Long-Term Ovarian Function In Rat After Heterotopic Autologous Transplantation Of Cryopreserved Ovarian Tissues

OBJECTIVE To assess the long-term efficacy of cryopreserved ovarian tissues after heterotopic autologous transplanted in rats.METHODS Seventy-eight Wistar adult female rats were divided into four groups randomly as follows:sham-operated group(group 1,n=15), fresh-transplanted group(group 2,n=24),frozen-thawed-transplanted group(group 3,n=24) and ovariectomized group(group 4,n=15).Group 1,underwent a sham operation;Group 2,underwent bilateral ovariectomy and their each ovarian were cut into 8 blocks(about 1mm×2mm×2mm),selected randomly 6 blocks of fresh ovarian tissues and autografted immediately to their each renal capsule;Group 3, underwent bilateral ovariectomy followed by ovarian tissue cryopreservation,then transplanted the ovarian tissues autologously(6 blocks of frozen-thawed ovarian tissues per capsule) under the renal capsules 2-4 weeks later;Group 4,underwent bilateral ovariectomy,and served as castration group.Vaginal irrigation samples were collected daily every two weeks until typical cornified epithelial cells were observed.One third of the animals in each group were killed and their uteri were weighted,and the ovaries and parts of the uteri were removed for morphologic analysis at the 5^th,8^th,or 10^th months after transplantation respectively.Before being killed,the serum concentrations of estradiol-17β(E₂) and progesterone(P) were measured during the period of estrus-like cytological variation according to completely cornified epithelial cells shown by the samples of vaginal irrigation.The follicles of each stage of folliculogenesis were counted under the light microscope. RESULTS Both fresh and frozen ovarian grafts survived in all the animal models.Blood vessels surrounding the grafts were visible.Daily vaginal irrigation showed completely comified epithelial cells in groups 2 and 3.In groups 2 and 3, the serum E₂ and progesterone concentrations during estrus remained comparable to the sham-operated rats' hormone levels(groupl),and significantly higher than the concentrations in group 4 at the 5^th,8^th,10^th months respectively(P＜0.01). Morphologically,there were each stage of follicles and lutea.No significant differences were observed in the proportion of each stage of follicles between frozen-thawed tissues and fresh ovarian tissues(P＞0.05).And the proportion of each stage follicles in both types of grafts was comparable to that in group 1 at the same time(P＞0.05).The number of primordial follicles in fresh grafts and frozen grafts were significantly lower than that in group 1.Grafts in group 2 contained 59.5%of the primordial follicles in sham-operated controls while frozen grafts contained 55.1%at the 5^th month after implantation.CONCLUSIONS Cryopreservation of small pieces of ovarian tissues is feasible.After autologous implanted under the renal capsule,the cryopreserved ovarian tissues contain each stages of follicles and lutea.The follicles survive and develop well,though the number of primordial follicles decreased.The secretary function of ovarian tissues could be preserved in a long term.