A Preliminary Study Of Ovulation Induction Mechanism And Application Of Bushen Tiaogan Chinese Herbal Medicine

BackgroundPloy cystic ovarian syndrome is the most common reason of ovulation failure, the main features are Anovulation, Polycystic Ovaries and Hyperandrogenism or the performance of Hyperandrogenism The clinical manifestations is various,Including Abnormal menstruation (oligomenorrhea, amenorrhea, dysfunctional uterine bleeding), infertility, hirsutism, acne, obesity, and often accompanied by insulin resistance, hyperinsulinemia and hyperlipidemia; some patients with elevated serum prolactin concentration. Correlation with early pregnancy loss, late pregnancy complications and increased risk of ovarian cancer. There is no concensus about the pathogenesis and diagnosis of the disease currently, the main measures to care about this disease including diet, medication and surgery.unfortunately, some patients are failure after standard management.We receive Good clinical efficacy when treated PCOS with herbs with Bushen and Tiaogan. Unfortunately,most herbs are applicating with experience,lack of experimental evidence and repeatability. previous experiment by Professor Xing Fuqi indicated that some kidney reinforcing herbs such as Curculigo, Morinda, Cistanche, Cuscuta, Epimedium, Rehmannia glutinosa has the capability of increasing FSH secretion in rat pituitary cells. And Paeonia, licorice, Curculigo, Morinda can significantly reduce the prolactin secretion of rat anterior pituitary cells. This study is based on the data from previous researchs and theory of treating PCOS from "kidney and liver" in traditional medical. We select Curculigo, Morinda, Cuscuta, Epimedium, white peony and licorice to explore the possible ovulation mechanism of Bushen Tiaogan herbs. observation the clinical efficacy of these drugs in patients with polycystic ovary syndrome.PartⅠstudy of Ovulation induction mechanism of Chinese herbal medicineObjectiveTo investigate the effects of the Bushen Tiaogan heabals which compose with Curculigo, Morinda, Cuscuta, Epimedium, white peony and licorice in anovulatory mouse. And study the effects of the expression of the growth differentiation factor -9 in follicles; then exploring the possible ovulation mechanisms of these herbs.Method1. building of Anovulation mouse model and subgroup:120 5-days-old female mice were enrolled, randomly 100 subcutaneous injection of testosterone propionate 0.04ml (1ml:25mg) in the neck, The others subcutaneous injection of 0.04mL of tea oil as control. The ones injected with testosterone mice were randomly divided into 5 groups, group A treated with low-dose herbs, group B with moderate dose herbs, group C with high-dose herbs, groups D with clomiphene citrate snd group E group as model contro. Since 8-weeks-old we observed the vaginal smears daily, ten consecutive days of a sustained smear tips made of keratinocytes suggest a successful model.2. Since Anovulation mouse model establishment, all the mouse were intragastric administration 0.5ml fluid daily ten days, Group A with 0.5g/ml herbs, B with 1.0g/ml herbs, C with 2g/ml herbs, d with 1.2mg/ml CC and distilled wate for E,F.3. all the mouse were weighed 24 hours after last administration, collected 1.0ml arterial and venous blood by removal of the eye, the serum was separated after centrifugation and stored at 4℃refrigerator, killed by cervical dislocation after blood collection, then cut the abdominal wall, peritoneum, along the uterus up to find mouse ovarian tissues, and separate the uterus and ovaries, removing the surrounding fascia and adipose tissue, rapid investment in fixed 4% formaldehyde solution after weighing. calculated the uterus index (mg/100g) and ovarian index (mg/100g).4. radioimmunoassay for the detection of serum estradiol and FSH concentration. immunohistochemistry (SABC) Determination of mouse ovary Expression of growth differentiation factor -9, setting one as control by replacement primary antibody with PBS. According to the following criteria for interpretation of immunohistochemical results with blind reading. Positive standard:Each stained slice were randomly selected five vision which with clear background,and color was brown cytoplasm as positive, according to the intensity of the positive particles and the proportion of positive cells following judgments were divided into (-),(+),(++),(+++) 4 levels.5. The serum estradiol concentration, index of uters and ovaries were demonstrated with Mean±standard deviation, and comparison the difference with one way ANOVA and LSD(if multiple comparison was needed),the expression of GDF-9 and the composition of follicles were assessed by K Independent Sample nonparametric test。