Therapeutic effects of vitamin D supplementation on COVID-19 aggravation: a systematic review and meta-analysis of randomized controlled trials

Background The therapeutic effects of vitamin D supplementation on Coronavirus disease 2019 (COVID-19) aggravation remain controversial and inconclusive. To probe into this contentious issue, we performed the present meta-analysis of randomized controlled trials (RCTs). Methods Literature published up to June 2023 was retrieved from Cochrane Library, PubMed, Web of Science and Embase. RCTs assessing mortality, intensive care unit (ICU) admission, mechanical ventilation (MV), length of hospitalization (LOH), and inflammatory markers containing C-reactive protein (CRP), D-dimer, interleukin-6 (IL-6), lactate dehydrogenase (LDH) were included. 19 RCTs were involved in the analysis and were conducted subgroup analyses on the baseline COVID-19 severity and vitamin D administration. Results In the severity subgroup, statistically significant effects in moderate to severe group were observed in ICU admission (OR 0.43, 95% CI 0.23, 0.80; p = 0.008), MV (OR 0.44, 95% CI 0.27, 0.72; p = 0.001) and LOH (SMD –0.49, 95% CI –0.92, −0.06; p = 0.027). In the administration subgroup, effects of ICU admission (OR 0.39, 95% CI 0.16, 0.97; p = 0.044), MV (OR 0.18, 95% CI 0.07, 0.46; p = 0.000) and LOH (SMD –0.50, 95% CI –0.96, −0.04; p = 0.034) were more pronounced in patients supplied with multiple-dose vitamin D than single-dose. Although the result of mortality showed no statistically significant effect, it indicated a reduced trend (OR 0.87, 95% CI 0.63, 1.12; p > 0.05). The results of inflammatory markers reached no statistical differences. Conclusion This meta-analysis revealed that moderate to severe COVID-19 patients supplied with multiple doses of vitamin D were less apt to need ICU admission, mechanical ventilation and have shorter hospital stays.


Introduction
A downward trend of the Coronavirus disease 2019  outbreak can be witnessed throughout the world in 2023, but the COVID-19 pandemic has not gone away, with an estimated 767 million confirmed cases and 6.9 million fatalities up to June 2023, according to epidemiological data on the Coronavirus Dashboard of World Health Organization (World Health Organization, 2023).COVID-19, due to the highly infectious SARS-CoV-2 virus, is a respiratory disease of which symptoms range from mild, moderate, and even severe and critical (COVID-19 Treatment Guidelines Panel, 2023).Despite the perception that COVID-19 is primarily a respiratory illness, certain research suggested that the nutritional status of infected individuals may influence the progression of COVID-19 (Li et al., 2021;Silverio et al., 2021).Reportedly, vitamin D insufficiency has come forth as a potential but modifiable risk factor with important implications, and vitamin D's significance in lowering the severity and incidence of COVID-19 is increasingly established (Im et al., 2020).Observational studies that underwent meta-analyses (Ben-Eltriki et al., 2022;Wang et al., 2022) revealed that COVID-19 patients had noticeably lower vitamin D concentrations in serum and greater odds of SARS-CoV-2 infection, and worse prognosis than healthy controls.Meanwhile, low vitamin D levels are closely tied to rising inflammatory marker levels (Hopefl et al., 2022).In the period of COVID-19, inadequate intake of vitamin D and the status of hypovitaminosis D has developed into public health concern that requires addressing.
During COVID-19, numerous randomized controlled trials (RCTs) have been stimulated to elucidate whether additional intake of vitamin D could prevent COVID-19 aggravation.However, the results were mixed, with some studies claiming statistically significant protective benefits and others reporting null results.Up to this point, published meta-analyses of non-RCTs on this topic account for a larger portion, whereas the number of RCTs in meta-analyses of vitamin D supplementation and COVID-19 is fairly limited.In the meta-analysis with 6 RCTs by Varikasuvu et al. (Varikasuvu et al., 2022), COVID-19 patients supplemented with vitamin D showed fewer rates but no statistically significant differences of ICU admission and mortality, which was consistent with another meta-analysis with 8 RCTs by Kümmel et al. (Kümmel et al., 2022).Intriguingly, the pooled analyses of ICU admission reached statistical significance in the meta-analysis with 9 RCTs by Zaazouee et al. (Zaazouee et al., 2023) and the metaanalysis with 9 RCTs and 14 non-RCTs by Hosseini et al. (Hosseini et al., 2022).Hence, it is urgent to conduct a new meta-analysis of RCTs with a larger sample size to collect emerging evidence and to provide more convincing and valuable information.In addition, evidence supporting the therapeutic effects of vitamin D supplementation with different doses (single dose/multiple doses) on COVID-19 patients with different severity (particularly in mild to moderate/moderate to severe COVID-19 patients) is still not entirely inconclusive.Based on these factors, we aim to collect updated published RCTs and conduct a meta-analysis with larger sample sizes to better illustrate the connection between COVID-19 and vitamin D supplementation.A high focus will be placed on the following investigative questions: 1) in COVID-19 patients with different severity, is vitamin D supplementation a new approach to mitigate the risk of mortality, ICU admission, and mechanical ventilation, and to reduce length of hospitalization and levels of inflammatory markers?2) in terms of administration, could singlehigh-dose vitamin D improve the curative effect of COVID-19 in comparison with multiple-dose vitamin D?

