Conclusions Vitamin D supplementation alone was not associated with all cause mortality in adults compared with placebo or no treatment. Vitamin D supplementation reduced the risk of cancer death by 15%. Additional large clinical studies are needed to determine whether vitamin D 3 supplementation is associated with lower all cause mortality.

Results 50 trials with a total of 74 655 participants were identified. Vitamin D supplementation was not associated with all cause mortality (risk ratio 0.98, 95% confidence interval 0.95 to 1.02, I 2 =0%), cardiovascular mortality (0.98, 0.88 to 1.08, 0%), or non-cancer, non-cardiovascular mortality (1.05, 0.93 to 1.18, 0%). Vitamin D supplementation statistically significantly reduced the risk of cancer death (0.85, 0.74 to 0.97, 0%). In subgroup analyses, all cause mortality was significantly lower in trials with vitamin D 3 supplementation than in trials with vitamin D 2 supplementation (P for interaction=0.04); neither vitamin D 3 nor vitamin D 2 was associated with a statistically significant reduction in all cause mortality.

Eligibility criteria for selecting studies Randomised controlled trials comparing vitamin D supplementation with a placebo or no treatment for mortality were included. Independent data extraction was conducted and study quality assessed. A meta-analysis was carried out by using fixed effects and random effects models to calculate risk ratio of death in the group receiving vitamin D supplementation and the control group.

Recently, additional trials 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 assessing the effect of vitamin D supplementation on mortality have become available, which have approximately doubled the number of trial participants. Among these trials, the Vitamin D and Omega 3 Trial (VITAL) did not confirm the benefit of vitamin D supplementation on mortality. 31 Because of the conflicting evidence, limitations of previous reviews, and availability of new data, we aimed to conduct a systematic review and meta-analysis of randomised controlled trials to evaluate the effect of vitamin D supplementation on all cause mortality.

Clinical data examining the effect of vitamin D supplementation on mortality reduction are inconsistent. Observational studies have revealed an inverse association of vitamin D status and mortality. 6 7 8 9 Previous systemic reviews and meta-analyses of randomised controlled trials suggested that vitamin D supplementation has a small effect on total mortality. 5 10 11 Interpretation of these reviews is difficult because they include trials of vitamin D administered with calcium, which has been associated with uncommon but important side effects (eg, cardiovascular events). 12 13 14 15 Additionally, these reviews lack sufficient detail (eg, community versus institution settings), and trial sequential analysis showed that the pooled sample size failed to meet the optimum size. 10 11

Vitamin D supplementation has been advocated for maintaining or even improving musculoskeletal health. Evidence from observational studies indicates that low vitamin D status is associated with higher mortality from life threatening conditions such as cancer and cardiovascular disease. 1 2 Therefore, supplemental vitamin D has been viewed as a potential strategy for preventing non-skeletal chronic diseases. 3 4 5 If adequate vitamin D concentrations were to reduce risk of death from a wide variety of medical conditions, vitamin D supplementation would be a safe, economical, and widely available method to reduce mortality.

No patients were involved in setting the research question or the outcome measures, nor were they involved in developing plans for design or implementation of the study. No patients were asked to advise on interpretation or writing of results. The results will be disseminated to a wide audience, including members of the public, patients, health professionals, and experts in the specialty through social media and networks.

We conducted sensitivity analyses by excluding trials with high or unknown risk of bias; excluding trials with high risk or unknown risk of bias of the different domains; excluding quasi randomised or cluster randomised trials; excluding the largest trial; excluding trials with a follow-up of less than one year; using random effect models; adding trials that had been excluded for using vitamin D administered with calcium; adding trials that had been excluded for using hydroxylated vitamin D or vitamin D analogues; and using trial duration rather than long term follow-up.

We performed several subgroup analyses to test interactions according to dose (≥2000 and <2000 IU/day); type of vitamin D (vitamin D 2 and vitamin D 3 ); timing of treatment (daily and intermittently); baseline 25 hydroxyvitamin D (≥50 and <50 nmol/L); and mean age (≥70 and <70 years). We conducted retrospective subgroup analyses based on length of follow-up (at least three years and less than three years); year of publication (before 2014 and in or after 2014); sex (female and both sexes); residential status (community and institution); bolus (yes and no); intervention (vitamin D and calcium with vitamin D); and latitude (≥40° and <40°).

