Table 1 Misconceptions regarding the estrous cycle

1. “Females are not more variable than males” means that the estrous cycle does not have an effect on the outcome of interest

This is an often used argument to rule out the importance of the estrous cycle’s effect on the outcome of interest. We show here that female variability is not predictive of the effect of the estrous cycle on variables from behavior to molecular phenotypes. Unless the study includes the information about the estrous/menstrual cycle stage or hormone levels measurements, it should not make any conclusions about the effects of cycling ovarian hormones

2. The estrous cycle tracking is a stressor and may represent a hidden variable in the data if incorporated in the study

If performed properly by trained individuals, tracking of the estrous cycle is not stressful to rodents. If researchers are concerned with more handling of females than of males, they can always handle males in parallel with the estrous cycle tracking. In any case, not having the information about the hormonal state of the animals is much more of a hidden variable than the stress imposed by vaginal smearing

3. The studies should start with smaller cohorts of mixed male and female animals and then proceed with bigger follow-up studies if any sex-biased “trends” in the data are observed

The under-powered studies can be misleading and even lead to further exclusion of female animals from the studies. As shown here, many times sex difference can be masked if the estrous cycle is not accounted for, and this is especially true when smaller numbers of animals are used. We warn that this practice may lead to inaccurate interpretation of lack of the influence of sex on the outcome of interest, followed by the use of male animals only

4. Monitoring the estrous cycle requires an expert in reproductive endocrinology

While systematic tracking and staging of female animals can be labor-intensive, the skills required are simple in comparison to the average techniques used in neuroscience, and the increased resolution of the data are worth the effort. Several resources are available to assist laboratories unfamiliar with the procedure (see Practical consideration and recommendations section). Further, if systematic tracking is not feasible, taking single smears at the time of data collection can be done quickly and cheaply with minimal training and can provide valuable information for the field