Materials and methods
This meta-analysis was performed and reported in strict accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) (Page et al., 2021).

Data retrieval and literature search
The research problem was put forward by the principal investigator (YWX).With the database including PubMed, EMBASE, Web of Science, and Cochrane Library, a thorough retrieval of relevant and available literature published up to 19 June 2023, was performed independently by two investigators (YYY and WLS).The search strategy is shown detailedly in Additional Supplementary Table S1.After deduplication, the title and abstract of each retrieved literature were evaluated independently by two co-authors (FY and SPS) to exclude articles that were not related to our study.Any differences were reconciled by consensus or by another two reviewers (GXZ and XYL).

Inclusion and exclusion criteria
To better establish the framework of the research questions and seek evidence, we used the PICOS strategy (patient, intervention, comparison, outcome, study).Finally, we adopted the inclusion as follows: 1) inpatients or outpatients diagnosed with COVID-19, severity at baseline ranged from asymptomatic, mild, moderate, and severe; no limitations on age, gender, or ethnicity; 2) comparing administration of single-dose or multiple-dose vitamin D to placebo or standardized therapy for COVID-19; no limitations on the route of administration, duration of medication, and type of vitamin D; 3) reporting baseline COVID-19 severity, endpoints including ICU admission, mortality, mechanical ventilation, length of hospitalization, and inflammatory markers before and after the intervention; 4) randomized controlled trials published with no restriction in language; exclusion criteria as follows: 1) pregnant or lactating women; 2) taking vitamin D supplementation before/at the recruiting time; 3) types of clinical trials other than RCT such as retrospective studies, observational studies, and pilot protocols.

Study outcomes
Prespecified primary outcomes were the following events encompassing need for ICU admission and MV, mortality in COVID-19 patients.The secondary outcomes were length of hospitalization and changes in the levels of inflammatory markers encompassing CRP, D-dimer, IL-6, and LDH.

Data extraction
Two reviewers (YYY and WLS) independently collected the eligible data from the included RCTs using a pre-designed table.Data comprised the source of study, publication year, location, study design, number of participants, baseline characteristics of participants (mean age, sex, vitamin D status), details between the intervention group and control group, and duration of follow-up.Discrepancies were settled by clear consensus.When the mean values and standard deviations (SD) of the provided outcomes were presented indirectly, we manage to derive the desired value by using an estimation formula based on the given numerical values, such as median, range, sample size, and quartile (Wan et al., 2014;Luo et al., 2016).

Quality assessment and publication bias
Two reviewers (FY and GXZ) independently conducted the quality assessment of the included studies, by the use of Cochrane Collaboration's bias risk tool.The risk of bias for each domain was categorized as low, high, or unclear by the criteria of the Cochrane Handbook of Systematic Reviews (Higgins et al., 2011).To evaluate potential publication bias, we combined the visual perception of the funnel plot and the values of Egger's test when the number of included studies was at least 10 ( Mavridis and Salanti, 2014).We defined significant publication bias as asymmetric funnel plots or the p-value of Egger's test <0.05.When the funnel plot asymmetry was caused by significant publication bias, we applied the trim and filling method to make the adjustment.

