Label-free based quantitative proteomics analysis to explore the molecular mechanism of gynecological cold coagulation and blood stasis syndrome.

Cold coagulation and blood stasis (CCBS) syndrome is one of the common traditional Chinese medicine (TCM) syndromes of gynecological diseases. However, the molecular mechanism of CCBS syndrome is still unclear. Thus, there is a need to reveal the occurrence and regulation mechanism of CCBS syndrome, in order to provide a theoretical basis for the treatment of CCBS syndrome in gynecological diseases. The plasma proteins in primary dysmenorrhea (PD) patients with CCBS syndrome, endometriosis (EMS) patients with CCBS syndrome, and healthy women were screened using Label-free quantitative proteomics. Based on the TCM theory of "same TCM syndrome in different diseases," the differentially expressed proteins (DEPs) identified in each group were subjected to intersection mapping to obtain common DEPs in CCBS syndrome. The DEPs of gynecological CCBS syndrome in the intersection part were again cross-mapped with the DEPs of gynecological CCBS syndrome obtained by the research group according to the TCM theory of "different TCM syndromes in same disease" theory in the early stage, so as to obtain the DEPs of gynecological CCBS syndrome that were shared by the two parts. The common DEPs were subjected to bioinformatics analysis, and were verified by enzyme-linked immunosorbent assay (ELISA). A total of 67 common DEPs were identified in CCBS syndrome, of which 33 DEPs were upregulated and 34 DEPs were downregulated. The functional classification of DEPs involved in metabolic process, energy production and conversion, immune system process, antioxidant activity, response to stimulus, and biological adhesion. The subcellular location mainly located in the cytoplasm, nucleus, and extracellular. Gene ontology (GO) enrichment analysis showed that the upregulated DEPs mainly concentrated in lipid transport, cell migration, and inflammatory reaction, and the downregulated DEPs mostly related to cell junction, metabolism, and energy response. Protein domain enrichment analysis and clustering analysis revealed that the DEPs mainly related to cell proliferation and differentiation, cell morphology, metabolism, and immunity. The Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis clustering analysis showed that the upregulated DEPs were involved in inflammation and oxidative damage, while the downregulated DEPs were involved in inflammation, cell adhesion, cell apoptosis, and metabolism. The results of ELISA showed significantly increased levels of Cell surface glycoprotein MUC18 (MCAM) and Apolipoprotein C1 (APOC1), and significantly decreased levels of Vasodilator-stimulated phosphoprotein (VASP), Fatty acid-binding protein 5 (FABP5), and Vinculin (VCL) in patients with CCBS syndrome compared with healthy women. We speculated that cold evil may affect the immune process, inflammatory response, metabolic process, energy production and conversion, oxidative damage, endothelial cell dysfunction, and other differential proteins expression to cause CCBS syndrome in gynecological diseases.

Clinical efficacy of Wenjing decoction in the treatment of ovulatory disorder infertility: A systematic review and meta-analysis.

OBJECTIVES: Wenjing decoction (WJD) was widely used in the treatment for ovulatory disorder infertility (ODI) in China, while its efficacy was not clearly known. In this study, we evaluated the clinical efficacy of WJD by meta-analysis. METHODS: Eight electronic databases including Cochrane Library, PubMed, Embase, Web of Science, China National Knowledge Infrastructure, WanFang Data, VIP Database, and China Biology Medicine were searched for randomized controlled trials (RCTs) published from the inception of each database to July 1, 2021, of which the interventions involve WJD and clomiphene. Outcomes included clinical efficacy rate, pregnancy rate, ovulation rate, dominant follicle diameter, endometrial thickness, estradiol, follicle-stimulating hormone, and luteinizing hormone. Meta-analysis and risk of bias were performed by RevMan 5.3 software. RESULTS: Eleven RCTs including 915 patients, of which 476 in the intervention group and 439 in the control group. Meta-analysis showed that WJD was better than clomiphene for patients with ODI in terms of clinical effective rate (odds ratio [OR] = 1.22, 95% confidence interval [CI]: 1.08-1.34), pregnancy rate (OR = 1.54, 95% CI: 1.15-2.07), ovulation rate (OR = 1.34, 95% CI: 1.07-1.67), endometrial thickness (mean difference [MD] = 1.50, 95% CI: 0.90-2.10), and dominant follicle diameter (MD = 1.85, 95% CI: 0.68-3.02). The estradiol level (MD = 91.0, 95% CI: 80.3-101.88) in patients taking WJD was significantly higher than those taking clomiphene, while the follicle-stimulating hormone level (MD = -0.93, 95% CI: -1.13 to -0.72) and the luteinizing hormone level (MD = -4.41, 95% CI: -4.80 to -4.03) in patients taking WJD was significantly lower than those taking clomiphene. Our results also indicated that WJD combined with clomiphene was better than clomiphene alone for patients with ODI in terms of pregnancy rate (OR = 1.79, 95% CI: 1.37-2.35). CONCLUSIONS: WJD may be effective in the treatment of patients with ODI. Due to the quality and quantity of literature, RCT with large sample size and high quality need to be performed to verify our conclusion.

