Expression In Myometrium Research Articles

20α-Hydroxysteroid Dehydrogenase Expression in the Human Myometrium at Term and Preterm Birth: Relationships to Fetal Sex and Maternal Body Mass Index

The mechanism by which human labor is initiated in the presence of elevated circulating progesterone levels remains unknown. Recent evidence indicates that the progesterone-metabolizing enzyme, 20α-hydroxysteroid dehydrogenase (20α-HSD), encoded by the gene AKR1C1, may contribute to functional progesterone withdrawal. We found that AKR1C1 expression significantly increased with labor onset in term myometrium, but not in preterm myometrium. Among preterm laboring deliveries, clinically diagnosed chorioamnionitis was associated with significantly elevated AKR1C1 expression. AKR1C1 expression positively correlated with BMI before labor and negatively correlated with BMI during labor. Analysis by fetal sex showed that AKR1C1 expression was significantly higher in women who delivered male babies compared to women who delivered female babies at term, but not preterm. Further, in pregnancies where the fetus was female, AKR1C1 expression positively correlated with the mother’s age and BMI at the time of delivery. In conclusion, the increase in myometrial AKR1C1 expression with term labor is consistent with 20α-HSD playing a role in local progesterone metabolism to promote birth. Interestingly, this role appears to be specific to term pregnancies where the fetus is male. Upregulated AKR1C1 expression in the myometrium at preterm in-labor with clinical chorioamnionitis suggests that increased 20α-HSD activity is a mechanism through which inflammation drives progesterone withdrawal in preterm labor. The link between AKR1C1 expression and maternal BMI may provide insight into why maternal obesity is often associated with dysfunctional labor. Higher myometrial AKR1C1 expression in male pregnancies may indicate fetal sex-related differences in the mechanisms that precipitate labor onset at term.

Integrating Transcriptomic and ChIP-Seq Reveals Important Regulatory Regions Modulating Gene Expression in Myometrium during Implantation in Pigs

The myometrium is the outer layer of the uterus. Its contraction and steroidogenic activities are required for embryo implantation. However, the molecular mechanisms underlying its functions remain unknown in pigs. The myometrium includes the inner circular muscle (CM) and the outer longitudinal muscle (LM) layers. In this study, we collected the CM and LM samples from the mesometrial side (named M) of the uterus on days 12 (pre-implantation stage) and 15 (implantation stage) of pregnancy and day 15 of the estrous cycle. The transcriptomic results revealed distinct differences between the uterine CM and LM layers in early pregnancy: the genes expressed in the LM layer were mainly related to contraction pathways, whereas the transcriptional signatures in the CM layer on day 15 of pregnancy were primarily involved in the immune response processes. Subsequent comparisons in the CM layer between pregnant and cyclic gilts show that the transcriptional signatures of the CM layer are implantation-dependent. Next, we investigated the genome-wide profiling of histone H3 lysine 27 acetylation (H3K27ac) and histone H3 lysine 4 trimethylation (H3K4me3) in pig uterine CM and LM layers. The genomic regions that had transcriptional activity and were associated with the expression of genes in the two layers were characterized. Taken together, the regulatory regions identified in the study may contribute to modulating the gene expression in pig uterine CM and LM layers during implantation.

Regulation of uterine function during estrous cycle, anestrus phase and pregnancy by steroids in red deer (Cervus elaphus L.)

Steroid synthesis and production in ruminant uterus is not obvious, especially in seasonally reproduced. We compared steroid production by investigating enzymes involved in red deer uterine steroid metabolism in reproductive seasons. Blood and uteri (endometrium and myometrium) were collected post mortem from hinds on 4th day (N = 8), 13th day of the cycle (N = 8), anestrus (N = 8) and pregnancy (N = 8). The expression of cytochrome P450 aromatase (P450), 3 -beta-hydroxysteroid dehydrogenase (3β-HSD), 17 -beta-hydroxysteroid dehydrogenase (17β-HSD), aldo–keto reductase family 1 C1 (AKR1C1), estrogen receptor alpha (ERα), and progesterone receptors (PRs), were analyzed using real-time-PCR and Western Blotting. Plasma samples were assayed for 17-beta-estradiol (E2), progesterone (P4), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T4) concentrations by EIA. Hinds at the beginning of the estrous cycle, mainly in endometrium, were characterized by a high mRNA expression of 3β-HSD, AKR1C1, PRs and ERα, contrary to the expression in myometrium during pregnancy (P < 0.05). For P4, E2, and FSH, concentration was the highest during the 13th day of the estrous cycle (P < 0.05). Uterine steroid production and output in hinds as a representative seasonally reproduced ruminant occurred mainly during the estrous cycle and sustained in anestrus.

