Analyzing Sex in Tissues and Cells

In 2001, the U.S. Institute of Medicine declared that “every cell has a sex” (Pardue et al., 2001). Progress has been made over the last two decades in understanding how to analyze sex in tissues and cells (Shah et al., 2014; Ritz et al., 2014; Clayton, 2016; Tannenbaum et al., 2016; Docherty et al., 2019).

General Points
  • 1. 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 in order to ensure that experiments are reproducible and that findings (in one sex) are not over-generalized (to the other sex) (Wizemann, 2012).
  • 2. 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. Cell media might influence cell behavior. For example, phenol red, a common pH indicator, is estrogenic and as such it can alter cell responses (Mauvais-Jarvis et al., 2017).
  • 3. Consider the basic question to be addressed and how the study design and outcomes might be affected by the sex of tissues and cells. Sex differences must be considered before they can be ruled out.
Identify problems and formulate hypotheses
  • • Distinguish between sex (biological) and gender (socio-cultural). Sex is genetically intrinsic to every cell of a sexually reproducing organism. In human tissues, 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 they are removed from a donor. In studies of bone tissue, for example, bone development is influenced by biological sex (in part through the action of sex steroid hormones) but also by gender (by diet and patterns of physical activity).
  • • “Sex” and “gender” are frequently and incorrectly used interchangeably in the literature. Therefore, 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 life sciences research, it may be necessary to use search terms related to the female/male distinction, such as sex steroid hormones, gonadal hormones, sex chromosomes, estrogens, androgens and steroid receptors.
  • • 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 other terms 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).
Research Design
  • • Determine how a given experiment will use sex as a biological variable. This decision can be based on prior sex-specific research or assumptions about biological plausibility.
  • • Cells and cell lines can be selected for study in different ways depending on the types of questions to be asked (see Table 1).
Table 1: Configuring Biological Experiments on Cell Lines and Tissue Samples
Cell Lines and/or Tissue SamplesStudy Characteristics and Considerations
Cells and tissues from female and male donors Cell lines of female or male cells will have different genetic characteristics and may exhibit differences in growth rate, metabolism and response to stimuli (Ritz, 2014).

If sex is used as a variable, other donor characteristics should be matched or differences must be controlled for.

Studies of both female and male cells give insight into specific forms of sexual dimorphism.

Analyzing other factors that may intersect with sex is critical in order to avoid overlooking sex differences as well as to avoid overemphasizing sex.

Single-sex Studies in cells or tissues from only one sex (female or male) may be useful in closing research gaps, investigating differences among cell types within a sex, or studying diseases or interventions that are female-specific or male-specific.

For example, female-only studies might be used to investigate how cells differ by hormonal status (pre-pubescent; pre-menopausal with normal ovulation; pre-menopausal with ovulation altered by hormonal contraceptives, drugs or stress; and menopausal), age, circadian cycle and other factors. Male-only cells or tissues may be useful in closing research gaps, investigating differences among cell types within a sex, or studying diseases or interventions that are male-specific (e.g. prostate cancer).

Results in single-sex studies should not be generalized to the general population.

Collect data

  • Sample Matching: When sex is used as a variable, tissues and cells should be matched by non-sex characteristics that might influence outcome (such as age, hormonal status or the reproductive history of the donor). Alternatively, when such matching is not feasible, results can be disaggregated by sex and then adjusted depending on statistically-significant differences between female-derived and male-derived tissues or cells by non-sex traits, presuming that such differences can be measured and their effects on outcome are known.
  • Cell Media: 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). Cell media might influence cell behavior. For example, phenol red, a common pH indicator, is estrogenic and as such it can alter cell responses (Mauvais-Jarvis et al., 2017).
Analyze data
  • • 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. Mixing cultures of cells from female and male animals. Do not mix cultures of cells from female and male animals, as cells may have different rates of cell cycle and proliferation and may respond differently to stimuli for apoptosis and growth factors added to the cell media. 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).
    • 3. Concluding that sex differences exist without accounting for confounding variables (see Method: Intersectional Approaches).
    • 4. Interpreting results in a sex-blind manner.
    • 5. Assuming that differences associated with sex also apply to gender.
Reporting results
  • • 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 in order to reduce publication bias, enable meta-analysis, and promote the identification of confounding variables.
  • • Check that sex differences are properly visualized in the tables, figures, and conclusions (see Rethinking Language and Visual Representations).
  • • Check that sex-related findings are presented correctly in the title, abstract and keywords.
  • • If significant sex differences emerge, describe the follow-up research required.
  • • When sex differences are identified, specify how these findings might be translated into preventive, diagnostic and therapeutic practices to improve patient outcomes.

Works Cited

Clayton, J. A. (2016). Studying both sexes: a guiding principle for biomedicine. FASEB J., 30, 519–524.

Docherty, J. R., Stanford, S. C., Panattieri, R. A., Alexander, S. P., Cirino, G., George, C. H., ... & Sobey, C. G. (2019). Sex: A change in our guidelines to authors to ensure that this is no longer an ignored experimental variable. British Journal of Pharmacology, 176(21).

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.

Mauvais-Jarvis, F., Arnold, A. P., & Reue, K. (2017). A guide for the design of pre-clinical studies on sex differences in metabolism. Cell metabolism, 25(6), 1216-1230.

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).

Messing, K., Parent, L., St-Pierre, J., Vaillancourt, C., & 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.

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.

Rich-Edwards, J. W., Kaiser, U. B., Chen, G. L., Manson, J. E., & Goldstein, J. M. (2018). Sex and gender differences research design for basic, clinical, and population studies: essentials for investigators. Endocrine Reviews, 39(4), 424-439.

Ritz, S. Antle, D., Côté, J., Deroy, K., Fraleigh, N., Shah, K., McCormack, C., & Bradbury, N. (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.



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