Analyzing Sex in Lab Animal Research

Animal research has been vital to science and medicine since its inception. Until the 1960s, however, researchers rarely reported the sex of animals except in experiments related to reproduction. Even today, the sex of animal subjects is “omitted in 22–42% of articles in neuroscience, physiology, and interdisciplinary biology journals” (Beery & Zucker, 2011). Analysis of animal studies in which sex is reported shows that females are underrepresented in most subfields except reproductive biology and immunology—see chart below. chart of male vs female animals

General Points: The Underrepresentation of Female Animals Single-sex experiments are the only option for sex-specific phenomena (ovarian cancer or prostate cancer, for instance) and can also be beneficial if one sex has been understudied (McCarthy & Becker, 2002). The majority of single-sex animal experiments, however, do not fall into these categories. Female animals are generally underrepresented in studies of conditions that affect both sexes and are underrepresented in research where evidence suggests that sex influences outcomes.

  • When can experiments be done in only one sex?
    • 1. When studying a sex-specific phenomenon, such as ovarian cancer or prostate cancer.
    • 2. To address inadequate published data for one sex in a particular area.
    • 3. Where there is statistically robust evidence that sex does not influence a trait or outcome.
In diseases where one sex predominates, such as breast cancer, both sexes may still need to be included, but researchers may choose not to use them in equal numbers. Things become more complicated in instances where a disease differs profoundly in women and men, but an animal model has been developed for only one sex. For instance, rodent models of hypertension do not accurately reflect the physiology of heart disease in women. In these instances, new models will need to be developed. It was discovered that patterns of disease differed in women where there was less occlusion of the coronary arteries but more microvascular disease due to changes in microvascular endothelial function (Ouyang, P. et al., 2016; Miller, V.M. et al., 2011; Mehta, L.S., 2016; Regensteiner, J., 2015). Animal models of coronary microvascular disease need to be established, including animal models that take into account potential interactions of pregnancy-related conditions such as preeclampsia or gestational hypertension, menopausal neurovascular dysregulation related to “hot flashes”, and metabolic conditions such as diabetes.

Sex and Lab Environment in Animal Studies

  • How can we best design animal studies to take into account sex (biological characteristics) and environmental factors and processes? The figure below shows the complex interdependency of sex and environment, including researcher sex, throughout the rodent life cycle.

    sex and lab environment interact
    Animal research includes the interaction between sex (biological characteristics, such as genes, hormones, age, reproductive phase, strain, etc.) and environmental conditions (such as caging practices, sex of researchers, attitudes and behaviors of researchers, room temperature, diet, etc.).

    Research Design
    A. Investigate the Role of Sex

    • • The influence of sex must be investigated before it can be ruled out.
    • • Research can be done stepwise. Male and female animals should be strain-matched (or strain-matched and genotype-matched) and age-matched, and should be reared under identical conditions (cages, bedding, diet, etc.). Females should not be breeders unless required for assessment of the phenotype.

      • Step 1. Total sample size (based on power calculations). Including females and males in a study often requires doubling the number of experimental subjects, but not always. Efficient experimental designs can incorporate sex while maintaining control over variance (Beery, 2018). For example, factorial designs require sample sizes to be increased by 14–33%, but not doubled (Buch et al., 2019; Miller et al., 2016). Analyzing data by sex and environmental factors helps detect meaningful effects that, in turn, help reduce confounding variables and the cumulative number of experiments required.
      • Step 2. Sex-based powering: test hypothesis in both males and females and power each to determine effect. Consider factorial designs to reduce sample size when investigating sex-treatment interactions in animals (Beery, 2018; Lazic, 2018).
      • Step 3. Comparison between sexes: power studies to determine the actual “sex effect.” Testing for sex effects has a financial cost. A demonstrated sex difference, however, justifies sex-specific research because harm in one sex is costly to society and individual patients. Overall, it is less expensive to understand sex in the basic science phase than during the more costly clinical trial phase (Klein et al., 2015). Analyzing sex may decrease the number of drugs that fail in development and also helps companies avoid being forced to remove drugs from the market due to adverse events in one sex.

