In this study, we observed a positive association between breastfeeding during infancy on mtDNA content at adolescent age. The longer the adolescent was postnatally breastfed, the higher the blood mtDNA content was at adolescent age, suggesting a duration-dependent effect of breastfeeding. The effect of breastfeeding was also dose-dependent, with longer duration and exclusivity increasing its effects32. The WHO recommended to further breastfed infants up to two years33, as children’s health and development continued to benefit from the provision of breast milk34. To our knowledge, no other study previously described this association and it might entail an important mechanism to explain the positive effects of breastfeeding on the energy metabolism35,36,37. There are several mechanisms through which breastfeeding can exert its positive effects on mtDNA, such as the energy metabolism, metabolic hormones, antioxidant compounds, and neurocognitive development, elucidated below.

Breastfeeding has a protective effect against obesity7. In contrast to formula feeding, breastfeeding is mainly on demand and less prone to overfeeding. Mothers in a lower SES of the household were less likely to breastfeed their child, which has been described by several other studies38,39,40,41. Although the epidemiologic evidence about the relationship between adiposity and mtDNA content was limited, an inverse association of mtDNA content with the visceral fat area and waist circumference has been reported42,43,44 and BMI has been linked with less mtDNA content in middle aged adults45. Civitarese et al.46 demonstrated, using a clinical trial, that patients who consumed 25% less calories than normal (i.e. caloric restricted patients), showed a lower amount of mtDNA damage, a higher mtDNA content, and increased levels of antioxidant enzymes. In addition, an experimental animal study in young and adult rats demonstrated that neonatal overfeeding caused dysfunction of the mitochondrial respiratory chain complex in the heart47. Caloric restriction might enhance mitochondrial functioning and expand their longevity via reducing oxidative stress30.

A positive association between metabolic hormones (i.e. leptin and insulin) and mtDNA content in early life has been reported26,27. Breast milk contains the hormone leptin, which may reduce psychosocial stress in infants48. Leptin is involved in energy homeostasis and could regulate the energy balance in child- and adulthood35. When administering leptin to leptin-deficient children, their energy intake and fat mass decreased, while lean body mass was unaffected49. In an in vitro study, leptin exposure increased PGC-1α expression, an important factor in mitochondrial biogenesis, as well as decreased the levels of H 2 O 2 . These data suggests that leptin plays a role in mitochondrial biogenesis by avoiding the production of ROS50.

Lower blood glucose and serum insulin concentrations in childhood and lower insulin levels in later life have been associated with breastfeeding. Breastfeeding has as such been associated with a decreased risk of type 2 diabetes mellitus6. The function of mitochondria is closely related to insulin secretion and possibly insulin activity51,52. mtDNA content may be linked with type 2 diabetes mellitus and can serve as an indicator of insulin sensitivity53. Lee et al.54 reported that blood mtDNA content was 25–35% lower in type 2 diabetes mellitus cases, compared with healthy individuals. Also in pre-diabetic subjects who progressed to type 2 diabetes mellitus within two years, a lower blood mtDNA content was reported.

Mitochondria play an important role in the energy metabolism and maintaining normal physiology in the cell. A loss in mitochondrial function is induced by aging and several studies reported a decline in mtDNA content with aging28,29,30,55. The study of Mengel-From et al.56 supported these findings by demonstrating that mtDNA copy number declines while ageing, with 5.4 mtDNA copies less every 10 years from the age of 48. In addition, age-related mitochondrial dysfunction has been reported to contribute to insulin resistance, a major factor in type 2 diabetes mellitus, in the elderly57. In a larger study with approximately 1000 subjects, mtDNA copy numbers were significantly reduced with age, but only in males58. Although the interaction of the sex by breastfeeding on blood mtDNA content was not significant, stratified analysis showed only significant associations in adolescent boys. Sex steroid hormones are able to regulate mitochondrial function. Moreover, mitochondria play an important role in the biosynthesis of these hormones59. Experimental studies in rats showed sex-related differences in mitochondrial function. Remarkably, male rats were more susceptible to mtDNA damage induced by ROS60. In contrast to our higher mtDNA content in boys, population based studies in adults reported lower peripheral blood mtDNA content in men compared with women61,62. Baseline sex-related differences in mtDNA content might explain potential higher susceptibility in mtDNA content changes in boys compared with girls. In addition, Lucas et al.63 observed that infants responded differently to their early nutritional environment dependent on their sex. Experimental studies reported that breast milk composition was determined by infant’s sex64,65,66. A better understanding of the mechanisms driving sex-dependent milk synthesis is needed to further investigate the potential different nutritional and hormonal intakes in males and females, and their possible effect on blood mtDNA content in later life.

