Abnormal Energy Metabolism Of Granulosa Cells In PCOS And The Mechanism Of Hypoxia Promoting Glycolysis

The follicle is composed of oocyte and multiple granulosa cells around it.The outer cell layer of the follicle is theca cell.The basement membrane separates the theca cell from the granulosa cell layer,and isolates all blood vessels from the interior of the follicle.With the action of gonadotropin?Gn?,the increase of follicle diameter was accompanied by the proliferation and functional differentiation of granulosa cells.The amount of fluid secreted and accumulated between granulosa cells increased,forming follicular antrum.Follicular fluid?FF?divides granulosa cells into two subpopulations,including mural granulosa cells?mGCs?lining the wall of follicle and cumulus granulosa cells?c GCs?surrounding oocytes.When the dominant follicle matured accompanied by mGCs increasing,the oxygen is consumed and prevented from the follicle cavity,the barrier effect of basement membrane and the decrease of blood vessel density in the cell layer of follicle membrane,the follicle development is immersed in a short-term hypoxia environment.During the development,maturation,fertilization and embryo develop-ment of oocytes,a large amount of adenosine triphosphate?ATP?is produced by oxidative phosphorylation of mitochondria to provide energy.However,due to the low efficiency of direct utilization of glucose by oocytes,pyruvate,a glycolysis pathway product of granulosa cells,needed to be transported to the mitochondria of oocytes through the monocarboxylic acid transport system as a substrate for ATP energy production of oocytes.Mitochondrial membrane potential?MMP?is an important index of cell viability.Mitochondrial DNA?mt DNA?is the key to maintain mitochondrial function and cell growth.Reactive oxygen species?ROS?produced by oxidative stress?OS?can damage mitochondrial function,through lipid peroxidation,protein oxidation and DNA damage,cells functions are affected.In the ovarian development,granulosa cells respond too much to oxidative stress,which can lead to the decrease of mt DNA copy number and MMP,more seriously it can lead to apoptosis.Polycystic ovarian syndrome?PCOS?is one of the most common endocrine diseases in women of childbearing age,which has a significant impact on reproductive function,characterized by persistent follicular development and maturation disorders in the ovary.In vitro fertilization and embryo transfer?IVF-ET?,the quality of oocytes and embryos in PCOS patients was decreased,and the pregnancy outcome was not ideal.At present,the cause of PCOS follicular maturation disorder and low oocyte development competence is not clear.Hypoxia inducible factor-1??HIF-1??is considered to play a key role in the regulation of cell oxygen homeostasis,which is involved in oxygen homeostasis,growth and development,hypoxia adaptation,energy metabolism and other important activities.We speculate that the damage of energy metabolism function of ovarian granulosa cells and then the interference of hypoxic environment promoting the proliferation of granulosa cells may be one of the mechanisms of the decline of oocyte development competence in PCOS patients.This study is divided into two parts.The first part compare clinical characteristics,outcome of oocyte and embryo development,oxidative/antioxidant stress index of follicular fluid including MDA,8-OHd G,SOD,pyruvate and glucose in follicular fluid were compared.Changes of MMP,ATP,ROS,mt DNA and glycolysis related genes GLUT1,LDHA and PFKP in mGCs of the PCOS patients and patients with tubal factor infertility under IVF-ET.The effects of oxidative stress of follicular fluid on the development,maturation,fertilization and early embryo development of oocytes were analyzed.It is suggested that the damage of mitochondria function and abnormal glycolysis of granulosa cells interfere with the stability of follicular fluid microenvironment and participate in the abnormal process of PCOS follicular development.In the second part,in vitro cultured human mGCs,we added mitochondrial inhibitor?CCCP?,glycolysis inhibitor?BA?and hypoxia inducer?CoCl2?,to study the relationship between the transformation of energy metabolism pathway and the change of cell viability in granulosa cells and the protective effect of mitochondrial function stability on hypoxia promoting metabolism transformation.Part one Relationship between oxidative stress and energy metabolism of granulosa cells and oocyte development in PCOSObjective:To analyze the clinical characteristics and IVF-ET treatment outcome of PCOS patients and simple tubal infertility patients,to detect the content of oxidative stress markers in follicular