Integrative serum metabolite prioritization and functional screening identify N-acetyl-L-glutamine as a protective candidate in premature ovarian insufficiency.

Background: Premature ovarian insufficiency (POI) is a major cause of female infertility and is increasingly associated with systemic metabolic dysregulation. However, whether circulating metabolic alterations contribute causally to POI development or primarily arise as secondary consequences of ovarian failure remains unclear. In this study, bidirectional Mendelian randomization (MR), cell-based screening, and exploratory target-prioritization analyses were integrated to identify POI-related metabolites and functionally relevant candidates. Methods: Two-sample MR was performed using genome-wide association studies (GWAS) summary data for 1,400 serum metabolites/metabolite ratios and POI. After instrumental variable filtering and harmonization, 1,352 exposures with valid inverse-variance weighted (IVW) estimates were retained for forward MR and multiple-testing correction. Both Benjamini-Hochberg false discovery rate (FDR) and Bonferroni correction were applied. Reverse MR was then conducted as a secondary directionality analysis to assess whether genetic liability to POI was also associated with circulating metabolic alterations. Experimentally tractable metabolites were screened in cyclophosphamide (CTX)-injured KGN cells using CCK-8 assays and Western blotting. For the prioritized metabolite, further functional validation was performed using SA-β-gal staining, ROS detection, and JC-1 assays. Proteome-wide MR, colocalization analysis, summary-data-based MR (SMR), drug prediction, and molecular docking were subsequently conducted as exploratory downstream analyses. Results: Among the 1,352 analysable exposures, 54 showed nominal associations with POI at Conclusion: This study identifies N-acetyl-L-glutamine as a biologically plausible and experimentally supported protective metabolite candidate that attenuates CTX-induced senescence, oxidative stress, and mitochondrial dysfunction in granulosa-like cells. By integrating metabolome-wide MR with bidirectional analyses, our findings support a metabolite-centred framework for investigating POI-related metabolic vulnerability and oncofertility-related ovarian injury. Sphinganine-1-phosphate emerged as a multiple-testing-corrected risk-associated metabolite, whereas LILRB1 and cianidanol generated exploratory hypotheses for future mechanistic and pharmacological studies.