| IVF-ET becomes more and more important for infertility treatment, but low embryo implantation rate is one of tough problems in the program.Some efforts have been made to improve clinical pregnancy rate technologically and theologically. The interaction between endometrium and embryo is determinant for successful implantation, and trophoblast of embryo is one of key factors.CsA is an agent used in patients who have been transplanted organ or suffer from autoimmune diseases, and CsA stimulates proliferation of malignant tumor cells and non-tumor cells. It has been shown that CsA induces maternal-fetal immunotolerance that leads to embryo absorb rate decrease in CBA/JxDBA/2 abortion-prone matings, and also improves proliferation and invasion, and inhibits apoptosis of trophoblast from 50-60 days gestation in women. It has been further demonstrated that the action of CsA on trophoblast was through combination with cyclophilin via API and Ca2+/calcineurin/NFAT of MAPK3/MAPK1 pathway.Synchronization of embryo and endometrium development is necessary condition for endometrium to receive embryo with normal trophoblast functions. However, we do not know now whether CsA can improve embryo implantation, and whether CsA may enhance embryo developing speed.In this study, effects and molecular mechanisms of CsA on embryo development, cellular differentiation, cellular proliferation and apoptosis, and blastocyst adhesion and invasion were studied in murine embryo in vitro to investigate the possible effects of CsA on embryo implantation.Part I The modulation of cyclosporine A on cell proliferation, apoptosis, differentiation and blastocyst formation in pre-implantation embryoObjective To investigate the modulation of CsA on cell proliferation, cell apoptosis, cell differentiation and blastocyst formation of pre-hatching embryos in the mouse model.Methods.① 2dpc embryos were retrieved and cultured to 4dpc in media containing 0,0.1,1.0 and 10μM of CsA. The embryo cells and cICM were counted after DAPI and Oct4 (immunofluorenscence) nucleus staining, respectively. cICM, TB were used as parameters to judge cell differentiation and allocation. ② 5dpc embryos were harvested to count embryo cells after staining with Hocehst 33258. The blastocyst formation was observed and blastocyst formation rates were calculated. Cell apoptosis was counted after TUNEL nucleus staining and observed under fluorescence microscope. On 5dpc, CDX2 and Oct4 mRNA of blastocysts were relatively quantified by RT-PCR.Results 2754 2dpc embryos were divided into 4 groups and cultured in media containing 0,0.1,1.0 and 10 μM of CsA. The blastocyst formation rates on 5dpc in groups were 68.1%(n=689),67.5%(n=682),66.4%(n=693) and 68.4 (n=690), respectively, and the differences were not significant among groups (P>0.05).2dpc embryos were cultured in media containing 0,0.1,1.0, 10μM of CsA to 5dpc, and 175 blastocysts in stage 3 and after were gained and used to set the cell number. Cell numbers per blastocyst among 0,0.1,1.0 and 10μM of CsA groups were 91.1±3.2 (n=34),82.9±2.1 (n=47),88.5±3.0 (n=45) and 93.3±2.9 (n=49). There were no significant differences among groups (P>0.05).2dpc embryos were cultured in media containing 0,0.1,1.0, 10μM of CsA to 4dpc, and 154 blastocysts in stage 3 and after were gained and used to cICM and TB analyses (n=47,36,39 and 32 respectively) were used for cell number of cICM and TB. The cICM per blastocyst were 10.6±0.5,12.3±0.7,12.3±0.6 and 12.2±0.7. The TBs per blastocyst were 38.6±1.3,40.5±1.8,42.1±1.9 and 45.0±2.7. There were no significant differences for cICM and TB among groups (P>0.05).When quantities of Oct4 and CDX2 mRNA in OμM CsA group(control)were defined as "1", mRNA for Oct4 were 1.00±0.07,1.42±0.08,1.63±0.18 and 0.60±0.07 for 0,0.1,1.0, 10μM of CsA groups respectively. Oct4 mRNA were up-regulated in 0.1 and 1.0μM compared to other groups (P<0.05), but down-regulation was observed in 10μM CsA groups (P<0.001). mRNA for CDX2 were 1.00±0.24,1.10±0.13, 1.14±0.22 and 0.77±0.17 for 0,0.1,1.0, 10μM of CsA groups respectively. The differences of CDX2 mRNA quantity were not significant among groups (P>0.05).2dpc embryos were cultured