GnRHa pretreatment is associated with improved follicular preservation and graft function compared with combined oral contraceptive pretreatment after ovarian tissue vitrification and autotransplantation in mice

Ovarian tissue cryopreservation and transplantation is an important fertility preservation strategy, but cryoinjury and post-transplantation ischemia-reperfusion injury lead to massive follicle loss and limited graft lifespan. This study was performed to compare the ovarian protective effects of gonadotropin-releasing hormone agonist (GnRHa) versus combined oral contraceptive (COC) pretreatment during vitrification and autotransplantation, and to evaluate selected molecular markers associated with follicular development, apoptosis, and graft revascularization. Seventy-two six-week-old female C57BL/6 mice were allocated to four pretreatment arms ( n = 18 per arm: untreated, GnRHa, COC, or saline vehicle) and further divided into pre-transplantation (fresh control, GnRHa-pretreated vitrification [GV], COC-pretreated vitrification [CV], vitrification-only [V]; n = 10 per group) and post-transplantation cohorts (fresh-transplanted [FT], GnRHa-pretreated vitrified-transplanted [GVT], COC-pretreated vitrified-transplanted [CVT], and vitrified-transplanted [VT]; n = 8 per group). Ovaries underwent 14-day GnRHa/COC pretreatment followed by two-step vitrification and subrenal capsule autotransplantation. Histomorphology, ultrastructure, and molecular markers were examined after warming, and follicular density, Ki-67 immunostaining of primordial follicles, apoptosis (cleaved caspase-3), graft vascular density (CD31), and serum hormones were assessed 4 weeks after grafting. Both GnRHa and COC pretreatment significantly reduced serum FSH and E₂ after 14 days. After warming, significant primordial follicle loss occurred in the CV and V groups (severest in the V group) but not in the GV group, with no significant difference between the GV and CV groups; folliculogenesis- and apoptosis-related markers did not differ among groups, although ultrastructural damage was mildest in the GV group. Four weeks post-autotransplantation, the GVT group exhibited a primordial follicle density not significantly different from the FT group and significantly higher than the VT group, with intermediate levels in the CVT group. Its proportion of Ki-67-positive primordial follicles did not differ significantly from that of FT and was significantly lower than in the CVT and VT groups; GDF9 and BMP15 protein levels did not differ significantly from FT but were significantly reduced in VT; AMH protein was lower than in FT in all vitrified-transplanted groups but highest in the GVT group. Cleaved caspase-3 scores in growing follicles remained statistically comparable to fresh controls in the GVT group but were significantly elevated in the CVT and VT groups; graft vascular density (CD31) did not differ among the transplanted groups. Serum E₂ and FSH did not differ significantly between the GVT and FT groups but were significantly impaired in the CVT and VT groups (E₂ was also significantly lower in GVT than in fresh controls); serum AMH decreased progressively across the FT, GVT, CVT and VT groups, remaining significantly higher in the GVT group than in the CVT and VT groups. GnRHa pretreatment is associated with preserved primordial follicular density, reduced post-transplantation primordial follicle activation, and attenuated apoptosis in growing follicles, leading to improved structural and functional outcomes relative to COC-pretreated and vitrification-only controls in the setting of ovarian tissue vitrification and autotransplantation.