| Part I:Effect of acupuncture on endometrial blood flow in women undergoing in vitro fertilization:a prospective randomized controlled studyObjectives:To evaluate the effect of manual acupuncture on endometrial three-dimensional(3D)power Doppler ultrasound parameters as markers of uterine receptivity in women undergoing In vitro fertilization embryo transfer(IVF-ET).Design:Patients undergoing IVF-ET were randomly received active or sham acupuncture treatment from the day of oocyte aspiration to embryo transfer.After treatment,the endometrial ultrasound parameters were compared between the two groups.The rates of implantation,clinical pregnancy,abortion and live birth were also compared.Location:Reproductive Medicine Center and Outpatient Department of Integrated Traditional Chinese and Western Medicine,Tongji Hospital,Wuhan,China.Subjects:Seventy patients were enrolled.Thirty-four patients in active acupuncture group and 35 in sham acupuncture group finished this study.Interventions:Patients in active acupuncture group were treated as traditional acupuncture therapy and patients in sham acupuncture group were only slightly stimulated at the non-acupoints on each shoulder and upper arm.Outcome Measures:The primary outcome measures of this study were the ultrasonic indexes such as the endometrial thicknesses and volumes,vascularization index(VI),flow index(FI),vascularization flow index(VFI),subendometrial VI(sVI),subendometrial FI(sFI)and subendometrial VFI(sVFI).Secondary outcome measurements included implantation rate,pregnancy rate,miscarriage rate,live birth rate and number of live births.Results:There were no significant differences in endometrial thickness,endometrial volume,sVI,sFI,sVFI,implantation rate,pregnancy rate,miscarriage rate,live birth rate and number of live births(P>0.05).Compared with the sham acupuncture group,the VI,FI and VFI of the active acupuncture group was significantly higher(P<0.05).Conclusions:For patients undergoing IVF-ET,acupuncture may have beneficial effects on ultrasound markers of VI,FI and VFI,as well as the uterine receptivity during embryo implantation.Part II:Establishment of superovulation rat modelObjectives:To observe the effect of different doses of pregnant horse serum(PMSG)on the mating rate,pregnancy rate,the pinopodes and nucleolus tubular system(NCS)of endometrial glandular epithelial cells and the leukocyte inhibitory factor(LIF)protein expression and explore which dose is suitable for clinical practice.The aim is also to provide a good experimental basis for subsequent acupuncture experiments.Methods:Seventy-five rats were randomly divided into normal group(N),model 10(M10),model 20(M20),model 40(M40)and model 100(M100).PMSG 10 IU,20 IU,40 IU and 100 IU were respectively intraperitoneal injected in the different modle groups.After 48 hours,the rats were given intraperitoneal injection injection of human chorionic gonadotropin(HCG)10 IU,20 IU,40 IU and 100 IU,and immediately caged.On the next day,vaginal smear of seeing a large number of sperms was treat as the first day of pregnancy(D1).On the D8 days,record the pregnancy rate and the number of implanted blastocysts.Another 25 rats were randomly divided into 5 groups as above.The rats in each group were mated and collected the samples on the D5 days.The expression of the pinopodes and NCS of endometrial glandular epithelial cells and the LIF protein expression were observed.Results:There was no significant difference in the mating rate between the five groups(P>0.05).Compared with N group,the pregnancy rate of M10 group was not significantly lower(P>0.05),while the pregnancy rate of M20,M40 and M100 groups were significantly lower than that of N group(P<0.01).The number of blastocysts of M20,M40 and M100 groups was significantly increased than that of N group(P<0.05).The results of electron microscopy showed that there were abundant pinopodes and NCS in endometrial glandular epithelial cells of N group;the expression of pinopodes and NCS in the M10 group was similar to that of N group;the expression of pinopodes and NCS in the M20 group were lower than that of N group;The expression of pinopodes M40 and M100 group were obviously lower than that of N group and there almost no NCS in the M40 and M100 group.The immunohistochemical results indicated that the expression of LIF protein in the endometrium of M10 group was not significantly lower than that of N group;the expression of LIF protein in M20,M40 and M100 groups was significantly lower than that in N group;the expression of LIF protein in M40 and M100 groups was significantly lower than that of M20 group.The west-blot results indicated that the expression of LIF protein in the endometrium of M10 group was not significantly lower than that of N group(P>0.05);the expression of LIF protein in M20,M40 and M100 groups was significantly lower than that in N group(P<0.05).The expression of LIF protein in M40 group and M100 group was less than that in M20 group,but the difference was not statistically significant(P>0.05).Conclusion:There was no significant difference in the pregnancy rate between the M10 and N group,the average number of blastocysts was not high,and the expression of LIF was not decreased,so the M10 was not the ideal model.The pregnancy rate of M20 group was significantly lower than that of normal group,the average number of blastocysts was higher and the expression of LIF was lower than that of N group,so we thought that M20 was the ideal model.The pregnancy rate of M40 and M100 rats was lower than that of the normal group,but the average blastocysts were not higher than the M20 group,so we thought that they were not ideal animal models.Part III:Effect of Acupuncture on Endometrial Angiogenesis and Uterus Dendritic Cells in COH Rats during Peri-Implantation PeriodObjective:Based on the previous studies and theoretical basis,we put forward a hypothesis that acupuncture could improve endometrial angiogenesis during the peri-implantation period and the further mechanism may be by regulating the number and function of uDCs.The purpose of this study is to verify the above hypothesis hoping to provide a scientific explanation for the application of acupuncture in IVF-ET.Methods:Early pregnant rats were randomized into normal group(N),COH group(M),acupuncture group(A),progesterone group(P)and acupuncture plus progesterone group(A+P).The model was established with the pregnant mare serum(PMSG)and human chorionic gonadotrophin(HCG).Samples of endometrium were collected on Day 4,6 and 8(D4,D6 and D8)of gestation.Results:Compared with group M,the number of embryos in the other groups was significantly lower(p<0.05)and pregnancy rate was significantly higher(p<0.05).The endometrial VEGF and FGF-2 protein and mRNA was significantly lower on the D4 and higher on the D6 and D8(p<0.05),except for VEGF of group P on the D8.The LIF protein was significantly higher on the D4 and D6.The endometrial TGF-β protein and mRNA was significantly higher on the D4 and D8 and lower on the D6(p<0.05),except for that of group A on the D6.The endometrial IL-15 protein and mRNA was significantly higher on the D4,D6 and D8(p<0.05),except for that of group P,A and A+P on the D4 and group A on the D6.The proportion of uDCs in the endometrium was significantly lower on the D4 and D6,and higher on the D8(p<0.05).In vitro,the proliferation,tube formation and migration of uDCs was significantly decreased on the D4 and increased on the D8(p<0.05),except for the proliferation of group P on the D8.Acupuncture and progesterone could also regulate the levels of VEGF,IL-15,TGF-β1 and IL-18 secreted by uDCs instead of sFLT-1.Conclusions:Acupuncture may take effect by regulating the endometrial angiogenesis and the number and function of uDCs. |
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