The Effects And Mechanisms Of Vitrification On Imprinting Genes In Fetuses And Placentas Of Mouse

Since the first live birth conceived using frozen-thawed embryos three decades ago,significant progress has been made in the cryopreservation techniques used in assisted reproductive technology(ART).Thus,freezing embryos has become a routine practice for ART units,and some units have recently even started to use the ‘freeze-all' strategy for certain patients.Genome-wide epigenetic reprogramming occurs in mammalian development in primary germ cells(PGCs)and gametogenesis.During the preimplantation period,epigenetics plays an important role in embryonic development and,thus,affects the life of offspring.ART techniques such as vitrification are performed during this epigenetic programming period.It has been reported that epigenetic disturbances can be found in offspring after vitrification-thawed embryo transfer.This evidence supports the conclusion that vitrification may increase the exposure of the epigenome to external influences.In epidemics,vitrification may result to adverse pregnancy events,the effect of vitrification on the long-term health of the resulting offspring is still unclear.The safety of this procedure has been questioned,and potential vitrification-induced risks require further exploration.Further studies are required to understand the effects of cryogenic technologies on genomic imprinting.In this study,we aim to investigate the epigenetic disturbance of fetuses and placentas derived from vitrified 8-cell embryos using a mouse model.Despite the routine use of embryo vitrification in clinical settings,the effect that this procedure may have on genomic imprinting deserves much greater attention.Part 1:The development of fetuses derived from vitrified 8 cell embryos in mice Objective: In this study,the effect of vitrification on the development of fetuses was investigated in a mouse model.Methods: E9.5 fetuses were collected form NC(natural mating)group,IVC(in vitro culture)group and VET(vitrified embryo transfer)group.The survival rate of fetuses in E9.5 was recorded and the crown-rump length of fetuseswere also measured in each group.Results:(1)It was found that the survival rate of fetuses at E9.5 was significantly decreased in IVC(40.2%)and VET(37.5%)groups compared to NC group(100%)(P<0.05).(2)The fetal crown-rump length was significantly reduced in both the IVC(0.210 ± 0.059 mm)and VET(0.205 ± 0.048 mm)groups compared with the NC group(0.288 ± 0.083 mm)(P<0.05).However,it was not statistically different between IVC and VET group(P>0.05).Conclusion: According to our results,both vitrification and cultivation conditions may lead to decrease the survival rate of fetuses.And both vitrification and cultivation conditions may lead to a reduced fetal crown-rump length in E9.5 mice.Part 2:The imprinting disorders and changes in DNA methylation levels in ICRs in E9.5 mouse fetuses and placentas derived from vitrified 8-cell embryos Objective: In this study,the effect of vitrification on imprinting disorders and changes in DNA methylation levels in ICRs was investigated in E9.5 mouse fetuses and placentas derived from vitrified 8-cell embryos.Methods: In this experimental study,E9.5 fetuses and placentas were collected from a natural mating(NC)group,an in vitro culture(IVC)group and a vitrified embryo transfer(VET)group.The expression of nine paternally expressed imprinted genes(Dlk1,Igf2,Kcnq1ot1,Mest,Ndn,Peg3,Plagl1,Sgce,Snrpn)and fifteen maternally expressed imprinted genes(Cd81,Cdkn1 c,Dcn,Gatm,Gnas,Grb10,Gtl2,H19,Igf2 r,Mash2,Osbpl5,Phlda2,Slc22a18,Ube3 a,Zim1)between the three groups was analyzed at E9.5 in the mouse model by reverse transcription-quantitative polymerase chain reaction(RT-qPCR).Moreover,the methylation levels of H19 and KvDMR1 were compared between groups by pyrosequencing.Results:(1)The expression of paternally expressed imprinted genes in fetuses.Compared to NC group,the Peg3(NC:0.746±0.233;IVC:1.396±0.592)in IVC and Igf2(NC:1.223±0.299;VET:2.048±0.537),Peg3(NC:0.746±0.233;VET:1.981±0.238)in VET was up-regulated,Ndn(NC:1.198±0.229;VET:0.699±0.227),Sgce(NC:1.112±0.147;VET:0.694±0.187)in VET was down-regulated,and it was statistically different between groups(P < 0.05).