Effects And Mechanism Of Vitrification On Mouse Embryo Development

The number of infertility patients worldwide is increasing in recent years,and assisted reproductive technology(ART)is one of the most effective methods of infertility treatment.Vitrification is a technique of embryo cryopreservation widely used in the clinical practice of ART.With the improvement of vitrification and pre-implantation genetic testing(PGT),“freeze-all”policy is increasingly being performed in the ART.Although frozen embryo transfer(FET)may be associated with better clinical outcomes in certain patient populations like women with polycystic ovary syndrome,some clinical researches have demonstrated several limitations of FET,including an increased risk of pre-eclampsia and large for gestational age(LGA).Moreover,live birth rates were significantly lower following FET compared with fresh ET when using fresh donor oocytes to undergo assisted reproduction.These findings implicated that the freeze-thaw process may lower developmental potential of embryos and effect the following pregnancy outcome.Therefore,it is important to explore the effects of vitrification on embryo development,gene expression and offspring safety.In this study,we simulated the embryo freezing strategy in human to vitrify 8-cell stage mouse embryos and analyzed the effects of vitrification on mouse embryo development.We also analyzed the changes of oxidative stress responses,DNA damage,epigenetic modification during vitrification,as well as the differences of transcriptome profiles in E18.5 fetuses and placentas between control group and vitrified group.Our research provides theoretical and experimental basis for revealing the influence of vitrification on embryo development and safety evaluation of this technology.The main research contents and results are as follows:1.Effects of vitrification on the development of pre-implantation and post-implantation mouse embryos:We vitrified mouse 8-cell stage embryos and cultured them in vitro,then transferred them into recipients.Then we analyzed the rate of embryo development in vitro and after implantation,which showed that:(1)There were no significant differences in blastocyst formation rate and hatching rate between vitrified 8-cell embryos and non-vitrified embryos,but the total cell number of blastocysts in vitrified group was lower than that in control group(control 60.92±2.39,vitrified 49.40±1.72,p<0.05);(2)The live pup rate(control 41.67%±4.58,vitrified 28.00%±4.07,p<0.05)and the mean litter size(control 4.17±0.56,vitrified 2.80±0.4,p<0.05)in the vitrified group were lower than that in the control group;(3)Vitrification did not significantly affect the fetal and placental weight or placental efficiency.2.Mechanism of vitrification affecting mouse embryo development:To analyze the mechanism of vitrification affecting embryo development,we tested the effects of vitrification on oxidative stress responses,DNA damage and epigenetic modification in blastocysts.The results showed that:(1)Vitrification increased ROS level and decreased mitochondrial activity in blastocysts;(2)Vitrification significantly increased the intensity ofγ-H2A.X and cellular apoptosis level in blastocysts;(3)Inhibition of non-homologous end-joining(NHEJ)but not Homologous recombination(HR)pathway significantly reduced the blastocyst rate of vitrified embryos(control 86.06±5.11,vitrified 59.60±6.01,p<0.05).Futhermore,NHEJ pathway inhibition caused severe DNA damage and reduced total cell number of blastocysts in vitrified embryos(control 60.92±2.39,vitrified 49.40±1.72,p<0.05);(4)vitrification increased the level of H3K4me2,H4K12/16 acetylation and decreased m~6A level in blastocysts.But there was no significant change of H3K9me3 in vitrified group.3.Effects of vitrification on transcriptome profiles of E18.5 placenta and fetus:E18.5placenta and fetus were collected and used for subsequent RNA sequencing respectively.The results showed that:(1)Vitrification significantly affected RNA profile in placentas,of which 286 and 428 differentially expressed genes were up-regulated and down-regulated,respectively.And the up-regulated genes are related to cell pyroptosis;(2)Vitrification significantly affected RNA profile in brains,of which 304 and 130differentially expressed genes were up-regulated and down-regulated,respectively.