Controlling litter effects to enhance rigor and reproducibility with rodent models of neurodevelopmental disorders

Abstract

Research with rodents is crucial for expanding our understanding of genetic and environmental risk factors for neurodevelopmental disorders (NDD). However, there is growing concern about the number of animal studies that are difficult to replicate, potentially undermining the validity of results. These concerns have prompted funding agencies and academic journals to implement more rigorous standards in an effort to increase reproducibility in research. However, these standards fail to address a major source of variability in rodent research brought on by the “litter effect,” the fact that rodents from the same litter are phenotypically more similar to one other than rodents from different litters of the same strain. We show that the litter effect accounts for 30–60% of the variability associated with commonly studied phenotypes, including brain, placenta, and body weight. Moreover, we show how failure to control for litter-to-litter variation can mask a phenotype in Chd8V986*/+ mice that model haploinsufficiency of CHD8, a high-confidence autism gene. Thus, if not properly controlled, the litter effect has the potential to negatively influence rigor and reproducibility of NDD research. While efforts have been made to educate scientists on the importance of controlling for litter effects in previous publications, our analysis of the recent literature (2015–2020) shows that the vast majority of NDD studies focused on genetic risks, including mutant mouse studies, and environmental risks, such as air pollution and valproic acid exposure, do not correct for litter effects or report information on the number of litters used. We outline best practices to help scientists minimize the impact of litter-to-litter variability and to enhance rigor and reproducibility in future NDD studies using rodent models.

Communication and social interaction in the cannabinoid‐type 1 receptor null mouse: Implications for autism spectrum disorder

“…Experiment 1a used male and female pups obtained from 11 litters, including as much as possible all three genotypes ( CB1 −/− , CB1 +/− , and CB1 +/+ ) . The approach used here, including a high number of litters together with the use of WT and mutant littermates and testing all litter members without sampling, has been suggested as the most appropriate to protect from the risk of false positives related to litter effects, by past (Chiarotti et al, 1987; Zorrilla, 1997) and recent (Jimenez & Zylka, 2021) guidelines on mouse neurodevelopmental studies. On PND 4 pups were marked after testing by paw tattoo, using a non‐toxic odor‐less tattoo ink (Ketchum permanent Tattoo Inks green paste, Ketchum MFG.…”

Detection of Azoxystrobin Fungicide and Metabolite Azoxystrobin-Acid in Pregnant Women and Children, Estimation of Daily Intake, and Evaluation of Placental and Lactational Transfer in Mice

“…The animal data were analyzed by using the litter as the statistical unit to minimize potential confounds associated with the litter effect. All offspring were used for each litter and analyzed by applying the nonlinear mixed-effects model approach ( Jiménez and Zylka 2021 ). For studies with human urine samples, the differences between groups were compared using the Mann-Whitney test.…”

Persistent autism-relevant behavioral phenotype and social neuropeptide alterations in female mice offspring induced by maternal transfer of PBDE congeners in the commercial mixture DE-71

“…values of offspring in each litter were averaged to represent one sample. This allowed us to control for large differences between litters if they existed (Jiménez and Zylka 2021 ). Based on our previous findings we estimate 5.8, 5.8, 7.2 pups/litter for VEH/CON, L-DE-71 and H-DE-71, respectively, with no differences in the secondary sex ratio (Kozlova et al 2020 ).…”