Relationship Between Oocyte Development Potential And Oxidative Stress And Mitochondrial Function With Ovarian Endometriosis Cyst

The development and maturation of follicles takes a long time,it begins from the primordial follicles and goes through the development process of pre-sinus follicles,sinus follicles and pre-ovulation follicles.The follicular fluid accumulated between the granulosa cells in the sinus increased the fusion and formed the follicular cavity.The follicular fluid microenvironment in the follicular cavity played a crucial role in the late development and final maturation of oocytes.Granulosa cells(GCs)are the largest cell group comprising the parietal GCs(mGCs)lining the follicle walls and the cumulus granulosa cells(cGCs)surrounding the oocytes.MGCs mainly produces ovarian hormones and secreted cytokines,which regulate oocyte development through paracrine and autocrine forms.cGCs enwraps around the oocyte,and the envelope of cGCs protrudes through the zona pellucida to form a gap junction with the envelope of the egg,thus forming a tightly bound syncytium with the oocyte and influencing the development of the follicle through a complex intercellular connection mechanism.Mitochondria is the main place to form ATP(adenosine triphosphate,ATP)and provide energy.Granular cell contains a large number of mitochondria,the energy for oocyte growth and development is also coming from mitochondria of granular cell mainly.The development,maturation,fertilization and embryonic development of oocytes also depend on the normal function of mitochondria.Oxidative stress(OS)seriously affects the quality of oocytes and embryos as well as embryo implantation through lipid peroxidation,protein oxidation and DNA damage.If the oxidative stress reaction is excessive and the production level of oxidative damage products is too high,the activity of ATPase will be inhibited,the mitochondria will be damaged,and the energy supply for follicular development will be reduced,thus the quality of oocytes and the potential of embryo development will be reduced.Endometriosis(EMs)is one of the most common diseases of women of childbearing age.Most of EMs occurred in the pelvic cavity,and the ovary was one of the most common invasive sites.Its typical lesion was ectopic endometriosis(Ovarian endometriosis cyst,OEC).It is known that multiple large cysts or OEC after operation can significantly damage the ovarian cortical structure,seriously affect the ovarian reserve and oocyte development,leading to adverse pregnancy outcomes.However,whether the ovarian reserve function and oocyte development potential of small non-surgical OEC patients are also affected remains to be determined.There have been many studies on oxidative stress in peritoneal fluid and heterotopic cyst tissues,but few reports on OEC patients’ follicular fluid oxygenated stress markers and their oocyte development potential.By detecting the content of oxidative damage markers in OEC patients’ follicular fluid and their correlation with oocyte development potential indexes,it can be speculated that the high oxidative stress response in the follicular fluid is an adverse influence factor of oocyte development,maturation and embryo development.Follicular fluid is also a microenvironment for the survival of granulosa cells.Therefore,we hypothesized that the high oxidative stress response in the follicular fluid may cause oxidative damage of granulosa cells,resulting in changes in mitochondrial structure,DNA damage and reduced ATP production,which may be one of the mechanisms for the decreased oocyte development potential in OEC patients.In the process of oocyte-corona-cumulu compounds in vitro culture with mitochondrial dysfunction,granular cell will release free mitochondrial DNA(Cyclic free mitochondrial DNA,cfmtDNA)into the culture solution.Therefor we hypothesized that cfmtDNA increasing in follicular fluid is an indicator for mitochondrial dysfunction.OEC caused mitochondrial function impaired,causing cfmtDNA increase in follicular fluid,further damage to the granular cell function,resulting in a decline in oocyte development potential.CfmtDNA in follicular fluid can be used as an predictor to predict oocyte development potential.This