We performed trial sequential analysis to explore whether cumulative data were adequately powered to evaluate outcomes. Trial sequential analysis (version 0.9.5.10) 39 was used to maintain an overall 5% risk of type I error and 80% power. We initially anticipated an intervention effect of a 10% relative risk reduction for all cause mortality. In additional analyses, we used progressively smaller thresholds (7.5% and 5%) until the optimum sample size exceeded the actual sample size.

We performed statistical analyses using RevMan (version 5.3.3; The Cochrane Collaboration) and the meta package in R (version 3.4.3; R Project for Statistical Computing). Analyses for all outcomes were conducted on an intention to treat basis. We used risk ratios and their associated 95% confidence intervals to assess outcomes, and considered a P value less than 0.05 to be statistically significant. We assessed heterogeneity using the I 2 test. 37 If significant heterogeneity was not present (I 2 <50%), we used fixed effects models to pool outcomes; we used random effects models when significant heterogeneity was present (I 2 ≥50%). The possibility of small study effects was assessed qualitatively by visual estimate of the funnel plot and quantitatively by calculation of the Egger test, the Begg test, and the Harbord test. 38

Two researchers (YZ and LJ) independently assessed the quality of all included trials by using the Cochrane Collaboration risk of bias tool. 35 They also examined the quality of evidence for outcomes using the grading of recommendations assessment, development, and evaluation (GRADE) approach. 36

Two independent researchers (YZ and LJ) used a standard data extraction form to extract data from the included trials. When randomised controlled trials had more than two arms, we pooled data from the separate treatment arms. When a study mentioned an outcome of interest without providing estimates, we contacted the author for the data. Disagreements were resolved by consensus.

After removal of duplicates, two independent researchers (YZ and LJ) screened all titles and abstracts. They obtained full texts and performed further screening when studies were deemed eligible. Disagreements were resolved by consensus.

One of the authors (PX) conducted the search of several databases: Medline (Ovid), Embase (Ovid), the Cochrane Central Register of Controlled Trials (CENTRAL), from inception to 26 December 2018. We also searched ClinicalTrials.gov and the World Health Organization International Clinical Trials Registry Platform to identify ongoing or unpublished eligible trials. To maximise the search for relevant articles, we reviewed reference lists of identified trials and systematic reviews. We did not apply language restrictions. Supplemental eTable 2 presents the search strategy.

We excluded studies if they were case reports, case series, or observational studies; if all the participants received vitamin D; if they included pregnant or lactating women, or critically ill patients; if they used hydroxylated vitamin D or vitamin D analogues (which could differ from native vitamin D in effect and safety, including lower risk of fall 34 and higher risk of hypercalcaemia 10 34 ).

We considered trials to be eligible if they enrolled adults (age ≥18) with any health condition; if they compared vitamin D supplements at any dose with placebo or no treatment (when other agents were also given (eg, calcium), they had to be the same dosage in all groups); if they provided information on deaths from all causes (non-accidental) or any cause reported separately; and if they were randomised controlled trials (including quasi randomised and cluster randomised trials).

Vitamin D supplementation was associated with significant reduction in cancer mortality (risk ratio 0.85, 95% confidence interval 0.74 to 0.97, I 2 =0%; fig 3 ). However, benefit was only seen in participants receiving vitamin D 3 supplementation, and no participants received vitamin D 2 supplementation (supplemental eTable 8). We found no statistically significant difference between groups in cardiovascular mortality (0.98, 0.88 to 1.08, I 2 =0%) or non-cancer, non-cardiovascular mortality (1.05, 0.93 to 1.18, I 2 =0%). Vitamin D supplementation did not reduce the risk of death from cerebrovascular disease (1.04, 0.84 to 1.29, I 2 =0%; supplemental eTable7) or ischaemic heart disease (0.96, 0.81 to 1.15, I 2 =0%; supplemental eTable 8).

