Effect Of Lowering Androgen Treatment Of Polycystic Ovary Syndrome On The Apoptosis Of Cumulus Granulosa Cells

Polycystic ovary syndrome (PCOS) is a heterogeneity of clinical syndrome, which is caused by a wide range of metabolic abnormalities, the endocrine disorders is main one. Both ovaries of PCOS patients contain a large number of antral follicle, but cannot form mature follicle. At present, though there are some pathological research, but its pathological mechanism is not very clear.The study that the pathogenesis of PCOS didn’t ovulate showed that PCOS may be caused by the primary and inner follicular growth.High androgen play a very important role in the process of PCOS pathogenesis. But high androgen can inhibit the growth and development of ovarian follicles in ovarian, which can prevent advantage mature follicle, lead to abnormal follicular atresia and cause in polycystic ovary change (PCO) eventually;At the same time, increased androgen caused the hypothalamus-pituitary-gonadotropin axis dysfunction and promote insulin increased continuously, thus stimulate luteinizing hormone (LH) is generated, PCOS patients high LH and promote ovarian and adrenal gland secretion of androgens, so form continued without a vicious cycle of ovulation.Scientific studies have shown that the cumulus granulosa cells apoptosis regulation disorder and internality of abnormal development of follicle there may be a connection.Based on the research of the PCOS patients, found that cumulus granulosa cells internal resistance to apoptosis and promote apoptosis exist between the two states of imbalance, the phenomenon with PCOS patients serum testosterone levels have close relationship.Granulosa cell apoptosis of mice can be induced by high androgen hematic disease, the study found that testosterone granulosa cell apoptosis can be induced mice, and increase active Caspase3expression, and in polycystic ovary syndrome rats model induced by excessive androgen experiment, found that the apoptosis rate cumulus granulosa cells of rats increased significantly.The latest research results show that the endoplasmic reticulum stress (ERS) involved in apoptosis pathway.To resume the function of cells, the biology through activating folded protein response (UPR) repair cell damage caused ERS, if the damage is serious, can’t recover in time, ERS will cause the cell apoptosis.Numerous studies have demonstrated that the apoptosis of cumulus granulosa cells, a direct impact on the corresponding oocytes at a later stage of growth, development, maturity and the quality of the fertilization and embryonic development.This suggests that the mound oocytes and the eggs between granular cell, there are some broad and complex mechanism of adhesive.In this mechanism, including gap junction intercellular interaction, in the process of oocyte development plays an important role in regulating.Lee, etc in early studies, reported the apoptosis of cumulus granulosa cells and PCOS patients age, fertilization rate, the number of eggs and IVF outcome, thus proposed, using the apoptosis of cumulus granulosa cells in predicting the conclusion of IVF outcome.Cao Yijuan will eggs for in vitro culture, such as human the apoptosis rate of cumulus granulosa cells and the relationship between the oocyte development potential, the research results show that the lower the oocyte maturation, instead of the apoptosis rate of cumulus granulosa cells is higher;And as the oocyte development potential and ratings decline, the apoptosis rate of cumulus granulosa cells will increase.The domestic research suggests the development potential of the egg and egg high granular cell growth condition is closely related;The apoptosis rate of cumulus granulosa cells effect of egg maturation, fertilization and cleavage, the apoptosis rate of cumulus granulosa cells and fertilize an egg, embryo quality and closely related to pregnancy outcomes.To sum up, this topic had studied the relationship between the apoptosis rate of cumulus granulosa cells and the pathogenesis of polycystic ovary syndrome on the basis of predecessors’ research of theory and experiment, and relationship between the FAI and the pathogenesis of polycystic ovary syndrome had been studied,and study the effect to the apoptosis of cumulus granulosa cells in patients with polycystic ovary syndrome by decreasing androgen levels,.Objective:1. To compare the apoptosis rate of cumulus granulosa cells among treatment group, control group and normal control group, and to explore the diagnostic value in the process of infertility of free testosterone index level in high androgen hematic disease and polycystic ovary syndrome (PCOS).2. To detect the expression level of apoptosis regulatory proteins Caspase3, Bcl-2and endoplasmic reticulum stress protein CHOP.3. The study the correlation between embryo quality and the apoptosis rate of cumulus granulosa cells after fertilization, understand the effect of the apoptosis rate of cumulus granulosa cells to embryo development potential of PCOS.Methods:1. The selection of nanfang hospital, southern medical university reproductive center first IVF-ET treatment of60patients with infertility.Through screening, will meet the conditions of the patients into three groups:(1) experimental group:male reduction treatment (oral DAYING-35) after free testosterone index levels were normal in patients with PCOS;[2] the control group:free testosterone index rise in patients with PCOS (do not take oral DAYING-35male);(3) the normal control group:normal ovarian response of infertility patients.All of the patients, after treatment with super stimulate ovulation, eggs and pick up the eggs, demolition of egg and egg quality assessment after IVF.2. The scheme of corpus luteum mid long in one menstrual cycle before mid luteal began to use the GnRH-a, play to fall after the adjustment, based on pituitary drop to adjust start designed.the Gn, vaginal ultrasound monitoring follicle number and follicle diameter, when the follicle diameter of2or more acuity18mm, injections of human chorionic gonadotropin (HCG),5000-10000-u prompt ovulation.Fertilization