| Part1 Literatures researchObjective:To elaborate Diminished Ovarian Reserve (DOR) on etiology、pathogenesis、 diagnosis、treatment and their research progress in detail by reviewing the existing literatures of. DOR in the last decade. It can provide evidences for diagnosis and treatment of DOR.Methods:By consulting the domestic and foreign literatures in CNKI and PUBMED nearly 10 years, it has elaborated detailedly disease name、epidemiology、 etiology、pathology and physiology、Clinical manifestation、diagnostic standard、therapeutic method of DOR, assessment methods of ovarian function and DOR、premature ovarian failure (POF) animal models from the perspective of modern medicine;it has also elaborated detailedly traditional chinese medicine(TCM)name of DOR、etiology and pathogenesis、treatment based on syndrome differentiation from the perspective of TCM.Results:DOR is a kind of disease which has complex etiology and pathogenesis, highly characteristic clinical manifestations. The standard of diagnosis and therapeutic plan have no reached a consensus. Age、AMH、AFC、INHB are importantly valuable indicators for evaluateing ovarian function。 The treatment of DOR and effect evaluation of ovarian function are the diffi-culties. As a result, the focus of DOR clinical research is to explore the ideal ways and methods to enhancing therapeutic effect and decreasing side-effect. TCM holds that the basic pathogenesis of DOR is kidney deficiency, combining liver stangation and/or spleen deficiency and/or blood stasis. Kidney deficiency and liver stangation syndrome (KDALSS) is the most common syndrome of DOR. Therefore, therapeutic principle of DOR is tonifying the kidney, smoothing the liver and strengthening the spleen, promoting the blood circulation.Conclusions:DOR is a kind of disease which has complex etiology and pathogenesis, highly characteristic clinical manifestations. TCM holds that the basic pathogenesis of DOR is kidney deficiency, combining liver stangation and/or spleen deficiency and/or blood stasis, involve kidney, liver, spleen, heart. Kidney deficiency and liver stangation syndrome(KDALSS) is the most common syndrome of DOR in clinical. The therapy of TCM on DOR have unique advantages and characteristics, and it is worthy of exploring and researching. Yijing decoction is a famous prescription of Fuqing in Qing period for treating menstruation delay and amenorrhea.Part2 Study of Jiajian Yijing decoction on ovarian function and Transform-ing growth factor-β superfami ly members in DOR patients of Kidney deficien-cy and hepatic depression syndromeObjective:Compare to artificial menstrual cycle therapy(AMCT), the clinical effe-cts of Jiajian Yijing decoction(JD) on KDALSS and DOR patients and the influence of JD on relevant members in Transforming growth factor-β (TGF-β)superfamily have been researched, the curative effect and possible mechanisms of JD have been elaborated. ALL provide evidence-based basis for widely using JD in clinical.Methods:The clinical trial has adopted prospective and randomized method.67 KDALSS DOR patients have been randomly divided into Chinese medicine treatment group(CG)33cases and Western medicine control group(WG) 34 cases. CG has treated with JD from the first day which menstrual blood clean for 3 menstrual cycles. (If menstruation did not come after continuously treating with JD for 40 days, substituting Dydrogesterone for JD,20mg/d×10d.) WG has treated with AMCT for 3 menstrual cycles(Progynova 2mg/d×10d+Dydroges-terone 20mg/d×10d). TCM symptoms integral, serum concentrations of dFSH、 bLH、bE2、AMH、TGF-β1、BMP-15, blood routine test, liver and kidney function test and Mammary gland ultrasonography have been compared in CG and WG before and after treatment.Results:(1) The baseline data before treatment comparison:The distribution of age、course、menstruation and sterility had no difference in two groups (p>0.05), the clinical datas in two groups were comparable.