PTGS2 mRNA Expression Research Articles

Eicosanoid pathway expression in bovine endometrial epithelial and stromal cells in response to lipopolysaccharide, interleukin 1 beta, and tumor necrosis factor alpha

During endometrial inflammation, bovine endometrium responds by increasing the production of pro-inflammatory mediators, such as interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNFα), and eicosanoids. The purpose of this study was to establish and characterize an in vitro model of endometrial inflammation using bovine endometrial epithelial (bEEL) and stromal (bCSC) cell lines. We evaluated the effects of the infectious agent (bacterial lipopolysaccharide; LPS) and pro-inflammatory mediators (IL-1β and TNFα) on eicosanoid biosynthesis pathway gene expression and production by bEEL and bCSC cells. Based on concentration-response experiments, the optimal concentrations for responses were 1 μg/mL LPS, 10 ng/mL IL-1β and 50 ng/mL TNFα. Real-time PCR results show that there was an upregulation of relative mRNA expression of PTGS2 when bEEL and bCSC were treated with LPS, IL-1β and TNFα. An increase in PTGES3 expression was observed when bEEL cells were treated with LPS and IL-1β and PTGES2 when treated with IL-1β. In bCSC cells, FAAH relative mRNA was decreased upon treatments. Rate of production of PGE2, PGF2α, PGE2-EA and PGF2α-EA were also determined using liquid chromatography tandem mass spectrometry. Our results show that eicosanoid production was increased in both cell lines in response to LPS, IL-1β, and TNFα. We suggest that the characteristics of bEEL and bCSC cell lines mimic the physiological responses found in mammals with endometrial infection, making them excellent in vitro models for intrauterine environment studies.

Abstract 138: White AdiposeTissue in the Preeclamptic-Like BPH/5 Mouse Has a Unique Inflammatory Molecular Signature During Early Pregnancy

Introduction: Preeclampsia (PE) is a hypertensive disease of pregnancy that affects an estimated 8% of women worldwide. The underlying mechanism is unknown; however, maternal obesity increases the risk of PE by 30% and may contribute to increased inflammation seen in preeclamptic mothers. Hypothesis: To better understand the role of white adipose tissue (WAT) signaling on adverse pregnancy outcomes, we used the obese PE-like BPH/5 mouse to test the hypothesis that visceral WAT has an inflammatory phenotype prior to the presentation of the maternal hypertensive syndrome Methods: Visceral WAT adjacent to the reproductive tract (“reproductive WAT”) was harvested from ad libitum fed control C57 and BPH/5 female mice (8 weeks of age) at embryonic day (e) 7.5. RNA was isolated and subjected to Next Generation Sequencing (NGS) using an Illumina platform. DAVID pathway analysis was performed to annotate differentially expressed genes. BPH/5 female mice were calorie restricted by 25% from e0.5 until e7.5 and reproductive WAT was collected for RNA isolation and quantitative real-time PCR. The calorie restriction paradigm has been shown to significantly reduce reproductive WAT accumulation throughout gestation in BPH/5 mice. Results: NGS of e7.5 reproductive WAT revealed 899 genes that were significantly dysregulated in BPH/5 vs C57. DAVID showed upregulation of lipid metabolic processes in both C57 and BPH/5 pregnant reproductive WAT, while 18 inflammation-related pathways were significantly upregulated in reproductive WAT of pregnant BPH/5 mice compared to pregnant C57 (FDR<=5%, adjP<=0.1, FC >=1.5). Quantitative real-time PCR confirmed upregulation of PE- related inflammatory genes, interleukin (IL)-6, IL-15 and Ptgs2, in e7.5 BPH/5 reproductive WAT vs C57 (n=6, p<0.05). Seven days 25% calorie restriction during pregnancy significantly reduced this elevated IL-6 and Ptgs2 mRNA expression in reproductive WAT (n=6, p<0.05) Conclusions: These data show that obese PE-like BPH/5 mice have elevated inflammatory gene expression in reproductive WAT before placenta formation. Further investigations are needed to confirm if the reduction in IL-6 and Ptgs2 in reproductive WAT by calorie restriction improves fetoplacental and maternal outcomes in BPH/5 mice

New insights into the efficacy of SR-16234, a selective estrogen receptor modulator, on the growth of murine endometriosis-like lesions.

