Abstract

Infertility is a worldwide health issue affecting more than10% of couples, with female-related causes representing more than half of cases. Ovarian disfunctions such as polycystic ovary syndrome (PCOS), diminished ovarian reserve (DOR), tubal factor, and unexplained infertility, are closely linked to granulosa cell (GC) health and mitochondrial function. Markers like cell-free mitochondrial DNA (cf-mtDNA) in follicular fluid (FF), primarily released from apoptotic GCs, has recently emerged as a potential biomarker for mitochondrial integrity, oocyte competence, and reproductive outcomes. This study investigated the levels of cf-mtDNA in FF from 154 Iraqi women undergoing intracytoplasmic sperm injection (ICSI) in the period between November 2023 and June 2024. Patients were devided into five groups: male factor infertility, which represent the control, n=35), PCOS (n=30), DOR (n=28), unexplained infertility (n=34), and tubal factor infertility (n=27). FF was collected at oocyte retrieval and analyzed for cf-mtDNA using quantitative PCR targeting the mitochondrial ND4 gene , with nuclear ?-globin as an internal nuclear DNA reference. Results demonstrated a consistent reduction in cf-mtDNA levels across all infertility groups compared to controls. The most pronounced deficits occurred in tubal factor (mean 0.38) and DOR (mean 0.37) cases, indicating significant mitochondrial disfunction in GCs. PCOS and unexplained infertility also showed reduced levels (means 0.52 and 0.67 , respectively), though with greater variability in the unexplained group. Results also revealed that patients averaged 34 years of age, had a median BMI of around 29.5, exhibited moderate oocyte yield and embryo quality. The current study findings suggested that mitochondrial deficiency is a commn pathological factor in different infertility disorders. cf-mtDNA in FF may serve as a sensitive and non-invasive biomarker for GC apoptosis with oocyte metabolic potential. Including cf-mtDNA analysis into infertility diagnostics could improve prognostic assessment and monitor personalized ICSI strategies.