Study On The Repair Effect Of Umbilical Cord Mesenchymal Stem Cell Derived Exosomes On Frozen-thawed Ovarian Tissue Based On In Vitro Culture System

Objective: Ovarian tissue cryopreservation and transplantation(OTCT),as an effective female fertility preservation technology,provides an important guarantee for the restoration of fertility and endocrine function.However,problems such as early ischemia and hypoxia after transplantation may cause acute loss of follicles.Mesenchymal stem cells(MSCs)can improve the ischemia and hypoxia of ovarian tissue in the early stage after transplantation,but their clinical application has potential immunogenicity and tumorigenicity.The therapeutic effect of MSCs depends to a large extent on the exosomes it secretes.Because of their low immunogenicity,no risk of tumorigenesis and easy storage,exosomes are expected to become a new "cell-free therapy" to replace MSCs.This study is to explore the repair effect of human umbilical cord mesenchymal stem cellderived exosomes(hUC-MSC-Exos)on frozen-thawed mouse ovarian tissue based on in vitro culture system in order to improve the functional reconstruction of frozen-thawed ovarian tissue.Methods: Collect the culture medium of hUC-MSCs,extract exosomes and identify them by transmission electron microscope and Western Blot.Bilateral ovaries were obtained from normal BALB/c female mice,and were cryopreserved by vitrification and then were thawed.HUC-MSC-Exos were added to the ovarian culture system constructed by extracellular matrix gel Matrigel.The frozen-thawed mouse ovaries were divided into Control group,Exos group,and Matrigel+Exos group.HUC-MSC-Exos were labeled with PKH26 to detect the uptake of hUC-MSC-Exos by the frozen-thawed mouse ovaries in Matrigel+Exos group.After 4 days of culture in vitro,the ovaries of each group were collected to detect the ovarian tissue morphology and follicle count with HE staining,the vascular endothelial cell marker CD31 was detected with immunohistochemistry to count the number of microvessels,the cell proliferation-related index Ki67 was detected with immunofluorescence,and the cell apoptosis was detected with TUNEL staining.The culture medium cultured for 2 and 4 days in vitro was collected,and the level of antiMullerian hormone(AMH)was detected with enzyme-linked immunosorbent assay.Results: Under the transmission electron microscope,the particle size of hUC-MSCExos was distributed between 20-300 nm.Western Blot showed that hUC-MSC-Exos were rich in the surface markers CD63,TSG101 and PDCD6 IP,but GAPDH was negative.HUC-MSC-Exos were detected in frozen-thawed mouse ovaries in in vitro culture system,which reached the peak after 3-4 days of in vitro culture.The results of HE staining showed that compared with the Control group,the number of morphologically unhealthy follicles in the Exos group and the Matrigel+Exos group was less(P<0.05),and the proportion of primary follicles in the Matrigel+Exos group was significantly reduced(P<0.05),but the proportion of antral follicles was significantly increased(P<0.05).In addition,the Ki67-positive cell rate in the Exos group was significantly higher than that in the Control group(P<0.001),and there was no significant difference in the mean fluorescence intensity of TUNEL in the three groups(P>0.05).After 4 days of in vitro culture,the number of microvessels in the Matrigel+Exos group was significantly higher than that in the Control group(P<0.01).AMH concentration in the Matrigel+Exos group was significantly higher than that in the Control group(P<0.05),and there was no statistical difference between the Matrigel+Exos and Exos group(P>0.05).Conclusion: In in vitro culture system,hUC-MSC-Exos could improve the structure of frozen-thawed mice ovaries,promote follicular development,microvascular formation and cell proliferation,and increase the concentration of AMH in culture medium.In summary,hUC-MSC-Exos could improve the structure and endocrine function of frozenthawed mice ovaries to a certain extent.