To evaluate the effects of SR-16234 (SR), a selective estrogen receptor modulator (SERM), on murine endometriosis-like lesions. BALB/c mice (n=53) were used to establish the murine endometriosis model. Ovariectomized, estradiol replaced, 6-week-old murine endometriosis model were injected with lipopolysaccharide (LPS) with or without SR (1mg/kg/d) or vehicle, over a period of 4weeks. Upon treatment completion, the endometriosis-like lesions that developed in the abdominal cavity of mice were counted, measured, and collected. Gene expression of inflammatory cytokines and estrogen receptor (ER) in the lesions was assessed by real-time RT-PCR. Immunohistochemical analysis was used to evaluate the effect of SR on cell proliferation, angiogenic activity, inflammation, and NF-κB phosphorylation. Treatment with SR significantly reduced the total number and size of lesions per mouse without inducing endometrial growth. In addition, SR downregulated LPS-enhanced Vegf, Il-6, Ptgs-2, and Ccl-2 and ER mRNA expression in endometriosis-like lesions. Immunohistochemical analysis demonstrated a decrease in percentage of positive cells of Ki67, and intensity and rate of positive cells of ERα, CD3, F4/80, PECAM by SR treatment. SR also decreased the expression of NF-κB p65 and phospho-NF-κB p65. SR has a regressive effect on the development of murine endometriosis-like lesions.

Calorie Restriction In The Preeclamptic‐like BPH/5 Mouse Reduces Adipose Tissue Gene Expression of Preeclampsia‐Related Inflammatory Genes

BackgroundPreeclampsia (PE) is a hypertensive disease of pregnancy that is a leading cause of fetal and maternal morbidity/mortality. Obesity is a risk factor for developing PE, however, the underlying mechanism is unknown. Using the PE‐like BPH/5 mouse model, we previously demonstrated that cyclooxygenase (Cox)‐2 and interleukin (IL)‐15 dysregulation at the maternal‐fetal interface plays a causal role in poor maternal and fetal outcomes. Because BPH/5 mice are obese, we hypothesized that the maternal reproductive white adipose tissue (rWAT) is a source of these pro‐inflammatory molecules, and that reducing rWAT mass by calorie restriction (CR) would decrease the inflammatory uterine milieu in this model.MethodsUsing qRT‐PCR, we measured mRNA expression of Ptgs2 (the gene encoding Cox2), IL‐15 and 18S (housekeeper gene) within the rWAT and implantation sites of non pregnant and pregnant (embryonic day (e) 7.5) ad libitum fed BPH/5 and C57Bl/6J control mice. In addition, we determined Ptgs2 and IL‐15 mRNA expression in BPH/5 rWAT and implantation sites after 7 days CR (pair‐feeding with pregnant C57Bl/6J mice) beginning the day of copulation (e0.5).ResultsPtgs2 expression in non‐pregnant rWAT was comparable between BPH/5 and C57Bl/6J. However, there was a 2‐fold increase in Ptgs2 expression at e7.5 in BPH/5 rWAT versus C57Bl/6J. After CR, Ptgs2 expression in BPH/5 rWAT was significantly lower compared to ad libitum fed BPH/5 mice (p=0.038, n=6). Also in implantation sites, the expression of Ptgs2 was significantly lower in CR as compared to ad libitum fed BPH/5 mice (p=0.046, n=6). IL‐15 expression was 30‐fold higher in BPH/5 rWAT and 8‐fold higher in implantation sites at e7.5 compared to C57Bl/6J control mice (p=0.0015, n=6), but this expression was not significantly affected by CR in either tissue.ConclusionThese data show that 7 days CR in BPH/5 obese mice during early pregnancy reduces the expression of Cox‐2 coding genes in rWAT and at the maternal‐fetal interface, suggesting that WAT‐derived inflammatory mediators might be involved in the development of PE and poor pregnancy outcomes in obese BPH/5 mice. Future studies should investigate the molecular phenotype of WAT in women that are at risk to develop PE, to provide further insight in the mechanisms that make obesity a risk factor and possible interventions to prevent PE.Support or Funding InformationThis study has been funded by the Lousiana Biomedical Collaborative Research Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NOD-like receptor pyrin domain-containing-3 (NLRP3) regulates inflammation-induced pro-labor mediators in human myometrial cells.

