Document Type

Article

Publication Date

12-2-2025

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This article is the author's final published version in Nutrients, Volume 17, Issue 23, December 2025, Article Number 3783.

The published version is available at https://doi.org/10.3390/nu17233783. Copyright © 2025 The Author(s).

Abstract

Background/Objectives: 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. Methods: 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). Results: 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 (p < 0.0001). Serum 24,25(OH)2D3 averaged 15.62 ± 10.99 nmol/L, compared with 11.26 ± 6.09 nmol/L in FF (p = 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, p < 0.0001) than for 25(OH)D3 (R2 = 0.69, p < 0.0001). After seven months of storage, 25(OH)D3 concentrations decreased significantly (serum −32%; FF −38%; both p < 0.0001), whereas 24,25(OH)2D3 levels remained stable (serum p = 0.24; FF p = 0.36). Conclusions: 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.

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English

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