fluid,mitochondrial function and glucose metabolism level in granulosa cells of the two groups of infertile people,and to explore the influence of PCOS on early embryonic development and the relationship between PCOS and the reduction of energy metabolism function of granulosa cells.Methods:1.158 age matched IVF-ET women from April 2018 to December 2018were selected,including 60 cases PCOS patients who met the Rotterdam diagnostic criteria?PCOS group?,and 98 cases infertile patients with simple fallopian tube factor?control group?.The oocyte and embryo development indexes and clinical pregnancy rate of the two groups were recorded.2.Follicular fluid was selected?40 cases in PCOS group and 50 cases in control group?.The levels of oxidative stress in follicular fluid were measured by ELISA.Pyruvate and Glucose in follicular fluid were detected by Kit.3.Two groups of mGCs were cultured in vitro.MMP,ATP and ROS were used to detect the mitochondrial function of the two groups of granulosa cells after the cells were attached to the wall.The experiment was repeated at least three times.4.q-PCR was used to compare the mt DNA copy number,RT-PCR was used to compare the glycolysis related gene GLUT1,LDHA,PFKP and hypoxia related gene HIF-1?m RNA expression of the two groups.The experiment was repeated at least three times.Results:1. Characteristics of general case data of patients:the body mass index?BMI?,basal LH,T and FGB levels in PCOS group were higher than those in control group?P<0.05?,and FSH levels were lower?P<0.05?.2. Controlled ovarian hyperstimulation indicators:there was no significant difference between PCOS group and control group in Gn dosage,duration and E₂level of h CG days?P>0.05?.3.Oocyte and embryo development indicators:the number of oocytes obtained and fertilized in PCOS group was higher than that in the control group?P<0.05?.There was no significant difference in the number of mature eggs,normal fertilized eggs,cleavage,available embryos and high-quality embryos between the two groups?P>0.05?.The rate of available embryo,high quality embryo,high quality blastocyst formation and clinical pregnancy in PCOS group were lower than those in control group?P<0.05?.4.Detect oxidative stress markers:the levels of MDA and 8-OHd G in follicular fluid of PCOS group were higher than those of control group?P<0.05?,the compensatory increase of SOD in PCOS group was statistically significant?P<0.05?.5.Detect glucose metabolism markers in follicular fluid:the content of pyruvate in follicular fluid of PCOS group was lower than that of the control group?P<0.05?,and the content of glucose was higher than that of the control group?P<0.05?.6.mGCs mitochondrial function:compared with the control group,the expression level of MMP,ATP and mt DNA in PCOS group decreased significantly?P<0.01?,and the level of ROS increased significantly?P<0.01?.7.mGCs glucose metabolism function:the expression of GLUT1,LDHA,PFKP and HIF-1?m RNA in PCOS group was lower than that in control group,and the expression of GLUT1,LDHA and PFKP was positively correlated with the expression level of low HIF-1?m RNA?P<0.05?.Conclusion:In PCOS patients,the rate of high-quality embryo formation,the rate of high-quality blastocyst formation and the rate of clinical pregnancy all decreased.There was over expression of oxidative stress in the microenvironment of follicular fluid.The oxidative damage of mGCs and the disorder of energy metabolism may interfere with the energy supply of granulosa cells to oocytes,and affect the development potential of oocytes and early embryos.Part two Relationship between hypoxia promote energy metabolism cellsObjective:The mitochondrial function of mGCs was observed by adding CCCP and BA;the mitochondrial function of mGCs was changed by adding CCCP,and the mitochondrial dysfunction model was established;the hypoxia environment was induced by adding CoCl2,and the hypoxia model was established.The glycolysis level and the change of cell viability were analyzed by the two models to study the promotion of hypoxia on the transformation of metabolism mode and verify the mitochondrial function of mGCs,which can stabilize the protective mechanism of hypoxia promoting metabolic transformation and cell proliferation.Methods:1.mGCs from infertile patients with simple fallopian tube factor were cultured in vitro.The granulosa cells were divided into control group?without drugs?,CCCP group,BA0.5 group and BA2.5 group.After the cells were attached to the wall,the mitochondrial inhibitor CCCP?10?m?and glycolysis inhibitor BA?0.5 mm/2.5 mm?were added respectively,MMP,ROS and ATP Kit were used to detect the levels of MMP,ATP and ROS in the four groups