The present study showed a slightly increased BC risk for moderate vitamin B1 consumption. In men, moderate intake of the vitamins B1, B2, and the vitamins related to energy metabolism and higher intake of vitamin B1 were associated with an increased BC risk, while in women, higher intake of all vitamins and vitamin combinations, except for the entire B group vitamin complex, showed an inverse association.
B group vitamins have essential roles in preventing transmission from a normal cell to a malignant cancer cell, by involving into the pathways of energy metabolism, oxidative stress reduction, and methylation regulation [39, 40].
B group vitamins and energy metabolism
Experimental studies show that vitamin B1 is needed for the metabolism of glucose , thereby delivering the fuel of our cells . Contrary, the present study showed a slight increased BC risk for moderate and high vitamin B1 intake among male, while among female high intake was associated with a decreased BC risk. No previous observational studies were conducted on the influence of vitamin B1 on BC risk, nonetheless, several attempts have been made to correlate vitamin B1 intake to other cancer types. However, results remain inconclusive [43, 44]. A possible explanation for the discrepant findings between male and female might be the different source from which participants retrieved their vitamin B1. While male mainly retrieved their vitamin B1 intake from meat consumption, the main vitamin B1 source for female are vegetables (Supplementary Table 2). Meat consumption has previously been associated with an increased BC risk [45,46,47,48,49,50], while high vegetable consumption showed a decreased BC risk [9, 10]. Moreover, after adjustment of the main food resources (i.e. meat and vegetables) results for men and women were similar and did not reach statistical significance, thereby strengthening the hypothesis that indeed the different food source might explain the observed gender difference. However, future research is needed to further clarify this gender difference.
Vitamin B2 is a coenzyme for many metabolic processes in the body [18, 41]. Although this suggests an inverse effect of vitamin B2 on BC risk, in current observational studies this effect is controversial . The present study also shows conflicting results; among men moderate vitamin B2 intake showed an increased BC risk, while among women a decreased risk was observed. This gender difference might be explained by the source. It is expected that men mainly derive vitamin B2 from meat, while women might derive it mainly from other sources. This is confirmed by the higher vitamin B2 meat-derived intake in men compared to women (Mmeat-B2 men: 0.70 mg, SD = 0.63, Mmeat-B2 women: 0.55 mg, SD = 0.50, t = 90.96, p < 0.001) (Supplementary Table 2). Meat has previously been associated with an increased BC risk due to its pro-carcinogenic components [45,46,47,48,49,50], which could abolish the positive effect of vitamin B2. However, adjustment for the main food sources did not significantly change the findings. Therefore, future research is needed to clarify this gender difference.
Vitamin B3 plays a role in energy metabolism, which is essential to maintain cellular metabolism and respiration  and it is important for genetic and epigenetic regulators . Studies of the consequences of DNA damage in cultured mouse and human cells as a function of vitamin B3 status have supported the hypothesis that vitamin B3 may be a protective factor in limiting carcinogenic events . In line with this, we observed a decreased BC risk for high dietary vitamin B3 intake among women. Since meat is a rich source of vitamin B3 , this again could explain the observed gender differences. In our study, men retrieved on average more vitamin B3 from meat than women (Mmeat-B3, men: 17.83 mg, SD = 15.71, Mmeat-B3 women: 14.54 mg, SD = 13.44, t = 78.49, p < 0.001) (Supplementary Table 2). However, adjustment for the main food sources did not significantly change the findings. Therefore, future research is needed to clarify this gender difference. When analyzing the effect of the vitamins B1, B2 and B3 (i.e. the vitamins related to energy metabolism) together, moderate intake among men showed an increased risk on BC and high intake a decreased risk among women.
B group vitamins and oxidative stress
Oxidative stress is an imbalance in the cell which leads to DNA damage , thereby increasing cancer risk . Experimental studies showed that the vitamins B6 and B2 are involved in oxidative stress reduction by catalyzing regulatory enzymes [55, 56]. Results of the present study confirm these findings by showing an inverse association of high intake of vitamin B6 on BC risk among women, in line with a previously conducted cohort study .
