Study On The Effect And Mechanism Of Resveratrol On Human Arrested Embryo Development And Oxidative Damage Of Aging Ovarian Granulosa Cells In IVF

Objective:1.This research aims to explore the effect of resveratrol on human embryos with developmental arrest during IVF.2.To study the molecular mechanism of resveratrol on improving oxidative damage of ovarian granulosa cells in elderly patients through PI3K/AKT pathway.Methods:1.The retrospective study of patients with arrested embryos and single cell transcriptome sequencing of arrested embryos.The clinical data of IVF/ICSI patients were disposed and divided into control group and arrested group.The number of retrieved oocytes,MⅡoocytes,fertilization,good-quality embryos,multi-PN embryos,and the pregnancy outcomes were analyzed.At the same time,the arrested embryos were collected for single-cell transcriptome sequencing to explore the mechanism of developmental arrest.Then,based on the gene expression and pathway analysis from the sequencing,the arrested embryos were cultured in vitro with different molecular compounds to select the befitting ones that could promote arrested embryos to continue develop.2.Molecular research of arrested embryos.Human embryos arrested on day 3 were cultured in vitro with or without resveratrol.Through the morphology observation by light microscope,Q-PCR technology and immunofluorescence technology,The effect of resveratrol on the important genes related to oxidative stress,cell cycle and PI3K/AKT pathway in arrested embryos were detected.3.Molecular research of ovarian granulosa cells from elderly patients.First,retrospectively analyse the effect of age on oocyte,embryo and pregnancy outcome.Then collect ovarian granulosa cells from young patients and elderly patients during IVF/ICSI,and compare the differences in the expression of key genes in the oxidation reaction,cell cycle and PI3K/AKT pathway between the two groups.H₂O₂was used to induce the KGN cell line to establish an ovarian granulosa cell senescence model,and the molecular regulation mechanism of resveratrol to improve the oxidative senescence of ovarian granulosa cells under the mediation of PI3K/AKT pathway was investigated by Q-PCR and WB technology.ROS detection,SOD detection and mitochondrial membrane potential detection were used to study the antioxidant protection of resveratrol on aging ovarian granulosa cells mediated by PI3K/AKT pathway.Results:1.The proportion of patients with embryo arrest was as high as 54.73%,and the proportion of cycles with embryo arrest was as high as 39.20%.The advanced age of both males and females,as well as the decrease in AFC of the woman,increased the incidence of embryo arrest（P<0.05）,and the rate of good-quality embryos in the arrested group decreased,and multi-PN increased in the arrested group（P<0.05）.2.Single-cell sequencing showed that,in arrested embryos,ribosomal proteins,transcription factors,cyclins proteins,embryonic developmental pluripotency genes were significantly down-regulated,cell cycle,cell stress,DNA repair and other pathways were down-regulated,The expression of 4EBP1,a downstream molecule of PI3K/AKT/m TOR pathway,was increased,and the expression of P53,a negative cell cycle regulator,was increased.3.The results of molecular experiments of arrested embryos showed resveratrol reduced the m RNAs expression of PI3K,AKT,P53 and P21 in the arrested embryos（P<0.05）,and enhanced the m RNAs of SIRT1,Nrf2 and FOXO3A in the arrested embryos（P<0.05）.Immunofluorescence showed that resveratrol reduced the protein expression of p-AKT,P53 and P21 in arrested embryos（P<0.05）,and enhanced the protein expression of SIRT1 and Nrf2 in arrested embryos（P<0.05）.4.Studies on ovarian granulosa cells in advanced age showed that,as the age increased,the number of retrieved oocytes,MⅡoocytes and good-quality embryos descended,followed by the decreasing clinical pregnancy rate,the live birth rate,and the cumulative live birth rate.（P<0.05）.Compared with ovarian granulosa cells in the young group,the m RNA expressions of SIRT1,Nrf2 and FOXO3A of the elderly group were all decreased（P<0.05）,and the m RNA expressions of P53,P21,PI3K and AKT of the elderly group were increased（P<0.05）.In the senescence-like KGN cells induced by H₂O₂,the m RNA expression of SIRT1,Nrf2 and FOXO3A was lower than that in the control group（P<0.05）,and the m RNA expression of PI3K,AKT,P53 and P21 was higher than that in the control group（P<0.05）.The gene expression pattern was the same as that of ovarian granulosa cells in elderly patients,so an oxidative senescence model of ovarian granulosa cells was successfully established.Then the mechanism of PI3K/AKT-mediated resveratrol improving oxidative aging was further studied through senescence-like KGN.The results were as follows.（1）Compared with the control group,the protein expressions of SIRT1,Nrf2 and FOXO3A in H₂O₂ group decreased（P<0.05）,and the protein expressions of p-PI3K/PI3K,p-AKT/AKT,p-P53/P53 and P21 increased in H₂O₂ group（P<0.05）.In LY294002 group,the protein expressions of SIRT1,Nrf2 and FOXO3A were higher than those in the H₂O₂group（P<0.05）,and the protein expressions of p-PI3K/PI3K,p-AKT/AKT,p-P53/P53and P21 were lower than those in the H₂O₂ group（P<0.05）.In the resveratrol group,the protein expressions of SIRT1,Nrf2 and FOXO3A were higher than those in the H₂O₂group（P<0.05）,and the protein expressions of p-PI3K/PI3K,p-AKT/AKT,p-P53/P53and P21 were lower than those in the H₂O₂ group（P<0.05）.In the LY294002+resveratrol group,the protein expressions of p-PI3K/PI3K,p-AKT/AKT,p-P53/P53 and P21 were lower than those in the resveratrol group（P<0.05）,and the protein expressions of SIRT1,Nrf2,FOXO3A,p-PI3K/PI3K,p-AKT/AKT,p-P53/P53 and P21 were not different from those of the LY294002 group（P>0.05）.The PI3K/AKT pathway mediated the molecular mechanism of resveratrol to improve the oxidative damage of aging ovarian granulosa cells.（2）Further study the effect of resveratrol on improving the oxidative damage of aging-like KGN showed that,the SOD and MMP of KGN in the H₂O₂ group were lower than those in the control group（P<0.05）,the SOD and MMP of the resveratrol group were higher than those in the H₂O₂ group（P<0.05）,the SOD and MMP of the LY294002+resveratrol group were lower than those of the resveratrol group（P<0.05）.and were same with the LY294002 group（P>0.05）,The ROS of KGN in the H₂O₂ group was higher than that of the control group（P<0.05）,the ROS of the resveratrol group was lower than that of the H₂O₂ group（P<0.05）.There was no difference on ROS in LY294002+resveratrol group compared with LY294002 group（P>0.05）.The PI3K/AKT pathway mediated the effect of resveratrol on the oxidative damage of ovarian granulosa cells.Conclusions:1.During IVF/ICSI,the incidence of embryo arrest is still very high.Age and ovarian function are important factors that cause embryonic developmental arrest.Insufficiency of congenital essence is the TCM pathogenesis of embryonic developmental arrest.The goog-quality embryo rates in cycle where arrest occurs is significantly reduced,which reduces the efficiency of IVF.2.Arrested embryos present a state of cell cycle arrest and senescence,resveratrol activats the cell cycle through regulation of antioxidant response and promotes partial arrested embryos develop further,and the process is related to the PI3K/AKT pathway.3.PI3K/AKT pathway-mediated oxidative damage is an important mechanism of granulosa cell aging in elderly patients.Resveratrol can regulate the redox reaction and cell cycle process in senescent granulosa cells through PI3K/AKT pathway,thereby prevents oxidative damage,activats cell cycle,and saves cell from senescence.