Inflammation plays a major role in preterm birth. Nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) plays a role in inflammatory diseases. The aims of this study were to determine the effect of term labor on the expression of NLRP3 in human myometrium and the effect of NLRP3 silencing on pro-labor mediators in myometrial cells. NLRP3 expression was assessed in myometrium from non-laboring and laboring women by qRT-PCR and Western blotting. Human primary myometrial cells were transfected with NLRP3 siRNA (siNLRP3), treated with pro-inflammatory cytokines and toll-like receptor (TLR) ligands, and assayed for pro-inflammatory mediators' expression. NLRP3 expression was higher in myometrium after term spontaneous labor and by TNF, IL1B, fsl-1, and flagellin. In siNLRP3-transfected cells, there was a significant decrease in the expression of pro-inflammatory cytokines (IL1A, IL6), chemokines (CXCL8, CCL2), and adhesion molecules (ICAM1 and VCAM1) stimulated with IL1B, TNF, or TLR ligands; decrease in IL1B-stimulated PTGS2 and PTGFR mRNA expression and PGF2α release; and increase in TNF-stimulated myometrial gel shrinkage as assessed by an in vitro cell contraction assay. NLRP3 is increased with labor in myometrial, and knockdown of NLRP3 is associated with an attenuation of inflammation-induced expression of pro-inflammatory and pro-labor mediators in human myometrium.

Lipopolysaccharide promotes the development of murine endometriosis-like lesions via the nuclear factor-kappa B pathway.

Is lipopolysaccharide (LPS) involved in the development of endometriosis? BALB/c mice (n=69) were used for the murine endometriosis model. Mice with surgically induced endometriosis were injected with LPS intraperitoneally. After 4 weeks of LPS injections with or without the nuclear factor-kappa B (NF-κB) inhibitor, the extent of endometriosis-like lesions was evaluated. Expression of inflammatory factors in the implants was evaluated using real-time RT-PCR. Cell proliferation, angiogenic activity, inflammation, and NF-κB phosphorylation were assessed by immunohistochemical staining. Lipopolysaccharide increased total number, size, and mRNA expression of Ptgs-2, Vegf, Ccl-2, and Il-6 in endometriosis-like lesions. LPS also increased the percentage of Ki67-positive cells and enhanced the intensity and rate of positive cells of CD3, F4/80, and PECAM. Intense expression of phospho-NF-κB p65 after LPS administration was observed. Treatment with the NF-kB inhibitor negated these LPS-induced effects. LPS-induced pelvic inflammation status enhanced the development of murine endometriosis-like lesions via NF-κB pathway.

Preclinical evaluation of drugs to block inflammation-driven preterm birth.

Intrauterine inflammation, the major cause of early preterm birth, can have microbial and sterile aetiologies. We assessed in a Transwell model the anti-inflammatory efficacies of five drugs on human extraplacental membranes delivered after preterm spontaneous labour (30-34 wk). Drugs [TPCA1 (IKKβ inhibitor), 5 z-7-oxozeaenol (OxZ, TAK1 inhibitor), inhibitor of NF-κB essential modulator binding domain (iNBD), SB239063 (p38 MAPK inhibitor) and N-acetyl cysteine (free radical scavenger free radicals)] were added after 12 h equilibration to the amniotic compartment. Concentrations of IL-6, TNF-α, MCP-1, IL-1β and PGE2 in the media, and IL6, TNFA and PTGS2 mRNA expression levels in membranes, were determined after 12 h. Data were analysed using mixed models analyses. Thirteen of the 28 membranes had histological chorioamnionitis (HCA+); five were positive for bacterial culture and six for fetal inflammatory reaction. Baseline PGE2 and cytokine production was similar between HCA- and HCA+ membranes. Anti-inflammatory effects were also similar between HCA- and HCA+ membranes. TPCA1 and OxZ were the most effective drugs; each inhibited amniotic secretion of 4/5 pro-inflammatory mediators and mRNA levels of 2/3, regardless of stimulus. We conclude that treatment with TPCA1 or OxZ, in combination with antibiotics, may minimise the adverse effects of intrauterine inflammation in pregnancy.

A Novel Role for SIRT3 in Regulating Mediators Involved in the Terminal Pathways of Human Labor and Delivery.

