Neuroscientists have long understood that sex-related mechanisms have the potential to influence both brain and behavior. However, whether the potential role for such mechanisms in brain and behavior was an opportunity or a problem depended on a researcher's point of view. For many, this role was clearly perceived as a problem. Female animals and humans were systematically excluded from most research for many years, based on a largely unfounded fear of reduced power to observe significant effects due to a perceived risk of increased variability in females, ascribed in large part to hormonal cycling. A significant body of literature has now repeatedly demonstrated that females are in fact not more variable than males, which has begun to turn the tide (Becker et al., 2016; Beery and Zucker, 2011; Prendergast et al., 2014; R. M. Shansky, 2019). As is well known, in 2016 the NIH mandated the “consideration of sex as a biological variable (SABV)”, which followed the mandated inclusion of women as well as men in clinical research in 1993 (Beery and Zucker, 2011). The goal of these mandates was to avoid losing valuable data about natural variability in humans and model species, as lack of this testing was believed to have contributed to several incidents of unanticipated and dangerous side effects of approved drugs in some women (Nunamaker and Turner, 2023; Shansky and Murphy, 2021) although whether these related at all to sex mechanisms per se remains contentious (Greenblatt et al., 2019; Lee et al., 2023; Rushovich et al., 2023; Zhao et al., 2023). Although the SABV mandate reduced sex bias against using female animals in neuroscience publications, significantly increasing the number of papers using both males and females, it did not eliminate it, as single-sex publications in neuroscience-relevant fields continued to be male- biased (Li et al., 2021; Mamlouk et al., 2020; Nunamaker and Turner, 2023). Moreover, we would argue that the concern about sex influences did not disappear, but began to be expressed differently, with many more publications subsequently including females in their experiments consistent with NIH policy, but not analyzing data to test for potential sex effects—either analyzing it incorrectly (Garcia-Sifuentes and Maney, 2021) or not at all (Mamlouk et al., 2020; Woitowich et al., 2020). In short, while data collection in behavioral neuroscience has shifted towards better incorporating biological diversity related to sex, data analysis and interpretation necessary to benefitting from this effort has lagged significantly behind. This review and commentary aims to address one aspect of what may be limiting these efforts: the use of binary frameworks to represent variability in brains and behaviors.

Due to trailblazing research prior to these mandates, and a wealth of data that has emerged subsequently when sex factors have been analyzed, we now know more than ever before about numerous sex mechanisms and the ways they potentially impact neural structure and function, especially in model species (Becker et al., 2005; Becker and Chartoff, 2019; Hodes and Kropp, 2023; McCarthy et al., 2012; Shansky and Murphy, 2021). “Sex-related mechanisms” in this research can refer to one or more factors that are typically required for the successful production of one kind of gamete (egg or sperm) and/or successful reproduction with that form of gamete in a species. These are not all necessarily active in an individual or in a sample at a given time, reflecting the contextual and life history influences on what constitutes sex (Griffiths, 2021; Richardson, 2022). In the domains most likely to influence neural function, at minimum these mechanisms include 1) sex chromosome genes and their regulation, and 2) gonadal effects, including both early life and pubertal gonadal steroid hormones and their regulation. Much of this research has elucidated intricate genetic and endocrine mechanisms regulating reproductive and other innate behaviors in rodent models (Gegenhuber et al., 2022; McCarthy, 2023; McCarthy and Arnold, 2011). Although for simplicity many scientists are used to “rounding up” sex into two binary categories of “male” and “female”, this literature has clearly described not two inflexible, binary “male and female” states, but instead a nonbinary spectrum of sex influences. This can be surprising, as one might expect sex mechanisms' role in regulating behavior to be the most dichotomous and/or least flexible in reproductive behaviors. However, even reproduction-relevant “innate” behaviors in rodents are in fact remarkably experience dependent (Karigo et al., 2021; Mei et al., 2023; Remedios et al., 2017), suggesting that even sex-biased behaviors relevant to reproductive roles are not inevitable or binary, but reflect interactions between an individual's unique sex-related variables and their environmental milieu. This complex, nonbinary relationship between sex mechanisms and behavior is even more likely to be the case in the context of higher-order cognition, but this has been understudied compared to binary frameworks, described below.

