| Objective:1.To evaluate the efficacy and safety of sequential therapy of promoting blood circulation-regulating liver-tonifying kidney for EM related infertility,and to evaluate its effect on ovarian function.2.Continuous dynamic observation of follicular development and apoptosis in the emotional cycle of EM rats,to preliminarily explore the effect of EM disease on follicular development.3.From the perspective of oxidative stress mediated apoptosis of granule cells,to preliminarily explore the mechanism of improving follicular development in rats by promoting blood circulation-regulating liver-tonifying kidney traditional Chinese medicine.Methods:Clinical study:Effect of sequential therapy on ovarian function in EM infertility patientsA randomized,double-blind,placebo-controlled,multicenter,prospective clinical study.204 cases of EM related infertility qi stagnation blood stasis type after laparoscopic surgery were randomly divided into 2 groups,according to the ratio of 1:1 by central randomization system for clinical research.The patient began to take preovulatory decoction 14 doses after conservative surgery for endometriosis.Take the medicine from day 2 to 5 after the arrival of the first postoperative menstrual tide,before ovulation take preovulatory decoction,and after ovulation take postovulatory decoction.If the follicular luteinization or no ovulation occurs,the preovulatory decoction is changed to the postovulatory decoction after 14 days.Continuous use of 6 menstrual cycles.Stop taking medication after pregnancy is found.TCM group was treated by sequential therapy of promoting blood circulation-regulating liver-tonifying kidney,the preovulatory decoction is Huoxuexiaoyi decoction,and the postovulatory decoction is Bushengutai decoction.In placebo group the preovulatory and postovulatory decoction were placebo.After the drugs are blinded by a third party,they are dynamically and randomly distributed by the central randomization system for clinical research.The study was blind to patients,patient recruiters,clinical researchers,drug managers,outcome assessors,data managers,and statisticians.During the 1st,3rd and 6th menstrual cycles after surgery,serum FSH,LH and E2 were tested on day 2-5 of the menstrual period,and serum E2 and P were detected on day 6-9 of the middle luteal body.Since the first menstrual cycle postoperative,each menstrual period of 2 to 5 days ultrasonic examination of antral follicle count(AFC),vaginal ultrasound monitoring ovulation,give birth guidance according to the ovulation,and fill in the TCM symptom assessment after each cycle is clean.Safety examinations(Blood routine,urine routine,liver and kidney function,electrocardiogram)are required before taking medicine and after completing 6 menstrual cycles of medication.If a pregnancy is found during the period of medication,take safety examinations within 7 days,and followe up the pregnancy outcome.During the study,adverse events of patients were closely observed and recorded at any time.Periodic clinical monitoring was conducted to ensure the quality of the study.After the completion of the blind state audit,the blindness was removed for the first time.The professional staff of the central randomization system for clinical research informed the statisticians of the groups A and B corresponding to the random Numbers.After completing the statistical analysis of the data,the blindness was removed for the second time.The professional staff announced that group A and group B were the TCM or placebo groups.Animal experiment:Effect of Chinese medicine on follicle development and apoptosis in EM model ratsAfter 2 weeks of vaginal smears in 225 female SD rats,210 rats with normal estrus cycles were screened.They were randomly divided into 4 groups,each 28 in the blank group,sham operation group and donor group,and 126 in the building modules group.The EM rat model was established by allogeneic uterus transplantation.After the successful decision,the building modules group was randomly divided into 4 groups,model group,huoxuexiaoyi group,tonify kidney group and sequential therapy group,30 in each group.The blank group,the sham operation group and the model group were given distilled water for 15 days.The huoxuexiaoyi group was given Huoxuexiaoyi decoction for 15 days.The tonify kidney group were given Bushengutai decoction for 15 days.The sequential therapy group was given 3 days for Huoxuexiaoyi decoction plus 2 days for Bushengutai decoction,a total of 15 days.In each group,the recovery of estrous period after operation was observed?the uterus and ovary tissues were taken at the pre-estrus,the estrus,the post-estrus and the anestrus,and calculate the organ index of uterus and ovary.HE and TUNEL staini technique used to observe the development of ovarian follicles,and the number of pre-sinus follicles,sinus follicles,mature follicles,apoptotic follicles and corpus luteum were calculated.The level of ROS,SOD,CAT