Our study is therefore the

Our study is therefore the first to show that direct exposure to low levels of both insulin and BCAA exclusively during preimplantation period is sufficient to induce an increment in both body weight gain and blood pressure during early postnatal life. It also gives support to our hypothesis that insulin and BCAA are important nutritional mediators involved in the programming of postnatal disease by protein undernutrition at the early embryo stage [4]. Our study further confirms in general terms the critical relevance of the mammalian preimplantation period as a developmental window where programming with long-term consequences for health and disease risk can be exerted and evident in both rodent and large animal species [5], [21], [22], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34]. In a broader perspective, this periconceptional window where embryo culture conditions may affect postnatal phenotype is also pertinent to the human and assisted reproduction treatment (ART). Although controversy exist on the role of the culture medium as determinant of birth weight in human IVF [35], [36], [37], [38], our experimental work here and a new randomised control trial in the human [39], strongly indicate that the composition of the culture medium is an important contributing factor for birth weight variation in assisted reproduction. ART children further display increased risk of cardiovascular dysfunction likely to derive to their in vitro treatments rather than factors associated with parental infertility, including relative hypertension [40], [41], [42] together with cardiovascular remodeling during pregnancy resulting in altered heart shape and chamber size [41], [43]. Thus, across small and large animal models, and in human ART, embryo culture conditions and composition are critical factors determining postnatal phenotype – yet, in clinical practice, commercial culture media composition is undisclosed [38]. We consider first the short-term outcomes of our treatments that had no effect on NSC232003 phenotype, in contrast to the Emb-LPD blastocyst phenotype. Studies in mice have shown that insulin and AA, including BCAA, added to culture medium can exert stimulatory effects. For instance, AA supplementation can promote blastocyst formation, cell proliferation [44], [45], [46], [47], glucose uptake [14] and outgrowth formation [11]. Similarly, supplementation with insulin can increase the formation [15] and cell number of blastocysts [48], [49] along with reduced protein degradation [50], increased protein synthesis [51], [52], [53] and enhanced endocytosis with resultant increased protein intake [50], [54]. These positive effects of insulin and AA supplementation act through the presence of preimplantation insulin receptors [51], [55], [56] and amino acid transporters (AAT) including system L and system b0,+ AATs responsible for BCAA transport [57], [58], [59]. Furthermore, it has been suggested that insulin can act synergistically with AA to improve embryo development in vitro [60]. The absence of effect of low insulin and/or BCAA on blastocyst phenotype in the current study suggests that these factors are not inductive for the increased trophectoderm proliferation seen in the Emb-LPD model. This is supported by the evidence that (i) non-essential AAs rather than BCAAs may have a predominant effect on mouse embryo proliferation in vitro [45] and that (ii) for insulin, whilst the concentrations used here match those used previously, the stimulatory effect has been on ICM rather than trophectoderm cell numbers [49]. Moreover, the effect of maternal Emb-LPD on increasing trophectoderm proliferation in the mouse is not matched in the rat where trophectoderm cell numbers decrease [61], suggesting it is not a conserved response across species. This view further suggests that different features of the compensatory response in extra-embryonic lineages activated by Emb-LPD treatment (see Introduction, second paragraph) may be separately regulated. Thus, in an earlier study, we found that the stimulation of mouse blastocyst trophectoderm endocytosis as occurring after Emb-LPD could be induced in vitro through low BCAA concentration (equivalent to that used here) in the presence of normalised insulin [9]. Alternative maternal environmental factors changing in response to Emb-LPD treatment should therefore be assessed in vitro for evidence of their role in the early programming events in this model; for example, this could include oestrogen, glucose or other AAs or their combination with insulin [8], or other factors found to alter in response to maternal undernutrition in other models [62].