Human milk oligosaccharides (HMOs) are a group of complex carbohydrates that are highly abundant in human milk and play an important role in supporting infant health [1]. More than 150 different HMO structures have been identified and HMO composition can vary significantly between individuals and over the course of lactation [2]. This variation is driven by both fixed and modifiable factors, including maternal nutrition and supplementation [2, 3]. For example, multiple micronutrient supplementation has been associated with HMO composition [3], but little is known regarding the effect of individual micronutrients.

North American guidelines recommend folic acid supplementation throughout pregnancy and lactation to support optimal growth and development. Folic acid is a synthetic folate form that must be reduced for use in the body; capacity for this is limited, resulting in unmetabolized folic acid (UMFA) [4], a biologically inactive form. Reduced folates serve as co-factors in one carbon metabolism, re-methylating homocysteine to methionine and producing numerous outputs including S-adenosylmethionine, the universal methyl donor [4].

There is an increasing interest in supplementation with (6S)-5-methyltetrahydrofolic acid (5-MTHF) as an alternative to folic acid, as this form is reduced and not metabolically limited [4, 5]. We previously reported that supplementation with folic acid altered folate forms present in human milk as compared to (6S)-5-MTHF, increasing the proportion of human milk UMFA by 14-fold, seemingly at the expense of reduced folate forms [6]. It is unknown whether maternal exposure to folic acid supplementation, resulting in higher UMFA in human milk, has further downstream effects on human milk composition. Thus, our aim was to evaluate the effect of supplementation with folic acid versus (6S)-5-MTHF on HMO composition, and mediation of this effect by folate forms present in human milk.