Subgroup analyses found that all cause mortality was significantly lower among trials with vitamin D 3 supplementation than in trials with vitamin D 2 supplementation (P for interaction=0.04; table 2 ), although neither group was associated with all cause mortality. Meta-regressions found that all cause mortality was significantly lower in trials with longer follow-up (P for interaction=0.04; supplemental eFigures 9 and 10).

All 50 trials reported all cause mortality. There was no statistically significant difference in all cause mortality between the vitamin D supplementation group and the control group (risk ratio 0.98, 95% confidence interval 0.95 to 1.02, I 2 =0%; fig 2 ). In trial sequential analysis, the information size of all cause mortality met the required size of 10% and 7.5% relative risk reduction; however, futility was not reached in our additional trial sequential analysis with 5% relative risk reduction (supplemental eFigures 3-5). Funnel plot analysis showed no asymmetry (supplemental eFigure 6); additionally the Egger test (P=0.50), Begg test (P=0.20), and Harbord test (P=0.36) detected no significant small study effects. The meta-analysis results for all cause mortality were robust in sensitivity analyses (supplemental eTable 7).

Supplemental eFigures 1 and 2 show risk of bias. Twenty trials had a low risk of bias, 18 trials had an unclear risk, and 12 trials had a high risk of bias. Using the GRADE summary of evidence, the quality of evidence for the primary outcome was high (supplemental eTable 6).

Discussion

In this meta-analysis of 50 randomised controlled trials with a total of 74 655 participants, vitamin D supplementation was not significantly associated with total mortality (risk ratio 0.98, 95% confidence interval 0.95 to 1.02). The findings suggest that vitamin D supplementation reduced cancer mortality by 15% (95% confidence interval 0.74 to 0.97), but not mortality from cardiovascular disease, cerebrovascular disease, or ischaemic heart disease.

