Variety Of Human Cytokines Expressed During Implantation And Effect Of Mifepristone On Them

Part I Spectrum of cytokines in endometrium decidualizationOBJECTIVETo investigate the changes of cytokines expressed in endometrium and early pregnant decidua and their relationship with implantation. METHODS1. Collected human normal endometrial specimens in "implantation window", digested with 0.25% collagnase I, filtrated and pasted wall to isolate, purify and culture the endometrial stromal cells . Immunohistochemical analysis for vimentin staining was used to identify the purity of ESC. 1×10⁶ESC were counted and cultured in 1ml serum free DMEM/F12 for 48h. An in vitro co-culture system was developed using isolated extravillous cytotrophoblasts and decidual stromal cells in human early pregnancy. Human endometrial stromal cell were also cultured in vitro. Then collected their culture medias and detected the concentration of cytokines by the the Luminex xMAP system simultaneously, elaborate a spectrum of cytokines.2. Collected samples of deciduas in normal human early pregnancy. The DSC were digested with Trypsin-EDTA and purified by discontinuous Percoll gradient, of Immunohistochemical analysis for PRL staining was used to identify the purity of DSC. 1×10⁶ DSC were counted and cultured in 1ml serum free DMEM/F12. 3. Luminex xMAP technology was used to measure the concentrations of TNF-α, IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, GM-CSF, G-CSF, MIP-1α, MCP-1, RANTES in DSC and ESC samples simultaneously, then compared their variety of diffrent temporo-spatial point., Data were evaluated using SPSS 10.0 software. Independent-samples T test was performed to analyse their variety in decidualization. Spearman's correlation was used to analyse their relationship. P＜0.05 was accepted as indicative of stastical significance.RESULTS1. Morphological features of two types of cells cultured in vitro were observed. Immunohistoehemistry (SABC) indicated DSC were positive for PRL expression, with the cytoplasm stained brown, and ESC were positive for vimentin expression, with the cytoplasm stained brown. The purity of these cells prepared for this experiment was＞95%.2. IL-8, IL-6, IP-10, MCP-1, MIP-1α, TNF-α, GM-CSF, G-CSF were produced in the greater quantity than other cytokinesin ESC and DSC, which may play specific roles in regulating embryo implantation during early gestation.3. Concentrations of TNF-α, IFN-γ, IL-1β, IL-6, IL-8, IL-10, IP-10, GM-CSF, G-CSF, MIP-1α, RANTES in decidual stromal cells culture medium were significantly lower than in endometrial stromal cells culture medium, while IL-4 was significantly higher. MCP-1, IL-2, IL-12 did not differ significantly between DSC and ESC.4. There were positive correlations between (1) IL-1βand TNF-α, IFN-γ, IL-6, IL-8, IL-10, GM-CSF, G-CSF, MIP-1α, RANTES, respectively; (2)IL-2 and IL-4, IL-12; (3)TNF-αand MCP-1 during decidualization. The positive relations also existed in TNF-α, IFN-γ, IL-6, IL-8, IL-10, GM-CSF, G-CSF, MIP-1α, RANTES and IP-10 each other. There were negative correlations between (1) IL-4 and TNF-α, IFN-γ, IL-1β, IL-6, IL-8, IL-10, GM-CSF, G-CSF, MIP-1α, RANTES, respectively: (2) MCP-1 and IL-2, IL-12; (3) TNF-αand IL-2 during decidualization. Cytokines may cooperate with each other and form a complex network.CONCLUSIONS1. Cytokines play an important rote in induction and maintenance of endometrium decidualization.Th1 type pro-inflammatory cytokines like IFN-γ, TNF-α, chemokines like IL-8, MCP-1, MIP-1α, RANTES, IP-10 and GM-CSF, G-CSF, IL-1βdecreased to inhibit immune exclusive responses and IL-4 increased to protect fetus. On ESC decidualization, favorable and deleterious cytokines cross-regulate and remodel the complex cytokine networks to induce decidualization and maintain the immunological success of pregnancy. Cytokines play an important role in proliferation and differentiation of the ESC. On ESC decidualization favorable and deleterious cytokines establish a balance to maintain the immunological success of pregnancy.2. A new technology, Luminex xMAP, facilitates the simultaneous evaluation of multiple immune mediators with advantages of higher throughput, smaller sample volume, rapider, more reliable, made it possible to measured lower concentration molecule and protein.Partâ…¡An in vitro model of human extravillous cytotrophoblasts and decidual stromal cells co-culture systemOBJECTIVETo develop a simple and satisfactory method to isolate and purify human extravillous cytotrophoblasts (EVCT) and decidualized stromal cells (DSC) and estabilsh a co-culture system.METHODS1. Collected samples of villous in normal human early pregnancy. The EVCT were digested with 0.125% Trypsin and deoxyribonucleaseI and purified by BSA gradient. Immunohistochemical analysis for cytokeratin7 staining and immunofluorescence analysis by TGF-β₂ were used to identify the purity of EVCT.2. The DSC were cultured in vitro as partâ… .3. 