This post-hoc analysis of the DO-HEALTH trial investigated the effects of vitamin D, omega-3, and exercise on biological aging, measured by four DNAm clocks. Omega-3 supplementation was associated with statistically significant reductions in age-acceleration or pace-of-aging values for PhenoAge (d = -0.16, 95% CI: -0.30 to -0.02), GrimAge2 (d = -0.32, 95% CI: -0.59 to -0.06), and DunedinPACE (d = -0.17, 95% CI: -0.31 to -0.04). Additive effects of all three interventions were observed on PhenoAge. Vitamin D and exercise alone showed no significant effects on the DNAm clocks.
The study provides evidence for a correlation between omega-3 supplementation and a slowing of biological aging, as measured by DNAm clocks. However, it is crucial to note that this is a post-hoc analysis of a subgroup within a larger trial, and the observed effects, while statistically significant, are relatively small. The study does not establish a causal relationship between the interventions and changes in DNAm clocks. Other unmeasured factors could contribute to the observed associations.
The practical utility of the findings is that they suggest a potential benefit of omega-3 supplementation, and possibly combined interventions, on biological aging. This aligns with previous research indicating the health benefits of omega-3s. However, the magnitude of the effect on DNAm clocks (equivalent to a few months over three years) needs to be considered in the context of overall health and aging. The findings are placed within the context of existing research, notably the CALERIE trial, highlighting both similarities and differences.
While the study suggests that omega-3 supplementation may be a beneficial strategy for promoting healthy aging, it is important to acknowledge the uncertainties. The long-term effects of these interventions on health outcomes remain to be determined. The study also emphasizes the potential for personalized approaches, suggesting that individuals with lower baseline omega-3 levels may benefit more from supplementation. This guidance is tentative and requires further investigation.
Critical unanswered questions include the precise mechanisms by which omega-3 influences DNAm clocks, the long-term clinical significance of the observed changes, and whether these findings are generalizable to other populations. The methodological limitation of using a healthier and more active subgroup may affect the generalizability of the conclusions. While the study's rigorous design (randomized, double-blind, placebo-controlled) strengthens the internal validity, the post-hoc nature of the analysis and the focus on a specific subgroup limit the extent to which these findings can be definitively interpreted. Further research is needed to confirm these findings and address these open questions.
The abstract clearly states the research question, addressing the lack of large clinical trials on the combined effects of vitamin D, omega-3, and exercise on biological aging.
The abstract concisely summarizes the study design, mentioning the post hoc analysis of the DO-HEALTH trial, the interventions, and the outcome measures (DNAm clocks).
The abstract highlights the key findings, including the effects of omega-3 and the additive benefits of the combined treatments on specific DNAm clocks.
The abstract provides a concise conclusion, indicating a small protective effect of omega-3 and the additive benefits of the combined interventions.
The abstract briefly introduces the concept of epigenetic clocks and their relevance to biological aging, providing context for the study.
This high-impact improvement would strengthen the abstract by providing more context to the reader and highlighting the clinical significance of the findings. The abstract is the first, and sometimes the only, section that readers encounter, so making the clinical implications clear is crucial for engaging the audience and demonstrating the study's value.
Implementation: Add a sentence before the final summary sentence stating something like: "These findings suggest potential clinical strategies for promoting healthy aging and reducing the risk of age-related diseases."
This medium-impact improvement will enhance the abstract's clarity and provide a more complete picture of the study's findings. While the abstract mentions standardized effects, specifying the direction (positive or negative) would help readers better understand the results.
Implementation: Change "standardized effects ranged from 0.16 to 0.32 units" to "standardized effects, indicating slower aging, ranged from -0.16 to -0.32 units". The negative sign is implied by the context, but making it explicit will prevent any ambiguity.
This medium-impact improvement would enhance the abstract's completeness by including the total number of participants in the original DO-HEALTH trial. While the abstract mentions the number of participants in the post hoc analysis (777), providing the original sample size gives important context.
Implementation: Modify the sentence to read, "Here, we report the results of a post hoc analysis among 777 participants, drawn from the larger DO-HEALTH trial of 2157 older adults, on the effect of...".
The introduction clearly establishes the context by referencing prior research on epigenetic clocks and their association with aging, morbidity, and mortality. It effectively differentiates between first, second, and third-generation clocks, highlighting the stronger evidence for the latter two.