    • • Researchers should also evaluate overlap between groups (similarities between males and females) and difference within groups (differences among males or among females). Overemphasizing sex differences should be avoided.
    • • Finding no sex effect should also be reported. To reduce publication bias, researchers should report when sex differences (main effects or interaction effects) are not detected or when data regarding sex differences are statistically inconclusive (Wizemann, 2012). Reporting null results is crucial for meta-analysis.
    • • For phenotypes that do not display sex difference, future experiments should be sex inclusive; that is, they should include equal numbers of randomly selected males and females for each test group studied. Not every experiment needs to be designed to evaluate sex differences. However, for every experiment, the sex of the animal test subjects should be reported to ensure that experiments are reproducible and findings in one sex are not over-generalized to the other sex (Wizemann, 2012).
    • • Analyze the Impact of Sex Hormones
      • Considering the estrous cycle (Byers et al., 2012). Research has shown that the majority of sex effects are not influenced by cycle (Becker et al., 2005). Researchers should compare the variability in female and male responses. Researchers do not need to monitor the estrus cycle unless females are more variable.
        • • In a meta-analysis of nearly 10,000 traits, Prendergast et al. (2014) found that, for most biological measurements, females are no more variable than males (Itoh & Arnold, 2015; Becker et al., 2016). Other factors, including group versus single animal housing, can have a greater impact on variability of a trait than stages of the estrous cycle.
      • Menopause Models. Menopause is an emerging area of laboratory animal research. In rodents, reproductive aging occurs and can be used in research to gain insight into menopause in women. Reproductive aging in female rats or mice begins towards the end of the first year, and the timing can vary depending on strain and environmental factors (Finch et al., 1984). When over one year old, female rodents show either low serum levels of gonadal steroids (persistent diestrus) and/or chronically elevated serum estrogen levels (persistent estrus) (Finch et al., 1984). Persistent estrus leads eventually to a diestrus condition, but there can be an intervening period of pseudopregnancy, depending on the species (Finch et al., 1984). Ovariectomy can simulate reproductive aging by rapidly reducing gonadal steroid levels, and this “acute menopause” has a multitude of effects. One area of increasing interest is the effects of menopause on the immune system. In rodents, ovariectomy “reduce[s] lymphocyte chemotaxis, mitogen-induced T cell proliferation responses, and [Interleukin-2] production” (Marriott & Huet-Hudson, 2006).
      • Pregnancy or Pseudopregnancy. Fewer than 10% of medications approved by the U.S. Food and Drug Administration since 1980 have enough information to determine risks for birth defects (Adam et al., 2011; Mishra & Mohanty, 2010). New animal research on drug safety should assess effects on the dam and the fetus during pregnancy and lactation (McDonnell-Dowling & Kelly, 2015).
      • Pharmacokinetics. The estrous cycle can also affect pharmacokinetics. For example, Kulkarni et al. (2012) found that the oral bioavailability of genistein, a soy isoflavone with antioxidant properties, was inversely correlated with estrogen level (which regulates hepatic disposition of a drug).

    B. Investigate How the Environment Interacts with Sex
    sex and lab environment interact in rats
    Animal research includes the interaction between sex (biological characteristics, such as genes, hormones, age, reproductive phase, strain, etc.) and the lab environment (e.g. caging practices, attitudes and behaviors of researchers, room temperature, diet, etc.). The double-ended arrows represent interactions between sex and environment.
    Environmental processes, such as caging practices or differential handling, may impact female and male animals differently. Researchers should not identify an effect as dependent on sex (or a biological trait) when, in fact, it depends on an environmental condition.

