Genetic Safety And Fertility Reserve Of Vitrification On Prepubertal Mouse Ovaries Using A Innovative Vitrification Solution

Parts1Studies of two vitrification protocols on prepubertal mouse ovariesObjective Low concentration and hypotoxic vitrification solution and suitable vitrification protocol are key to perform successful cryopreservation ovaries by vitrification to restore ovarian function. We used an innovative vitrification solution (EG5.5/30) which was successfully used to vitrificate rat and sheep ovarian tissue and ED20solution to compare the cryogenic protective effects of vitrified-warmed prepubertal mouse ovaries. Osmotic equilibrium was performed for the first time to immerse prepubertal mouse ovaries in two vitrification solutions to observe osmotic behavior of ovaries to design suitable vitrification protocols.Methods In this part,12-day-old prepubertal mouse ovaries were randomly divided into three groups:none treated ovaries were taken as the control group (n=18), ovaries that were vitrified with ED20and EG5.5/30solutions were taken as the ED20vitrified group (n=24), and EG5.5/30vitrified group (n=24). Osmotic equilibrium was performed to imerse prepubertal mouse ovaries in the two vitrification solutions prior to vitrification. We observed osmmotic dehydration behavior of prepubertal mouse ovaries in this two vitrification solutions by taking photoes at different time under invert microscope. The osmotic behaviors were examined by surface areas and then cryopreserved by vitrification. Respectively to assess the cryogenic protective effects of two vitrification solutions on oocytes surviving ratio and fluorescence intensity quantitative analysis of microfilament and microtubules of GV oocytes retrieval from fresh and vitrificated-warmed juvenile mouse ovaries by immunofluorescence and confocal microscopy.Results Osmotic shrinkage occurred when prepubertal mouse ovaries were transferred into pre-equilibrium medium which lasted for15minutes until prepubertal mouse ovaries were transferred into two vitrification solutions. And osmotic shrinkage occurred acutely again in ED20and EG5.5/30solutions. The27.7%of ovarian osmotic dehydration rate for2minutes in ED20solution and23.1%of ovarian osmotic dehydration rate for4minutes in EG5.5/30solution were regarded as the optimal osmotic dehydration status. Vitrified-warmed prepubertal mouse ovaries allowed a high surviving ratio of oocytes although a significant reduction from92.6%(fresh) to87%(ED20group) and84.6%(EG5.5/30group) respectively following cryopreservation (P<0.05). And no significant difference was observed between the two cryopreserved groups (P>0.05). Results of quantitative analysis of micro filament and microtubules of GV oocytes decreased significantly in the two cryopreserved groups when compared to that of control group (P<0.05). There was not statistically significant between the two cryopreserved groups (P>0.05).Conclusion The innovative vitrification solution (EG5.5/30) suits for cryopreservation prepubertal mouse ovaries by vitrification, similar to ED20solution. Our study made us to suggest that observing osmotic behavior of ovaries help us design optimal vitrification protocols.Parts2:DNA methylation of H19/Snrpn imprinted genes in cryopreserving juvenile mouse ovaries by vitrificationObjective Characteristics of vitrification such as in vitro operation, and high concentration dependent unavoidably bring to toxic and osmotic injury. Whether vitrification may cause modifications to different methylation regions of imprinted genes and affect the future development of the oocytes and fetus is still not clear. This study for the first time investigated the effects and mechanism of vitrification of juvenil mouse ovaries on the methylation status of the H19and Snrpn differentially methylated regions with the desire to warn the vitrification-induced genetic safety problem, benefit to infertility people and their offsprings.Methods Cryopreservation of juvenile mouse ovaries by vitrification were performed using ED20and EG5.5/30solutions. Total156ovaries were randomly divided to three groups:the fresh group; the EG5.5/30vitrified group; the ED20vitrified group (n=52×3). Retrieval DNA of fresh and vitrified-warmed juvenile mouse ovaries was for epigenetic analyses. The DNA methylation status of differentially methylated regions of a maternally (Snrpn) and a paternally (H19) imprinted genes were detected by bisulfite sequencing PCR. Retrieval RNA of fresh and vitrified-warmed juvenile mouse ovaries and obtained cDNA were analyzed by reverse transcription. Real