Total 25-hydroxyvitamin D was measured in 101,104 nonpregnant and 15,938 pregnant women, total 1,25-dihydroxyvitamin D in 5,952 nonpregnant and 183 pregnant women, and free 25-hydroxyvitamin D in 304 nonpregnant and 60 pregnant women. No participant had valid results for all three metabolites simultaneously, so the study could not determine whether individual women experienced coordinated changes across the full panel. Maternal age was available consistently enough to serve as the prespecified adjustment variable, but information on supplementation, diet, season, body mass index, ethnicity, kidney and liver function, comorbidities and pregnancy complications was unavailable or incomplete.

After adjustment for age, pregnancy was associated with a total 25-hydroxyvitamin D concentration 22.07 nmol/L higher than among nonpregnant women, with a 95% confidence interval of 21.50–22.63 nmol/L and P<0.001. The corresponding association for total 1,25-dihydroxyvitamin D was considerably larger: pregnant women had concentrations estimated to be 125.20 pmol/L higher, with a 95% confidence interval of 115.39–135.02 pmol/L and P<0.001. Directly measured free 25-hydroxyvitamin D showed no statistically significant difference, with an adjusted difference of −2.63 pmol/L, a 95% confidence interval of −8.99 to 3.74 pmol/L and P=0.417.

Log-transformed analyses produced the same overall pattern, and repeated equal-size subsampling did not materially change the estimates. The cross-sectional gestational patterns also differed among the metabolites. Median total 25-hydroxyvitamin D was 57.50 nmol/L in nonpregnant women, 67.50 nmol/L in the first trimester and 90.00 nmol/L in the third trimester.

Compared with nonpregnant women, the age-adjusted difference was 10.60 nmol/L in the first trimester, 30.08 nmol/L in the second and 37.40 nmol/L in the third. A nonlinear model estimated lower concentrations after the earliest gestational weeks, higher values later and an apparent plateau near term. Total 1,25-dihydroxyvitamin D was already higher in the first trimester than in nonpregnant women, with an age-adjusted difference of 67.37 pmol/L and a 95% confidence interval of 48.69–86.04 pmol/L. The estimated differences increased to 137.99 pmol/L in the second trimester and 167.42 pmol/L in the third, while the median concentration in the third trimester was 279.60 pmol/L, compared with 126.72 pmol/L among nonpregnant women.

Free 25-hydroxyvitamin D did not show a statistically significant difference in any trimester, but the relevant groups were very small: 34 pregnant participants in the first trimester, 15 in the second and only 11 in the third. The nonsignificant results therefore cannot establish that free concentrations were equivalent between pregnant and nonpregnant women or remained stable across pregnancy. Among participants with results for both total metabolites, the molar ratio of total 25-hydroxyvitamin D to total 1,25-dihydroxyvitamin D was lower during pregnancy: the median ratio was 289.80, with an interquartile range of 210.9–394.5, compared with 392.9, with an interquartile range of 260.4–575.6, among nonpregnant women.

The age-adjusted ratio was 0.754, with a 95% confidence interval of 0.684–0.831, and remained below the nonpregnant reference in all three trimesters. The researchers described this as an indirect metabolic index rather than a direct measure of CYP27B1 activity, because circulating concentrations are also influenced by substrate availability, protein binding, clearance, and renal and extrarenal metabolism. A complementary systematic review and meta-analysis broadly supported the elevation in total 1,25-dihydroxyvitamin D. Nine studies, including the present cohort, contributed to the primary pooled comparison and yielded a ratio of means of 1.88 for pregnant versus nonpregnant women, with a 95% confidence interval of 1.67–2.12.

Excluding the current cohort left the estimate nearly unchanged at 1.87, with a 95% confidence interval of 1.62–2.16 across eight studies. Heterogeneity was substantial, with I²=76.63%, which the investigators linked to differences in assay platforms, gestational timing, supplementation, renal function and population characteristics. The result establishes a consistent association in the pooled observational evidence, not a causal effect of pregnancy.

Evidence for free 25-hydroxyvitamin D was less certain. A random-effects analysis of three studies suggested modestly lower concentrations during pregnancy, with a pooled ratio of means of 0.78 and a 95% confidence interval of 0.63–0.97. After the present cohort was removed, only two independent studies remained; their ratio was 0.74, but the 95% confidence interval widened to 0.41–1.33 and crossed 1.

Those two studies used calculated rather than directly measured free 25-hydroxyvitamin D, and calculated and directly measured concentrations are not analytically interchangeable. The laboratory cohort itself contained only 60 pregnant women with directly measured free values, making this comparison exploratory and less precise than the findings for total metabolites. Several limitations constrain the conclusions.

The samples came from an indication-driven clinical laboratory rather than a population-based cohort, and the clinical reasons for testing were not recorded in a standardized way. Selective ordering of the specialized assays created markedly different and potentially unrepresentative subgroups, particularly for free 25-hydroxyvitamin D. The cross-sectional design also prevents conclusions about within-person changes: later gestational stages were represented by different women, not repeated measurements in the same participants. The study also lacked vitamin D-binding protein and albumin measurements, which prevented direct evaluation of how binding-protein changes contributed to the relationship between total and free 25-hydroxyvitamin D. Clinical outcome data were unavailable, so the analysis could not determine whether any metabolite predicted maternal, placental, fetal or neonatal outcomes.

For laboratory interpretation, the results support paying close attention to the specific metabolite, assay and gestational stage. Because total 1,25-dihydroxyvitamin D is substantially higher during pregnancy, a value that appears elevated against a nonpregnant reference interval may be physiologically expected, while a value within a conventional nonpregnant interval may be lower than expected for a particular stage. Higher total 25-hydroxyvitamin D cannot automatically be interpreted as a proportional increase in free 25-hydroxyvitamin D, and circulating concentrations do not by themselves establish greater tissue activity.

The authors emphasized that any pregnancy-specific reference intervals would need assay-specific validation and prospective links to meaningful outcomes. Current guidance described in the study supports empiric supplementation during pregnancy and advises against routine biochemical testing in generally healthy pregnant people without a clinical indication; the findings do not establish new testing thresholds or clinical recommendations.

Subject of Research: Pregnancy-associated concentrations of circulating total and free vitamin D metabolites

Article Title: Pregnancy-associated alterations in circulating vitamin D metabolites: higher total 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D without increased free 25-hydroxyvitamin D

Article References: Zhang, H.-F., Chen, X., Xiang, J.-Y., Zhang, Z.-Y., Liu, Y., Huesker, K., von Baehr, V., Elitok, S., Krämer, B. K., Reichetzeder, C., & Hocher, B. (2026). Pregnancy-associated alterations in circulating vitamin D metabolites: higher total 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D without increased free 25-hydroxyvitamin D. Frontiers in Endocrinology, 17, Article 1957418. https://doi.org/10.3389/fendo.2026.1957418

Image Credits: AI Generated

DOI: 10.3389/fendo.2026.1957418

Keywords: pregnancy, vitamin D metabolism, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, free vitamin D, vitamin D-binding protein, cross-sectional study, meta-analysis, laboratory medicine, gestation, Pregnancy-associated, alterations