The effect of calcium supplementation in people under 35 years old: A systematic review and meta-analysis of randomized controlled trials

Essential revisions:

(1) Please describe the analysis software and its version used in the Methods.

Thank you for your careful review. According to your suggestions, we have described the information in details in the methods section, as follows:

Data extraction and integration were done on Microsoft Office Excel (version 2011). Meta-analysis, subgroup analysis and sensitivity analysis were all performed by Comprehensive Meta Analysis (version 3.3.070, Biostat, Englewood, NJ). (See Lines 144-147 on Page 7 in the Main Text)

2) Please provide information regarding vitamin D supplementation and perform a sub-analysis to see if there are any differences with or without vitamin D supplementation in the results.

Thank you very much for your comments and suggestions. According to your suggestions, we have added the corresponding analyses regarding calcium supplementation with or without vitamin D supplementation. Among the included RCTs, 32 trials used calcium-only supplementation (without vitamin D supplementation) and 11 trials used calcium plus vitamin D supplementation. The detailed information are provided in the Author response table 1 and Author response table 2. We have added subgroup analyses by vitamin D supplementation as you suggested, and the corresponding results are provided in Author response table 3 and Author response table 4.

When we pooled the data from the two subgroups separately, we found that calcium supplementation with vitamin D had greater beneficial effects on both the femoral neck BMD (MD: 0.758, 95% CI: 0.350 to 1.166, P < 0.001 VS. MD: 0.477, 95% CI: 0.045 to 0.910, P = 0.031) and the femoral neck BMC (MD: 0.393, 95% CI: 0.067 to 0.719, P = 0.018 VS. MD: 0.269, 95% CI: -0.025 to 0.563, P = 0.073) than calcium supplementation without vitamin D. However, for both BMD and BMC at the other sites (including lumbar spine, total hip, and total body), the observed effects in the subgroup without vitamin D supplementation appeared to be slightly better than in the subgroup with vitamin D supplementation.

We have added the relevant parts in the sections of Method, Results and Discussion in the main text of the revised manuscript, as follows:

Method: We further conducted some post hoc subgroup analyses according to the level of calcium intake at baseline (< 714 vs. ≥ 714 mg/day, based on the median value), the calcium supplementation dose (<1000 versus ≥1000 mg/day, based on the median value) and vitamin D supplementation (with or without vitamin D). (See Lines 128-131 on Page 6-7)

Results: In order to compare the effect of the presence or absence of vitamin D on the effect of calcium supplementation, we divided all the data into two groups and ran the calculations separately. Calcium supplementation with vitamin D showed greater beneficial effects on femoral neck BMD and BMC (0.758, 95% CI: 0.350 to 1.166, P<.001; 0.393, 95% CI: 0.067 to 0.719, P=0.018). However, for BMCs of lumbar spine and total body, as well as total body BMD, only calcium supplementation without vitamin D had a significant improvement effect. (see Lines 260-265 on Page 13)

Discussion: To explore whether the effect of improving BMD or BMC is due to calcium alone or calcium plus vitamin D, our subgroup analyses found that calcium supplementation with vitamin D had greater beneficial effects on both the femoral neck BMD and BMC than calcium supplementation without vitamin D. However, for both BMD and BMC at the other sites (including lumbar spine, total hip, and total body), the observed effects in the subgroup without vitamin D supplementation appeared to be slightly better than in the subgroup with vitamin D supplementation. Therefore, these results suggested that calcium supplementation alone could improve BMD or BMC, although additional vitamin D supplementation may be beneficial in improving BMD or BMC at the femoral neck. (see Lines 369-376 on Page 23)

(3) Please correct the first paragraph of the discussion. Comments on femoral neck anatomy, fracture, and mortality in the elderly do not seem to fit the point of this manuscript.

Thank you for your kind comments. According to your suggestions, we have removed these sentences about femoral neck anatomy, fracture and mortality in the elderly, which are not applicable to be mentioned in the first paragraph of the discussion. The first paragraph of the revised discussion is showed as follows:

This meta-analysis comprehensively summarized the evidence for the efficiency of calcium supplementation in young people before the peak of bone mass and at the plateau period. The findings indicated significant improvement effects of calcium supplements on both BMD and BMC, especially on the femoral neck. (See Lines 309-312 on Page 21 in the Main Text)

Please tell us directly if it needs further corrections, we will be very grateful and truly appreciate it, and try our best to revise it.

