Expression of epithelial neutrophil-activating peptide 78 in cultured human endometrial stromal cells

It has been demonstrated that human endometrial stromal cells (ESC) produce a variety of chemokines in vivo and in vitro. To evaluate the expression of epithelial neutrophil-activating peptide 78 (ENA-78) in the endometrium, concentrations of ENA-78 in cyclic endometrial tissues were measured using enzyme-linked immunosorbent assay. The expression of ENA-78 was also detected in cyclic endometrium by immunohistochemistry. Endometrial tissues in the secretory phase contained higher amounts of ENA-78 protein than did those in the proliferative phase. Immunofluorescence staining revealed that ENA-78 protein was localized mainly in the stroma of endometrium. In addition, to evaluate the involvement of inflammatory mediators and ovarian steroid hormones in the production of ENA-78 by ESC was evaluated by in-vitro studies. Unstimulated ESC constitutively secreted ENA-78. Progesterone, lipopolysaccharide, tumour necrosis factor-alpha, and interleukin-1beta significantly stimulated the expression of ENA-78 by ESC. It is suggested that the production of ENA-78 by ESC is regulated by progesterone as well as by the inflammatory mediators. The modulation of ENA-78 concentration in the local environment by these mediators may contribute to the normal and pathological processes of human reproduction through regulation of leukocyte trafficking into the endometrium.     

Expression of interferon-gamma-inducible protein-10 in human endometrial stromal cells

Human endometrial stromal cells (ESC) can produce a variety of chemokines, especially after inflammatory stimulation. Interferon-gamma-inducible protein-10 (IP-10) is a potent chemoattractant for lymphocytes, and belongs to the family of non-ELR CXC chemokines. The expression of IP-10 in ESC after stimulation with interferon-gamma (IFN-gamma), interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), or lipopolysaccharide (LPS) was evaluated using an enzyme-linked immunosorbent assay and Northern blot analysis. A small amount of IP-10 protein was detected in the culture media of unstimulated ESC. The expression of IP-10 mRNA was detected in ESC. IFN-gamma, IL-1 beta, TNF-alpha and LPS significantly stimulated the expression of IP-10 mRNA and protein in ESC. These results suggest that the production of IP-10 by ESC is regulated by inflammatory mediators. The modulation of IP-10 concentrations in the local environment may contribute to the normal and pathological processes of human reproduction by regulating leukocyte trafficking in the endometrium.     

Expression of macrophage inflammatory protein-3alpha in an endometrial epithelial cell line,HHUA, and cultured human endometrial stromal cells

It has been demonstrated that human endometrial epithelial cells (EEC) and stromal cells (ESC) produce a variety of chemokines in vivo and in vitro. To evaluate the expression of macrophage inflammatory protein (MIP)-3alpha in endometrial cells, the production of MIP-3alpha by an EEC line, HHUA, and cultured ESC stimulated with various inflammatory mediators was evaluated by ELISA. Unstimulated HHUA and ESC constitutively secreted MIP-3alpha. Tumour necrosis factor-alpha and interleukin-1beta significantly stimulated the secretion of MIP-3alpha by HHUA and ESC. Lipopolysaccharide also stimulated the secretion of MIP-3alpha by ESC, but not by HHUA. These results show that the concentration of MIP-3alpha in the endometrium is modulated by these inflammatory mediators. MIP-3alpha may contribute to the normal and pathological processes of human reproduction by regulating the trafficking of immature dendritic cells and memory T lymphocytes into the endometrium.     

17β-雌二醇和二噁英对子宫内膜间质细胞表达趋化因子IL-8及其受体CXCR1的调控作用

解析内外环境因素对子宫内膜间质细胞表达IL-8及其自分泌作用的调控。采用免疫组化法比较子宫内膜异位症患者异位灶和在位内膜CXCR1翻译水平表达;流式细胞术分析17β-雌二醇和二噁英单独或联合作用对子宫内膜间质细胞表面CXCR1表达的调控作用;ELISA法分析17β-雌二醇和二噁英单独或联合作用对子宫内膜间质细胞分泌IL-8的影响。结果显示CXCR1在子宫内膜异位症患者异位灶组织高表达。17β-雌二醇和二噁英单独作用均抑制子宫内膜间质细胞表面CX-CR1的表达以及IL-8的分泌。二者联合作用能够上调CXCR1的表达,上调幅度与雌二醇浓度呈正相关;但进一步抑制了IL-8的分泌。雌激素与二噁英对子宫内膜间质细胞复合作用抑制其IL-8的分泌及其自分泌作用;子宫内膜异位症患者腹腔液高水平IL-8并非由内外雌激素样物质直接作用于异位灶子宫内膜间质细胞所致。     