Principal findings and comparison with other studies The results of this study on all cause mortality differ from two previous systematic reviews.51011 A Cochrane review in 2014 found that vitamin D supplementation decreased all cause mortality in analyses of 56 trials with a total of 95 286 participants (relative risk 0.97, 95% confidence interval 0.94 to 0.99, P=0.02).10 In the same year, a systematic review by Bolland and colleagues that included 40 trials with a total of 81 173 participants also suggested a small effect on all cause mortality (0.96, 0.93 to 1.00, P=0.04).11 The previous reviews probably reached more optimistic conclusions as a result of different selection criteria and newly published trials. Compared with these reviews,1011 we excluded more than 10 trials totalling approximately 50 000 participants of vitamin D administered with calcium, six trials73747576777879 of hydroxylated vitamin D or vitamin D analogues, and one trial80 retracted in 2017. To determine whether the null finding was driven by excluding trials which had been included in previous reviews, we performed two sensitivity analyses by adding trials that were originally excluded, and confirmed the results of the overall analysis. Moreover, this study additionally included 17 randomised controlled trials1617181920212223242526272829303132 published after 2014, so that the more recent trials accounted for 50.3% (37 568/74 655) of the total number of participants. In contrast to the results for total mortality, this study found that vitamin D supplementation reduced cancer mortality by 15%. The results of previous reviews on cancer mortality have been inconsistent. In 2014, a Cochrane review by Bjelakovic and colleagues presented low quality evidence that vitamin D supplementation resulted in a decrease in cancer mortality (relative risk 0.88, 95% confidence interval 0.78 to 0.98), but suggested that the required information size was not reached.81 In parallel, two systematic reviews published similar results.1182 However, their meta-analyses were limited by the number of trials (n≤4), administration of a generally low dose of vitamin D (≤1100 IU/day), and mixed interventions (vitamin D plus calcium). In 2018, a meta-analysis by Goulão and colleagues did not find evidence to suggest that vitamin D supplementation alone reduced cancer mortality (1.03, 0.91 to 1.15).83 After we submitted our current study for initial review by TheBMJ, an additional meta-analysis by Keum and colleagues was published.84 Their review found that vitamin D supplementation significantly reduced cancer mortality (0.87, 0.79 to 0.96).84 Our findings on cancer mortality are consistent with those of Keum and colleagues, but some of the methods used in the two studies differ. The study by Keum and colleagues included trials of hydroxylated vitamin D, vitamin D analogues, and vitamin D administered with calcium, which were excluded in our study. Moreover, our study provided absolute and relative risks, evaluated the quality of the evidence by using the GRADE approach, and explored the optimum sample size with trial sequential analysis. More importantly, our study found that reduced cancer mortality was only seen with vitamin D 3 supplementation, not with vitamin D 2 supplementation. An important finding from our subgroup analysis was that the effect of vitamin D differs for vitamin D 2 and D 3 supplementation. We found that all cause mortality was significantly lower among trials with vitamin D 3 supplementation than in trials with vitamin D 2 supplementation; however neither supplement was associated with statistically significant reduced risk. Similarly, vitamin D 3 supplementation reduced the risk of cancer mortality, but vitamin D 2 did not. The different effect on mortality of vitamin D 2 and D 3 might be explained by the diverse effect on raising 25 hydroxyvitamin D concentrations. Historically, vitamin D 2 and vitamin D 3 were considered to be equally effective at raising 25 hydroxyvitamin D concentrations. Currently, the comparative efficacy of vitamins D 2 and D 3 has been investigated in several intervention trials, with most indicating that vitamin D 3 increases 25 hydroxyvitamin D concentrations more efficiently than vitamin D 2 .8586 A Cochrane review in 2014 found that vitamin D 3 seemed to reduce total mortality (risk ratio 0.94, 95% confidence interval 0.91 to 0.98), whereas vitamin D 2 had no statistically significant beneficial effects on total mortality (1.02, 0.96 to 1.08).10 However, the Cochrane review did not reveal heterogeneity between vitamin D 2 and D 3 . Therefore, we should be cautious about the strength of the evidence that vitamin D 3 reduced all cause mortality (0.95, 0.91 to1.00, P=0.07). Vitamin D 3 is the most widely used type of vitamin D supplementation and has a clinically relevant effect of reducing all cause mortality by 5%, with the P value and 95% confidence interval close to the level of formal statistical significance. The current study is not a positive study, but it is also not an unambiguously negative study. In addition, subgroup analyses are observational by nature and are not based on randomised comparisons.87 Therefore, the effect of vitamin D 3 on all cause mortality requires additional evidence, preferably gathered by future large randomised controlled trials. A further important finding from meta-regression was that all cause mortality was statistically significantly lower in trials with longer follow-up. Sensitivity analysis found a potential effect of vitamin D supplementation on all cause mortality after trials with a follow-up of less than one year were excluded (risk ratio 0.97, 95% confidence interval 0.93 to 1.00). However, subgroup analysis did not find a statistically significant difference in the effect of vitamin D supplementation on mortality in trials with a follow-up of less than three years and more than three years (P=0.26). Additionally, the previous meta-analysis did not find a subgroup difference according to the length of follow-up.1011 The VITAL trial reported increasing benefit over time.31 Although no significant differences relate to cancer mortality (risk ratio 0.83, 95% confidence interval 0.67 to 1.02) or all cause mortality (0.99, 0.87 to 1.12), after excluding the first one and two years of follow-up, the risk ratio was significantly reduced to 0.75 for cancer mortality (95% confidence interval 0.59 to 0.96) and was slightly reduced to 0.96 for all cause mortality (0.84 to 1.11). Therefore, the length of follow-up could modify the effect of vitamin D supplementation on all cause mortality.