(4) I just suggest making a meta-PCA, as demonstrated before in this paper: https://www.nature.com/articles/ncomms13357.

Thank you very much for your excellent and thoughtful suggestions. Following your suggestion, we have thoroughly and carefully read your recommended paper and some related papers using meta-PCA (such as doi: 10.1002/14651858.CD013229.pub2; doi: 10.1016/j.numecd.2022.03.010; doi: 10.1371/journal.pcbi.1005890; doi: 10.1093/bioinformatics/btx765. etc.). Janina S Ried and Janina M Jeff et al. have developed this PCA-based meta-analysis approach to capture variations in multiple traits simultaneously across multiple studies in a uniform way. This approach can be applied to discover what single-trait analysis cannot capture.

We must admit that this is the first time we have heard and learned about this method, which will be of great help to us in our future research. However, after careful discussions among all members of our research team, we were unable to make good use of the meta-PCA method in our current study to analyse the available data in a short period of time. We apologise very sincerely for this and hope that you would understand our plight.

Thanks again for your insightful and thoughtful comments. We have made our best efforts to implement all your suggestions in the revised manuscript. However, there may remain some deficiencies. Please tell us directly if it needs further corrections, we will be very grateful and appreciate it, and try our best to revise this manuscript.

Reviewer #1 (Recommendations for the authors):

The outcomes and clinical lessons are very clear and the methodology to draw conclusions is appropriate. The strengths and the limitations of this systematic review are clearly outlined.

The biggest limitation of this study is that the heterogeneity is large and the random effect model is used appropriately.

Thank you for your kind comments. We are also aware of the large inter-trial heterogeneity and have attempted to compensate for this. As you mentioned, the biggest limitation of this study was indeed the large intertrial heterogeneity. Because of this, we have compulsorily chosen the random-effects model, which, as you said, would have been better suited to obtaining more conservative results. In addition, we performed meta-regression and meta-subgroup analyses in order to explore possible sources of the heterogeneity and eventually found that the observed heterogeneity may be due to the differences in age, sexes, regions of subjects, doses, intervention duration, and types of calcium supplementation, dietary calcium intake levels at baseline, and with or without vitamin D supplementation.

We have updated the results and discussions about potential sources of heterogeneity in the revised manuscript, as follows:

In general, the heterogeneity between trials was obvious in the analysis for BMD (P < 0.001, I² = 86.28%) and slightly smaller for BMC (P < 0.001, I² = 79.28%). The intertrial heterogeneity was significantly distinct across the sites measured. Subgroup analyses and meta-regression analyses suggested that this heterogeneity could be explained partially by differences in age, duration, calcium doses, types of calcium supplement, with or without vitamin D supplementation, baseline dietary calcium intake levels, sexes and regions of participants. (See Lines 295-300 on Page 20 in the Main Text)

Several limitations need to be considered. First, there was substantial intertrial heterogeneity in the present analysis, which might be attributed to the differences in dietary calcium intake at baseline, population regions, age, intervention duration, calcium doses, types of calcium supplements, with or without vitamin D supplementation and sexes according to subgroup and meta-regression analyses. To take heterogeneity into account, we used random effect models to summarize the effect estimates, which could reduce the impact of heterogeneity on the results to some extent. (See Lines 396-401 on Page 24 in the Main Text)

It would be better to describe the analysis software used in the Methods.

Thank you very much for your careful review. According to your suggestions, we have described the information in detail in the methods section, as follows:

Data extraction and integration were done on Microsoft Office Excel (version 2011). Meta-analysis, subgroup analysis and sensitivity analysis were all performed by Comprehensive Meta Analysis (version 3.3.070, Biostat, Englewood, NJ). (See Lines 144-147 on Page 7 in the Main Text)

Thanks again for your great patience and considerate suggestions.

These insightful and thoughtful comments really did help us to improve the quality and clarity of this manuscript. However, there may remain some deficiencies. Please tell us directly if it needs further corrections, we will be very grateful and appreciate it, and try our best to revise this manuscript.

Reviewer #2 (Recommendations for the authors):

I recommend that the authors provide information regarding vitamin D supplementation and perform a sub-analysis to see if there are any differences with or without vitamin D supplementation in the results.