Preterm birth remains the major cause of neonatal mortality and morbidity, mediated largely by an inflammatory process. The sirtuin (SIRT) family of cellular regulators has been implicated as key inhibitors of inflammation. We have previously reported a role for SIRT1, SIRT2, and SIRT6 in regulating inflammation-induced prolabor mediators. In this study, we determined the effect of term labor and pro-inflammatory cytokines on SIRT3, SIRT4, SIRT5, and SIRT7 expression in human myometrium. Functional studies were also used to investigate the effect of small interfering RNA (siRNA) knockdown of SIRTs in regulating inflammation-induced prolabor mediators. Western blot analysis and qRT-PCR were used to determine SIRT3, SIRT4, SIRT5, and SIRT7 mRNA and protein expression in human myometrium. Small interfering RNA knockdown of SIRT3 in myometrial primary cells determined its role in response to inflammatory stimuli IL1B and TNF. SIRT3 mRNA and protein expression levels were significantly lower in term laboring myometrium compared with term nonlaboring myometrium. There was no effect of labor on SIRT4, SIRT5 or SIRT7 protein expression. The pro-inflammatory cytokines IL1B and TNF significantly decreased levels of SIRT3 mRNA and protein expression. SIRT3 knockdown by siRNA significantly augmented IL1B- and TNF-stimulated IL6, CXCL8, and CCL2 mRNA expression and release; PTGS2 mRNA expression and subsequent PGF2alpha release; the mRNA expression and secretion of the adhesion molecule ICAM1 and the extracellular matrix remodeling enzyme MMP9; and nuclear factor kappa B1 (NFkappaB1) transcriptional activity. In human myometrium, SIRT3 expression decreases with term labor and regulates the mediators involved in the terminal effector pathways of human labor and delivery through the NFkappaB1 pathway.

The Transcription Factor Interferon Regulatory Factor-1 (IRF1) Plays a Key Role in the Terminal Effector Pathways of Human Preterm Labor.

Preterm birth is the largest single cause of neonatal death and morbidity. By activating cytokine- and Toll-like receptor (TLR)-signaling pathways, infection and/or inflammation are strongly associated with preterm delivery. Interferon regulatory factor-1 (IRF1) is an important regulator of the inflammatory response. The aims of this study were to establish the effect of 1) labor on IRF1 expression in human fetal membranes and myometrium, 2) prolabor mediators on IRF1 expression and activity, and 3) IRF1 small interfering RNA on the expression of prolabor mediators. IRF1 expression was higher in fetal membranes and myometrium after spontaneous term labor and in preterm fetal membranes with infection. The proinflammatory cytokine IL1B, the bacterial product fsl-1, and viral analog polyinosinic:polycytidylic acid (poly [I:C]) significantly increased IRF1 mRNA expression and transcriptional activity in human primary myometrial cells. In addition, IL1B increased IRF1 activity in primary amnion cells. IRF1 silencing in myometrial cells decreased IL1B-, fsl-1-, and poly (I:C)-induced cytokine (IL6, TNF, IL1B) and chemokine (CXCL8, CCL2) mRNA expression and IL6, CXCL8, and CCL2 release. IL1B-, fsl-1-, and poly (I:C)-induced PTGS2 mRNA expression and IL1B-induced prostaglandin release was also decreased by IRF1 silencing. In conclusion, IRF1 upregulation in fetal membranes and myometrium after term labor indicates a proinflammatory role for IRF1 in human parturition. IRF1 is involved in TLR- and cytokine-mediated signaling in human myometrium. These data provide new insights into the mechanisms associated with inflammation- and infection-associated preterm birth. IRF1 inhibitors as therapeutics for the management of spontaneous preterm birth warrants further investigation.

Effects of maternal n-3 fatty acid supplementation on placental cytokines, pro-resolving lipid mediators and their precursors.

