Incorporating Sex Influences into Today’s Brain Research

Each sperm carries with it an X or a Y chromosome. Each unfertilized egg, an X. When conception occurs, and the egg and sperm come together, the sex of the offspring is determined by the comingling of those two chromosomes. XX will become female and XY will become male. This one difference is expressed in every single cell in the body—including the ones that make up the brain. Yet, historically, most in the medical and scientific communities have believed that meaningful differences only manifested themselves in the “bikini zones,” or the parts of the body specifically involved with reproduction. As such, most biomedical research, to date, has been conducted solely on male tissues, animals, and human participants.

But we are learning that meaningful differences are not limited to the sexual organs. In fact, over the past two decades, scientists across disciplines have found a variety of important sex differences in the size, connectivity, and activity of different brain regions; in the prevalence and severity of disorders such as depression and Alzheimer’s disease; and in the response to different medications and medical interventions. Such findings, argues Jennifer Plank-Bazinet, a Health Scientist Administrator who contributes to the Sex as a Biological Variable (SABV) efforts at the National Institutes of Health’s (NIH) Office of Research on Women’s Health (ORWH), are showing today’s scientists that both sexes need to be included in future research to not only further our understanding of the body and brain—but also to develop more effective treatments for disease.

“We now know that women metabolize nicotine faster than men, so nicotine replacement therapies may be less effective in women. We now know that men and women respond differently to pain—recent work suggests that different cells drive pain processing in male and female mice” she says. “Without studying both sexes, we would never know if we’re taking the right steps towards appropriate clinical interventions for both men and women. So you can understand why we need to make sure that biomedical research is considering SABV in future studies.”

To that end, the NIH announced a sweeping policy change in 2014 mandating that all future biomedical research funded by the agency address sex influences. But while the list of studies showing that sex differences exist outside the “bikini zone”—and the acceptance of how important these sex differences may be to our understanding of both brain and behavior—is growing, how the research community should address SABV remains hotly debated.

His and Her Brains?

In 2014, Larry Cahill penned an essay for Cerebrum, entitled, “Equal ≠ The Same: Sex Differences in the Human Brain.” He made a strong case that sex matters quite a bit in most areas of medicine—and that continuing to ignore these differences was doing a great disservice to the health and wellbeing of women and men. While a lot has changed in the two years since Cahill wrote the piece—including the NIH mandate, he says that there is still a fight ahead.

“The new regulations whereby all grants at NIH are supposed to actively consider sex influences is important. Now it’s something that people have to deal with, when before they could just ignore it,” he says. “And while it is forcing science to turn a corner that cannot and will not be unturned, it does not mean the game is over. The status quo has not changed. The majority of the field still does not understand the incredible importance of the issue.”

It’s a valid concern. At Neuroscience 2016, the annual meeting of the Society for Neuroscience, less than 300 of the more than 15,000 scientific abstracts presented at the meeting explicitly investigated sex differences. And that may be because many young scientists are still trying to avoid dealing with them—often for political reasons. Emily Jacobs, a neuroscientist at the University of California Santa Barbara, says she tried to avoid sex differences herself—until her work with dopamine kept showing important estrogen variations that needed to be taken into account. Her research suggested that 17 β-estradiol, or the estrogen that fluctuates across a woman’s menstrual cycle, affected dopamine release and performance on a working memory task. She said it was an “a-ha” moment for her and her body of work.

“Initially, I was averse to studying sex differences. It seemed like a politically prickly topic that I didn’t want to engage with. But I realized that the dopamine system was ripe with sex-specific mechanisms that existed whether I took the time to look or not,” she says. “It is critical for the field to understand the way males and females do—and do not—differ. That knowledge carries profound clinical implications for the treatment of diseases and for the theories that shape our basic understanding of science.”

