## 6 Critical Appraisal and Considerations for Future Trials
For instance, gestational age is a crucial consideration for intervention studies since repletion needs to be restored prior to observing changes in the outcome of interest. Although blood 25(OH)D levels may promptly respond to supplementation (assuming sufficient doses), peripheral cellular deficiency may require more time. Intervention studies designed during late pregnancy may therefore not have sufficient time to restore the physiological conditions required for the prevention of dysfunction, or indeed, GDM [94]. The short durations of many of the existing trials (i.e., weeks or months) may also be ineffective in altering the pathogenic pathways of disorders such as GDM, which is thought to have initial manifestations from the very early stages of pregnancy [94]. This may explain some of the null or inconsistent findings from existing studies since, of the 18 included trials, only three [40; 79; 83] started supplementation in the first trimester (i.e., from ~12 weeks), while the majority (n = 12/18 RCTs) commenced at or after 20 weeks gestation, with many lasting less than three months. When considering the above limitations, coupled with small sample sizes, lack of statistical power, open-label designs, and the use of low dose vitamin D supplements in the control groups (which is largely unavoidable in pregnancy), the equivocal findings presented in this review are not surprising. This is further compounded by a lack of defined knowledge around the appropriate doses or optimal concentrations of vitamin D, and the duration of supplementation necessary to appreciate any benefits, particularly during pregnancy when the vitamin D system undergoes substantial adaptations to support increased maternal and fetal requirements. Co-supplementation was also often used, making it impossible to isolate the effects of vitamin D alone. This is particularly important when considering co-supplementation with magnesium since magnesium is an integral cofactor in the enzymatic conversion of vitamin D to 25(OH)D and 1,25(OH)D, and is present in most pregnancy multivitamins [95]. Studies also varied in their supplementation regimens, ranging between daily, weekly, biweekly, monthly or intermittent supplementation of varying doses. As mentioned earlier, these regimens may influence the measured outcomes since bolus doses can be absorbed rapidly and become undetectable, while a steady state can be achieved by supplying constant doses of vitamin D for 3-4 months [96]. In addition, as outlined in this review, there are many uncertainties regarding whether vitamin D is only effective in certain subgroups or whether a certain threshold exists at which vitamin D ceases to incur additional benefits. It is possible that vitamin D may have differential effects in women of different ethnicities, age groups or baseline risk profiles, but this is yet to be fully delineated. Finally, there is a need to ascertain the long-term effects of vitamin D supplementation during pregnancy to determine whether improvements in glycaemic control following supplementation may translate into a reduced risk of type 2 diabetes or recurrent GDM in subsequent pregnancies. Taken together, while the existing research indicates potential benefits of vitamin D for glycaemic control in pregnancy but not for GDM prevention per se, more conclusive evidence is needed to confirm these findings and to determine the mechanistic pathways underlying the protective features of vitamin D, if any.