## 6 Critical Appraisal and Considerations for Future Trials
This contributes to a high risk of contamination among control participants, should they take supplements outside the trial protocol [86]. Contamination can in turn influence 25(OH)D levels and, as described above, alterations in 25(OH)D levels can lead to a number of problems with analysing and interpreting RCT results. It can be argued that self-purchased vitamin D is likely to be similar in both intervention and control arms of a double-blind trial, thereby cancelling out any differences in effects between groups. In reality however, this is not the case since taking supplements will reduce the proportion of vitamin D-deficient participants in the control arm (without really impacting the intervention arm) and hence reduce the chances of detecting a beneficial effect, particularly if the association between vitamin D status and disease risk is non-linear as has been demonstrated [86]. Participants can also have their vitamin D levels tested relatively easily, which is especially problematic among pregnant women who often have their vitamin D levels checked as part of routine antenatal care. This can risk unblinding and further exacerbate contamination should they decide to take supplements or make lifestyle changes such as increasing their sun exposure or intake of vitamin D-fortified foods to self-treat their deficiency. These limitations can reduce the internal validity of RCTs and influence their findings, and may in part explain some of the discrepancies demonstrated in the literature. To counter these issues, trials need to incorporate close monitoring of 25(OH)D status, with frequent sampling and testing over the course of a trial to assess potential protocol violations, but this comes with substantial costs and time commitments. Of note, none of the included trials measured vitamin D status at different time points across the course of pregnancy, hence the above problems could have been encountered and remained undetected. Fourth, the clinical trials reviewed herein demonstrate substantial heterogeneity and variations in methodological rigour that should be considered in the interpretation of their findings. For instance, there are considerable differences in study designs, populations and settings, including varying cut-off points used to define vitamin D sufficiency and/or deficiency, different diagnostic criteria for GDM (ADA, IADPSG, etc. ), different assays used to determine 25(OH)D levels (with only two studies using the gold-standard LC/MS), and different geographies and population groups studied, with the majority of trials (n = 11/18) being conducted in Iran, limiting generalisability (Table 1). Participants also had different risk profiles at baseline, with some having GDM or being classified as high-risk and others being relatively healthy, hence, direct comparisons become challenging. Some studies [78; 84] noted significant differences in baseline glycaemic parameters between intervention and control groups (e.g., the placebo group was less insulin-resistant at baseline), making their results difficult to interpret. It is also important to consider the selection of factors to be controlled in an experimental setting, since some aspects such as body mass index, ethnicity, and increased maternal age can increase the risk of both GDM and vitamin D deficiency [93]. Other factors strictly related to vitamin D, such as endogenous biosynthesis (sun exposure) and amounts of dietary intake, should be taken into account when interpreting results from vitamin D trials. Yet, lifestyle factors such as these have seldom been incorporated in the existing trials and may explain some of the variations observed in the evidence. Finally, there remain uncertainties regarding the functions of vitamin D at different stages of pregnancy, how these functions may be related to different clinical outcomes, and the appropriate 'critical windows' for measuring and treating deficiency or insufficiency in line with relevant pregnancy outcomes.