Dual agonist–antagonist effect of ulipristal acetate in human endometrium and myometrium

ABSTRACT The aim of this study was to assess the molecular effect of ulipristal acetate (UPA) on gene expression in myometrium and endometrium of patients with symptomatic fibroids. Tissues isolated from four women treated preoperatively with UPA (5 mg) were compared to those from untreated controls using NanoString platform to assess the expression of 75 candidate genes modulated by UPA and ovarian steroids. Deregulated genes were then validated by real-time PCR. In myometrium, UPA exerted an antagonistic effect similar to that observed in fibroids. In UPA-treated endometrium, six genes were identified as highly and significantly upregulated, including matricellular genes CCN1 (54-fold, P = 0.0018) and CCN2 (11-fold, P = 0.00044), Krüppel-like factor 4 (>3-fold, P = 0.0036), and mast cell markers including tryptases TPSAB1/TPSB2 (31-fold, P = 0.023) and carboxypeptidase A (CPA3, 17-fold, P = 0.05). In endometrium, UPA induced the expression of genes involved in fibrogenesis and mast cell function—some of them being widely involved in hepatic injury, which could explain the marked fibrosis and inflammatory cell infiltration observed in explanted livers from patients under UPA treatment.

Regulatory Influence of Galanin and GALR1/GALR2 Receptors on Inflamed Uterus Contractility in Pigs.

Uterine inflammation is a very common and serious pathology in domestic animals, the development and progression of which often result from disturbed myometrial contractility. We investigated the effect of inflammation on the protein expression of galanin (GAL) receptor subtypes (GALR)1 and GALR2 in myometrium and their role in the contractile amplitude and frequency of an inflamed gilt uterus. The gilts of the E. coli and SAL groups received E. coli suspension or saline in their uteri, respectively, and only laparotomy was performed (CON group). Eight days later, the E. coli group developed severe acute endometritis and lowered GALR1 protein expression in the myometrium. Compared to the pretreatment period, GAL (10−7 M) reduced the amplitude and frequency in myometrium and endometrium/myometrium of the CON and SAL groups, the amplitude in both stripes and frequency in endometrium/myometrium of the E. coli group. In this group, myometrial frequency after using GAL increased, and it was higher than in other groups. GALR2 antagonist diminished the decrease in amplitude in myometrium and the frequency in endometrium/myometrium (SAL, E. coli groups) induced by GAL (10−7 M). GALR1/GALR2 antagonist and GAL (10−7 M) reversed the decrease in amplitude and diminished the decrease in frequency in both examined stripes (CON, SAL groups), and diminished the drop in amplitude and abolished the rise in the frequency in the myometrium (E. coli group). In summary, the inflammation reduced GALR1 protein expression in pig myometrium, and GALR1 and GALR2 participated in the contractile regulation of an inflamed uterus.

TBX2, a Novel Regulator of Labour.

Background and Objectives: Therapeutic interventions targeting molecular factors involved in the transition from uterine quiescence to overt labour are not substantially reducing the rate of spontaneous preterm labour. The identification of novel rational therapeutic targets are essential to prevent the most common cause of neonatal mortality. Based on our previous work showing that Tbx2 (T-Box transcription factor 2) is a putative upstream regulator preceding progesterone withdrawal in mouse myometrium, we now investigate the role of TBX2 in human myometrium. Materials and Methods: RNA microarray analysis of (A) preterm human myometrium samples and (B) myometrial cells overexpressing TBX2 in vitro, combined with subsequent analysis of the two publicly available datasets of (C) Chan et al. and (D) Sharp et al. The effect of TBX2 overexpression on cytokines/chemokines secreted to the myometrium cell culture medium were determined by Luminex assay. Results: Analysis shows that overexpression of TBX2 in myometrial cells results in downregulation of TNFα- and interferon signalling. This downregulation is consistent with the decreased expression of cytokines and chemokines of which a subset has been previously associated with the inflammatory pathways relevant for human labour. In contrast, CXCL5 (C-X-C motif chemokine ligand 5), CCL21 and IL-6 (Interleukin 6), previously reported in relation to parturition, do not seem to be under TBX2 control. The combined bioinformatical analysis of the four mRNA datasets identifies a subset of upstream regulators common to both preterm and term labour under control of TBX2. Surprisingly, TBX2 mRNA levels are increased in preterm contractile myometrium. Conclusions: We identified a subset of upstream regulators common to both preterm and term labour that are activated in labour and repressed by TBX2. The increased TBX2 mRNA expression in myometrium collected during a preterm caesarean section while in spontaneous preterm labour compared to tissue harvested during iatrogenic preterm delivery does not fit the bioinformatical model. We can only explain this by speculating that the in vivo activity of TBX2 in human myometrium depends not only on the TBX2 expression levels but also on levels of the accessory proteins necessary for TBX2 activity.

