Reducing Storage-Related Bias in Reproductive Vitamin D Research: Towards a Stable and Reliable Biomarker of Ovarian Vitamin D Status

<b>Background/Objectives:</b> One of the main reasons for discrepancies in the role of vitamin D in ART could be the measurement of the conventional biomarker 25(OH)D₃. It is known that this value is affected by multiple factors, such as tissue origin, assay variability, classification criteria, and potential storage-related degradation. In this study, we investigate 24,25(OH)₂D₃ as a new biomarker to improve vitamin D assessment in women's reproductive health, particularly regarding oocyte development. <b>Methods:</b> A prospective cohort study including 35 oocyte donors undergoing controlled ovarian stimulation, who were recruited between October and November 2017, was conducted. Vitamin D metabolites were measured at the baseline and after seven months of storage at -80 °C. Paired serum and pooled follicular fluid (FF) samples were collected at oocyte retrieval. 25(OH)D₃ and 24,25(OH)₂D₃ were quantified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Statistical analyses included paired tests (serum vs. FF; baseline vs. stored) and Pearson's correlations (two-sided α = 0.05). <b>Results:</b> At the baseline, the mean serum 25(OH)D₃ concentration was 91.56 ± 39.01 nmol/L and the mean FF concentration was 58.13 ± 19.55 nmol/L (<i>p</i> < 0.0001). Serum 24,25(OH)₂D₃ averaged 15.62 ± 10.99 nmol/L, compared with 11.26 ± 6.09 nmol/L in FF (<i>p</i> = 0.004). In both fluids, 25(OH)D₃ and 24,25(OH)₂D₃ were strongly correlated (serum R² = 0.92; FF R² = 0.91). Across fluids, the serum-FF correlation was stronger for 24,25(OH)₂D₃ (R² = 0.77, <i>p</i> <0.0001) than for 25(OH)D₃ (R² = 0.69, <i>p</i> < 0.0001). After seven months of storage, 25(OH)D₃ concentrations decreased significantly (serum -32%; FF -38%; both <i>p</i> < 0.0001), whereas 24,25(OH)₂D₃ levels remained stable (serum <i>p</i> = 0.24; FF <i>p</i> = 0.36). <b>Conclusions:</b> Serum 24,25(OH)₂D₃ is a more reliable and minimally invasive biomarker for assessing ovarian vitamin D status than the current gold standard, 25(OH)D₃. Incorporating this metabolite into research studies and storage quality control may improve the reliability of retrospective analyses based on cryopreserved material, contributing to a better understanding of the role of vitamin D in human reproduction.