Effects of Bushen-Tiaojing-Fang on the pregnancy outcomes of infertile patients with repeated controlled ovarian stimulation.

Bushen-Tiaojing-Fang (BSTJF) is commonly used to treat infertility. This study investigated the effects of BSTJF on the pregnancy outcomes of patients with repeated controlled ovarian stimulation (COS), on mitochondrial function, and on oxidative stress in ovarian granulosa cells (GCs) and follicular fluid (FF). The samples and clinical data of 97 patients, including 35 in the control group, 29 in the placebo group and 33 in the BSTJF group, were collected for this study. The mitochondrial ultrastructure, ATP content, mitochondrial DNA (mtDNA) number, 8-hydroxy-2-deoxyguanosine (8-OHdG), Mn-superoxide dismutase (Mn-SOD), glutathione peroxidase (GSH-Px) activity levels, and mRNA expression levels of Mn-SOD, GSH-Px, and nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2) were analyzed. The high-grade embryo (P < 0.001), implantation (P = 0.033), and clinical pregnancy (P = 0.031) rates, as well as the ATP content (P = 0.014), mtDNA number (P = 0.035), GSH-Px activity (P = 0.004 in GCs and P = 0.008 in FF) and mRNA expression levels (P = 0.019), were significantly lower in the placebo group than in the control group, whereas the 8-OHdG content was significantly (P = 0.006 in FF) higher in the placebo group than in the control group. Compared with those in the placebo group, the high-grade embryo rate (P = 0.007), antioxidant enzyme activity (P = 0.037 and 0.036 in Mn-SOD; P = 0.047 and 0.030 in GSH-Px) and mRNA level (P < 0.001 in Nrf2, P = 0.039 in Mn-SOD and P = 0.002 in GSH-Px) were significantly higher in the BSTJF group, as were changes in mitochondrial ultrastructure, ATP (P = 0.040) and mtDNA number (P = 0.013). In conclusion, BSTJF can improve oxidative stress in patients with repeated COS and pregnancy outcomes.

Bushen Tiaojing (II and III) Decoctions Activate MAPK14 and MAPK3/1 to Promote the Expression of Cumulus Expansion-Related Factors in Mice.

BACKGROUND: Bushen Tiaojing Decoctions (BSTJ-II-D and BSTJ-III-D) are used to assist pregnancy in clinical practice. In this study, we explored the ability of sequential administration of BSTJ-II-D and BSTJ-III-D to promote cumulus cell (CC) expansion and its underlying mechanisms in controlled ovarian hyperstimulation (COH) mice. METHODS: Kunming mice were randomly divided into three groups. The normal group was injected intraperitoneally with saline, and distilled water was administered orally by gavage. As the COH model, mice were injected with GnRHa, eCG, and hCG. Subsequently, the BSTJD group received BSTJ-II-D and BSTJ-III-D orally by gavage, while the control group received distilled water. We evaluated CC expansion and oocyte first polar body (PB1) extrusion under a stereomicroscope. Serum levels of follicle-stimulating hormone (FSH) were detected by radioimmunoassay. The expression of the CC expansion-related factors PTX3 and PTGS2 was detected by immunofluorescence, western blot, and quantitative real-time-polymerase chain reaction analyses (qRT-PCR). Expression of p-MAPK14, p-MAPK3/1, MAPK14, and MAPK3/1 was detected by western blot analysis. RESULTS: Sequential administration of BSTJ-II-D and BSTJ-III-D promoted cumulus expansion and oocyte PB1 extrusion and upregulated PTX3 and PTGS2 expression at the mRNA and protein levels. Furthermore, the levels of p-MAPK14/MAPK14, p-MAPK3/1/MAPK3/1 proteins, and serum FSH in the BSTJD group were higher than those in the normal and control groups. CONCLUSIONS: Sequential administration of BSTJ-II-D and BSTJ-III-D promotes cumulus expansion and oocyte maturation in COH mice by increasing FSH expression and activating the MAPK14 and MAPK3/1 signalling pathways, thereby increasing expression of PTX3 and PTGS2.