Identification and validation of suitable reference genes for quantitative real-time PCR gene expression analysis in pregnant human myometrium

Accurate quantification of quantitative PCR (qPCR) data requires a set of stable reference genes (RGs) for normalisation. Despite its importance to mechanistic studies, no evaluation of RG stability has been conducted for pregnant human myometrium. A systematic search of the literature was performed to identify the most used RGs in human myometrial gene expression studies. The stability of these genes, and others, was then evaluated using geNorm and NormFinder algorithms, in samples of myometrium from singleton or twin pregnancies (n = 7 per group) delivering at term or preterm. The most frequently cited RGs were GAPDH, ACTB, B2M and 18s. There was strong agreement between algorithms on the most and least stable genes: Both indicated CYC1, YWHAZ and ATP5B were the most stably expressed. Despite being some of the most used RGs, B2M, 18s and ACTB expression was least stable and was too variable for use as accurate normalisation factors. Pairwise variation analysis determined that the optimal number of RGs for accurate normalisation is two. Validation of the choice of RGs by comparing relative expression of oxytocin receptors (OXTR) using the least stable 18s and B2M, with the most stable, CYC1 and YWHAZ, erroneously demonstrated significantly increased OXTR expression in myometrium in singleton pregnancies compared to twins. This study demonstrates the importance of appropriate RG selection for accurate quantification of relative expression in pregnant human myometrium qPCR studies. For normalisation, the geometric mean of CYC1 and YWHAZ or ATP5B is suggested. The use of ACTB, 18s and B2M, is not recommended.

The role of somatostatin and its receptors (sstr2, sstr5) in the contractility of gilt inflamed uterus

We studied the inflammation effect on somatostatin receptors subtypes 2 (sstr2) and 5 (sstr5) expression in myometrium and somatostatin influence alone or with sstr2 and sstr5 antagonists on the contractility of gilt inflamed uterus. On day 3 of the estrous cycle, either E.coli suspension (E.coli group) or saline (SAL group) were injected into uterine horns. In the control pigs (CON group), only laparotomy was performed. Eight days later, in the E.coli group developed severe acute endometritis. In this group, myometrial sstr2 mRNA expression lowered and protein expression increased compared to other groups. Compared to period before somatostatin administration, somatostatin did not change tension in myometrium and endometrium/myometrium of three groups, reduced amplitude and frequency in the CON and SAL groups, and increased amplitude and decreased frequency in the E.coli group. In this group, amplitude was increased by somatostatin compared to other groups. In the CON and SAL groups, sstr2 eliminated inhibitory somatostatin effect on amplitude, while sstr5 antagonist reversed inhibitory somatostatin effect on amplitude. In the E.coli group, sstr2 antagonist reversed stimulatory somatostatin effect on amplitude, while in sstr5 antagonist presence stimulatory somatostatin effect was more deepened compared to somatostatin action alone. After using sstr2 antagonist more deepened inhibitory somatostatin effect on frequency in the CON and E.coli groups was found. Sstr5 antagonist partly eliminated inhibitory somatostatin effect on frequency in the SAL group. Summarizing, the uterine inflammation increases the myometrial sstr2 protein expression; somatostatin raises amplitude of the inflamed uterus acting by sstr2, while drops this parameter by sstr5.

