A new funding requirement from the Medical Research Council (MRC) came into play from September 2022, necessitating that both sexes are incorporated into the experimental design of grant applications involving animals, tissues and (non-immortalised) cells. This initiative was introduced following consultation with a group of experts in biomedical research, statistics and experimental design (chaired by the co-author, Mandy MacLean). The MRC is the first UK funding body to implement such a change, following in the footsteps of the National Institutes of Health (NIH) in the US, which introduced a ‘Sex as a Biological Variable’ (SABV) policy in 2016.
Sex matters
The importance of considering sex as a biological variable is underscored by notable discrepancies in the expression of some physiological and pathological traits by males and females. Whilst some of these phenomena are easily classified as sex-specific (e.g., ovarian cancer), others are not so obvious. Characteristics shared by both sexes can differ in magnitude, such as the more vigorous response of the female immune system to an inflammatory stimulus. Divergences in the prevalence of some traits have also been noted, including the higher incidence of heart disease in men. Moreover, some characteristics are seemingly equally expressed in both sexes, yet the underlying biological processes are distinct. A notable example is the behavioural response to drugs of abuse, which is broadly similar between males and females, despite vast differences in the underlying neurological changes.
The benefits of studying both males and females
Many interesting and clinically significant biological phenomena have likely gone undiscovered due to a historic overreliance on male animals for pre-clinical investigations. In our laboratory, novel observations made in female cells and animals have led to substantial grant funding and contributed significant evidence for ongoing clinical trials.
We study a type of pulmonary hypertension (PH) known as pulmonary arterial hypertension (PAH), which affects up to four-fold more women than men. From the late 1960’s through to the 1980’s, diet pills containing indirect serotonergic agents resulted in a flurry of PAH cases that resulted in the deaths of many women. However, male mice dosed with these drugs did not develop PH, stalling pre-clinical investigations. We later demonstrated that female mice treated with serotonergic diet pills or over-expressing the serotonin transporter go on to develop PH, allowing us to establish some of the causative mechanisms. We subsequently studied human pulmonary artery smooth muscle cells (PASMCs) isolated from PAH patients, which play a key role in the progression of the disease. We found that the basal expression of proliferative signalling pathways is distinct between male and female human PASMCs, and that serotonin induces a proliferative response in female PAH patient cells only. Inhibitors of serotonin are now being investigated in clinical trials for PH.
Our work on serotonin revealed an interplay with oestrogens that predisposes females to PAH. We subsequently discovered that anastrozole, which inhibits oestrogen synthesis, reverses experimental PH in female animals but has no effect in males. A clinical trial examining anastrozole in PAH patients is underway. These examples suggest that encouraging the study of female animals, tissues and cells will lead to clinically important observations and new models of disease that might otherwise be missed.
Addressing the concerns
In the past there has been considerable trepidation that routine inclusion of female animals will considerably increase the cost of a research project. In recognition of this fact, the MRC has renewed its commitment to funding rigorously designed projects, irrespective of any increase in costs resulting from the study of both sexes. Furthermore, the new policy only mandates that sex is considered as a basic biological variable and does not necessitate that sex differences are explicitly investigated.
Another common concern is that the female reproductive cycle introduces variability to the data collected. This may be true for some physiological traits, but a growing body of evidence suggests that for most characteristics, randomly cycling females can be studied without introducing additional variability. Indeed, a meta-analysis covering behavioural, morphological, physiological, and molecular traits found no evidence for greater female variability in neurological function; in fact, male variability was substantially higher for several of the characteristics examined.
Of course, it must be recognised that there are some cases in which studying a single sex has a clear scientific rationale, or is scientifically appropriate, as is the case when investigating reproductive physiology, sex-specific diseases (e.g., Klinefelter syndrome, pre-eclampsia, breast cancers), or purely molecular interactions. The use of a single sex may also be warranted by the availability of resources (e.g., samples from patients with rare diseases, or studies requiring the use of non-human primates). As such, the MRC will still consider funding single-sex studies where justified.
Experimental design is key
It is hoped that an end to over-reliance on male animals, tissues and cells will improve the reproducibility and applicability of pre-clinical research and inform future clinical studies. Furthermore, studying both sexes as default may provide preliminary evidence for a sexual dimorphism which can then be followed-up by interested parties in a larger-scale study with adequate power to detect statistically significant effects. However, it must be recognised that a shift towards the inclusion of both sexes in all studies will not be without difficulties.
Simply incorporating female animals, tissues and cells into a study does not guarantee a deeper understanding of the field of research under investigation. Whilst the new MRC policy does not require that males and females be compared directly with one another, the fact that most MRC-funded researchers must now study both sexes will inevitably lead to an increase in the frequency of such comparisons. However, a recent analysis found that many reports of a sex difference were not backed up by an appropriate factorial statistical test. In these cases, any conclusions drawn from the data may be misleading, illustrating the importance of optimising the design and analysis of studies including both sexes. As such, the successful implementation of the new MRC policy will require appropriate training, consultation with statisticians, and tools such as The National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) experimental design assistant.
This change in requirement from the MRC is an important step towards improving the rigour of pre-clinical studies. Appropriate experimental design will be crucial to unlock biological sex differences, and to successfully translate pre-clinical findings to a clinical setting.
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