research is divided into two parts,the first part compared the patients age,infertility fixed number of year,body mass index(BMI),AFC,basic FSH,CA125,AMH,the hormone levels on HCG-day(E2,P,LH),number of oocyte obtained,mature oocyte number,mature oocytes rate,normal fertilized oocyte number,normal fertilization rate,cleavage number,cleavage rate,high quality embryo number,rate of high quality embryo and follicular fluid in 3-NT,8-OHdG,,4-HNE,AOPP and SOD level of the OEC patients and patients with tubal factor infertility.Analysis of follicle melt oxidation stress of oocyte development,maturation,fertilization and early embryo development,it is suggested that OEC patients with high follicular oxygenation stress may be a factor affecting oocyte development potential.In the second part,the effects of OEC on mitochondrial function of mural granular cells were studied by observing and detecting mitochondrial ultrastructure,abnormal mitochondrial rate,ATPase,IF1 and OXPHOS complex contents in mural granular cells of OEC patients.By detecting the changes of cfmtDNA content in follicular fluid,mtDNA content in mGCs and cGCs and their correlation with transplantable embryo rate and embryo predominance rate,to study the effect of OEC on mtDNA in granulosa cells and the correlation between cfmtDNA in follicular fluid and mtDNA in mGCs and cGCs,suggesting that cfmtDNA in follicular fluid can be used as an indicator to predict oocyte development potential.Part one Markers of oxidative stress and oocyte development in follicular fluid of OEC patients Correlation study of potential indicatorsObjective:By detecting the content of markers of oxidative stress in the follicular fluid of OEC and oviduct infertility patients,we analyzed the effects of changes in the state of oxidative stress in the follicular fluid of endometriosis on the development of oocytes and early embryos。Methods:From Apri1 2018 to December 2018,30 patients with unilateral or bilateral ovarian endometriosis cyst(OEC)(number of OEC 2 on each side,OEC diameter<4cm,part of cyst puncture was chocolate solution)diagnosed by multiple b-ultrasonography were selected.30 patients with simple tubal infertility during the same period were selected as the control group.oocyte and embryo development indexes,clinical pregnancy rate of the two groups were recorded,and the content of oxidative stress markers in follicular fluid was detected by ELISA.Results:1.Characteristics of general case data of patientsAge composition of patients:the average age of the OEC group was 31.1±3.9 years;The average age of the control group was 29.3±3.6 years.The infertility years were 3.1±2.1 years and 3.1± 2.2 years,respectively,and the body mass index was 22.5±3.0kg/m2和23.2±3.0kg/m2,respectively.There was no significant difference between the two groups.The AFC of the two groups was 10.4±4.4 and 11.6±3.5,basic FSH level was 5.7± 1.6mU/mL and 5.1 ± 1.8mU/mL,CA125 was 34.4±9.0IU/L and 29.9±8.6IU/L,AMH was 2.5±0.9ng/mL and 2.8±0.8ng/mL,Gn dosage was 2642.5±883.7IU and 2495.5±729.9IU,and Gn days were 12.1 ± 2.3d and 11.8±2.4d,respectively.The hormone levels(E2,P,LH)of endometriosis group on HCG-day were 2951.6 ±1428.4pg/mL,1.2± 1.0ng/mlL,1.1±0.9 mU/mL,and the control group were 3541.9± 1397.0 pg/mL,0.9±0.7 ng/mlL,and 0.8±0.5 mU/mL.There was no statistical difference between the two groups.Endometrial thickness was 12.0±3.2mm and 12.4±23mm,with no statistical difference between the two groups,showing a contrastable effect.2.Oocyte and embryo development indicatorsThe number of oocytes obtained,mature oocytes,normal fertilized oocytes,cleavage embryos in the OEC group were all lower than those in the control group(P<0.05).However,there was no significant difference in the rate of mature oocytes,normal fertilization and cleavage between between the two groups(P>0.05).The optimal embryo rate and clinical pregnancy rate in the OEC group were lower than those in the control group(P<0.05).3.Detection of markers of oxidative stressThere was no significant difference in 3-NT concentration between the two groups(P>0.05).In the observation group,the levels of markers of oxidative damage in the follicular fluid,including 8-OHDG,4-HNE and AOPP,were all higher than the corresponding