| The polycystic ovary syndrome (PCOS) is the most common endocrine metabolic disorder in women of reproductive age with a high prevalence of 5-10%. The manifestations are highly heterogeneous, as follows: reproductive disorders (oligomenorrhea, amenorrhea, infertility, hirsutism, increased pregnancy complications, etc.); metabolic disorders (abnormal glucose tolerance, type 2 diabetes mellitus, hypertension, cardiovascular disease and abnormal lipid metabolism); mental illness (depression, anxiety and reduced life quality). From the 1960s, a large amount of research has been undertaken on its etiology and pathophysiologic change, but its pathogenesis is still not clear. Exist in the following hypothesis: 1, ovarian or adrenal steroid hormone enzyme synthesis defect; 2, abnormalities of hypothalamic GnRH secretion; 3, insulin resistance and compensatory hyperinsulinemia. In recent 20 years, insulin resistance and compensatory hyperinsulinemia are considered important in the pathogenesis of PCOS. Insulin resistance can cause not only chronic low-grade inflammatory changes, metabolic disorders, but also reproductive disorders. Insulin levels increase: interaction with LH to increased ovarian androgen synthesis and secretion; decrease the liver secretion of sex hormone binding globulin, increase free testosterone concentrations correspondingly; and increased synthesis of steroid hormones by ovarian cells with insulin-like growth factor (IGF) synergy. Under physiological conditions, insulin binds to a subunit of its receptor, induces tyrosine base phosphorylation ofβsubunit, activates phosphorylation of multiple tyrosine residues of the insulin receptor (InsR) tyrosine kinase and insulin receptor substrate (IRS) and contributes to cascade reaction of the downstream signaling. Such as the insulin receptor and its substrate enhanced serine phosphorylation, tyrosine phosphorylation decreased and insulin signaling blocked which means the insulin resistance. Studies have shown that insulin resistance exists in adipose tissue, muscle, liver and other organs of PCOS patients, manifested as insulin receptor and its substrate phosphorylation change, which causes insulin signaling pathways blocked. Granulosa cells of PCOS patients show resistance in the synthesis of glycogen, and which can be corrected by insulin sensitizer. PCOS ovarian luteinized granulosa cells and insulin-stimulated glucose metabolism lactate production decreased, while pyruvate consumption increased. This tells the action of insulin in ovarian granulosa cells is damaged. Whether insulin receptor substrate Serine phosphorylation (IRS-1 SerP) increase which leads to insulin signaling obstacles exists in PCOS granulosa cells? Research has not been reported at home and abroad.Metformin (Met), as an insulin sensitizer treats PCOS by increasing peripheral tissue insulin sensitivity, reducing blood insulin levels, and indirectly reducing androgen, which conduces to the ovary restore ovulation, correcting metabolic disorders. The molecular mechanism of Met treating PCOS is unknown. Whether Met can improve granule cell insulin action damage, directly inhibit the expression of estrogen synthase cytochrome P450 aromatase (P450arom) of granulosa luteal cells and reduce the estrogen production, these need to be further confirmed by experiments.AMPK signaling pathway, also called "cellular energy regulator", is the central link that regulates cellular energy state. AMPK is a trimeric enzyme by one catalytic subunitαand two regulatory subunitsβ,γ. AMPK activity is fromα-subunit phosphorylation of tyrosine 172 residues. AMPK enzymes can be activated by tissue hypoxia, hormones and drugs. Whether Met increases AMPK phosphorylation (pMAPK) and the energy supply in granulosa cells? These are worth studying. Objective: 1. Whether Met can improve clinical outcomes of PCOS patients assisted by reproductive technology; 2. Whether Met can turn down expression of granulosa cells insulin receptor substrates serine phosphorylation (IRS-1SerP) in PCOS ovary? 