B group vitamins and DNA stability
Vitamin B9 plays a pivotal role in cell metabolism which is necessary for DNA synthesis and repair, as well as for methylation . Previous epidemiological studies and a meta-analysis confirm this protective role of vitamin B9 in the development of BC . The present study also shows a reduced BC risk among women (for both vitamin B9 separate as well as combined with vitamin B12). In men, this protective effect could not be observed. This is possibly the result of the fact that men are less responsive to vitamin B9 than women, due to a lower body mass in which the folate dose distributes over a larger volume . The difference might also be explained by androgens, which are involved in one-carbon metabolism . In our study, men did consume on average more vitamin B9 than women (MB9, men: 194.27 µg, SD = 128.66, MB9 women: 182.12 µg, SD = 96.28, t = 37.76, p < 0.001).
Vitamin B12 is essential for the reduction of DNA damage . Experimental studies show that vitamin B12 deficiency mimics radiation damage to DNA, possibly leading to cancer . However, observational studies showed no effect of high intake of vitamin B12 on BC risk . The present study only observed a significant protective effect of high vitamin B12 intake among women. The main sources of vitamin B12 is meat . Since meat is mainly consumed by men (MB12, meat men: 3.86 µg, SD = 4.30, MB12 meat women: 3.35 µg, SD = 3.85, t = 43.03, p < 0.001) (Supplementary Table 2), it might be argued that the observed effect in men are caused by the earlier mentioned negative effects of other (micro)nutrients in meat . However, adjustment for the main food sources did not significantly change the findings. Therefore, future research is needed to clarify this gender difference.
Although BLEND is one of the largest known pooled cohort studies investigating the association between dietary B group vitamin intake and the risk of developing BC, allowing for detailed analyses with enough statistical power, it has several limitations. First, limited information was available for possible BC risk factors, such as body mass index, physical activity, socioeconomic status, and occupational exposures. Nevertheless, current literature shows only a small proportion of BC cases can be attributed to these factors [23, 64,65,66]. In addition, no information was available on comorbidities that may make people alter their diet , or of which the drugs may influence the bioavailability of B group vitamins in the body . At last, information on alcohol was lacking which might influence B vitamins’ absorption .
A second limitation arises from the use of FFQs, which could lead to recall bias, systematic and random error when estimating vitamin intake. However, since the dietary intake of all included studies were validated, recall bias has likely only played a minor role. In addition, measurement error could be negligible, considering our large sample size.
Thirdly, although people are less likely to change their dietary habits at an older age, most of the included studies only measured their participants at baseline and we were, therefore, unable to take possible changes of dietary habits over time into account. This could have led to misclassification of long-term exposure . However, the included NLCS study repeated the questionnaire 5 year after baseline, and showed only a minor decline in average intake for all food items .
Fourthly, most of the included studies did not provide information on supplement use. Therefore, we were unable to take supplemental vitamin intake into account, which may have led to an underestimation of the true effect of B group vitamins.
Fifthly, a single database was used for the conversion of food into nutrient intake. Since the food composition of similar food items and the food fortification may differ between different countries, the use of country specific food composition tables might be more accurate. Previous studies, however, showed that the use of a common food composition database advantages over the use of country-specific food composition databases in that errors are consistent between the countries . In addition, all our main regression analyses were study centre stratified.
Besides, results obtained from cohort studies on diet and cancer risk cannot always rule out the possibility of reversed causality. Since there is no evidence that people are likely to alter their diet in the period before BC diagnosis, we decided to not exclude study participants who received a BC diagnosis within a short period of follow-up.
Finally, in view of multiple testing, it could indeed be debated whether, for instance, Bonferroni p value adjustments should have been applied. However, it previously has been argued that the use of Bonferroni p value adjustments is impractical and likely too conservative when testing a priori hypotheses . Since we were able to formulate plausible a priori hypotheses regarding all the included analyses, based on data from previous studies, we did not apply Bonferroni correction in our analyses. Moreover, if we had adjusted for the number of main analyses being performed (n = 10) the significance level would have been 0.005. In that case, most of the observed associations between the vitamin B intake and BC risk would still be statistically significant.