Strengths and limitations This systematic review and meta-analysis has several methodological strengths. We followed the recommendations of the Cochrane Collaboration and PRISMA statement, including a priori protocol. This study also included a rigorous assessment of the quality of evidence using the GRADE approach (the quality for the primary outcome was high) and of the minimum information size required in trial sequential analysis (the study met the optimum size). Our study has important limitations. The study was based solely on published trials that reported mortality outcomes. However, most trials of vitamin D supplementation did not report mortality, which suggests that substantial selective reporting was likely. Also, all cause mortality reported among all included trials was the secondary outcome of the trials. Data for this secondary outcome might have been collected differently than data for the primary outcome in the trials. Most included trials allowed personal supplementation with low dose vitamin D in the control group. In the VITAL trial,31 for example, 42.5% of participants in the control group used vitamin D supplementation (≤800 IU/day). The high prevalence of vitamin D supplementation in the control group made it more difficult to distinguish between the treatment and control groups. The dose of vitamin D used in included trials varied. Our study could not accurately compare equivalent daily vitamin D supplementation dose in the included trials because they all had different treatment regimens and dosing intervals (daily, weekly, monthly, or bolus doses). This might be one of the reasons why this study did not determine an effective daily dose of vitamin D supplementation. Furthermore, the vitamin D status before, during, and after treatment is useful to determine the effectiveness of vitamin D supplementation in improving the actual vitamin D status. Long term vitamin D status is expected to be a much more accurate, reliable, and important clinical parameter compared with a daily dose of vitamin D supplementation. However, previous trials were limited in providing such data. These limitations and uncertainties associated with vitamin D supplementation dose and vitamin D status in treatment and control groups warrant further investigation. The baseline 25 hydroxyvitamin D concentrations of trial participants have not been low enough, which could partly contribute to the null finding on the association of vitamin D supplementation and all cause mortality. Observational studies have indicated an increased mortality risk only at low 25 hydroxyvitamin D concentrations. An individual participant data meta-analysis of observational studies showed that the adjusted hazard ratio (95% confidence interval) for mortality in the 25 hydroxyvitamin D groups with concentrations less than 30, 30-40, and 40-50 nmol/L were 1.67 (1.44 to 1.89), 1.33 (1.16 to 1.51), and 1.15 (1.00 to 1.29), respectively, compared with participants with 25 hydroxyvitamin D concentrations of 75-100 nmol/L.8 In this study, more than half of participants (50 466/66 546) from trials reported a baseline mean 25 hydroxyvitamin D concentration of more than 50 nmol/L.

Implications Mortality is the most important clinical outcome. Our study size met the optimum sample size of 7.5% relative risk reduction and the pooled risk ratio was close to 1 with a narrow confidence interval. Our findings suggest that vitamin D supplementation did not have a clinically relevant effect on all cause mortality, and so there is little evidence that vitamin D supplementation reduces all cause mortality. However, vitamin D supplementation reduced cancer mortality by 15%. Therefore, this analysis supports the concept that the risk of cancer death could be reduced by vitamin D supplementation, and a more targeted intervention for this role might be appropriate. The current study found that all cause mortality was significantly lower among trials with vitamin D 3 supplementation than in trials with vitamin D 2 supplementation, with a trend towards reduced all cause mortality in those taking vitamin D 3 (P=0.07). Similarly, vitamin D 3 supplementation reduced the risk of cancer death, but vitamin D 2 did not. Another finding from subgroup analysis suggested that all cause mortality was significantly lower in trials with longer follow-up, and that the benefit of reduced cancer mortality was seen in trials with longer follow-up (more than three years) but not in those with a shorter follow-up. According to these findings, supplementation with vitamin D 3 for at least three years should be considered. Additional large randomised controlled trials are needed to confirm the results from our subgroup analyses. Several large ongoing trials have the potential to corroborate or refute our findings. In the D-Health trial (Australian New Zealand Clinical Trials Registry: ACTRN12613000743763), high dose vitamin D supplementation (60 000 IU/month) is being used to prevent mortality and cancer in Australian adults aged 60-79. The D-Health trial recently completed the recruitment of almost 21 315 participants, with a minimum of five years of follow-up. Using a similar study design, the VIDAL trial (Vitamin D and Longevity trial; ISRCTN46328341) is analysing the effect of intermittent high dose vitamin D supplementation (60 000 IU/month) on all cause mortality in adults aged 65-84 with a corrected serum calcium level of 2.65 mmol/L. The DO-HEALTH trial (Vitamin D3-Omega3-Home Exercise-Healthy Ageing and Longevity Trial; ClinicalTrials.gov identifier: NCT01745263) has recruited 2152 participants from five European countries aged 70 years and older. The specific aim is to establish whether vitamin D will prevent disease at an older age. The final results of the DO-HEALTH trial will be available in autumn 2019. Although none of these trials have screened for low baseline 25 hydroxyvitamin D for eligibility, all trials have used vitamin D 3 as the intervention.