levels in the control group(P<0.05 or P<0.01).The SOD concentration in the follicular fluid of the OEC group was significantly lower than that of the control group,and the difference was statistically significant(P<0.01).Conclusion:The optimal embryo rate and clinical pregnancy rate of OEC infertility patients were decreased,and the excessive expression of oxidative stress in the follicular fluid microenvironment could affect the oocyte and early embryonic development potential.Part two The mitochondrial function of granulosa cells in OEC patients and the expression of cfmtDNA in follicular fluid and the expression of mtDNA in granulosa cellsObjective:Changes in mitochondrial ultrastructure,ATPase,IF1 and OXPHOS complex in mural granulosa cells were detected to analyze changes in mitochondrial function of mural granulosa cells with ovarian endometriosis cyst.By detecting the expression levels of cfmtDNA in OEC patients’follicular fluid,mtDNA in mGCs and CCs,the content changes of cfmtDNA and granular cell mtDNA in the follicular fluid and the correlation with the development potential of oocytes were analyzed.Methods:As in the first part,mGCs was retained from the follicular fluid of the enrolled patients,mitochondrial structure was observed by transmission electron microscopy,ATPase and IF1 contents were detected by qPCR,and OXPHOS complex contents were detected by Western method.30 OEC infertility patients with ICSI assisted pregnancy during the same period were selected as OEC group,ICSI assisted pregnancy was required due to severe oligospermia of the male partner or previous IVF without fertilization history.And 30 patients who need ICSI assisted pregnancy with or without fallopian tube factors were selected as the control group.Oocyte and embryo development indicators and clinical pregnancy rate of the two groups were recorded.The expression levels of cfmtDNA,mGCs and cGCs in follicular fluid were detected by qPCR.Results:1.Transmission electron microscopy ultrastructure and abnormal mitoc-hondrial rateThe results of transmission electron microscopy showed that compared with the control group patients,the number of mitochondria in mGCs in OEC patients was significantly reduced,and mitochondria of different sizes and shapes could be seen,mitochondrial ridge expansion,partial fusion of ridge fracture,mitochondrial swelling and vacuolar formation.Electron microscopic ultrastructure observation of mitochondria in mGCs of OEC patients with infertility and control group patients showed that the rate of abnormal mitochondria in mGCs of the OEC group was significantly higher than that of the control group,and the difference was statistically significant(P<0.01).2.Content of ATPase and IF1Through the detection of mitochondrial ATPase and IF 1 levels in mGCs of OEC patients with infertility and control group patients,the results showed that the mitochondrial ATPase level in the OEC group was significantly lower than that in the control group,and the IF 1 level was significantly higher than that in the control group,and the difference was statistically significant(P<0.01).3.OXPHOS complex contentThe levels of protein products of ATP5A1,MTCO1 and UQCRC2 in mitochondrial OXPHOS complex of murine granular cells in the observation group were all higher than those in the control group,and the differences were statistically significant(P<0.01).There was no significant difference in the levels of NDUFB8 and SDHB protein products in mitochondria between the two groups(P>0.05).4.Characteristics of general case data of ICSI patientsThe age composition of the patients was 24 to 36 years in the OEC group,with an average age of 30.6±3.5 years.The control group was 25 to 37 years old,with an average age of 30.5±3.3 years.For the two group,the infertility years were 4.2±2.0 years and 4.1 ± 1.8 years,respectively,and the body mass index were 23.1±3.0kg/m2 and 22.9±3.0kg/m2.There was no statistical difference between the two groups.The AFC of the two groups was 9.4±3.7 and 10.2±4.3,basal FSH levels were 5.8± 1.5mu/mL and 5.6± 1.7mu/mL,CA125 was 35.3± 10.9lU/L and 30.2±9.0IU/L,AMH was 2.3± 1.0ng/mL and 2.5±0.9ng/mL,Gn dosage was 2534.4±607.6IU and 2360.4±626.8,and Gn days were 11.7±2.6d and 