The aim of this study was to determine whether supplementation with fish oil-derived n-3 polyunsaturated fatty acids (n-3 PUFA) during pregnancy modifies placental PUFA composition, the accumulation of specialised pro-resolving lipid mediators (SPMs, specifically resolvins (Rv), protectins (PD) and upstream precursors) and inflammatory gene expression. Placentas were collected from women (n=51) enrolled in a randomised, placebo controlled trial of n-3 PUFA supplementation from 20-week gestation. Lipids were extracted for fatty acid analysis and SPMs were quantitated by mass spectrometry. Gene expression was determined by qRT-PCR. Using multiple regression analysis, data were correlated for placental n-3 PUFA and SPM levels with PUFA levels in maternal and cord blood erythrocytes. Supplementation with n-3 PUFAs increased placental docosahexaenoic acid (DHA) levels, but not eicosapentaenoic acid (EPA) levels (P<0.05), and increased the levels of the SPM precursors 18-hydroxyeicosapentaenoic acid and 17-hydroxydocosahexaenoic acid (17-HDHA) by two- to threefold (P<0.0005). RvD1, 17R-RvD1, RvD2 and PD1 were detectable in all placentas, but concentrations were not significantly increased by n-3 PUFA supplementation. Placental DHA levels were positively associated with maternal and cord DHA levels (P<0.005), and with placental 17-HDHA concentrations (P<0.0001). Placental mRNA expression of PTGS2, IL1β, IL6 and IL10 was unaffected by n-3 PUFA supplementation, but TNFα expression was increased by 14-fold (P<0.05). We conclude that n-3 PUFA supplementation in pregnancy i) enhances placental accumulation of DHA and SPM precursors, ii) does not alter placental EPA levels, and iii) has no stimulatory effects on inflammatory gene expression. Further studies are required to ascertain the biological significance of SPMs in the placenta and the potential immunomodulatory effects of elevating placental SPM levels.

Stimulatory effect of fibroblast-derived prostaglandin E₂ on keratinocyte stratification in the skin equivalent.

Epidermal-dermal interaction plays important roles in physiological events such as wound healing. In this study, we examined a double paracrine mechanism between keratinocytes and fibroblasts through interleukin-1 (IL-1) and an IL-1-induced inflammatory mediator prostaglandin E₂ (PGE₂) using the skin equivalent. The epidermal layer of the skin equivalent expressed high levels of IL-1α mRNA (IL1A mRNA) and relatively low levels of IL-1β mRNA (IL1B mRNA). IL1A mRNA was not detected in fibroblasts. Fibroblasts also expressed low but not negligible levels of IL1B mRNA only in the presence of keratinocytes. Expression of prostaglandin-endoperoxide synthase 2 mRNA (PTGS2 mRNA) and production of PGE₂ in three-dimensionally cultured fibroblasts were noticeably stimulated by co-culture with keratinocytes, whereas PTGS2 mRNA expression in the epidermal layer was very low. In addition, hydroxyprostaglandin dehydrogenase 15-(NAD) mRNA was highly expressed in keratinocytes but not in fibroblasts, and exogenous IL-1β stimulated PTGS2 mRNA expression in the dermal equivalent. The thickness of the epidermal layer and the number of MKI67-positive keratinocytes in the skin equivalent were decreased by treatment with indomethacin, and the decrease recovered when exogenous PGE₂ was added. These results indicate that keratinocytes stimulate their own proliferation through a double paracrine mechanism mediated by IL-1 and PGE₂.

Abstract 422: DNA hypermethylation or upregulated miRNA21 expression potentially leads to decreased mRNA expression of COL1A2, SFRP2, SOCS3, BCL2, MAL and PTGS2 in left-sided colorectal adenoma and cancer