[Expert consensus statement on Kangfu Xiaoyan Suppository in treatment of pelvic inflammatory in clinical practice].

Kangfu Xiaoyan Suppository is widely used in the treatment of gynecological inflammatory diseases. Long-term clinical application and a certain amount of research evidences show that Kangfu Xiaoyan Suppository can alleviate the clinical symptoms of pelvic inflammatory diseases,reduce the recurrence rate,and relieve sequelae,with a better safety and economic characteristics. As a type of nationally protected traditional Chinese medicine and type B medicine included in medical insurance,it has been selected as a Chinese patent medicine for rectal administration. It was included in the Guidelines for diagnosis and treatment of common gynecological diseases of traditional Chinese medicine published by the Chinese Academy of Traditional Chinese Medicine in 2012,the Pelvic inflammatory diseases diagnosis and treatment guidelines issued by the Infectious Diseases Collaborative Group of the Obstetrics and Gynecology Branch of the Chinese Medical Association in 2014,and the group standard of Single use of traditional Chinese medicine/combined antibiot guidelines for clinical practice-pelvic inflammatory diseases of the Chinese Academy of Traditional Chinese Medicine in 2017. To further enhance clinicians' understanding of the drug and better guide its rational clinical use,experts from the field of gynecology of traditional Chinese and Western medicine were invited to develop and compile this expert consensus. This consensus takes full account of clinical evidences and expert clinical experience,and form recommendations for clinical problems based on evidences and consensus recommendations for clinical problems without evidence by nominal grouping method. The expert consensus is mainly formed in the consideration of six factors: quality of evidence,economy,efficacy,adverse reactions,patient acceptability and others. Based on clinical research evidences and expert experience,this consensus provides a preliminary reference for the clinical use of the drug in a concise and clear format. However,evidence-based support is still required in a large number of high-quality studies,and this consensus will be revised in the future according to new clinical problems and the update of evidence-based evidence in practical application.

[Effects of Cangfu Congxian Decoction on Oxidative Stress in Polycystic Ovary Syndrome Patients].

OBJECTIVE: To observe the effect of Cangfu Congxian Decoction (CCD) on oxidative stress in granulosa cells of polycystic ovary syndrome (PCOS) patients. METHODS: Forty PCOS patients underwent in vitro fertilization-embryo transfer (IVF-ET) were assigned to the treatment group and the control group 1 according to random digit table, 20 in each group. Patients in the treatment group took CCD (200 mL, once in the morning and once in the afternoon) 2 months before IVF-ET, while those in the control group 1 took no Chinese medical decoction. Recruited were another 20 patients undergoing IVF-ET for tubal factors (as the control group 2). The clinical effect of IVF-ET were observed, including oocyte retrieval number, 2 pronuclear (2PN) fertilization rate, good quality embryo rate, clinical pregnancy rate, and ovarian hyperstimulation syndrome (OHSS) induced transplantation cancel rate. The expression of relative oxygen species (ROS) in granulosa cells was detected using cell immunofluorescence combined with confocal microscopy and FCM. RESULTS: Compared with the control group 1, occyte retrieval number, 2PN fertilization rate, and good quality embryo rate increased in the control group 2 and the treatment group (P <0. 05). OHSS induced transplantation cancel rate decreased in the control group 2 (P < 0.05). Fluorescence intensity of ROS decreased in the treatment group and the control group 2, as compared with the control group 1 (P < 0.01). CONCLUSION: CCD increased good quality embryo rate by down-regulating the expression of ROS protein in ovarian granulosa cells, and correcting in vivo oxidative stress.

[Bushen Tiaojing Recipe Regulated Expressions of BMPR I[/ALK6-Smads in Mouse Oocytes Cul- ture in vitro].

