In 2001, the U.S. Institute of Medicine declared that 'every cell has a sex' (Pardue et al, 2001). In the past decade, progress has been made in understanding how to analyze sex and, to a lesser extent, gender in tissues and cells (Shah et al., 2014; Ritz et al., 2014; Clayton, 2016; Tannenbaum et al., 2016).
This checklist is intended as an aid for researchers, grant writers, project directors, evaluators, and funding organizations as they address and assess the incorporation of sex and gender analyses into bioscience research utilizing tissues and cells derived from animals, including humans. As such, it should be read in conjunction with the methodology described in Designing Health & Biomedical Research and in the Checklist Health & Medicine (applicable to humans and animal models).
General Points1. Not every experiment needs to be designed to evaluate sex differences. However, for every experiment, the sex of the tissues or cells needs to be noted and reported, to ensure that experiments are reproducible and findings (in one sex) are not over-generalized (to the other sex) (Wizemann, 2012). 2. Sex differences must be investigated before they can be ruled out (see Not Considering Sex Difference as a Problem). Not all observed sex differences are biological in origin (see Overemphasizing Sex Differences as a Problem). Consider the basic question to be addressed and how the study design and outcomes might be affected by gender and the sex of tissues and cells.
3. It is important to consider whether the expression of genes on the sex chromosomes or autosomes of a cell or tissue under study is influenced by sex hormones. The expression of genes may be influenced by sex steroidal hormones in the cell culture media, or by the hormonal environment of the donor animal (Veilleux et al., 2012). Cells removed for in vitro experiments may behave differently than in vivo.
Step 1: Establishing the research question and hypotheses:
- ● Understand the analytical distinctions between sex (biological) and gender (socio-cultural).
- ● Sex: Sex is genetically intrinsic to every cell of a sexually-reproducing organism. Even if a cell is removed from its normal bodily environment and is no longer subject to the effects of sex hormones, "complement of sex chromosomes in cells studied in culture has the potential to affect expression of proteins and 'mechanistic' signaling pathways" (Taylor et al., 2011; Yoon et al., 2014). ● Gender: Recognize that gender has explanatory power (see Analyzing Gender Assumptions). Gender can influence tissue and cell experiments in many ways, even though tissues and cells are not themselves gendered—for instance, gender influences research priorities, research designs, and the development of cells and tissues before being removed from a donor. For example, in studies of bone tissue, bone development is influenced by biological sex (in part through the action of sex steroid hormones) but also by diet and patterns of physical activity, both of which are influenced by gender roles. Differences between women's and men's bones cannot be attributed to biology alone (Fausto-Sterling, 2005). Similarly, researchers have observed differences in genetic expression between lung tumors derived from women and from men. Such differences may be created by "constitutional genetic differences", (biological sex differences) and/or factors modulated by gender, including smoking behavior (Planchard et al., 2009).
Step 2: Literature Search
- ● Perform a literature and database search with adequate terms for "sex" and "gender." MeSH (Medical Subject Headings)—the U.S. National Library of Medicine controlled-vocabulary thesaurus used for indexing articles for PubMed)—does not distinguish consistently between sex and gender (in large part because authors do not). Hence, researchers need to develop search strategies to identify the full range of previously documented sex and gender differences. In basic life sciences research, search terms related to the female/male distinction, such as sex steroid hormones, gonadal hormones, sex chromosomes, estrogens, and androgens or steroid receptors may be required.
- ● Several studies suggest combining the name of a condition or biomedical research topic with standardized MeSH terms, such as “sex factors" and "sex characteristics," or text words, such as "gender differences" and "sex differences." Researchers have developed search strategies that yield better results than "sex" or "gender" alone (Oertelt-Prigione et al., 2010; Moerman et al., 2008). For example, useful MeSH terms include:
MeSH termDefinition (Scope Note)
Sex factorsMaleness or femaleness as a constituent element or influence contributing to the production of a result. It may be applicable to the cause or effect of a circumstance. It is used with human or animal concepts but should be differentiated from SEX H terms includeCHARACTERISTICS, anatomical or physiological manifestations of sex, and from SEX DISTRIBUTION, the number of males and females in given circumstances.
