A Study On The Morphology And Function After Cryopreservation, Thawing, And Subcutaneous Autotransplantation Of Ovarian Tissue In Rats

Objectives: To study the restoration of ovarian steroid secretion and the development of follicles after subcutaneous autotransplantation of cryopreserved rat ovarian tissue. Find out a convenient and practical method of its cryopreservation and storage.Methods: The adult female SD rat ovarian pieces were suspended in a cryoprotectant solution containing 40% (V/V) ethylene glycol and 0.35 mol/1 sucrose. The Cryopreservation of pieces was accomplished by direct plunging into liquid nitrogen. Then the pieces were stored in liquid nitrogen for up to 28 day and fast thawed. Histological sections of fresh (non-cryopreserved) and cooled ovarian pieces (immediately after dilution of cryoprotectants ) were analyzed and the number of viable and damaged primordial follicles was counted. Fresh and frozen-thawed ovarian pieces were autotransplanted heterotopically (in the back subcutis) to the bilaterally ovariectomized adult female SD rats. Vaginal smears were taken daily after transplantation to observe the restoration of oestrous cycles. Serum levels of E₂ were determined on day 0 (the day of surgery or baseline) and day 7, 14, 21, 28 and 35. Serum levels of P were determined on day 28 and 35. On day 35 after transplantation the grafted ovarian pieces and the uteruses were removed and underwent histological examination. The weights of uteruses were measured in the same day.Results: 83.3% of the recipients in fresh group and 60.9% in frozen-thawed group resulted in restoration of regular oestrous cyclic activity; this value was not significantly different between the two groups (P>0.05). The vaginal cytology showed trophic maturation within 8.75 ± 1.97 day or 18.83 ± 2.33 day after transplantation in the fresh and frozen-thawed groups, respectively; statistically significant difference (P<0.05) was demonstrated between the two groups. The mean oestrous day of the...