rate of fertilization and the ratio of the number of eggs. Line3. After fertilization, the following Index detection and statistics:age, BMI (Body Mass Index, BMI), FSH of foundation and designed.the Gn (Gonadotropin) days, a number of eggs, fertilization rate, high quality embryo rate, estradiol (estradiol, E2), prolactin (prolactin, PRL), total testosterone (testosterone, T) and sex hormone binding globulin (sex hormone-binding globulin, SHBG), Free testosterone Index (Free androgen Index, FAI).4. All of the patients after surgery for cumulus granulosa cells, the following experiments:(1)3-end in situ labeling method (TUNEL) and flow cytometry analysis the apoptosis rate of cumulus granulosa cells, comparing three sets of samples apoptosis rate difference;(2) the Real-Time PCR and Western Blotting detection of endoplasmic reticulum stress protein CHOP, apoptosis regulatory proteins, the Bcl-2and Caspase3expressed in cells high granular cell, compare the differences between the three groups.(3) to form embryos after observation, embryo transfer, luteal support and follow-up clinical pregnancy, the pregnancy rate of statistics.Results:Through screening, accord with a standard into the group of60cases of patients, treatment group, control group and normal control group20cases:(1) experimental group:oral taie-35, free testosterone index levels were normal in patients with PCOS;(2) in the control group:free testosterone index rise in PCOS patients, do not take oral taie-35male;(3) the normal control group using normal ovarian response of infertility patients.Experimental results were as follows:1. The basic clinical data and the status of assisted reproductionThere was no statistically significant difference between three groups of patients with basic characteristics, including age, BMI, basic FSH, designed.the Gn days (P>0.05).2. Cells and embryos(1) The comparison of cell number between control group, experimental group and normal control group:compared with control group, normal control group and experimental group received cell number there was no significant difference between the two groups (P>0.05);Compared with normal control group and experimental group, the cells there was no significant difference between the array (P>0.05).2group, control group and normal control group, high quality embryo rate, M II period the number of cells, and fertilization rate comparison:compared with control group, experimental group was higher than the control group, the difference was statistically significant (P<0.05);Normal control group is higher than the control group, the result is statistically significant (P<0.05), suggesting that the experimental group and normal control group of oocyte quality is higher than the control group.FAI (3) experimental group and control group and normal control group comparison: compared with control group, experimental group is lower than the control group, the difference was statistically significant (P<0.05);But FAI group and normal control group, there was no statistically significant difference (P>0.05).3. TUNEL method is used to check the apoptosis rate of cumulus granulosa cells, flow cytometry analysis verified the apoptosis.(1) control group, experimental group and normal control group the apoptosis rate of cumulus granulosa cells results show that compared with the control group, experimental group apoptosis rate is lower than the control group, the difference was statistically significant (P<0.05);Control group was higher than normal control group, the difference was statistically significant (P<0.05);Experimental group compared with normal control group, there was no statistically significant difference (P>0.05).Real-Time PCR detection CHOP,[2] the Bel-2and Caspase3mRNA expression level shows that between groups have CHOP on granulosa cells, the Bcl-2and Caspase3mRNA expression There was no statistical difference of three groups of the Bcl-2expression meaning;Compared with the control group, experimental group and normal control group Caspase3, CHOP expression significantly lower (P<0.05);Compared with normal control group, experimental group the Bcl-2, Caspase3and CHOP expression differences has no statistical significance (P>0.05).(3) Western Blotting test CHOP, Bcl-2and Caspase3protein expression levels showed that each group have CHOP on granulosa cells, the Bcl-2and Caspase3protein expression.Three groups of the Bcl-2protein expression level differences had no statistical significance;Compared with the control group, experimental group and normal control group CHOP, Caspase3protein expression levels significantly lower (P<0.05);Compared with normal control group, experimental group the Bcl-2, Caspase3and CHOP expression had no significant difference (P>0.05).Conclusion:This topic through the experiment, the direct observation after fall male after treatment in patients with PCOS and FAI rise in PCOS patients and normal ovarian reactivity of infertility patients embryo quality and the apoptosis rate of cumulus granulosa cells after fertilization through the comparison, found that after fall male therapy, the patients with PCOS after in vitro fertilization, fertilization rate and embryo quality got improved significantly, improve the quality of oocyte and the apoptosis rate of cumulus granulosa cells significantly reduced.Show on the pathogenesis of PCOS, serum free testosterone index that can affect the apoptosis rate of cumulus granulosa cells and high quality embryo rate and pregnancy outcome after fertilization, the higher serum free testosterone index, the apoptosis rate of cumulus granulosa cells is higher, after fertilization embryo rate, the lower quality, clinical pregnancy rate is lower.Biochemical index in the diagnosis of PCOS preferred FAI, because it can not only accurately reflect free testosterone levels, also can indirectly reflect the apoptosis rate of cumulus granulosa cells, high quality embryos after fertilization rate and clinical pregnancy rate. There are great advantages and clinical application value compared with pure check total testosterone and free testosterone.