The difference was significantif the P value less than 0.05. All the data were statistically treated by adopting statistical package of SPSS 19.0.Results1. The uterine index, ovarian index, serum FSH concentration and E2 concentration of mouse were significantly different among each group. The surum FSH concentration were significantly high than PCOS model group when comparison with LSD. And the serum E2 level were significantly different between every two group, which clomiphene citrate group> high dose herbs group>mid-dose herbgroup> low-dose herbs group> control group> the PCOS model group; the uterine index are similar when high-dose ones vs mid-dose ones and CC group vs control groupof (P values were 0.838 and 0.974 respectively),and the distinction between other groups are significantly (all P value less than 0.05), which high-dose group> low-dose group> clomiphene citrate group and the control group> the model group; the ovarian index were significantly high in PCOS model group when comparison with other 5 groups.2. In model group, there are more primordial follicles and cystic expansion and less growing follicles. On contrast,with significantly increased mount of dominant follicle and mature corpus luteum in Chinese herbs medicine groups and clomiphene group. The constituent ratio of follicle follicles were significantly different (chi-square value 22.561, P value 0.000).3. In each groups, the immunohistochemical staining intensity of GDF-9 was significant difference, the Chinese herbal medicine groups were higher than PCOS model group. Conclusion1. The Curculigo, Morinda, Cuscuta, Epimedium, white peony and licorice can promote follicle development in anovulation mice2.3. Curculigo, Morinda, Cuscuta, Epimedium, white peony and licorice increased the expression of GDF-9 in follicles of anovulation mice, and it is the possible mechanism of the follicle development promotion of Bushen Tiaogan herbs.3. anovulation model in mice was successfully builded by injection with testosteronePartⅡStudy of the effects of Bushen Tiaogan Chinese herbal medicine in patients with PCOSObjective: To investigate the clinical effects of Curculigo, Morinda, Cuscuta, Epimedium, white peony and licorice in patients with PCOS, and explore the difference between those herbs and other medicines suck as clomiphene citrate and letrozole.Methods1. Subjects:patients who visit ART center of Nanfang hospital between Dec 2008 and Dec 2010.Diagnosed with ploycystic ovary syndrome (according to the Diagnostic criteria of Japan Society of Obstetrics and Gynecology 2007), required inducing ovulation. All patients are confirmed that at least one side normal tube with hysterosalpingography or laparoscopy, the density of sperm are more or equal to 20×106/ml, the ratio of a plus b is more or equal to 30 percent. 111 patients who met all the condition were included to our study.2. Subgoup:All patients were randomized (random table method) is divided into A, B, C three groups. Group A with Bushentiaogan Chinese herbal medicine (Cuscuta seed 20g, Morinda 20g, Cuscuta 20g, Epimedium 10g, paeoniae alba 20g, Glycyrrhiza uralensis 10g); Group B with clomiphene citrate (100mg×5d) and Group C with letrozole (5mg×5d). there are 38,37 and 36 in group A,B and C respectively. And complete 101,102,101 ovulation induction cycle in group A,B and C.3. Primary outcome:Srum estradiol concentration, serum PRL concentration, endometrial thickness and the number of mature follicles (follicle diameters≥14mm) on the day of hCG administration. clinical pregnancy, multiple pregnancy rate and the incidence of ovarian hyperstimulation syndrome.4. Statistical Analysis:The serum hormone concentration, BMI, HOMA-IR and other Enumeration data were demonstrated with Mean±standard deviation, and comparison the difference with one way ANOVA and LSD(if multiple comparison was needed), the measurement data were documented with percentage(%) and assessed by K Independent Sample nonparametric test。