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Mind the Gap: Sex Bias in Basic Skin Research Betty Y. Kong1,2, Isabel M. Haugh1,2, Bethanee J. Schlosser1, Spiro Getsios1 and Amy S. Paller1

Given the recent National Institutes of Health proposal for balanced use of male and female cells and animals in preclinical studies, we explored whether sex bias exists in skin research. We surveyed 802 dermatological research articles from 2012 through 2013. No information about the sex of studied cells or animals was provided in 60% of papers. Among keratinocytes of known sex, 70% were male. Few studies compared male versus female cells or animals. Disclosure of sex and comparative studies contribute to our understanding of the biologic basis of sex differences. Addressing sex-specific differences in preclinical research informs subse- quent clinical trial design and promotes individualized therapy. Journal of Investigative Dermatology (2016) 136, 12e14; doi:10.1038/JID.2015.298

More than 20 years ago, the US Comparative studies are needed and cutaneous SCC progression in mice National Institutes of Health estab- to discover and elucidate differences (Brooks et al., 2014), as well as the lished the Office of Research on in disease prevalence, impact, and lower catalase levels in the skin and Women’s Health, aimed in part at response to interventions, which could ultraviolet B-induced myeloid cells of increasing representation of women in have a genetic, epigenetic, hormonal, male mice (Sullivan et al., 2012)may clinical trials. A parallel call to action, and/or behavioral (e.g., sun protection contribute to the two- to threefold however, for male and female sex rep- habits) basis. Several results from increase in nonmelanoma skin cancers resentation in cell- and animal-based in vitro and in vivo comparative in men and the larger, histologically research was only raised in October research show that differences originate advanced, more numerous, and less 2014, when the National Institutes from intrinsic cellular differences and inflamed UVB-induced SCCs in male of Health announced its intention are further dichotomized by the hairless mice (Thomas-Ahner et al., to develop guidelines requiring in- different hormonal environments to 2007). Finally, sex-specific differences vestigators to report plans for balancing which they are exposed. For example, in the expression of glucocorticoid male and female cells and animals in CD4þ T cells from women produce receptors exist in the liver, central preclinical studies (Clayton and Collins, more IFNg (Th1 bias) and from men nervous system, and immune system, 2014; Sandberg et al., 2015). produce more IL-17A (Zhang et al., suggesting that male skin may also More women than men seek derma- 2012). Male gut microbiota transferred be more sensitive to corticosteroids tological consultation for skin disease; to female nonobese diabetic mice pro- (Chrousos, 2010; Duma et al., 2010; our analysis of 149,614 patients seen tects against the development of Quinn et al., 2014). for cutaneous issues (based on ICD-9 autoimmunity in an androgen receptor- To explore how often discovery in diagnosis codes) in outpatient clinics dependent manner (Markle et al., cutaneous biology stems from the study at Northwestern Medicine revealed a 2013). These differences begin to of one sex and whether sex is dis- female to male ratio of 1.8:1. Although explain the predominance of autoim- closed, we evaluated a cohort of social behavior may influence seeking mune disorders in women. research publications from 1 January consultation, biological differences may In cutaneous biology, comparative 2012 to 31 December 2013 that underpin this imbalance. For example, studies are beginning to define the distinct included all “Original Articles” in three women exhibit a greater prevalence of action of estrogen on estrogen alpha and dermatological science-focused jour- rosacea, lupus, and scleroderma with beta receptors and their relationships nals (Journal of Investigative Derma- a different distribution of androgenetic with the IGF-1 receptor in understanding tology, Journal of Dermatological alopecia in comparison with men. In wound re-epithelialization, macrophage Science, and Experimental Derma- addition, melanoma incidence rates polarization, and why skin wound heal- tology). Of 802 original articles are higher in younger females and ing is slower in men than in women reviewed, one or more animal model older males (Liu et al., 2013), with a (Campbell et al., 2010; Emmerson et al., or cell type was studied in 549 higher death rate from melanoma in 2012, 2013; Markiewicz et al., 2013; (Table 1). Both animals and cells were males (De Giorgi et al., 2011; Joosse Campbell et al., 2014). The impact of studied in 143 papers, more than one et al., 2011). estrogen on keratinocyte differentiation cell type in 126, and more than one