and GSH in serum was measured by colorimetry.Results:Clinical study:1.A total of 204 patients were included in the study,each 102 in the TCM group and placebo group.10 cases(9.8%)of TCM group were lost,1 case was removed,and 91 cases were completed.13 patients(12.7%)in the placebo group were lost,2 were removed and 87 were completed.There was no emergency breaking blindness during the study.2.There was no statistically significant difference between the two groups in demographic general condition and baseline condition of disease before treatment,P>0.05.The mean age of TCM group was 29.1± 3.24 years,placebo group was 29.76±2.96 years.In the two groups BMI was in the normal range,college degree or above and brainworkers are mainly.There were 57 cases of primary infertility(62.6%)in TCM group and 55 cases(63.2%)in the placebo group.Before the operation,most of the two groups were combined with ovarian endometriosis and dysmenorrhea.Ovarian endometriosis was mainly unilateral.The duration of ovarian endometriosis was 12.75±15.07 months in TCM group and 16.71± 18.19months in placebo group.The duration of dysmenorrhea was 94.45±80.77months in TCM group and 68.25±65.52months in placebo group.The age of menarche in TCM group was 13.48± 1.31 years,the menstrual cycle was 29.54±2.84 days,and the menstrual period was 5.91±1.42 days.In the placebo group,the age of menarche was 13.14±1.11 years,the menstrual cycle was 29.24±12.73 days,and the menstrual period was 5.94±1.22 days.In the two groups r-AFS stage III and EFI scores greater than 4 points were mainly.The r-AFS score of TCM group was 32.20±25.87,and EFI score 7.75±1.22.The r-AFS score of placebo group was 29.46±26.92,and EFI score 7.95±1.25.The r-AFS stage I,II,III and IV in TCM group was 9 cases(9.9%),15 cases(16.5%),and 44 cases(48.4%),23 cases(25.3%)and placebo groups 11 cases(12.6%),19 cases(21.8%),37 cases(42.5%),20 cases(20.0%).3.In terms of clinical pregnancy,45 pregnancies in TCM group(49.5%)and 30 pregnancies in the placebo group(34.5%),the pregnancy rate of TCM group was significantly higher than that of the placebo group P<0.05,OR 1.859,95%CL 1.065-3.396,RR 1.434,95%CL 1.035-2.173.In the postoperative 3 months,32 cases(35.2%)in TCM group and 25 cases(28.7%)in the placebo group were pregnant.There were 13 pregnancies(14.3%)in TCM group and 5 pregnancies(5.7%)in the placebo group between 4 to 6 months after surgery.In both groups,the number of pregnancy cases within 3 months after surgery was significantly higher than that between 4 and 6 months after surgery,and the difference was statistically significant,P<0.05.4.In terms of early pregnancy loss,there were 10 cases(22.2%)in the TCM group and 9 cases(30%)in the placebo group.Among them,there were 2 cases of biochemical pregnancy(4.4%),1 case of ectopic pregnancy(2.2%),3 cases of embryo sterilization(6.7%)and 4 cases of spontaneous abortion(8.9%)in the TCM group.In the placebo group,there were 3 cases(10%)of biochemical pregnancy,2 cases of ectopic pregnancy(6.7%),2 cases of embryo sterilization(6.7%)and 2 cases of spontaneous abortion(6.7%).There was no statistically significant difference between the two groups,P>0.05.5.In terms of continuing pregnancy,after 12 weeks of pregnancy,35 cases(38.5%)in the TCM group and 21 cases(24.1%)in the placebo group continued pregnancy.The rate of continuous pregnancy in the TCM group was significantly higher than that in the placebo group,and the difference was statistically significant,P<0.05,OR 1.964,95%CL 1.028-3.754;RR 1.593?95%CL 1.012-2.510.6.In terms of basic ovarian function,the levels of FSH,LH,FSH/LH and E2 were mostly within the normal range in the first,third and sixth menstrual cycles after surgery,the difference between the two groups was not statistically significant,P>0.05.The difference between the two groups at different time points was not statistically significant,P>0.05.At the 1st to 6th menstrual cycles after surgery,the number of AFC in each cycle between the two groups was not statistically significant,P>0.05.The difference between the two groups at different time points was not statistically significant,P>0.05.7.1n terms of follicular development,in TCM group the incidence of accumulated cycle dominant follicle was 94.43%,the cumulative cycle ovulation rate was 80.06%,the ovulatory rate of mature follicle was 64.15%,and all of them were significantly higher than those in the placebo group90.43%,69.7%,54.35%,P<0.05.The cumulative incidence of periodic follicular lutein in the TCM group was 12.08%,significantly lower than placebo group 18.18%,P<0.05.The 1 st to 6th menstrual cycle after surgery,per menstrual cycle incidence of dominant follicle and the ovulation rate of mature follicle was compared between two groups,the difference was not statistically significant,P>0.05.The 1st to 5th menstrual cycle after surgery,per menstrual cycle ovulatory rate and the incidence of follicular luteinization was compared between