Maja Ramljak, Ottawa ON – A summary of a recently published Nature Perspective.
A Perspective published in Nature Neuroscience as of March 1 2021, has succinctly and firmly made the call for a shift in science culture – one where sex is finally considered as a biological variable and “male” is no longer the default. This article will provide a summary and breakdown of the Perspective.
See the full Perspective here: Considering sex as a biological variable will require a global shift in science culture
What is a Perspective?
The high-impact journal Nature publishes a variety of other material alongside the original research articles that most people are familiar with and think of when they hear “Nature” or “Nature Neuroscience”. Other forms of submission include News and Commentary, Correspondence, Reviews, and Perspective articles. Perspective articles act as a forum for authors to discuss models and ideas from a personal viewpoint. Both Perspective and Review articles follow the same formatting guidelines, undergo peer-revision as well as substantial editing by Nature’s editors in consultation with the author. However, Perspectives are more forward looking and/or speculative than a Review and may take a narrower field of view. Perspectives may be opinionated – their intent is to stimulate discussion and new experimental approaches.
In Brief
Rebecca Shansky and Anne Murphy of Northeastern University and Georgia State University respectively, are the authors of this Perspective piece published in Nature Neuroscience.
Male rodents have been the default model organism in preclinical neuroscience research for decades. This has negative consequences for the rest of the population with regards to diagnosis and treatment. There is a recognized need for study of both sexes, but this is often neither an easy nor economical choice when publishing and career advancement come into play. Funding agency initiatives have been introduced in attempts to alleviate sex disparity in research, but are lacking in ways to hold funding recipients accountable.
This perspective highlights several areas of behavioural, cellular, and systems neuroscience in which fundamental sex differences have been identified. The authors declare that “true rigorous science” must include males and females; male-centric research must no longer be passed as exemplary. In order to do this, a cultural and structural change in how we conduct research and evaluate scientific progress must be made.
Shansky & Murphy also note that an equitable and more representative body of knowledge is a more translational one. They identify experimental standards and professional reward systems as a starting point for change.
Some Background:
- In recent years, there has been increased public awareness of discrepancies in diagnostic practices. These discrepancies stem from a focus on male symptomatology. Male symptoms are “textbook” diagnostic criteria. Under- or misdiagnosis in women occurs because their symptom profiles lack one or more of the male-derived criteria. This leads to delayed treatment, misdiagnosis, or a complete lack of diagnosis (of which consequences include permanent disability, depression, and suicide)
- Even when accurately diagnosed, women experience a greater frequency and number of negative side effects from the available pharmacological treatments. This is a result of using the male response as a default in preclinical and clinical research, without exploring the female response
- Our underdeveloped understanding of etiology, symptomatology, and treatment of mental and neurological disease in women stems in part from a bias that presents itself even in preclinical neuroscience research
- A 2011 study evaluated biomedical publications and found that neuroscience studies used male animals six times more often than they used females. A 2017 follow-up suggested that this imbalance had only barely improved
- US National Institutes of Health (NIH) introduced the Considering Sex as a Biological Variable (SABV) mandate in 2016. This is a policy that states that grant applications must include male and female subjects and/or cells in experimental design and analysis – a move to broaden and refine understanding of how and in whom basic science findings will translate into clinical settings/applications
Note: This perspective does not touch on ethnicity or race, but these factors must also be considered during discussion of etiology, symptomatology, and treatment – especially since bias continues to contribute to and complicate diagnostic practice and treatment. The call to shift science culture to include sex as a biological variable is an important one but is merely a small step towards true inclusivity and true generalizable translational research. Research, treatment, treatment-seeking, and bias are all intrinsically linked. See the following examples of racial and ethnocultural disparities in healthcare:
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non-Black healthcare practitioners who score higher on an implicit bias test will spend less time with Black patients and answer fewer of their questions
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racial bias in pain assessment and treatment recommendations – many white doctors see Black patients as less sensitive to pain, resulting in inadequate treatment
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ethnocultural minorities in Ontario report higher levels of psychosocial distress and many do not seek mental health care
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African immigrants were more likely to underuse mental health services compared to White Canadian-born people in Montreal.
