By Aparna Shah
A little over a year ago, the NIH Director, Dr. Francis Collins and the Director of NIH Office of Research on Women’s Health, Dr. Janine Clayton expressed their concern about the stark imbalance in the sex of animals used in preclinical research and the implications of this imbalance on clinical practice. While the NIH has released a notice to fix this issue, more awareness is needed to encourage preclinical scientists to genuinely acknowledge the value of considering both sexes in their experimental designs.
With this on my mind, I participated in an informal discussion led by Dr. Catherine Woolley at one of the several fantastic ‘Meet-the-Expert’ sessions at the recent SfN annual meeting titled, ‘Sex Differences in the Brain: What Are They, What Aren’t They, and When Do They Matter?’
In the early stages of her career, Dr. Woolley investigated the effects of estrogen on hippocampal synapses in female rats. Upon learning the topic of her research, people often asked if she was studying sex differences in the brain – a label she actively resisted being identified with. For one, she associated this label with studies on sexual and reproductive behavior. But more importantly, she grew weary of the questions that followed (A typical example: ‘Is this why women are smarter than men?’). This unwarranted public attitude, she said, stems from studies that magnify small differences between sexes, inappropriately translate molecular differences to behavioral differences or over interpret data to imply that one sex is better or worse than the other.
It wasn’t until members of her team discovered sex-specific effects of a drug, URB 597 (that was already being used in clinical trials!), that Dr. Woolley acknowledged that investigating sex differences had become a salient feature of the research conducted in her lab. Here’s the unedited version of this story: Dr. Woolley shared that her lab tested the drug’s effects, failed to replicate results from published reports, and tested the drug again from a different source, only to realize that the main difference between procedures in literature and in her lab was that they had been using female rats! Upon realizing this, she added male rats to her protocol and the rest is…published.
Further, Dr. Woolley emphasized the importance of considering sex as a biological variable in a study. “There are some things that appear to work very similarly in both males and females but we are finding more differences than similarities between sexes at the level we work at – molecular regulation of synaptic plasticity,” she pointed out. “These molecules could one day be targets for therapeutics. If sex differences do, in fact, exist at the target level we need this information before drugs reach the clinical trials stage.”
Several important take-home messages about experimental design emerged from the discussion. Here’s my attempt at summarizing these:
- It is not enough to casually mention in the Methods section of a paper that a few males and females were tested and no sex differences were observed. A well-powered study is necessary to investigate this. More importantly, results should be reported whether you see sex-differences or not.
- Reasons (Excuses?) for choosing male animals usually revolve around confounding effects of the estrous cycle. However, this statement warrants supporting data showing more variability in females than in males. According to a meta-analysis, this isn’t necessarily true.
- When choosing between ovariectomized and intact female animals for a study, while the latter is obviously more physiologically relevant, one can’t argue that it is always the better choice. Both come with some caveats. The right choice depends on the questions you’re asking and the experimental design.
- Be careful with how you interpret data. Your finding may be one piece in a big puzzle so avoid over-interpretation. One observed sex difference could be an adaptation to compensate for another. Different behavioral assays testing apparently similar modalities may give different results depending on underlying features of the test. Avoid using the word ‘outperform’ when describing one sex versus the other.
As Dr. Woolley reflected upon her career, she shared some advice for young scientists. I made a note of a few things I wish I was reminded of more often!
- Learn to be comfortable with a certain degree of uncertainty. Things don’t and won’t necessarily go as planned. But that does not imply failure.
- Even if things seem to be falling apart, don’t give up – be observant – pay attention to details!
- In the context of the project or experiments you decide to work on, be personally responsible for what you’re doing in the lab. Don’t just follow orders.
- As a woman investigating sex differences, don’t be afraid of the data, do not try to fit it to a preconceived model. No one benefits by believing there are no differences if they truly exist.
This discussion enhanced my way of thinking about the issue of considering sex as a variable for preclinical research (with the bonus advice that I am grateful for). However, I still wondered how to approach other scientists and convince them to pay attention to this issue and incorporate female animals in their designs. So, I asked Dr. Woolley this question. Her answer, “Persuade scientists with science! Publish good science and let the data speak.” On that note, I should probably get to the lab!
Oh! I almost forgot to mention, I did ask her to recommend a good book with a neuroscientist’s perspective suitable for a wider audience. No commissions for either her or me if you read this book…but I do hope you enjoyed this post!
Clayton, J. A., & Collins, F. S. (2014). Policy: NIH to balance sex in cell and animal studies. Nature, 509(7500), 282-283
Woolley, C. S., & McEwen, B. S. (1992). Estradiol mediates fluctuation in hippocampal synapse density during the estrous cycle in the adult rat. J Neurosci, 12(7), 2549-2554
Tabatadze, N., Huang, G., May, R. M., Jain, A., & Woolley, C. S. (2015). Sex Differences in Molecular Signaling at Inhibitory Synapses in the Hippocampus. J Neurosci, 35(32), 11252-11265. doi: 10.1523/JNEUROSCI.1067-15.2015
Prendergast, B. J., Onishi, K. G., & Zucker, I. (2014). Female mice liberated for inclusion in neuroscience and biomedical research. Neurosci Biobehav Rev, 40, 1-5. doi: 10.1016/j.neubiorev.2014.01.001
Eliot L. (2009) Pink Brain, Blue Brain, How Small Differences Grow Into Troublesome Gaps- and What We Can Do About It. New York, NY: Houghton Mifflin Harcourt ISBN: 978-0-618-39311-4
Any views expressed are those of the author, and do not necessarily reflect those of PLOS.
Aparna Shah is a postdoctoral researcher at Johns Hopkins University. She is fascinated by neurophysiological and behavioral changes associated with mood and substance use disorders, and is passionate about understanding potential sex differences in the brain.