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  • In contrast to the preimplantation blastocyst phenotype we h


    In contrast to the preimplantation blastocyst phenotype, we have found that embryo culture in depleted insulin and BCAA replicates well the postnatal phenotype programmed by Emb-LPD. How might these long-term outcomes be so induced by periconceptional metabolite levels? Increased fetus and birth weight have been reported in mice derived from the transfer of blastocysts that were in vitro-exposed to insulin from the 2-cell stage [15], [16]. Embryo culture with AngiotensinI is also a positive factor in fetal development [14]. However, in the current study, increased gestational growth occurs when preimplantation metabolites are depleted. Our Emb-LPD studies show that the low insulin and BCAA maternal environment associates with altered blastocyst mTORC1 signalling and compensatory extra-embryonic responses that collectively enhance maternal nutrient supply and lead to increased fetal-placental efficiency [5], [8], [10]. Moreover, these extra-embryonic responses include epigenetic modifications that can be traced back to the preimplantation period [63]. Thus, we propose that preimplantation insulin and BCAA depletion, through activation of extra-embryonic responses, can lead to increased gestational growth and birth weight as shown here. Moreover, in the Emb-LPD model, after release from the dietary challenge, the embryonic somatic lineages activate a separate mechanism to stimulate ribosome biogenesis through increased rRNA expression, also epigenetically regulated [64]. This latter mechanism provides the opportunity to maximise the benefit of extra-embryonic adaptations and forms the basis for ‘catch-up’ growth to occur, a common feature in many developmental programming models [2]. The resulting increased birth weight and early postnatal growth rate may then directly associate with increased chronic disease risk in adulthood as found in the Emb-LPD model [5]. The molecular and signalling pathways linking preimplantation insulin and BCAA levels with the increased fetal growth trajectory after Emb-LPD release are yet to be defined but convergence through the mTORC1 pathway is a prime candidate. Thus, the ribosome factor identified in stimulating ribosome biogenesis through reducing rDNA methylation after release from maternal Emb-LPD, Rrn3 (TIF-1A), is sensitive to nutrient levels and mTORC1 signalling [65], [66]. It is also worth highlighting that, similar to the Emb-LPD model [5], after depletion of BCAA and insulin preimplantation, early age body weight is a strong predictor of metabolic health measured several weeks later, especially in female offspring. In contrast, offspring from single or no nutrient depletion preimplantation show no or weak relationships between body weight at any age and AngiotensinI glucose handling capability (Table 3). This suggests that depletion of two key nutrients preimplantation may induce a tight co-regulation of metabolism and body weight very early on in life. Underlying mechanisms warrant further investigation. It is relevant to highlight that our postnatal data were subjected to a robust statistical analysis in which factors such as the random mother effect (i.e. embryo recipient) and gestational litter size were included where appropriate and considering interpretation complications through collinearity. However, litter size can be critical for the proper analysis of in utero programming data from litter-bearing animals [67] and often is ignored in the statistical analyses of DOHaD-related studies working with polyovulatory species, which can result in type 1 errors. Here it is also important to consider some factors that could have influenced our results. Our in vitro embryo culture was carried out under atmospheric O2 conditions (~20%), and it is known that a low O2 environment (i.e. 5%) promotes a better utilization of AA in mouse preimplantation embryos [68]. Also, unlike microfluidic systems where a constant flow of medium can be regulated [69], in our static in vitro system consumption and degradation of culture medium components took place without renewal, hindering a constant exposure of nutrients tested in our study. Although it remains to be determined if these refinements (i.e. low O2 and microfluidics) could exacerbate the postnatal phenotype observed in our study, we believe our data come from a robust in vitro embryo culture model.