Multifactor Regulation on Expressions of MMP-9 and TIMP-1 in Endometrial Stromal Cells

Objective To investigate the regulatory effect of multifactor on the matrix metalloproteinases-9 （MMP-9） and the tissue inhibitor of metalloproteinase-1 （TIMP-1） in endometrial stromal cells. Methods The endometrial stromal cells separated from the proliferative endometrial tissues were incubated with medium alone, 17-β estradiol （E2,10^-8 mol/L）, medroxyprogesterone acetate （MPA, 10^-6 mol/L）, E2（10^-8 mol/L）＋MPA （10^-6 mol/L）, E2 （10^-8 mol/L）＋MPA （10^-6 mol/L）＋RU486 （10^-5 mol/L） or HB-EGF （10 ng/ml） for 48 h respectively. The expressions of MMP-9 and TIMP-1 were detected by in situ hybridization, immunocytochemistry, reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting. Results Compared with control group [mRNA, 0. 729 ± 0. 090 （MMP-9） and 1.056± 0.154 （TIMP-1）; protein, 0.545 ±0.086 （MMP-9） and 0.745 ±0.154 （TIMP-1）], expressions of MMP-9 and TIMP-1 in E2 alone, progestin alone or E2 combined with progestin group were respectively：mRNA, 0.413 ± 0.069, 0.402 ± 0.073 and 0.407 ± 0.039; 0.487 ± 0.093, 0.503 ± 0.093 and 0.468 ± 0.075：protein, 0.294 ± 0.076, 0.331 ±0.064 and 0.265 ±0.049; 0.425 ±0.085, 0.397 ±0.065 and 0.435 ± 0.099. RU486 weakened the expression level of down-regulation, while HB-EGF elevated the level of MMP-9 and TIMP-1 after 48 h treatment （mRNA, 0.955 ± 0.068 and 1.396 ± 0.238; protein, 0. 780 ± 0.109 and 0.985 ± 0.165）. Conclusions 1） Both E2 and progestin can down-regulate the expressions of MMP-9 and TIMP-1 in endometrial stromal cells, but RU486 can inhibit the effect. 2） HB-EGF can elevate the level of MMP-9 and TIMP-1. 3） E2, progestin and HB-EGF have effect on the ratio of MM-P/TIMP-1.

The influence of insulin on secretion of IGF-Ⅰand IGFBP-Ⅰin cultures of human endometrial stromal cells

s To study the influence of insulin on IGF Ⅰ and IGFBP Ⅰ secretion of the hum an endometrial stromal cells Methods Late proliferative phase endometrial stromal cells were isolated from endometriu m tissues and then cultured for 24 h in Hams F 12 only as a control and in Hams F 12 with different concentrations of estradiol (E2) and insulin (INS) as trea ted groups Simultaneously, the endometrial stromal cells from late secretory phase endometrium were cultured for 24 h in Hams F 12 only as a control and in Hams F 12 supplemented with different concentrations of progesterone (P) and i nsulin as treated groups After 24 h of culturing, the mediums were collected f or either IGF Ⅰ or IGFBP Ⅰ assays Result The concentrations of IGF Ⅰ in medium from cultured endometrial stromal cells i n the proliferative phase were 0 78±0 47 ng/ml in the hormone free control g roup; 1 44±0 59 ng/ml and 1 39± 0 33 ng/ml in 100 pg/ml E2 group and 20 μU /ml INS group, which was higher than that of the control group ( P <0 05 and P <0 01, respectively) The IGF Ⅰ concentration in the 100 μU/ml INS group was 2 03±0 53 ng/ml, which was higher than that of the 20 μU/ml INS group ( P <0 01) Levels of IGF Ⅰ in the 100 pg/ml E2 plus 20 μU/ml INS gro up was 2 18±0 36 ng/ml, which was significantly higher than that of the 20 μU/ml INS and 100 pg/ml E2 group ( P <0 01), but lower than that of the 100 pg/ml E2 p lus 100 μU/ml INS group (3 42±0 75 ng/ml), P <0 01 The concentration o f IGFBP Ⅰ in medium from cultured endometrial stromal cells in the secretory ph a se was 2 50±1 39 ng/ml in the hormone free control group and 5 44±2 09 ng /ml in the 10 pg/ml P group, which was significantly higher than that of the con trol ( P <0 01) IGFBP Ⅰ concentration in 20 μU/ml INS group was 0 1 6±0 58 ng/ml, which was lower compared with control, but higher compared with the 100 μU/ml INS group ( P <0 01) The level of IGFBP Ⅰ in the 10 ng/ml P plu s 20 μU/ml INS group was 2 10±1 17 ng/ml, lower compared with the 10 ng/ml P gr oup, but higher compared with the 10 pg/ml P plus 100 μU/ml INS group, P <0 01 Conclusions Insulin can stimulate basal (without hormone) and E2 stimulated IGF Ⅰ secreti on in cultured stromal cells from human late proliferative endometrium in a dose dependent manner Insulin can suppress basal (without hormone) and P stimula ted IGFBP Ⅰ secretions in cultured stromal cells from human secretory endomet rium in a dose dependent manner

Roles of Regulatory T Cells in Pathogenesis of Endometriosis

Numerous studies have shown aberrant immune cell function in endometriosis,including T cells,B cells,natural killer cells,and macrophages(MΦ).These alterations are thought to be induced by various mechanisms that promote the disease.Regulatory T cells(Tregs)may account for a decreased ability of newly recruited leukocytes to initiate effective immune responses against viable endometrial fragments,permitting their survival.Tregs differentiate during the development of endometriosis,which confer immunosuppression or play other roles in disease progression.In this review,we provide an overview of the regulation and roles of Tregs in endometriosis.These data provide further scientific evidence for the altered immune response in endometriosis,which could be a potential target in the treatment of endometriosis.This review could create new diagnostic strategies and effective immune-targeted therapies for this highly prevalent disease.Recent progress in the field indicates that these goals may be achieved in the future.