(2)The expression of paternally expressed imprinted genes in placentas: Compared to NC group,the Mest in IVC was down-regulated,Igf2,Ndn,Sgce in VET was down-regulated,Kcnq1ot1 in VET was up-regulated,and it was statistically different between groups(P < 0.05).(3)The expression of maternally expressed imprinted genes in fetuses: Compared to NC group,there were ten maternally expressed imprinted genes up-regulated(Cd81,Gatm,Gnas,Grb10,H19,Igf2 r,Mash2,Obspl5,Slc22a18,Ube3a)and one maternally expressed imprinted genes down-regulated(Cdkn1c)in IVC group.In VET group,there were nine maternally expressed imprinted genes up-regulated(Gnas,Grb10,Gtl2,H19,Igf2 r,Mash2,Obspl5,Phlda2,Slc22a18)and two maternally expressed imprinted genes(Cdkn1c,Dcn)down-regulated,and it was statistically different between groups(P<0.05).(4)The expression of maternally expressed imprinted genes in placentas: Compared to NC group,Slc22a18(NC: 0.964 ± 0.071;IVC: 0.426 ± 0.093),Ube3a(NC: 1.049 ± 0.098;IVC: 0.583 ± 0.116)in IVC group were down-regulated,Cdkn1c(NC: 0.916 ± 0.118;VET: 0.590 ± 0.245),Gnas(NC: 0.911 ± 0.269;VET: 0.562 ± 0.257),Slc22a18(NC: 0.964 ± 0.071;VET: 0.376 ± 0.080),Ube3a(NC: 1.049 ± 0.098;VET: 0.362 ± 0.171)in VET were down-regulated,and it was statistically different between groups(P<0.05).(5)DNA methylation changes of imprinting gene H19 were investigated in both placentas and fetuses: Alteration of the methylation level of H19 was not found in three groups both in fetuses and placentas(P>0.05).(6)DNA methylation changes of imprinting gene Kcnq1ot1 ICR--KvDMR1 were investigated in both placentas and fetuses: In fetuses,the methylation level of KvDMR1 in IVC and VET were not statistically different(P>0.05),however,compared to IVC group(0.364 ± 0.129),the methylation level of KvDMR1 in VET group(0.464 ± 0.109)was statistically increased.In placentas,compared to NC group(0.425 ± 0.062),the methylation level of KvDMR1 in IVC(0.358 ± 0.114)and VET group(0.343 ± 0.094)was statistically decreased.Conclusion: First,a majority of maternally expressed genes were upregulated in IVC group and VET group.According to the parental conflict hypothesis,maternally expressed genes repress fetal growth in the uterus.According to our research,the upregulation of maternally expressed genes in the fetuses of the IVC and VET groups may have resulted in reduced growth of the fetuses.Then,in placentas,Kcnq1ot1 expression was slightly increased in the IVC group and significantly increased in the VET group,which was consistent with the changes in global DNA and KvDMR1 methylation levels.Furthermore,maternally expressed genes including Osbp15,Phlda2,Slc22a18,Cdkn1 c and Cd81 also presented decreased expression in both the IVC and VET groups in placentas.Despite the routine use of embryo vitrification in clinical settings,the effect that this procedure may have on genomic imprinting deserves much greater attention.Part 3: The global DNA methylation level and disorders of methylation-related enzymes in E9.5 mouse fetuses and placentas derived from vitrified 8-cell embryos was explored in this part.Objective: In this study,the global DNA methylation level and disorders of methylation-related enzymes was investigated in E9.5 mouse fetuses and placentas derived from vitrified 8-cell embryos.Methods: E9.5 fetuses and placentas were collected from a natural mating(NC)group,an in vitro culture(IVC)group and a