Statistical analysis
In this meta-analysis of RCTs, we performed all our analyses by applying Stata 17.0 (StataCorp, College Station, TX, USA).Treatment effects were summarized as odd ratios (OR) with 95% confidence intervals (CI) for dichotomous outcomes and standard mean differences (SMD) with 95% CI for continuous outcomes.Besides, we used the I 2 statistic to identify the heterogeneity across studies, and we viewed I 2 > 50% as statistically significant heterogeneity.When significant statistical heterogeneity was noted, we reported OR/SMD using the random effects model.When I 2 < 50%, we used the fixed effects model.In conducting all the analyses, we considered the result reaching statistical significance if the p-value <0.05.Regarding sensitivity analysis, we undertook a one-study leaveout method for each outcome by eliminating one RCT at a time and by analyzing repeatedly.

Search results
According to the search strategy, we initially identified 780 articles.After removing duplicates and non-RCTs (reviews, meta-analyses, protocols, case reports, Mendelian randomization studies, etc.), 203 articles remained eventually.Of them, 153 articles were filtered as irrelevant articles after viewing the titles and abstracts; 50 articles were assessed for eligibility; 31 articles were excluded for the below reasons: articles retracted (n = 2), lack of relevant outcomes (n = 17), no vitamin D intervention (n = 7), unexpected study design (n = 3) and baseline COVID-19 severity is severe to critical (n = 2).After undergoing the above screening, 19 RCTs were included in the final metaanalysis.The flowchart diagram of this study selection is displayed in Figure 1.

Study characteristics
The main characteristics of the 19 RCTs (Castillo et al., 2020;Maghbooli et al., 2021;Murai et al., 2021;Sabico et al., 2021;Sánchez-Zuno et al., 2021;Annweiler et al., 2022;Cannata-Andía et al., 2022;Cervero et al., 2022;De Niet et al., 2022;Elamir et al., 2022;Fernandes et al., 2022;Karonova et al., 2022;Mariani et al., 2022;Rastogi et al., 2022;Said et al., 2022;Sarhan et al., 2022;Soliman et al., 2022;Torres et al., 2022;Zurita-Cruz et al., 2022) are summarized in Table 1, with a total of 2,435 participants incorporated.Of these 19 RCTs, there are 7 RCTs making comparisons between the effects of vitamin D and placebo, 7 RCTs between the effects of vitamin D and standard of care, and 5 RCTs between the effects of different dosages of vitamin D supplementation.The majority of studies took Cholecalciferol as an intervention, and the majority of participants are vitamin D-deficient and even vitamin D-insufficient at baseline.Vitamin D was dispensed with a single-high dose or multiple doses.The baseline severity of COVID-19 patients varied across the included studies.According to the National Institutes of Health classification (COVID-19 Treatment Guidelines Panel, 2023), COVID-19 infection was categorized into mild disease (defined as non-pneumonia and pneumonia cases, such as mild respiratory symptoms and fever), moderate disease (defined as a lower respiratory disease in clinical evaluation or medical imaging manifestations and the pulse oxygen saturation (SpO 2 ) ≥ 94% on indoor air at sea level), severe disease (defined as SpO 2 < 94% on indoor air at sea level, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO 2 /FiO 2 ) < 300 mmHg, breathing rate >30 times/min, or pulmonary infiltration >50%), and critical disease (defined as respiratory failure, infectious shock, and/or multiple organ dysfunction).We conducted two subgroup analyses based on the baseline severity of COVID-19 (mild to moderate group, moderate to severe group) and administration of vitamin D (single-dose group, multiple-dose group).

