Discussion
Main findings: This longitudinal study of pregnancy in T1DM is the first to report multiple detailed measures of vitamin D (total, bioavailable and free concentrations of 25(OH)D and 1,25(OH)2D; VDBP; relevant ratios), and their associations with subsequent PE. We were surprised to find that at V2 and V3, elevated plasma ‘active’ 1,25(OH)2D, low VDBP, and elevated 1,25(OH)2D:VDBP ratios were associated with subsequent PE.
In contrast, 25(OH)D, the standard metric of vitamin D, did not predict PE; of note, however, insufficient or deficient levels were almost universal in our diabetic cohort. Reported associations of vitamin D deficiency in diabetic pregnancy include preterm birth, increased T1DM rates in offspring of women with T1DM, and poor glycaemic control.11, 16, 19, 20, 23 Some studies of non-diabetic pregnant women have suggested associations between low total 25(OH)D and contemporaneous9 or subsequent PE,8, 11 perhaps limited to early-onset disease.12 Our present finding of no significant association is consistent with our previous study (that used different methodology to measure 25(OH)D),24 and with a study by Vestgaard et al ..16 The present work extends those findings by showing that, like total 25(OH)D, neither bioavailable nor free forms of 25(OH)D were predictive of PE. Again, there is the caveat that our cohort did not contain vitamin D-sufficient women.
Maternal ‘active vitamin D’ (1,25(OH)2D) concentrations are known to increase markedly throughout normal pregnancy compared with normal, non-pregnant values,38 reaching levels that would be toxic in other circumstances. This phenomenon is recognized but not fully understood. Other studies of active vitamin D throughout pregnancy are sparse, and there is none in the context of maternal diabetes. A smaller longitudinal case-control study of PE in non-diabetic pregnancy (10 PE cases vs 40 controls) found no association between serum 1,25(OH)2D in late second and early third trimesters and subsequent PE.7 In contrast, we found that bioavailable and free 1,25(OH)2D both predict PE at the second and early third trimesters, while total 1,25(OH)2D predicted PE at the second trimester. The pregnancy-associated increase in active vitamin D is known to be accompanied by, but disproportionate to, an increase in VDBP, and a change in binding affinity has been suggested.38, 39High 1,25(OH)2D is thought to reflect the calcium needs of the developing foetus, increasing calcium absorption and up-regulating trans-placental transport.39
Lower VDBP concentrations were predictive of PE at the third trimester, and the ratio of 1,25(OH)2D/VDBP was predictive at V2 and V3. A larger study is needed to determine whether this ratio is a better predictor than 1,25(OH)2D alone. VDBP, like many plasma proteins, significantly increased as pregnancy advanced, independent of diabetes or PE status.
Strengths and Limitations: In this study, we measure not only 25(OH)D, but also active 1,25(OH)2D and VDBP in a longitudinal study of T1DM women with and without (late-onset) PE. We include estimates of bioavailable and free concentrations. Our cohort, although small, was rigorously phenotyped, and was free of proteinuria and hypertension at enrolment. Gestational time-points were well-defined, and all study visits were prior to PE onset. A non-diabetic control group provided reference values for normal pregnancy.
Limitations include reliance on affinity constants to estimate free forms of vitamin D: these constants may vary between individuals, and may be altered by pregnancy.38 Our cohort was predominantly Caucasian, and there are ethnic differences in vitamin D metabolism and function, including variation of VDBP allelic forms according to race.40, 41 Sunshine exposure affects total 25(OH)D levels, and vitamin D deficiency may have seasonal and geographical variations. In our subset, 51% of the women were from Norway, 29% from Australia, and 20% from USA. Small numbers precluded stratification by season or location, but most participants, regardless of origin, were vitamin D insufficient or deficient. The absence of vitamin D-sufficient women is a limitation.
Interpretation: Vitamin D is converted from pro-hormone, 25(OH)D, to active hormone by 25(OH)D-1α-hydroxylase, predominantly in the kidney, but also in macrophages and, during pregnancy, in the placenta.42 Higher levels of ‘active vitamin D’ in diabetic women with subsequent PE could reflect early subclinical renal or placental dysfunction. The kidney is the major site of its formation, and furthermore, VDBP is filtered through the glomerulus and is reabsorbed by the proximal tubules.2, 36 We may hypothesise that early renal dysfunction, prior to PE onset, perturbs both 1,25(OH)2D and VDBP.43, 44 Women were excluded from our study if they had microalbuminuria or more severe albuminuria at V1. Nevertheless, other evidence from the MAMPED cohort supports the concept that subtle, early renal abnormalities confer PE risk: specifically increased first trimester urinary neutrophil-gelatinase associated lipocalin (creatinine corrected) (uNGALcc) and elevated ‘estimated glomerular filtration rates’ (eGFR).29 Relating current data to these prior findings, we observed, specifically in DM+PE+ women at first trimester, that total, bioavailable and free 25(OH)D were negatively correlated with eGFR (all p<0.05), while total and free 1,25(OH)2D at V2 were positively correlated with uNGALcc (p<0.05). Regarding VDBP, any renal insult during or even before pregnancy could alter glomerular and tubular processing. Overall, these lines of evidence support the notion that subtle subclinical renal dysfunction, preceding microalbuminuria, is associated with PE.
An alternative possibility is that the association between active vitamin D and subsequent PE is ‘defensive’, a response to early stresses initiating disease. Active vitamin D has protective functions for the fetoplacental unit, inhibiting inflammatory cytokines45 and inducing anti-microbial peptide synthesis.46 In a rat model (reduced utero-placental perfusion, RUPP), treatment of animals with 1,25(OH)2D early in gestation ameliorated PE, apparently by reducing oxidative and ER stresses;47 and in a cross-sectional study in non-diabetic humans, plasma 1,25(OH)2D was lower in those with than without PE,48 perhaps reflecting defeat of protective responses.
Associations between vitamin D and PE may differ between ‘mild, late onset’ PE and ‘early-onset severe’ form of the disease, and Bodnaret al. have suggested that the association between vitamin D deficiency and PE is limited to the latter.12 In the present prospective study, an overwhelming majority (~90%) of PE cases in T1DM women were ‘mild, late onset’. A prospective study of early-onset, severe disease was beyond the scope of MAMPED.
Whether any of the associations we have identified reflect a causal relationship, and how they might be affected by vitamin D supplementation is unknown. Currently, during pregnancy, all women are recommended to take 600 IU vitamin D daily,6 but a recent study shows that 4,000 IU daily is more effective at maintaining sufficiency without toxicity.3 Whether supplementation reduces PE risk for women with diabetes is unknown: a recent multicentre study to assess efficacy in preventing GDM showed a marginal reduction in fasting glucose, but was underpowered to address PE.49 In that study, and in contrast to our T1DM patients, women were largely vitamin D sufficient at study entry.50 Studies of effects of vitamin D on human hypertension have yielded generally negative results.49
Conclusion: This is the first longitudinal, observational study to investigate associations of vitamin D metabolites and VDBP with PE in women with T1DM. In the late second trimester, 1,25(OH)2D and 1,25(OH)2D/VDBP ratio were good predictors of PE. Further studies should address the value of these biomarkers, the significance of differential changes of 25(OH)D and 1,25(OH)2D during pregnancy, mechanistic implications, and whether optimising vitamin D status during pregnancy is effective in reducing the high prevalence of PE in T1DM women.