Comparative Study Of Intervention About Bushenantai Recipe And Its Two Components On Embryonic Implantation Of Controlled Ovarian Hyperstimulation

Objective:To investigate the roles of Bushenantai recipe and its two components during implantation in mice of COH (controlled ovarian hyperstimulation), and definite selective or cooperative effects between Bushen and Huoxue components; we observe the effects of the recipe and its two components on blastocysts and the expression of MMP-2 (Matrix Metalloproteinase-2), MMP-9(Matrix Metalloproteinase-9), TIMP-3(Matrix Metalloproteinase Inhibitor-3), TGF-β1 (Transforming Growth Factor-beta1) and integrinαvβ3 in mice, furthermore, provide experimental bases of kidney-reinforcement and blood-activation for the treatment of infertility.Method:All mice were randomly divided into normal group, model group, bushenantai group, bushen group and huoxue group. Model and TCM groups were intraperitoneal injected with PMSG 10IU at 5pm, then mated with fertile males (day1=vaginal plug) after intraperitoneal injected with HCG 10IU 48 hours later. Normal group would be mated without COH. And observed vaginal plug rate at pregnancy day 1(Pd1), and the number of implantation sites at Pd5,6,7. Uterus were collected on Pd5,6 and7. Detected the expression of MMP-2, MMP-9,TIMP-3 TGF-β1 and integrinαvβ3 mRNA by real time RT-PCR, expression of MMP-2,MMP-9,TIMP-3 TGF-β1and integrinαvβ3 were analyzed by immunohistochemistry.Result:1. Comparison of number of implantation sitesThe number of implantation sites in model group and TCM groups were significantly higher than the normal group, and TCM groups were higher than model group, but there is no statistical difference between two gropes above. Respectively, bushenantai group were higher than bushen and huoxue group, but there is no significantly difference between above.2. Comparison of RT-PCR resultsOn the expression of MMP-2 mRNA, compared to the normal group, the model group increased significantly on Pd5 and 6(p<0.05), but, decreased significantly (p<0.05) on Pd7; compared to the model group, the TCM group increased in the different degree, especially in the Huoxue group (p<0.01). On the expression of MMP-9 mRNA, from Pd5 to Pd7, the normal group increased gradually, while the model group show trends which first inclined but soon after high-up; on Pd7, compared to the model group , the TCM group increased significantly (p<0.05). On the expression of TIMP-3 mRNA, it is increased from Pd5 to Pd7 in the normal group, while there is no rise trend in model group. Compared to normal group, there are significant differences on Pd5, 6 and 7, and the TCM group show the same tendency with normal group, however, the TCM group increased significantly compared to model group on Pd7 (p<0.05). On the expression of TGF-βmRNA, on Pd5 and 6, there is significant increase in model group compared to normal group (p<0.05). On Pd5 and 7, there is no difference between TCM group and normal group, and the tendency of Huoxue group is same with normal group completely. On the expression of integrinαvβ3 mRNA, in the normal group, it is similar on Pd5, 6 and 7; compared to normal group, the model group increase significantly on Pd5 and 6(p<0.05), while decreased on Pd7, compared to modal group(p<0.05), but, the TCM group show different tendency.3. Comparison of ImmunohistochemistryMMP-2,MMP-9,TIMP-3 were expressed on the endometrial glandular and Luminal epithelium cell during implantation, with the development of deciduas, expressed on the stromal cell decidualization. Pd5, TGF-β1 expressed on Primary decidual zone, and gradually expressed on Secondary decidual zone and extracellular matrix, meanwhile expressed on the endometrial glandular and Luminal epithelium cell. Integrinαvβ3 expressed on the endometrial glandular and Luminal epithelium cell, and have a small mount of expression on the stromal cell decidualization. Immunohistochemistry observation suggests that there was no remarkably difference on MMP-2, MMP-9, TIMP-3, TGF-β1 and integrinαvβ3 spatial distribution in the five groups. Normal groups have compact matrix and rich glands, contrastly, the model group have sparse matrix and poor glands, the state of TCM groups improve the model group's situation.Conclusion:Bushenantai recipe and its bushen,huoxue components could up-regulation the implantation sites and normalize MMP-2, MMP-9 and TIMP-3's mRNA, There is no obvious difference between the expression of TGF-β1 and integrinαvβ3, bushen and huoxue components had cooperation in embryo implantation, and Huoxue Medicine dose not show a negative impact on the mice.