Objective To observe the effects of Bushen Tiaojing Recipe (BTR) on the counts of survival preantral follicles and the bone morphogenetic protein receptor II (BMPR II )/activin receptor- like kinase 6-drosophila mothers against decapentaplegic proteins (ALK6-Smads) signal pathway in oocytes cultured in vitro, and to study its mechanism for improving the quality of oocytes. Methods Prean- tral follicles were mechanically isolated from 65 female 12-day old healthy Kunming mice, which were inoculated by normal rats' serum (as the control group) , high, medium, low dose BTR containing serums (as Shen-supplementing groups) , high dose BTR containing serum + K02288 (as the inhibitor group) , respectively. All were cultured by common method in vitro. On the 6th day the counts of survival preantral follicles were compared between each Shen-supplementing group and the control group respectively. mR- NA expressions of BMPR II, ALK6, Smad1 , Smad5, and Smad8 were detected by Real-time fluorescence quantitative PCR. The protein expressions of indices mentioned above and phospho-Smadl/5/8 (p- Smadl/5/8) were detected by cellular immunofluorescence test. Results Compared with the control group, the quantity of survival preantral follicles increased in the high dose BTR containing serum group; mRNA expressions of BMPR II, ALK6, Smad5, and Smad8 were elevated, protein expressions of indi- ces mentioned above and p-Smadl/5/8 were increased in the 3 Shen-supplementing groups (P <0. 05) ; mRNA and protein expressions of Smad1 were increased in high and medium dose BTR containing serum groups (P<0.05). Compared with the high dose BTR containing serum group, protein expressions of Smad1/5/8 were reduced in the inhibitor group (P <0.05). Conclusion BTR could elevate the quantity of survival preantral follicles cultured in vitroand improve the quality of oocytes, which might be possibly as- sociated to regulating the BMPR II/ALK6-Smads signal pathway in oocytes.

[Effects of Bushen Wenyang Huayu Recipe on Expressions of HIF-1α, PHD2, and VHL in Endometriosis Rats with Shen Yang Deficiency Blood Stasis Syndrome].

OBJECTIVE: To observe the effect of Bushen Wenyang Huayu Recipe (BWHR) on hypoxia inducible factor-1α (HIF-1α), proline hydroxylase2 (PHD2), von Hippel Lindau disease (VHL) suppressor gene expressions in endometriosis (EM) rats with Shen yang deficiency blood stasis syndrome (SYDBSS), and to explore the pathogenesis of EM and the mechanism of BWHR for treating EM. METHODS: Totally 50 SD rats were randomly divided into five groups, i.e., the blank control group, the sham-operation group, the model group, the Chinese medicine (CM) group, and the Western medicine (WM) group, 10 in each group. Rats in the blank control group and the sham-operation group were fed routinely. Rats in the rest 3 groups received 30-day "extended refrigerator freezing and ice water immersion" and combined with " autotransplantation" to establish EM rat model with SYDBSS. One Milliliter BWHR at 3.33 g/mL was administered to rats in the CM group by gastrogavage. Gestrinone at the daily dose of 0. 5 mg/kg was administered to rats in the WM group by gastrogavage. Equal volume of normal saline was administered to rats in the model group, the blank control group, and the sham-operation group. The size and morphology of ectopic foci in rats were observed after 4 weeks of medication. Expressions of serum CA125, plasma cyclic adenosine monophosphate (cAMP), and plasma cyclic guanosine monophosphate (cGMP) were detected by radioimmunoassay. Morphological changes of eutopic endometrium and ectopic tissue were observed under the optical microscope by HE staining. Protein expressions and contents of HIF-lα, PHD2, and VHL were detected by immunohistochemical SABC method and Western blot. mRNA expressions of HIF-1α, PHD2, and VHL were detected by RT-PCR. RESULTS: The ectopic foci grew significantly in the model group. Their volumes were obviously contracted after treated by CM and WM. Compared with the blank control group and the sham-operation group, serum CA125 and plasma cGMP obviously increased, cAMP obviously decreased (P < 0.05); expressions and contents of HIF-1α mRNA and protein all decreased (P < 0.05); mRNA and protein expressions and contents of PHD2 and VHL all decreased in the model group (P < 0.05). Compared with model group, levels of CA125 and cGMP obviously decreased; cAMP levels obviously increased, expressions and contents of HIF-1α mRNA and protein all increased, mRNA and protein expressions and contents of PHD2 and VHL all increased in the WM group and the CM group (P < 0.05). Compared with the CM group, PHD2 protein contents were higher in the WM group (P < 0.05). HIF-1α was negatively correlated with PHD2 (r = -0.799, P = 0.00). HIF-1α was negatively correlated with VHL (r = -0. 625, P = 0.003). CONCLUSIONS: BWHR could effectively treat EM. Its mechanism might be associated with reducing contents of HIF-1α, serum CA125, and plasma cGMP, and up-regulating expressions of PHD2, VHL, and cAMP.

