| Objective:1.To explore the clinical effect of Dingkun Dan in the treatment of thin endometrial infertility during ovulation induction cycle,and to analyze the efficacy of Dingkun Dan in assisting pregnancy,so as to provide a simple and effective treatment for patients with thin endometrial infertility.2.A rat model of kidney yang deficiency thin endometrium was established by animal experiments to observe the regulatory effect of Dingkun Dan on serum hormone level,endometrial thickness and endometrial related markers on Wnt/B-catenin signaling pathway in female rats,and to explore the pharmacodynamic mechanism and target of Dingkun Dan in the treatment of thin endometrium.Methods:1.120 patients with thin endometrial infertility at the reproductive medicine department of Jiangsu Provincial Hospital of Traditional Chinese Medicine were selected and divided into dingkundan group,progynova group and combined drug group by random number table method,40 patients in each group.All treatment groups were treated with drugs based on the periodic treatment of induced ovulation(LE+HMG+HCG).Each treatment group started medication on the 9th day of the menstrual cycle,and all of them were taken orally for 15 days consecutively.In the Dingkun Dan group,10.8g/time was taken,twice daily.Progynova group took progynova 1mg/d;The combination group took progynova and DingkunDan at the same time.After ovulation,patients in the three groups were given 20mg of dafentone per day for 15 days.Three menstrual cycles were observed.The changes of menstrual volume,TCM syndrome score,endometrial thickness,endometrial morphology and blood flow grading,endometrial volume,uterine artery blood flow parameters(VI,FI,VFI)and ovulation induction results(E2 level,ovulation rate,pregnancy rate,etc.)were compared before and after treatment in each group.2.Female SD rats with regular estrous cycles were selected and divided into a blank group,a model group,a DingkunDan group,a progynova group and a combined drug group according to the random number table method,with 8 rats in each group.A thin endometrial model with kidney Yang deficiency was established in all groups except the blank group,namely,95%ethanol was used to perform chemical damage to the endometrium of the rats in a surgical method,and the rats were given hydroxyl urea suspension(450mg·kg-·-d-1)by gavage on the next day after the operation for 8 consecutive days.On the second day after the operation,the blank group was given water and food,the model group was given distilled water,and the other treatment groups were given progynova,Dingkun Dan,progynova+Dingkun Dan respectively.After three estrous cycles,serum E2,LH,P,OPN,VEGF and MMP-9 levels were detected by ELISA.HE staining was used to observe the distribution of glands and vessels in each layer of the uterus,morphological changes of luminal or glandular epithelial cells,and endometrial thickness.Immunohistochemical analysis was used to study the expression of CK19,Vimentin and VEGF in the endometrium of rats during estrus.The expression characteristics of β-catenin,VEGF and MMP-9 mRNA in rat endometrium were quantitatively analyzed by RT-PCR.And the expressions of β-catenin,VEGF and MMP-9 protein in the endometrium of rats in each group were detected by Western-blot.Results:1.Clinical ResearchChanges of menstrual volume before and after treatment were compared in each group.The increase of menstrual blood loss chart score in DingkunDan group,progynova group and combined drug group was 14.05±4.80,10.15±3.50 and 15.25±5.30,respectively,with statistically differences(P<0.01).There were statistically differences between the DingkunDan group,the combined drug group and the progynova group(P<0.01).In TCM syndrome scores,there were statistically significant differences in TCM syndrome scores before and after treatment in this group(P<0.01).After treatment,there were significant differences(8.75±6.44 vs 12.35±5.6,P<0.05)between the DingkunDan group and the combined drug group and progynova group(6.64±6.16 vs 12.35±5.6,P<0.01).In endometrial thickness,compared before and after treatment in this group,the increase of endometrial thickness was 1.97±0.36mm,2.03±0.47mm and 2.43±0.64mm respectively in DingkunDan group,progynova group and the combined treatment group,with statistically significant differences(P<0.01).There was a statistically significant difference in intimal thickness between the DingkunDan group and the combined treatment group(P=0.027).In endometrial morphology,the proportion of type A endometrium in the DingkunDan group and the combined treatment group was significantly increased compared with that before and after treatment(22.5%vs 45.0%,P<0.05;15.0%VS 57.5%,P<0.01).In endometrial volume,after treatment,the proportion of endometrial volume≥2.0ml increased significantly in each group,from 10%to 57.5%in the Dingkun Dan group,and from 15%to 72.5%in the progynova group.Among them,the combination group accounted for the largest proportion,reaching 77.5%,but there was no difference between the groups(P>0.05).In endometrial blood flow grading,the proportion of endometrial blood flow grade Ⅲ in this group before and after treatment increased in Dingkun Dan group and progynova group(22.5%vs7.5%,12.5%VS 5.0%,respectively),yet without statistical difference(P>0.05),but there was significant increase in the combined treatment group(35.0%VS 0.0%,P<0.01).In uterine artery blood flow parameters,subendometrial blood flow parameters(VI,FI,VFI)were significantly increased in each group after treatment with significant differences(P<0.01).After treatment,blood flow VI in each treatment group increased compared with that before treatment.Compared with the DingkunDan group,the combination group and