vitrified embryo transfer(VET)group.Global methylation levels were measured by ELISA,and disorders of methylation-related enzymes,as well as gene imprinting,were investigated by reverse transcription-quantitative polymerase chain reaction(RT-qPCR)in these groups.Results:(1)In fetuses,we found that the methylation level was significantly reduced in the IVC group(0.0146 ± 0.0144)compared to the NC group(0.0324 ± 0.0185)(P<0.05).However,after vitrification and warming,the methylation level was increased in the VET group(0.0321 ± 0.0209)compared to the IVC group(0.0146 ± 0.0144)(P<0.05).Additionally,the methylation level showed no significant difference in the NC group(0.0324 ± 0.0185)and the VET group(0.0321 ± 0.0209)(P>0.05).In placentas,the methylation levels of both the IVC(0.0049 ± 0.0033)and VET(0.0117 ± 0.0073)groups were significantly reduced compared to the NC group(0.0292 ± 0.0221)(P<0.05).Additionally,the methylation level was slightly increased in the VET group(0.0117 ± 0.0073)but did not significantly differ compared to the IVC group(0.0049 ± 0.0033).(2)In fetuses,Dnmt1 and Dnmt3 b showed an increase in the VET(Dnmt1: 14.337 ± 6.373;and Dnmt3b: 5.825 ± 2.456)groups compared to the NC group(Dnmt1: 0.637 ± 0.267;and Dnmt3b: 0.898 ± 0.218)and the IVC group(Dnmt1: 3.343 ± 1.037;and Dnmt3b: 2.625 ± 1.168)(P<0.05).Additionally,Tet2 was significantly increased in the IVC group(1.528 ± 0.702)compared to the NC group(0.941 ± 0.133)and VET group(0.334 ± 0.104)(P<0.05).When Tet3 was analyzed,we found that it was significantly reduced in the VET group(0.300 ± 0.056)compared to the NC group(0.958 ± 0.088)and the IVC group(1.386 ± 0.700),which revealed a possible mechanism underlying the decreased global methylation level of the IVC group(P>0.05).In placentas,Dnmt1 was increased significantly in the VET group(3.928 ± 1.236)compared to the NC group(1.105 ± 0.604)(P<0.05).Conclusion: vitrification changed the methylation pattern in fetuses,and these change was correlated with disorders of methylation-related enzymes.Part 4: Effects of vitrification on the development potential of embryo in preimplantation period Objective: To evaluate the effect of vitrification on the development potential of embryo in preimplantation period Methods:Eight cell embryo and blastocyst were collected from a natural mating(NC)group,an in vitro culture(IVC)group and a vitrified embryo transfer(VET)group.The blastocyst formation rate and the blastocyst hatching rate were recorded.The expression of development potential factors were investigated by reverse transcription-quantitative polymerase chain reaction(RT-qPCR)in these groups.Results:(1)The blastocyst formation rate was 89.02%(73/82)in IVC group which is higher than VET group but without statistically difference(P>0.05).The blastocyst hatching rate was 93.15%(68/73)in IVC group which is statistically higher than VET group 80.88%(55/68)(P<0.05).(2)In 8 cell embryos.the expression of Nanog in VET was statistically up-regulated compared to NC group(P<0.05).The expression of Lif was statistically down-regulated in IVC and VET group compared to NC group(P<0.05).(3)In blastocyst,the expression of Pou5f1,Nanog,Sox2 in IVC and VET group were decreased but without statistically difference compared to NC group.The expression of Tead4 was statistically up-regulated in IVC group while statistically down-regulated in VET group(P<0.05).The expression of Lif,Cdx2,Eomes and Elf5 in IVC and VET group were significantly decreased compared to NC group(P<0.05).The expression of Gata6,Sox17,Fgfr2 and Fgfr4 in IVC and VET group were decreased but without statistically difference compared to NC group(P>0.05).Conclusion : These results suggest that both in vitro culture and vitrification leads to a decline in the quality of the blastocyst by affecting the development potential factor.