The Research On The Effect Of Multi-channel Intervention Of Traditional Chinese Medicine In The Elderly Patients With IVF-ET And The Mechanism Of Tonifying Kidney-activating Blood Recipe Through Retention Enema To Improve The Ovarian Function Of Kidney De

Objective:To study the clinical efficacy of multi-channel intervention of traditional Chinese medicine（TCM）in elderly infertility patients undergoing in vitro fertilization-embryo transfer（IVF-ET）again（kidney-yin deficiency-liver stagnation-blood stasis type）.The specific mechanism of improving ovarian function of kidney deficiency and blood stasis-diminished ovarian reserve（DOR）model rats by tonifying kidney and promoting blood circulation through retention enema was studied.Methods:1.Clinical research:a total of 121 elderly patients（second or third class in The novel POSEIDON stratification）with secondary IVF-ET（kidney Yin deficiency-liver stagnation-blood stasis type）were enrolled by two-way cohort study.In the treatment group,a total of 61 patients were treated with multi-channel intervention of TCM（oral Zishen Zhuyun recipe+retention enema+auricular point application）.A total of 61 in the control group was treated with DHEA,and the two groups were treated for three menstrual cycles.The treatment was able to observe the changes of natural pregnancy rate,TCM syndrome score,reproductive hormone level（FSH,LH,FSH/LH,E2）,Gonadotropin（Gn）use（Startup dose,total dose,days used）,embryo quality related indicators（number of follicles obtained,number of mature follicles,rate of mature follicles,number of fertilization,rate of fertilization,number of normal fertilization,rate of normal fertilization,number of superior embryos,rate of superior embryos）and pregnancy outcome（clinical pregnancy rate,biochemical pregnancy rate,ectopic pregnancy rate and early abortion rate）in the two groups.2.Experimental research:the SPF 6～7 weeks SD female rats were duplicated kidney deficiency-blood stasis-DOR model rats by tripterygium wilfordii polyglycosides combining with adrenaline,and randomly divided into blank group,model group,tonifying kidney-promoting blood circulation through retention enema（TKPBRE）high/medium/low dose group,TCM control group and western medicine control group,according to the requirements of the experimental intervention,then observation the change of each group rats,including generally situation,ovarian wet weight,HE staining was used to observe the morphological and pathological changes of ovarian tissues.Hormone levels（FSH,AMH,INHB,E2）and blood coagulation function were determined by Elisa.The expression of BMP-15 protein was detected by immunohistochemistry,and the m RNA expressions of TGF-beta1,smad3 and p38MAPK were tested by Real-time Quantitative PCR Detecting System.Results:1.Clinical research:（1）the natural pregnancy rate in the treatment group was 8.19%（5/61）,the clinical pregnancy rate was33.33%（12/36）,the biochemical pregnancy rate was5.55%（2/36）,the early abortion rate was 16.67%（2/12）,and the ectopic pregnancy rate was0%（0/36）,The recovery rate of IVF-ET in elderly patients was 27.86%（17/61）,significantly better than the control group（P<0.05）.（2）Improving abnormal condition of menstrual cycle or volume and,the score of main/secondary symptoms,the disappearance rate of single symptom and the total effective rate of TCM syndromes in the treatment group were better than that in the control group（P<0.05）.（3）Comparison within the treatment group reduced FSH,FSH/LH,and increased LH（P<0.05）,and improved E2 and FSH/LH better than the control group（P<0.05）.（4）Comparison within the treatment group reduced the Gn start-up dose and the total amount of Gn（P<0.05）,and there was no difference with the control group（P>0.05）.（5）the number of follicles obtained,the number of mature follicles obtained,the number of fertilizationwere increased in the treatment group（P<0.05）,and the number of follicles obtained and superior embryos were significantly improved in the treatment group compared with the control group（P<0.05）.（6）The incidence of adverse reactions in both groups was 0%,and the safety grade was level one,and both types of intervention are safe.2.Experimental research:（1）Compared with the blank group,the general situation of rats was poor,the wet weight of ovary and the index of ovarian viscera decreased（P<0.05）,the follicles at all levels decreased significantly（P<0.05）,the atretic follicles increased（P<0.05）,the values of AMH,INH-B and E2 decreased（P<0.05）,the values of FSH increased（P<0.05）,the values of APTT,PT and TT decreased（P<0.05）,the values of FIB increased（P<0.05）in the model group.（2）the number of follicles at all levels in the TKPBRE medium dose group and the western medicine control group increased significantly,the number of atretic follicles decreased,the wet weight of the ovary and the index of ovarian organs increased（P<0.05）.（3）the FIB value decreased and the APTT,PT,TT values increased in the TKPBRE medium dose group（P<0.05）.Except the TKPBRE medium and low dose group,the AMH expression increased in the other groups.The expression levels of INHB and E₂increased in each group,while the expression levels of FSH decreased（P<0.05）.（4）in the TKPBRE medium and high-dose group,the TCM control group and the western medicine control group,the m RNA expressions of TGF-β1,Smad3 and p38MAPK were significantly up-regulated（P<0.05）.In the TKPBRE high,medium and low-dose group and the western medicine,TCM control group,the protein content of BMP-15 in ovarian tissues was increased（P<0.05）.（5）TGF-β1/smad3,p38MAPK signaling pathways were positively correlated with AMH,INHB,E₂,and negatively correlated with FSH.However,from the correlation coefficient analysis,TGF-β1 was strongly correlated with AMH,INHB,FSH and E₂.Smad3 was strongly correlated with AMH and INHB,and strongly correlated with FSH and E₂.P38MAPK was strongly correlated with AMH,INHB,FSH and E₂.Conclusions:1.Clinical research:（1）Multi-channel intervention of TCM in elderly patients with secondary IVF can improve the clinical symptoms of kidney yin deficiency-liver stagnation-blood stasis,reduce the TCM syndrome score,and improve the natural pregnancy rate.（2）Multi-channel intervention of TCM achieves the effect of assisting pregnancy by improving reproductive endocrine level and the ovarian response,reduction the Gn usage amount,improving embryo quality,and increase the clinical pregnancy rate,reduce the abortion rate.2.Experimental research:（1）Tripterygium wilfordii polyglycosides combined with epinephrine successfully replicated the DOR rat model of kidney deficiency and blood stasis.（2）Tonifying kidney and activating blood can improve the general condition,ovarian wet weight,ovarian hormone level and coagulation function of model rats;It positively regulated the expression of BMP-15 protein,TGF-β1m RNA,smad3m RNA and p38MAPKm RNA.（3）The method of tonifying kidney and activating blood circulation can improve the ovarian function of the decreased ovarian reserve function of kidney deficiency-blood stasis model rats,which may be realized by regulating TGF-β1/smad3 and p38MAPK signaling pathways.