Inflammation changes the expression of neuropeptide Y receptors in the pig myometrium and their role in the uterine contractility.

In the experiment the influence of inflammation on neuropeptide Y (NPY) receptor subtype 1 (Y1Rs) and 2 (Y2Rs) expression pattern in the gilt myometrium and NPY effect alone or with Y1Rs and Y2Rs antagonists on contractility of an inflamed uterus were recognized. On Day 3 of the estrous cycle, either E.coli suspension (E.coli group) or saline (SAL group) were administered into uterine horns. In the control gilts (CON group), only laparotomy was carried out. Eight days later, E.coli treatment evoked severe acute endometritis, significantly reduced Y1Rs mRNA and protein expression and increased Y2Rs protein expression in myometrium in relation to the CON and SAL groups. Compared to period before NPY application, NPY (10−7 M) significantly reduced amplitude in myometrium and endometrium/myometrium and frequency in myometrium of the CON and SAL groups and amplitude in endometrium/myometrium and frequency in strips of the E.coli group. In this group, after using NPY (10−7 M), the amplitude rose significantly in both kinds of strips, while frequency fell in endometrium/myometrium in relation to the SAL group. In the CON and SAL groups, NPY (10−7 M) with Y1Rs antagonist significantly increased the amplitude in endometrium/myometrium, while with Y2Rs antagonist caused a rise in myometrium. In the E.coli group after using Y1Rs or Y2Rs antagonist and NPY (10−7 M) amplitude did not significantly change in endometrium/myometrium, and this parameter was significantly lower after using the NPY and Y1Rs antagonist than in other groups. Y1Rs antagonist and NPY (10−8, 10−7 M) significantly increased frequency in endometrium/myometrium of the CON and SAL groups. In the E.coli group, Y1Rs or Y2Rs antagonists and NPY (10−7 M) significantly increased frequency in myometrium and it was significantly higher than in the CON group. Inflammation changes Y1Rs and Y2Rs expression in myometrium of the inflamed pig uterus and NPY reduces this organ contractility by Y1Rs and Y2Rs.

Downregulates of ICAM1 expression in Myometrium from pregnant Rattus novergicus infected with Tachyzoite of Toxoplasma gondii with Hyperbaric Oxygen Therapy

&lt;p align="center"&gt;&lt;strong&gt;Abstract&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Background:&lt;/strong&gt; To find the effect of Hyperbaric Oxygen Therapy in the myometrium expressions of ICAM1 from pregnant Rattus norvegicus infected with Tachyzoite &lt;em&gt;Toxoplasma gondii.&lt;/em&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Material and Methods :&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;This is an experimental study with a post-test only design on 37 pregnant &lt;em&gt;Rattus norvegicus&lt;/em&gt; &lt;em&gt;Sprague Dawley&lt;/em&gt;, then the rats were divided into 4 groups. The HBOT group A is pregnant rats infected with tachyzoite received therapy 10 sessions of HBOT 2.4 ATA in 3x30 minutes; the group B is Pregnant only and get no HBOT; the group C is Pregnant and infected with &lt;em&gt;tachyzoite T.gondii&lt;/em&gt; but not received HBOT, and the group D is normal pregnant rats. Each infected pregnant rats were given a 10&lt;sup&gt;3 &lt;/sup&gt;Tachyzoite of &lt;em&gt;Toxoplasma gondii&lt;/em&gt; via intraperitoneal. Examinations of ICAM1 expressions were performed on day-5 after HBOT (twice a day). Rats were killed and the myometrial ICAM1 levels were measured with Immunohistochemistry examination. All data were analyzed with the ANOVA test with SPSS 21 Program.&lt;strong&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results: &lt;/strong&gt;The results show that Hyperbaric Oxygen Therapy can downregulate ICAM1 in the administered of HBO 2.4 ATA for 3x30 minutes in 10 sessions over 5 days of therapy.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusion:&lt;/strong&gt; The study concludes that HBOT can downregulate the expressions of ICAM1 in the myometrium, in the provision of HBO 2.4 ATA for 3x30 minutes in 10 sessions.&lt;/p&gt;

Influence of cycle stage, age and endometrial biopsy score on oxytocin receptor distribution and gene expression in the cervix and uterus of non-pregnant mares