(2) The TCM symptoms total integrals comparison:TCM symptoms total integrals after treatment in two groups were significantly lower than integrals before treatment (p<0.01).Compared to WG, the total integrals D-value (before treatment integrals-after treatment integrals)in CG decreased obviously (p<0.01).(3)The Main TCM symptoms integrals comparison:The TCM symptoms integrals of menstrual cycle disorder、menstrual quantity changes、menstrual qualita-tive changes after treatment in CG and WG were significantly lower than integrals before treatment (p<0.01).Compared to WG, the TCM symptoms integrals D-value of menstrual quantity changes and menstrual qualitative changes in CG increased distinctly (p<0.05). Compared to CG, the TCM symptoms integrals D-value of menstrual cycle disorder in WG increased distinctly (p<0.05). The two main TCM symptoms integrals of lumbosacral pain, chest and breast tenderness after treatment in CG were significantly lower than integrals before treatment (p<0.01),but there were no differene in WG (p>0.05). This two main TCM symptoms integrals D-value in CG are significantly lower than integrals in WG (p<0.01)(4)The secondary TCM symptoms integrals comparison:The secondary TCM symptoms integrals of dizziness tinnitus、hyposexuality、physical and mental fatigue、lower abdominal pain and distension、stomach swelling and sigh after treatment in CG were significantly lower than integrals before treatment (p <0.01). Compared to WG, this five secondary TCM symptoms integrals D-value in CG decreased distinctly (p<0.05). The secondary TCM symptoms integrals of hyposexuality、stomach swelling and sigh after treatment in WG were significantly lower than integrals before treatment (p<0.05).(5)The serum bFSH、bLH、bE2 concentrations comparison:The serum bFSH、 bLH concentrations after treatment in CG and WG were significantly lower than concentrations before treatment (p<0.01). The serum bE2 concentrations after treatment in CG and WG were significantly higher than concentrations before treatment (p<0.01). The serum bFSH、bLH、bE2 concentrations D-value before and after treatment had no differene between CG and WG (p>0.05)(6)The serum AMH、BMP、TGF-β1 concentrations comparison:The serum AMH、BMP-15 concentrations after treatment in CG and WG were significantly higher than concentrations before treatment (p<0.01).while comparing to concen-trations before treatment, the serum TGF-β1 concentrations after treatment in CG and WG had no differene (p>0.05). The serum AMH、BMP-15、 TGF- β1 concentrations D-value before and after treatment had no differene between CG and WG (p>0.05)(7)The AFC comparison pre and post therapy:The AFC after treatment in CG and WG were significantly more than the AFC before treatment (p<0.01) The AFC D-value pre and post therapy had no differene between CG and WG (p >0.05).(8)Pregnancy rate comparison; The clinical pregnancy rate in CMG and WMG have no statistically significant (P>0.05)。(9)Comprehensive curative effect comparison:The comprehensive effective rate in CMG and WMG have no statistically significant (P>0.05); The comp-rehensive curative effect in CMG and WMG have no statistically significant (P>0.05)。Conclusions:(1)The JD and AMCT (Progynova 2mg/d10d+Dydrogesterone 20mg/d×10d) were effective methods for treating KDALSS DOR patients. The two treatments had similarly comprehensive curative effect, could apparently improve ovarian function on KDALSS DOR patients.The curative effects showed as follows: relieving clinical symptoms, reducing bFSH, bLH, elevating bE2, AMH, increasing the AFC, BMP-15. JD is better than AMCT in relieving clinical symptoms and increasing pregnancy rate. The JD probablly had better clinical efficacy than AMCT. AS to patients with contraindications to hormone using and fertility requirements, the JD was better choice.