Sex characteristicsThose characteristics that distinguish one SEX from the other. The primary sex characteristics are the OVARIES and TESTES and their related hormones. Secondary sex characteristics are those which are masculine or feminine but not directly related to reproduction.
Sex distributionThe number of males and females in a given population. The distribution may refer to how many men or women or what proportion of either in the group. The population is usually patients with a specific disease but the concept is not restricted to humans and is not restricted to medicine.
SexThe totality of characteristics of reproductive structure, functions, PHENOTYPE, and GENOTYPE, differentiating the MALE from the FEMALE organism.
Sex ratioThe number of males per 100 females.
Gender identitySocially-constructed identity of male or female. NOTE: psychological; do not confuse with SEX CHARACTERISTICS (physiological); do not confuse with SEXUAL ORIENTATION see SEX BEHAVIOR: gender identity is knowing that one is male or female; sexual orientation is preferring heterosexual or homosexual behavior; no qualif.
- ● In addition, a database has been established that includes a survey of sex and gender related references in major clinical disciplines and biomedical basic research (see http://bioinformatics.charite.de/gender/. This website has two login prompts. At the first prompt, enter User: "gender" and Password: "ppgm". At the second prompt, enter User: "guest" and Password: "guest"). In basic life sciences research, it may also be helpful to use search terms related to the female/male distinction, such as sex hormones or reproductive biology.
Step 3: Establishing Research Questions and Hypotheses (see Formulating Research Questions)
- ● Determine if a given experiment will utilize sex as a variable. This decision can be based on prior basic science research, which may allow researchers to estimate the likelihood of significant sex differences in regard to the experimental intervention used and the outcome measured. Epidemiological research may also be informative; a disease with significantly different prevalence, prognosis, etc. in women and men might imply sex differences on the cellular level.
- ● Cells and cell lines can be selected for study in different ways depending on the types of questions to be asked:
Step 4: Establishing Research Methods and Planning Data Analysis (see Designing Health & Biomedical Research)
- ● Sample Matching: If sex is used as a variable, tissues and cells should be matched according to non-sex characteristics which might influence outcome (such as age, hormonal status, or reproductive history); alternatively, if such matching is not feasible, results can be adjusted according to statistically-significant differences between female- and male-derived tissues or cells according to non-sex traits, presuming that such differences can be measured and their effects on outcome are known.
- ● Cell Media: Mixed cultures of cells from female and male animals, such as those used to study neonatal tissues, must be validated to assure that viability, rate of cell cycle and proliferation, and stimuli for apoptosis and growth factors added to the cell media affect cells of both sexes the same. For example, female and male cells have different sensitivity to certain apoptotic agents—and these differences are modulated by cell type and age (Penaloza et al., 2009).
Step 5: Interpreting Data through Concepts and Theoretical Models
- ● Analyze all concepts and theoretical models for unfounded assumptions (see Rethinking Concepts and Theories).
- ● Studies should take care to avoid:
- 1. Assuming that findings in one sex apply to the other.
- 2. Concluding that sex differences exist without accounting for confounding variables (see factors intersecting with sex and gender).
- 3. Interpreting results in a sex- or gender-blind manner.
Step 6: Reporting Findings (see Analyzing Sex).
- ● Report the sex of cells and tissues used in research, even in single-sex experiments. (Wizemann, 2012).
- ● Report null findings. Researchers should report when sex differences (main or interaction effects) are not detected in their analyses to reduce publication bias, enable meta-analysis, and promote the identification of confounding variables
- ● Check that sex or gender differences are properly visualized in the tables, figures, and conclusions (see Rethinking Language and Visual Representations).