Persistent breeding-induced endometritis (PBIE) or delayed uterine clearance (DUC) are major causes of mare subfertility. Oxytocin and its receptor are thought to play significant roles in the pathogenesis of DUC but the specific roles of oxytocin receptor (OR) distribution and gene expression remain undefined.In this study both OR distribution and gene expression in the endometrium, myometrium and cervix during both luteal and non-luteal phases in non-pregnant mares (n = 27) of differing age (young: 2–9 years, n = 17; old: > 10 years, n = 10) and endometrial biopsy score were described using immunohistochemistry (IHC) and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively.Immunohistochemistry showed a similar pattern of OR distribution in uterus and cervix, with the exception of the glandular epithelium, absent in the cervix. Uterine ORs were localized in endometrial luminal and glandular epithelia, transmural vascular endothelium, sub-epithelial and peri-glandular stromal cells and myometrial smooth muscle cells. The OR labeling intensity was consistently greatest in the vascular endothelium. Real-time qPCR showed a higher OR gene expression in myometrium compared to cervix (P = 0.001) and endometrium (P = 0.009). There was no difference in OR gene expression between cervix and endometrium (P = 1.0). Oxytocin receptor gene expression was significantly higher during the non-luteal phase in both combined uterine tissues (endometrium and myometrium) and myometrium. Oxytocin receptor distribution and gene expression were not influenced by a mare's age or endometrial biopsy score. As endometrial biopsy score and mare age were not predictors of OR gene expression, deficient OR gene expression is unlikely to be associated with DUC.

Expression of alpha and beta adrenergic receptors in the pig uterus during inflammation

Under physiological conditions, noradrenaline (NA) and adrenergic receptors (ARs) are implicated in the function of the uterus. The role of NA and the expression of ARs in the inflamed uterus is not fully understood. The aim of the present study was to determine the effect of inflammation on the levels of α1 (A, B, D)-, α2 (A, B, C)- and β (1, 2, 3)-ARs mRNA and protein expression and the localization of these receptors in the porcine uterus. On Day 3 of the estrous cycle (Day 0 of the study), 50 ml of either saline (group SAL) or E. coli suspension (109 colony-forming units/ml, group E. coli) were injected into each uterine horn. In the control pigs (group CON), only laparotomy was performed. Eight days later, α1D-ARs mRNA (P < 0.001) and protein (P < 0.05) levels and α2A-ARs protein level (P < 0.05) were increased in the inflamed endometrium, while the α2C-ARs protein level (P < 0.001) was lowered, as compared to the SAL and CON groups. In the inflamed endometrium, β2-ARs mRNA (P < 0.01) and protein (CON: P < 0.01, SAL: P < 0.001) expression was lower than in the other two groups, and β1-ARs mRNA (P < 0.001) and protein (P < 0.01) expression was higher compared to the SAL group. After bacterial treatment, α2A- (P < 0.001) and α2B (P < 0.05) -ARs protein levels and β2-ARs mRNA (CON: P < 0.01, SAL: P < 0.05) and protein (CON: P < 0.01, SAL: P < 0.05) expression in myometrium were found to be increased compared to both groups. In turn, in myometrium following E. coli infusion, the α2C-ARs protein level was lower (P < 0.01) than in the CON group. All studied receptors were present in the luminal and glandular epithelium, blood vessels and myometrial muscular cells of the gilt uteri in the E. coli, SAL and CON groups. The data show that inflammation changes the ARs expression in porcine uterus, suggesting their importance in the course/consequences of uterine inflammation. Those affected ARs may constitute a therapeutic target in an inflamed uterus.

Insemination Dose and Site With Frozen Semen Affects the Sperm Transport and Inflammatory Response in Mares?