(2)The probable mechanism of JD for improving clinical symptoms、ovarian function and increasing pregnancy rate as follows: ①It had multi-system、 multi-link、multi-target overall moderating effects, and it improved clinical symptoms and ovarian function, promoted menstruation and pregnancy on KDALSS DOR patients possibly by adjusting or rebuilding the function of HPOA;② It can adjust ovarian local autocrine and paracrine cytokine, such as TGF-β super-family members:AMH、BMP-15、TGF-β1, and it also could improve ovarian local micro-environment, restore ovarian function.Part3 Study of Jiajian Yijing decoct ion on ovarian function and Transform-ing growth factor-β superfamily members in DOR SD adult female rats induced by cytoxanObjective:(1)To establish DOR SD model through single intraperitoneal injecting 75 mg/kg cytoxan (CTX), and confirm the success and reversibility of the model.(2) In comparison with Progynova (P), to study the effects of high、middle、 low dose of JD on improving ovarian function and adjusting TGF-β super-family relevant members in DOR SD rats which had treated by CTX. To elaborate the curative effects and possible mechanism of JD. To provide experimental evidences for widely using JD in clinical study.Methods:The animal experiment had adopted prospective and randomized methods. After built model,360 SPF adult female SD rats had been randomly divided into blank control group (NG)、model control group (MG)、Progynova group (PG)、JD high-dose group(JHG)、JD middle-dose group (JMG)、JD low-dose group (JLG) (Except rats in NG had been induced by the same volume of saline, the other rats had been induced by 75mg/kg CTX). NG、MG、PG、JHG、JMG、JLG had been administrated respectively by gavage of the same volume of saline、the same volume of saline、0.09mg/kg Progynova、23.58g/kg JD、11.79g/kg JD、5.895g/kg JD for 4 weeks continuously.10 rats at diestrus in every group had been broken the neck to death in the 1st、2nd、3rd、4th、6th、8th week after administration. The general situation、vaginal exfoliative cell、the serum concentrations of FSH、LH、E2、AMH, INHB, ovary index, ovarian tissue pathology structure, follicle count, the expression of AMH、TGF-β1、TGF-β1R、BMP-15 in ovarian tissue of rats all had been observed in different groups and different weeks.Results:(1) The general situation of model:The MG rats had loose hair, activity decreases、dispiritedness、limb weakness.After 4 weeks administration, the appearance signs、activities、mental state、feeding、drinking water、hair、breaths、mouth、eyes、nose、ears、 stools and urine color of every treatment group rats had been returned to normal and had showed no significant difference to NG.(2) The impact on estrus cycle (EC):After molding, the MG rats regular ECs that had 4-5d had disappeared, it extended to 7-16d and the anestrus extended to 5-7d froml.5-2.5d. The ECs in MG had regained after molding for 6w. The ECs in PG、JHG、JMG had regained after molding for 4w. The ECs in TG、JLG had regained after molding for 6w.(3) The impact on ovarian pathological structure(OPS):After molding, Ovaries in MG had atrophied medulla、cortex hyperplasia、 Fibrosis、vessel hypertrophy、 decreased granular cell layer、granular cell loosely arranged and even cavity、reduced primitive and mature follicles count、increased the primary follicles、secondary、atretic follicles count. The largest damages on OPS in MG had happened after molding for 2w. The OPS in MG had regained after molding for 8w. The OPS in JHG、JMG、PG had regained after molding for 4w. The OPS in JLG had regained after molding for 6w.