Title:SEX: What’s the Difference?

In laboratory studies, researchers have long considered and accounted for differing features of their subjects such as age and weight. But only since last year did the National Institutes of Health (NIH) require scientists seeking research funding to also consider the role of sex as a biological variable. The policy change has led to exciting new research at Georgetown that demonstrates the importance of studying differences between male and female cells, animals, and humans. But there is still a lot of work to do—for researchers studying sex differences, and for policymakers striving to address the impact of sex disparities in medical research.

As director of the Center for the Study of Sex Differences at Georgetown, Kathryn Sandberg, PhD, has served as a leading national advocate for the inclusion of sex as a biological variable. She saw that a gap persisted in the use of female cells and animals in basic research, and took action. In 2013, Sandberg and Scott Fleming, associate vice president for federal relations at Georgetown, visited the offices of Rep. Nita Lowey (D-NY) and Rep. Rosa DeLauro (D-Conn), encouraging them to support the inclusion of females in publicly funded basic research. In 1993, Lowey and DeLauro had been actively involved in efforts to pass the NIH Revitalization Act, advocating for provisions that required the inclusion of women in federally funded Phase III clinical trials (using human subjects).

Lowey and DeLauro then wrote to NIH Director Francis Collins, MD, PhD, and subsequently provided guidance in the form of language in the Appropriations Committee’s report accompanying the NIH funding bill. The report urged specific steps to address the problem, stating that the exclusion of female subjects undermined the credibility of research. Collins responded with a commentary in the May 2014 journal Nature, where he and Janine Clayton, MD, who directs the NIH Office of Research on Women’s Health, pledged to improve the balance of male and female subjects in preclinical research supported by NIH.

In January of 2016, NIH began requiring all applications for research funding to “account for the possible role of sex as a biological variable in vertebrate animal and human studies.” At the time, women made up roughly half of all participants in NIH-supported clinical research but most basic and preclinical research focused exclusively on male subjects, an NIH announcement said, producing findings that failed to reflect the role of sex as a biological variable.

“It was a step forward,” Sandberg says of the NIH policy.

Sex differences in hypertension

Why do men typically develop hypertension at an earlier age than women? As a professor of nephrology and hypertension in the department of medicine, questions like this sparked Sandberg’s interest in the study of sex differences.

The hypertension difference was evident in female animal models as well. “Because that was such a striking difference and because I’m a molecularly trained person, I wanted to understand what was behind that,” Sandberg says. She began to investigate the renin angiotensin system, a key regulator of blood pressure.

Receiving an invitation to a meeting of the Society for Women’s Health Research only solidified Sandberg’s interest. “It was on sex differences, but not just limited to my specialty,” she says. “It was everything: neuroscience and psychiatric conditions, to very basic cell biology or cancer biology. I realized that there’s a group of people who are interested in why the sexes are different, and what the causes of those sex differences are.”

In addition to pursuing her own research on sex differences, Sandberg promotes the study of sex differences on campus at Georgetown and at national meetings, where she often chairs sessions on the topic. “We promote it within our community and support our colleagues who are studying it,” she says. “If there’s a really good paper that I’ve reviewed, I offer to write an editorial to try to help promote it. And I sit on study sections and I bring it up when appropriate there. You have to go from all sides.”

The estrous cycle myth

Researchers frequently argue that they need to use male-only subjects in basic medical research because monthly hormonal variations caused by the estrous cycle in female subjects will produce skewed results. Sandberg challenges that assumption. Several different studies have found that there is actually more variation in male subjects than females, including one paper that Sandberg cites where researchers studied more than 700 different measures of metabolism in significant numbers of male and female subjects. Despite the research, the myth about variability caused by the estrous cycle persists, she says.

“I think people are still convinced that we can’t include females in animal studies because you must control for the estrous cycle,” Sandberg says. “While the cycle does impact different functions and different measures, variability is also present in the male. We just don’t understand it.”

