In order for basic and preclinical research to be translated to the clinic, it is not enough to simply include females in experiments—potential effects of sex must also be examined
Until relatively recently, basic biomedical research was almost exclusively conducted with male human, animal, and cell models. It was widely assumed that the research findings and medical treatments developed from these studies could be generalized to the whole population. However, there are sex differences in key biological pathways and processes that can influence a person’s disease risk, experience of symptoms, and response to treatment. The lack of female representation in preclinical biomedical research has resulted in gaps in our medical knowledge, with important consequences for women’s health. In the past decade, efforts to remedy this historic exclusion have increased, but opportunities remain to make basic biomedical research more equitable, reproducible, and applicable to all people.
Excluded from experiments
First, it’s important to differentiate between the biological construct of sex and the societal construct of gender. All animals, including humans, have sex, which is defined by a set of physical and physiological traits. In most biological research, sex is categorized as male or female (although variations do occur). To the best of our current knowledge, gender is relevant only for research with humans and can include the categories of men, women, and nonbinary people [1]. This article focuses on fundamental and preclinical studies, which are typically conducted in human, animal, or cell culture models, and thus will use the terms male and female to refer to research subjects.
The common and preferential practice of studying only male research subjects is largely due to the assumption that females are the more variable sex. It was a longstanding belief among (historically male) biomedical researchers that the hormonal fluctuations associated with menstrual cycles in women and estrous cycles in female mammals would make data collected from these subjects more variable [2].
However, whether such sex differences in variability even exist was not experimentally addressed until recently, says Nicole Woitowich, a research assistant professor in the Department of Medical Social Sciences, Northwestern University’s Feinberg School of Medicine (Figure 1). “Eventually, a small but mighty contingent of biomedical scientists began pushing against the exclusion of females simply because it would ‘complicate’ data,” she says. “And in fact, there have now been many studies that show female animals are not intrinsically more variable than males.”
Figure 1. Dr. Nicole Woitowich of Northwestern University. (Photo courtesy of Nicole Woitowich.)
Why sex matters
The exclusion of female subjects was further justified by a belief that results found in males could be generalized to both sexes. This erroneous assumption has had dire consequences.
Séverine Lamon, a research associate professor and a molecular biologist at Deakin University (Figure 2), says that this sex bias has been especially pronounced in preclinical research, which informs our knowledge of human biology and disease and may eventually be applied to human health. “It’s like building a house without the right foundation,” she says. “If the foundational research is not balanced, then the clinical applications will be unbalanced.”
Figure 2. Dr. Séverine Lamon, a molecular biologist at Deakin University. (Photo courtesy of Deakin University.)
This has been apparent in drug pharmacokinetics, for instance. Eight out of ten of the drugs removed from the U.S. market between 1997 and 2000 were withdrawn due to side effects occurring mainly or exclusively in women [3]. Females and males may metabolize drugs differently, leading to differences in blood concentrations, drug elimination times, and side effects. This was the case for the sleeping pill zolpidem (Ambien), first approved by the Food and Drug Administration (FDA) in 1992. After decades of reports of cognitive side effects in women, the FDA adjusted its recommended dosage based on sex [2].
Addressing the sex bias in biomedical research has the potential to not just improve quality of care for women but for all people. For example, researchers have known for decades that men are more likely than women to develop a form of brain cancer called glioblastoma, and that the standard therapy for this disease is more effective in women than men. In a new study, researchers identified different molecular signatures in the tumors of men and women that help explain the disparities in patients’ response to treatment and survival. The findings suggest that considering the sex of the patient and the molecular subtype of their tumor may improve survival for all patients [4].
Funding organizations step up
Pressure to include females in biomedical research grew in the 1990s and early 2000s, as more researchers called attention to the limitations of sex-biased studies. The movement was galvanized by a 2009 review of the scientific literature that quantified the extent of sex bias across several biological disciplines. The review revealed a male bias in eight of the ten biological disciplines surveyed. The ratio of studies reporting on only male subjects versus only females was most skewed in the fields of neuroscience (5.5–1) and pharmacology (5–1)—two disciplines with strong preclinical relevance [2], [5].
“It wasn’t until 2016 when the National Institutes of Health (NIH) implemented a policy requiring investigators to consider sex as a biological variable in federally funded research,” says Woitowich. “Now, investigators are asked to consider how sex might be a key variable in their studies and requires them to provide a significant justification for using only a single sex.”
The policy puts forth expectations that researchers will factor sex as a biological variable into research questions, study designs, analyses of data, and reporting of findings. The policy states that “Consideration of sex may be critical to the interpretation, validation, and generalizability of research findings. Appropriate analysis and transparent reporting of data by sex may therefore enhance the rigor and applicability of preclinical biomedical research” [6].
The Canadian Institutes of Health Research (CIHR) have also responded to calls for sex-inclusive research practices and policies with their Sex and Gender-Based Analysis in Research Action Plan. Like NIH, CIHR expects all investigators to integrate sex and/or gender into their research designs and analyses when appropriate [7].