(4) The impact on ovarian index (OI):Comparing with NG, the OI in MG significantly reduced in the 1st、2nd、 3rd、 4th、6th week after modeling (P<0.01), but had no difference in the 8th week after modeling (P>0.05).It had difference in every week in MG (P<0.01).Comparing with the 1st week in MG, the 3rd、4th、6th week OI in MG distinctly went down (P<0.05 or P<0.01). Comparing with NG and MG, the 3rd week OI in JHG JMG began to go up apparently and returned to normal, until the 8th week (P<0.01); the 4th week OI in PG、JLG began to go up visibly until the 8th week, returned to normal in the 8th week (P<0.05 or P<0.01) The OI in JHG、JMG was apparently higher than the PG OI in the 4th、6th、 8th week (P<0.05 or P<0.01). The OI in JHG was apparently higher than the JLG OI in the 4th week (P<0.05). The 01 in JMG was apparently higher than the JLG OI in the 8th week (P<0.01)(5) The impact on all levels of follicle count (OI):Comparing with NG, the number of primitive follicles and mature follicles in MG in the 1st、2nd、3rd、4th、6th、8th week decreased significantly (P <0.01). They had difference in every week in MG (P<0.05). Comparing with the 1st week in MG, the 8th week number of primitive follicles and mature follicles in MG distinctly increased (P<0.05).Comparing with NG and MG, the 2nd week primitive follicles and mature follicles in JHG、JMG began to go up apparently, until the 8th week(P<0.05 or P<0.01), primitive follicles in JHG、JMG began to return to normal in the 4th week (P>0.05), mature follicles in JHG returned to normal in the 6th week, mature follicles in JMG returned to normal in the 8th week (P>0.05). Comparing with NG and MG, the 3rd week primitive follicles and mature follicles in PG、JLG began to go up apparently, primitive follicles constant went up until the 8th week, returned to normal in the 4th week (P<0.05 or P<0.01), mature follicles constant went up until the 6th week (P<0.05 or P<0.01).The number of primitive follicles in JHG was apparently higher than the PG、TG、JLG in the 3rd、 8th week (P<0.05 or P<0.01). The number of primitive follicles in JMG was apparently higher than the PG, JLG in the 3rd week (P<0.01).The number of mature follicles in JHG was apparently higher than the number of mature follicles in PG in the 6th week (P<0.05), and apparently higher than the number of mature follicles in JLG in the 4th、6th、8th week (P<0.05 or P <0.01).Comparing with NG, the primary、secondary、atretic follicles count in MG in the 1st、2nd、3rd、4th、6th、8th week increased significantly (P< 0.01). They had obvious difference in every week in MG (P<0.01). Comparing with the 1st week in MG, the 3rd week primary follicles count in MG began to distinctly increase, the 4th week secondary、atretic follicles count in MG began to distinctly increase, all until the 8th week (P<0.05 or P<0.01). Comparing with NG and MG, the 2nd week primary、secondary、atretic follicles count in JHG, JMG began to go down apparently, until the 8th week (P<0.05 or P<0.01);the 4th primary follicles returned to normal in JHG, the 8th week primary follicles returned to normal in JMG (P>0.05), the 3rd week atretic follicles began to return to normal in JHG、JMG (P>0.05); the 3rd week primary follicles count in PG, JLG began to go down apparently, until the 8th week (P<0.05 or P<0.01), returned to normal in 6th week (P>0.05). The number of primary follicles in JHG、JMG was apparently less than the PG、 JLG in the 3rd、6th、8th week (P<0.01). The number of primary follicles in PG was apparently less than the JLG in the 4th week (P<0.01). The number of secondary follicles in JHG、JMG was apparently less than the PG、JLG in the 8th week (P<0.05 or P<0.01)(6) The impact on serum concentrations of FSH, LH、E2:Comparing with NG, serum concentrations of FSH in MG in the 1st、2nd、 3rd、4th、6th、8th week increased significantly (P<0.01). They had obvious difference in every week in MG (P<0.01).Comparing with the 1st week in MG, the 1st week FSH serum concentrations in MG began to distinctly decrease, until the 8th week (P<0.05 or P<0.01). Comparing with NG and MG, the 1st week FSH serum concentrations in JHG、JMG began to go down apparently, until the 8th week (P<0.05 or P<0.01), began to return to normal in the 4th week (P>0.05); the 2nd week FSH serum concentrations in JPG、JLG began to go down apparently, until the 4th