3. Whether Met can stimulate granulosa cells pMAPK to increase lactate synthesis; 4. Whether Met can be inhibit expression of granulosa cell P450 aromatase and indirectly low estrogen synthesis, which can provide a theoretical basis for clinical treatment.Method: 1. Collect 150 cases of PCOS patients treated by in vitro maturation (IVM) in reproductive center of Anhui medical university first affiliated hospital from January 2005 to December 2009 for 92 cycles, 44 cases were given metformin (1000mg / day×3 Months) pre-treatment; Collect 124 cases of PCOS patients treated by in vitro fertilization and embryo transfer (IVF-ET), 66 cases were given metformin (1000mg / day×3 months) pre-treatments. Analysis of two groups of clinical data: the basis of sex hormone levels, fasting blood glucose and insulin, the number of oocytes, in vitro maturation rate, fertilization rate, implantation rate, endometrial thickness, clinical pregnancy rate, Ovarian Hyper-Stimulation Syndrome (OHSS) rate.2. Real PT-PCR is detected gene expression of IRS-1 and MAPK, in PCOS ovarian granulosa cells after 36h incubation by adding Met: 10uM,100 uM and 200uM. Western Blot is used to detect phosphorylation expression of IRS-1Ser in metformin pre-treatment PCOS granulosa cells.3. Protein and mRNA expression of P450arom of PCOS luteinized granulosa cells by metformin is also tested in vivo and in vitro.Results:1. Pre-treatment metformin of PCOS can increase the clinical pregnancy rate in IVM group and IVF group (34.1%±12.5%,39.40%±20.69%. P<0.05 ), and also significant decrease the incidence of OHSS in IVF PCOS patients group (9.09%±22.41%, P<0.05). 2. IRS-1 gene expression of ovarian granulosa cells in PCOS in vitro is 2.1401E5±32365 in Met 10 uM(group A ), 1.7560E4±1362 in Met 100 uM (group B) and 1.2849E4±1118 in Met 200 uM (group C). There are statistic difference between group A and group B or group C (P<0.05). AMPK gene expression is 1.5547E3±172 in Met 10uM group, 1.1886E4±2509 in Met 100uM group and 1.0716E6±1.61560E5 in Met 200uM group. The means of three groups are not same in variance analysis, F equals 825.325, and p<0.05. There are statistic difference between Met 10uM group and 200uM group(P<0.05).3. There are no significant difference about IRS-1 gene expression of ovarian granulosa cells of PCOS patients obtained after Met pre-treatment compared with control group (33986±7174 vs 34534±7796, t=1.262, P=0.224). AMPK gene expression is statistic difference compared Met group with PCOS group (1.1055E6±1.34019E5 vs1.4205E3±196, t=34.938, p<0.05). IRS-1Ser phosphoprotein expression of Met group is lower than PCOS group with statistical difference(0.30±0.07vs1.17±0.15, t=22.87, p<0.05).4. P450 gene expressions of luteinized granulose cell in PCOS group, pre-treatment of Met group in vivo and adding Met group in vitro are 1.0225E7±2.56166E6, 1.6154E4±1446 and 1.4817E4±1452 respectively. Compared with other two groups, PCOS group shows statistic difference in variance analysis (p<0.05). P450 protein expressions of three groups are 1.47±0.30, 0.57±0.23 and 0.58±0.21 respectively. Group A and group C, group A and group B have statistic differences.Conclusion:1. Pre-treatment metformin for PCOS patients can increase clinical pregnancy rate and decrease the occurrence of OHSS in IVM-ET and IVF-ET.2. IRS-1 gene expression of PCOS ovarian granulosa cells in vitro shows negative effect on dependent of Met dose, but AMPK shows positive. IRS-1Ser phosphorylation decreased in PCOS granulosa cells with Met pre-treatment. Therefore Met can improve insulin sensitivity in ovarian granulosa cells, activate AMPK pathway and increase cellular energy synthesis.3. In vivo and in vitro experiments, Met decrease expression of P450arom protein and mRNA of PCOS's ovarian luteinized granulosa cells, which lead to low estrogen synthesis and prevent the occurrence of OHSS.
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