The difference was significantif the P value less than 0.05. All the data were statistically treated by adopting statistical package of SPSS 19.0.Results1. basic information:there are no significant difference between A, B, and C groups such as age, body mass index, basic serum sex hormone(luteinizing hormone, follicle-stimulating hormone, testosterone, prolactin, estradiol) on the day 2 or 3 of the menstruation cycle.2. The concentration of serum estradiol on the day of hCG administration were 381.1±134.5pg/ml,759.0±506.4pg/ml,560.3±259.9pg/ml in group A, B and C respectively,and the serum prolactin were:16.75±9.29pg/ml,24.48±17.72pg/ml,25.22±18.93pg/ml; mature follicles (≥14mm) were 1.35±0.71, 2.77±1.69,1.89±0.91; endometrial thickness were:10.01±2.19mm,8.69±2.04mm,8.81±2.07mm respectively. there are significantly different between 3 groups including serum estradiol and prolactin levels, the number of mature follicles and endometrial thickness on the day of hCG administration. the number of mature follicles in group A was significantly lower than other groups, on contrast, endometrial thickness in Bushentiaogan Chinese herbal medicine group was significantly higher than the clomiphene citrate group and the letrozole group (P<0.01);3. comparison of ovulation rate and clinical pregnancy rate The cumulative ovulation rate was 67.3%,69.6%,63.4% in group A, B and C respectively, cumulative pregnancy rate was:31.6%,35.1%,30.6%,4. it is no significant difference when analysis with chi-square test (chi-square value is 0.192, P value is 0.908); there were no significant difference too concerning about ova5. Medicines, clomiphene citrate and letrozole group, chi-square value of 0.994, P value to 0.608, no significant difference; cumulative pregnancy rate was:31.6%, 35.1%,30.6%, chi-square value of 0.192, P value of 0.908, no significant difference. Ovulation rate per cycle and clinical pregnancy rate was no significant difference; Chinese patients without multiple pregnancies occurred in patients with B-1 under the confirmed ectopic pregnancy, patients with clomiphene citrate multiple pregnancy rate was 23.1%(3), of which 7.7%(1 case) were three pregnancies, letrozole patients multiple pregnancy rate of 18.2%(2 cases),-3 groups had no significant difference (chi-square value is 0.975, P value is 0.614).Conclusion1. Curculigo, Morinda, Cuscuta, Epimedium, white peony and licorice for patients with PCOS can be achieved successfully ovulation, the ovulation rate, pregnancy rate and clinical pregnancy rate per cycle is equal to clomiphene citrate and letrozole groups;2. comparison with CC or LE, there were such adventages:highly Single follicular development rate, little effect on the endometrium;3. There is no evidence that the herbs is superior than CC and LE, and the use of the herbs is unclear.so we do not recommend those herbs as first-line medicine for patients with PCOS.4. Within patients that recuurent failure by CC or LE, expecially ones who were cancelled because of multi-follicular development, the thickness of the endometrial is too thin and drug resistance., the administration of those herbs maybe give us astonish outcomes.Full-text Summary1. anovulation model in mice was successfully builded by injection with testosterone2. The Curculigo, Morinda, Cuscuta, Epimedium, white peony and licorice can promote follicle development in anovulation mice3. Curculigo, Morinda, Cuscuta, Epimedium, white peony and licorice increased the expression of GDF-9 in follicles of anovulation mice, and it is the possible mechanism of the follicle development promotion of Bushen Tiaogan herbs.4. Curculigo, Morinda, Cuscuta, Epimedium, white peony and licorice for patients with PCOS can be achieved successfully ovulation, the ovulation rate, pregnancy rate and clinical pregnancy rate per cycle is equal to clomiphene citrate and letrozole groups; as well as the thickness of the endometrium is thick than others5. This is no evidence that Bushen Tiaogan herbs are better than CC or LE, and lackness of the secrurity of herbs aderministration, we did not suggest those herbs take the position of CC as the first-line ovulation drugs. However, Within patients that recuurent failure by CC or LE, expecially ones who were cancelled because of multi-follicular development, the thickness of the endometrial is too thin and drug resistance., the administration of those herbs maybe give us astonish outcomes.