Expression of Cytokeratin,Actin and Endocrine Hormones in Human Hatched Blastocyst Implantation Model in vitro

Objective To investigate the expression of cytokeratin,actin and hCG,E2 and P of human hacthed blastocysts in the model.Methods Human hatched blastocysts were co-cultured with human endometrial decidualization stromal cell monolayer.The process of orientation,attachment,outgrowth and invasion in morphology were observed.Immunofluorescence staining for cytokeratin and actin,immunofluorescence measurement of hCG and radioimmunoassay measurement of E2 and P were performed.Results Blastocysts attached to stromal cell layer after 5h in co-culture.After 24h in co-culture,the trophoblast protruded from two opposite poles of the blastocyst and underwent outgrowth into the stromal cell monolayer,blastocyst became bigger and invaded finally into the stromal cells.After 48h in co-culture,cytokeratin staining was only visible in trophoblast but not stromal cells,actin staining was visible in both of trophoblast and stromal cells with distinct conformation and structure.Stromal cells had prominent linear actin filaments,aligned along the long axis of the cells.Cytokeratin staining in trophoblast cells was localized in short filaments arranged in a mesh.hCG,E2 and P levels in the supernate of stromal cell-blastocyst co-culture were higher than the supernate from blastocyst cultured only （P〈0.01）.Conclusion An implantation model for the reflection of the process of human blastocysts attachment,outgrowth and invasion into stromal cells has been established in vitro.Cytokeratin,actin and hCG,E2 and P take place corresponding changes in the human implantation blastocyst cells.

Unique and independent role of the GABAB1 subunit in embryo implantation and uterine decidualization in mice

Embryo implantation and decidualization are crucial for successful pregnancy,which include multiple genes and signaling pathways,while the precise mechanism regarding embryo implantation and decidualization has yet to be explored.The GABA which activates GABA_(A)or GABA_(B)receptors has been found playing an important role in early pregnancy.Here we seek to investigate whether GABA_(B)receptors participate in embryo implantation in mice.This study first characterized the spatiotemporal expression pattern of GABA_(B)receptors in the uterus during the peri-implantation period and found that GABA_(B1)expression was drastically upregulated in stromal cells on days 4e6,a period of embryo implantation and early stages of decidualization.Embryo delayed implantation and oil-induced decidualization models were further used to confirm that the GABA_(B1)was associated with embryo implantation and decidualization.We also found estrogen or progesterone had no directly effect on expression of GABA_(B1)in ovariectomized model.Because we were unable to detect significant GABA_(B2)which couples with GABA_(B1)to form whole GABA_(B)receptors,and the agonist and antagonist of whole GABA_(B)receptors had weak effect on the proliferation and differentiation of stromal cells as well,we excluded the possibility whole GABA_(B)receptors function,and concluded it should be non-classical signals of GABA_(B1)involving in embryo implantation and decidualization.Future studies should focus on investigating the roles and mechanisms of GABA_(B1)during embryo implantation and decidualization.

Indoleamine 2,3-Dioxygenase in Endometriosis

Endometriosis(EMS)is a chronic inflammatory and estrogen-dependent gynecological disease characterized by the presence of endometrial tissue outside the uterine cavity.Although it is a benign disease,EMS is tumor-like in several aspects,which include unrestrained growth,decreased apoptosis,and aggressive invasion.EMS involves endocrine disorders and immunological factors.Indoleamine 2,3-dioxygenase(IDO)is an intracellular enzyme that catalyzes the initial and rate-limiting step of the metabolism of tryptophan.IDO is a potential candidate facilitating EMS development.Increased IDO expression in both eutopic and ectopic endometria of women with EMS is biologically important in aspects,which include regulation of endometrial stromal cell function and modulation of adjacent local immunocytes to generate a supportive microenvironment.In turn,the expression of IDO can be regulated by the complex endocrine-immune microenvironment networks in endometrial lesions.Here,we systematically review the roles of IDO in EMS to explore its pathological implications and treatment potential.

Decidualization and Related Pregnancy Complications

Decidualization is the differentiation of endometrial stromal cells into secretory decidual stromal cells.Human decidualization involves some amount of signaling molecules and pathways as well as genetic reprogramming,which is driven by the postovulatory rise in progesterone levels and local cyclic adenosine monophosphate production.Decidualization extends from the primary decidual zone to the secondary decidual zone,and then exits through apoptosis.Evidences support that decidual fibroblasts function as the pool of decidual stromal cells during pregnancy.Decidualization undergoes an acute inflammatory phase,an anti-inflammatory secretory phase to the final recession phase.The decidualization of the inner layer of endometrium,termed decidua,is the most critical determinant of pregnancy success,which can promote placenta formation,modulate immune tolerance,foster resistance to oxidative stress,sense embryo quality,and control labor.Failure to adequate decidualization in terms of hormones,biochemistry,and immunology leads to adverse pregnancy outcomes,including diseases such as preeclampsia,miscarriage,premature labor,repeated implantation failures,and some age-related decline in reproductive capacity.The development of animal models and in vitro culture systems combined with emerging technologies provides a powerful system to explore the mechanism of decidualization.However,decidualization is a dynamic,multi-step process,and translating of current research progress into disease predictions and interventions for pregnancy complications remains to be achieved.The study of periodic regeneration and spontaneous decidualization of the endometrium will be beneficial to the diagnosis and treatment of pregnancy diseases.