(315) Novel Mitochondrial Variants in Idiopathic Premature Ovarian Insufficiency: Insights from Exome Sequencing and Mouse Models

Novel Mitochondrial Variants in Idiopathic Premature Ovarian Insufficiency: Insights from Exome Sequencing and Mouse Models

Kajal Sihag¹, Rajni Sharma³, JB Sharma², Ashutosh Halder¹, Mona Sharma^1*
1.Department of Reproductive Biology, AIIMS, New Delhi
2.Department of Obstetrics and Gynecology, AIIMS, New Delhi
3.Department of Paediatrics, AIIMS, New Delhi

* Corresponding Author

Additional Professor, Department of Reproductive Biology, Room no. 2087, Teaching Block, AIIMS, New Delhi.                              

dr.mona18sharma@gmail.com

Abstract Text:

Mitochondria serve as the primary energy source for cells, playing a critical role during normal folliculogenesis. Oocytes require substantial ATP to support their growth and maturation, with mitochondria generating this energy predominantly through the β-oxidation of fatty acids via the mitochondrial fatty acid shuttle (MFAS). MFAS include key enzymes like CPT1 (Carnitine Palmitoyltransferase 1) and CPT2 (Carnitine Palmitoyltransferase 2), CACT, OCTN2 and L-carnitine. This process is crucial for key cellular functions such as spindle formation, chromosomal segregation, and meiotic progression—all of which are vital for oocyte competence and successful embryo development post-fertilization. A significant portion of the energy needed for these processes is derived from β-oxidation within the MFAS pathway. Therefore, proper functioning of the MFAS, efficient β-oxidation, and adequate ATP production are essential for the healthy development of follicles and oocytes. Despite this, the contribution of MFAS dysfunction to POI remains unexplored. Understanding how mitochondrial dysfunction influences follicular dynamics in POI may provide valuable insights into potential therapeutic strategies that support mitochondrial function, thereby delaying ovarian failure. To investigate this, our study aimed to understand the impact of MFAS on follicular dynamics and their role in the onset and progression of premature ovarian insufficiency. The objectives of this study were to explore genetic aberrations in genes regulating MFAS in women with POI and to develop mouse models of POI using MFAS inhibitors, to examine their effects on ovarian folliculogenesis. Whole Exome Sequencing (WES) of 10 women with idiopathic POI, along with in-silico analyses and protein modelling, was used to predict the impact of identified variants on protein structure and function. The novel mitochondrial variants, for which inhibitors are available, were utilized to create the POI model.C57BL/6 adolescent mice (3–5 weeks) were treated with MFAS inhibitors (Cyclophosphamide, Etomoxir, Omeprazole, L Amino Carnitine, Etoposide and Mildronate) and organs (ovaries, kidneys, liver, spleen) were collected for H&E analysis. A novel OCTN2 (SLC25A5) variant and VOUS in CPT1A, CPT1B, and CPT2 were identified in MFAS-related genes. In mitochondrial function, two novel pathogenic variants (ARHGEF28, AOX1) and six likely pathogenic variants (MT-ND2, MT-ATP6, MT-CYB, MT-ND4, MT-ND5, MT-CO3) were found. Additionally, 18 known POI-related pathogenic variants and 29 VOUS in mitochondrial and cell-cycle genes were found. POI model displayed reduced body weight, ovarian shrinkage, fewer follicles, and increased atresia. They further showed kidney, liver, and spleen abnormalities, confirming MFAS inhibition impacts ovarian and systemic health. This study identified VOUS in CPT1A, CPT1B, and CPT2 and pathogenic mitochondrial variants suggest MFAS dysfunction and severe energy metabolism issues, particularly in young POI cases. Adequate ATP production is crucial for ovarian folliculogenesis, and any impairment in ATP generation can lead to lipotoxicity. Additionally, the kidney, liver, and spleen, key organs involved in metabolic regulation, also appear to be affected. Our mouse model revealed altered ovarian histology, accompanied by changes in these metabolic organs, further supporting systemic involvement. In a nutshell, our findings identify novel gene variants that affect metabolism, meiosis, and ovulation, supporting the notion of POI as a multifactorial condition. Disruption in MFAS and fatty acid oxidation appears to have significant implications for both ovarian and systemic health. Investigating MFAS dysfunction could offer critical insights into the etiopathogenesis of POI.