Abstract Background: It is well known that beside genetic factors such as mutations, epigenetic mechanisms can also contribute to colorectal cancer formation and progression. Although several genes influenced by DNA methylation and miRNA expression alteration have been identified, our knowledge of the background of gene expression alterations during CRC development remains incomplete. Aims: Our aims were to identify DNA methylation markers and miRNAs playing role in left-sided CRC development on the basis of gene expression alterations along the adenoma-carcinoma formation. Materials &amp; methods: Whole genome expression profiling was performed by using HGU133 Plus 2.0 microarrays (Affymetrix) on healthy colonic (n=49), colorectal adenoma (n=49) and left-sided CRC (n=49) biopsy samples and also on laser microdissected (LCM) epithelial and stromal cells from healthy (n=6) and CRC (n=6) samples. Methylation status of genes with gradually decreasing or increasing expression along the adenoma-carcinoma sequence were analyzed on macrodissected (n=10) and LCM (n=5) healthy colonic, adenomatous biopsy (n=10) and LCM (n=5), macrodissected (n=10) and LCM (n=5) left-sided colorectal cancer samples using bisulfite-sequencing PCR (BS-PCR) followed by pyrosequencing. In silico miRNA prediction for the selected genes with miRWALK algorithm, miRNA expression was analyzed on colorectal cancers (n=3), adenomas (n=3) and normal tissue adjacent to tumor (NAT)(n=3) samples using the Human Panel I + II with the miRCURY Universal RT microRNA PCR protocol (Exiqon). In order to investigate the potential impact of DNA methylation on the protein levels PTGDR and SFRP1 immunohistochemistry experiments were performed. Results: A set of transcripts (18 genes including MAL, SFRP1, SULT1A1, PRIMA1, PTGDR) showed decreasing expression (p≤0,01) in the biopsy samples along the adenoma-carcinoma sequence. COL1A2, SFRP2, SOCS3 showed hypermethylation and THBS2 showed hypomethylation both in the adenomas and tumor samples compared to NAT, while BCL2, PRIMA1 and PTGDR showed hypermethylation only in the CRC group. miR-21 was found to be significantly (p&amp;lt;0,01) upregulated in the adenoma and tumor samples compared to the healthy colonic tissue controls that can potentially influence the expression of genes without remarkable DNA methylation alteration (e.g. BCL2, MAL, PTGS2). Decreasing protein levels of PTGDR and SFRP1 could be observed along the adenoma-carcinoma sequence. Conclusion: Genome-wide gene expression-based screening was found to be a suitable approach for the identification of genes, that can be potentially downregulated by DNA hypermethylation or miRNA upregulation. Hypermethylation of the selected markers (COL1A2, SFRP2, SOCS3) or miR21 upregulation might result in reduced expression and may contribute to the formation of colorectal cancer. Citation Format: Bela Molnar, Balint Peterfia, Alexandra Kalmar, Peter Hollosi, Zsofia Brigitta Nagy, Barnabas Wichmann, Ilona Kovalszky, Zsolt Tulassay. DNA hypermethylation or upregulated miRNA21 expression potentially leads to decreased mRNA expression of COL1A2, SFRP2, SOCS3, BCL2, MAL and PTGS2 in left-sided colorectal adenoma and cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 422. doi:10.1158/1538-7445.AM2014-422

Altering n-3 to n-6 polyunsaturated fatty acid ratios affects prostaglandin production by ovine uterine endometrium

Consumption of n-3 polyunsaturated fatty acids (PUFAs) is considered beneficial to health but effects on fertility remain uncertain. This study investigated the effect of n-3 PUFA supplementation on endometrial prostaglandin (PG) production. Ovine uterine endometrial cells were cultured to confluence in DMEM/F12 medium containing 10% foetal bovine serum. Stromal and epithelial cell populations were confirmed by immunocytochemistry. Cultures were supplemented with 0, 20 or 100μM of α-linolenic acid (ALA), stearidonic acid (SDA), eicosapentaenoic acid (EPA) with lipopolysaccharide (LPS) at 0 and 0.1μg/ml, or different combinations of EPA with arachidonic acid (AA) in serum-free medium for 24h. PGs were quantified using radioimmunoassay and PG-endoperoxide synthase (PTGS) isoforms, PGE and PGF synthase (microsomal PGES1 and PGFS) mRNAs by qPCR. LPS increased PGE2 production significantly without changing PGF2α production, causing increased PGE2:PGF2α ratios. ALA and SDA increased PGE2, PGF2α and PGE2:PGF2α ratios (P<0.05–0.01) while EPA alone did not affect PG generation. AA significantly stimulated PTGS1 and PTGS2 mRNA expression and PGE2 and PGF2α production (P<0.01). The stimulatory effect of AA was attenuated by up to 80% (P<0.05) when AA was combined with EPA. The PGE2:PGF2α ratio was not affected by AA or EPA alone, but increased when these two PUFAs were combined (P<0.05). SDA and EPA decreased PTGS1 mRNA expression (P<0.05) but did not alter PTGS2 expression. EPA and AA up-regulated mPGES1 expression (P<0.05) without affecting PGFS expression. Since AA is preferentially incorporated in uterine endometrium to produce 2-series PGs, alteration of PG production by EPA may affect many reproductive processes.

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus–oocyte complexes

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2.

Association between a functional variant at PTGS2 gene 3′UTR and its mRNA expression in lymphoblastoid cell lines

PTGS2 genetic 3' untranslated region (3'UTR) miRNA binding sites variants are significantly associated with cancer risk; however, the roles of genetic variants in PTGS2 gene 3'UTR and post-transcriptional regulation have not been elucidated. We report that rs689470 and rs5275 in the PTGS2 3'UTR have potential miRNA-binding sites by using bioinformatics analysis. However, only the rs689470 was significantly associated with PTGS2 mRNA expression in lymphoblastoid cell lines (P = 0.026), but not for rs5275 (P = 0.626). rs689470 might be putative variants mediating the post-transcriptional regulation of target PTGS2 gene. Better understanding of how 3'UTR variants regulate PTGS2 activity will pave the way to targeting the PTGS2 pathway in cancer therapy.