The NIH and CIHR are leading the way with their sex- and gender-inclusive policies. The Australian equivalent, the National Health and Medical Research Council (NHMRC), for example, only indirectly recommends that researchers include both sexes in the design and analysis of their studies [8], [9]. Lamon, who is involved in efforts to make this a requirement of federally funded research in Australia, says that without enforcement to incentivize researchers to include female subjects, the country lags behind the U.S. and Canada in this important effort. “I am surprised that Australia hasn’t tackled this issue earlier, and now we have some serious catching up to do,” she says. “Implementing a policy like this can be a lengthy process. And I think we are late to the party.”
Progress, and room for improvement
In 2019, Woitowich and colleagues conducted follow-up analyses to assess the impact of NIH’s Sex as a Biological Variable policy. Overall, the number of studies including females grew to 49%, up from 28% ten years earlier. The researchers noted increased inclusion of female subjects in six out of nine biological disciplines surveyed [5].
However, the findings were not all good news. The majority of studies using only a single sex failed to provide evidence-based rationale. In fact, several studies explicitly stated that they only used one sex to prevent experimental variability, now known to be a misconception and unsound research practice [5].
Further troubling was the finding that many of the studies that used both sexes did not specify whether they had analyzed the results for sex differences [5]. “When you do not consider sex as a variable in your analyses, it causes statistical inaccuracies that down the road lead to wrong interpretations of the results,” says Lamon.
In order for basic and preclinical research to be translated to the clinic, it is not enough to simply include females in experiments. Researchers must also examine the potential effects of sex. The inclusion of sex as a factor in the analysis allows the identification of sex differences if they are present. Researchers should not pool data from males and females without screening for sex differences and reporting the results. Otherwise, other researchers are left to assume either that there are no sex differences or that any sex differences have yet to be studied. Either way, this may result in redundant studies requiring additional time, money, and biological resources [5].
Woitowich says it “boggles her mind” that so many researchers are either not analyzing data by sex or failing to report it in their methods. “It’s wasteful and it’s not good science,” she says.
Training the next generation
While the biomedical sciences have made strides in including female subjects in research, there are still gaps in analyses and reporting of data by sex. Lamon says that funding organizations and scientific journals, as gatekeepers for this research, can advance sex-inclusive science by requiring researchers to consider sex as a variable.
“I also think as researchers, we have an individual responsibility in not only designing research that includes equal representation of sex and/or gender, but in designing studies that allow comparisons between males and females,” she says. “In addition, we have an educational duty. As a group leader, it’s important to me that my students understand and carry on this message of including both sexes and/or gender in research design and data analysis.”
Woitowich agrees on the importance of educating the next generation of scientists. “I’m already seeing a change in early career scientists, where they are invested in equity in research and put that at the forefront when they design studies,” she says. “I think it’s phenomenal that this has become a part of standard scientific training. We will see the impacts of how young investigators today are being trained in the years to come.”
To build on the progress that has been made, there is a continued need for education, awareness, and advocacy regarding sex-based research practices. With concerted efforts at the institutional, funding, and publishing levels, the consideration of sex as a biological variable may become standard practice in basic and preclinical research, contributing to a more complete understanding of health and disease for all people.
References
- National Institutes of Health: Sex & Gender. Accessed: May 25, 2023. [Online]. Available: https://orwh.od.nih.gov/sex-gender
- I. Zucker, B. J. Prendergast, and A. K. Beery, “Pervasive neglect of sex differences in biomedical research,” Cold Spring Harbor Perspectives Biol., vol. 14, no. 4, May 2022, Art. no. a039156, doi: 10.1101/cshperspect.a039156.
- K. Burrowes, “Gender bias in medicine and medical research is still putting women’s health at risk,” The Conversation, Mar. 7, 2021. [Online]. Available: https://theconversation.com/gender-bias-in-medicine-and-medical-research-is-still-putting-womens-health-at-risk-156495
- W. Yang et al., “Sex differences in GBM revealed by analysis of patient imaging, transcriptome, and survival data,” Sci. Transl. Med., vol. 11, no. 473, Jan. 2019, Art. no. eaao5253, doi: 10.1126/scitranslmed.aao5253.
- N. C. Woitowich, A. Beery, and T. Woodruff, “Meta-research: A 10-year follow-up study of sex inclusion in the biological sciences,” eLife, vol. 9, Jun. 2020, Art. no. e56344, doi: 10.7554/eLife.56344.
- National Institutes of Health Policy on Sex as a Biological Variable. Accessed: May 25, 2023. [Online]. Available: https://orwh.od.nih.gov/sex-gender/nih-policy-sex-biological-variable
- Canadian Institutes of Health Research: Sex and Gender in Health Research. Accessed: May 6, 2023. [Online]. Available: https://cihr-irsc.gc.ca/e/50833.html
- S. Lamon and O. Knowles, “Why are males still the default subjects in medical research?’’ Conversation, Oct. 4, 2021. [Online]. Available: https://theconversation.com/why-are-males-still-the-default-subjects-in-medical-research-167545
- B. Whitehead, “Male participants will no longer be the norm in medical research,” InSight, May 8, 2023. [Online]. Available: https://insightplus.mja.com.au/2023/16/male-participants-will-no-longer-be-the-norm-in-medical-research/