10.5±2.3d,respectively.respectively.The HCG-day hormone levels(E2,P,LH)in the OEC group were 3536.5± 1528.5pg/mL,1.1±0.5ng/mlL,1.2 ± 0.6mu/mL,and the control group were 3489.6±1469.3pg/mL,0.9±0.4ng/mlL,and 1.0±0.5mu/mL.There was no statistical difference between the two groups.The thickness of endometrium was 12.2±3.0mm and 12.5±2.8mm,and there was no statistical difference between the two groups.5.Oocyte and embryo development indicatorsThe number of transplantable embryos(rate)and optimal embryos(rate)in the observation group were all lower than those in the control group(P<0.05).However,there was no significant difference in oocytes(rate),mature oocytes(rate),2PN number(rate)and cleavage number(rate)between the two groups(P>0.05).6.Expression of cfmtDNA in follicular fluidThrough the detection of cfmtDNA expression in follicular fluid of OEC patients with infertility and control group patients,the results showed that the expression level of free mtDNA in follicular fluid of the OEC group was higher than that of the control group,and the difference was statistically significant(P<0.05).7.mtDNA expression quantity in parietal granular cellsmtDNA expression quantity of mtDNA in mural granulosa cells of OEC patients with infertility and control group patients was detected.The results showed that mtDNA expression level of mtDNA in mural granulosa cells of the OEC group was lower than that of the control group,and the difference was statistically significant(P<0.01).8.mtDNA expression quantity in cumulus granulosa cellsThe mtDNA expression quantity of cumulus granulos in OEC patients and control group patients was detected.The results showed that the mtDNA expression quantity of cumulus granulosa oocyte in the OEC group was lower than that in the control group,and the difference was statistically significant(P<0.01).9.Correlation analysis between the expression of cfmtDNA in follicular fluid and the expression of mtDNA in granulosa cellsCorrelation analysis of the expression level of cfmtDNA in follicular fluid with mtDNA in mural granular cells and cumulus granular cells showed that the expression level of cfmtDNA in follicular fluid was negatively correlated with mtDNA in mural granular cells and cumulus granular cells(rs were-0.798 and-0.690,P<0.01).Conclusion:There are structural and functional changes in mGCs mito-chondria in OEC patients.Decreased ATPase expression and increased IF1 expression affect oxidative phosphorylation and lead to decreased OXPHOS complex content.This affects the energy supply of oocytes,and thus adversely affects oocytes and the development potential of early embryos.The expression level of cfmtDNA in OEC patients follicles increased,while the expression level of mtDNA in mGCs and cGCs decreased.The rate of transplantable embryos and the rate of optimal embryos decreased,The cfmtDNA of follicular fluid was negatively correlated with the mtDNA expression of mural granulosa cells and cumulus granulosa cells.The cfmtDNA in follicular fluid could indirectly predict the development potential of oocytes and embryos.Conciusion:1.The optimal embryo rate and clinical pregnancy rate of OEC infertility patients were decreased,and the excessive expression of oxidative stress in the follicular fluid microenvironment could affect the oocyte and early embryonic development potential.2.The structure and function of mitochondria in mural granulosa cells of OEC patients are changed.The decrease of ATPase expression and the increase of IF1 expression affect oxidative phosphorylation and lead to the decrease of OXPHOS complex content.This affects the energy supply of oocytes,and thus adversely affects oocytes and the development potential of early embryos.3.The expression of free mtDNA in the follicular fluid of OEC patients increased,the expression of mtDNA in the mural granulosa cells and the cumulus granulosa cells decreased,the transplantable embryo rate and embryo optimization rate decreased.4.The cfmtDNA of follicular fluid was negatively correlated with the mtDNA expression of mural granulosa cells and cumulus granulosa cells.These suggest that follicular fluid cfmtDNA could be used as an indicator to predict the development potential of oocytes and early embryonic development.