Where Are All the Female Test Subjects?

The animals and human cells used in medical research are overwhelmingly male—meaning that as new therapies are developed, some biological sex differences may go unaddressed.

In 1987, the National Institutes of Health made a bold update to its grant guidelines, encouraging scientists seeking funding to include women and minorities in their clinical research. Six years later, the U.S. Congress took things a step further by passing the NIH Revitalization Act, which legally mandated the inclusion of women and minorities “in numbers adequate to allow for valid analyses of difference in intervention effect.”

Inclusion and equality, however, are far from interchangeable—as the Boston Globe reported earlier this year, women still make up only one-third of all clinical trial participants. But earlier along in the scientific pipeline, the disparity is even more pronounced: Test subjects in pre-clinical studies—those involving animals or human cells, before human bodies get involved—skew overwhelmingly male.

In a study recently published in the journal Surgery, researchers from Northwestern University’s medical school surveyed papers published between 2011 and 2012 in five major surgical journals: Surgery, Annals of Surgery, American Journal of Surgery, JAMA Surgery, and Journal of Surgical Research. For studies involving animals, 22 percent did not specify sex at all; of those that did, 80 percent included only male subjects. For cell research, 76 percent neglected to mention sex; those that did used only male cells 71 percent of the time.

“The obvious issue is, if you’re only studying males, you don’t know if that therapy is going to work in females,” says lead researcher and Northwestern surgery professor Melina Kibbe, who has since been named editor-in-chief of JAMA Surgery. “The problem is not studying males per se—the problem is studying only one sex. Research really needs to be conducted in both sexes.”

Human biology backs up her argument. Past research has suggested that the health benefits of aspirin, for example, may differ by sex, helping to prevent heart attacks in men and strokes in women. Last year, the Food and Drug Administration issued its first (and, to date, only) set of sex-specific dosage guidelines, halving the recommended dose for women of the prescription sleep aid Ambien. And as a 60 Minutes report on the issue noted in February, “Drugs are just the beginning. Sex differences have been found in pain receptors, liver enzymes, even the wiring of the brain.”

So why do such stark differences still persist in basic science research?

Partly because of ignorance within the scientific community, Kibbe explains, adding, “I think I’m a perfect example of that.” Her own interest in sex disparities in research subjects arose only after Teresa Woodruff, who heads Northwestern’s Women’s Health Research Institute (WHRI), asked if Kibbe had used rats of both sexes in her vascular therapy research. She hadn’t: “I really wasn’t thinking about the issue. It wasn’t something that was on my radar.”

When Kibbe accepted WHRI funding to support additional research on female rats, “There was a dramatic difference in how female animals responded to my therapy compared to male animals. And that’s how I became a convert and very, very aware of the issue.”

But the reason Kibbe originally used only males—and the reason why male animals are vastly more common as test subjects in general—is one that presents a hurdle for those looking to level the scientific playing field. The more dramatic hormone fluctuations of female animals mean that they’re generally considered more difficult, Kibbe explains: “It’s a variable that is not held constant throughout the experiment.”

Money, too, is a deterrent for many researchers; including animals or cells of both sexes means more subjects and more work to be funded.

“I think some people, if they actually did think about it, would say, ‘Oh, I’m duplicating costs, I’m just making it more expensive,’” says Woodruff, a professor of gynecology and molecular biosciences at Northwestern and a co-author on the Surgery study. But, she adds, “I actually think that’s the wrong economics.”

“In the end, it serves the public better that we understand about sex at the cheaper end of the equation, before it gets so much more expensive as you go closer to clinical trials,” she explains. “It is going to be better in the long run, less costly, for us to have inclusion of both sexes in basic science.”

Woodruff and her colleagues are currently working to increase the reach of their crusade beyond surgery: Several researchers affiliated with the WHRI have written yet-to-be-published studies that survey existing literature in the areas of anesthesiology, neuroscience, and dermatology (“We’re very confident in the derm field that it’s almost all male cells,” Woodruff says, “because most of the cells for skin disease [research] come from foreskin.”)

Recently, steps towards parity have grown increasingly larger: Of the five journals surveyed in the Surgery study, three have since revised their author guidelines, requiring that researchers identify the sex makeup of their test subjects and, in studies involving only one sex, provide their rationale for excluding the other; editors of the remaining three journals have said they will also make the change. Last year, the NIH’s Office of Research on Women’s Health launched a program to provide supplemental funding to currently-funded single-sex projects so that researchers could add subjects of the missing sex. And in a column published in May in the journal Nature, NIH director Francis Collins announced that the agency would begin implementing new sex-balancing policies for pre-clinical research later this fall.

“It’s really an exciting time for all of us who have been advocating for sex inclusion science,” Woodruff says. “If we can fundamentally study the way sex influences biology, I think we’re going to learn a great deal … We’re going to have a whole new era of scientific work that’s going to change, frankly, our health.”