[Effects of Bushen Wenyang Huayu Recipe on TRPV1 and Sensitization Factor NGF in Experimental Endometriosis].

OBJECTIVE: To study the effect of Bushen Wenyang Huayu Recipe (BSWYHYR) on nerve growth factor (NGF) and transient receptor potential vanilloid receptor I (TRPV1) in experimental endometriosis (EMT), and to explore its mechanism for treating EMT-induced pain. METHODS: Totally in-bred line BALB/c 75 female mice were divided into five groups, i.e., the sham-operation group, the model group, the high dose BSWYHYR group, the low dose BSWYHYR group, the gestrinone group, 15 in each group. Writhing response was observed in each group. Serum contents of NGF were detected using ELISA. Expression levels of NGF and TRPV1 in uterus and ectopic foci were detected using immunohistochemical staining SP and Western blot. mRNA expression levels of NGF and TRPV1 in uterus and ectopic foci were detected by Real-time PCR. RESULTS: The serum NGF content in the model group was higher than that in the sham-operation group (P < 0.01), and there was positive correlation between NGF and the writhing frequency (r = 0.574, P < 0.01). Compared with the model group, serum levels of NGF significantly decreased in the 3 treatment groups (P < 0.05). Compared with the sham-operation group, mRNA and protein expression levels of NGF and TRPV1 increased significantly in the model group (P < 0.01). Protein expression levels of NGF and TRPV1 decreased significantly in the 3 treatment groups, when compared with the model group (P < 0.01). mRNA expression levels of NGF and TRPV1 decreased most in the high dose BSWYHYR group (P < 0.01). NGF in uterus and ectopic foci was positively correlated with protein and mRNA expression levels of TRPV1 (P < 0.01). CONCLUSIONS: NGF and TRPV1 participated in the occurrence of pain in EMS. BSWYHYR played an important role in inhibiting EMT-induced pain through reducing the up-regulation of NGF on TRPV1.

[Effect of bushen tiaojing recipe on the quality of the oocytes and reproductive hormones in the follicular fluid in IVF-ET patients].

OBJECTIVE: To explore the effect of Bushen Tiaojing Recipe (BTR) on the quality of oocytes, reproductive hormones, and the expression of bone morphogenetic protein-15 (BMP15) of in vitro fertilization-embryo transfer (IVF-ET) patients. METHODS: Sixty infertility patients who prepared for IVF-ET were assigned to two groups according to the treatment order, the treatment group [20 cases, treated with BTR + controlled ovarian hyperstimulation (COH)] and the control group (treated with COH alone, 40 cases). Age, the time limit for infertility, basal follicle-stimulating hormone (bFSH) concentration, usage days and the dosage of gonadotropins (Gn), serum levels of estradiol (E2), luteotropic hormone (LH), and progesterone (P) on the HCG injection day, the number of retrieved occytes, the fertilization rate, the number of embryos, the high quality embryo rate, and the clinical pregnancy rate were compared. Concentrations of follicle-stimulating hormone (FSH), LH, E2, testosterone (T), and P in the follicular fluid were detected via chemiluminescence microparticle immunoassay. The mRNA and protein expression of BMP-15 in mature granulosa cells was detected by real-time fluorescent PCR and Western blot. RESULTS: Thirty-two patients were pregnant and the total pregnancy rate was 53.3%. Of them, 19 were pregnant and the total pregnancy rate was 47.5% in the control group, while 20 were pregnant and the total pregnancy rate was 65.0% in the treatment group. But there was no statistical difference between the two groups (P > 0.05). Compared with the control group, the Gn dosage was lower and the high quality embryo rate was higher in the treatment group, showing statistical difference (P < 0.05). There was no statistical difference in serum concentrations of E2, LH, or P on the HCG injection day, the number of retrieved oocytes, or the fertilization rate (P > 0.05). Compared with the control group, FSH concentrations in the follicular fluid were significantly lower and LH concentrations were significantly higher in the treatment group (P < 0.05). The LH concentrations in the follicular fluid were significantly higher in pregnant patients than non-pregnant patients, showing statistical difference (P < 0.05).There was no statistical difference in E2, T, or P concentrations (P > 0.05). The mRNA and protein expression of BMP-15 in granulosa cells was higher in the treatment group than in the control group (P < 0.05). It was also higher in pregnant patients than non-pregnant patients, showing statistical difference (P < 0.05). CONCLUSION: During the IVF-ET process, BTR could elevate the quality of oocytes, and increase the sensitivity of ovarian follicles to exogenous Gn, which was correlated with the mRNA and protein expression of BMP-15 in granulosa cells, and changing concentrations of FSH and LH.

