The Sex Inclusive Research Framework: Challenging misconceptions and fostering change

Introduction

Reduction in the 3Rs is about ensuring the minimum number of animals are used where in vivo models are required to answer a scientific question. It is essential for reduction that studies with animals are appropriately designed and analysed so every animal used generates reliable data that add to the scientific knowledge base. Sex inclusive research that uses males and females is one part of designing a robust in vivo study that produces reproducible, generalisable and meaningful results using the fewest number of animals. 

Currently most preclinical research uses only one sex, typically males. This means many female animals are bred but are not used for research, and many males are used in studies that are not generalisable to the whole human population. This has also created a sex-bias in the scientific knowledge base where information about female biology is lacking. The NC3Rs and others have been driving change to make sex inclusive research the standard. For example our grant holders are required to use females and males in any animal studies they perform, unless there is a strong scientific justification to use only one sex. We have also contributed to the Sex Inclusive Research Framework (SIRF), launched earlier this year and published in Nature Communications this month.

Dr Natasha Karp led the SIRF project and has published a number of papers on the importance of using males and females simultaneously in research. We spoke to Natasha to find out why SIRF was developed and how you can use it to ensure your research is sex inclusive. 

What made you want to tackle sex bias in preclinical research? 

As a biostatistician, I know that sex inclusive research is fundamental to produce reproducible and generalisable research. My role is to support researchers to conduct in vivo experiments in line with the 3Rs, yielding robust findings that genuinely contribute to our collective knowledge. I found myself in a unique position where I had an opportunity to combine my statistical expertise and insights from collaborating with in vivo scientists to address the impact of sex in preclinical research. 

Numerous funding bodies have introduced policies mandating the inclusion of both males and females in preclinical research as the default position. There has been resistance from some groups in the research community who questioned the value of inclusive research. These views are typically based on misconceptions that including female and male animals in experiments could increase overall animals use, lead to unnecessary duplication and slow scientific progress. Among this discourse, an insight from Johnson et al. [1] struck me: we were facing a significant "knowledge gap" due to the lack of comprehensive studies involving females and males.  

At that time, I was providing statistical support to the International Mouse Phenotyping Project, an initiative aimed at mapping the genotype to phenotype relationship by generating knockout mice. This required in-house breeding programmes generating male and female animals. However, the data analysis was conducted separately for each sex. It dawned on me that the sheer scale of our data presented an opportunity to explore the extent to which sex explains variation in the data and influences the effect of interventions. This study found that sex frequently explained variation in the response [2], sparking my interest and involvement in sex inclusive research. 

What is SIRF?

SIRF is a set of resources to assess in vivo or ex vivo research proposals through the lens of sex inclusive research. The framework consists of a decision tree with a series of questions culminating in a traffic light classification of sex inclusivity. This signals if a research proposal aligns with best practices (green), presents potential risks (amber) or is sex biased (red). Each question is accompanied by supporting information and insights into why it is relevant to sex inclusive research.  

SIRF is designed for both those developing research proposals and reviewers evaluating them. While the framework has been tailored for in vivo and ex vivo studies, many of its components are also relevant to in vitro investigations and clinical trials. 

Why did you create SIRF?

Of course, researchers want to do the best science and generate meaningful data but many think of sex inclusive research as an insurmountable challenge. I realised that many of the barriers to tackling sex biased research were misconceptions endemic throughout the research hierarchy. For example, that female animals could not be used as the estrous cycle introduced unacceptable levels of variability [3], or that including female and male animals in experiments would increase overall animal use [4] – neither are true! Funders were providing the impetus for change, moving from encouraging to mandating and requiring a justification for sex exclusion. While I was helping to develop the Medical Research Council’s (MRC) ‘Sex in experimental design’ policy, a key coordinator expressed the need for resources to help grant reviewers put the policy intro practice. SIRF was borne from change management theories, recognising the need to win the hearts and minds of the scientific community to make sex inclusive research the new standard way of working.

How was SIRF developed?

I assembled a working group of community leaders including representatives from industry, academia, animal ethical review bodies and funding review committees. We developed the framework based on our collective experience of reviewing research proposals, funders’ policies on sex in experimental design, and common questions and misconceptions we had encountered from preclinical researchers. Our goal was to create a concise, robust and user-friendly framework that was applicable to a full spectrum of research questions that would guide researchers and reviewers toward more inclusive research practices. SIRF went through an iterative process of usability testing to ensure it was fit for purpose. We evaluated a collection of 30 published rationales for single sex experiments or lack of sex-based analyses and applied SIRF to 36 research proposals to check it was easy to use and helpful in practice, seeking feedback from eight UK animal ethical review bodies and the NC3Rs as a funder.

How does SIRF help the research community?

The framework is designed to move researchers away from culturally embedded misconceptions and help them to understand how they can make their experiments sex inclusive. It provides a transparent assessment of proposals based on current best practice. For those involved in funding and reviewing research, SIRF can be used as a tool for training and guidance to help them implement sex inclusive policies. We have developed an interactive version of the tool that returns a report which can be submitted along with funding proposals to encourage sex inclusive reflection early on in the research process. 

Ultimately, SIRF helps to ensure that scientific research is inclusive, meaningful and impactful.

Resources and support 

• MRC/NC3Rs webinar: Using male and female animals in research.
• NC3Rs guidance from the Experimental Design Assistant: How to account for different sexes in the design and analysis of an in vivo experiment.
• Paper: Best practices to promote rigor and reproducibility in the era of sex-inclusive research. Discusses considerations when designing sex inclusive experiments.
• Paper: Analysis of sex differences in pre-clinical and clinical data sets. Includes a flow chart with example analysis paths in different research scenarios.

References