The Role And Mechanism Of Cumulus Cells In Vitrification Of Immature Oocytes

Part I: Effects of cumulus cells on the developmental potential of mouse oocyte after vitrification Objective1.To investigate the effects of cumulus cells on fertilization and embryonic development of immature oocytes in mice during vitrification.2.To investigate whether the immature oocyte should be in vitro maturation before or after vitrification.MethodsImmature mouse oocytes were collected and divided into four groups: group 1: COC vitrified after in vitro maturation;group 2: DO vitrified after in vitro maturation;group 3: DO maturation in vitro;group 4: COC vitrified after in vitro maturation;group 5: DO vitrified after in vitro maturation.Group 3 was used as the control group;mature oocytes were obtained from five groups after in vitro culture and fertilized in vitro with male mouse sperm of the same species and cultured to blastocyst stage.The thawing survival rate,maturation rate,fertilization rate,cleavage rate and blastocyst formation rate of the five groups were observed and compared,and the data were statistically analyzed.Results1.Compared with the other four groups,the MII rate,thawing survival rate,fertilization rate,cleavage rate and blastocyst formation rate of DO in vitro maturation group were higher(P < 0.05).2.The MII rate(72.33% VS 60.39%)and thawing survival rate(79.13% VS 52.46%)of COC group after in vitro maturation were higher than those of DO group after in vitro maturation(P < 0.05),fertilization rate(53.85% VS 46.88%),cleavage rate(61.22% VS 60.00%)and blastocyst formation rate(14.29% VS 3.33%)although there was no statistical difference between the two groups,the trend of COC group after in vitro maturation was still higher than that of DO group after in vitro maturation.3.The MII rate(45.57% VS 62.50%)and thawing survival rate(48.47% VS 68.38%)of the DO cryopreserved in vitro maturation group were lower than those of the COC cryopreserved in vitro maturation group(P < 0.05).4.There were no significant differences in MII rate(72.33% VS 62.50%),thawing survival rate(79.13% VS 68.38%),fertilization rate(53.85% VS 64.00%)blastocyst formation rate(14.29% VS 25.00%)and in vitro maturation group(P > 0.05).5.The thawing survival rate(52.46% VS 48.47%,P < 0.05)of DO cryopreservation group was higher than that of DO cryopreservation group,but the blastocyst formation rate(3.33%)was lower than that of DO cryopreservation group(12.00%)and DO direct in vitro maturation group(26.98%).Conclusion1.The maturation rate of nude immature oocyte is better than that of frozen immature oocyte.Compared with unfrozen oocyte,the freezing process significantly reduces the development potential of oocyte and subsequent embryo.2.Cumulus cells have protective effects on oocyte freezing,which is beneficial to improving the subsequent fertilization and embryo development potential,whether in vitro maturation culture followed by freezing or in vitro maturation.Part II: Effects of cumulus cells on the spindle and chromosome of human immature oocytes during vitrificationObjectiveBy comparing the distribution of spindles and chromosomes of human immature oocytes with or without cumulus cells after vitrification,the effect of cumulus cells on Vitrification of immature oocytes was discussed.MethodsImmature oocytes collected from ICSI patients in our center were collected,and human immature oocytes were divided into first frozen and then IVM group and first IVM refreed group,and then divided into two groups according to the presence or absence of cumulus cells.For the denuded oocytes,cumulus cell-oocyte complex(COC) group,fresh naked eggs were directly subjected to IVM as a control,and the five groups of oocytes were vitrified and thawed for spindle and chromosome immunity.Fluorescent staining was used to calculate the normal morphology rate of the five groups of spindles and chromosomes.Results1.Compared with the first frozen IVM group,the normal rate of spindle(60.00% VS 11.76%)and chromosome(45.00%% VS 11.76%)in the IVM COC group was significantly increased;2.Compared with the fresh DO group,the normal rate of the spindle of the IVM DO group(78.26% VS 11.76%)and chromosome(65.22% VS11.76%)was significantly reduced;and the spindle of the DO group was frozen by the IVM(55.56% VS 78.26%)and chromosome(61.11% VS 65.22%)was not statistically different from the fresh DO group.3.Compared with the first IVM and then frozen COC group,there was no significant difference in the normal rate of spindle(85.71% VS 60.00%)and chromosome(66.67% VS 45.00%)in the IVM COC group.ConclusionIn DO immature oocyte,the spindle damage is less than that of frozen oocyte and matured oocyte in vitro.In cumulus cell-enclosed oocyte,the morphology of spindle is normal,cumulus cells can protect immature oocyte from freezing and slow down the cooling.The rate of penetration of cryoprotectants into oocytes reduces the toxicity of cryoprotectants.Part III:Effect of vitrification/thawing on genome expression of human immature oocytesObjectiveBy comparing the differences in genome expression between in vitro maturation culture after vitrification and direct in vitro maturation of human immature oocytes,the key genes affecting oocyte freezing and corresponding regulatory pathways were screened to explore the effect of vitrification on gene expression profiles of immature oocytes.MethodsSome immature oocytes from patients with intracytoplasmic sperm injection in our center were directly cultured in vitro,some were vitrified and then matured in vitro,the whole genome of this two groups was analyzed.Results1.In the whole-genome expression microarray analysis of oocytes after vitrification,765 differentially expressed genes were obtained,including 103 up-regulated genes and 662 down-regulated genes;2.cellular process,metabolic process,biological regulation,biological regulation,response to stimulus,cellular component organization or biogenesis,multicellular organismal processes,localization,developmental process,Signaling,positive regulation of biological process,negative regulation of biological process,The immune system process are involved in the regulation of biological processes that downregulate gene expression after vitrification;3.Cell,cell part,organelle,organelle part,membrane,membrane part,membraneenclosed lumen,macromolecular complex,extracellular region,extracellular region part are involved in the cellular regulation of gene expression after vitrification;Binding,catalytic activity,molecular function regulator,transcription regulator activity transporter activity,signal transducer activity,molecular transducer activity,structural molecule activity and other molecular functions involved in down-regulation of gene expression after vitrification;4.Pathways in cancer,Human papillomavirus infection,Metabolic pathways,Autophagy-animal,Mitophagy animal,RNA degradation,HTLV-I infection,Proteoglycans in cancer,Gastric cancer,Hepatocellular carcinoma,Breast cancer,Hippo signaling pathway,MTOR signaling pathway,wnt signaling pathway,signaling pathway regulating stem cell pluripotency,and ECM receptor interaction pathway are involved in the regulation of genes after vitrification.ConclusionCompared with the oocytes matured directly in vitro,there are many differentially expressed genes in the oocytes matured in vitro after vitrification at immature stage,which are related to oocyte quality,organelle composition and various regulatory pathways.