Considering Sex as a Biological Variable in Biomedical Research

In June 2015, the National Institutes of Health (NIH) announced a new policy highlighting the expectation that sex as a biological variable (SABV) be factored into research designs, analyses, and reporting of vertebrate animal and human studies. NIH-funded research grants and career-development grants are now under this new policy and the first scientific reviews are complete. Since implementation of this policy, the research community has voiced concern about exactly how to study males and females, particularly in basic research. Investigators are asking: ‘‘What does it mean to consider SABV?’’ This commentary serves to provide some perspective. Purpose and Background The Sex as a Biological Variable (SABV) policy is part of the National Institutes of Health’s (NIH’s) reenergized focus on the importance of rigor and transparency to reproducibility, including appropriate accounting for the potential influence of sex on experimental outcomes in preclinical research. Just like randomization, blinding, sample-size calculations, and other basic design elements, consideration of sex is a critical component of experimental design. Specifically: The NIH expects investigators to explain how relevant biological variables, such as sex, age, weight, and underlying health condition, are factored into research designs and analyses of studies in vertebrate animals and humans. This applies to basic, preclinical, and clinical research. Studies proposing to use only one sex should provide strong justification from the scientific literature or preliminary data to support this decision. Cost alone and absence of known sex differences are inadequate justifications for not addressing SABV. These are not new expectations. The NIH has always sought the most rigorous science—strict application of the scientific method to ensure robust and unbiased experimental design, methodology, analysis, interpretation, and reporting of results—and assessment of these factors has always been implicit in peer review. However, the NIH has now formalized these expectations for grant applications and peer review. It is important to point out that the language of the policy is purposefully broad and not prescriptive. The intrinsic goal is to encourage—never limit—the creative thinking and innovative ideas within each investigator. How researchers consider SABV must be driven by the science and context of the individual research question. Sex is a fundamental biological variable with profound consequences. Underrepresenting female cells and animals in preclinical research has resulted in a poorer understanding of the biological, physiological, and pathophysiological mechanisms in the female compared with the male. Without data from females, it is impossible to determine whether results obtained in male cells and animals also apply to female cells and animals. Historical reliance on male vertebrate animals (e.g., rats, mice) in preclinical research has resulted in the generation of incomplete data available to inform translation to clinical trials enrolling both men and women. And, these issues are not limited to the basic biological fields. A report of studies from the surgical literature revealed that for female-prevalent diseases, of those studies that stated the sex of the animals, only 12% studied female animals. We are asking investigators to consider the potential influence of sex and to address sex in the design and analysis of biomedical research. We would like to ensure that, from the very first idea about a biomedical research area, sex influences are examined. This will lead to a stronger foundation on which to build clinical research and trials and inform the community as to whether such influences will Office of Research on Women’s Health, National Institutes of Health, Bethesda, MD. Office of the Center Director, Center for Devices & Radiologic Health, U.S. Food and Drug Administration, U.S. Department of Health and Human Services, Silver Spring, MD. GENDER AND THE GENOME Volume 1, Number 2, 2017 a Mary Ann Liebert, Inc. DOI: 10.1089/gg.2017.0006 89 D ow nl oa de d by P E N N S T A T E M U L T IS IT E f ro m o nl in e. lie be rt pu b. co m a t 0 7/ 10 /1 7. F or p er so na l u se o nl y. need to be factored into the design and power calculations in clinical trials. Sex Is a Basic Biological Variable Since the landmark Institute of Medicine report entitled Exploring the Biological Contributions to Human Health: Does Sex Matter?, there has been an exponential expansion of basic science evidence and clinical data to support the concept that sex is a basic biological variable and that every cell has a sex. There are distinct differences in global gene expression patterns between male and female animals. In mice, the majority (50%–75%) of genes have been shown to be sexbiased (i.e., expressed at a different level in the two sexes) in tissues such as liver, fat, and muscle. Seventy-two percent of active genes in the liver had sexually dimorphic expression, 68% in adipose tissue, 55% in muscle tissue, and 14% in brain tissue. While sexually dimorphic genes only displayed a mean difference of 8%–9% in expression level between males and females, this sex difference in the majority of genes could reflect fundamental sex differences in physiology. In humans, DNA methylation patterns in whole blood specimens are different for men and women for genes implicated in metabolic and cardiovascular disease. CYP1A1, known to play a prominent role in metabolism of polycyclic aromatic hydrocarbons, is more active in women; this may have bearing on the fact that female smokers have been shown to have higher levels of aromatic/hydrophobic DNA adducts in lung tissue than do male smokers. Epidemiological studies suggest that women may be at greater risk for developing lung cancer than men. Also, human female liver cells have more cytochrome CYP3A compared to male liver cells. This is particularly significant as CYP3A actions account for metabolism of half of pharmacopeia drugs. Clinically, we have learned that, even though men and women should pay attention to the risk factors that affect heart health, the cardiovascular risks and responses to intervention differ between women and men. Low-dose aspirin has different preventive effects in men and women. In women, aspirin reduces risk of ischemic stroke, whereas, in men, lowdose aspirin therapy reduces risk of heart attack. Cholesterol plaque in women might not build up into major artery blockages, but instead would spread evenly throughout artery walls. Consequently, artery blockages can be more difficult to detect on coronary angiography in women, who may also present with subtle symptoms of ischemia but are still at high risk for myocardial infarction. And, women experience higher rates of adverse drug reactions than men. Although many sex differences likely stem from a differential of X and Y genes, sex hormones act directly on genes throughout the genome. Testosterone can cause significant brain sexual dimorphism. Widespread areas of the cortical mantle are significantly thicker in women than in men, ratios of gray:white matter also differ, and there are sex differences in every brain lobe. Cell-culture studies have demonstrated that male (XY) and female (XX) neurons respond differently to various stimuli. Male neurons are more sensitive to stress from reactive oxygen species and excitatory neurotransmitters, and do not have the capacity to maintain intracellular levels of reduced glutathione. Female neurons are more sensitive to some stimuli that prompt apoptosis. These data have potential implications in treatments for stroke, traumatic brain injury, neurodegenerative diseases, cerebral ischemia, and other sex-skewed neurological conditions, such as Parkinson’s disease and schizophrenia. In addition, genomic imprinting affects the development of several mental disorders in a sexually dimorphic manner, and there is accumulating evidence for effects of other inherited epigenetic mechanisms including DNA methylation, histone modifications, nucleosome repositioning, higher-order chromatin remodeling, noncoding RNA mechanisms, and RNA and DNA editing. What Does It Mean to Consider SABV? Appropriate strategies that consider SABV depend on the context of the research question, existing knowledge about male and female biology and behavior in a given area of research, available methodology, and other factors. For this reason, the language of the policy is intentionally broad. We would like investigators to consider how sex plays a role in the work that they are doing—not to adhere to a policy just to adhere to a policy. We would like investigators to truly consider, to rethink, how (and if at all) sex may be playing a role in their work, in their observations, in their experimental materials, in their study designs, in the data, in how the data are analyzed, in how the data are interpreted, and, of course, in how the data are reported. Accounting for SABV in applications for NIH-funded research could be reflected in the ways discussed in the following sections. Consider the influence of sex in study design Research findings may be influenced by sex and/or gender, as women and men are characterized by both. Factors that contribute to biological sex differences include biological and physiological characteristics encoded in DNA. These effects may include chromosomal or biochemical interactions, hormonal cycles and reproductive stages, and pathways and clinical presentations in health. Consider the role of sex chromosomes. Consider the role of sex hormones. Review available literature for the influence