17β-雌二醇和二噁英对子宫内膜间质细胞表达趋化因子IL-8及其受体CXCR1 的调控作用

解析内外环境因素对子宫内膜间质细胞表达IL-8及其自分泌作用的调控。采用免疫组化法比较子宫内膜异位症患者异位灶和在位内膜CXCRI翻译水平表达；流式细胞术分析17β-雌二醇和二嗯英单独或联合作用对子宫内膜间质细胞表面CXCRI表达的调控作用；ELISA法分析17β-雌二醇和二嗯英单独或联合作用对子宫内膜间质细胞分泌IL-8的影响。结果显示CXCRI在子宫内膜异位症患者异位灶组织高表达。17β-雌二醇和二嗯英单独作用均抑制子宫内膜间质细胞表面CX—CR1的表达以及IL-8的分泌。二者联合作用能够上调CXCR1的表达，上调幅度与雌二醇浓度呈正相关；但进一步抑制了IL-8的分泌。雌激素与二嗯英对子宫内膜间质细胞复合作用抑制其IL-8的分泌及其自分泌作用；子宫内膜异位症患者腹腔液高水平IL-8并非由内外雌激素样物质直接作用于异位灶子宫内膜间质细胞所致。     

Progesterone induces the fibulin-1 expression in human endometrial stromal cells

BACKGROUND: By using microarray analysis with human endometrial stromal cells (ESCs), we previously reported that the mRNA for fibulin-1, an extracellular matrix as well as a plasma glycoprotein, is up-regulated by progesterone. In the present study, we tried to clarify the spatial and temporal regulation mechanism of fibulin-1 in the human endometrium. METHODS AND RESULTS: Quantitative analysis with real-time PCR experiments on human endometrial tissues showed significantly higher fibulin-1 mRNA expressions in secretory phase endometria than in proliferative phase. Immunohistochemical studies revealed that the fibulin-1 protein is expressed in the glandular epithelium in proliferative phase endometria, and that expression switched to the stroma in secretory phase endometria. In culture experiments with ESCs, a significant increase of fibulin-1 mRNA expression was observed in cells treated with 6 alpha-methyl-17 alpha-hydroxy-progesterone acetate (MPA) or 8 bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP). MPA stimulated the fibulin-1 mRNA expression in a dose-dependent manner, and a progesterone antagonist, RU-486, inhibited the stimulatory effect almost completely. By contrast, beta-estradiol alone did not increase the fibulin-1 mRNA expression. CONCLUSIONS: These results suggest that fiblin-1 is an important molecule that mediates progesterone action in human ESC differentiation towards implantation.     

Temporal and site-specific expression of transforming growth factor- beta4 in human endometrium

We recently identified a novel member of the transforming growth factor (TGF)-beta superfamily and showed that this gene, designated as endometrial bleeding associated factor (ebaf), or TGFbeta4, has a unique expression pattern in human endometrium. By Northern blot analysis, we showed that this gene was expressed in human endometrium during the late secretory and menstrual phases and was absent in proliferative, early and mid-secretory endometria. In this report, we show by in-situ hybridization that the mRNA of the TGF-beta4 is not expressed in the proliferative endometria. On the other hand, focal expression of the TGFbeta4 mRNA first appears in some endometrial glands in the mid-secretory phase. The TGFbeta4 mRNA is strongly expressed in the endometrial stroma during the late secretory and menstrual phases of the cycle. We raised a polyclonal rabbit antiserum against a peptide at the C terminal of the protein. Western blot analysis using affinity purified antiserum shows that the TGFbeta4 precursor detected in the endometrium as well as placenta is 41 kDa. Bands in the range of 45-51 kDa are also present in human endometrium, more predominantly during the late secretory phase. Immunohistochemical staining shows a low level of immunoreactivity for TGFbeta4 in the early, mid- and late proliferative and early and mid-secretory endometria. A strong immunoreactivity for TGFbeta4 is present in the stroma and to lesser extent in the endometrial glands in late secretory and menstrual endometria. The specificity of staining was shown by neutralizing the activity of the antibody with the synthetic peptide used for raising the antibody and by omitting the antibody. The findings show that TGFbeta4, both at the mRNA and protein levels, exhibits temporal and site specific expression in human endometrium.      