    Environmental Processes that May Interact with Sex:

    • Caging: Individual vs. group?
      • • To avoid aggressive behaviors, male rodents are often caged in small groups or alone. Rodents housed alone “expend more energy maintaining body temperature, which can cause differences in parameters such as caloric intake, muscle activity, metabolic rate, fat distribution, or body size, with a plethora of potential downstream effects on bodily and cellular activity” (Ritz et al., 2014). In contrast, females are more often housed together to lower costs. Rodents housed together often sleep clustered and, as a result, expend less energy to keep warm. In this scenario a “sex difference” may be identified where, in fact, differences result from different housing conditions.
      • • A same-sized group may create different stressors for females and males. Being caged alone may itself cause stress (Ritz et al., 2014), but single housing reduces trait variability in both males and females (Prendergast et al., 2014).
      • • Group caging can also result in self-induced or social hair loss, also called barbering (Kaleuff et al., 2006). Barbering (1) often reflects social hierarchies in same-sex group cages (both females and males); (2) may result from the stress of overcrowding; (3) takes place in breeding groups (females barber males); and (4) occurs among lactating rodents (pups barber mothers). Barbering occurs in some strains more than others.
      • • Cage size can limit animal behavior. For example, many cages cannot accommodate the full range of female sexual behavior. In the wild, females may dart, approach and solicit males (Birke, 2011). For these reasons, articles should specify housing conditions.
    • Researcher/Staff. Experimenters may be a confounding variable in rodent research where stress is a significant factor. One study found that rats and mice demonstrated a reduced pain response in the presence of a male experimenter compared to a female experimenter. Both male and female rodents showed this response, but females showed it more. The researchers identified this “male observer effect” as a stress response to androstenone and androstadienone, axillary secretions found in higher concentrations in males than females. In addition to stress-induced analgesia, the presence of these compounds resulted in increased plasma corticosterone levels (Sorge et al., 2014).
    • Handling. Control rodents should undergo similarly stressful procedures as those of the experimental rodents, such as sham surgeries. Taking vaginal smears to establish the stage of the estrous cycle in female rodents can be stressful; male rodents should be handled in similar ways (Becker et al., 2005). Without these controls differences in stress responses can be mistaken for other sex differences.
    • Circadian Cycling. Testosterone varies seasonally and with circadian rhythms. Similarly, hormone concentrations in females can fluctuate over the course of a single day of the estrous cycle. Hypothalamo-pituitary-adrenal (HPA) secretions, which in turn affect gonadal secretions, also vary over the course of the day (Becker et al., 2005). Researchers should specify the photoperiod in the colony and the time of day at which measurements are taken.
    • Social Dynamics. Edelmann et al. (2013) found that rat maternal behavior mediates sex differences in play among juveniles. Simulated maternal grooming, in addition to normal maternal care, reduced play in males but not in females. This effect may be mediated by increased serotonin signaling, as maternal licking also increased serotonin receptor mRNA.
    • Temperature. Laboratory mice are typically housed at temperatures below their thermoneutral zone. Gaskill et al. (2009) found that when mice were able to move among three cages with different temperatures, mice of both sexes preferred warmer environments for inactive and maintenance behaviors (with no preference for active behaviors). Females preferred the highest temperature; males showed no preference between the medium and highest temperatures. As with day length, ambient temperatures in the research colony should be reported consistently.
    • Diet. Diets impact weight, metabolism, hormone levels and immune functions; hence diet formulation should be reported (Bhupathy et al., 2010; Luczak et al., 2011). Glover & Assinder (2006) found that diets rich in phytoestrogens may have sex-specific effects on cardiac health. In males, soy-based diets significantly decreased cardiac function, increased myocellular disarray and led to an increase in β-MyHC, a myosin motor protein associated with heart failure. This latter effect was also observed to a lesser degree in females.
    • Bedding. Bedding might also have an impact on hormonal status. Corncob bedding has been reported to reduce aggressive behavior compared to cardboard-based bedding by influencing estrogen receptor alpha expression in the brain (Landeros et al., 2012). Elevated levels of non-estrogenic mitogens have also been previously reported in mice housed on corncob bedding (Markaverich et al., 2002).
    Analyzing sex and environment—and how they interact—is important to increasing the translational value of animal models. The cost of developing a drug ranges between $350 million and $5 billion—the average cost has been estimated at between $2 and $3 billion; 95% of drug candidates fail (DiMasi et al, 2016; Arrowsmith, 2011; Herper, 2013). Including sex and gender as research variables may help bring down those costs, promote discovery of disease mechanisms, and save lives.

    Works Cited

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