Exploration Of A Mid-long Term Cryopreservation Technology Of Human Oocytes Based On The Application Of Melatonin

Objective:To explore the effect of cryopreservation time on the developmental competence of huamn oocytes,and whether the addition of melatonin（MT）into the vitrification-warming medium can improve the effect of mid-long term cryopreservation in oocytes as well as its mechanism of action,aiming to establish a novel mid-long term cryopreservation technology system for human oocyte.Subjects:The immature oocytes（GV or MI）discarded in the controlled superovulation cycle were cultured and matured by in vitro maturation（IVM）technology as the source of mature oocytes（IVM-MII oocytes）for this study.Methods and indicators1.The collected IVM-MII oocytes were randomly divided into five groups:Fresh group（group F）;no-MT-treated cryopreservation for 0 months group（n MC-0 group）;no-MT-treated cryopreservation for 6 months group（n MC-6 group）;10^-9M MT-treated cryopreservation for 0 months group（MC-0 group）and 10^-9M MT-treated cryopreservation for 6 months group（MC-6 group）.2.The morphological characteristics of oocytes after cryopreservation were observed under a inverted microscope（400×）;3.The fresh and vitrified-warmed oocytes underwent insemination by intracytoplasmic single sperm injection（ICSI）technology followed by 5-6 days of early embryo culture in vitro,during which the fertilization and embryo development status of each group were recorded and analyzed;4.Protein expression levels in oocytes were detected by microproteomics;5.Fluorescent staining was used to detect and compare indicators related to oocyte mitochondrial function and oxidative damage,such as ATP,ROS,GSH,and ROS/GSH levels.Results:1.The morphological observation of each group of oocytes after vitrification and warming showed that cryopreservation could lead to structural damage of oocytes and the involvement of 10^-9M MT could maintain a normal morphology similar to that of fresh oocytes;2.By analyzing the ICSI of five groups of oocytes and the development of oocytes cultured in vitro,it was found that cryopreservation decreased the developmental ability of oocytes,and the involvement of 10^-9M MT effectively improved the subsequent development of oocytes;3.The results of single-oocyte proteomic assays showed that,except for the MC-6 group,the mitochondria-related differentially expressed proteins were down-regulated in the F group compared with other groups.Compared with the non-MT-treated group,the mitochondrial-related differential proteins were mostly up-regulated in the 10^-9M MT-treated group.With the extension of cryopreservation time,there was no difference in the up-and down-regulation of mitochondrial-related differential proteins between the n MC-6 group and then MC-0 group;4.Fluorescence staining revealed significant differences in the ATP,ROS and GSH levels between the n MC-0 group or the n MC-6 group and the F group,and between the MC-0 and n MC-0 groups and between the MC-6 and n MC-6 groups,as well as in the ATP and ROS levels between the n MC-0 and the n MC-6 groups.The ratio of ROS/GSH in oocytes in n MC-0 group and n MC-6 group were significantly higher than that in F group;5.ROS/GSH ration analysis showed that the ratio of ROS/GSH in non-MT-treated group oocytes was significantly higher than that of the F group and 10^-9M MT-treated group.With the extension of cryopreservation time,the ratio of ROS/GSH in the n MC-0 group was significantly lower than that in the n MC-6 group.However,there was no significant difference in the ROS/GSH ratio between the MC-0 and MC-6 groups,and both were comparable to the F group;6.A preliminary set of human oocyte freezing-thawing medium products has been produced,as well as an effective technological system for mid-long term cryopreservation of human oocytes has been established.Conclusion:1.For human oocytes cryopreservation,the extension of cryopreservation time aggravates the damage to the developmental competence of human oocytes.2.The involvement of 10^-9M MT may effectively protect the expression of mitochondria-related proteins and maintain mitochondrial functions,such as ATP synthesis and inhibition of oxidative damage and then achieve protection of the developmental capacity of vitrified oocytes.3.Based on the application of MT,an effective technological system for mid-long term cryopreservation of human oocytes can be established.