XX Marks the Spot: Addressing Sex Bias in Neuroscience

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Neuroscience emerged as a defined discipline more than half a century ago, enthralling scientists to study the most enigmatic organ: the brain. Researchers explored animal models and probed the mechanisms behind behavior, cognition, and neurological disorders. As their understanding of the brain expanded, so did the sex gap.

Sex differences extend well beyond reproductive organs and hormones.¹ For many neurological and psychiatric diseases, sex-linked differences affect disease prevalence and response to intervention.^2,3

Many endpoints do not change across the estrous cycle, but the neuroscience field went male, male, male, and females just dropped out completely.

 ­—Margaret McCarthy, University of Maryland

Historically, most researchers believed that the preferential model, focused on male humans, animals, and cells, was a no-brainer. “Nobody said to include females unless you were specifically asking questions about sex differences,” said Rebecca Shansky, a neurobiologist at Northeastern University. This bias stemmed from a seemingly pragmatic view that male data was less fussy and variable, while female data contained higher variability due to the estrous cycle.⁴ It was not until a string of studies in the mid-1990s rattled researchers’ beliefs about the brain.

A Call to Explore Sex as a Biological Variable

In the early 1990s, neuroscientist Bruce McEwen and his group at The Rockefeller University published a series of studies that showed how estradiol, a major female sex hormone, modulated the density of pyramidal cell synapses in the rat hippocampus throughout the estrous cycle.^5,6 “It was like a bomb going off in the field because the hippocampus is not involved in reproduction,” remarked Margaret McCarthy, a neuroscientist at the University of Maryland who studies sex differences in the developing brain. Pyramidal cells are involved in motor function, memory, and learning. These findings demonstrated that gonadal hormones were not solely relevant for reproduction, but they could alter an adult rat’s brain.⁷ “At first, nobody would believe it. Synapses were supposed to be permanent, and we didn’t consider the brain to be plastic then; it was immutable.” 

Further breakthroughs in hippocampal research highlighted sex differences. However, many researchers continued to believe that excluding female subjects would reduce or eliminate experimental variability. While these biases were not intentionally malicious, the lack of female representation remained a significant concern in the field. This gap drastically limited researchers’ understanding of diseases and led to a call for inclusion to rectify this long-standing imbalance of sex representation in biological research. 

In 1993, in a major step forward against sex-biased research, the United States Congress passed the National Institutes of Health (NIH) Revitalization Act, mandating the inclusion of women in clinical trials. This spurred researchers to tackle the road less traveled to redefine the narrative on sex bias: female animals.

“If you believe in science, you should practice science according to the data that we have. This is what I have really been trying to communicate to my peers over the last 10–15 years,” remarked Shansky. “Many of these assumptions that are baked into how we think science is going to work are not true, and we should take a minute to introspect on where those biases come from.”

To debunk the belief that the estrous cycle rendered female animals more variable in behavioral and neurological outcomes, researchers conducted studies in mice and rats demonstrating that female animals are not more variable than male animals.^8-10 “This variability depends on the input, such as housing conditions and even dominance hierarchies,” said McCarthy. “Many endpoints do not change across the estrous cycle, but the neuroscience field went male, male, male, and females just dropped out completely.”

While clinical trials began including women, this representation did not extend to basic research for another quarter of a century. It was not until 2016 that another major milestone for sex inclusion in basic research emerged to address the sex imbalance. The NIH enforced the “Sex as a Biological Variable” (SABV) research policy, which mandated that sex be factored into research designs, analyses, and reporting in human and animal studies across biomedical research when applicable. This was a deliberate approach to turn the dial towards equitable representation in understanding biological differences and shaping precision medicine.

However, the NIH mandate was met with mixed opinions.¹¹ While many lauded the policy, some researchers remained reluctant to incorporate female models into their work and raised concerns about the increase in experimental durations and variability. Despite this reluctance, the next question many researchers sought to answer was whether this mandate truly alleviated sex bias in neuroscience studies.