of granulosa cells.The experiment was repeated at least three times.2. The change of red/green fluorescence intensity of mGCs MMP was recorded by fluorescence microscope after adding CCCP?10?m?and BA?0.5mm?.3. mGCs from 15 cases of infertile patients with simple fallopian tube factor were cultured in vitro.RT-PCR was used to detect HIF-1?and GLUT1,LDHA and PFKP m RNA expression levels related to glycolysis after adding CCCP?10?m?.The experiment was repeated at least three times.4.mGCs were cultured in vitro once again,the granulosa cells were divided into control group?without drug?,CCCP group and CoCl2 group.After the cells were attached to the wall,the mitochondrial inhibitors CCCP?10?m?and CoCl2?100?were added respectively,continue to culture for 24,48 and 72 hours,use CCK8 kit to detect cell viability,glucose kit to detect glucose consumption,RT-PCR to detect HIF-1?and GLUT1,LDHA and PFKP expression levels related to glycolysis,and repeat the experiment at least3 times.Results:1.Compared with the control group,MMP and ATP decreased significantly after CCCP?10?m?and BA?0.5 mm/2.5 mm?treatment?P<0.01?.2.Compared with the control group,after CCCP?10?m?and BA?0.5mm/2.5 mm?treatment,the ROS level of CCCP group and BA?0.5 mm?group slightly increased,but there was no statistical difference?P>0.05?,while the ROS level of BA?2.5 mm?group significantly increased?P<0.05?.3.Using mitochondrial dysfunction model,the expression of HIF-1?,GLUT1 and LDHA m RNA in mGCs after CCCP?10?m?treated was significantly higher than control group?P<0.05?,while the expression of PFKP m RNA was increased,but there was no significant difference?P>0.05?.4. Using hypoxia model:The expression of GLUT1,LDHA and PFKP m RNA in mGCs was significantly higher than that in the control group?P<0.05?,and the expression of HIF-1?m RNA had no significant change?P>0.05?.5. mGCs treated with CCCP?10?m?and CoCl2?100?m?were cultured for 24,48 and 72 hours,the glucose consumption of mGCs increased,and the highest glucose consumption was found in 72 hours?P<0.05?.The glucose consumption of CCCP group and CoCl2 group in 72 hours was significantly higher than that of the control group?P<0.05?.6. mGCs treated with CCCP?10?m?and CoCl2?100?m?were cultured for 24,48 and 72 hours,and the cell viability of the two groups increased gradually,the most significant increase was 48 and 72 hours after treatment?P<0.05?.Compared with the control group,the cell viability of mGCs treated with CCCP decreased at 24 and 48 hours,but there was no significant difference after 72 hours;the cell viability of CoCl2 group increased at 24,48and 72 hours,which was significantly higher than that of the control group and CCCP treated group?P<0.05?.Conclusion:In the later stage of follicular development and maturation,granulosa cells have two energy metabolism pathways:mitochondrial oxidative phosphorylation and glycolysis,which are jointly involved in maintaining the stability of the internal environment of the follicle;the stable mitochondrial function and hypoxia environment promote the transformation of the energy mode of granulosa cells to glycolysis mode in the process of follicular development.Conciusion:1.In PCOS patients,the rates of high-quality embryo formation,high-quality blastocyst formation and clinical pregnancy were all decreased.The over expression of oxidative stress and the disorder of glucose metabolism in follicular fluid microenvironment may affect the development potential of oocytes and early embryos.2.In PCOS patients,mitochondrial function of granulosa cells was damaged,MMP and ATP were decreased,ROS was increased,mt DNA copy number was decreased,and glycolysis related gene expression of granulosa cells was decreased.The oxidative damage of granulosa cells and the disorder of energy metabolism may interfere with the energy supply of granulosa cells to oocytes,which may be one of the mechanisms of the decline of oocyte development potential in PCOS patients.3.The changes of mitochondrial function after adding CCCP and BA suggest that oxidative phosphorylation and glycolysis are two energy metabolism ways to regulate the stability of granulosa cells.4.CCCP decreased mitochondrial membrane potential,changed mitochondrial function,and then decreased cell viability.However,with the increase of HIF-1?m RNA expression,the activity of glucose metabolism in granulosa cells gradually increased,and promoted cell viability.5.In CoCl2 group,the activities of glucose metabolism and cell proliferation were the highest,which indicated that the stability of mitochondria function of granulosa cells had a certain protective effect on hypoxia promoting metabolism switch and cell proliferation.