week (P<0.05 or P<0.01), began to return to normal in the 4th week in PG, began to return to normal in the 6th week in JLG (P>0.05).Comparing with NG, serum concentrations of LH in MG in the 1st、2nd、 3rd、4th、6th week increased significantly (P<0.01), retured to normal in the 8th week (P>0.05). They had obvious difference in every week in MG (P<0.01).Comparing with NG and MG, the 2nd week LH serum concentrations in all treatment group began to go down apparently, LH serum concentrations constant went down until the 8th week in JHG、JMG(P<0.05 or P<0.01) and began to return to normal in the 2nd week (P>0.05); LH serum concentrations constant went down until the 6th week in PG (P<0.05 or P<0.01) and began to return to normal in the 2nd week (P>0.05), began to return to normal in the 2nd week in PG (P>0.05);LH serum concentrations constant went down until the 4th week in JLG (P<0.05) and began to return to normal in the 3rd week(P>0.05). The 2nd week LH serum concentrations in PG、JMG were apparently lower than LH serum concentrations in JLG (P<0.05)Comparing with NG, E2serum concentrations in MG in the 1st、2nd、3rd、 4th、6th、8th week decreased significantly (P<0.01).They had obvious difference in every week in MG (P<0.01). Comparing with the 1st week in MG, the 4th week E2 serum concentrations in MG began to distinctly increase, until the 8th week (P<0.05 or P<0.01). Comparing with NG and MG, the 1st week E2 serum concentrations in JHG、JMG、PG began to go up apparently, until the 8th week (P<0.05 or P<0.01), began to return to normal in the 4th week (P>0.05);the 4th week E2 serum concentrations in JLG began to go up apparently, until the 6th week (P<0.05 or P<0.01).The 1st week E2 serum concentrations in PG were higher than E2 serum concentrations in the other treatment groups (P<0.05 or P<0.01)(7) The impact on serum concentrations of AMH、INHB:Comparing with NG, AMH、INHB serum concentrations in MG in the 1st、 2nd,3rd,4th,6th,8th week decreased significantly(P<0.01). They had obvious difference in every week in MG (P<0.01). Comparing with the 1st week in MG, the 3rd week AMH serum concentrations in MG began to distinctly increase, until the 8th week(P<0.05 or P<0.01), the 6th week INHB serum concentrations in MG began to distinctly increase, until the 8th week (P<0.05 or P<0.01)Comparing with NG and MG, the 1st week AMH serum concentrations in JHG, JMG, PG began to go up apparently, until the 8th week (P<0.05 or P<0.01), began to return to normal in the 4th week in PG (P>0.05), began to return to normal in the 8th week in JHG (P>0.05);the 3rd week AMH serum concentrations in JLG began to go up apparently, until the 8th week (P<0.05 or P<0.01).The 2nd week AMH serum concentrations in PG, JHG, JMG were obviously higher than AMH serum concentrations in JLG (P<0.05 or P<0.01).Comparing with NG and MG, the 1st week INHB serum concentrations in JHG, JMG began to go up apparently, until the 8th week (P<0.05 or P<0.01), began to return to normal in the 8th week (P>0.05); the 4th week INHB serum concentrations in PG, JLG went up apparently (P>0.05)(8) The impact on ovarian AMH、BMP-15、TGF-β1、TGF-β1R IOD value:Comparing with NG, ovarian AMH、BMP-15、TGF-β1、TGF-β1R IOD value in MG in the 1st、2nd、3rd、4th、6th、8th week decreased significantly (P< 0.01). They had obvious difference in every week in MG (P<0.01). Comparing with the 1st week in MG, the 4th week ovarian AMH IOD value in MG began to distinctly increase, until the 8th week (P<0.05 or P<0.01); the 8th week BMP-15 IOD value in MG distinctly increased (P<0.05); the 3rd week ovarian TGF-β1 IOD value in MG began to distinctly increase, until the 8th week (P<0.05 or P<0.01); the 8th week ovarian TGF-β1R IOD value in MG distinctly increased (P<0.05).Comparing with NG and MG, the 1st week ovarian AMH IOD value in JHG、 JMG began to go up obviously, until the 8th week (P<0.05 or P<0.01), began to return to normal in the 6th