Research On Effectiveness Of Improving Embryo Implantation Of Bushenantai Recipe

Objective1. To investigate the improving effect of Bushenantai recipe on pregnancy rate and number of implanted blastocyst of mice with implantation dysfunction induced by indomethacin and mechanism.2. To observe the effect of Bushenantai Recipe on the expression of endometrial LIF in the mouse with embryonic implantation dysfunction (EID).3. To observe the effect of Bushenantai Recipe on the expression of endometrial HB-EGF and EGF-R in mouse with embryonic implantation dysfunction (EID).MethodPart one Establish Mice with Embryonic Implantation Dysfunction Model Induced by Indomethacin and Observe Effect of Bushenantai Recipe on the Implantation of These MiceAll pregnancy mice were divided into three groups in random: normal group, model group and traditional Chinese medicine group (TCM group). Mice of normal group was injected subcutaneously twice on pregnancy day 3 and 4 with gingeli, indomethacin (4.33mg/kg)was administrated subcutaneously twice on pregnancy day 3 and 4 to establish animal models of implantation dysfunction in Model group and TCM group. Subsequently, the fifth and eighth day of pregnancy, the pregnancy rate, number of implanted blastocyst was compared in 3groups. Afterwards, endometria which were dyed by hematoxylin and eosin (HE)could be observed respectively by light microscope on Pd5,6,8.Part Two Study of Regulation Effect of Bushenantai Recipe on the level of Estrogen and Progesterone in Mice with Blastocyst Implantation DysfunctionModeling method is similar with the method of part one. All pregnancy mice were divided into three groups in random: normal group, model group and traditional Chinese medicine group (TCM group), each group had 22.The plasma Level of estrogen (E2) and progesterone (P4) level on the fifth, eighth day of pregnancy and the Estrogen receptor and Progesterone receptor of endometrium of uterus were dyed by immunohistochemistry on the eighth day of pregnancy.Part Three Effect of Bushenantai Recipe on the Expression of Endometrial LIF in Mice with Embryonic Implantation DysfunctionModeling method was similar with the method of part one. 120 Kunming mice post coition were randomly assigned to normal group, model group and traditional Chinese medicine group. On Pd4,5,6, the uteruses of mice in each group were obtained while the endometrium was dyed by Evan's blue. Afterwards, the expression of LIF mRNA and protein was analyzed by means of RT- PCR and immunohistochemistry respectively.Part Four Effect of Bushenantai Recipe on the Expression of Endometrial HB-EGF and EGF-receptor (EGFR) in Mice with Embryonic Implantation DysfunctionModeling method was similar with the method of part one. 30 Kunming mice post coition were randomly assigned to normal group, model group and traditional Chinese medicine group. On Pd5,6, the uteruses of mice in each group were obtained while the endometrium was dyed with Evan's blue. Subsequently,the expression of HB-EGF mRNA was analyzed by means of RT- PCR on Pd5,6. Afterwards, the expression of HB-EGF and EGF-R protein was analyzed by means of immunohistochemistry on Pd5,6.ResultsPart one Establish Mice with Embryonic Implantation Dysfunction Model Induced by Indomethacin and Observe Effect of Bushenantai Recipe on the Implantation of These Mice On Pd5, compared with the normal group, the coloration of endometrium dyed by Evan's blue as well as the mean points of response was remarkably lowered in model group; The difference between normal group and TCM group was insignificant. On Pd8, The pregnancy rate and number of implanted embryo was lower in model group(27.3%,5.3±0.7) than in normal group(90.9%,13.3±2.8) significantly ( all P<0.01), but was higher in TCM group(72.7%,10.7±2.2) than in model group(all P<0.05). On Pd5,6,8, contrasted to the model group, endometria blood vessels of TCM group increased obviously and the decidualization of interstitial cell progressed orderly; The difference between normal group and TCM group was insignificant.Part Two Study of Regulation Effect of Bushenantai Recipe on the level of Estrogen and Progesterone in Mice with Blastocyst Implantation DysfunctionThe plasma level of Estrogen on Pd5 of TCM group was higher than model group, on Pd8 was the highest in three group;The plasma level of Progesterone on Pd5 in model group and TCM group were lower than normal group(P<0.01),but on Pd8 the plasma level of Progesterone in TCM group was higher than model group, though it was lower than normal group. Immunohistochemistry observation showed that there was more Estrogen receptor and Progesterone receptor in endometrium of mice in TCM group than model group.Part Three Effect of Bushenantai Recipe on the Expression of Endometrial LIF in Mice with Embryonic Implantation DysfunctionThe expression of LIF mRNA and protein in the EID mouse endometrium was delayed in a temporal and cell specific manner. Bushenantai Recipe could help to normalize the expression of LIF mRNA and protein on Pd4,5,6.Part Four Effect of Bushenantai Recipe on the Expression of Endometrial HB-EGF and EGF-receptor (EGFR) in Mice with Embryonic Implantation DysfunctionThe expression of HB-EGF and EGF-receptor in EID mice endometrium were delayed in a temporal and cell specific manner,at the same time the quantity was decreased. Bushenantai Recipe could help to normalize the expression of HB-EGF and EGF-R on Pd5,6.Conclusions1. Bushenantai recipe can improve the pregnancy rate and number of implanted of mice with Embryonic Implantation Dysfunction.2. The results demonstrate: Bushenantai recipe can increase the pregnancy rate and number of implanted embryo of model mice significantly. The mechanism is: First, this recipe can regulate the contents of Estrogen, Progestogen and their receptors in blood-serum during the implantation of embryo. It can not only induce the expression of LIF, HB-EGF and their receptors at the right time, but also profit the decidualization of endometrium and the dialogue between endometrium and embryo.The results mentioned above manifest that Bushenantai recipe may play an important role in the process of apposition and adhesion of the blastocyst to the uterine lumen, penetration of the epithelium and decidualization of the stromal cells...