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.