Sex differences in cognition have been of longstanding interest because of the potential for sex mechanisms to influence differential neuropsychiatric risk and resilience between individuals. Essentially all neuropsychiatric diagnoses show some impact of sex and/or gender in their etiology, diagnosis, or prognosis (Grissom and Reyes, 2019). A prominent subdomain of cognition that has been appealed to as potentially influenced by sex mechanisms are executive functions, as alterations in executive functions are found in high-profile neuropsychiatric challenges that show an apparent sex and/or gender bias, especially neurodevelopmental diagnoses and addictions (Becker et al., 2017; Becker and Chartoff, 2019; Santos et al., 2022). Available evidence suggests there is a role for sex-related mechanisms in these diagnosis biases (Kiraly et al., 2018; Mossa and Manzini, 2019; Shansky and Murphy, 2021; Werling and Geschwind, 2013), but we note that this does not exclude a role for gender roles and experiences in the development and expression of these diagnoses (Ellemers, 2018).

One domain of executive function, value- based decision making, has been shown to be sensitive to sex-related factors (Grissom and Reyes, 2019). During this type of task, individuals choose between two or more options that differ in expected value as well as in some risk or cost (Grissom and Reyes, 2019; Orsini and Setlow, 2017; Weafer and de Wit, 2014). In humans and rodent models, these findings can be roughly summarized as showing that males on average are less avoidant of rewarding choices that may also result in negative outcomes (punishment, nonreward, or reduced reward). This is a binary framing of the literature, however, that is inconsistent with the complex, nonbinary relationship we already know exists between sex mechanisms and “less cognitive” behaviors. This is a challenge for our field for two reasons. First, systemic and implicit sex biases have led to a literature which often describes the behavior of female animals and people as less optimal than male “defaults”, an interpretation which can only exist if male and female are pitted against each other as binary opposites. Descriptions of performance on tasks measuring cognition, executive functions, and learned behaviors are often imbued with value judgements reflected in terms like “correct performance”, “errors”, “superior”, and “deficit”, implying that neutral mechanisms of variation, such as those related to sex, are necessarily incorrect or nonoptimal (Grissom and Reyes, 2019). Second, framing females and males as opposites of a binary ignores the enormous overlap in fundamental neural mechanisms across all mammals, and obscures important variation that can occur not only across sexes but within them. Sex differences in brain and behavior reported in the literature reflect differences in group means, but significant overlap between groups tells us that important variability in an individual may not be captured by knowing their overall sex category (Joel et al., 2018; Maney, 2016). Given interest in sex mechanisms in executive functions and evidence for these mechanisms in affecting value based decision making, we propose to use this literature to offer evidence against a sex binary and an alternative to binary interpretations of sex in data, to avoid sexism and increase researcher comfort and interest in understanding sex mechanisms in their own work.

We offer this review and commentary to provide evidence for a key fact - sex influences on cognition are nonbinary. By “nonbinary”, we mean simply that sex mechanisms in mammals do not exist in one of two discrete and immutable states. A nonbinary view of sex influences is now supported by overwhelming evidence from multiple fields including psychology, neuroscience, and behavioral ecology and evolution (Hyde et al., 2019; Joel, 2021; McLaughlin et al., 2023), providing a key conceptual framework for understanding sex in the context of brain and behavior. In this framework, “sex” is a proxy term for numerous mechanisms which can sometimes correlate, but not always, because they are dissociable from each other. In other words, sex is multidimensional. This does not mean that sex mechanisms do not influence neural systems - far from it. However, it does mean that we cannot conclude that an observed average sex difference in behavior is the result of two separate ‘patterns’ of neural mechanisms acting in a single dimension to create two opposed sex-behavior states. Instead, available evidence suggests that multidimensional sex variables are “tuning factors” which interact with non-sex genetic and environmental variability to influence neural systems and behavior in an individual. Using value-based decision making as a key example, we can demonstrate how this may serve to create useful variation in strategies within and across individuals to navigate decisions in a restless, non-optimizable world.

There are several broad dimensions by which sex is widely understood to influence neural function. At a cell-autonomous level, sex chromosome copy number and gene variants influence development and function across the lifespan. At an organismal level, gonadal steroid hormones such as estrogen, progesterone and testosterone exert endocrine effects throughout the body, including the brain. This endocrine function is enriched in the brain by local neurosteroid synthesis and metabolism. Lastly, endocrine mechanisms and sex chromosome mechanisms can mutually influence each other, via the transcriptional role of steroid hormone receptors on both autosomal and sex-linked loci. We will first review current understanding for how any of these dissociable sex mechanisms can influence reward-guided decision making as a subdomain of executive function, including motivated behavior, outcome processing, exploratory, habitual, and perseverative behaviors, and relevant neuroanatomical findings. Following this, we will use this evidence to offer two suggestions to reduce bias and binary framings in research that are intended to be adoptable by any laboratory without needing to measure sex mechanisms directly (Fig. 1). We offer these approaches in the service of interpreting sex influences as contributors of species-wide variation in the neural mechanisms of cognition across individuals.