The Research Of GDF-9GDF-9B Expression And Regulation In Superovulation Cycle

The development of assisted reproductive technology, especially in vitro fertilization-embryo transfer is the most effective method to therapy infertility. Controlled ovarian hyperstimulation is one of the reasons that pregnancy rate during IVF-ET is gradually rising, however, people will generate different ovarian response when they apply ectogenic gonadotropin. The incidence rate of poor ovarian response is about12%-30%. Poor ovarian responders obtain fewer oocytes and lower serum estrogen level after superovulation, which always induce lower pregnancy rate and treatment cancellation. So it is very urgent problem to find an effective method to evalue and treat poor ovarian response to controlled ovarian hyperstimulation.Growth differentiation factor (GDF)-9and GDF-9B, as differentiation factors derived from oocytes, play a critical regulation role in the process of folliculogensis. Both of them belong to TGF-β family. GDF-9and GDF-9B effect oocytes, granulose cells and theca cells by autocrine and paracrine manners. They are involved in regulating cellular differentiation, proliferation via the formation of dominant follicle and oocyte maturation. This study will explore the relationships and effect mechanism between GDF-9, GDF-9B and poor ovarian response in patients who undergo controlled ovarian hyperstimulation, which will provide the theory support to increase dominant follicles and to improve ovarian function.Section I The relationship between GDF-9GDF-9B expression and poor ovarian responseObjective:To investigate the relationship between the expression of GDF-9, GDF-9B and poor ovarian response, to determine whether two factors are related to the mechanism of poor ovarian response.Methods:According to the number of ovarian follicles which average diameter were larger than fourteen millimeter,70patients undergoing in vitro fertilization treatment were divided into three groups,21poor responders,25moderate responders and24 high responders. Real-time PCR was performed to detect the levels of GDF-9GDF-9B mRNA in mural and cumulus granulose cells obtained from different response groups. The relationship was analyzed between the level of GDF-9, GDF-9B and the poor ovarian response.Results:There were no significant differences in the patient age, years of infertility, the serum levels of basal follicle stimulating hormone, basal estradiol and luteinizing hormone in different ovarian response groups(P>0.05). The expression of GDF-9mRNA was lower in poor ovarian response group than that of moderate and high response groups (P<0.05). In contrast, the expression of GDF-9B mRNA was the highest in poor ovarian response group. The expression of GDF-9, GDF-9B were respectively positive and negative correlated with the number of follicles (r=0.685,0.648;-0.737,-0.601; P<0.001).Section Ⅱ Human GDF-9gene eukaryotic expression and purificationObjectives:To construct the eukaryotic expression vector of human GDF-9gene and induce expression in stable transfection cell line, to obtain purified recombinant GDF-9protein.Methods:Human GDF-9gene was cloned by PCR method using the complementary DNA, and was inserted into pcDNA3.1(+) vector to construct recombinant eukaryotic expression plasmid pcDNA3.1(+)/GDF-9, which was transfected into human embryonic kidney293T cell line by liposome mediated to be induced expression. Interest protein was purified by immunoaffinity chromatography.Results:Recombinant plasmid was successfully constructed which was confirmed by double enzyme digestion and gene sequencing result. Recombinant protein GDF-9was successfully obtained which was verified by western-blotting.Section Ⅲ Effect of recombinant protein GDF-9on mouse superovulation Objectives:To study the influence of recombinant protein GDF-9on superovulation in vivo animal models, to analyze the effect on follicular development.Methods:30eugamic KM mice were divided into three groups, which received HMG10IU, HMG101U+recombinant protein GDF-910μg and equal volume normal saline i.p. respectively.48hours later, HCG10IU was injected into each mouse.16～20hours later30mice were killed, their ovary weight and the number of follicles were compared. Enzyme linked immunosorbent assay process to examine the level of serum estrogen (E2) in mice, their oocytes were fertilized in vitro, and fertilization information was observed.Results:The number of follicles, ovary weight and the level of serum estrogen (E2) after superovulation in HMG group, HMG+recombinant protein GDF-9group were significantly higher than those of the control group (P<0.05), especially, the group injected both recombinant protein GDF-9and HMG. There were no significant differences in fertility rate of different groups (.P>0.05).Conclusion:1. The expression of GDF-9、GDF-9B genes can be detected in granulose cells obtained from the patients who undergo superovulation. The lower expression of GDF-9and the higher expression of GDF-9B were associated with the possible mechanism of poor ovarian response. There was a positive correlation between GDF-9mRNA level and the number of mice follicles after superovlation, and there was a negative correlation between GDF-9B and the number of follicles.2. The eukaryotic expression vector pcDNA3.1(+)/GDF-9was successfully constructed. Recombinant GDF-9protein was successfully obtained.3. Recombinant protein GDF-9combined with gonadotropin could effectively improve KM mice ovarian response to superovulation, and increase ovulation rate. Utilizing recombinant protein GDF-9combined with gonadotropin had no influence on mice oocytes fertilizability.