Cyclophosphamide as a reproductive hazard: disrupted riboflavin metabolism and circadian rhythms drive ovarian decline

Cyclophosphamide (CTX), a widely used chemotherapeutic agent, is notorious for its severe gonadal toxicity, leading to premature ovarian insufficiency and infertility. However, the underlying molecular pathways, particularly those beyond DNA damage, remain incompletely understood. In this study, we investigated the effects of multiple exposures to CTX on ovarian injury in rats and their potential molecular mechanisms. We first evaluated the expression of circadian rhythm genes in the ovaries of rats exposed to CTX, and found that the expression of circadian rhythm genes was abnormal in the ovaries exposed to CTX. We also found through proteomic analysis that the expression of riboflavin kinase (RFK) in the ovaries of CTX exposed rats significantly decreased, accompanied by an increase in degradation of clock proteins cryptochrome 1 and 2 (CRY1/2). The deficiency of RFK can cause riboflavin metabolism defects, thereby inhibiting the synthesis of flavin adenine dinucleotide (FAD). FAD competes with CRY protein for binding to ubiquitin E3 ligase, reducing CRY degradation and maintaining the stability of circadian rhythm. Further molecular experiments showed that knocking down RFK not only led to a decrease in FAD synthase levels, but also exacerbated the degradation of CRY1/2, disrupted circadian rhythm regulation, and affected the function of ovarian granulosa cells, accelerating ovarian aging. Crucially, supplementation with FAD mitigated these defects. All in all, these research findings suggest that CTX can impair ovarian function by causing dysregulation of riboflavin metabolism, leading to abnormal circadian rhythms.