the two groups,the difference was not statistically significant,P>0.05.The 6th menstrual cycle after surgery,the ovulation rate of the TCM group was 82.5%?significantly higher than placebo group 62.5%;the incidence of follicular lutein in TMC group was 5%,significantly lower than the placebo group by 22.92%,the difference was statistically significant,P<0.05.8.In terms of luteal function,E2 and P levels in the middle luteal body of patients with the 1st,3rd and 6th menstrual cycles after surgery were compared between the two groups,and the difference was not statistically significant,P>0.05.The difference between the two groups at different time points was not statistically significant,P>0.05.At the 1st,3rd and 6th menstrual cycles after surgery,the incidence of luteal insufficiency in ovulation patients with each menstrual cycle was compared between the two groups,and the difference was not statistically significant,P>0.05.The incidence of luteal insufficiency in patients with cumulative cycle ovulation in the Chinese medicine group was 20.5%,lower than that in the placebo group by 32.9%,and the difference was statistically significant,P<0.05.9.In terms of TCM symptom score,there was no significant difference between the two groups in preoperative and postoperative scores and total scores at the first month,P>0.05.The scores and total scores of patients in the two groups were significantly lower in the first month after surgery than before surgery,with statistically significant difference P<0.05.In TCM group postoperative 6 menstrual poor ratings,postoperative 3-6 menstrual breast pain score before and postoperative 4-6 months before menstruation upset irritability score,postoperative 3-6 months total score were significantly lower than that of the placebo group,the difference was statistically significant,P<0.05.For the rest of the time points and symptom scores,there was no statistically significant difference between the two groups,P>0.05.The postoperative Chinese medicine group experienced dysplasia,anterior breast distention,anterior lower abdomen distention,abdominal pain,peritoneal irritability score and total score gradually decreased with the treatment time,the difference between measurements at different time points was statistically significant,P<0.05.There was no statistically significant difference between the postoperative placebo group at different time points,P>0.05.10.In terms of safety,28 cases(27.7%)of adverse events occurred in the Chinese medicine group and 20 cases(19.8%)in the placebo group.There was no statistically significant difference between the two groups,P>0.05.Adverse events mainly included cold,vulva pruritus,upper respiratory tract infection,and no serious adverse events occurred in both groups.20 patients in the traditional Chinese medicine group(19.8%)had combined medication,and 11 patients in the placebo group(10.9%)had no statistically significant difference between the two groups,P>0.05.The combined drugs include antibiotics,painkillers and antifungal drugs.Six patients in the Chinese medicine group had adverse reactions(5.6%),mainly manifested as mild diarrhea and nausea,which were tolerable and disappeared after drug withdrawal.No significant adverse reactions occurred in the placebo group.Animal experiments:1.Comparison of estrous cycle recovery:After the interference of estradiol valerate,modeling and surgery,the estradiol of the blank group was the fastest to recover the estrous phase,and the model group was the last to recover the estrous phase,showing statistical significance compared with other groups,P<0.05.The sham operation group,sequential therapy group,huoxue xiaoyi group and tonify kidney group gradually recovered their estrous phase,and the comparison difference between the four groups was not statistically significant,P>0.05.2.Comparison of celiac adhesion:The peritoneal adhesion was the most serious in the tonify kidney group,followed by the model group.The difference between the two groups was not statistically significant,P>0.05.There was no statistically significant difference between the sequential treatment group and the huoxue xiaoyi group in the comparison of abdominal adhesion between the two groups,P>0.05.However,the difference was significantly less than that of the tonify kidney group and the model group,and the difference was statistically significant,P<0.05.There was no obvious adhesion between the blank group and the sham operation group.3.Comparison of ovarian and uterine conditions:There was no statistically significant difference in ovarian index and uterus index between each period of estrus cycle and each group,P>0.05.4.Comparison of follicular development:Number of pre-sinus follicles:The blank group was the largest,significantly higher than other groups,and the difference was statistically significant,P<0.05.Comparison of the number of pre-sinus follicles between the sham operation group and huoxue xiaoyi group