The National Institutes of Health (NIH) issued a policy in 2015 to promote inclusion of both male and female subjects in preclinical biomedical research [1]. As guidance to address this policy, NIH has articulated “The Four Cs of Studying Sex to Strengthen Science” (https://orwh.od.nih.gov/sex-gender/nih-policy-sex-biological-variable): (1) Consider (design studies that take sex into account, or explain why it is not incorporated), (2) Collect (tabulate sex-based data), (3) Characterize (analyze sex-based data), and (4) Communicate (report and publish sex-based data). This article describes a general, multi-step approach to address The Four Cs when investigation of sex differences is not the primary aim, and sample sizes required to detect sex differences are not known. We acknowledge that there are many instances where sex may be a top priority; however, as written, the NIH policy and associated “Four C’s” guidance recognize that this is not invariably the case.  The factors that may contribute to an individual researcher’s prioritization of sex differences among all possible independent variables are beyond the scope of this brief article.

(1) IP acid alone was tested in one group of 12 mice, ketoprofen + IP acid was tested in a separate group of 12 mice, and each group contained equal numbers of male and female mice (N = 6/sex). The total group size, which was modestly larger than the N = 6–10 mice used in our previous study with only males [3], was selected based on the observation that confidence-interval widths for any normally distributed population decline with increasing sample size and approach an asymptote at approximately N = 12 [4]. We further chose to constitute this N = 12 group size with equal numbers of N = 6 mice per sex in part to comply with published guidance by the British Journal of Pharmacology for use of equal sample sizes of N ≥ 5 across groups [5]. However, it is important to note that NIH guidance does not specify minimum group sizes or allocations by sex, and our approach could be used regardless of the numbers of males and females included in a study.

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