Effects Of Icariin On The Proliferation And Secretion Of Ovarian Granulosa Cells To Improve The Outcome Of IVF Of DOR Patients With Kidney Yang Deficiency

Objective:To investigate the pregnancy outcome and clinical interventions to DOR patients with Kidney Yang Deficiency under IVF treatment,to explore the biological mechanism of Icariin affecting the proliferation and secretion of ovarian granulosa cellsMethods:In the clinical study,the DOR patients with Kidney Yang Deficiency were treated by IVF-ET were selected as the study objects.The patients with normal ovarian function were selected as the control group.The patients with normal ovarian function were treated with a short-acting long protocol for ovarian stimulation by GnRH-agonist,and the DOR patients with Kidney Yang Deficiency were treated with micro ovarian stimulation protocol.The observation indexes included the general characteristic of the patients,the markers for evaluating ovarian function,and the KPIs in the IVF treatment cycle.Further,the DOR patients with kidney Yang Deficiency were randomly divided into two groups:Wen Shen Bu Yang treatment group and the control group.Observing the TCM syndrome score of kidney yang deficiency before and after treatment.In the experimental study,human granulosa cell like tumor cells(KGN cells)were cultured and divided into six groups:?control group(PBS treatment);?ICA treatment group(5ug/L);?ICA treatment group(10ug/L);?ICA treatment group(25ug/L);?ICA treatment group(50ug/L);?ICA treatment group(75ug/L);analysing the mechanism of ICA affecting the proliferation of granulosa cells and steroid hormone secretionResults:1)Compared with the patients with normal ovarian reserve function,the age of DOR patients with Kidney Yang Deficiency was increased significantly,the evaluation indexes of ovarian reserve function(bFSH,AMH level and AFC)and the KPIs in IVF treatment cycle,the number of obtained oocytes,the number of M II oocytes,the rate of normal fertilization,the rate of cleavage,and the rate of high-quality blastocysts were all decreased significantly.The number and the quality of oocytes of DOR patients with Kidney Yang Deficiency crucially affect the clinical pregnancy outcome.2)Wen Shen Bu Yangt reatment can relieve the clinical symptoms of TCM in the DOR patients with Kidney Yang Deficiency.The pre-treatment score of the clinical symptoms of TCM was 21.71±2.07,and the post-treatment score was 18.06±2.15,with significant statistical difference.After Wen Shen Bu Yang treatment,the D3 high-quality embryo rate and the blastocyst formation rate were significantly improved of the DOR patients with Kidney Yang Deficiency.3)Icariin can remarkably affect the level of estradiol secretion and proliferation of granulosa cells of the ovary.When icariin is treated at 5ug/L,the effect was the most obvious,but it had little effect on progesterone secretion.Further analysis the expression of CREB protein in granulosa cells,it shows that Icariin at 5ug/L can up-regulate the level of CREB protein expression in granulocytes.Conclusions:1)The age of the DOR patients with Kidney Yang Deficiency is increased significantly,the number of M II oocytes obtained decreased significantly,and the rate of high-quality blastocysts decreased significantly,which suggested that the developmental potential of oocytes,especially the development potential of high-quality blastocysts is decreased significantly of the DOR patients with Kidney Yang Deficiency.Early evaluation and effective intervention are very significant.2)Wen Shen Bu Yang treatment can relieve the clinical symptoms of TCM in DOR patients with Kidney Yang deficiency.Wen Shen Bu Yang treatment can significantly improve The D3 high-quality embryo rate and the blastocyst formation rate.So Wen Shen Bu Yang treatment provides a strong guarantee for improving the clinical pregnancy outcome.3)Icariin can remarkably improve the estradiol secretion of granulosa cells and the proliferation ability of granulosa cells.When icariin is treated at 5ug/L,the effect is the most obvious.4)Icariin can remarkably up-regulate the expression of CREB protein,which suggests that Icariin may improve the proliferation mechanism of granulosa cells by up-regulating the aromatase gene transcription molecular pathway of granulosa cells.