Risk of bias and quality assessment
The funnel plots and the results of Egger's test, generated from the data of the included RCTs in the meta-analysis, are listed in Additional Figures 1-3.It was important to note that Egger's test of ICU admission in the subgroup of severity and administration (p = 0.026), mortality in subgroup of administration (p = 0.046), showed significant publication bias.However, after further analyses with the trim-and-fill method, the publication bias did not impact the estimates (no trimming performed and no data changed), indicating that publication bias had little effect and verifying the robustness of our results (Duval and Tweedie, 2004).Cochrane Collaboration's bias risk tool was used to assess the quality of the methodology of included RCTs.The quality assessment of included studies is summarized in Figure 2.Among the 19 RCTs, 47.4% were assessed as high risk of bias, which could be largely attributed to ambiguity blinding setting and possible selective reporting from multiple outcomes.Likewise, 26.3% were assessed as unclear of risk bias, mainly due to problems in the implementation of blinding.26.3% were assessed as low risk of bias.

Results of meta-analysis
Pooled results calculated by Stata display as shown in Tables 2, 3. Analyses of primary outcomes (ICU admission, mechanical ventilation, mortality) and secondary outcomes (length of hospitalization, inflammatory markers) were assessed, disclosing results as described below.

Sensitivity analysis
We conducted a sensitivity analysis to identify the impact of each trial on the effective index, and ultimately the significant effects of any individual study were unobserved (Additional Supplementary Figures S4-S11).

Discussion
Centering on the question of whether vitamin D supplementation could diminish the progression of COVID-19, this meta-analysis of 19 RCTs including a distinctly larger sample size than ever before is the first to explore the efficacy of vitamin D in COVID-19 patients with different baseline severity.More convincing than previously published results, the pooled analyses disclosed newfound and statistically significant results that vitamin D supplementation reduced the likelihood of admission to ICU, the need for mechanical ventilation, and the length of hospitalization, especially in moderate to severe COVID-19 patients and those administrated with multiple doses of vitamin D. Nevertheless, the intervention did not significantly transform into decline in mortality or decreased level of inflammatory markers.
Worldwide, a very high prevalence of hypovitaminosis D status has been reported in many countries (van Schoor and Lips, 2017).As an essential nutrient for the human body, vitamin D, of which active metabolite is 1,25(OH) 2 D 3 , plays a participating role in regulating immunoreaction and inflammatory responses to microorganism infections, such as Epstein-Barr Virus, Human Immunodeficiency Virus, Hepatitis B Virus, Human Papilloma Virus, Influenza (Teymoori-Rad et al., 2019), and SARS-CoV-2 (Bilezikian et al., 2020).Malnutrition such as hypocalcemia, hypovitaminosis D in patients has been consistently linked to COVID-19 progression and a worsened prognosis.In a retrospective study conducted by Minasi et al. (Minasi et al., 2023), the relationship between hypocalcemia and adverse clinical outcomes in COVID-19 patients was investigated.The study revealed a significant correlation between serum calcium levels and circulating 25(OH)D.However, the researchers hypothesized that the observed association with the severity of COVID-19 was not directly related to the role of vitamin D in regulating calcium homeostasis.Instead, they suggested that vitamin D might influence the immune response and the production of proinflammatory cytokines.With a direct antiviral effect, vitamin D induces antimicrobial peptides (part of the innate immune system) against enveloped/nonenveloped viruses (Hansdottir et al., 2008;Youssef et al., 2011), and reinforces the barriers made up of cells to help fight off invasive viruses via E-cadherin (Oh et al., 2019).Additionally, vitamin D can suppress cytokine storms which account for acute respiratory distress syndrome (ARDS), by decreasing the production of proinflammatory T-Helper-1 cells (Th-1) and T-Helper-17 cells (Th-17) (Tomaszewska et al., 2022) and increasing the expression of antiinflammatory cytokines under the regulation of inflammationrelated genes (Wöbke et al., 2014).Under most circumstances, the immunopathogenesis of COVID-19 infection involves disruptions in inflammatory mediators.While these disturbances may not directly cause the disease, they contribute to its progression.Some studies claimed that level  et al., 2020), indicating the interaction of IL-6 and Th-17 cells in the pathogenesis of COVID-19 with poor prognosis.Most importantly, SARS-CoV-2 infects host cells by utilizing angiotensin-converting enzyme 2 (ACE2) as its receptor (Hoffmann et al., 2020) and leads to the downregulation of ACE2 expression (Lu et al., 2022).Meanwhile, vitamin D can reduce the pulmonary permeability of ARDS by means of mediating the renin-angiotensin system (Malek Mahdavi, 2020).Hence, in COVID-19 patients, downregulation of ACE2 tends to trigger an inflammatory chain reaction and the cytokine storm complicated by ARDS.Vitamin D could be a promising therapeutic approach in patients during COVID-19.
It is noteworthy that vitamin D supplementation shows no significant linkage with mortality and inflammatory markers according to our results, which is consistent with some previous meta-analyses (Hosseini et al., 2022;Kümmel et al., 2022;Varikasuvu et al., 2022;Zaazouee et al., 2023).In accordance with previous evidence, pre-existing vitamin D deficiency predisposes COVID-19 patients to suffer from a worse  prognosis.Reportedly, decreased synthesis of vitamin D-binding protein tends to be more common in critical illness, potentially on account of inflammation, injury, disrupted metabolism, and hepatic dysfunction (Jeng et al., 2009;Madden et al., 2015), suggesting that long-term supplemental vitamin D rather than single high-dose vitamin D is preferable.What's more, a recent study revealed that immune responses of the first line of the human body against viral replication or spread turned out to be slight and delayed, especially in critical COVID-19 patients (Liu et al., 2021).This may help to explain why in the administration subgroup multiple-dose group had more effective results in ICU admission, mechanical ventilation, and length of hospitalization than single-dose group.It is regular long-term vitamin D supplementation that takes a protective effect and offers the human body a relatively suitable circumstance allowing the various beneficial effects to be manifested and reinforced in preventing COVID-19.
Owing to pre-designed study protocols and rigorous screening methods, participants in the majority of previous    Forest plots of RCT for the association of vitamin D supplementation and CRP levels in the severity subgroup (A) and administration subgroup (B).

