Fig 2: Summary scheme comparing placental miR-340 expression and the adult outcome in the different experiments. Female offspring born from control dams present high levels of placental miR-340, while the male levels are endogenously low. MiR-340 targets Nr3c1/GR, Cry2, and H3f3b programming susceptibility to activity-based anorexia (ABA) specifically in females (left panel). Prenatal stress downregulated miR-340 through gene-body DNA methylation, upregulating Nr3c1/GR, Cry2, and H3f3b, thus are protecting the female offspring from sensitivity to ABA (central panel). Upregulation of placental miR-340 through lentiviral infection of blastocysts reduced GR/CRY2/H3F3b protein levels, and robustly increased ABA susceptibility in both sexes (right panel). CV control virus, GR glucocorticoid receptor, OE overexpression

Fig 3: MiR-340 is associated with differential expression of nutrient transporters both in mouse and human female placenta. a Heat-map comparing placental nutrient transporters between female mice endogenously expressing high or low levels of miR-340. b The expression of Slc7a5, Slc7a11, Slc38a1, Slc16a3, Igf2, and Igf2bp1 is inversely correlated with miR-340 in mice. c Eosin-hematoxylin staining of the mouse placenta show a thicker junctional zone in the high miR-340 group (t(6) = 3.07, p = 0.022). d STRING pathway analysis linking the miR-340 targets Nr3c1, Cry2, and H3f3b with selected nutrient transporters (https://string-db.org). e Expression of miR-340 in the human placenta in central, medial, and lateral areas. Girls present higher levels in the central area compared to boys (two-way ANOVA F(1,104) = 14.78, p < 0.0001). Data are presented as mean and s.e.m. f The expression of SLC7A5, SLC7A11, SLC38A1, SLC16A3, IGF2, and IGF2BP1 are inversely correlated with miR-340 expression in the central area of human samples. Scale bar: 1 mm

Fig 4: MiR-340 overexpression (OE) decreases GR, CRY2, and H3F3b protein in the placenta and in BeWo cells. a Experimental design for generation of placenta-specific transgenes. b Representative pictures out of about 20 transfers of placenta-specific infection. c Placental miR-340 was highly expressed in the female transgenes of both groups compared to males (two-way ANOVA Fsex(1,23) = 47.39, p < 0.0001) and was increased by the miR-340 OE lentivirus in both sexes (Ftreatment(1,23) = 28.78, p < 0.0001, N = 6). Infection of BeWo cells with the miR-340 OE lentivirus led to increase in miR-340 expression levels (Unpaired ttest t(6) = 1.77, p = 0.019). Fetal hypothalamic gene expression of anorexia candidate genes differed between the CV and miR-340 OE groups (MANOVA F(6,5) = 7.08, p = 0.024), with high expression of AgRPand low expression of Htr1a. d In miR-340 OE transgenes, placental GR mRNA inversely correlated with miR-340 (N = 6) and placental protein tended to be reduced (t(6) = 2.414, p = 0.052, N = 4). e In BeWo cells, Nr3c1/GR mRNA x miR-340 correlation showed a similar pattern to the placenta and miR-340 OE decreased GR protein levels (t(6) = 3.09, p = 0.011, N = 4). f Placental Cry2 mRNA inversely correlated with miR-340 (N = 12) and showed decreased protein levels after miR-340 OE (Unpaired ttest t(6) = 2.451, p = 0.049, N = 4). g In BeWo cells, miR-340 OE significantly decreased CRY2 protein levels (t(6) = 3.381, p = 0.0148). h–i MiR-340 OE did not affect H3f3b mRNA expression but decreased H3F3b protein levels in the placenta (t(6) = 2.452, p = 0.0451, N = 4) (h) and in BeWo cells (t(6) = 4.25, p = 0.0054, N = 4) (i). Data presented as mean and s.e.m. CV control virus. Scale bar: 1 mm