Clinical Researches On The Effects Of Bushen Tiaojing Recipe On Outcome Of IVF-ET And Its Effects On Apoptosis Of Granulosa Cell And Oocyte-secreted Factors

In vitro fertilization-embryo transfer (IVF-ET), one of the most scientific and effective methods in treating infertility a common and frequent disease (morbidity of 7%-10%) in human reproductive health, has made great progress recent years. While the success rate still remains low with relatively high rate of multiple pregnancy and abortion. Effort to optimize the protocols of controlled ovarian hyperstimulation (COH) is still the major method to improve oocyte quality, which plays an important role in IVF-ET.Researches on Traditional Chinese Medicine (TCM) used to improve the quality of oocyte and embryo has made great progress recently.Our research team studied the effects of Bushen Tiaojing Recipe on the reproductive function of anovulatory rats and concluded that this recipe can induce follicular development and ovulation in many paths and targets, and can benefit embryo implantation by promoting uterus development and regulating the microenvironment of endometrium.The purpose of our research is to study the effects of Bushen Tiaojing Recipe Combined with IVF-ET on treatment of infertility more deeply, on the basis of preliminary research.It is known that bidirectional interactions between the oocytes and surrounding somatic cells through gap junctions and paracrine signaling are pivotal in maintaining the growth and development of both cell types during folliculogenesis. The oocyte plays an active role throughout folliculogenesis via secretion of oocyte-secreted factors(OSFs) that effects cumulus cell proliferation , expansion, differentiation, steriodogenesis, gene expression and maintain an appropriate microenvironment for the acquisition of its developmental competence. It is reported that the apoptosis of cumulus cells and OSFs are associated with oocyte development directly. Therefore, in this study we observed the effects of Bushen Tiaojing Recipe on IVF-ET, granulosa cell apoptosis and OSFs and discussed the mechanisms involved in order to supply theoretical and experimental basis for traditional Chinese medicine used in infertility and assistant therapy for IVF-ET.Part one: Clinical application of Bushen Tiaojing Recipe in IVF-ET Objective: To observe effects of Bushen Tiaojing Recipe on pregnancy outcome in patients undergoing IVF-ET.Methods: Sixty infertile patients with tubal factors combined with kidney deficiency undergoing IVF-ET were divided into two groups at random, experimental group (Bushen Tiaojing Recipe +GnRHa/FSH/hCG) and control group (GnRHa/FSH/hCG).The content of hormones(E2,LH,P) on HCG day, days and dosage of gonadotropin, number of oocytes and mature oocytes, fertilization rate, high quality embryos rate ,pregnancy rate and complication were observed.Results: (1) The content of E2 was significantly higher in experimental group than in control group(P<0.05).The level of P,LH was no statistical significance between two groups(P>0.05).(2) The time and dosage of gonadotropin were no statistical significance between two groups(P >0.05),while the oocyte number was significantly larger in experimental group than in control group(P<0.05).(3) The rate of mature oocyte and oocyte cleavage were no statistical significance between two groups(P>0.05),while the rate of fertilization , high quality embryo were significantly higher in experimental group than in control group(P<0.05).(4) The clinical pregnancy rate of two groups respectively were 73.33%(22/30) and 46.67%(14/30). In experimental group, the pregnancy rate was significantly higher than in control group(P<0.05).(5) No differences were observed between the two groups in complications including OHSS and abortion.Conclusions: It comes into the conclusion that combined use of Bushen Tiaojing Recipe during COH of IVF-ET can increase oocyte number and improve fertilization rate, high quality embryo rate and clinical pregnancy rate.Part two: The relationship human granulosa cells apoptosis and OSFs to oocyte development potentialObjective: To investigate the incidence of apoptosis in human single oocyte's granulosa cells and the level of GDF-9 and BMP-15 in corresponding follicular fluid(FF)and evaluate the relationship granulosa cells apoptosis and OSFs to oocyte development potential.Methods: A total of 69 occytes and their corresponding FF from women undergoing IVF-ET because of tubal infertility were examined. FF was sampled by trans-vaginal ultrasound-guided puncture and aspiration of >18 mm diameter follicles. Fluid from the first aspirated follicle of each ovary was carefully collected in a tube, and the oocyte-cumulus- complex (COC) that was used for IVF was identified in the fluid sample coming from the same follicle and maintained in isolation from that coming from other follicles. Individual oocytes were inseminated and subsequently assessed on the basis of their maturation , fertilization, cleavage and preimplantation development. The 69 occytes were divided into good quality oocytes group which divided into high-quality embryos and low-quality oocytes group including immature, nonfertilized, developed bad embryos. FF samples were centrifuged and collected granulosa cells used for the measurement of apoptosis rate by TUNEL.The GDF-9 and BMP-15 levels in FF were measured by western-blot.Results: (1)69 oocytes were observed including 36 high-quality oocytes and 33 low-quality oocytes. The average of apoptosis rate in granulosa cells was 12.746±3.73 in high-quality oocytes group and 19.88±6.36 in low-quality oocytes group. The apoptosis rate in high-quality oocytes group was significantly lower than in low-quality oocytes group (P<0.01)(.2)The level of GDF-9 in FF was 2.4983±0.1328 in high-quality oocytes group and 1.3052±0.406 in low-quality oocytes group. The level of GDF-9 in high-quality oocytes group was significantly higher than in low-quality oocytes group (P<0.01).