1×10⁵ EVCT were placed in Matrigel-coated (5g/L) Transwell upper chamber (8μm pore) and 6×10⁵ DSC were placed in Transwell lower chamber for invasion assays.RESULTS1. Immunohistochemistry (SABC) indicated EVCT were positive for CK7 expression, with the membrane and cytoplasm stained brown. DSC were positive for PRL expression, with the cytoplasm stained brown.. The purity of these cell prepared for this experiment was greater than 95%.2. Immunofluorescence analysis indicated＞95% EVCT were positive for TGFβ2 expression, with the membrane and cytoplasm stained green. EVCT in upper Transwell chamber keep the invasive ability. The invasion index of EVCT was 3.22±0.04.CONCLUSIONSHigh purity of DSC and ESC can be obtained by proper culture method. Cultured EVCT can keep the invasive potential to invade Matrigel-coated Transwell filters. This co-culture model will be useful to study the trophoblast invasion and embyo implantation mechanisms.Partâ…¢Speetrura of eytokines in trophoblast invasionOBJECTIVETo study the variations of multiple cytokines expressed during trophoblast invasion and their relationship with implantation.METHODS1. The EVCT and EVCT/DSC co-culture system were cultured in vitro as Partâ…¡.2. 2×10⁵ EVCT were placed in Transwell upper chamber(0.4μm pore) and 6×10⁵ DSC were placed in the lower chamber, then added 0.8ml serum free DMEM/F12 in the dual chamber and cultured cells for 48h. The same amount EVCT and DSC were also mixed together in 24-well culture dish and incubated in the same condition. 1×10⁶ EVCT were counted and cultured in 24-well culture dish in lml serum free DMEM/F12. Luminex system was used to measure the concentrations of 15-cytokines (as above) in each group. Multiple comparisons within EVCT,DSC and EVCT/DSC groups to indicate the interactions between trophoblast and deciduas during EVCT invasion. Compared EVCT/DSC in different early pregnancy phase and co-culture mode (Transwell and mixed model), which implied the complex status of cytokines secretion. Data were analysed by oneway-ANOVA, independent-samples T test and Spearman's correlation. RESULTS1. IL-8, IL-6, IP-10, MCP-1, GM-CSF, G-CSF were produced in the greater quantity than other cytokines in EVCT and EVCT/DSC, which may play specific roles in regulating embryo implantation during early gestation.2. Compared with simple EVCT cultured medium, IFN-γ, IL-6, IL-10, IL-12, IP-10, G-CSF, GM-CSF, MCP-1, RANTES decreased in EVCT/DSC co-culture system. But IL-1β, MIP-1α, IL-4, TNF-α, IL-2, IL-8 did not differ significantly between them.3. Compared with simple DSC cultured medium, IL-1β, IL-6, IFN-γ, MIP-1α, RANTES, IP-10 increased and MCP-1, IL-4, IL-12 decreased in EVCT/DSC co-culture systerh. But IL-10, G-CSF, GM-CSF, TNF-α, IL-2, IL-8 did not differ significantly between them.4. IL-6, IL-8, IL-10, G-CSF, MIP-1α, IP-10 secreted by co-cultured EVCT/DSC in mixed model were significantly decreased than those of same amount cells in Transwell chamber.5. TNF-α, IL-8, IFN-γ, G-CSF, MIP-1αof EVCT/DSC decreased while RANTES increased when pregnancy went on. No difference was found in IL-1β, IL-2, IL-4, IL-6, IL-I2, IL-10, IP-10, MCP-1,GM-CSF.CONCLUSIONSCytokines play an important role in regulating trophoblast proliferation, differentiation and invasion. IL-1β, MCP-1, RANTES, IL-6 may up-regulate trophoblast invasive ability. G-CSF, GM-CSF, IL-8, IP-10, MIP-1αmay inhibit immune rejective response. Co-cultured with deciduas can diminish deleterious cytokines and establish a complicated balance to maintain the immunological success of pregnancy.Partâ…£Effect of mifepristone on cytokines expressed during implantationOBJECTIVETo investigate the effect of mifepristone on cytokines during trophoblast invasion.METHODS1. The EVCT and EVCT/DSC co-culture system were cultured in vitro as Partâ…¡. 2. Both EVCT and EVCT/DSC co-culture system from the same source were divided into two groups, one was incubated with 1×10^-6mol/L RU486 for 48h, another was cultured without RU486(as control). Collected the culture media and detected the concentration of 15-cytokines by Luminex xMAP. Paired-samples T test was performed to demonstrate mifepristone's effect on cytokines of fetal-maternal interface.RESULTS1. Mifepristone down- regulated the levels of IL-4, IL-6, IL-10, GM-CSF, G-CSF, MIP-1α, RANTES, MCP-1 and up-regulated the levels of TNF-α, IL-1β, IL-8, IL-12, IFN-γsecreted by EVCT/DSC co-culture system. Mifepristone had no effect on IL-2 and IP-10.2. Mifepristone down- regulated the levels of IL-2,IL-10,GM-CSF,G-CSF,MIP-1αand up-regulated the levels of TNF-α. IL-1β. IFN-γsecreted by pure EVCT group. No difference was found in IL-4,IL-6,IL-8,IL-12,MCP-1,RANTES and IP-10.CONCLUSIONSMifepristone can affect the levels of cytokines and induce the predominance Th1 type cells, which may enhance immune rejection to conceptus. Mifepristone can also cease growth of trophoblasts and deciduas. Such lead to the termination of pregnancy.