Our findings showed a duration effect for the positive association between being breastfed and blood mtDNA content, and in a stratified analysis we observed this from 11 weeks of breastfeeding onwards. This is in concordance with a multitude of studies describing beneficial effects of longer breastfeeding7,67. Although many studies focused on long-term breastfeeding (i.e. for at least six months), our study demonstrated that also shorter term breastfeeding has an impact at the biomolecular level, such as the mtDNA content in peripheral blood.

Melatonin, also present in human milk, is a well-known antioxidant. Due to its small size, it can reach various cellular components, in particular mitochondria. Several studies reported that melatonin had a protective role in mitochondrial homeostasis68,69. During pregnancy, the placenta produces melatonin, while after delivery, it is passed to the child through breastfeeding70, where it can exert an important role in antioxidant defence sustaining the integrity, stability, and function of mitochondrial membranes71. However, to elucidate the protective role of melatonin, further research is necessary.

Breastfeeding is known to have a beneficial effect on the cognitive development of the child3,4,5 and to be related to improved performance in intelligence tests72. Since mitochondrial dysfunction plays an important role in neurodegenerative disorders73, it may be possible that an increase of mtDNA content has beneficial effects on brain development. The brain is sensitive to mitochondrial protein synthesis during early postnatal development, probably due to the massive mitochondrial biogenesis occurring at that stage74. Maternal milk is a rich source of fatty acids and other bioactive components that are essential for brain development. In mitochondria, fatty acids serve as energy sources, molecules for post-translational modifications of proteins, membrane components and as signalling factors75. The study of Oddy et al.76 also reported that a shorter duration of breastfeeding was associated with increased mental health morbidity throughout a period spanning early childhood to adolescence. Future studies may elucidate the role of mtDNA in the association between breastfeeding and cognition.

The study has several limitations and strengths. As this study was part of FLEHS III, it is representative of the population living in Flanders77. Since it is a retrospective study and the data on breastfeeding was collected several years after the mother breastfed her child, it might suffer from a recall bias. On the other hand, the fact that there are 14 or 15 years between breastfeeding and the analysis of mtDNA content, gives an indication of the long-term effect of breastfeeding. Since the turnover rate of mtDNA is estimated to range from ~2 to 350 days78, these data suggest a possible metabolic alteration influencing the mitochondrial biogenesis on longer term. Wachsmuth et al.55 analysed the mtDNA copy numbers from different tissues and reported that each tissue of an individual seemed to regulate their mtDNA copy number in a tissue-related manner. Because the total number of mtDNA copies per cell varies between different tissues of the same individual, our findings are limited to peripheral blood. However, a correlation between myocardial DNA content and peripheral blood mtDNA content was reported79. While this study focussed on mtDNA content as a proxy for energy metabolism, also other mitochondrial (e.g. oxidative phosphorylation) or other energy-related processes (e.g. amount of fatty acids) might be involved. Mitochondrial function can be altered by environmental or life style factors, such as nutrition. Unfortunately, we did not have a food frequency questionnaire. We additionally adjusted our models for leukocyte counts, since changes in the leukocyte cell proportions might influence the mtDNA content. In addition, as stated by Hurtado-Roca et al.80, platelet counts should also be taken into account when measuring mtDNA content. However, these data were not available.