36Overweight and obesity affect over 20% of children worldwide. Emerging evidence shows that 37 nonnutritive sweeteners (NNS) could adversely influence weight gain and metabolic health, particularly 38 during critical periods of development. Thus, we aimed to investigate the impact of prenatal NNS 39 exposure on postnatal growth and adiposity. Among 2298 families participating in the CHILD cohort 40 study, children born to mothers who regularly consumed NNS during pregnancy had elevated body mass 41 index and adiposity at 3 years of age. In a complementary study designed to eliminate confounding by 42 human lifestyle factors and investigate causal mechanisms, we exposed pregnant mice and cultured 43 adipocytes to NNS (aspartame or sucralose) at doses relevant to human consumption. In mice, maternal 44 NNS exposure caused elevated body weight, adiposity and insulin resistance in offspring, especially in 45 males. Further, in 3T3-L1 pre-adipocyte cells, sucralose exposure during early stages of differentiation 46 caused increased lipid accumulation and expression of adipocyte differentiation genes (e.g. C/EBP-α, 47 FABP4, FAS). The same genes were upregulated in the adipose tissue of male mouse offspring born to 48 sucralose-fed dams. Together, these clinical and experimental findings provide evidence suggesting that 49 maternal NNS consumption induces obesity risk in the offspring through effects on adiposity and 50 adipocyte differentiation. 51Introduction 52 53 Globally, over 20% of children are overweight or obese, with rates exceeding 50% in some countries (1). 54The obesity epidemic has arisen alongside a surge in over-nutrition, which stimulates adipocytes to 55 expand and store excess calories. Mounting evidence shows that obesity originates early in life, perhaps 56 even in utero. The Developmental Origins of Health and Disease (DOHaD) hypothesis postulates that 57 prenatal and early postnatal exposures can "program" lifelong metabolism, weight gain, and other 58 endocrine pathways (reviewed in (2)). Moreover, in early life, environmental exposures can stimulate 59 adipocyte precursor cells to induce the process of adipocyte differentiation, creating a large reservoir of 60 adipocytes to support the development of obesity in response to over-nutrition later in life (3-7). 61 62 Excess energy intake from sugar, especially sugar-sweetened beverages, is strongly associated with 63 obesity (8-11); hence, sugar substitutes or "non-nutritive sweeteners" (NNS) including aspartame and 64 sucralose are marketed as healthier alternatives (12, 13). NNS are widely consumed, including by 65 pregnant women. Almost 30% of mothers in the Canadian CHILD cohort consumed NNS during 66 pregnancy (14), and similar rates have been reported in the USA (24% (15)) and Denmark (45% (16)). 67Contrary to their intended benefits, NNS have been inconsistently associated with metabolic 68 derangements and adverse effects on cardiometabolic health in adults (17)(18)(19) and children (20); 69 however, few studies have investigated the m...