Considering Litter Effects in Preclinical Research: Evidence from E17.5 Acid-Sensing Ion Channel 2a Knockout Mice...

Background: The reproducibility of research findings continues to be a challenge in many fields, including neurosciences. It is now required that biological variables such as sex and age be considered in preclinical and clinical research. Rodents are frequently used to model clinical conditions; however, litter information is rarely presented. Some studies utilize entire litters with each animal treated as an independent sample, while others equally assign animals from each litter to different groups/treatments, and others use averaged data. These methods can yield different results. Methods: This study used different analysis methods to evaluate embryo and placenta weights from E17.5 acid-sensing ion channel 2a (ASIC2a) mice with or without seizure exposure. Results: When each embryo was treated as an individual sample, fetal and placental weight significantly differed following seizures in the ASIC2a heterozygous (+/-) and homozygous (-/-) groups. Differences in fetal weight were driven by females in the ASIC2a+/- group and both sexes in the ASIC2a-/- group. These differences were lost when an average per sex/genotype/litter was used. There was no difference in placental weight when treated individually; however, female ASIC2a-/- placentas weighed less following seizures. This difference was lost with averaged data. ASIC2a-/- fetuses from -/- dams had reduced weights post-seizure exposure. Position on the uterine horn influenced embryo and placental weight. Conclusions: Our results indicate that using full litters analyzed as individual data points should be avoided, as it can lead to Type I errors. Furthermore, studies should account for litter effects and be transparent in their methods and results.