Study On The Mechanism Of TSH Regulating The Secretion Function Of Granular Cells

ObjectiveTo study the secretion function of ovarian granulosa cells in infertile women with subclinical hypothyroidism.The effects and mechanism of TSH on the secretion function of granular cells were investigated by exploring the changes of the expression of TSHR,FSHR and CYP19A1 on ovarian granular cells in infertile women with SCH.MethodsPart one:Isolation and identification of ovarian granulosa cellsCollect the follicular fluid on the day of egg extraction of infertile women who received the ⅣF/ICSI assisted fertility treatment at the Reproductive center of the first hospital affiliated to Suzhou University from September 2018 to January 2019,and to separate the granulosa cells from it with the Percoll density gradient separation solution,Granulosa cells were identified by flow cytometry and cellular immunofluorescence in the detection of granular cell specific protein FSHR.Part Two:Correlation study on the effect of TSH on the secretion function of granular cells1.Using the research method of cohort research,for infertile women who performedⅣF/ICSI assisted fertility treatment at the reproductive center of the first hospital affiliated to Suzhou University from September 2018 to January 2019,Set the concentration of TSH<2.5 mIU/L in the menstrual period as a normal group,while the other ones are set as an Elevated group.The follicular fluid was collected on the day of egg extraction,and the granulosa cells were isolated by Percoll density gradient separation solution.The mRNA expression levels of FSHR,TSHR and CYP19A1 on two groups of granulosa cells were detected by RT-PCR.2.Four women with ⅣF/ICSI assisted therapy were randomly selected,the follicle solution was collected on the day of egg extraction,and the granular cells were separated by Percoll density gradient separator.Then 0,2.5,5.0 and 10mIU/L TSH stimulation was added after culture in vitro for 24h.2.1 Collect the supernatant to detect the concentration of estradiol and progesterone by radioimmunoassay in the Nuclear medicine Department of of the first hospital affiliated to Suzhou University.2.2 Detection of mRNA levels in cell TSHR and CYP19A1 by RT-PCT.2.3 Detection of cell TSHR protein expression levels by Western Blot.ResultsPart one:Isolation and identification of ovarian granulosa cells1.Flow cytometry have detected the expression of FSHR in almost all cells,and 15.3%of cell FSHR expression strongly positive.2.The expression of FSHR in cytoplasm and membrane was detected by cellular immunofluorescence.Part Two:Correlation study on the effect of TSH on the secretion function of granular cells Compared with the normal group,the mRNA expression of the elevated group incresas with statistically significant The primary ovarian granular cells increased with the concentration of TSH stimulation,and the secretion of estradiol and progesterone level decreased.Primary granular cells increased with the concentration of TSH stimulation,and the protein level of TSHR increased,and the difference was statistically significant.Conclusion1.The cells obtained from follicular fluid by Percoll density gradient separation are identificated as ovarian granular cells,by the expression of FSHR protein both from the membrane and cytoplasm.2.The infertile population whose basic concentration of TSH are greater than 2.5mIU/L,have significantly higher mRNA expression of TSHR compared to the less ones.3.The secretion of estradiol and progesterone level decreased while the concentration of TSH added exogenously in primary ovarian granulosa cells,with the increase of the concentration of TSH protein and TSH mRNA,and the reduction of CYP19A1 mRNA.4.High levels of TSH reduce the secretion of Steroid hormone in granulosa cells,which may be related to the increase of TSHR and the reduction of granular aromatic enzymes,as the reaction of the stimulation of high levels of TSH.