The macrophage stimulating protein/RON system: a potential novel target for prevention and treatment of endometriosis

Our recent DNA microarray analysis using tissue obtained by laser capture microdissection (LCM) identified up-regulation of RON (a tyrosine kinase receptor) during the late secretory phase in eutopic endometrial epithelial cells from patients with deep endometriosis compared with control endometrium from women with macroscopically normal pelvic cavities. In the present study, we further investigated mRNA expression of RON and its ligand, macrophage stimulating protein (MSP), in deep endometriotic lesions, eutopic endometrium from patients with deep endometriosis and control endometrium by using LCM and quantitative real-time RT-PCR. MSP mRNA expression in endometrial epithelial cells was significantly up-regulated in endometriosis patients during the late secretory phase compared with expression in controls. Furthermore, we detected up-regulation of MSP mRNA in ectopic endometrial epithelial cells compared with matched eutopic endometrial epithelial cells within the same patients regardless of the menstrual phase. MSP has an intrinsically dual functional nature through its receptor RON-it is a trophic cytokine preventing apoptosis and a scatter factor promoting invasion, both of which may be necessary for the initial development and growth of endometriosis. The present findings suggest that the MSP/RON system may be involved in the pathophysiology of endometriosis.     

A quantitative evaluation of alpha1, alpha4, alphaV and beta3 endometrial integrins of fertile and unexplained infertile women during the menstrual cycle. A flow cytometric appraisal.

The expression of integrin molecules alpha1beta1, alpha4beta1 and alphaVbeta3 within endometrial tissue has been proposed as a marker of uterine receptivity during the implantation window. The present investigation examines by flow cytometric analysis the concentrations of alpha1, alpha4, alphaV and beta3 integrin subunits in endometrial stromal (ESC) and epithelial cells (EEC) in two groups of women throughout the menstrual cycle: normal fertile women (n = 27) and women with unexplained infertility (n = 26). Integrin concentrations in endometrial cells were calculated in relative fluorescence units against a negative cellular control. The assessment of integrin subunits detected the protein in ESC and EEC from the late proliferative to the late secretory phase. In both groups of women, the alpha1 was the highest integrin expressed in ESC and EEC throughout the menstrual cycle. All women exhibited low concentrations of alpha4-EEC at the time of the implantation window. Infertile women expressed lower concentrations of the alpha4-ESC during the proliferative and early secretory phase while lower concentrations of the alpha1-ESC were seen during the late secretory phase. Interestingly, the infertile women expressed lower concentrations of beta3-EEC in the early, mid-secretory and late secretory phases (P < 0.05). Infertile women also expressed lower concentrations of alpha1-EEC and alphaV-EEC during the late secretory phase (P < 0.05). It can be concluded that the quantitative determination of beta3-EEC by flow cytometry confirmed its potential feature as a marker of endometrial receptivity at the time of the implantation window. In addition, the defective expression of the alpha1-ESC found in the late secretory phase might be associated with the poor fertility outcome of women with unexplained infertility.     

