The Functions And Mechanism Of Melatonin On Human Oocytes During Cryopreservation

Objective:Cryopreservation of human oocytes is an important technology in the field of assisted reproduction,but it still needs to improve.At present,many studies have shown that cryopreservation can cause damages to human oocytes and impair their developmental competence.Melatonin can serve as an antioxidant to improve the effect of cryopreservation on animal oocytes.However,there has been no report on whether the same protective effect applies to human oocytes.In this study,oocytes,vitrified and warmed in media supplemented different concentrations of MLT,were detected mitochondrial function to determine the optimal function concentration.Then it was proved that the optimal concentration of MLT could inhibit the oocytes from ultrastructure damage,maintain the permeability of the oolemma,reduce the incidence of early apoptosis and thus improve the developmental competence.Finally,the mechanism involved in protection of MLT on cryopreserved human oocytes was further researched.Material and Methods:Recruitment From February 2019 to January 2020,a total of236 women suffered infertility under 35 years of age were recruited from our assisted reproduction center for ICSI treatment.oocytes at GV and MI stage from ICSI operation were performed IVM culture,and those matured were used in this study.（1）The morphological characteristics of oocytes after cryopreservation were observed under a stereomicroscope（400x）,and the survival rate of oocytes in the MLT treated group(10^-11,10^-9,10^-7and 10^-5M MLT)and non-MLT treated group（0M）was recorded.（2）The levels of ROS,MMP and Ca²⁺in oocytes of the vitrification groups and the fresh group were detected by fluorescence staining.（3）The ultrastructures of oocytes in three groups were observed by TEM.（4）The membrane permeability of oocytes in three groups was compared by cell shrinkage and reswelling experiment.（5）Fluorescence staining was used to compare the early apoptosis rate of oocytes in three groups.（6）The developmental condition of oocytes in three groups was recorded after ICSI and time-lapse culture.（7）Single-cell RNA sequencing technology was used to determine the transcriptome levels of three groups of oocytes.（8）Immunofluorescence was used to compare the protein expression levels of target genes in three groups of oocytes.Result:（1）Morphological results showed that cryopreservation caused structural damage to oocytes,and supplementing 10^-9M MLT to vitrification and warming media could maintain the morphology of oocytes,which was similar to those from the fresh group.（2）The fluorescence staining results showed that cryopreservation caused high levels of intracellular ROS and Ca²⁺concentration as well as low level of MMP in oocytes.However,supplementing 10^-9M MLT to vitrification and warming media could obtain lowest levels of ROS and Ca²⁺concentration as well as highest level of MMP.Based on the above results,it can be concluded that 10^-9M is the optimal concentration of MLT for protecting the mitochondrial function of human oocytes during cryopreservation.And this optimal concentration was applied to the following experiments.（3）The results of TEM showed that cryopreservation damaged the ultrastructure of oocytes（including mitochondria,cortical granules and microvilli）,while 10^-9M MLT could maintain the normal ultrastructure of oocytes,with no significant difference from the fresh group.（4）The membrane permeability experiment results showed that cryopreservation reduced the water permeability of oocyte membrane,while 10^-9M MLT could maintain the membrane function.（5）The fluorescence staining results showed that cryopreservation increased the early apoptosis rate of oocytes,while 10^-9M MLT significantly reduced this rate,and there was no significant difference from the fresh group.（6）ICSI experimental results showed that cryopreservation decreased oocyte developmental competence,while 10^-9M MLT reserved the declining,with no significant difference from the fresh group.（7）The results of single cell RNA sequencing showed that the differentially expressed genes related to membrane permeability were AQP1,AQP2 and AQP11.（8）Immunofluorescence experiment results showed that cryopreservation down-regulated the expression level of AQP1 gene,while 10^-9M MLT inhibited the reduction of this protein level,and there was no significant difference between the expression levels of AQP2 and AQP11 among these three groups.Conclusions:（1）Appropriate concentration of MLT can protect the mitochondrial function of cryopreserved human oocytes by inhibiting oxidative stress,and the optimal concentration is 10^-9M.（2）10^-9M MLT can maintain the ultrastructure and membrane permeability of oocytes after vitrification and warming,reduce the early apoptosis rate,and thus improve their developmental competence.（3）MLT may maintain the permeability of oocyte membrane by inhibiting the decrease of AQP1 expression in oocytes caused by cryopreservation.