Negative effect of vitrification of whole rat ovarian tissue and its subsequent autotransplantation into omentum

ABSTRACT

This study was designed to examine the viability of follicles in whole rat ovary after vitrification-warming and transplantation into omentum. Sixteen 6-8 week old female Wistar rats were randomly divided into four groups, with four 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). In group 3 (fresh grafts), ovarian tissues were dissected out and implanted into the omentum. In group 4 (vitrified grafts), ovarian tissues were dissected out and used for autologous transplantation into omentum after vitrification and warming. Ovaries in the vitrified groups were exposed sequentially to two vitrification solutions 10% ethylene glycol and 10% DMSO in holding medium [HM; human tubal fluid (HTF) medium with 20% FBS] for 15 min and then 20% ethylene glycol, 20% DMSO and 0.5 M sucrose in HM for 2 min before plunging into liquid nitrogen. After warming at 37°C, cryoprotectants were diluted with 1.0 M sucrose in HTF for 10 min. All of the collected ovaries underwent hematoxylin and eosin-stained paraffin serial sections. Follicles at each developmental stage were counted and divided according to the morphological changes. Among the morphologically changed follicles after vitrification, developing and antral follicles significantly accounted for high proportions compared to the fresh control group. A significant decrease in the percentage of healthy follicles was observed in the vitrified ovarian tissues implanted into the omentum in comparison with the fresh omental graft (8.3% vs. 46.3%, P < 0.05). Cryopreservation of whole rat ovarian tissue by the vitrification method used had detrimental effect on follicles. Subsequent autotransplantation of the ovaries into the omentum was not able to support viability of follicles and also insufficient blood supply associated ischemia caused more damage and significant decrease in the number of follicles.