Study Of Autologous Transplantation Of Mitochondrial From Mesenchymal Stem Cells In Assisted Reproductive Techniques

Backgroud and Object:The reproductive ability of females reaches the peak at 25 years old, and it begins to decrease significantly over 35. In ART (assisted reproductive technology), the pregnancy rate of infertility patients over 37 years old is only about 20%. A number of studies have shown that the age-related decline in pregnancy rates and repeated IVF implantation failure is due to mitochondrial dysfunction in eggs, reducing the ability to generate ATP and affecting the normal separation of chromosomes and other physiological functions. Therefore, through the experimental study, the transferation of mitochondrial from HADSCs (human adipose derived mesenchymal stem cells) into oocytes can improve the quality of embryo development and increase the success rate of IVF.Methods:First Part: Human adipose tissue was collected after liposuction surgery from the cosmetic and plastic surgery center, digested with 0.01 % collagenase I. Adherent culture could obtain HADSCs. Cell proliferation curve was drawn. Cell surface antigens were measured by flow cytometry. HADSCs were underwent osteogenic and adipogenic differentiation.Second Part: Compare mitochondrial function in HADSCs and adipocytes by measuring the levels of mitochondrial ATP, reactive oxygen species (ROS) and mitochondrial membrane potential using flow-cytometry. he data were analyzed by paired t test, P < 0.05 was statistically significant.Third Part : The first case of mitochondrial transplantation from autologous HADSCs. Mitochondria were extracted by differential centrifugation. On the day of oocyte retrieval, mitochondria were injected into the cytoplasm of the egg cells with the brake sperm. Then, we observed the fertilization and embryo development, and conduct preimplantation genetic screening and mitochondrial DNA copy number determination.Results:First Part: HADSCs exhibited fibroblast-like morphology, well proliferation, with obvious exponential growth phase, and could survive in vitro for a long time and maintain a undifferentiated state. Flow cytometry has shown that cells were highly positive for CD90, CD105, and weakly positive for CD34 and CD45. Cells could differentiate into osteogenic and adipogenic direction.Second Part: With the increase of age, the function of mitochondrial in fat cells and ADSCs decreased. However, in the same adipose tissue, the level of mitochondrial ATP and membrane potential of ADSCs were higher than that of adipose tissue, and the level of reactive oxygen species (ROS) was lower than that of adipocytes, P < 0.05.Third Part: There was no obvious abnormality of embryo fertilization and embryo development in the first case of mitochondrial transplantation. PGS showed that the chromosomes were abnormal. In the experimental group, the copy number of mitochondrial DNA was higher than that of the control group.Conclusion:Adipose tissue derived mesenchymal stem cells were isolated and cultured stably.The study showed that the mitochondrial function was better in ADSCs. To carry out the first clinical mitochondrial transplantation of autologous ADSCs, it showed that the copy number of mitochondrial DNA increased, and mitochondria transplantation inIVF has some feasible.