Persistent breeding-induced endometritis (PBIE) or delayed uterine clearance (DUC) are major causes of mare subfertility. Oxytocin and its receptor are thought to play significant roles in the pathogenesis of DUC but the specific roles of oxytocin receptor (OR) distribution and gene expression remain undefined.In this study both OR distribution and gene expression in the endometrium, myometrium and cervix during both luteal and non-luteal phases in non-pregnant mares (n = 27) of differing age (young: 2–9 years, n = 17; old: > 10 years, n = 10) and endometrial biopsy score were described using immunohistochemistry (IHC) and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively.Immunohistochemistry showed a similar pattern of OR distribution in uterus and cervix, with the exception of the glandular epithelium, absent in the cervix. Uterine ORs were localized in endometrial luminal and glandular epithelia, transmural vascular endothelium, sub-epithelial and peri-glandular stromal cells and myometrial smooth muscle cells. The OR labeling intensity was consistently greatest in the vascular endothelium. Real-time qPCR showed a higher OR gene expression in myometrium compared to cervix (P = 0.001) and endometrium (P = 0.009). There was no difference in OR gene expression between cervix and endometrium (P = 1.0). Oxytocin receptor gene expression was significantly higher during the non-luteal phase in both combined uterine tissues (endometrium and myometrium) and myometrium. Oxytocin receptor distribution and gene expression were not influenced by a mare's age or endometrial biopsy score. As endometrial biopsy score and mare age were not predictors of OR gene expression, deficient OR gene expression is unlikely to be associated with DUC.

Pellino 1 is a novel regulator of TNF and TLR signalling in human myometrial and amnion cells

Preterm birth is the primary cause of neonatal deaths and morbidities. Pathological processes causally linked to preterm birth are inflammation and infection. Pellino-1 (Peli1) has previously been found to regulate the inflammatory response in non-gestational tissues in response to toll-like receptor (TLR) ligands and pro-inflammatory cytokines. The aims of this study were to determine the effect of labor on Peli1 expression in myometrium and fetal membranes, and the effect of Peli1 silencing by siRNA (siPELI1) on the production of pro-inflammatory and pro-labor mediators. The expression of Peli1 was found to be higher in myometrium and fetal membranes with term labor, compared to non-laboring samples. Peli1 mRNA and protein expression was also higher in amnion from women with preterm histological chorioamnionitis. In human primary myometrial cells, siPELI1 transfected cells showed a decrease in pro-inflammatory cytokine IL6, chemokines (CXCL8, CCL2) and adhesion molecule ICAM1 when in the presence of pro-inflammatory cytokine TNF, TLR2/6 ligand fsl-1, TLR5 ligand flagellin, and TLR3 ligand poly(I:C). Similarly in primary amnion cells, siPELI1 transfected cells decreased IL1B-induced expression and secretion of IL6 and CXCL8. In siPELI1 transfected myometrial cells, there was a decrease in prostaglandin PGF2α and its receptor, PTGFR mRNA expression when treated with TNF. There was a decrease in NF-κB RELA transcriptional activity in siPELI1 transfected cells in the presence of TNF, fsl-1 and flagellin, but not poly(I:C). Our study suggests a novel role for Peli1 in regulating pro-inflammatory and pro-labor mediators through TNF and TLR signalling.

Runt-related transcription factor 1 (RUNX1) deficiency attenuates inflammation-induced pro-inflammatory and pro-labour mediators in myometrium.

Identifying new targets that regulate myometrial activation are required to develop effective treatments to stop preterm labor. Inflammation, which can be induced by sterile or infective insults, plays a role in initiating and maintaining uterine contractions. Several high throughput transcription screening studies have identified an upregulation of runt-related transcription factor 1 (RUNX1) mRNA expression in myometrium with labor. The role of RUNX1 in labor, however, is not known. We report increased RUNX1 during late gestation which was further augmented in labor, suggesting that RUNX1 may be involved in the transition of the myometrium from a quiescent into a contractile state in preparation for labor. By inhibiting the expression of RUNX1, we have established that RUNX1 induces the expression of pro-inflammatory cytokines, chemokines, adhesion molecules, contraction-associated proteins OXR and PTGFR, the uterotonic PGF2α, and the ECM remodelling enzyme MMP9. Targeting RUNX1 may be a novel approach to prevent preterm labor.