[Effect of Bushen Tiaojing Recipe containing serum on FSH/cAMP-PKA pathway in in vitro cultured human ovarian granular cells].

OBJECTIVE: To explore the potential molecular mechanisms for Bushen Tiaojing Recipe (BTR) improving the endocrine function of ovarian granular cells by observing the effect of BTR containing serum on follicle stimulating hormone/cyclic adenosine monophosphate-protein kinase A (FSH/ cAMP-PKA) pathway in in vitro cultured human ovarian granular cells. METHODS: The primary ovarian granular cells collected from in vitro fertilization-embryo transfer patients were cultured for 24 h. The human and rat serum containing different concentrations of BTR (low, medium, high dose), and their normal serums were co-incubated with ovarian granular cells for 48 h respectively, and then they were divided into the low, medium, high dose BTR groups and the control group. The levels of estradiol (E2), progesterone (P), and cyclic adenosine monophosphate (cAMP) in the culture medium were measured by radioimmunoassay. The protein expression of FSHR in ovarian granular cells was detected by Western Blot. The mRNA expression of follicle stimulating hormone receptor (FSHR) and P450 aromatase (P450arom) in ovarian granular cells were detected by Real-time PCR. RESULTS: In human BTR containing serum groups: Compared with control group, the levels of E2 and cAMP in the culture medium were higher (both P < 0.05) in the medium and high dose BTR groups; the levels of P in the culture medium decreased in the medium dose BTR group (P < 0.01). The protein and mRNA expression of FSHR in ovarian granular cells increased (all P < 0.01), the mRNA expressions of P450arom in ovarian granular cells were higher (P < 0.05, P< 0.01) in the medium and high dose BTR groups. In rat BTR containing serum groups: Compared with the control group, the levels of E2 in the culture medium were higher (all P < 0.01), cAMP in the culture medium were higher (P < 0.05, P < 0.01) in the medium and high dose BTR group; the levels of P in the culture medium decreased in the medium dose BTR group (P < 0.01). The protein and mRNA expression of FSHR in ovarian granular cells were higher (all P < 0.01), the mRNA expression of P450arom in ovarian granular cells increased in the medium and high dose BTR groups (P < 0.05, P < 0.01). CONCLUSION: BTR could possibly improve the endocrine function of ovarian granular cells by regulating main effector molecules FSHR, cAMP, P450arom, and E2 in FSH/cAMP-PKA pathway of ovarian granular cells.

[Effect of Bushen Tiaojing Recipe and Xiaoyao Pill on adenohypophysis and ovary in androgen-induced sterile rats: a comparative study].

OBJECTIVE: To observe the effect of Bushen Tiaojing Recipe (BTR) and Xiaoyao Pill (XYP) on the morphology and sex hormones secretion of adenohypophysis and ovaries in androgen-induced sterile rats (ASR). METHODS: Fifty 9-day old SD female rats randomly recruited from total 60 rats were subcutaneously injected with testosterone propionate to establish the ASR model. And the rest 10 rats were recruited as the normal group. Thirty successfully modeled rats were recruited and randomly divided into the model group, the BTR group (administered with BTR suspension), and the XYP group (administered with XYP suspension), 10 in each group. Five weeks later, rats were decapitated in the proestrus. Serum levels of estradiol (E2), progesterone (P), testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were detected by radioimmunoassay. The morphologies of adenohypophysis and ovary were observed after HE staining. RESULTS: Compared with the normal group, serum E2 and T levels increased, while FSH and LH levels decreased in the model group (all P < 0.01). The morphology of adenohypophysis and ovary was abnormal in the model group. Compared with the model group, serum E2 and T levels decreased, while FSH and LH levels increased in the BTR group and the XYP group (P < 0.05, P < 0.01). Besides, E2 and T levels in the BTR group and FSH levels in the XYP group restored to normal (all P > 0.05). The damaged structure of adenohypophysis and ovary got restored to different degrees. CONCLUSION: BTR and XYP both could improve ovulation failure.

