Title: The effectiveness of vitamin B6 in reducing mastalgia: a systematic review and meta-
analysis
Version: 1 Date: 16 Jun 2025
Reviewer:  Fateme Tahmasbi
Reviewer's report:
A. Introduction
The background would benefit greatly from a more disciplined paragraph structure that
separates concepts thematically and maintains a logical progression from problem definition to
intervention rationale and study justification.
B. The Methods section provides a comprehensive overview of the systematic review process;
however, its paragraph structure is often overly dense and at times disorganized, which impairs
clarity and readability.
It is important to note that the manuscript does not mention the inclusion of a completed
PRISMA checklist in the supplementary materials.
Also, while the authors define mastalgia broadly to include both cyclic and non-cyclic types,
they later state that only women with cyclic mastalgia were included in the analysis. This
contradiction between the inclusive definition and the actual inclusion criteria introduces
ambiguity and should be clarified.
The inclusion criteria encompass both RCTs and quasi-experimental designs, yet the Cochrane
Risk of Bias (RoB) tool used for quality assessment is not appropriate for evaluating non-
randomized studies. This creates a serious methodological flaw, as quasi-experimental studies—
by definition—lack elements like random sequence generation or allocation concealment, which
are central to the RoB tool. Applying this tool uniformly to both RCTs and quasi-experimental
studies can result in misleading or unfair quality assessments.
For quasi-experimental designs or non-randomized studies, the authors should have used a more
appropriate tool such as ROBINS-I (Risk of Bias In Non-randomized Studies - of Interventions)
Quality assessment (Risk of bias)
The study by Soltany et al. [35] was at high risk for most of the Cochrane quality assessments,
given its before-and-after design. The quality assessment of the other four studies found that the
risk of random assignment bias was low in all four studies [24-26, 34]. The risk of randomization
concealment was unclear in three studies [24, 26, 34] and low in only one study [25]. The risk
of bias due to the lack of blinding participants and assessors was low in all four studies [24-26,
34]. The risk of attrition bias was low in all four studies, and the risk of reporting bias was low
in two studies [25, 34] and high in two studies [24, 26] (Figure 2 and Table S1).
C. The Results section provides a generally clear narrative of the study selection process,
study characteristics, and meta-analytic findings; however, there are notable methodological
and reporting concerns that undermine its robustness. While the search strategy is well-
documented and includes manual searching, only five studies met the inclusion criteria for
the systematic review, with just three eligible for meta-analysis. The rationale for excluding
two studies from meta-analysis is inadequately explained and should be more clearly justified.
Study characteristics are described in detail, but this level of granularity—particularly regarding
blinding and group design—is repetitive and would be better summarized in a table. A major
concern lies in the high heterogeneity reported (I² = 99.57%), which severely limits the
interpretability of the pooled results. The authors acknowledge this but do not critically discuss
potential sources of heterogeneity or consider whether a meta-analysis was appropriate under
such conditions. Sensitivity analysis via the one-out method appropriately demonstrates the
impact of a single outlier study (Ghazanfarpour et al.), reducing heterogeneity and slightly
stabilizing the effect estimate, though heterogeneity remains high. Publication bias testing is
statistically inappropriate with such a small number of studies and should not be relied upon;
this limitation is acknowledged but not adequately emphasized. Finally, the GRADE assessment
is superficial—while the evidence is rated as low or very low quality, the reasoning is not based
on a structured evaluation of GRADE domains (e.g., risk of bias, inconsistency, imprecision).
Moreover, the authors refer to the included studies as observational, contradicting their earlier
classification of them as clinical trials. Overall, the Results section would benefit from improved
clarity, critical interpretation of heterogeneity, and a more rigorous application of GRADE
methodology.
D. The Discussion section shows commendable effort in interpreting the study’s findings, but
it suffers from several critical limitations in both analytical depth and structure. The authors
appropriately acknowledge the main statistical finding—i.e., that the pooled effect size of
vitamin B6 on mastalgia is large but not statistically significant due to wide confidence intervals
and extreme heterogeneity. However, their discussion of this heterogeneity remains superficial.
While they note its existence and the results of the sensitivity analysis, they fail to explore in
detail the potential clinical or methodological sources of this inconsistency (e.g., variation in
dosage, outcome measures, or patient populations). This lack of deeper reflection limits the
reader’s understanding of what factors may have influenced the pooled results.
The integration of prior literature is helpful but poorly synthesized. The studies cited—some
showing effect and others not—are presented in a piecemeal fashion without critical comparison
to the current findings. There is also an overreliance on older studies (e.g., Doll et al., 1989),
and the authors miss the opportunity to contextualize their results within a more up-to-date or
mechanistic understanding of vitamin B6’s role in pain modulation. Furthermore, the mention of
potential anti-inflammatory and antioxidant properties of vitamin B6 lacks appropriate linkage
to the current clinical findings and does not account for why such effects might have been
inconsistent across trials.
The limitations are adequately listed, but many are already evident earlier in the manuscript and
could be more incisively discussed. For example, the authors note the geographic homogeneity
of studies (all from Iran) but fail to discuss potential cultural, dietary, or clinical practice
variations that may affect external validity. They also mention methodological weaknesses
of included studies, but do not relate these back to their GRADE findings or provide
concrete examples of bias domains compromised. Additionally, the claim that they excluded
patients with PMS or malignancy to reduce psychological confounding is poorly justified and
methodologically unclear—especially since many women with mastalgia do have PMS, making
this exclusion potentially problematic for real-world applicability.
While the conclusion appropriately calls for further high-quality research, the authors could
strengthen it by offering more actionable recommendations for future trials—such as the need
for standardized outcome measures, longer follow-up, or comparison with alternative therapies.