Does the Sex of Test Animals Matter?
Sex differences in human hearing loss have long been a topic of intense research interest by hearing scientists. On average, women of all ages have better hearing at higher frequencies than men (Gates et al., 1990), but, interestingly, as they age, women lose high frequency hearing faster than men (Moller, 1981). Better high frequency hearing in women is likely due to multiple factors, including direct and indirect effects of estrogen and progesterone (Hultcrantz et al., 2006). But the issue is complicated by the fact that the hobbies, weekend activities, and workplaces of males are notoriously noisier (Lie et al., 2016), and are also sometimes filled with other threats to hearing beyond noise, such as chemical exposure (Estill et al., 2017).
It is therefore logical to conclude that preclinical animal research, in auditory function or any discipline, should include both male and female animals. However, female animals have historically been underrepresented in all preclinical research and particularly in neuroscience research, in which single-sex male studies outnumbered female-only studies by a ratio of 5.5 to 1 in 2009 (Beery et al., 2011).
Many reasons are cited for the reluctance to use female animals. Some scientists are concerned that hormonal cycles decrease the homogeneity of study populations, add variability to a study, and increase confounding variables. Others believe that the results of studies with male animals can simply be applied to female animals (Beery et al., 2011). Practical concerns are also raised, as the variability introduced by using both sexes might require a large number of animals to provide appropriate statistical power, and therefore could be practically and financially difficult (Fields, 2014).
Animals in Turner Scientific Studies
Study sponsors at Turner Scientific frequently ask if we use both sexes in our studies, and the answer usually depends on the goals of the sponsor. In general, we encourage the use of BOTH male and female mice, rats, or other species. Given the complicated relationship between sex and hearing briefly described above, it makes sense to design preclinical studies with both sexes to more accurately model the human condition. There is also much specific evidence that sex can influence the results of preclinical hearing research, such as the following sampling of studies:
Female rats experienced more profound deterioration of hearing than male rats when exposed to cisplatin, likely due to more significant deterioration of the spiral ganglion and brainstem (Kirkim et al., 2015).
Otoacoustic emissions were greater in female rhesus monkeys compared to males, and varied in both sexes based on hormone levels related to breeding season (McFadden et al., 2006).
If estrogen receptors were knocked out, severe progressive hearing loss occurred in female mice (Hultcrantz et al., 2006).
Aged male and female rats responded differently to chronic noise in their home cage environments. Females showed worsened hair cell loss and hearing thresholds after noise compared to males, and opposite effects were also seen on an inhibitory neurotransmitter marker in the auditory cortex (Turner et al., 2013).
Sertraline (Zoloft) relieved the consequences of prenatal stress in female mice only, as measured by pre-pulse inhibition (Pereira-Figueiredo et al., 2017).
We realize, however, that using both sexes does increase the complexity and therefore the time and cost for a study. But we also believe that many possible treatments for serious human conditions are missed when preclinical drug development focuses its efforts primarily on males. Therefore, for sponsors who need early, proof-of-concept studies, in which the goal is just to determine if their test article has any effect on hearing or a related condition like tinnitus, we have an important conversation about whether to use males only, females only, or both sexes. Budgetary constraints often drive such decisions by sponsors but we feel it is important to recognize the many variables that contribute to the success of a study, and this is an important one that we think all-too-often has been ignored by those of us involved in the work. While early proof-of-concept studies may be less critical, for larger studies that may be GLP-level and used for IND enabling purposes, and where there is no scientific justification to do otherwise, we generally recommend using both male and females of the species being studied.
Beery, A.K., & Zucker, I. (2011). Sex bias in neuroscience and biomedical research. Neurosci Biobehav Rev, 35(3), 565-72.
Estill, C.F., Rice, C.H., Morata, T., & Bhattacharya, A. (2017). Noise and neurotoxic chemical exposure relationship to workplace traumatic injuries: a review. J Safety Res, 60, 35-42.
Fields, R. (September 1, 2014). Testing males and females in every medical experiment is a bad idea. Retrieved from Scientific American: https://www.scientificamerican.com/article/testing-males-and-females-in-every-medical-experiment-is-a-bad-idea/
Gates, G.A., Cooper, J.C., Kannel, W.B., & Miller, N.J. (1990). Hearing in the elderly: the Framingham cohort, 1983-1985. Ear Hear, 11(4), 247-256.
Hultcrantz, M., Simonoska, R., & Stenberg, A.E. (2006). Estrogen and hearing: a summary of recent investigations. Acta Otolaryngol, 126(1), 10-14.
Lie A., Skoogstad M., Johannessen H.A., & Tynes T. (2016). Occupational noise exposure and hearing: a systematic review. Int Arch Occup Environ Health 89, 351-72.
Kirkim, G., Olgun, Y., Aktas, S., Kiray, M., Kolatan, E., Altun, Z., Ercetin, P., Bagriyanik, A., Yilmaz, O., & Ellidokuz, H. (2015). Is there a gender-related susceptibility for cisplatin ototoxicity? Eur Arch Otorhinolarnygol, 272(1), 2755-2763.
McFadden, D., Pasanen, E.G., Raper, J., Lange H.S., & Wallen, K. (2006). Sex differences in otoacoustic emissions measured in rhesus monkeys (Macaca mulatta). Horm Behav, 50(2), 274-284.
Moller, M.D. (1981). Hearing in 70 and 75 year old people: results from a cross sectional and longitudinal population study. Am J Otolaryngol, 2(1), 22-29.
Pereira-Figueiredo, I., Castellano, O., Riolobos, A.S., Ferreira-Dias, G., Lopez, D.E., & Sancho, C. (2017). Long-term sertraline intake reverses the behavioral changes induced by prenatal stress in rats in a sex-dependent way. Front Behav Neurosci, 11(99), 1-11.
Turner, J.G., Parrish, J.L., Zuiderveld, L., Darr, S., Hughes, L.F., Caspary, D.M., Idrezbegovic, E., & Canlon, B. (2013). Acoustic experience alters the aged auditory system. Ear & Hearing, 34(2), 151-159.