Metabolomic analysis of follicular fluid in women with unexplained infertility

Unexplained infertility (UI) affects ∼10% of infertile couples, yet standard diagnostic protocols fail to identify a cause. Follicular fluid (FF), which supports oocyte development, contains metabolites that may reflect underlying molecular disturbances. In this exploratory study, we investigated the FF metabolome of women with UI and compared it with controls to explore metabolic alterations associated with UI. FF was collected during oocyte retrieval from 20 women undergoing IVF (ten with UI, ten with male factor infertility), matched for age, BMI, stimulation, and fertilization protocols. Metabolomic profiling was performed using hydrophilic interaction and reversed-phase liquid chromatography coupled to Q-TOF-MS/MS, followed by metabolite identification (XCMS Online and MetaboAnalyst) and KEGG pathway analysis. Approximately 2000 features were detected. Differential metabolites were identified by OPLS-DA (VIP > 2) and validated using univariate metrics such as fold change (|log₂FC| > 1), statistical significance (p < 0.05), and ROC analysis (AUC > 0.8). Twelve metabolites, including diacylglycerols, phosphatidic acids, vitamin D3 derivatives (VitD3-glucosiduronate, 1α-hydroxy-2β-(5-hydroxypentoxy)-VitD3), asparaginyl-asparagine, 3α-hydroxy-6-oxo-5β-cholan-24-oic acid, Leu-Pro-Ala-Ser-Phe, triacylglycerols, phosphatidylcholine, and lactosyl-ceramide were significantly decreased, while Ile-Lys-Val-Val was increased in women with UI. Pathway analysis highlighted disruptions in glycerophospholipid, glycerolipid, steroid, and linoleic acid metabolism. Consistent with the untargeted findings, targeted analysis demonstrated significantly reduced levels of follicular 25-hydroxyvitamin D [25(OH)D] in women with UI despite uniform oral supplementation, indicating dysregulated follicular vitamin D availability. Whilst the study was limited by sample size, the metabolome analysis was performed in triplicate for each sample, thus providing preliminary insights into the metabolic disruptions in FF from women with UI.