The aim of this study was to determine a suitable cryoprotectant combination for whole rat ovarian vitrification and evaluate hormonal changes, the ultrastructure of the initial follicle, and follicle maturational, angiogenic and apoptotic gene expression of vitrified ovary following autotransplantation.
In the first part of this study we sought to determine the suitable cryoprotectant combination. Wistar rat ovaries were randomly divided into 13 groups as follows: control (nonvitrified); VI and TI (EG + DMSO); VII and TII (EG + PROH); VIII and TIII (DMSO + PROH); VIV and TIV (EG + DMSO + 0.25 mol/l sucrose); VV and TV (EG + PROH + 0.25 mol/l sucrose); and VVI and TVI (EG + PROH + 0.25 mol/l sucrose). Groups were evaluated by standard histology techniques, EM and immunohistochemistry.
In the second part of this study we investigated the effects of ovarian autotransplantation by using the appropriate previously determined cryoprotectant combination. The six groups of rat ovaries were divided as follows: control (nonvitrified nontransplanted); nVTnG (nonvitrified transplanted nongonadectomized); VTnG (vitrified transplanted nongonadectomized); nVTG (nonvitrified transplanted gonadectomized); VTG (vitrified transplanted gonadectomized); and BLG (bilateral gonadectomized). These groups were evaluated by standard histology techniques, EM and immunohistochemistery. Expressions of follicle maturational, angiogenic and apoptotic genes were studied by Real-time PCR. The levels of gonadotropins (LH, FSH) and steroid hormones (estradiol, progesterone and testosterone) in blood serum were compared to the control and BLG groups.
The results showed that vitrified groups had lower numbers of intact follicles and higher numbers of apoptotic follicles compared to the control group. Between vitrified groups, the VIV group had the best survival rate, particularly for initial follicles. This group had a lower incidence of apoptosis. Ultrastructural changes of the last group were considerable compared to the control but this result did not differ compared to the sucrose-free (VI) group.
Vitrified and nonvitrified ovarian autotransplantation caused restoration of the hormone cycle and ovarian function; these results approximated the controls in both of the nVTG and VTG groups. In the recent groups, the percentage of follicular maturation and ultrastructure of transplanted ovaries were in better condition. In these groups, however, the incidence of apoptosis in primordial and antral follicles was higher, whereas the incidence of apoptosis in primary and preantral follicles was lower than the VTnG and nVTnG groups. Also the rate of expressions of angiogenic factors (CD31 and CD34) in all of the transplanted groups, particularly in the nVTG and VTG groups, were comparable with the control.
The results of this study showed that a combination of EG + DMSO and sucrose was more suited for follicular preservation, particularly at the initial stage could relatively restore ovarian function after vitrification and autotransplantation.