Research
Sex-dependent Behavioural Strategies
In behavioural neuroscience, aspects of an animal’s physical activity are used to make inferences about the animal’s cognitive or psychological state. Many common behavioural tests and metrics used in behavioural neuroscience were designed and validated decades ago – when exclusive use of male animals was standard practice. As such, when male and female rodents produce comparable but different responses in these behavioural paradigms, inaccurate conclusions may be drawn.
For example, spatial navigation paradigms are traditionally considered to be hippocampus dependent and decades of research have produced results to suggest that both sexes perform the task at comparable levels – but males tend to learn to navigate more quickly than females. However, later research has found that females actually tend to switch to a striatal-based strategy in an estrous-dependent manner. Meaning, changes across the estrous cycle will affect behavioural strategy and the brain areas involved. This may therefore result in females traveling farther distances as they complete the task, producing longer latencies.
When examining this sex difference, the original explanation of “males learn better, females are spatially impaired” is expanded to “males and females use different strategies”. Indeed, males navigate to a target using nearby landmarks; females use a strategy based on their own position in space. Male rodents may have been faster, but female rodents navigated in ways that minimized exposure to predators/other dangers. If not careful, sex bias inherent in experimental design may also shape the way we interpret the outcome, and what is deemed to be the “right” way to solve a problem or task. It is necessary that we go beyond simply assessing females in behavioural paradigms that were designed for males and expand upon current behavioural parameters to include appropriate quantitative and qualitative multidimensional measurements.
Sex-dependent structural and synaptic plasticity
For decades, it was believed that “fundamental” biological processes such as neural transmission were the same across sexes. Whatever was discovered in males was simply generalized to females. In other words – if the male and female brain are the same, there is no need to study both. (See Box 1 – SABV myths in the paper for more myths that have contributed to sex bias in research). Indeed, there are volumetric sex differences in discrete subregions of the brain, conserved from rodents to humans. Moreover, researchers have identified sex differences in structural plasticity of individual neurons in response to experience.
Rodent studies of chronic stress in male rats have shown effects localized to areas such as the hippocampus, prefrontal cortex (PFC), and the basolateral amygdala. Repeated stress in male rats elicited apical dendritic atrophy and spine elimination in the hippocampal and prefrontal pyramidal cells, while dendritic and spine growth was observed in the basolateral amygdala. Intuitively, such observations make sense. Stress-related illnesses such as post-traumatic stress disorder or depression are characterized by a hypoactive PFC and hippocampus (reflecting the observed atrophy) and a hyperactive amygdala (reflecting the observed growth).
Interestingly, when the same experiment was done in female rats, the results showed the exact opposite of what was reported in males. Chronic stress in female rats elicited only a slight change in the basal branch number in the hippocampus, PFC dendrites and spine density increased, and the amygdala showed dendrite and spine loss.
Were female rodents omitted completely from this area of research, the results from the male rodents would have been incorrectly generalized to the female rodents. This has drastic consequences considering that women are 2x as likely as men to develop stress-related disorders. If the translational work had continued exclusively in males, the search for a treatment to reverse the impact of stress on brain structure would have been achieved for males – and possibly inadvertently exacerbated the effects of stress in females.
These differences also bring about questions such as – what is ‘good’ for the brain? What is ‘bad’? Are these stress-induced structural alterations across the brain reflective of some sort of adaptive response or more of a disease state? To answer these questions, one must also ask – How does all this change across males and females?
“Importantly, even when preclinical results do not directly translate to humans along a biological sex divide, gaining a deeper understanding of the brain’s mechanistic diversity increases the likelihood of treating people of all sexes and genders.”
