Background.

The intestinal microbiota has coevolved with the human genome, and numerous studies suggest that the gut microbiome and human physiology and metabolism are integrated. The composition of the gut microbiota has been associated with immune development and regulation (1), lipid deposition (2), and plasma glucose levels (3). In humans, treatment with vancomycin is associated with development of adiposity (4) and exposure to antibiotics in early life was recently linked to increased body mass index in childhood (5).

Potential effects of the gut microbiota on bone metabolism have only been investigated in a limited number of studies. In mice, absence of gut microbiota leads to increased bone mass and fewer osteoclasts in the trabecular bone as well as a lower number of CD4 positive cells and osteoclast precursor cells in the bone marrow. Colonisation of germ free mice normalised bone mass and the number of CD4 positive cells in the bone marrow (6). Furthermore, treatment of mice with penicillin, vancomycin or the combination of vancomycin and ampicillin is associated with increased bone mass and size (7).

Effects on bone health of increasing activity of selective gut microbes, through the use of prebiotics, have been assessed in a number of small laboratory and clinical studies. Compared to placebo, intake of a prebiotic (non-digestible oligosaccharides derived from lactose) increased trabecular bone mass in rats, possibly due to improved utilization of calcium and magnesium (8). In humans, treatment with prebiotics (short- and long-chain inulin-type fructans) has been shown to increase calcium absorption and bone mineralization during pubertal growth, and the effects appears to be modulated by common genetic variations in the vitamin D receptor (9). In addition, prebiotic and antibiotic treatments in humans are associated with changes in the secretion of glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (10, 11) and these gut hormones may play a role in the regulation of bone formation and resorption (12, 13).

The modulation of gut microbiota by antibiotics could potentially affect clinically relevant measures of bone mass and quality in humans, in particular attainment of peak bone mass, and, consequently, the risk of fragility fractures in adulthood.

Aims.

* To investigate if exposure to antibiotics in early life is associated with changes in total body bone mass in childhood.

* To determine if associations between early antibiotic use and subsequent total body bone mass persist after adjustment for potential confounding factors such as altered body composition.

* To examine if exposure to antibiotics in early life is likely to affect the risk of clinical events related to osteoporosis in later life, such as hip fracture, based on associations with hip BMD.

* To study the relative contribution of alterations in cortical bone size, thickness, density and turnover to relationships between early life exposure to antibiotics and bone mass which we observe, based on tibial pQCT scan measurements and CTX results.

Hypothesis.

Early life exposure to antibiotics influences gut microbiota, which changes bone metabolism. These alterations cause increases in bone mass in late childhood.

Exposure variables.

Antibiotics in the first 24 months of life.

Outcome variables.

* Indices of total body bone mass as measured by dual-energy x-ray absorptiometry at ages 9.9, 15.5 and 17.8 years.

* Hip BMD as measured at age 13.5 and 15.5

* Cortical bone indices as measured by tibial pQCT at age 15.5 years and 17.8 years

* CTX at age 15.5 years

Confounding variables.

Sex, height, weight, fat mass, lean mass, smoking in first trimester, breast feeding, Tanner stage, physical activity, vitamin D status, socio-economic status/maternal education, insulin, glucose, lipids, leptin, adiponectin, CRP

References.

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2. Backhed F, Ding H, Wang T, et al. The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci U S A 2004; 101(44): 15718-23.

3. Karlsson FH, Tremaroli V, Nookaew I, et al. Gut metagenome in European women with normal, impaired and diabetic glucose control. Nature 2013; 498(7452): 99-103.

4. Thuny F, Richet H, Casalta JP, Angelakis E, Habib G, Raoult D. Vancomycin treatment of infective endocarditis is linked with recently acquired obesity. PLoS One 2010; 5(2): e9074.

5. Blustein J, Attina T, Liu M, et al. Association of caesarean delivery with child adiposity from age 6 weeks to 15 years. Int J Obes (Lond) 2013.

6. Sjogren K, Engdahl C, Henning P, et al. The gut microbiota regulates bone mass in mice. J Bone Miner Res 2012; 27(6): 1357-67.

7. Cho I, Yamanishi S, Cox L, et al. Antibiotics in early life alter the murine colonic microbiome and adiposity. Nature 2012; 488(7413): 621-6.

8. Weaver CM, Martin BR, Nakatsu CH, et al. Galactooligosaccharides improve mineral absorption and bone properties in growing rats through gut fermentation. Journal of agricultural and food chemistry 2011; 59(12): 6501-10.

9. Abrams SA, Griffin IJ, Hawthorne KM, et al. A combination of prebiotic short- and long-chain inulin-type fructans enhances calcium absorption and bone mineralization in young adolescents. Am J Clin Nutr 2005; 82(2): 471-6.

10. Cani PD, Lecourt E, Dewulf EM, et al. Gut microbiota fermentation of prebiotics increases satietogenic and incretin gut peptide production with consequences for appetite sensation and glucose response after a meal. Am J Clin Nutr 2009; 90(5): 1236-43.

11. Francois F, Roper J, Joseph N, et al. The effect of H. pylori eradication on meal-associated changes in plasma ghrelin and leptin. BMC gastroenterology 2011; 11: 37.

12. Tsukiyama K, Yamada Y, Yamada C, et al. Gastric inhibitory polypeptide as an endogenous factor promoting new bone formation after food ingestion. Mol Endocrinol 2006; 20(7): 1644-51.

13. Yamada C, Yamada Y, Tsukiyama K, et al. The murine glucagon-like peptide-1 receptor is essential for control of bone resorption. Endocrinology 2008; 149(2): 574-9.