Inclusive research: Women left out of drug research, clinical trials

Neha Powar (35) was put on blood thinners and medicines for cholesterol and hypertension after testing positive for a cardiac stress test.

However, she consulted another cardiologist when she gained weight abnormally. Much to her relief and surprise, her chest pain was diagnosed as an anxiety attack and not a cardiovascular issue.

Curiously, several reports of women being misdiagnosed with stress or anxiety when they actually had heart disease have also emerged.

Research shows women are more likely to be misdiagnosed than men when it comes to heart attacks. This is mostly attributed to ‘atypical’ symptoms of a heart attack exhibited by women, including fatigue, sleep disturbance, shortness of breath, nausea and non-chest pain symptoms such as back and abdominal pain.

In addition to misdiagnosis, a study conducted across 17 Indian hospitals between 2011 and 2015 found significant gender disparities in cardiovascular care. Despite having higher comorbidities such as hypertension, women were less likely to get the correct treatment. As a result, women in India as well as globally fare worse in the event of cardiovascular diseases, with mortality being higher for them.

It is an established fact that one of the main reasons for the worse outcomes is that the female subjects are underrepresented in research that studies disease mechanism and treatment. This can be traced to a male-default bias, its roots going all the way back to Greece of the fourth century BCE, where Aristotle referred to women as “mutilated males”, thus setting precedence for the male body being the default.

In his biological work, On the Generation of Animals, he said, “The first departure indeed is that the offspring should become female instead of male”. He admitted that this is a “natural necessity”, reducing the role of females to reproduction and carrying the progeny forward.

Though researchers in the 21st century no longer see females as mutilated males, there is gross underrepresentation of the female body in medical textbooks and biomedical research. A study found that when sex was specified, 80 per cent publications studied only male animals, 17 per cent studied only female animals, and only 3 per cent studied both sexes. Experiments are often conducted on male animals and the results are extrapolated to females.

Historically, male animals were used more as they were thought to be less variable due to the absence of monthly cycles. But one cannot exclude female models in research because of their monthly cycles as women who will benefit from such a research do face monthly cycles.

During her PhD at the National Centre for Biological Sciences, Bengaluru, Dr Kanika Gupta realised how male animals were preferred for research.

She says, “I had also been using only male animals like everybody else. Contrary to the spirit of scientific questioning, I had adopted the excuse that females cannot be used because their hormonal cycle would introduce variability in results.”

When she looked up literature to understand the bias, she found limited but impactful studies that showed the female hormonal cycle was not entirely responsible for sex differences. The studies highlighted the fundamentally flawed expectation of researchers that females will exhibit male-like behaviour in response to the same stimulus, which motivated her to study sex-specific responses.

“It also cautioned me on unfounded theories that prevail in the scientific community,” she says. Her guide and labmates were supportive towards challenging the norm of using primarily male animals.

“My guide and labmates helped at whatever stages they could. Instead of challenges, I found it great to work on sex differences because there was very little literature existing and so one could explore so many areas,” she says. Being a relatively new area of research, there were very limited research articles from India that could be cited Her PhD work published in 2021 discussed how male and female rats responded to a single traumatic experience. She was surprised by both similarities and differences in the results. While male rats showed anxiety-like behaviour and neuronal activation in the amygdala region of the brain 10 days after a trauma, female rats did not have any of these effects.

“Until we do not find the changes that female rats undergo upon a traumatic experience, it will not be fair to extend our findings from male studies to develop interventions for human disorders such as Post-Traumatic Stress Disorder prevalent in both men and women,” says Dr Gupta.

After her PhD, Dr Gupta, now a lead neuroscientist in a team working on smart EEG-wearables in India, further worked towards equal representation of males and females in her postdoc laboratory’s projects at UCLA.

Though there is an increase in research about sex differences in physiological response, such research may not be free of gender bias. According to Dr Gupta, there are some male scientists doing pathbreaking research in sex differences, whose papers she has used as a basis for her research.

“I do not have the numbers or statistics to back this up,” she says, “but somehow I have always felt that a more aggressive voice and approach to bring out this change is coming from female scientists.”

In short, exclusion of females from research may eventually have clinical translations, which can have far-reaching consequences. This includes, but is not limited to, misdiagnosis and wrong medical treatment of women. The gender gap in healthcare causes prolonged suffering and delay in the onset of correct treatment.

Many research groups are now calling for equal inclusion of women and men in clinical trials and female and male animals in research. The National Institutes of Health have made it mandatory for researchers to include both male and female animals in pre-clinical research.

The Canadian Institutes of Health Research have introduced a requirement that grant applicants answer questions regarding whether their research is inclusive of sex.

Journal editors have also recognised the ethical and practical problems of excluding females.

The European Association of Science Editors established a Gender Policy Committee assigned to develop guidelines for reporting of sex and gender equity in research, recommending journal editors to ask authors to report their research segregated by sex and gender.

“I have not heard of any such regulatory guidelines in Indian research funding agencies yet,” says Dr Gupta, whose former guide confirmed the same.

“Journals request a statement on the sexes of animals used in experiments, as was the case in our studies,” says Bhavana Muralidharan, a faculty member at Bengaluru’s Institute for Stem Cell Science and Regenerative Medicine. Her lab focuses on studying the mechanistic basis of neural development, particularly the role of chromatin regulation in this process and its disruption in neurodevelopmental disorders.

Dr Muralidharan’s group utilises mouse embryos for their experiments. Since sexing is not possible through external observation during embryonic stages, it is expected that the embryos consist of a combination of both sexes (probability being 50:50).

In order to reflect this fact, they include a statement regarding the composition of the embryos used.

“When we use newborn pups that are up to five days old, we make sure that we have adequate male and female representation,” says Dr Muralidharan.

She has not observed different experimental results for different sexes so far. She says that unless there is a scientific need to study only one sex for specific sex-linked disorders, both male and female models should be used. “You cannot do the study in male animals and make a generic statement,” she says.

Dr Gupta cites the example of Ambien, a drug tested only on male animals. When it became available in the market, it was prescribed to both men and women at the same dosage. Only after women reported severe side effects like disorientation in the morning and around 700 road accidents in the US, it was found that women needed only half the dose that men were prescribed.

Even at the level of clinical trials, there has been gross underrepresentation of female subjects. After the Thalidomide tragedy of 1962, the US Food and Drug Administration in 1977 essentially banned women of child-bearing potential in that country from participating in phase one and early phase two clinical trials, unless the trial was to test a drug for a life-threatening condition.

Though it was eventually reversed in 1993, countless drugs were put in the market through clinical research, with drugs studied only in male bodies, and the results extrapolated to females, with dosage often adjusted to body weight. However, other sex differences such as endogenous hormone levels, weight, muscle mass and metabolic enzymes also impact the pharmacokinetic and pharmacodynamic properties of drugs, implying that drugs could have drastically different effects in males and females.

Even when females are included in clinical trials, it happens only in phase three, where the study is done on 1,000 to 3,000 participants. This is long after phase one and two tests (with a male bias), where the treatment is given to fewer participants to evaluate the drug’s safety, required dosage and effectiveness. Other than unknown adverse drug reactions, the exclusion also means that clinicians are ruling out drugs that could potentially work in women, but do not show effects in men during the early clinical trials. Inclusive research is important not only for doing better science, but also to ensure that biomedical research helps women. “When female [subjects] are rejected from biological research, it is similar to denying the female kind their equal share of benefits.