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Bridging the Sex Bias Gap Sex differences are crucial for understanding the brain, yet neuroscience has long favored male models, skewing insights and clinical outcomes. Now, researchers integrate sex as a biological variable in their studies, paving the way for more balanced and inclusive neuroscience research.           1990s: Historically, most of the basic research and clinical studies predominantly focused on male models (humans, animals, and cells). It wasn’t until 1993 that the U.S. Congress passed a law in requiring inclusion of women in National Institutes of Health (NIH)-sponsored clinical trials. However, this policy failed to encourage the same standards in basic research. Early 2000s: The field was influenced by a long-standing belief: fluctuating hormones of the estrous cycle complicated female studies. However, studies showed that female mice and rats were not more variable in non-neurological and neurological outcomes than male animals. This led to a call for female animal inclusion scientific experiments, as neuroscience had a stark sex gap due to male-focused studies. Late 2010s: In 2016, the NIH enforced the “Sex as a Biological Variable” (SABV) policy, requiring sex to be factored into research designs, analyses, and reporting in biomedical research when applicable. More researchers incorporated female animals, rodents and nonhuman primates, and cell lines, such as induced pluripotent stem cells, to better understand sex differences in neurological disease. 2016–Present: Since SABV, the number of neuroscience studies including both sexes significantly increased. Now, researchers are reframing their analyses of female data to better understand biological differences and shape developing therapeutics.

Exploring the Nuances of Sex as a Biological Variable

To assess the extent of male bias in neuroscience and biomedical research, a meta-analysis conducted in 2009 found that a vast majority did not use both sexes.¹² In follow-up analyses examining the number of studies that included both sexes between 2009–2019, with SABV solidly in place, neuroscience greatly benefitted as the research papers incorporating both sexes steadily increased by roughly 30 percent compared to 2009.^13,14

A lot of the things that we just kind of assumed would be the same in basic neuroscience are turning out not to be true. That’s an exciting point to be at in terms of discovery because we're really blowing up established dogmas about how the brain works. ­

 —Rebecca Shansky, Northeastern University

However, further breakdown of the studies showed that while most papers included both sexes, with a majority using rodents, only 19 percent included studies using an optimal design for finding possible sex differences, and only five percent included sex as a discovery variable. 

This oversight is significant as there is a clear pattern for sex-specific prevalence rates for various mental and physical disorders and differences in behavioral responses to stress, pain, and fear.^15-17 While the mandate tipped the scale to include female cohorts, there is still room for improvement in updating researchers’ experimental designs. For instance, Shansky who studies sex differences in rats’ responses to fear, believes that there is much work to be done in terms of validating models.^18,19 “Males may express that they have a memory or that they are aggressive, stressed, or sad with a certain repertoire of behaviors that females don't do,” said Shansky. “So, if you're just throwing a female into a male-derived test and looking for the same behavior, it's not necessarily going to tell you what you want to know, and that behavior could mean something else and that’s something to explore.”

Studying sex differences may also reveal no differences. “Finding no differences is just as important and just as interesting, but the truth is, they don't get as much attention,” said McCarthy. She studies the reproductive system and reproductive behavior, naturally comparing males and females, but she is also interested in how the brain develops differently in male and female rats.²⁰ While male and female brains share commonalities in the fundamental parameters of brain development, they can differ in the sizes of certain brain regions or the number and types of synapses.²¹ Beyond these macro and micro differences, male and female brains can diverge due to hormonal influences.²² Researchers have identified epigenetic contributions and differences in responses to psychological or pathogenic stresses.^23,24

         

While many neuroscientists examine sex differences in mice and rats, nonhuman primate models are even more underserved in examining sex differences. In Alzheimer’s disease (AD), two-thirds of affected individuals are women. Researchers employ transgenic mouse models to develop abnormal proteins that mimic AD. However, Agnès Lacreuse, a primatologist at the University of Massachusetts Amherst, prefers nonhuman primate models as their genetic makeup, brain, behavior, and aging processes more closely resemble those of humans. However, these studies can be cost prohibitive, making it even more difficult to account for a balanced sex comparison. 

Lacreuse studies cognitive aging in marmosets, which live around 10–12 years and are good candidates for longitudinal studies as they naturally develop neuropathology relevant to AD. Like humans, she found that aging marmosets displayed intra-individual variability in aging pathologies, such as cognition, neurodegeneration, and neuronal aging. Female marmosets experienced cognitive decline earlier and steeper than male marmosets.²⁵

“Even though the marmoset is not a mini human, they are similar and different to humans in many ways. So, what is important is to do comparative studies between different species of primates, including humans, to better understand how and why these differences exist and their mechanisms,” remarked Lacreuse. 