DREAM Is Involved in the Genesis of Inflammation-Induced Prolabour Mediators in Human Myometrial and Amnion Cells

Preterm birth is the primary cause of perinatal morbidity and mortality worldwide. Inflammation induces a cascade of events leading to preterm birth by activating nuclear factor-κB (NF-κB). In nongestational tissues, downstream regulatory element antagonist modulator (DREAM) regulates NF-κB activity. Our aims were to analyse DREAM expression in myometrium and fetal membranes obtained at term and preterm and to determine the effect of DREAM inhibition on prolabour mediators in primary myometrial and amnion cells. DREAM mRNA expression was significantly higher in fetal membranes obtained after spontaneous labour compared to nonlabour and in amnion from women with histological preterm chorioamnionitis when compared to amnion from women without chorioamnionitis. In primary myometrial and amnion cells, the effect of DREAM silencing by siRNA was a significant decrease in the expression of proinflammatory cytokine IL-6, the chemokines IL-8 and MCP-1, the adhesion molecule ICAM-1, MMP-9 mRNA expression and activity, and NF-κB transcriptional activity when stimulated with the proinflammatory cytokine IL-1β, the bacterial products fsl-1 or flagellin, or the viral dsRNA analogue poly(I:C). These data suggest that, in states of heightened inflammation, DREAM mRNA expression is increased and that, in myometrial and amnion cells, DREAM regulates proinflammatory and prolabour mediators which may be mediated via NF-κB.

Seipin deficiency leads to defective parturition in mice.

Seipin is an integral endoplasmic reticulum membrane protein encoded by Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2/Bscl2) gene. Seipin deficiency results in lipodystrophy, diabetes, muscle hypertrophy, and male infertility in both human and mouse. Seipin function in female reproduction is unknown. Bscl2-/- dams had normal embryo implantation and body weight gain during pregnancy but reduced delivery rates from 2nd to 4th pregnancies and reduced numbers of pups delivered from 1st to 4th pregnancies. Characterization of first pregnancy revealed increased gestation period and parturition problems, including uterine prolapse, difficulty in delivery, undelivered fetuses, and undelivered tissues in Bscl2-/- females. Bscl2-/- uterine weight was comparable to control at 3 weeks old but significantly increased with myometrial hypertrophy at 10 months old. In situ hybridization revealed relatively low level of Bscl2 mRNA expression in myometrium throughout pregnancy and postpartum but high level of expression in uterine luminal epithelium, suggesting that systemic effect (e.g. elevated glucose and insulin levels) rather than local seipin-deficiency in myometrium might be a main contributing factor to myometrial hypertrophy. On near-term gestation day 18.5 (D18.5), Bscl2-/- females had normal levels of serum progesterone and 17β-estradiol, indicating functional ovary and placenta. Proliferating Cell Nuclear Antigen (PCNA) staining showed minimal myometrial cell proliferation in both D18.5 Bscl2+/+ and Bscl2-/- uteri. There was strong LC3 immunostaining in Bscl2+/+ and Bscl2-/- peripartum myometrium and increased LC3 staining in Bscl2-/- peripartum uterine luminal epithelium, suggesting a potential role of seipin in regulating autophagy in uterine luminal epithelium but not myometrium. This study demonstrates an association of seipin with myometrium and parturition.

SMAD7 regulates proinflammatory and prolabor mediators in amnion and myometrium.

Preterm birth continues to be a significant public health problem. Infection (bacterial and or viral) and inflammation, by stimulating proinflammatory cytokines, adhesion molecules, and matrix metalloproteinase 9 (MMP9), play a central role in the rupture of membranes and myometrial contractions. SMAD7 has been implicated in regulating the inflammatory response; however, no studies have been performed with regard to human labor. In this study, we determined the effect of spontaneous human labor and prolabor mediators on SMAD7 expression in myometrium and fetal membranes. Functional studies were employed to investigate the effect of siRNA knockdown of SMAD7 (siSMAD7) in regulating infection and inflammation-induced prolabor mediators. SMAD7 mRNA and protein expression were significantly higher with spontaneous term labor, compared to no labor, in myometrium and fetal membranes. SMAD7 expression was also significantly higher in amnion from women with preterm chorioamnionitis. The proinflammatory cytokines IL1B and TNF, the bacterial product fsl-1, and the viral dsRNA analog poly(I:C) significantly increased SMAD7 in myometrial cells and amnion cells. In myometrial cells, siSMAD7 cells significantly decreased cytokine (IL6) and chemokine (CXCL1, CXCL8, CCL2 are also known as GRO-alpha, interleukin (IL)-8 and monocyte chemotactic protein-1 (MCP-1)) production induced by IL1B, TNF, and fsl-1. There was also a decrease in the expression of adhesion molecules intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) in siSMAD7 cells, and MMP9 expression. In amnion, siSMAD7 cells treated with IL1B also decreased cytokine and chemokine production, ICAM1 and MMP9 expression. In conclusion, we report a proinflammatory role for SMAD7 in human gestational tissues, with SMAD7 silencing attenuating the inflammatory response.