in media containing 0,0.1,1.0,10μM of CsA to 4dpc, and 97 embryos in stage 3 and after were gained and stained with TUNEL.The apoptosis indexes(%) were 4.51±0.30 (n=14),5.14±0.17 (n=32),5.56±0.23 (n=24) and 3.79±0.12 (n=27) respectively. The differences among groups were not significant.Conclusion We concluded from above:1. In the concentration from 0.1 to 10μM of CsA, blastocyst formation rate and cell apoptosis are not affected by CsA in vitro in mouse embryo in 5dpc.2. In the concentration from 0.1 to 10μM of CsA, The total cell number (5dpc), cICM, and TB (4dpc) are not affected by CsA in vitro in mouse embryo.3. In the concentration from 0.1 to 1.0μM of CsA, Oct4 mRNA transcription is up-regulated, but down-regulation is observed in 1.0μM group. Transcription of CDX2 mRNA, a differential marker of TB, is not affected.Part Ⅱ Modulation mechanisms of cyclosporine A on hatching and implantation of blastocystObjective To investigate regulation and mechanisms of CsA on hatching and implantation of blastocyst.Methods ① The mouse blastocysts, gained from 2dpc to 4dpc culture in vitro, were retrieved and cultured in media containing 0,0.1,1.0 and 10μM of CsA to 6dpc. Blastocyst hatching was observed and ISP1 mRNA, key enzyme for hatching, was relatively quantified with RT-PCR.② The mouse hatched blastocysts, gained from 2dpc to 5dpc culture, cultured in media containing 0,0.1,1.0 and 10μM CsA and LN coated dishes further to 7 or 8dpc. Embryo adhesion and stretch growing were observed respectively. The mRNAs of Itgβ3 and MMP-9 transcription were relatively quantified with qRT-PCR. Their protein expression was evaluated with immunofluorenscence and observed under confocal laser scanning microscope.Results 799 embryos were used to test their hatching. The hatching rates in 0, 0.1,1.0 and 10μM of CsA groups were 90.15%(n=203),89.57%(n=211),96.25% (n=108) and 95.98%(n=199) respectively, P>0.05.The ISP1 mRNA were 1.00±0.31, 0.68±0.13,0.73±0.18 and 1.01±0.33.No significant differences were observed (P>0.05).360 embryos were used to test their adhesion. The adhesion rates in 0,0.1,1.0 and 10μM of CsA groups were 76.0%(n=79),91.8%(n=97),93.8%(n=80) and 83.7%(n=104) respectively, which showed adhesion in 0.1 and 1.0pM CsA groups were significantly higher than that of the control (P<0.01).126 embryos were used to test their stretch-growing ability. The areas occupied by embryos on dish floor (×104μm2) were 4.14±0.39 (n=29),4.32±0.37 (n=34), 5.76±0.37 (n=34) and 4.83±0.34 (n=29) respectively. The area in 1μM group was significantly higher than that of the control (P<0.05).When quantities of itgβ3 and MMP-9 mRNA of OμM CsA group (control) were defined as "1", the relative quantities of itgβ3 mRNA in 0,0.1,1.0 and 10μM CsA groups were 1.00±0.09,0.51±0.20,1.98±0.37 and 0.76±0.10 respectively. Itgβ3 mRNA transcription in 1μM was significantly higher than that of the control (P<0.05). Transcriptions of MMP-9 were 1.00±0.20,1.44±0.23,2.37±0.23 and 1.68±0.28 respectively. MMP-9 mRNA in 1μM and 10μM were significantly higher than that of the control (P<0.01 and P<0.05 respectively). Immunofluorenscence assay showed that the expression of itgβ3 and MMP-9 was up-regulated.Conclusion We concluded from above:1. CsA does not affect blastocyst hatching and its ISP1 mRNA transcription.2. CsA enhances adhesion of hatched blastocyst on the LN-coated dish in concentration of 0.1μM and 1.0μM, and stretching growth is up-regulated in 1.0μM. Up-regulations of integrin αvβ3 and MMP-9 mRNA transcription and peptides translation in trophoblast are related to the molecular mechanisms of CsA improving implantation ability of blastocyst.Summary1. CsA does not affect developmental procedure of pre-implantation embryo, including blastocyst formation, cell proliferation, apoptosis and differentiation (cICM and TB) in 0-10βM concentration. The mRNA of CDX2 transcription is not affected, but that of Oct4 is up regulated.2. CsA improves implantation of peri-implantation embryo by enhance its TB invasion ability, including adhesion, migration, digestion of ECM, via up-regulating expression of integrin αvβ3 and MMP-9. |
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