1Department of Dermatology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA 2 The first two authors contributed equally to this work. Correspondence: Amy S. Paller, Department of Dermatology, Northwestern University Feinberg School of Medicine, 676 North St. Clair Street, Suite 1600, Chicago, Illinois 60611, USA. E-mail: [email protected] Received 9 April 2015; revised 14 June 2015; accepted 14 July 2015; corrected proof published online 20 August 2015

12 Journal of Investigative Dermatology (2016), Volume 136 ª 2015 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology. BY Kong et al. Sex Bias in Basic Skin Research

Table 1. Published reports using cells and animals in skin research Mice of both sexes were used in 4.3% of papers, but few compared male and Journal No sex stated Male only Female only Both sexes Total female mice in the same experiments. Cells1 J Invest Dermatol 119 112 14 9 254 Our findings of sex bias in basic J Dermatol Sci 104 26 4 0 134 science skin research are echoed by Exp Dermatol 100 34 4 9 147 studies in other fields (Beery and Total 323 172 22 18 535 Zucker, 2011; Taylor et al., 2011; Yoon Mice2 J Invest Dermatol 124 11 44 5 184 et al., 2014), in which the sex of the J Dermatol Sci 21 12 19 3 55 origin of cells and animals was largely Exp Dermatol 14 8 15 4 41 unreported and, if known, was primarily Total 159 31 78 12 280 male. Tissue from noncutaneous sour- 1Cells included primarily normal human keratinocytes, melanocytes, and fibroblasts. ces is no more accessible in males than 2 ¼ ¼ ¼ ¼ Other animal models used included rat (N 8), zebrafish (N 5), rabbit (N 4), pig (N 2), guinea pig females; hence, this sex bias most likely (N¼2), horse (N¼1), hamster (N¼1), chicken (N¼1), and sheep (N¼1). originates from the historical conven- tion of using male-only models in biomedical research. Concerns about animal type in 74 (generally more than male patient (Boukamp et al., 1988). hormonal effects and added financial one mouse strain). The preponderance of studies with male costs to adequately power both male Among 535 papers utilizing cultured cells is not surprising, given the easy and female cohorts, coupled with an cells, 60.4% disclosed no information availability of male foreskins as a source insufficient appreciation for potential about the sex of origin, whereas 32.1% of cultured cells. However, our knowl- differences in outcome, may further used only male cells and 4.1% only edge from these male sex-biased studies exacerbate the bias. Moreover, the age female cells (Figure 1a). Only 3.3% of may not be generalizable if inherent cell of the source of cells (i.e., neonatal studies examined male and female cells differences related to sex exist. versus adult) makes a difference bio- but rarely in the same experiments. Of Animal models were studied in logically (Gilchrest, 1983; Gilchrest 139 studies utilizing normal human 305 papers, with mice used in 92.1% et al., 1982; Gilchrest et al., 1984). keratinocytes (most common cell type), (Table 1). Mouse sex was unspecified in Thus, testing hypotheses in both male sex was unstated in 60.4%, only male in 56.8% of the papers, but 27.9% re- and female adult skin-derived cells not 29.5%, only female in 4.3%, and both ported only females and 11.1% only only addresses sex bias but also more sexes in 5.8% (Figure 1b). Forty-four males (Figure 1a). This relatively greater closely models downstream clinical publications utilized the HaCaT kerati- use of female mice may reflect the applications. Male and female skin for nocyte line, which originated from a greater aggressiveness of male mice. culture is currently available from abdominoplasties, reduction mammo- plasties, rhytidoplasties (face lifts), and normal volunteers. Why is it important to perform comparative analyses? Limiting studies to only one sex may lead to discoveries that are only relevant for one sex. Furthermore, having preclinical studies in mice of a single sex increases the risk of designing clinical trials that are inappropriate or even harmful for the non-studied sex. Of the 10 prescription drugs withdrawn from the US market between 1997 and 2000, eight “posed greater health risks for women than for men,” and half of these were widely prescribed to both sexes (http:// www.gao.gov/products/GAO-01-286R). Perhaps the most widely publicized case of sex differences in medication response is the use of zolpidem (Ambien) for insomnia. More than 20 years after its initial approval, the recommended dose for women was Figure 1. Underreporting of cell and animal sex in basic skin research. (a)N¼ total number of publications with cells or mice. “Both sexes” largely represents different sex used in different decreased after new data revealed experiments, rather than comparative research. (b) Sex distribution in normal human keratinocytes and slower medication metabolism among keratinocyte cell lines. HaCaT cells, a spontaneously transformed keratinocyte line, are of male origin. females (Greenblatt et al., 2000). At

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