| At present, embryo implantation rate is low in in vitro fertilization and embryo transfer (IVF-ET). Clinic pregnancy's crucial factor is embryo implantation of wich capacity is decided by the preimplantaion embryo development in some degree. To improve pregnancy outcome, the clinic was improving the controlled ovarian hyper stimulation (COH) protocals, while embryo lab was exploring appropriate conditions and favorable factors for embryo development in vitro.Cyclosporine A (CsA) is immune depressant which is widely used to suppress immunological rejection in organ transplantation and treat immunological disease's. Researches reveal that appropriate dosage of CsA induce the maternal-fetal immunity tolerance and enhance proliferation, movement, migration, invasion and inhibit apoptosis in trophoblasts. So CsA regulates early pregnancy from two aspects above. Placental trophoblast cell originat from trophectoderm cell. CsA can promote cell proliferation of many other kinds besides trophoblast cells, So we infer CsA can play a role in preimplantation embryo development.ObjectivesIn this research,2-cell embryos development in vitro were observed when they were cultured in medium with CsA at different stage and medium with different consentration CsA respectively to discuss the effect and outset stage of CsA on early embryo development and the optimum dosage which is favorable for embryo development. The aim is to explore an effective way which can improve profound implantation capacity and promote implamtation rate of embryos.Methods1.2-cell embryos were obtained dissectively from ICR female mice (4 weeks after birth) after controlled ovulation hyperstimulation (COH) and mated naturally. Embryos obtained were divided into several groups and cultured subsequently in cleavage medium (CM) and blastocyst medium (BM). Embryo development were observed every day until hatching.2. To test the effect of 1μmol/L of CsA on embryos of different stage. The embryos were divided into 3 groups:CsA was added in medium from 2-cell stage, morula stage and medium without CsA. Embryos development at every stage were observed.3. To observe the effect of different concentration of CsA on early embryo development, the embryos were divided into 4 groups:2-cell embryos were cultured in medium with 10μmol/L,1μmol/L,0.1μmol/L and 0μmol/L (control) respectively. Blastocyst formation and hatching were observed.4. Total cell number of was counted in expanded blastocysts. Using Hoechst 33258 fluorescent dying. under fluorescence microscope.5. Statistical analysis was done by SPSS13.0 software package. Qualitative data were testes by x2, whereas quantitative data were tested by analysis of variance.Results1. Effect of 1μmol/L of CsA on embryos of different stageGroups added CsA from 2-cell stage, morula stage and without CsA,4-8cell embryo formation rates were 71.9%,62.5% vs 63.6% respectively; morula formation rates were 70.8%,62.5% vs 62.5% respectively; blastocyst formation rates were 62.9%,58% vs 53.4% respectively; blastocyst hatching rates were 37.5%,25.5% vs 21.3% respectively, all differences were not significant(P>0.05).2. The effect of different concentration of CsA on preimplantation embryosGroups added with 10,1,0.1,0μmol/L CsA, the total blastocyts formation rates were 82.1%,73.6%,70.7%, and71.3% respectively, difference was not significant (P>0.05). the expanded blastocyst formation were 65.5%,50.6%,47.7% and 48.2% respectively, there was no significant difference except for comparison between G1 and other three groups (P<0.05). the blastocyts hatching rates were 44.9%,42.2%, 34.4% and 38.7% respectively, difference was not significant(P>0.05). the linear regression formation between concentrations of CsA(X) and hatching rates(Y) was Y=0.05251gX+0.405 (r=0.9629).3. Effect of different concentration of CsA on total number of blastocyst cell.The total number of blastocyst cell(x±s) in G1, G2, G3, and G4 were 79±4.5, 78±4.2,74.6±4.3, and 77.2±5.3 respectively, significant difference only existed between G1 and G3 (P<0.05).ConclusionThe tendency of early embryo development in vitro reveals that appropriate dosage's CsA improves embryo development from 2-cell stage. In the range between 1μmol/L and lOμmol/L, the CsA may dose-dependently promote blastocyst formation and hatching.
|
PDF Full Text Request