In a practical sense, the variation caused by the estrous cycle isn’t always relevant. In a recent study of traumatic brain injury (TBI) that included male and female mice, Mark Burns, PhD, associate professor of neuroscience at Georgetown, did not control for the estrous cycle. “We justified that decision by saying, females playing sports aren’t all on the same cycle, it’s random, therefore we used random as well,” he says.

Considering the role of sex, not just doubling up

The requirement that researchers consider the role of sex as a biological variable means that those submitting grant applications must justify why they chose their particular research subjects.

“If they’re interested in hypertension, they need to talk about what’s known about the differences between men and women in terms of hypertension and why they are selecting the model they’re choosing,” Sandberg says. “If hypertension is equally prevalent in both males and females, why are you only studying males? You’d have to make an argument for why you’re only studying that model, only in males, and then the reviewers would look and see whether it made sense or not.”

However, complying with the requirement isn’t just about conducting research on equal numbers of male and female subjects. Burns pilots his neuroscience studies with both genders to see if there’s variation. When he discovers a potential difference, he says, “then we power the studies appropriately to detect that.”

Sex differences in TBI

In a study published June 13 in the neuroscience journal GLIA, Burns showed that male mice have much greater brain inflammation and nerve cell death one week after experiencing a TBI than female mice. The study looked specifically at the way that sex affects inflammation in the brain after TBI, and demonstrates the importance of including female subjects in basic research.

“We were very surprised at the different inflammatory profiles—just such a different inflammatory signature for TBI, different infiltration of peripheral inflammatory cells into the brain, different activation of the brain’s resident immune system. We were surprised at the results, and how strong the results were,” says Burns.

The study also demonstrates the challenges in drug development to treat TBI. Researchers conducting basic science typically use male cells or animals. However, the findings from that basic research become the basis for the clinical trial of the treatment, a phase that since 1993 requires the inclusion of both male and female human subjects.

Researchers have known that biological sex can impact the recovery from TBI, Burns explains, but basic science has typically excluded female subjects from research. This may be part of the reason that treatments developed to help TBI patients, such as anti-inflammatory drugs, have failed in clinical trials when both sexes are included.

This is a really important finding, says Burns. “If there are completely different inflammatory profiles in males and females, why would we expect a drug targeting inflammation to work the same in males and females?”

Burns praises the NIH and Sandberg for promoting the importance of including both sexes in basic research. “NIH has done an excellent job of really pushing this idea forward, and Kathryn Sandberg was one of the people advising the NIH to make sure that sex was included in preclinical trials,” he says. “To me, that’s what government policy is about—identifying a need or gap and saying, this is what we’re interested in.”

Impact of the NIH policy

When researchers apply for NIH funding, their applications are scored by study sections, and those scores determine in part whether their grant application will receive funding. Failing to address the role of sex as a biological variable in a grant application can now lead to a lower score, and in today’s hypercompetitive funding environment, that can tank the grant.

“A great grant can still end up with a score that makes it no longer fundable,” Sandberg says. Researchers seem to be taking the study of sex difference more seriously, as evidenced by an increase in the number of conferences that address the topic. But Sandberg is still waiting to see an increase in the publication of preclinical research that includes females before declaring that the NIH policy was effective.

In the August issue of the American Journal of Physiology – Renal Physiology, Sandberg writes that only 15 percent of the animal studies published in the journal from January to July 2017 included both sexes. For every study with female subjects, five studies were conducted solely in males. The journals Kidney International and the Journal of the American Society of Nephrology were even more lopsided, she says.

“The basic science leads the way for drug discovery,” Sandberg says. Pharmaceutical companies carefully review medical literature to identify treatments for potential investment, she adds. “If you’re only looking at studies in males, then you’re biasing all drug discovery toward what’s perfect for the male. So you’re missing out on drugs that could be beneficial in the female.”

“It turns out that women exist in real life,” Burns says with a smile. “And publicly funded science should absolutely be working for them too.”