progynova group,VI blood flow increased(P=0.005;P=0.004,respectively).As to ovulation induction results,the E2 level of the Dingkun Dan group was lower than that of the progynova group and the combined drug group,with significant statistical difference(P<0.01),while the E,level of the progynova group was lower than that of the combined drug group,with significant statistical difference(P=0.026).Clinical pregnancy rates in Dingkun Dan group and combined drug group were 22.5%and 52.5%,showing a statistically significant difference(P=0.006).Comparing the clinical curative effect,cure rates in dingkundan group and combined drug group were 22.5%,50%respectively,with a statistically significant difference(P=0.011),and the obvious efficiency of Dingkun Dan group was higher than that of the combined drug group(P<0.05).2.Experimental ResearchIn serological test,compared with the blank group,serum E2 level of the model group and each treatment group significantly increased(P<0.01).Compared with the model group,the difference in serum E2 level between the progynova group and the combined drug group was statistically significant(P<0.01,P<0.05,respectively).There was no significant difference in serum LH level between the two groups(P>0.05).Compared with the blank group,the serum P of the model group and each treatment group increased to different degrees,and the difference was statistically significant(P<0.01).Compared with the blank group,serum Levels of OPN,VEGF and MMP-9 in the model group significantly increased(P<0.01).Compared with the model group,OPN and MMP-9 levels in the Dingkun Dan group were decreased(P<0.05,P<0.01,respectively).The serum MMP-9 level in the combined drug group was decreased(P<0.01).Compared with progynova group,serum OPN level of Dingkun Dan group was lower than that of progynova group(P<0.05).Compared with the combined drug group,OPN level and MMP-9 level were lower in Dingkun Dan group(P<0.05).In endometrial morphology and thickness,compared with the blank group,the endometrial stromal cells in the model group were reduced,glands and vessels were sparse,and the endometrium was significantly thinner(P<0.01).Compared with the model group,the glands and blood vessels of each treatment group increased,stromal cells were dense,and the intima was significantly thickened(P<0.01).In immunohistochemical detection,compared with the blank group,the expression area of CK19 in model group significantly reduced(P<0.01).Compared with the model group,the expression area of CK19 in the Dingkun Dan group significantly increased(P<0.05),while the expression area of CK19 in the progynova group and the combined drug group significantly increased(P<0.01).Compared with the blank group,the IOD value of Vimentin in the model group was decreased(P<0.05),while the IOD value of Vimentin in the combined drug group significantly increased(P<0.01).Compared with the blank group,the AOD of VEGF in the model group was significantly decreased(P<0.01).Compared with the model group,the AOD of VEGF in the Dingkun Dan group and the combined drug group showed different degrees of increase(P<0.05,P<0.01).In RT-PCR detection,compared with the blank group,the levels of β-catenin and MMP-9 mRNA in the endometrium of the model group significantly increased(P<0.05),while the levels of VEGF mRNA were significantly decreased(P<0.05).MMP-9 mRNA was significantly decreased in progynova group and the combined drug group(P<0.05).Compared with progynova group,the MMP-9 mRNA of DingkunDan group increased(P<0.05).Compared with the combined drug group,the MMP-9 mRNA of the Dingkun Dan group increased(P<0.05).In Western-blot analysis,compared with the blank group,the levels of β-catenin,VEGF and MMP-9 protein in the model group significantly increased(P<0.01).Compared with model group,β-catenin protein level in each treatment group significantly reduced(P<0.01).The levels of MMP-9 protein in the Dingkun Dan group and the combined drug group were significantly decreased(P<0.01).Compared with progynova group,the level of β-catenin protein was higher and the level of MMP-9 protein was lower in DingkunDan group(P<0.05,P<0.01,respectively).The levels of β-catenin and MMP-9 protein in the combined drug group were significantly decreased(P<0.01).The level of β-catenin protein was higher in the Dingkun Dan group than in the combined drug group(P<0.01,P<0.05,respectively).Conclusion:1.DingkunDan can regulate the function of vegetative nerve and hypothalamic-pituitary-ovarian axis,increase E2 level,produce effects similarly to estrogen.2.Dingkun Dan combined with progynova can increase the menstrual volume of patients with thin endometrial infertility,improve the symptoms of kidney Yang deficiency syndrome,thicken the endometrium,increase the proportion of type A endometrium and blood flow Ⅲgrade endometrium,improve endometrial blood flow perfusion,increase cycle ovulation rate,reduce cycle cancellation rate,and optimize pregnancy outcome.3.Dingkun Dan may reduce OPN and MMP-9 levels,increase the expression of CK19,Vimentin and VEGF in damaged endometrial tissues of rats with kidney Yang deficiency,and promote the repair and regeneration of damaged endometrium in rats after operation.4.The expression of β-catenin and VEGF mRNA in the endometrium of rats with multiple damage was up-regulated by DingkunDan combined with progynova,and the expression of MMP-9 mRNA was down-regulated to improve endometrial tolerance.5.By up-regulation of VEGF expression,down-regulation of β-catenin and MMP-9 protein levels in endometrium of rats with kidney Yang deficiency and thin endometrium,Dingkun Dan combined with progynova promoted the repair and remodeling of damaged endometrium. |
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