The Adaptor Protein p62 Mediates Nuclear Factor κB Activation in Response to Inflammation and Facilitates the Formation of Prolabor Mediators in Human Myometrium.

Preventing spontaneous preterm birth is one of the most important issues facing perinatal medicine today. The pathophysiology of preterm labor, the single biggest cause of preterm birth, is poorly understood. Inflammation, however, plays a significant role in the terminal processes of human labor, which include myometrial contractions. Nuclear factor κB (NF-κB) drives the transcription of proinflammatory mediators involved in the terminal effector pathways of human labor and delivery. Recent studies in nongestational tissues have shown that the adaptor protein p62 interacts with NF-κB to induce inflammation. The aim of this study was to determine the role of p62 in the genesis of NF-κB-induced proinflammatory and prolabur mediators. Human spontaneous term labor was associated with increased p62 messenger RNA (mRNA) and protein expression in myometrium. Myometrial cells treated with proinflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor alpha (TNF-α) also significantly increased p62 mRNA and protein expression. Functional studies using p62 small interfering RNA (siRNA) demonstrated a significant attenuation of TNF-α- and IL-1β-induced proinflammatory cytokines (IL-6) and chemokine (IL-8 and monocyte chemoattractant protein 1 [MCP-1]) mRNA expression and secretion, expression of cyclooxygenase 2, release of prostaglandin F2α (PGF2α), and expression of the prostaglandin F receptor (FP). In addition, siRNA knockdown of p62 significantly suppressed IL-1β- and TNF-α-induced NF-κB activation. Collectively, these studies suggest that p62 is involved in the genesis of NF-κB-induced proinflammatory and prolabor mediators.

Lactate produced during labor modulates uterine inflammation via GPR81 (HCA1)

Uterine inflammatory processes trigger prolabor pathways and orchestrate on-time labor onset. Although essential for successful labor, inflammation needs to be regulated to avoid uncontrolled amplification and resolve postpartum. During labor, myometrial smooth muscle cells generate ATP mainly via anaerobic glycolysis, resulting in accumulation of lactate. Aside from its metabolic function, lactate has been shown to activate a G protein-coupled receptor, GPR81, reported to regulate inflammation. We therefore hypothesize that lactate produced during labor may act via GPR81 in the uterus to exert in a feedback manner antiinflammatory effects, to resolve or mitigate inflammation. We sought to investigate the role of lactate produced during labor and its receptor, GPR81, in regulating inflammation in the uterus. We investigated the expression of GPR81 in the uterus and the pharmacological role of lactate acting via GPR81 during labor, using shRNA-GPR81 and GPR81-/- mice. (1) Uterine lactate levels increased substantially from 2 to 9 mmol/L during labor. (2) Immunohistological analysis revealed expression of GPR81 in the uterus with high expression in myometrium. (3) GPR81 expression increased during gestation, and peaked near labor. (4) In primary myometrial smooth muscle cell and exvivo uteri from wild-type mice, lactate decreased interleukin-1β-induced transcription of key proinflammatory Il1b, Il6, Ccl2, and Pghs2; suppressive effects of lactate were not observed in cells and tissues from GPR81-/- mice. (5) Conversely, proinflammatory gene expression was augmented in the uterus at term in GPR81-/- mice and wild-type mice treated intrauterine with lentiviral-encoded shRNA-GPR81; GPR81 silencing also induced proinflammatory gene transcription in the uterus when labor was induced by endotoxin (lipopolysaccharide). (6) Importantly, administration to pregnant mice of a metabolically stable specific GPR81 agonist, 3,5-dihydroxybenzoic acid, decreased endotoxin-induced uterine inflammation, preterm birth, and associated neonatal mortality. Collectively, our data uncover a novel link between the anaerobic glycolysis and the control of uterine inflammation wherein the high levels of lactate produced during labor act on uterine GPR81 to down-regulate key proinflammatory genes. This discovery may represent a novel feedback mechanism to regulate inflammation during labor, and conveys a potential rationale for the use of GPR81 agonists to attenuate inflammation and resulting preterm birth.