Fig 1.
Comparative timelines of neurodevelopment and reflex maturation in mice and humans.
Shown is the timeline for major neurodevelopmental events and reflex maturation in mice (A) and in humans (B) and a schematic representation for the experimental design in mice (A). Pregnant dams received ART from embryonic day 0.5 to E20 (till birth of pups). Developmental milestones were assessed in pups daily from P1 to P21. Being altricial in nature, a considerable amount of neurodevelopment occurs in mice pups postnatally. Timing of neurogenesis, maturation and migration of neuronal processes have been depicted using shaded rectangles for both species. Postnatal days on which mature responses are normally expected for specific reflex tests in mice have been shown with pointers in (A). Comparable reflexes in human infants and the timing for maturation and disappearance of these reflexes have been shown in (B).
Fig 2.
Pups exposed in-utero to ART are born small and weigh less for gestational age.
Pup weight and length were assessed daily from postnatal day 1–21. Weight for the first 5 day is shown in (A) for males and (C) for females. Weight for the entire course is shown in (B) for males and (D) for females. Length is shown in (E) for males and (F) for females. Data are mean ± SEM (n = 11–34 pups/group). A mixed effects model was used to examine differences at each day between control and treatment arms (fixed effect) accounting for litter effects (random effect). *p<0.05 vs. control. ATV/r, ritonavir-boosted atazanavir; ABC, abacavir; 3TC, lamivudine; TDF, tenofovir; FTC, emtricitabine; CTRL, control.
Fig 3.
In-utero exposure to ART delays somatic development.
Postnatal day of pinna detachment (A), fur appearance (B), eye opening (C), incisor eruption (D) for male and female pups exposed in-utero to control (CTRL, white bars), ATV/r/ABC/3TC (light grey bars), or ATV/r/TDF/FTC (dark grey bars). Data are mean ± SEM (n = 11–34 pups/group). A mixed effects model was used to examine differences between control and treatment arms (fixed effect) accounting for litter effects (random effect) stratified by sex. *p<0.05 compared to control males. $p<0.05 compared to control females. ATV/r, ritonavir-boosted atazanavir; ABC, abacavir; 3TC, lamivudine; TDF, tenofovir; FTC, emtricitabine; CTRL, control.
Fig 4.
In-utero exposure to ART delays development of certain primitive reflexes.
Postnatal day of successful acquisition of negative geotaxis reflex (A), cliff aversion reflex (B), rooting reflex (C), ear twitch reflex (D), and forelimb grasp reflex (E) for male and female pups exposed in-utero to control (CTRL, white bars), ATV/r/ABC/3TC (light grey bars), or ATV/r/TDF/FTC (dark grey bars). Data are mean ± SEM (n = 11–34 pups/group). A mixed effects model was used to examine differences between control and treatment arms (fixed effect) accounting for litter effects (random effect) stratified by sex. *p<0.05 compared to control males. $p<0.05 compared to control females. Percentage of pups that successfully completed or failed the olfaction homing test on postnatal day 11 is shown in (F). * p<0.05 vs. control by chi-squared test. ATV/r, ritonavir-boosted atazanavir; ABC, abacavir; 3TC, lamivudine; TDF, tenofovir; FTC, emtricitabine; CTRL, control.
Fig 5.
In-utero exposure to ART does not disrupt the development of every primitive reflex.
Postnatal day of successful acquisition of surface righting reflex (A), auditory reflex (B), air righting reflex (C), and open field traversal (D) for male and female pups exposed in-utero to control (CTRL, white bars), ATV/r/ABC/3TC (light grey bars), or ATV/r/TDF/FTC (dark grey bars). Data are mean ± SEM (n = 11–34 pups/group). A mixed effects model was used to examine differences between control and treatment arms (fixed effect) accounting for litter effects (random effect) stratified by sex. No statistically significant differences were observed. ATV/r, ritonavir-boosted atazanavir; ABC, abacavir; 3TC, lamivudine; TDF, tenofovir; FTC, emtricitabine; CTRL, control.
Table 1.
Summary of differences in developmental milestone findings for each ART regimen compared to control.
Fig 6.
In-utero exposure to ART alters behaviour in the open field test in adulthood.
Speed of locomotion (A), total distance moved (B), distance moved in the centre of the arena (C), distance moved in the periphery (D), ambulatory time in the centre of the arena (E), ambulatory time in the periphery (F), rearing time (G), and resting time (H) for male and female mice exposed in-utero to control (CTRL, white bars), ATV/r/ABC/3TC (light grey bars), or ATV/r/TDF/FTC (dark grey bars). Data are mean ± SEM (n = 12–17 mice/group). One-way ANOVA with Bonferroni multiple comparisons tests was used to examine differences between control and treatment arms for each sex. $ indicates p<0.05 compared to control females. ATV/r, ritonavir-boosted atazanavir; ABC, abacavir; 3TC, lamivudine; TDF, tenofovir; FTC, emtricitabine; CTRL, control.