[Effect of bushen tiaojing recipe on the expressions of Smad1, Smad5, Smad8, and Smad4 in human mural granulosa cells].

OBJECTIVE: To explore the effect of Bushen Tiaojing Recipe (BTR) on the expressions of drosophila mothers against decapentaplegic protein (Smadl), Smad5, Smad8, and Smad4 on human mural granulosa cells. METHODS: Sixty-six patients undergoing in vitro fertilization-embryo transfer (IVF-ET) were randomly assigned to two groups in the ratio of 1:2, the treatment group and the control group. Twenty-three patients in the treatment group were treated with BTR and GnRHa/FSH/hCG, while forty-three patients in the control group were treated with GnRHa/FSH/hCG. The mRNA expressions of Smad1, Smad5, Smad8, and Smad4 on mural granulosa cells of the mature follicle were detected by real-time PCR on the ovum retrieval day. The expressions of Smad1, Smad5, Smad8, and Smad4 at the protein level were observed using cell immunofluorescence method. RESULTS: The mRNA and protein expressions of Smadl in the granulosa cells were significantly higher in the treatment group than in the control group (P <0.05). There was no statistical difference in the mRNA and protein expressions of Smad5, Smad8, and Smad4 between the two groups. CONCLUSION: The mechanisms of BTR for improving the pregnancy rate and the ovarian functions might be correlated with up-regulating mRNA and protein expressions of Smadl of human mural granulosa cells.

[Effects of Modified Wenjing Decoction on the ovarian oxidative damage of cold coagulation blood stasis model rats].

OBJECTIVE: To observe the effects of Modified Wenjing Decoction (MWJD) on the ovarian oxidative damage of cold coagulation blood stasis (CCBS) model rats, thus consummating the action mechanisms of MWJD for treating gynecopathy of CCBS syndrome. METHODS: Female SD rats were randomly divided into the normal group, the model group, and the treatment group. Rats in the model group and the treatment group were placed in the ice water (0 -1 degrees C) to establish the rat model of CCBS syndrome. Meanwhile, MWJD was given by gastrogavage to rats in the treatment group for 2 weeks. Rats were decapitated during the oestrous cycle. The serum levels of estradiol (E2), progestone (P), testosterone (T) were detected. The bilateral ovarian tissues were taken out and operated on ice. The total bilirubin (TBIL), superoxide dismutase (SOD), malondialdehyde (MDA), total antioxygen capacity (T-AOC) were detected after homogenate. RESULTS: Compared with the normal group, serum levels of E2, P, T, and ovarian levels of TBIL, SOD, T-AOC all obviously decreased in the model group, and the ovarian MDA level increased, showing statistical difference (P <0. 05, P <0.01). After treated by MWJD, when compared with the model group, serum levels of E2, P, T, and ovarian levels of TBIL, SOD, T-AOC increased, the ovarian MDA level decreased, showing statistical difference (P <0.05, P <0.01). All indices except serum T level and the ovarian MDA content restored to the normal levels (all P>0.05). CONCLUSIONS: The ovarian functions are abnormal in CCBS model rats. MWJD could restore the ovarian functions by improving the ovarian oxidative damage state, thus treating gynecopathy of CCBS syndrome.

[Effect of bushen tiaojing recipe on growth differentiation factor-9 in tubal infertility patients undergoing in vitro fertilization-embryo transfer].