The introduction succinctly summarizes the existing evidence linking vitamin D, omega-3, and exercise to the modulation of epigenetic clocks, citing relevant studies. This sets the stage for the current study's hypothesis.
The introduction clearly states the study's goal and hypothesis, which is to test whether the interventions, individually and in combination, would slow biological aging in a larger clinical trial.
The introduction provides a brief overview of the DO-HEALTH trial and the specific subgroup analyzed in this study, including key participant characteristics. It also acknowledges the limitations of the subgroup compared to the total population.
The introduction clearly explains the rationale for focusing on second and third-generation clocks and also reporting results for first-generation clocks. It also justifies the use of PC versions of some clocks and the original versions of others.
The introduction clearly explains the need for residualization for biological age clocks (all but DunedinPACE), contrasting it with DunedinPACE, which measures the pace of aging and does not require it. It also provides the interpretation of values for both types of clocks.
This medium-impact improvement would strengthen the introduction by providing a more explicit connection between the DO-HEALTH trial's previously reported clinical outcomes and the current analysis of epigenetic clocks. The Introduction section should build a clear bridge between prior findings and the current investigation. This would enhance the rationale for the current study and highlight its contribution to the overall DO-HEALTH research program.
Implementation: Add a sentence after the paragraph describing the DO-HEALTH trial's previous findings, such as: "Building upon these clinical findings, the current analysis aims to investigate the underlying molecular mechanisms, specifically changes in DNA methylation, that may contribute to the observed benefits of these interventions."
This medium-impact improvement would enhance the introduction by providing a brief explanation of *why* DNA methylation is a suitable biomarker for studying biological aging. This would provide crucial context for readers unfamiliar with epigenetics. The Introduction section's role is to provide necessary background, and this addition would strengthen that aspect.
Implementation: Add a sentence or phrase to the first sentence describing epigenetic clocks. For example: "Epigenetic clocks are DNA methylation (DNAm) algorithms—DNAm being a key mechanism of gene regulation that changes with age—that combine information from measurements across the genome to quantify variations in biological versus chronological aging."
This low-impact improvement would improve the flow and clarity of the introduction. The transition between describing the DO-HEALTH trial and introducing the epigenetic clocks could be smoother. This is important for maintaining reader engagement and comprehension.
Implementation: Add a transitional sentence or phrase. For example, after describing the DO-HEALTH trial and participant characteristics, add: "To investigate the potential mechanisms underlying these observed benefits, we analyzed changes in DNA methylation-based measures of biological aging."
The Methods section clearly describes the study design as a randomized, double-blind, placebo-controlled trial with a 2x2x2 factorial design. This design allows for the evaluation of both main effects and interactions between the three interventions.
The section provides detailed information about the study participants, including inclusion criteria, recruitment, and randomization procedures. This level of detail is crucial for assessing the study's internal validity and generalizability.
The Methods section clearly outlines the procedures for data collection, including follow-up duration, frequency of visits and calls, and blinding of participants and researchers. This information is essential for evaluating the study's rigor and minimizing bias.
The section provides a comprehensive description of the DNAm data collection, processing, and quality control procedures. This includes details about the DNA extraction method, the methylation array used, and the normalization and quality control steps. This level of detail is crucial for reproducibility.
The Methods section clearly defines the epigenetic clocks and DNAm-based protein measures used in the study, referencing the original publications for each. This allows readers to understand the specific measures used and their established properties.
The section specifies the statistical analysis plan, including the use of analysis of covariance and the covariates included in the models. This transparency is important for assessing the validity of the statistical inferences.
This medium-impact improvement would strengthen the Methods section by providing a clearer justification for *why* the Swiss subgroup was selected for this analysis. The Methods section should explain the rationale behind key methodological choices, and this addition would fulfill that purpose. Currently, it is mentioned that this subgroup had available samples, but a deeper explanation of the implications of using this subgroup would be beneficial.
Implementation: Add a sentence or two explaining the rationale for focusing on the Swiss subgroup. For example: "The Swiss subgroup was selected for this analysis due to the availability of biospecimens at both baseline and year 3, as well as the logistical feasibility of conducting the DNAm assays within this cohort. While this subgroup is generally healthier and more active than the overall DO-HEALTH population, it provides a valuable opportunity to investigate the effects of the interventions on DNAm measures of biological aging."
