For years, neuroscience research overlooked female subjects, creating a significant bias. Today, researchers actively rebalance the scales with more inclusive and diverse studies.
Sex differences extend well beyond reproductive organs and hormones. For many neurological and psychiatric diseases, sex-linked differences affect disease prevalence and response to intervention.
The neuroscience field went male, male, male, and females just dropped out completely. —Margaret McCarthy, University of Maryland
“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 oestrous cycle, until studies in the mid-1990’s rattled researchers’ beliefs about the brain.
Exploring Sex as a Biological Variable
In the early 1990’s, 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 oestrous cycle. “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. “At first, nobody would believe it. Synapses were supposed to be permanent and the brain was not understood 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 female 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) Revitalisation Act, mandating the inclusion of women in clinical trials. This spurred researchers to tackle the road less travelled 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,” said 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 oestrous cycle rendered female animals more variable in behavioural and neurological outcomes, researchers conducted studies in mice and rats, demonstrating that female animals are not more variable than male animals. “This variability depends on the input, such as housing conditions and even dominance hierarchies,” said McCarthy. “Many endpoints do not change across the oestrous cycle, but the neuroscience field went 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, mandating 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. 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.
