Background: Female fertility declines with obesity and advancing age, yet the mechanisms underlying this remain poorly understood. Mitochondrial dysfunction and associated oxidative stress have been identified within the ovarian follicular microenvironment of obese and aged individuals, but how these specific alterations impair ovulation and oocyte quality remains unclear. This study aims to determine how mitochondrial dysfunction disrupts periovulatory follicle function, ovulation, and subsequent oocyte developmental competence.

Methods: To investigate the acute effects of an altered follicular microenvironment, an ex-vivo mouse follicle maturation model was used, followed by assessment of ovulation and oocyte developmental competence using IVF. Antral follicles were dissected from ovaries of 22-26 day-old mice and cultured for 24h before undergoing hCG-induced ovulation and exposure to mitochondrial complex I inhibitor rotenone (5μM-8nM). Ovulation rates were assessed 12-15h following hCG stimulation. Mitochondrial reactive oxygen species were measured using MitoSOX Red.

Results: Ex-vivo cultured follicles undergo cumulus expansion in response to hCG and ovulate within 14h. Fertilisation of the of the ovulated oocytes by IVF produced blastocyst stage embryos with normal morphology. Treating follicles with titrated rotenone doses of 5μM, 1μM, 0.2μM, 40nM, 20nM, and 12nM completely inhibited ovulation. Follicles treated with 8nM rotenone trended towards reduced ovulation (~20%) compared controls (P=0.4). Interestingly, oocyte mtROS levels detected by MitoSOX Red staining were unaffected by this rotenone treatment (P=0.4). Ongoing studies are investigating downstream cellular mechanisms and oocyte developmental competence through meiotic spindle integrity, mitochondrial membrane potential and fertilisation rates.

Conclusion: These results highlight that mitochondrial function is essential for successful ovulation and suggests mitochondrial dysfunction may primarily affect surrounding follicular somatic cells rather than the oocyte during pre-ovulatory maturation. These findings establish a model for investigating how obesity- and age-associated alterations in the follicular microenvironment impair follicle development, ovulation, and oocyte developmental competence, with future applications in identifying potential therapeutic targets.