This medium-impact improvement would enhance the reproducibility of the study by providing more detail about the strength-training exercise program (SHEP). The Methods section should provide sufficient detail for other researchers to replicate the study, and this is a key component of the intervention. The current description is brief and lacks specifics about the exercises performed.
Implementation: Expand the description of the SHEP intervention. Include information about the specific exercises included, the intensity, sets, repetitions, and any progression guidelines. For example: "The SHEP program consisted of X exercises targeting major muscle groups, including Y and Z. Participants were instructed to perform A sets of B repetitions with C intensity. The program was designed to be progressively challenging, with participants encouraged to increase the intensity/repetitions/sets as they gained strength."
This low-impact improvement would enhance the clarity of the Methods section by explicitly stating whether any imputation was performed for missing data, and if so, what method was used. Missing data is a common issue in longitudinal studies, and the Methods section should address how it was handled. The current description mentions excluding probes with missing values, but it's unclear if any imputation was done for participant-level data.
Implementation: Add a sentence clarifying whether imputation was used for missing data. For example: "No imputation was performed for missing participant data. Participants with missing data for any of the covariates or outcome measures were excluded from the analysis." or "Missing data for covariates were imputed using [method]."
The Results section clearly presents the main findings, stating that omega-3 supplementation reduced age-acceleration or pace-of-aging values for three of the four primary clocks (PhenoAge, GrimAge2, and DunedinPACE). This direct presentation of results aligns with the section's purpose.
The section provides specific effect sizes and confidence intervals for the primary findings, allowing for a quantitative assessment of the treatment effects. This level of detail is crucial for interpreting the magnitude and significance of the results.
The Results section reports on the additive effects of omega-3 supplementation with the other interventions (vitamin D and SHEP), particularly for PhenoAge. This addresses the study's aim of investigating both individual and combined effects.
The section includes an analysis of the DNAm-based surrogate markers of plasma proteins underlying GrimAge, providing insights into potential mechanisms and supporting the primary findings.
The section reports on pre-defined subgroup analyses, investigating whether treatment effects differed based on baseline characteristics such as sex, age, BMI, and baseline vitamin D and omega-3 levels. This adds to the robustness of the findings.
The section appropriately references figures and tables (Fig. 2, Extended Data Table 3, Fig. 3, Extended Data Table 4, Extended Data Figs. 3-5) that provide further details and visual representations of the results. This enhances the clarity and transparency of the findings.
This medium-impact improvement would enhance the clarity and completeness of the Results section. While the section mentions that vitamin D and SHEP were not associated with changes in any of the clocks, it would be beneficial to explicitly state the effect sizes and confidence intervals for these null findings. This is crucial for a comprehensive understanding of all results. The Results section should report all findings, both positive and negative, with sufficient detail.
Implementation: Add a sentence explicitly stating the effect sizes and confidence intervals for the null findings related to vitamin D and SHEP. For example: "Vitamin D supplementation and SHEP were not associated with changes in any of the clocks (vitamin D: d = [effect size], 95% CI: [lower bound, upper bound]; SHEP: d = [effect size], 95% CI: [lower bound, upper bound])."
This medium-impact improvement would strengthen the Results section by providing a more concise and direct summary of the main findings at the beginning of the section. Currently, the section starts by describing the analysis approach before immediately presenting the main findings. A brief introductory sentence summarizing the overall results would improve the flow and clarity. The Results section should begin with a clear and concise statement of the most important findings.
Implementation: Add a brief introductory sentence summarizing the main findings before delving into the details. For example: "The primary analysis revealed that omega-3 supplementation was associated with a reduction in age acceleration or pace of aging across multiple epigenetic clocks, while vitamin D and SHEP showed no significant effects."
This low-impact improvement would improve the clarity and organization of the Results section. The paragraph describing the analysis of GrimAge plasma proteins could be more clearly separated from the preceding paragraph, which focuses on the primary epigenetic clock findings. This is important for logical flow and reader comprehension.
Implementation: Start a new paragraph for the section describing the analysis of GrimAge plasma proteins. Add a brief subheading, such as "Analysis of GrimAge Plasma Proteins," to further delineate this section.
Table 1 | Baseline characteristics of the study population overall and by treatment group
Fig. 1 | Flowchart of the DO-HEALTH Bio-Age trial in the Swiss subset of DO-HEALTH. The flowchart shows the allocation of participants across the eight treatment arms.
