P-626 Mitochondrial Fatty Acid Shuttle: Novel Targets in the Pathogenesis of Premature Ovarian Insufficiency

Abstract Study question Do mitochondrial variants play a role in the pathogenesis of Premature Ovarian Insufficiency (POI)? Summary answer Genetic variants in mitochondrial and fatty acid oxidation pathways are key contributors to the pathogenesis of POI, underscoring its intricate and multifactorial etiology. What is known already Mitochondria are essential for energy production in oocytes, facilitating folliculogenesis and oocyte maturation by generating ATP through the β-oxidation of fatty acids via the mitochondrial fatty acid shuttle (MFAS). This pathway, regulated by key enzymes such as CPT1, CPT2, CACT, OCTN2, and L-carnitine, is essential for spindle assembly, chromosomal segregation, and meiotic progression-factors critical for oocyte competence and embryo development. Disruptions in MFAS can impair β-oxidation and ATP production, thereby compromising follicular and oocyte health. Despite its fundamental role, the contribution of MFAS dysfunction to the pathophysiology of Premature Ovarian Insufficiency (POI) remains unexplored. Study design, size, duration Whole Exome Sequencing (WES) was conducted on blood DNA samples from idiopathic POI women (n = 10) to identify genetic variants. Serum reproductive hormone levels (E2, FSH, AMH, PRL) were measured, followed by in-silico analyses and protein modeling to assess the impact on protein structure/function. C57BL/6 adolescent mice (3–5 weeks, n = 4/group) were treated with MFAS inhibitors. Histological analysis of ovaries and other metabolic organs was performed post-treatment. The study lasted for 2 years. Participants/materials, setting, methods WES was performed on POI women, with data analyzed using our custom pipeline and in-silico tools (CADD, SIFT, PP2, CONDEL, FATHM) for predicting the impact of mitochondrial variants on protein function and structure, utilizing protein modeling tools like Pymol and ProteinPaint. Mice were treated with MFAS inhibitors (Etomoxir, Omeprazole, L-Amino Carnitine, Etoposide, Mildronate), along with cyclophosphamide (positive control) and saline (negative control), followed by collection of organs (ovaries, kidneys, liver, spleen) for H&E analysis. Main results and the role of chance A novel variant in OCTN2 (SLC25A5) and variants of uncertain significance (VOUS) in CPT1A, CPT1B, and CPT2 were identified within genes related to the mitochondrial fatty acid shuttle (MFAS). In terms of mitochondrial function, two novel pathogenic variants (ARHGEF28 and AOX1) along with six likely pathogenic variants (MT-ND2, MT-ATP6, MT-CYB, MT-ND4, MT-ND5, MT-CO3) were discovered. Notably, CPT1, CPT2, SLC25A5, AOX1, and ARHGEF28 exhibited high predicted local distance difference test (pLDDT) scores, suggesting significant structural alterations.SLC25A5 and AOX1 displayed very high pLDDT scores (>90), while ARHGEF28 scored between 70 and 90.Functionally, CPT1, CPT2, and SLC25A5 contribute to ATP production, AOX1 regulates ROS generation, and ARHGEF28 affects cell cycle progression. Furthermore, 18 pathogenic variants and 29 VOUS were found in mitochondrial and cell-cycle genes, many of which regulate meiosis, follicular development, and ovulation, indicating their potential impact on fertility. In POI mouse models, MFAS inhibition and a positive control led to ovarian shrinkage, follicular atresia, and systemic organ abnormalities (kidney, liver, spleen), resembling cyclophosphamide POI-induced histological changes. These findings highlight the impact of MFAS dysfunction on ovarian folliculogenesis and systemic health, providing new insights into the mitochondrial contribution to Premature Ovarian Insufficiency (POI). Limitations, reasons for caution Small sample size of human participants (n = 10), which may limit the generalizability of the findings, as well as the inability to obtain human ovarian tissue due to ethical constraints. Additionally, the lack of long-term treatment analysis restricts our understanding of the chronic effects of MFAS inhibition. Wider implications of the findings Exploring mitochondrial dysfunction alongside existing diagnostic criteria could facilitate early detection of POI and offer multiple avenues for early disease diagnosis. Integrating this approach with established hormonal diagnostic methods may provide more comprehensive insights, provided that similar studies are conducted in larger cohorts. Trial registration number No