Considering Sex as a Biological Variable in Basic and Clinical Studies: An Endocrine Society Scientific Statement

Abstract

In May 2014, the National Institutes of Health (NIH) stated its intent to "require applicants to consider sex as a biological variable (SABV) in the design and analysis of NIH-funded research involving animals and cells." Since then, proposed research plans that include animals routinely state that both sexes/genders will be used; however, in many instances, researchers and reviewers are at a loss about the issue of sex differences. Moreover, the terms sex and gender are used interchangeably by many researchers, further complicating the issue. In addition, the sex or gender of the researcher might influence study outcomes, especially those concerning behavioral studies, in both animals and humans. The act of observation may change the outcome (the "observer effect") and any experimental manipulation, no matter how well-controlled, is subject to it. This is nowhere more applicable than in physiology and behavior. The sex of established cultured cell lines is another issue, in addition to aneuploidy; chromosomal numbers can change as cells are passaged. Additionally, culture medium contains steroids, growth hormone, and insulin that might influence expression of various genes. These issues often are not taken into account, determined, or even considered. Issues pertaining to the "sex" of cultured cells are beyond the scope of this Statement. However, we will discuss the factors that influence sex and gender in both basic research (that using animal models) and clinical research (that involving human subjects), as well as in some areas of science where sex differences are routinely studied. Sex differences in baseline physiology and associated mechanisms form the foundation for understanding sex differences in diseases pathology, treatments, and outcomes. The purpose of this Statement is to highlight lessons learned, caveats, and what to consider when evaluating data pertaining to sex differences, using 3 areas of research as examples; it is not intended to serve as a guideline for research design.

Keywords: brain-gut; cardiovascular disease; chromosome complement; gender; sex differences; steroid hormones.

Figure 1.

Simplified view of the factors influencing sex differences in the brain. Three broad groups of factors influence the sexually dimorphic brain, as indicated by the broad, colored arrows. 1) Genes and genetic factors that influence the brain include both those on sex chromosomes and autosomes, and include both the DNA itself (represented by the classic double helix) but also chemical modification of DNA (eg, methylation) and modifications of proteins associated with DNA to form chromatin, including histones, and also changes in proteins that bind to DNA. 2) Hormones clearly influence sexual dimorphism in the brain; these are represented by the principal sex steroids, estradiol and testosterone, but also include other steroid and protein hormones (progestins, MIH, oxytocin, prolactin, etc). 3) The environment includes a wide spectrum of influences, including perinatal nutrition and familial support, socioeconomic and demographic factors, intrinsic factors of brain development, age, and gender, and larger environmental factors, such as education, profession, and societal expectations (the “gendered environment”). In addition to each class of factor influencing the brain (bold arrows), the human brain also reciprocally influences each of these groups of factors. Furthermore, each group of factors influences the other, as represented by the dotted arrows. Some examples include: the environment influences genes via epigenomics and genes influence the environment by population sizes and domains; the environment influences hormones by seasonal variations and the actions of xenobiotics, and hormones influence the environment by promoting reproduction and consumption of foodstuffs; genes directly influence hormones by regulating their production and action, and many hormones, including all steroid hormones, regulate gene transcription.

Figure 2.

Schematic diagram of the Four Core Genotypes mouse model. The testis-determining gene Sry is deleted from the Y chromosome, producing the Y^‒ chromosome. An Sry transgene is inserted onto chromosome 3. Thus, the type of gonad is no longer linked to the sex chromosomes. The model produces XX and XY mice with Sry and testes, and XX and XY mice without Sry, with ovaries. Sex differences in phenotype can be attributed to an effect of gonadal hormones, comparing mice with ovaries and testes, or to an effect of sex chromosomes, comparing XX and XY mice with the same type of gonad. [Modified with permission from Arnold AP & Chen X. Front Neuroendocrinol, 2009; 30(1) © Elsevier Inc. (112)].

Figure 3.

Developmental trajectories for total brain tissue volume, gray matter volume, and white matter volume in men and women over Development. Person-level data are shown for women (red) and men (blue) as points, with lines linking measures from the same person over time. Note the large interindividual variation in volumes within each sex, and the overlap of these distributions, between the sexes. Superimposed on these person-level data are group-level best fit volume trajectories (bold lines with shaded 95% confidence intervals). The developmental window covered is 5 to 25 years of age. For all plots, there are statistically significant sex differences in both trajectory shape (ie, sex differences in the tempo of volume change, P < 0.00001), and trajectory “height” (ie, sex differences in absolute volume across ages, P < 0.00001). [Adapted with permission from Giedd JN et al. Neuropsychopharmacology, 2015; 40 © Springer Nature (153)].

Figure 4.

Sex differences in structural connectomes across development. Connectomes representing the white matter structural connectivity in the brain, with nodes indicating the brain regions and edges between the nodes representing the structural connectivity between the nodes. Node colors representing respective brain regions are as follows: dark blue, frontal; cyan, temporal; green, parietal; red, occipital; white, subcortical. The depicted edges shown are those that survived permutation testing at P = 0.05. A, shows increased intrahemispheric connectivity in men (Upper, in blue) and increased inter-hemispheric connectivity in women (Lower, in orange) on average. B-D: Connectivity differences shown in A separated by age groups are shown: B, under 13 years, C, adolescent (13-18 years), and D, young adults (18-22 years). Left image: Men/Boys; Right image: Women/Girls. [Adapted with permission from Ingalhalikar M et al. Proc Natl Acad Sci U S A, 2014; 111(2) © National Academy of Sciences (163)].

Figure 5.

Heart and kidney functions are linked. Sex differences exist in many aspects of heart and kidney function at baseline and in CVD and CKD, as shown. Both organs feed-forward and influence each other’s function. Genes, hormones, and age are some known factors that modulate this relationship in a sex-specific manner. Abbreviations: CKD, chronic kidney disease; CVD, cardiovascular disease.