time PCR were applied to analyze the expression of H19and Snrpn imprinted genes. The expression of Dnmtl and IGF-2protein in follicles embedding in different groups was detected by immunohistochemistry.Results Supermethylation was found in the H19-DMR of the juvenile mouse ovaries from the ED20and EG5.5/30vitrified groups, and there were significant alterations when compared to that of fresh group (P<0.05). Morover the H19-DMR methylation ratio of the juvenile mouse ovaries from the EG5.5/30vitrified group increased significantly than that of ED20vitrified group (P<0.05); Methylation status of Snrpn-DMR showed a trend towards hypomethylation, and there were significantly difference in the two vitrified-warmed groups as compared with the control group(P <0.05). But there was not statistically significant between the two cryopreserved groups (P>0.05). H19expression decreased after cryopreservation, whereas no significant difference in the ED20and EG5.5/30vitrified groups when compared with the control group (P>0.05). The Snrpn expression was not detected in the fresh and vitrified-warmed groups. Dnmtl and IGF-2protein all appeared in the nucleus of granulosa cell and oocytes of fresh and vitrified-warmed juvenile mouse ovaries. The expression of Dnmtl protein of follicles of various stage in two cryopreserved groups remarkably decreased than that of fresh group (P<0.05). And there was not statistically significant between the two cryopreserved groups (P>0.05). The expression of IGF-2protein increased in the two vitrified-warmed groups when compared with the control group. But except for the ED20group there were significantly difference in the EG5.5/30group compared with the control group (P<0.05). There was not statistically significant between ED20vitrified group and the EG5.5/30vitrified group (P>0.05).Conclusion Our finding suggests that vitrification of juvenile mouse ovaries may alter methylation status of H19/Snrpn genomic imprinting to bring to adverse influence. Vitrification injuried the enzyme activity of Dnmtl, served as DNA methylation capital regulation enzyme. And there are some epigenetic risks of vitrification-induced DNA methylation aberration such as supermethylation of the H19-DMR, hypomethylation of Snrpn-DMR and the exact mechanism needs further investigation.Parts3Studies on the development and fertility potential of prepubertal mouse ovaries following cryopreservation and allotransplantationObjective To investigate the effects of oocytes retrieved from vitrified-wanned surviving ovarian grafts after allotransplantation by in vivo maturation, fertilization and blastoeyst formation.Methods To cryopreserve prepubertal mouse ovaries by vitrification treated with ED20(n=64) and EG5.5/30solutions (n=58). Fresh prepubertal mouse ovaries were served as control group (n=78). Then fresh and cryopreserved prepubertal mouse ovaries were allotransplanted under the kidney capsule of ovariectomized female mice recipients (n=100). Percentage of surviving ovarian grafts and serum levels of estradiol (E2) in recipients was measured by vaginal smear to observe onset of estrous cycle. At day15after the allotransplantation, superovulation and in vitro fertility (IVF) were performed to evaluate the fertility potential of the fresh and vitrified-warmed surviving ovarian grafts.Results Notwithstanding the day of initiating estrous cycles in two cryopreserved transplanted groups was later than the control group (P<0.05), puberty as well as endocrine function was restored when juvenile mouse ovaries were transplanted to ovariectomized recipients. And no significant differences were observed in recipients surviving rate, ovarian surviving rate and estradiol (E2) evels between the ED20and the EG5.5/30vitrified groups (P>0.05). The ratio of primordial, primary and antral follicles were not different between the ED20and the EG5.5/30cryopreserved transplanted groups. Mature oocytes derived from vitrified-warmed surviving ovarian grafts by superovulation protocol can achieve their fertility and develop to blastula in vitro at an even similar fertilization ratio and blastocyst formation ratio compared with the control group (P>0.05).Conclusion These results indicate that heterotopic transplantation of vitrified-warmed prepubertal mouse ovaries can restore ovarian function of endocrine and fertility. The oocytes obtained from vitrified-warmed prepubertal mouse ovaries after allotransplantation can be fertilized and developed into blastocysts, which shows that vitrification does not affect the developmental and fertility potential.