Integrated network toxicology and multi-omics profiling reveal cell cycle, DNA damage repair, and metabolic alterations in cyclophosphamide-induced premature ovarian insufficiency

Abstract

Cyclophosphamide (CTX)-induced chemotherapy injury is a leading iatrogenic cause of premature ovarian insufficiency (POI) and a major barrier to fertility preservation. Yet the systems-level mechanisms underlying its reproductive toxicity remain poorly defined. An integrated network toxicology framework incorporating machine learning and Mendelian randomization (MR) was applied to identify candidate targets, evaluate genetic associations, and establish a predictive model. Ovarian transcriptomic and metabolomic profiling, together with in vivo validation, were subsequently performed in a CTX-induced POI rat model. Single-cell-based in silico perturbation analysis was further conducted to investigate the potential cellular functions and mechanisms of key candidate targets in POI. Integrated bioinformatic analyses and machine learning prioritized six candidate targets associated with CTX-induced ovarian injury, with PLS regression consistently identified as the optimal predictive model. SHAP analysis revealed CXCL1 and COL4A4 as the major contributors to model prediction. MR supported genetic associations between genetically predicted CXCL1/COL4A4 expression and POI risk. Immunohistochemical validation confirmed protein expression patterns consistent with transcriptomic findings. Multi-omics profiling revealed a convergent ovarian injury signature characterized by impaired cell-cycle regulation (G2/M checkpoint), defective DNA damage repair and mitotic function (mitotic spindle), enhanced apoptotic signaling, and coordinated suppression of folate metabolism, ubiquinone biosynthesis, and cytochrome P450-mediated drug metabolism pathways. Single-cell-based in silico perturbation analysis further suggested that CXCL1 and COL4A4 may contribute to ovarian dysfunction through regulation of inflammatory responses and extracellular matrix remodeling. This study shows that CTX-induced ovarian injury is accompanied by coordinated alterations in immune-inflammatory signaling, cell-cycle and DNA damage responses, and metabolic homeostasis. CXCL1 and COL4A4 were prioritized as candidate biomarkers associated with POI risk, providing potential targets for further mechanistic investigation and fertility-preserving interventions.