Sex and the Lab: An Alcohol-Focused Commentary on the NIH Initiative to Balance Sex in Cell and Animal Studies

Abstract

In May 2014, Dr. Francis Collins, the director of U.S. National Institutes of Health (NIH), and Dr. Janine Clayton, the director of the U.S. National Institutes of Health Office of Research on Women's Health, published a commentary in the journal Nature announcing new policies to ensure that preclinical research funded by the NIH considers both males and females. While these policies are still developing, they have already generated great interest by the scientific community and triggered both criticism and applause. This review provides a description and interpretation of the NIH guidelines, and it traces the history that led to their implementation. As expected, this NIH initiative generated some anxiety in the scientific community. The use of female animals in the investigation of basic mechanisms is perceived to increase variability in the results, and the use of both sexes has been claimed to slow the pace of scientific discoveries and to increase the cost at a time characterized by declining research support. This review discusses issues related to the study of sex as a biological variable (SABV) in alcohol studies and provides examples of how researchers have successfully addressed some of them. A practical strategy is provided to include both sexes in biomedical research while maintaining control of the research direction. The inclusion of sex as an important biological variable in experimental design, analysis, and reporting of preclinical alcohol research is likely to lead to a better understanding of alcohol pharmacology and the development of alcohol use disorder, may promote drug discovery for new pharmacotherapies by increasing scientific rigor, and may provide clinical benefit to women's health. This review aims to promote the understanding of the NIH's SABV guidelines and to provide alcohol researchers with a theoretical and practical framework for working with both sexes in preclinical research.

Keywords: Cell and Animal Studies; Ethanol; Gender; Policy; Preclinical Research.

Figure 1

Flow-chart of a potential course of action when designing experiments that use animals of both sexes. A major misconception that has contributed to the exclusion of female animals in research studies is that results in females are much more variable and that tracking of vaginal cytology across the estrous cycle is necessary to reduce this variability. Results from meta-analyses indicated that variability in females on most biological measurements is not significantly greater than males (Becker et al., 2015, Mogil and Chanda, 2005, Prendergast et al., 2014). Researchers that work with both sexes advise to initially ignore the estrous/menstrual cycle and to first determine whether there is a basic sex difference in one male group versus one female group (McCarthy et al., 2012). This flow-chart shows how research can be developed after collecting data from males and females on any given end-point of interest. Ovals represent the start (red) or the end (green) points of the chart; the parallelograms represent outputs; the rectangle represents a process (action); and the diamonds indicate decisions to be made. After obtaining results from males and females (in which estrous cycle has not been monitored), the results are analyzed to determine whether females are more variable than males. More likely, females and males will present similar variability; at this point, data will be analyzed to identify whether there is a sex difference in the response. If no sex differences are found, data are reported. If sex differences are identified, the investigator is presented with the decision (left diamond) of whether to pursue them, in which case she/he will formulate new hypotheses and apply for new funding, or whether to simply report them and pursue other research. In the less-likely event that data from females present a much higher variability than data from males, the investigator is presented with the decision (right diamond) of whether to pursue the biological mechanism behind this variability, consider the estrous cycle and hormonal fluctuations and apply for new funding for this research, or whether to not pursue this finding and analyze and report the data obtained from males and females as described before. This figure intends to emphasize how the researcher retains the power to decide whether to investigate sex differences and/or estrous cycle-dependent changes in females. After analyzing the results for sex differences and reporting them, the investigator has the choice of using one or both sexes for future studies by providing a convincing rationale based on scientific findings, questions, and interests. In this contest, the NIH SABV guidelines discourage the use of a given sex as the “default” model to be used in research.

Figure 2

Pharmacological manipulation of binge drinking reveals that male and female mice have similar sensitivity to a metabotropic glutamate receptor 5 (mGlu5) antagonist at the receptor level (B) but different sensitivity to rapamycin (C) an inhibitor of mTOR, consistent with the sexually divergent changes in phosphoinositide 3-kinase (PI3K) and downstream signaling molecules following repeated binge drinking (A). Panel A: Depicted is a simplified diagram of mGlu1/5 intracellular signaling. Downstream signaling molecules that differ by sex and influence alcohol binge drinking are highlighted in bold font. Notably, 24-hour withdrawal from repeated binge drinking significantly altered the phosphorylation of PI3K, mTOR, 4E-binding protein 1 (4EBP1) and p70 ribosomal protein S6 kinase (P70 S6K) and tended to alter phosphoinositide-dependent protein kinase 1 (PDK) in the nucleus accumbens of male but not female mice (results in Cozzoli et al., 2016). Panel B: Binge ethanol consumption, averaged across seven 30-min sessions, following intraperitoneal injection of saline or different doses of MTEP. The efficacy of MTEP to decrease binge drinking was similar in the male and female mice, as the 20 mg/kg MTEP dose significantly decreased binge ethanol intake by 33% or 31% in the male and female mice, respectively. Values are the mean ± SEM for the number of animals on the figure. **p<0.01 versus respective saline group; adapted from (Cozzoli et al., 2014a). Panel C: Intra-accumbens rapamycin dose-dependently decreased binge ethanol intake during a 30-min binge session in male but not female mice, with a 28% decrease in binge ethanol intake following the 100 ng/side dose in male mice. Mice received bilateral infusions of vehicle or rapamycin prior to a binge ethanol session in a within-subjects design. Values are the mean ± SEM for the number of animals on the figure. *p<0.05, **p<0.01 versus vehicle infusion; line represents significant ANOVA; adapted from (Cozzoli et al., 2016).