Incorporating sex as a biological variable in preclinical research

In the spring of 2015 the National Institutes of Health announced new guidelines for the incorporation of sex as a biological variable in any research they fund. Chromosome compliment (XX for female, XY for male in all mammals), gonadal phenotype, and gamete size define sex as a biological parameter. (In contrast, gender is a human construction based on an individual or society’s perception of sex.) A mandate for inclusion of women and minorities in clinical trials had been successfully established long ago, but the gender balance did not trickle down to studies on animals and cells, or indirect human research (preclinical research).

We spoke with Margaret M. McCarthy, PhD, author of “Incorporating Sex as a Variable in Preclinical Neuropsychiatric Research” in a recent issue of Schizophrenia Bulletin, about the issue of separating sex and gender, considering the biological parameter of sex, flawed studies, and future research findings.

Why is the NIH requiring consideration of sex as a biological variable in preclinical research?

Because sex matters. Several analyses of published articles in multiple fields of biomedical research have found that the overwhelming majority of studies on animal models either use exclusively males or don’t report the sex of the animals they are studying (which usually means males). Preclinical research is used to inform clinical trials and 8 out of the last 10 clinical trials that were halted early were due to adverse events in women, some of them life-threatening.

Did any of those clinical trials involve treatments for schizophrenia?

No, but it is well established that schizophrenia is a very different disease in men versus women. Jill Goldstein at Harvard has studied this extensively and notes that at younger ages males are more likely to be diagnosed with schizophrenia and have stronger negative symptoms and less depressive symptoms than women. At older ages the prevalence switches so that women are more likely to be diagnosed. There is a mix of evidence as to why there are these differences, suggesting we need more fundamental research on the topic, and that begins with animal models.

Why don’t most researchers study both males and females?

For several reasons. One is a long standing bias that females are more complicated than males because their hormones cycle over days, weeks, or months. There is also a view that the only sex differences between males and females are those directly associated with reproduction. And lastly, a lot of researchers just think males are the norm, after all the researcher is more likely to be a man than a woman due to the paucity of women in science.

How is the NIH going to fix redress this imbalance?

By requiring that all research funded by them and that involves either animals or cells must both report the sex of the subjects and must analyze for the effects of sex on the endpoints they are measuring. This means that scientists must no longer use exclusively males, they must mix males and females in their groups and conduct a statistical analyses that determines if there is a difference. The NIH hopes to assure this becomes a routine component of research by including it as what is called a “review criteria,” meaning when a grant is peer-reviewed to determine if it is suitable for funding (only ~10-20% of grant proposals are awarded funding), the degree to which sex as a biological variable has been effectively incorporated into the experimental design will be assessed. This does not mean that everyone has to start studying sex differences. Indeed, if the researcher finds there is no difference in the response of males and females they can now include both sexes in all of their research going forward, which saves a lot of animals from going to waste. (Think about what happened to all those female littermates in the past).

Why is the NIH facing such resistance to this new requirement?

Because change is hard. Some believe they are going to have to double or even quadruple the number of animals they use (not true), so they can control for the estrus cycle (even more not true), and that this will cost a fortune but nobody is giving them any more money. Some are also sure, without any evidence, that whatever they are studying will not differ between males and females. They might be right, and so they only need to demonstrate that definitively once, and then they can move on using males and females in their research with confidence that there is no greater variability introduced by including females. Their sample size can remain exactly as it always has. Indeed their variability might even go down as it has been found that group housed male mice and rats form a dominance hierarchy which greatly impacts the physiology of the dominant and subordinate animals in very different ways. So in the end, males may be introducing just as much variability into a study, or even more, than females with all their pesky hormones ever do.

How will we know if it worked?

A few of ways. One result will be that the same types of analyses of published research that reveals the huge discrepancy in representation of females in animal and cell tissue studies can be repeated in five years and verify that the imbalance has been corrected. If it’s not, that means that the peer-review process is failing and the evaluation of sex as a biological variable has not been properly done. Second, no clinical trials would be started unless the preclinical findings were thoroughly vetted for males and females. Third, we should begin to see sex-specific treatments or dosing regimes approved by the FDA. This has already begun with the recommendation for lower doses of Ambien for woman, but unfortunately this was only after adverse events had occurred. The goal is to prevent or reduce this type of post-hoc modification going forward.

Featured Image: Lab rat. (c) JanPietruszka via iStock.