Endocrine and inflammatory factors and endometriosis-associated infertility in assisted reproduction techniques

Our research aimed to evaluate the effect of endometriosis on folliculogenesis and pregnancy, and to assess the involvement of inflammatory factors (IL1b, PGE2, PGF2α, and TGFβ2) in follicular fluid. A total of 65 follicular fluid aspirates were collected. Concentrations of inflammatory factors (IL1b, PGE2, PGF2α, and TGFβ2) and steroid hormones (E2, progesterone, FSH, and LH) within follicular fluid as well as serum E2 and LH concentrations were measured. The mRNA expression of IL1b, Ptgs2, aromatase, and PPARγ in granulosa cells was determined. The outcome of ART was monitored and recorded. The oocyte retrieval, rate of metaphase II oocyte, cleavage rate, effective embryo rate, and pregnancy rates of patients with endometriosis were all significantly lower than those of the control patients. In those with endometriosis, serum E2 concentrations were lower than those observed in controls. Aromatase levels in the granulosa cells of the endometriosis group were lower while concentrations of PGE2 in follicular fluid were higher than in the control group. Concentrations of PGE2, PGF2α, TGFβ2, and IL1b were significantly correlated with each other. These results suggest that the outcomes of ART, in relation to serum E2 concentration, were adversely affected by the presence of endometriosis. Furthermore, the results supported that, among the endocrine and inflammatory factors, PGE2 within the follicular fluid impairs the number and quality of oocytes.

Human cumulus cell gene expression as a biomarker of pregnancy outcome after single embryo transfer

To identify the cumulus cell gene expression associated with oocyte developmental competence, specifically live birth, after single ET (SET) assisted reproductive technology. Retrospective gene expression analysis in human cumulus cells from oocytes that established a pregnancy resulting in live birth versus no pregnancy after SET. Independent IVF clinic and research institute. Women undergoing IVF/intracytoplasmic sperm injection with SET. Quantitative reverse-transcriptase-polymerase chain reaction analysis was performed on cumulus masses collected before insemination. Oocytes and embryos were cultured and transferred independently in 38 patients undergoing elective SET. Paired cumulus samples from oocytes that developed into high- versus low-grade embryos also were compared. Gene expression profiles of metabolic (ALDOA, LDHA, PFKP, PKM2), signaling (AHR, GREM1, PTGS2, STS), extracellular matrix (HAS2, PTX3, TNFAIP6, VCAN), and loading control GAPDH in individual cumulus masses. VCAN and PTGS2 mRNA expression was significantly higher in cumulus cells from oocytes yielding a pregnancy resulting in a live birth, while PTX3 mRNA expression trended toward higher expression in pregnant samples. Cumulus cell levels of VCAN, GREM1, and PFKP correlated with birth weight in patients at 38 weeks of gestation. No genes correlated with clinical embryo morphology scores. Cumulus cell VCAN, PTGS2, GREM1, and PFKP expression may identify oocytes with high developmental potential, leading to enhanced implantation rates and greater developmental capacity throughout gestation.

Possible involvement of oxytocin and its receptor in the local regulation of prostaglandin secretion in the cat endometrium