Thanks for your insightful and thoughtful comments which really did help us to improve the quality and clarity of this manuscript. According to your suggestions, we have added the corresponding analyses regarding calcium supplementation with or without vitamin D supplementation. Among the included RCTs, 32 trials used calcium-only supplementation (without vitamin D supplementation) and 11 trials used calcium plus vitamin D supplementation. The detailed informations are provided in the Author response table 1 and Author response table 2. We have added subgroup analyses by vitamin D supplementation as you suggested, and the corresponding results are provided in Author response table 3 and Author response table 4.

When we pooled the data from the two subgroups separately, we found that calcium supplementation with vitamin D had greater beneficial effects on both the femoral neck BMD (MD: 0.758, 95% CI: 0.350 to 1.166, P < 0.001 VS. MD: 0.477, 95% CI: 0.045 to 0.910, P = 0.031) and the femoral neck BMC (MD: 0.393, 95% CI: 0.067 to 0.719, P = 0.018 VS. MD: 0.269, 95% CI: -0.025 to 0.563, P = 0.073) than calcium supplementation without vitamin D. However, for both BMD and BMC at the other sites (including lumbar spine, total hip, and total body), the observed effects in the subgroup without vitamin D supplementation appeared to be slightly better than in the subgroup with vitamin D supplementation.

We have added the relevant parts in the sections of Method, Results and Discussion in the main text of the revised manuscript, as follows:

Method: We further conducted some post hoc subgroup analyses according to the level of calcium intake at baseline (< 714 vs. ≥ 714 mg/day, based on the median value), the calcium supplementation dose (<1000 versus ≥1000 mg/day, based on the median value) and vitamin D supplementation (with or without vitamin D). (See Lines 128-131 on Page 6-7)

Results: In order to compare the effect of the presence or absence of vitamin D on the effect of calcium supplementation, we divided all the data into two groups and ran the calculations separately. Calcium supplementation with vitamin D showed greater beneficial effects on femoral neck BMD and BMC (0.758, 95% CI: 0.350 to 1.166, P<.001; 0.393, 95% CI: 0.067 to 0.719, P=0.018). However, for BMCs of lumbar spine and total body, as well as total body BMD, only calcium supplementation without vitamin D had a significant improvement effect. (see Lines 260-265 on Page 13)

Discussion: To explore whether the effect of improving BMD or BMC is due to calcium alone or calcium plus vitamin D, our subgroup analyses found that calcium supplementation with vitamin D had greater beneficial effects on both the femoral neck BMD and BMC than calcium supplementation without vitamin D. However, for both BMD and BMC at the other sites (including lumbar spine, total hip, and total body), the observed effects in the subgroup without vitamin D supplementation appeared to be slightly better than in the subgroup with vitamin D supplementation. Therefore, these results suggested that calcium supplementation alone could improve BMD or BMC, although additional vitamin D supplementation may be beneficial in improving BMD or BMC at the femoral neck. (see Lines 369-376 on Page 23)

Please correct the first paragraph of the discussion. Comments on femoral neck anatomy, fracture, and mortality in the elderly do not seem to fit the point of this manuscript.

Thank you for your suggestion. As you mentioned, we have corrected the first paragraph of the discussion and deleted relevant parts about femoral neck anatomy, fracture and mortality in the elderly. The first paragraph of the revised discussion is showed as follows:

This meta-analysis comprehensively summarized the evidence for the efficiency of calcium supplementation in young people before the peak of bone mass and at the plateau period. The findings indicated significant improvement effects of calcium supplements on both BMD and BMC, especially on the femoral neck. (See Lines 309-312 on Page 21 in the Main Text)

Again, thanks a lot for your great patience and considerate suggestions. Your insightful and thoughtful comments really did help us to improve the quality and clarity of this manuscript.

Reviewer #3 (Recommendations for the authors):

In order to elevate these results, I just suggest making a meta-PCA, as demonstrated before in this paper: https://www.nature.com/articles/ncomms13357

Just consider if it's not time-consuming.

Thank you for your suggestion. As you mentioned, we have corrected the first paragraph of the discussion and deleted relevant parts about femoral neck anatomy, fracture and mortality in the elderly. The first paragraph of the revised discussion is showed as follows:

This meta-analysis comprehensively summarized the evidence for the efficiency of calcium supplementation in young people before the peak of bone mass and at the plateau period. The findings indicated significant improvement effects of calcium supplements on both BMD and BMC, especially on the femoral neck. (See Lines 309-312 on Page 21 in the Main Text)

Again, thanks a lot for your great patience and considerate suggestions. Your insightful and thoughtful comments really did help us to improve the quality and clarity of this manuscript.