Expression of cystic fibrosis transmembrane conductance regulator in human endometrium

BACKGROUND: As a cAMP-regulated Cl- channel, cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in the active secretion of electrolytes and fluid in epithelial cells. Women with CFTR gene mutations are less fertile, generally assumed to be due to cervical factors. However, there is little known about CFTR protein expression in human endometrium and its possible roles in reproduction. METHODS AND RESULTS: CFTR protein and mRNA levels in human endometrium were analysed using immunohistochemical and in situ hybridization methods, respectively. Significant expression of CFTR protein was only seen in the glandular cells from late proliferative to all secretory phases, consistent with western blot analysis. High levels of CFTR mRNA were present only around the ovulatory period. In cultured glandular cells, the production of CFTR protein and mRNA was stimulated by estradiol and inhibited by progesterone. A forskolin-activated Cl- current in endometrial epithelial cells with a linear I-V relationship was detected by the whole-cell patch-clamp technique. CONCLUSIONS: (i) CFTR mRNA and protein were localized in human endometrial epithelial cells and the amounts varied in a cyclic manner; (ii) CFTR expression in cultured glandular cells was up- and downregulated by estradiol and progesterone, respectively; and (iii) CFTR in human endometrium functions as a cAMP-activated Cl- channel.     

Secretory endometrium highly expresses urocortin messenger RNA and peptide: possible role in the decidualization process

BACKGROUND: Urocortin (UCN) gene expression and synthesis have been reported in epithelial and stromal cells of the human endometrium. In this study we evaluated (i) UCN messenger RNA (mRNA) expression and peptide production in uterine specimens collected throughout the endometrial cycle, (ii) UCN secretion after decidualization of cultured human endometrial stromal cells (HESCs) and (iii) the effect of UCN on endometrial decidualization. METHODS: HESCs were isolated from samples of human endometrium collected from healthy patients with normal menstrual cycle and cultured in presence of cAMP, 17-beta-estradiol (E(2)) + medroxyprogesterone acetate (MPA) and UCN. UCN levels were measured in endometrial extracts by an enzyme immunoassay, and changes of endometrial UCN mRNA expression were measured by RT-PCR analysis. RESULTS: UCN peptide concentrations and mRNA expression were highest in the secretory phase of the menstrual cycle (P < 0.001, late secretory versus early and late proliferative phase) and higher in the late than the early secretory phase (P < 0.01). After decidualization of HESC with cAMP or E(2) + MPA, UCN levels rose in parallel with prolactin concentrations by days 6 (P < 0.01, for all). Finally, the addition of UCN to HESCs, with or without E(2) + MPA, induced the release of prolactin. CONCLUSIONS: The evidence that (i) UCN is highly expressed in the secretory phase of the endometrial cycle; (ii) cAMP and E(2) + MPA modulate secretion of UCN and (iii) UCN induces HESCs decidualization together suggest a possible role for UCN in endometrial physiology.     

The expression profile of micro-RNA in endometrium and endometriosis and the influence of ovarian steroids on their expression

MicroRNAs (miRNAs), through mRNA degradation or repression, act as key regulator of gene expression. Our aim was to identify specific miRNAs that are expressed in endometrium of women with and without endometriosis. We profiled the expression of 287 miRNAs in paired eutopic and ectopic endometrium and isolated endometrial cells using microarray and validated the expression of selected miRNAs using real-time PCR. On the basis of global normalization, 65 of these miRNAs were identified to be expressed above the threshold levels set during the analysis in the endometrium of women without endometriosis with a progressive decline in expression in paired eutopic and ectopic endometrium. Statistical analysis (ANOVA) identified 48 of these miRNAs as differentially expressed among these tissues and 32 miRNAs between isolated endometrial stromal cell (ESC) and glandular epithelial cell (GEC) (P < 0.05). The expression of hsa-miR20a, hsa-miR21, hsa-miR26a, hsa-miR18a, hsa-miR206, hsa-miR181a and hsa-miR142-5p, predicted to target many genes, including TGF-betaR2, ERalpha, ERbeta and PR, respectively, was validated in these tissues and cells using real-time PCR. Treatment of ESC and GEC with 17beta-estradiol and medroxyprogesterone acetate (10(-8) M) differentially regulated the expression of hsa-miR20a, hsa-miR21 and hsa-miR26a, which in part reversed following co-treatment with ICI-182780 and RU-486 (10(-6) M), respectively (P < 0.05). In conclusion, we provided evidence for the expression of a number of differentially expressed miRNAs in eutopic/ectopic endometrium and isolated endometrial cells, opening up the possibility that aberrant/altered expression of some miRNAs whose expression is regulated by the ovarian steroids may influence the expression of specific target genes with central roles in normal endometrial cellular activities and pathogenesis of endometriosis.