Ovarian originated oxytocin (OT) is involved in several reproductive process, amongst them its role in the regulation/modulation of the estrous cycle in several species has been demonstrated. Although the systemic role of endometrial originated prostaglandins (PGs), especially prostaglandin F 2α (PGF 2α), is equivocal in cats, their possible involvement in the local regulation of uterine events during the estrous cycle is uncertain. We examined the spontaneous and LH-stimulated OT production in cultured luteal cells, the spatial and temporal arrangement of OT receptors (OTR) in a cat endometrium and, finally the effects of OT on PG secretion and prostaglandin-endoperoxide synthase (PTGS2) expression in the feline cultured endometrial cells. Uteri together with ovaries were collected from adult domestic cats ( n = 27) at different stages of the estrous cycle, after routine ovariohisterectomy procedures. The endometrial and luteal cells were separated enzymatically. Luteinizing hormone (LH) augmented OT secretion in cultured luteal cells 2-fold compared with control ( P < 0.05). Oxytocin receptor was abundantly expressed in different ovarian structure, as well as in uterine tissues collected at early/developing and mid-luteal phase. The secretion of PGF 2α by endometrial epithelial cells was increased by OT at a dose 10 −7 M ( P < 0.001). Atosiban (specific OTR blocker) alone did not affect PG secretion but atosiban in combination with OT abolished the stimulating effect of OT on PGF 2α secretion. Oxytocin augmented PGE 2 secretion at a dose 10 −7 M and 10 −6 M in the endometrial stromal cells ( P < 0.001). The treatment with atosiban did not abrogated positive effect of OT on PGE 2 production in the stromal cells. Effect of OT on PTGS2 mRNA expression, the rate-limiting enzyme in PG production, was examined by Real Time-PCR and PTGS2 mRNA expression was significantly affected by OT in both epithelial and stromal cell cultures ( P < 0.01). The present observations have shown that OT is locally produced by the early/developing corpora lutea and that corpora lutea delivered OT may regulate PG secretion in a cat endometrium especially at early- and mid-diestrus, by affecting PTGS2 mRNA expression.

Chemical Composition of Five Commercial Gynostemma pentaphyllum Samples and Their Radical Scavenging, Antiproliferative, and Anti-inflammatory Properties

Five Gynostemma pentaphyllum (GP) samples were investigated and compared for their chemical compositions and their antioxidant, antiproliferative, and anti-inflammatory effects. Extracts (50% acetone, 75% ethanol, and 100% ethanol) of the five GP samples (GP1-5) differed in their total phenolic, saponin, and flavonoid contents and in their rutin and quercetin concentrations. The highest level of total flavonoids was 63.5 mg of rutin equiv/g in GP4, and the greatest total phenolic content was 44.3 mg of gallic acid equiv/g in GP1 with 50% acetone as the extraction solvent. GP2 had the highest total saponin content of 132.6 mg/g with 100% ethanol as the extraction solvent. These extracts also differed in their scavenging capacity against DPPH and hydroxyl radicals, although they all showed significant radical scavenging capacity. The 100% ethanol extracts also showed dose-dependently strong inhibition on IL-6 and Ptgs2 mRNA expression and weak inhibition on TNF-α mRNA expression. In addition, GP1 had the highest antiproliferative activity at 3.2 mg equiv/mL concentration in HT-29 human colon cancer cells. The results from this study will be used to promote the application of G. pentaphyllum for improving human health.

SIRT1 Is a Novel Regulator of Key Pathways of Human Labor1

Human sirtuin (SIRT) 1 and SIRT2, which possess nicotinamide adenosine dinucleotide (NAD(+))-dependent deacetylase activity, exhibit anti-inflammatory actions. However, there are no data available on SIRT1 and SIRT2 expression and regulation in human intrauterine tissues. Thus, the aim of this study was to characterize the localization and expression of SIRT1 and SIRT2 in 1) placenta and fetal membranes before and after term spontaneous labor onset, 2) prelabor fetal membranes from the supracervical site (SCS) and a distal site (DS), and 3) in response to proinflammatory stimuli. Further, the effect of SIRT activation using resveratrol and SRT1720 on prolabor mediators was also assessed. SIRT1 and SIRT2 were localized in the syncytiotrophoblast layer and the cytotrophoblasts of the placenta, amnion epithelium, trophoblast layer of the chorion, and decidual cells. Additionally, SIRT2 was found within the endothelial walls of placental vessels. SIRT2, but not SIRT1, staining was significantly lower in amnion and chorion obtained from the SCS compared to a DS. On the other hand, SIRT1, but not SIRT2, gene and/or protein expression was significantly lower in placenta, amnion, and chorion obtained after labor compared to prelabor. SIRT1 expression, but not SIRT2, was down-regulated by lipopolysaccharide (LPS) and proinflammatory cytokines TNF and IL1B. The SIRT1 activators resveratrol and SRT1720 significantly decreased LPS-induced TNF, IL6, and IL8 gene expression and release and PTGS2 mRNA expression and resultant prostaglandin (PG) E(2) and PGF(2α) release from human gestational tissues. In conclusion, SIRT1 possesses anti-inflammatory actions and thus may play a role in regulating pregnancy and parturition.