Fig. 2 | Treatment effects of vitamin D, omega-3 and SHEP individually and in combination on changes in DNAm measures from baseline to year 3. a-d, Treatment effects are expressed as standardized estimates of the change in DNAm measures from baseline to year 3 at the respective 95% CI.
Fig. 3 | Treatment effects of vitamin D, omega-3 and SHEP individually and in combination on changes in DNAm-based surrogate biomarkers of plasma proteins based on GrimAge. a-g, For the seven DNAm-based surrogate markers of plasma proteins underlying GrimAge, we analyzed versions constructed from DNAm PCs.
The Discussion section effectively summarizes the main findings of the study, reiterating the significant effects of omega-3 supplementation on three of the four DNAm clocks and the additive benefits of combined interventions on PhenoAge. This reinforces the key results for the reader.
The section places the findings within the context of previous research, comparing the results to those from the CALERIE trial and other studies on vitamin D and omega-3 supplementation. This contextualization is crucial for understanding the study's contribution to the existing body of knowledge.
The Discussion section appropriately acknowledges the limitations of the study, including the lack of a gold standard measure of biological aging, the use of DNAm measurements as a partial view, and the limited follow-up duration. This transparency is essential for a balanced interpretation of the results.
The section discusses the implications of the findings for personalized approaches to interventions, noting that individuals with lower starting levels of omega-3 exhibited larger epigenetic shifts. This highlights the potential for tailoring interventions based on individual characteristics.
The section connects the findings to the broader goals of geroscience, highlighting the potential for slowing biological aging to contribute to preventing chronic diseases. This link to the larger field is important for demonstrating the study's significance.
The Discussion section appropriately motivates further research, suggesting additional analyses of the DO-HEALTH trial data and broader application of biological aging measurements in evaluating interventions. This forward-looking perspective is important for advancing the field.
This medium-impact improvement would strengthen the Discussion section by providing a more in-depth discussion of the *mechanisms* by which omega-3 supplementation might influence epigenetic aging. While the Results section mentions some potential pathways (PAI-1, leptin, TIMP-1), the Discussion should elaborate on these and other possible mechanisms, drawing on existing literature. The Discussion section's role is to interpret the findings and speculate on underlying mechanisms, and this addition would fulfill that purpose.
Implementation: Add a paragraph or several sentences discussing potential mechanisms. For example: "The observed effects of omega-3 supplementation on DNAm clocks may be mediated through several pathways. As noted in the Results, omega-3 modified DNAm-based levels of PAI-1, leptin, and TIMP-1, all of which are involved in inflammation and metabolic regulation. Omega-3 fatty acids are known to have anti-inflammatory properties, and it is plausible that these effects are reflected in changes in DNA methylation patterns. [Cite relevant literature on omega-3 and inflammation/epigenetics]. Furthermore, omega-3 may influence DNAm through..."
This medium-impact improvement would enhance the Discussion section by providing a more nuanced discussion of the *differences* in findings between the DO-HEALTH and CALERIE trials. While the section mentions the differences, it could benefit from a deeper exploration of *why* these differences might exist. This is crucial for a comprehensive understanding of the field and for guiding future research. The Discussion section should compare and contrast the study's findings with those of other key studies, and this addition would strengthen that aspect.
Implementation: Expand the discussion of the differences between DO-HEALTH and CALERIE. Consider factors such as the different interventions (caloric restriction vs. omega-3/vitamin D/exercise), different populations, and different methodologies. For example: "While both the CALERIE and DO-HEALTH trials investigated the effects of interventions on DNAm clocks, the observed differences in results may be attributable to several factors. CALERIE focused on caloric restriction, a potent intervention known to affect multiple aging pathways, whereas DO-HEALTH tested specific nutritional supplements and exercise. The populations also differed, with CALERIE involving younger, non-obese individuals and DO-HEALTH focusing on generally healthy older adults. These differences in intervention type, intensity, and participant characteristics may contribute to the variations in observed effects on specific DNAm clocks."
This low-impact improvement would improve the clarity and flow of the Discussion section. The paragraph discussing the limitations of the study could be more clearly separated from the preceding paragraph, which focuses on comparing the results to the CALERIE trial. This is important for logical organization and reader comprehension.
Implementation: Start a new paragraph for the section discussing the limitations of the study. Add a brief subheading, such as "Study Limitations," to further delineate this section.