Sex-dependent pain pathways
Similar to trends in chronic stress research, chronic pain disproportionately affects women but preclinical work in this field is overwhelmingly conducted in males. Shansky & Murphy note that even clinical studies which often present a mix of male and female patients will rarely include sex as a factor for analysis. Meaning, interpretations of results are too often lacking.
An example of why such an oversight is dangerous lies in opioids – the “gold standard” for pain management treatment. Specifically, research has indicated that opioid potency is greater in males. Clearly there are mechanistic sex differences – and research has identified the midbrain periaqueductal gray (PAG) as a key player. The PAG is a hub of endogenous and exogenous pain modulation and contains dense populations of opioid receptors, the preferred receptor for morphine. It is thought that there is lower opioid receptor binding efficiency and G-protein activation in females when morphine is administered, as well as a sex-specific role of PAG microglia in regards to morphine efficacy.
The mechanisms underlying pain and opioid treatment are highly sex-specific. Chronic pain affects more women but the gold standard is a treatment that is appropriate for male patients. The authors report that recent literature analysis suggests that pain and pain management research is one area in which SABV initiative has been effective at increasing the use of female subjects – which is appropriate, considering the necessity of sex as a biological variable, especially in this area of research.
Moving forward
It is important to remember that NIH grants are non-binding. Meaning – you may claim that you will conduct experiments to address sex differences and improve translational potential, but awardees are not required to conduct the exact experiments they propose. In fact, there is no explicit language from the NIH to state that following the SABV is mandatory and will be enforced once funds are awarded.
Shansky & Murphy also address an important aspect of research culture. By studying males only, a lab may avoid the implicit “risk” of discovering sex differences – and dodge having to invest in and produce follow-up studies of males and females in parallel (as opposed to mixed cohorts), allowing them to quickly advance their work. This sets them up for progress and future funding opportunities. Moreover, the current publishing culture has seen a rapid rise in the number of data figures per paper. The demands for papers with more “mechanistic insight” have increased and the criteria for what is considered a “complete story” continues to grow and expand.
This contrasts strongly against the goals of SABV to include both male and female subjects and/or cells in experimental design. For example: a group of researchers spend X amount of dollars on study #1 in male rodents only. Following up on study 1 results, they then they spend the next X amount of dollars for study #2 in males again to flesh out and further understand the mechanisms and neurological underpinnings of the previous results. The final result is a well-rounded and more appealing “story” for publication. Compare this to a more inclusive but less mechanistically detailed scenario of study #1 in male rodents, then study #2 in female rodents, to see if findings hold true for both sexes. In the competitive culture of research, publication, and career advancement, the authors note that a male-centric research route is understandable – but it is no longer defensible.
To conclude the paper, Shansky & Murphy note that the use of both sexes in basic neuroscience research is an essential step in rectifying sex and gender-based health disparities – including life-threatening misdiagnoses in women. Current and future initiatives will fail unless there is a change in scientific publishing priorities and careful analysis of a phenomenon in males AND females is valued. Instead of extended research on a phenomenon in males only, the notion of a truly complete story should be one that produces an ‘ending’ for both sexes.
SABV policies are part of larger initiatives to improve the reproducibility, rigor, and inclusivity in publicly funded research – and use of both sexes should be standard in assessing high-impact work supported by these funds. A 2017 evaluation of SABV efforts by the European Commission reported that applications considering SABV rose only by 3% over 2 years – from 16% to 19% of all applications. A recent survey also found that male NIH study section members were less likely than their female counterparts to believe SABV to be an important policy. It’s important to note that men make up two-thirds of panel membership. It is clear that the dismantling of “male as the default” as best practice in research must occur across multiple levels of science – from the levels of researcher, funding agency, journal editor, and research consumer – in order to prioritize and encourage research conducted in both sexes.
“Indeed, how can we conduct rigorous research in both sexes while maintaining the pace of scientific progress? The two goals are only at odds if it is considered ‘progress’ to answer a biomedical question in a way that ignores over half the population.”