OBJECTIVE: To study the effect and mechanism of Bushen Tiaojing Recipe on improving oocyte and embryo qualities in patients undergoing in vitro fertilization-embryo transfer (IVF-ET) at the super-ovulatory cycle. METHODS: Fifty-eight tubal infertility patients undergoing IVF-ET were randomly assigned to two groups. Thirty patients in the treatment group were treated with Bushen Tiaojing Recipe and GnRHa/FSH/hCG, and twenty-eight patients in the control group were treated with GnRHa/FSH/hCG. Contents of GDF-9 in the mature follicular fluid were detected by Western blot. The expressions of GDF-9 in granulose cells were detected by Real-time PCR. The dose of gonadotropin (Gn), the number of oocytes obtained, the fertilization rate, the oocyte cleavage rate, the high quality embryo rate, and the pregnancy rate were compared. RESULTS: The contents of GDF-9 in the follicular fluid and its expression in granulosa cells were significantly higher in the treatment group than in the control group (P<0.05). The number of oocytes obtained, the fertilization rate, the high quality embryo rate, and the pregnancy rate were significantly higher in the treatment group than in the control group. There was no significant difference in the dose of Gn or the oocyte cleavage rate. CONCLUSIONS: Bushen Tiaojing Recipe could improve the pregnancy rate of IVF-ET. Its mechanism might be possibly through regulating the GDF-9 contents in the follicular fluid and granulosa cells.

[Effect of bushen tiaojing recipe and xiaoyao pill on expression of cathepsin-L mRNA in gonadotropin-primed mice].

OBJECTIVE: To study the effect of Bushen Tiaojing Recipe (BTR) and Xiaoyao Pill (XP) on cathepsin-L (Cat-L) mRNA in mice. METHODS: Immature mice were randomly divided into the normal group, the control group, the BTR group and the XP group, three in each group. Cat-L mRNA expression in mice was detected using reverse transcription polymerase chain reaction (RT-PCR) at 0, 4, 8 and 12 h after injecting 5 IU (human chorionic gonadotropin, HCG). RESULTS: Cat-L mRNA expression increased gradually after HCG injection, the relative levels in the control group at 0, 4, 8 and 12 h were 0.066 +/- 0.005, 0.383 +/- 0.045, 0.737 +/- 0.024 and 1.036 +/- 0.073 respectively, comparisons between different time-points showed significant difference (P < 0.01). Compared with the control group, the Cat L mRNA expression was higher at 4 h in both BTR and XP groups (P < 0.01), at 8 h in the XP group (P < 0.05), and at 12 h in BTR group after injecting HCG (P < 0.05). Compared with the control group, cat L mRNA expression showed no statistic difference at 8 h in BTR group and at 12 h in XC group. CONCLUSIONS: BTR promoted the ovulation by enhancing the expression of CatL gene, and that of XP by advancing the peak of CatL gene expression.

[Establishment of gynecological asthenia cold syndrome model].

OBJECTIVE: To establish the model of gynecological asthenia cold syndrome by simulating the etiology of gynecological asthenia cold syndrome depending on 'pathogenic cold impairing yang" in Chinese medicine theory. METHODS: The female SD rats were randomly divided into the model group and the normal group by randomized digital table, 20 in each group. Rats in the model group were placed in 0 degrees C - 1 degree C ice water, and the ice water was placed in 4 degrees C refrigerator, twice daily, 20 min each time, for a total of 30 days. The body temperature was determined and the changes of the estrous cycle were observed every day. When the body temperature decreased (with statistical difference from those of the normal group), and vaginal smears showed disordered estrous cycle, the model was successfully established. Rats were sacrificed during the diestrus period, the correlative indices including reproductive endocrine hormones in blood [follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol ( E2 ), testosterone (T), and progestone (P)], the thyroid function [triiodothyronine (T3), tetraiodothyronine (T4), thyrotropic-stimulating hormone (TSH)], the adrenal function [plasma adrenocorticotropic hormone (ACTH) and cortisol (Cor)], the cellular immune function [serum interleukin-2 (IL-2)], and energy tests [including plasma cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), the ratio of cAMP/cGMP, lactate dehydrogenase(LDH)], an the index of thymus, spleen, uterus, and ovary were detected. RESULTS: Compared with the normal group, rats in the model group appeared chill, dim-color hair, purple and dark ears and claw. They were depressed, scrunched, quiet, and clumsy. They liked to stay together. Their water intake and appetite were reduced, body weight lost, body temperature significantly lowered. They passed loose stool. Their estrous cycle and diestrus were prolonged. Their plasma cGMP content, cAMP/cGMP ratio, LDH, serum IL-2 content, E2, P, T, T3, LH, TSH were significantly lowered to some extent. Their thymus index and the ovary index significantly decreased, showing significant difference (P < 0.05). CONCLUSION: The rat model of gynecological asthenia cold syndrome prepared by extending the frozen time at refrigerator and ice water immersion was in line with clinical features of gynecological asthenia cold syndrome.