Conclusion
In conclusion, multiple-dose vitamin D supplementation is closely linked with significantly lower odds of ICU admission, mechanical ventilation, and shorter hospital stays in patients with moderate to severe COVID-19.In the unending era of COVID-19, long-term adherence to a daily intake of vitamin D is recommended to stimulate the immune system and promote anti-inflammatory effects for the purpose of preventing aggravation and poor prognosis after infection with the SARS-CoV-2 virus.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.Forest plot of RCT for the association of vitamin D supplementation and IL-6 levels in the severity subgroup (A) and administration subgroup (B).

FIGURE 1 PRISMA
FIGURE 1 PRISMA flowchart of search strategy.

FIGURE 2
FIGURE 2Summary of risk of bias.

FIGURE 4
FIGURE 4Forest plots of RCT for the association of vitamin D supplementation and mechanical ventilation in the severity subgroup (A) and administration subgroup (B).

FIGURE 3
FIGURE 3Forest plots of RCT for the association of vitamin D supplementation and ICU admission in the severity subgroup (A) and administration subgroup (B).

FIGURE 6
FIGURE 6Forest plots of RCT for the association of vitamin D supplementation and length of hospitalization in the severity subgroup (A) and administration subgroup (B).

FIGURE 5
FIGURE 5Forest plots of RCT for the association of vitamin D supplementation and mortality in the severity subgroup (A) and administration subgroup (B).

FIGURE 8
FIGURE 8Forest plot of RCT for the association of vitamin D supplementation and D-dimer levels in the severity subgroup (A) and administration subgroup (B).

FIGURE 10 Forest
FIGURE 10Forest plot of RCT for the association of vitamin D supplementation and LDH levels in the severity subgroup.

TABLE 1
Descriptive summary of included patients and randomized trials characteristics.

TABLE 1 (
Continued) Descriptive summary of included patients and randomized trials characteristics.

TABLE 1 (
Continued) Descriptive summary of included patients and randomized trials characteristics.

TABLE 2
Results of primary outcomes calculated by Stata.
of Th-17 cells increased in critical COVID-19 patients on account of their body releasing excessive amounts of IL-6 (Xu