week in JHG、JMG (P>0.05); the 2nd week ovarian AMH IOD value in PG、JLG began to go up obviously, constantly increased in PG until the 8th week (P<0.01), constantly increased in JLG until the 4th week (P<0.01), began to return to normal in the 6th week in JHG, JMG (P <0.05 or P<0.01).The 2nd week ovarian AMH IOD value inJHG、JMG were obviously higher than ovarian AMH IOD value inJLG (P<0.05). The 3rd week ovarian AMH IOD value in JMG were obviously higher than ovarian AMH IOD value in JLG (P<0.05).Comparing with NG and MG, the 1st week ovarian BMP-15 IOD value in JHG、 PG began to go up obviously, constantly increased in JHG until the 6th week, constantly increased in PG until the 4th week (P<0.05 or P<0.01). The 2nd week ovarian BMP-15 IOD value in JMG began to go up obviously, until the 6th week (P<0.01); The 4th week ovarian BMP-15 IOD value in JLG began to go up obviously, until the 6th week (P<0.01). The 1st、2nd、3rd week ovarian BMP-15 IOD value in JHG were obviously higher than ovarian AMH IOD value in JLG (P<0.05 or P<0.01);the 1st,2nd week ovarian BMP-15 IOD value in PG were obviously higher than ovarian AMHIOD value in JLG (P<0.05 or P<0.01); the 2nd week ovarian BMP-15 IOD value in JMG were obviously higher than ovarian AMHIOD value in JLG (P<0.05)Comparing with NG and MG, the 1st week ovarian TGF-β1 IOD value in all treatment groups began to go up obviously (P<0.05 or P<0.01), constantly increased in JHG, JMG until the 8th week, constantly increased in PG, JLG until the 4th week (P<0.05 or P<0.01); the 8th week ovarian TGF-β1 IOD value in JHG began to return to normal (P>0.05), were obviously higher than ovarian TGF-β1 IOD value in JMG、JLG、PG (P<0.05 or P<0.01)Comparing with NG and MG, the 1st week ovarian TGF-β 1R IOD value in JHG、 PG began to go up obviously (P<0.05 or P<0.01), until the 8th week; the 2nd week ovarian TGF-β1R IOD value in JMG began to go up obviously (P< 0.05 or P<0.01), until the 6th week; the 3rd week ovarian TGF-β 1R IOD value in JLG began to go up obviously (P<0.05 or P<0.01), until the 6th week。 The 1st week ovarian TGF-β1R IOD value in JHG were obviously higher than ovarian TGF-β1RIOD value in JMG (P<0.05)Conclusions:(1)The model induced by single intraperitoneal injecting 75mg/kg CTX was successful. This method had advantage of simplity and convenientness、 highest success rate、shortest building time、lowest mortality.The model ovarian function could recover which was different from POF model.(2) JD、P could improve obviously ovarian function of DOR rats, decrease FSH、LH concentrations, increase E2 concentrations and OI, add primitive follicles count and mature follicles count, cut down primary、secondary、 atretic follicles count. As to improving ovarian function, the efficacies of JHG、JMG were more quickly、 great、 durable than the efficacies of JLG、 PG. The efficacies of JD presented a certain dose-response.(3)The curative mechanism of JD、P on improving ovarian function might be realized partly by adjusting related menmbers of TGF- β super-family which were ovarian local autocrine and paracrine cytokines. After drug intervene-tion, the treatment groups could decrease AMH、INHB concentrations, add the expression of AMH、TGF-β1、TGF-β1R、BMP-15 in ovary. As to improving related menmbers of TGF-β super-family, the efficacies of JHG、JMG were more quickly、 great、durable than the efficacies of JLG、TG. The efficacies of JD presented a certain dose-response.(4) JD could improve ovarian local autocrine and paracrine cytokines, such as AMH、INHB、TGF-β1、TGF-β1R、BMP-15, inhibit ovarian follicles to raise, promote follicle growth、maturity, improve ovarian function. The efficacies of JD on improving ovarian function were better、safest, had no side effects. It could be researched and developed into a chinese patent medicine, and apply to clinic popularly. JMD might be the optimal dose. |
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