Starting in 2016, the National Institutes of Health mandated that researchers applying for grants factor sex as a variable into experiment design and data analyses. But one neurobiologist argues that outdated gender stereotypes, many of which are not evidence-based, are still finding their way into scientific experiments.

In a perspective published Thursday in Science, Northeastern University neurobiologist Rebecca Shansky said that one of the biggest misconceptions among her colleagues and many male and female scientists is that female hormones — and the estrous cycle in mice, which corresponds to the menstrual cycle in women — are “messy” and complicate matters for research.

“It seems that we are applying all of these higher standards for rigor to female animals that we haven’t had for male animals, and it’s all essentially based around this idea of ovarian hormones, and that to me just seems really wrong.”

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As a result, basic research — unless it sex-specific research — has largely used only male animal models. Results from these studies form the foundation of more rigorous clinical trials, and ultimately lead to drugs that are used by both men and women.

“I would hope that if a researcher considers their research to be clinically relevant, then they would want it to be clinically relevant for everyone and not just men,” Shanksy said in a conference call this week with reporters.

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Ambien is an example frequently cited by proponents of sex parity in research. The insomnia drug was approved in 1992, a year before the Food and Drug Administration lifted a ban on women’s routine participation in clinical trials. After reports of dangerous side effects, including women being involved in car crashes due to what’s known as “sleep driving,” the FDA in 2013 required the manufacturer to halve the recommended standard dose for women. Women seem to metabolize the drug more slowly, the FDA said, and that caused the effects of Ambien to stick around until the next morning.

And that’s just the most famous example. “Immunology research has discovered that there are differences in how men and women respond to vaccines, and it’s also well-known that symptoms of heart attacks are different,” said Margaret McCarthy, a neuroscientist and pharmacologist at the University of Maryland School of Medicine. “Pretty much at this point, there isn’t a common organ that men and women share that doesn’t have differences.”

But beyond the regular inclusion of women in clinical trials, even the inclusion of female animals in research continues to be a problem. McCarthy, who helps review intramural grant applications for the NIH, said one of the common themes is the failure to incorporate sex as a biological variable into applications.

“The message is really not getting out nearly as well as it should,” McCarthy said.

And so Shansky wanted to investigate the origins of what she calls a “dogma” about female hormones being inherently problematic for clean, uncomplicated research.

Her research took her to Victorian times, when the idea of women being unstable and at the whim of their emotions had a stronghold in the scientific community.

“This perception that women are emotional and hormonal was sort of a deliberate narrative, constructed in the Victorian era by biologists and psychologists to essentially preserve the patriarchy,” Shansky said in the conference call.

Her research also found that the perception that variability only exists in female mice may not actually be rooted in fact. A 2014 meta-analysis of some 300 mouse studies found that physiological, behavioral, and other biological measures in male and female subjects did not vary much regardless of the estrous cycle. Another such analysis from 2016 looked at rat studies and found similar results.

“If you think that females are more variable than males, that is not based in reality,” Shansky told STAT. “That is based on your perception of what you think ovarian hormones do to a female animal, and that’s sexist.”

In a statement Thursday, Chyren Hunter of the NIH’s Office of Research on Women’s Health refuted the validity of the idea that female hormones are more variable than male hormones.

“The belief that non-human female mammals are intrinsically more variable than males and therefore should be excluded from routine inclusion in research protocols is without foundation and has been refuted in peer-reviewed publications,” Hunter told STAT.

In fact, at least one study found that depending on whether animals were housed alone or in groups, males — because of dominance-induced changes — could actually have high variability in testosterone, a variability that Shansky said is not taken into account on a regular basis.

Even cortisol, the so-called stress hormone, fluctuates throughout the day. “No one has said you have to do two experiments twice a day, once in the daytime and once in the nighttime, for male studies of drug efficacy,” she said.

And even though the NIH’s 2016 mandate was intended to correct for some of this one-sidedness in research, Shansky said that may not be enough. The mandate doesn’t explicitly say how scientists ought to go about including both male and female animals, for instance, and Shansky writes in her paper that this lack of direction means researchers often still go to male animal models first and then try to replicate the findings in the female models.

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Shansky also suggests instead that researchers, especially in neuroscience, start with an equal number of male and female animals and conduct their experiments without the assumption of sex-based differences. If differences emerge that suggest that sex could be a variable, then the scientists could follow up with larger studies examining each sex more closely.

“I think the suggestion that one needs to have an equal number of females and males and do statistical analysis to find outliers is a good one,” said Catherine Dulac, a Howard Hughes Medical Institute investigator and neurobiologist at Harvard University. “People have to get used to having sex as a variable — for many studies I bet it won’t matter and for some I bet it will.”

Dulac also said that the NIH’s mandate may not be going far enough, since there is no requirement to use female animals — it only asks researchers to account for sex in some way.

“The problem is that [researchers] are going to think about it for 30 seconds to say why they can’t use female mice,” Dulac said. “You hope that they have that tinge of guilt [from not using female mice] because the situation is really becoming unbearable.”

Another way forward may be to focus on the next generation of scientists. “There’s shaming, and there’s training,” Dulac said. “I think training is the way to go forward.”

Nicole Woitowich, associate director for the Women’s Health Research Institute at Northwestern University, agreed, saying there may be resistance from current scientists.

“With folks who are set in their ways, I just don’t know that they are going to change their practices,” she said. “Training is our best hope for changing our attitudes towards sex and gender-inclusive research.”

But some are concerned that the NIH’s mandate and even experiments that pay more attention to female models might reinforce the idea sex is the only way to explain biological differences.

“It’s just important for us to not repeat the idea that sex is the ultimate variable that’s going to explain everything,” said Rebecca Jordan-Young, a sociomedical scientist at Barnard College. “The way sex actually relates to multiple other variables across animals is not the same [as in humans].”

With the Ambien example, for instance, some recent research has suggested that it may be body size and not necessarily sex that’s behind why some people may need a different dose of the drug.

Shansky agreed that sex isn’t the only variable. Even the strain of rat that a researcher uses can influence the results. “I do not think ‘rat strain as a biological variable’ is something NIH will add soon, but those are things worth thinking about when scientists choose experimental design,” Shansky said.

Because these basic biomedical studies do carry weight in the real-world, she said, it’s ultimately important to be careful about variables and pay attention to any differences that they bring.

“What we hope is that the things that work their way out into the actual world and into translational-level studies are the things that are really robust effects and that shouldn’t necessarily be so sensitive to things like sex,” Shansky said. “If it’s going to work in a human, maybe it should work in more than one strain of rat.”