The Effects Of GnRH Antagonist,Letrozole And Mifepristone On Cultured Human Granulosa Cell In Vitro

Objective:To investigate the effect of Gonadotropin-releasing hormone antagonists（GnRH-A）,Letrozole and Mifepristone on the human primary ovarian granulosa cell viability,apoptosis,the secretion of steroid hormones and the expression of Vascular Endothelial Growth Factor（VEGF）.Methods:1.The ovarian luteinizing granulosa cells were cultured and identified:Postovulatory granulosa cells were collected from follicular fluid obtained from patients undergoing in vitro fertilization-embryo transfer from March 2018 to October 2018 in Center for Reproductive Medicine of Renmin Hospital of Wuhan University.The granulosa cells were isolated and purified by 50%Percoll density gradients and identified by FSHR.Moreover,this study observed the growth behavior and adherence of granulosa cells.2.The effect of different concentration of GnRH-A,Letrozole and Mifepristone on the granulosa cells viability:After granulosa cells were cultured with different concentrations of GnRH-A,Letrozole,Mifepristone for 24 hours,the cells viability was detected by CCK-8 method.3.The effect of 10nM GnRH-A,10μM Letrozole,1μM Mifepristone and the combination of three drugs on the granulosa cells viability,cell apoptosis rate,the secretion level of steroid hormone and the expression of VEGF:After isolated granulosa cells were cultured for 24 hours,it was divided into blank control group,GnRH-A group,Letrozole group,Mifepristone group and the Combination of three drugs group.After the treatment for 24 hours,the study detect the cell viability through CCK-8 method,the cell mitochondrial membrane potential by JC-1 kit,the apoptosis rate by flow cytometry and VEGF mRNA levels by Real time PCR.The level of VEGF,Estradiol（E₂）and progesterone（P）in the supernatant of each group were also detected by enzyme-linked immunosorbent assay（ELISA）.Results:1.The survival rate and the cell purity of human primary ovarian granulosa cells obtained by Percoll density gradient centrifugation were over 90%.The growth state and adherence of cells were good after 48 hours of cell culture.2.The results of cell viability of granulosa cell treated with different concentrations of GnRH-A,Letrozole,Mifepristone for 24 hours:Different concentrations of GnRH-A,letrozole and mifepristone inhibited granulosa cells at different levels compared with the control group.The cell viability of 10nM GnRH-A,10μM Letrozole and 10μM Mifepristone group were most significantly inhibited,and the difference was statistically significant（p<0.05）.To avoid large number of cells death,our study choose 10nM GnRH-A,10μM Letrozole and 1μM Mifepristone for the following experiments.3.The effect of GnRH-A,Letrozole,Mifepristone and the Combination of three drugs on cell viability,mitochondrial membrane potential,cell apoptosis,secretion level of E₂ and P,and the expression of VEGF of granulosa cells:（1）Cell viability:Compared with the control group,GnRH-A,Letrozole,Mifepristone and the Combination group significantly inhibited the viability of granulosa cells.The inhibitory effect of Combination group was the most significant and the difference with the other group was statistically significant（p<0.05）.（2）Mitochondrial membrane potential:Compared with the control group,Mifepristone and Combination of three drugs significantly reduced the mitochondrial membrane potential of granulosa cells.The level of Combination group were the lowest in all groups and the difference was statistically significant compared with the other group.（p<0.05）.（3）Cell Apoptosis:Compared with the control group,Mifepristone and Combination of three drugs significantly promoted granulosa cell apoptosis.The apoptosis rate of the Combination group was the highest,and the difference with the control group was statistically significant（p<0.05）.（4）E₂and P level:The E₂ level of GnRH-A,Letrozole and the Combination group were significantly lower than the control group.And the E₂ level of the Combination group was the lowest and the difference with the other group was statistically significant（p<0.05）;the levels of P in the Letrozole,Mifepristone and the Combination group were significantly lower than the control group,and the P levels of the Combination group was the lowest and the difference with the GnRH-A group and the letrozole group was statistically significant（p<0.05）.（5）The expression of VEGF:Compared with the control group,the expression of VEGF mRNA in GnRH-A,Letrozole and the Combination group were significantly decreased,and the level of Combination group were the lowest and the difference with control group was statistically significant（p<0.05）.The level of VEGF in the supernatant of GnRH-A,Letrozole,Mifepristone and the Combination group were significantly lower than the level of the control group.The Combination group had the lowest VEGF level and had a statistically significant difference compared with GnRH-A group and Letrozole group（p<0.05）.Conclusion:Aftergranulosacellstreatedwith10nMGnRH-A,10μM Letrozole,1μM Mifepristone and the Combination of three drugs separately for 24hours,the cell viability and secretion level of steroid hormone and VEGF decreased at different level.Compared with GnRH-A and Letrozole,the Mifepristone group had higher cell apoptosis and lower level of P but higher level of E₂ and VEGF.The Combination group has the most significant effect on inhibiting granulosa cells viability,promoting cell apoptosis,decreasing VEGF mRNA expression and reducing the secretion levels of VEGF,E₂ and P.It is suggested that the combination of the three groups of drugs is more effective than the single drug group in the dissolution and degeneration of the corpus luteum.