showed no statistically significant difference,P>0.05,but both significantly higher than sequential treatment group,model group,tonify kidney group,the difference was statistically significant,P<0.05.Number of sinusoidal follicles:The blank group was significantly more than the other 5 groups,and the model group was significantly less than the other 5 groups.The difference was statistically significant,P<0.05.The sham operation group,sequential therapy group,huoxue xiaoyi group and tonify kidney group decreased successively,and the comparison difference between the groups was not statistically significant,P>0.05.Number of mature follicles:Sequential therapy group,huoxue xiaoyi group,sham operation group,blank group,tonify kidney group and model group decreased successively,and there was no statistically significant difference between each group,P>0.05.Number of apoptotic follicles:The sham operation group,the blank group,the sequential therapy group,the huoxue xiaoyi group,the tonify kidney group and the model group increased successively,and the comparison difference between the groups was not statistically significant,P>0.05.Number of corpus luteum:There was no statistically significant difference between the number of corpus luteum in the blank group and the sham operation group,P>0.05.The difference was statistically significant,P<0.05.The number of corpus luteum decreased successively in the tonify kidney group,the sequential therapy group,the huoxue xiaoyi group and the model group,and there was no statistically significant difference between the groups,P>0.05.5.Comparison of serum ROS,SOD,CAT and GSH contents:ROS:The serum ROS content of the model group and the tonify kidney group was similar,with no statistically significant difference,P>0.05;but significantly higher than that of the other four groups,P<0.05.Serum ROS content in the huoxue xiaoyi group,sham operation group,blank group and sequential therapy group decreased successively.Serum ROS content in the huoxue xiaoyi group was higher than that in the sequential therapy group,P<0.05.The difference between the other two groups was not statistically significant,P>0.05.SOD:The serum SOD content in sequential therapy group was significantly higher than that in the other 5 groups,and the difference was statistically significant,P<0.05.The blank group,the sham operation group,and the huoxue xiaoyi group were all higher than those of the tonify kidney group and the model group,P<0.05.There was no statistically significant difference between the blank group,the sham operation group and the huoxue xiaoyi group(P>0.05).There was no statistically significant difference between the two groups(P>0.05).CAT:The serum CAT content in the model group was significantly lower than that in the other 5 groups,and the difference was statistically significant,P<0.05.The difference between the two groups was statistically significant(P<0.05).GSH:The serum GSH content in the model group was significantly lower than that in the other 5 groups,and the difference was statistically significant,P<0.05.The serum GSH content of the sequential therapy group was significantly higher than that of the sequential therapy group(P<0.05).The difference between the other groups was not statistically significant,P>0.05.The content of ROS,SOD,CTA and GSH in each group at different stages of estrus cycle was not statistically significant,P>0.05.Conclusion:Clinical studies have found that Chinese medicine promoting blood circulation-regulating liver-tonifying kidney sequential therapy can effectively improve the Qi stagnation blood stasis type EM infertility patients with laparoscopic postoperative pregnancy rate,improve follicular development,promote the ovulation,reducing the occurrence of follicular luf,improve ovulation cycle luteal function,improve symptoms of traditional Chinese medicine,and does not increase the risk of early pregnancy loss,good security,worth extending application.It was found that EM affected follicular development and apoptosis in all stages of rat estrus cycle.huoxue xiaoyi decoction have strong antioxidant effect,can significantly reduce the number of EM model rats abdominal cavity adhesion,reduce the post-estrus and the anestrus period follicle apoptosis,promote anestrus period sinus follicle development,improve follicular development.The development of sinus follicles can be enhanced by tonify kidney decoction,but the anti-oxidation effect is weak.The use alone will aggravate the peritoneal adhesion of EM model rats and accelerate the apoptosis of oocytes during estrous period.The anti-oxidation effect of TCM sequential therapy is better than that of the single huoxue xiaoyi decoction,which can effectively reduce the abdominal adhesion,promote the number of sinus follicles,reduce the apoptosis of follicles during estrous and anestrus periods,and improve the development of follicles. |
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