Exploring Sex Differences in Cell Lines 

While SABV addressed sex bias concerns in human and animal studies, it posed challenges in the context of cell lines. Cell lines derived from reproductive tissue inherently denote their sex, yet the sex of other cell lines often remained unspecified in research. Researchers explored the advancement of cell lines, notably the emergence of mouse and human induced pluripotent stem cells (iPSCs) as promising models for studying neurological disease.^26-28 With four transcription factors, researchers could convert somatic cells into pluripotent stem cells, bypassing the need for embryos and the controversy surrounding their use, and readily create patient-specific cell lines from both male and female donors.²⁹ Therefore, iPSCs emerged as a key focal point for implementing SABV, to ensure a more balanced representation of cells from both sexes in rigorous studies.

         

For Tracy Young-Pearse, a stem cell neuroscientist at Harvard University and Brigham and Women’s Hospital, using iPSCs in a dish was paradigm-shifting for neurological disease.³⁰ She investigates sex differences primarily in the context of AD and the genetic factors driving heterogenetic phenotypes.

“[With iPSC models], we can capture the human genome from many different types of people, and we can understand how genetic variants, even when they're not super strong or fully penetrative mutations, interact with one another to affect cell biology in different molecular pathways,” explained Young-Pearse. 

iPSCs provide context for studying male versus female differences in mice and humans. Researchers can explore roughly 70 AD-associated loci and the differences in gene and protein levels on sex and somatic chromosomes, as well as study hallmark signs of amyloid beta (Aβ) plaques and tau tangles in the brain. 

The varying degrees of accumulation between Aβ plaques and tau tangles provide a foundation for making lines from various people who had different trajectories and ages of onset. “What’s really exciting is that we can not only understand those molecularly, but we can understand and disentangle those different molecular roads by using these cells in a dish,” said Young-Pearse.

The NIH also invested in these cell models for studying neurological disease through initiatives such as the iPSC Neurodegenerative Disease Initiative (iNDI) to generate hundreds of iPSC genetic variants to study different forms of dementia.³¹ The project employed various gene editing technologies, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems.³² Currently, iNDI mainly consists of iPSCs from male donors, due to the concern that random X-chromosome inactivation could skew gene expression in female lines. However, the project aims to include a more robust representation of female cell lines moving forward. 

Tangentially, a team of scientists at the Hadassah University Medical Center developed a line of human iPSCs (hiPSCs) from one donor with an unusual case of Klinefelter syndrome to study sex differences without confounding variables such as interpersonal differences.³³ This disorder results in an additional X chromosome in males (XXY), and researchers leveraged this unique model to generate hiPSC lines with different sex chromosome makeups (XX, XY, X0, and XXY) that were otherwise genetically identical. The use of this model provides a promising platform for better understanding the similarities and differences between the sexes in a wide range of health and disease contexts in neuroscience and beyond.

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Looking Ahead with SABV 2.0 

Nearly ten years after the SABV mandate, researchers reflect on where neuroscience research stands in terms of equitable representation and accurate interpretation of female findings to better understand biological differences and shape precision medicine. McCarthy believes that with the 10th anniversary of SABV approaching in 2026, it would be beneficial to revisit its requirements and possibly develop SABV 2.0.³⁴ 

“It's a paralyzing and controversial subject, but it’s very dangerous to think that we should not study sex differences,” remarked Lacreuse. As sex can influence multiple aspects of brain behavior and disease, “It is really important to include women in all studies, and while our studies speak about differences, it doesn't mean that one is better than the other.”

         

For Shansky, a balanced representation of female animals is only one aspect of improvement, and reevaluating how data is interpreted between male and female animals is another crucial consideration. She remains optimistic about continuously improving tools. “For the next wave of the behavioral neuroscience renaissance, all these machine learning tools do careful and high-resolution behavior analysis, and that’s the next step that’s going to allow us to better figure out what we should be looking at. It’s an evolving scene.”

While sex inclusion was just one checkbox on the laundry list of improving neuroscience research, there are still improvements to be made. Aside from data analysis, the next wave of progress may also create a more complex model such as gender identity. “One of the things we need to work on now is incorporating those environmental factors that affect health and disease that's relevant in terms of male versus female. That can really help propel us forward in having good human experimental systems for understanding responses to therapeutics,” said Young-Pearse.

Many are optimistic that SABV continues to be viewed as a boon rather than a burden and that the mandate will steadily reap benefits at the basic and translational levels with continued improvements. “A lot of the things that we just kind of assumed would be the same in basic neuroscience are turning out not to be true. That’s an exciting point to be at in terms of discovery because we're really blowing up established dogmas about how the brain works,” said Shansky.