Ultrastructural Analysis of Vitrified Rat Ovarian Tissue Follicles after Heterotopic Autotransplantation

Objective To evaluate and compare the efficiency of vitrification technique and heterotopic autotransplantation with different duration on morphology and ultrastructure of rat ovarian tissue follicles. Methods Twenty 5-6-week-old female SPF SD rats were randomly divided into two groups, with ten rats in each group. Freshly isolated ovaries saved as a control (group 1; fresh ovaries) in formalin-fixed or vitrified immediately after dissection (group 2; vitrified ovaries). Ovaries in vitrified groups were processed into thin slices then vitrified in cryotubes using vitrification medium and kept in liquid nitrogen for 21 days, rapidly thawed and transplanted into back muscles for 2 and 4 weeks. All rats in vitrified experimental group were sacrificed and tissue grafts were collected and fixed in 4 % formaldehyde solution. Then non-vitrified and vitrified heterotopic autotransplanted ovarian tissue was compared by light and transmission electron microscopic morphology of the follicles within the tissues. Results There was no significant difference between the percentages of normal primordial (28 % vs 41 %), primary (41 % vs 33 %), secondary (23 % vs 29 %) and antral (30 % vs 28 %) follicles in both fresh control and vitrified transplanted groups (pgreater than0.05). No clear differences in the structures of the follicles, the stromal cells and bundles of collagen fibers were well preserved. By mean of TEM, ovarian tissue follicles showed a well-preserved ultrastructure however the number of mitochondria-SER aggregates and Golgi apparatus were highest in 2 weeks vitrified transplanted group than the 4 weeks vitrified transplanted group. The intact contact between granulosa cells was highly visible in 4 weeks vitrified transplanted tissue whereas irregular shaped mitochondria with no cristae and widened gap junction with slight edema between granulosa cells were only observed in 2 weeks vitrified transplanted group. Conclusions vitrification technique with heterotopic autotransplantation of ovarian tissue is efficient to maintain their morphological and ultrastructural features at different durations (2 and 4 weeks) similar with fresh tissue, while mild ultrastructural changes in preantral follicles in 4 weeks transplanted tissue was noticed. Although these data are encouraging, further studies are necessary to optimize vitrification protocol and to confirm the accuracy of these observations.