But, beyond politics, some worry that sex is a bit of a loaded variable—and mandates like the NIH’s regarding SABV may be letting the pendulum swing too far in the other direction by placing too much emphasis on differences between males and females when there are many, many similarities. Daphna Joel, a neuroscientist at Tel-Aviv University in Israel, as well as one of a group of researchers who wrote a response to Cahill’s Cerebrum essay, says that there is a danger in thinking of male and female brains as two distinct entities. Rather, she argues each individual brain, regardless of sex, is a “mosaic” of features based on the interaction of genetic and environmental factors. That mosaic will influence both the development of disease and the outcomes of specific treatments. Simply focusing on one sex or the other will not provide the precise, personalized treatments that so many patients need.

Furthermore, she argues, one must be careful with correlation/causality issues. Differences observed between males and females may correlate with a sex category but may actually reflect a separate risk factor. She offers cardiovascular disease as an example.

“It would be easy to say that the risk is different because of sex. But when you look deeper, you see that it is not about sex. It’s about height. Women, on average, are shorter than men, and therefore have a different risk,” she says. “We are learning that how sex may affect the brain depends on other factors. It is an interaction of biology and environment that can manifest itself in many different ways. So you need to look beyond just sex as a categorical variable. You need to look at the other variables associated with sex, too.”

Just Add Females?

Another concern for researchers like Geert de Vries, director of the Neuroscience Institute at Georgia State University, is methodological. He says successfully considering SABV in studies is not as simple as just adding females to existing study paradigms—one has to appreciate that some differences show an amazing spread between the two sexes while others do not. In fact, depending on what you study, there is often more variability within one sex than between the two. As such, methods matter, and will matter greatly moving forward.

“This is not as simple as just including both males and females in a study. Studying sex differences is complex,” he says. “And while the NIH mandate is a good one, even they would tell you that getting the right guidance on how to interpret this mandate, and how to do a good study, is important.”

Jill Goldstein, director of research at the Connors Center for Women’s Health and Gender Biology at Brigham and Women’s Hospital, agrees. “Just adding more women into a study does not address many of the critical methodological issues involved in designing studies to identify sex effects,” she says. “Sex impacts every level of analysis and therefore must be systematically incorporated into the beginning of the study. Understanding how to design a study that incorporates sex effects begins with education about these strategies.”

She and others at the Connors Center offer workshops on how to design good sex difference studies—with guidance on how to manage and control for issues like reproductive status and regular hormonal changes across the menstrual cycle. And Plank-Bazinet says that the ORWH is working closely with the NIH Office of Extramural Research and the NIH Institutes and Centers to provide their own guidance as well—a variety of tools and resources are available to researchers on the Office of Extramural Research’s web page on Rigor and Reproducibility.

“We’ve published papers, we’ve given presentations to a variety of different scientific societies, and we give workshops. And we are continuing to find ways to reach out and provide this information to the community,” she says. “There is actually a lot of information out there on how to design experiments to effectively study sex differences already. But we want to make sure that scientists have the resources they need to do these studies in the right way.”

The Time Is Now

While Cahill says there is still quite a bit of work to do, he believes the pieces are starting to move into place to make the required “sea change” happen, including the NIH mandate and continuing changes in journal and peer review policies to address sex influences. And Jacobs believes with those changes, tomorrow’s studies will help pave the way for better medical interventions in the future.

“The best thing we can do right now is to be honest about what we know and what we don’t know. And we don’t know a lot,” she says. “We need to be explicit in the way we design and interpret studies. Scientists also need to be careful in the way we operationalize the phenomenon we’re studying.”

She says she hopes to see a revolution in women’s health—and a greater understanding of how sex influences the brain, behavior, and health in the process. Goldstein says that, after more than thirty years in the field, she is very optimistic about what the future holds.

“Our time is now. Historically, there has been so much push-back when it comes to studies of sex differences. The current climate feels like there is greater acceptance. Not just that sex differences exist but that they do matter in terms of health and medicine,” she says. “We are finally in a place where we are being encouraged to do this kind of research. Now that we are having these conversations, now that we have some real investment in these studies, it’s my hope that we can capitalize on this in order to translate our work into the development of more effective and precise therapeutics.”