First follicular fluid cf-mtDNA sequencing unveils ovarian stimulation-induced mutations impacting oocyte quality and IVF success

PURPOSE: Cell-free mitochondrial DNA (cf-mtDNA) has emerged as a promising non-invasive method to predict embryo implantation potential. This study presents the first sequencing of cf-mtDNA in follicular fluid, aiming to explore the impact of ovarian stimulation treatment on the accumulation of mtDNA mutations and assess their implications for IVF outcomes. METHODS: We selected 24 women aged 27 to 35 from the Assisted Reproduction Unit based on inclusion criteria requiring them to be undergoing their first IVF cycle without known infertility issues. For each participant, blood samples were collected prior to ovarian stimulation, and follicular fluid samples were obtained at the time of oocyte retrieval. Long-PCR amplification and next-generation sequencing were used to identify mtDNA mutations. RESULTS: Ovarian stimulation-induced mtDNA mutations in follicular fluid were identified primarily in the D-loop region of the mitochondrial genome. These mutations were correlated with low oocyte counts and poor embryo quality, ultimately reducing IVF success rates. The accumulation of these mutations followed a time-dependent pattern, with longer stimulation durations resulting in higher mutation rates and lower pregnancy outcomes. Prolonged stimulations may exacerbate oxidative stress, negatively impacting oocyte quality. CONCLUSIONS: By introducing the first sequencing of follicular fluid cf-mtDNA, this study establishes a direct link between ovarian stimulation-induced mtDNA mutations and reduced IVF efficacy, highlighting them as non-invasive biomarkers for predicting IVF outcomes. Future research may focus on optimizing stimulation protocols or incorporating antioxidants to minimize mtDNA mutations, improving oocyte quality and overall IVF success rates for patients.