Advancing fertility preservation in prepubertal mice: Efficacy of ovarian tissue culture and in vitro growth in mature oocyte development.

Aim: This study aimed to evaluate the ovarian tissue culture and in vitro follicle growth as safer alternatives to cryopreservation for generating in vitro fertilization (IVF)-ready mature oocytes from prepubertal mice without the risk of cancer cell contamination.

Methods: Ovaries from prepubertal B6D2F1 mice were cultured in α-minimum essential medium supplemented with an estrogen receptor antagonist, ICI 182780. Culture duration was investigated to identify the optimal timeframe for follicle growth and oocyte maturation. Follicles were isolated mechanically or using 1 mg/mL collagenase and cultured in Matrigel matrix or polyvinylpyrrolidone. Oocyte development at metaphase II was induced by in vitro maturation, followed by IVF.

Results: The optimal culture duration was 2-4 days, and tissues cultured beyond this period showed significant follicular degeneration. ICI 182780 supplementation resulted in the recovery of 20.5 follicles per ovary compared with 9.5 follicles in non-supplemented cultures (p < 0.05). Of the 452 isolated follicles, 237 (52.4%) showed growth, 150 (33.2%) underwent germinal vesicle breakdown, and 18 (4.0%) reached metaphase II. However, none of the metaphase II oocytes were successfully fertilized after IVF. Matrigel demonstrated a significantly higher in vitro maturation rate compared with polyvinylpyrrolidone in a comparative analysis of culture matrices (p < 0.001).

Conclusions: This study highlighted ovarian tissue culture and in vitro growth as effective strategies for producing mature oocytes from prepubertal mice. Further studies are required to overcome fertilization hurdles and understand the mechanisms that improve post-IVF embryo viability.