiles and simultaneous quantification of Aurantii fructus by use of HPLC-Q-TOF-MS combined with GC-MS and HPLC methods

Aurantii fructus (AF) is a traditional Chinese medicine that has been used to improve gastrointestinal motility disorders for over a thousand years, but there is no exhaustive identification of the basic chemical components and comprehensive quality control of this herb. In this study, high-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (HPLC-Q-TOF-MS) and gas chromatography coupled mass spectrometry (GC-MS) were employed to identify the basic chemical compounds, and high-performance liquid chromatography (HPLC) was developed to determine the major biochemical markers from AF extract. There were 104 compounds belonging to eight structure types, including 13 amino acids or peptides, seven alkaloids, 18 flavanones, 14 flavones, 15 polymethoxyflavonoids, six triterpenoids, nine coumarins, and 18 volatile oils, as well as four other compounds that were systematically identified as the basic components from AF, and among them, 41 compounds were reported for the first time. Twelve bioactive ingredients were chosen as the benchmark markers to evaluate the quality of AF. The analysis was completed with a gradient elution at a flow rate of 0.7 mL/min within 55 min. This efficient method was validated showing good linearity, precision, stability, repeatability and recovery. Furthermore, the method was successfully applied to the simultaneous determination of 12 chemical markers in different samples of AF. This study could be applied to the identification of multiple bioactive substances and improve the quality control of AF

Interactions between inflammatory signals and the progesterone receptor in regulating gene expression in pregnant human uterine myocytes

The absence of a fall in circulating progesterone levels has led to the concept that human labour is associated with ‘functional progesterone withdrawal’ caused through changes in the expression or function of progesterone receptor (PR). At the time of labour, the human uterus is heavily infiltrated with inflammatory cells, which release cytokines to create a ‘myometrial inflammation’ via NF-κB activation. The negative interaction between NF-κB and PR, may represent a mechanism to account for ‘functional progesterone withdrawal’ at term. Conversely, PR may act to inhibit NF-κB function and so play a role in inhibition of myometrial inflammation during pregnancy. To model this inter-relationship, we have used small interfering (si) RNA-mediated knock-down of PR in human pregnant myocytes and whole genome microarray analysis to identify genes regulated through PR. We then activated myometrial inflammation using IL-1β stimulation to determine the role of PR in myometrial inflammation regulation. Through PR-knock-down, we found that PR regulates gene networks involved in myometrial quiescence and extracellular matrix integrity. Activation of myometrial inflammation was found to antagonize PR-induced gene expression, of genes normally upregulated via PR. We found that PR does not play a role in repression of pro-inflammatory gene networks induced by IL-1β and that only MMP10 was significantly regulated in opposite directions by IL-1β and PR. We conclude that progesterone acting through PR does not generally inhibit myometrial inflammation. Activation of myometrial inflammation does cause ‘functional progesterone withdrawal’ but only in the context of genes normally upregulated via PR