(3) The level of BMP-15in FF was 3.3785±0.4823 in high-quality oocytes group and 1.336±0.2139 in low-quality oocytes group. The level of BMP-15 in high-quality oocytes group was significantly higher than in low-quality oocytes group (P<0.01).Conclusions: It comes into the conclusion that the incidence of apoptosis in human granulosa cells shows negative correlation with oocyte development potential ; and GDF-9 and BMP-15 level with good oocyte quality show significant positive correlation. The present study demonstrates that the incidence of apoptosis in human granulosa cells and the GDF-9 and BMP-15 level in FF appears to be potential factors in predicting oocyte quality and subsequent embryo development.Part three: The effect of Bushentiaojing Recipe on apoptosis in human granulosa cells in Patients Undergoing in Vitro FertilizationObjective: To investigate the effect of Bushentiaojing Recipe on apoptosis in human granulosa cells and to explore the mechanisms of improvement on oocyte and embryo quality in Patients Undergoing IVFMethods: Sixty infertile patients with tubal factors undergoing IVF-ET were divided into two groups at random, 30 cases in the study group treated with Bushentiaojing Recipe +GnRHa/FSH/hCG, and 30 cases in control group treated with GnRHa/FSH/hCG. FF was sampled by trans-vaginal ultrasound- guided puncture and aspiration of all≥18–20 mm diameter follicles. FF samples were centrifuged and collected granulosa cells. The apoptosis rate of granulosa cells measured by TUNEL. The immunocytochemistry was performed to determine the expression of apoptosis-related protein Caspase 3.The DNA of apoptosis cell was detected by flow cytometry .Results: (1) The average of apoptosis rate in granulosa cells was 13.61±4.53 in experimental group and 19.41±5.95 in control group. The apoptosis rate in experimental group was significantly lower than in control group (P<0.01 ).(2) The expression positive rate of Caspase 3 was 48.08±11.24% in experimental group and 68.58±10.31% in control group. The expression positive rate of Caspase 3 in experimental group was significantly lower than in control group (P<0.01).(3) The average value of the DNA of apoptosis cell in experimental group and control group was 1.26±0.24%,3.61±1.6% respectively, and the former were significantly lower than the latter.Conclusions: Bushen Tiaojing Recipe can decrease the apoptosis rate of granulosa cells and the content of DNA in apoptosis cells and can inhibit the expression of apoptosis-related protein Caspase 3. All of the above indicates that the mechanisms of Bushen Tiaojing Recipe can decrease apoptosis rate in granulosa cells is related to inhibit the expression of apoptosis-related protein Caspase 3.Part four:Effects of Bushen Tiaojing Recipe on GDF-9 and BMP-15 in Patients Undergoing in Vitro FertilizationObjective: To explore the mechanisms involved in the improvement of oocyte and embryo quality in patients undergoing IVF by investigating the effects of Bushen Tiaojing Recipe on OSFs GDF-9 and BMP-15.Methods: Sixty infertile patients with tubal factors undergoing IVF-ET were divided into two groups at random, 30 cases in the study group treated with Bushentiaojing Recipe +GnRHa/FSH/hCG, and 30 cases in control group treated with GnRHa/FSH/hCG. FF was sampled by trans-vaginal ultrasound-guided puncture and aspiration of all≥18-20 mm diameter follicles. FF samples were centrifuged and collected granulosa cells. The level of GDF-9 and BMP-15 in FF was analyzed by western-blot. The expression of GDF-9 and BMP-15 in granulosa cells was measured by RT-PCR.Results: (1) Compared with control group, The average value of GDF-9 in FF of experimental group(2.7305±0.1260) was higher than in control group(2.0564±0.3055) (P<0.05). (2) The expression of GDF-9 mRNA in human granulosa cells of experimental group and control group were 21807.0±1998.6 and 10177.0±2571.4 respectively. The expression of GDF-9 mRNA in experimental group was significantly higher than in control group(P<0.01). (3) Compared with control group, BMP-15 in FF of experimental group (2.6454±0.4008) was higher than in control group (1.6252±0.4371). (P<0.05).(2) The expression of BMP-15 mRNA in human granulosa cells of experimental group and control group were 11894.0±865.6 and 6441.0±794.6 respectively. The expression of BMP-15mRNA in experimental group was significantly higher than in control group(P<0.05).Conclusions: Bushen Tiaojing Recipe can increase the level of GDF-9 and BMP-15 in FF and the expression of GDF-9 and BMP-15 in granulosa cells.All of the above demonstrates that Bushen Tiaojing Recipe can improve oocyte quality, increase fertilization rate, high quality embryo rate and clinical pregnancy by decreasing granulosa cells apoptosis rate and increasing OSFs GDF-9 and BMP-15. Bushen Tiaojing Recipe may be the adjuvant treatment during IVF.