Fig. 1: E-cigarette exposure causes accumulation of erythrocytes and similar embryo elongation at post-implantation.

Implantation sites were scored at day 6.5 after exposure to SHAM or e-cigarette vapors with nicotine (VAPE NIC) and without nicotine (VAPE). Total litter number per dam was counted per exposure group and analyzed with one-way ANOVA with Tukey’s multiple comparisons test (a) (N = 10 SHAM, N = 17 VAPE, N = 13 VAPE NIC). Serum cotinine levels were measured across groups and analyzed via one-way ANOVA with Tukey’s multiple comparisons test (b) (N = 9 SHAM, N = 12 VAPE, N = 9 VAPE NIC). Longitudinal hematoxylin & eosin-stained representative implant site regions are depicted (c-e) (scale bar=0.1 mm) and erythrocyte presence was blindly scored per embryo with red bars indicative of high erythrocyte numbers, orange with low erythrocyte numbers, and grey with no erythrocyte presence (f) (N = 3 dams per group, implant site number N = 29 SHAM, N = 29 VAPE, N = 26 VAPE NIC). Elongation measurements of developing embryos were obtained by staining for hematoxylin and eosin and measuring the length of the embryo capsule using Fiji. Example embryo measurement images are depicted with double-sided arrows indicating the measured length of the embryo capsule (g, h). Length of elongating embryos was determined (i) (N = 3 dams per group, implant site number N = 25 SHAM, N = 29 VAPE, N = 23 VAPE NIC) and difference from the mean was measured with one-way ANOVA with Tukey’s multiple comparisons test (j) (N = 3 dams per group, implant site number N = 25 SHAM, N = 29 VAPE, N = 22 VAPE NIC). Grubbs outlier test was used where appropriate. Error bars indicate mean ± SEM. Red font p-values indicate significance with a p-value\(\le\)0.05. SHAM depicted as white circles on black bars (a, b, i, j); e-cigarette vapors without nicotine (VAPE) depicted as grey circles on grey bars (a, b, i, j); e-cigarette vapors with nicotine (VAPE NIC) depicted as pink circles on pink bars (a, b, i, j). p=adjusted p-value.