- ● Check that sex and gender related findings are presented correctly in the title, abstract, and keywords.
Step 7: Establishing Conclusions and Making Recommendations
- ● Where significant sex differences emerge, what follow-up research is necessary?
- ● When sex differences are identified, how can these findings be translated into preventive, diagnostic, and therapeutic practices to improve patient outcomes?
Recommendation: Require Researchers to Report the Sex of Tissues and CellsGranting agencies and journal editors can require researchers to report and analyze the sex of the tissues and cells used in order for research to be funded or findings published.
Clayton, J.A. (2016). Studying Both Sexes: A Guiding Principle for Biomedicine. The FASEB Journal, fj-15.
Fausto-Sterling, A. (2005). The Bare Bones of Sex, Part 1: Sex and Gender. Signs: Journal of Women in Culture and Society, 30 (2), 1491-1527.
Moerman, C., Deurenberg, R., Haafkens, J. (2009). Locating Sex-Specific Evidence on Clinical Questions in MEDLINE: A Search Filter for Use on OvidSP. BioMed Central Medicine Medical Research Methodology, 9 (25).
Oertelt-Prigione, S., Parol, R., Krohn, S., Preissner, R., & Regitz-Zagrosek, V. (2010). Analysis of Sex and Gender-Specific Research Reveals a Common Increase in Publications and Marked Differences between Disciplines. BioMed Central Medicine, 8, 70-80.
Pardue, M., & Wizemann, T. (Eds.) (2001). Exploring the Biological Contributions to Human Health: Does Sex Matter? Washington D.C.: National Academy Press.
Penaloza, C., Estevez, B., Orlanski, S., Sikorska, M., Walker, R., Smith, C., Smith, B., Lockshin, R., & Zakeri, Z. (2009). Sex of the Cell Dictates its Response: Differential Gene Expression and Sensitivity to Cell-Death-Inducing Stress in Male and Female Cells. Journal of the Federation of American Societies for Experimental Biology, 23 (6), 1869-1879.
Planchard, D., Loriot, Y., Aoubar, A., Commo, F., & Soria, J. (2009). Lung Cancer in Women: Differential Expression of Biomarkers in Men and Women. Seminars in Oncology, 36 (6), 553-565.
Ritz, S. Antle, D., Côté, J., Deroy, K., Fraleigh, N., Messing, K., Parent, L., St-Pierre, J., Vaillancourt, C., and Mergler, D. (2014). First Steps for Integrating Sex and Gender Considerations into Basic Experimental Biomedical Research. Journal of the Federation of American Societies for Experimental Biology 28, 4-13.
Shah, K., McCormack, C., & Bradbury, Neil, (2014). Do you Know the Sex of Your Cells? American Journal of Physiology - Cell Physiology, 306, C3-C18.
Tannenbaum, C., Schwarz, J., Clayton, J., de Vries, G., & Sullivan, C. (2016). Evaluating Sex as a Biological Variable in Preclinical Research: The Devil in the Details. Biology of Sex Differences, 7(1), 1.
Taylor, K., Vallejo-Giraldo, C., Schaible, N., Zakeri, R., & Miller, V. (2011). Reporting of Sex as a Variable in Cardiovascular Studies using Cultured Cells. Biology of Sex Differences, 2 (11), 1-7.
Veilleux, A., & Tchernof, A. (2012). Sex Differences in Body Fat Distribution. In Symonds, M. (Ed.), Adipose Tissue Biology, pp. 123-166. New York: Springer Science and Business Media.
Wizemann, T. (Ed.) (2012). Sex-Specific Reporting of Scientific Research: A Workshop Summary. Washington, D.C.: National Academies Press.
Yoon, D., Mansukhani, N., Stubbs, V., Helenowski, I, Woodruff, T., & Kibbe, M. (2014). Sex Bias Exists in Basic Science and Translational Surgical Research. Surgery, 156 (3), 508-516.