Sex still matters in cardiovascular research

In October 2021, we published an editorial letting authors, reviewers, and readers know that starting in January 2023, manuscripts submitted to the American Journal of Physiology-Heart and Circulatory Physiology (AJP-Heart and Circ) must include details on how sex as a biological variable was considered (1). We also produced an episode of the AJP-Heart and Circ podcast in which the editors discussed goals and expectations for optimizing rigorous approaches to the consideration of sex as a biological variable in cardiovascular research (https://ajpheart.podbean.com/e/sex-and-gender-use-for-ajp-heart-and-circ/). Our expectations are that for all new research articles, rapid reports, and innovative methodology article submissions, authors will have considered the use of sex and gender in the experimental design and execution of their studies and that strong scientific justification will be provided for any exemption requests not to include female/women and male/men groups (Table 1).

Table 1.

Expectations for AJP-Heart and Circ research-type articles

1. We expect authors to consider use of more than one sex or gender in experimental design, analysis, and reporting. Studies using only one sex or gender will be reviewed and published only if strong scientific justification is provided.

2. The introduction and discussion will report any known sex or gender differences or will state if no information on differences is available in the literature.

3. The abstract, methods, results, and discussion will provide details on sex or gender. results should provide combined and separated analyses when statistically appropriate. discussion should provide rationale if the analysis was not performed by sex or gender. For combined analysis, sample sizes by sex or gender should be reported and visualization should allow one to distinguish between sexes or genders (e.g., use of different colors or symbols to indicate individual sex or gender.

Rigor and reproducibility expectations for use of sex and gender in cardiovascular research, beginning January 2023 (1).

The purpose of this editorial is to remind you of the upcoming transition date and to provide a list of resource guides to help facilitate this transition (Table 2). Considering sex as a biological variable is a crucial component of strong experimental design, and studies with the highest rigor and reproducibility will include female/women and male/men representation. We acknowledge aspects of intersex biology, that gender as nonbinary, and that incorporating all gender as a spectrum in research is an area that is continually evolving in terms of best practices for variable inclusivity. One great resource for perspectives and example research articles can be found in our recent call for papers on Considering Sex as a Biological Variable in Cardiovascular Research (https://journals.physiology.org/topic/ajpheart-collections/sex-as-biological-variable?seriesKey=&tagCode=), which yielded 49 publications from over 100 submissions. The tremendous success of this call highlights that the cardiovascular community is ready for prioritizing sex as a biological variable.

Table 2.

Resources available to assist authors in considering sex as a biological variable

Resource	Link
Article Collection: Considering Sex as a Biological Variable in Cardiovascular Research	https://journals.physiology.org/topic/ajpheart-collections/sex-as-biological-variable?seriesKey=&tagCode=
A primer on incorporating sex as a biological variable into the conduct and reporting of basic and clinical research studies (2)	https://journals.physiology.org/doi/full/10.1152/ajpheart.00605.2021
Is “not different” enough to conclude similar cardiovascular responses across sexes? (3)	https://journals.physiology.org/doi/full/10.1152/ajpheart.00687.2021
When it’s time for the sex talk, words matter (4)	https://journals.physiology.org/doi/full/10.1152/ajpheart.00556.2021
In pursuit of scientific excellence: sex matters (5)	https://journals.physiology.org/doi/full/10.1152/ajpgi.00101.2012
Insufficient sex description of cells supplied by commercial vendors (6)	https://journals.physiology.org/doi/full/10.1152/ajpcell.00396.2014
Does sex matter?: an update on the implementation of sex as a biological variable in research (7)	https://journals.physiology.org/doi/full/10.1152/ajprenal.00575.2019
APS Journals Rigor and Reproducibility Checklist	https://journals.physiology.org/author-info.rigor-and-reproducibility-checklist
The Sex and Gender Equity in Research (SAGER) Guidelines (8–10)	https://researchintegrityjournal.biomedcentral.com/articles/10.1186/s41073-016-0007-6
Reporting sex as a biologic variable in research published in the Journal of Vascular Surgery Publications (11)	https://pubmed.ncbi.nlm.nih.gov/30554746/
Sex and gender differences research design for basic, clinical, and population studies: essentials for investigators (12)	https://pubmed.ncbi.nlm.nih.gov/29668873/
Increasing the statistical power of animal experiments with historical control data (13)	https://pubmed.ncbi.nlm.nih.gov/33603229/
Reduction by Prior Animal Informed Research (RePAIR) tool	https://utrecht-university.shinyapps.io/repair/

To specifically help with rigor and reproducibility, we highlight three perspectives published in the call. Denfield et al. (2) provide practical guidance and practical how-to examples on ways to incorporate sex as a biological variable into experimental design for basic and clinical research. The call also includes a perspective regarding the applicability of integrating equivalence testing when evaluating sex differences in cardiovascular research (3). Equivalence testing is used to determine whether group means are sufficiently close enough to be considered essentially equivalent. Investigators can use equivalence testing across multiple study designs, including cross-sectional comparisons and repeated-measures intervention. When used in conjunction with traditional hypothesis testing, equivalence analyses strengthen the ability to interpret findings.

As a third resource, Robinson et al. (4) provide a perspective on the topic of terminology (man/woman vs. male/female) for human subject research. This article highlights the importance of gender identity and related topics to psychological, emotional, and physical health. Pronouns and terminology accuracy are important to add clarity to the methodology and relevance of the science. Although the authors had some divergence of opinions regarding some specific qualifications, they provide consensus recommendations on how to use sex and gender terminology. Importantly, they all agreed that the most vital aspect is the broader focus on sex as a biological variable and appropriate inclusion of biological sex in all areas of cardiovascular research.

The overarching goal to incorporate sex as a biological variable in cardiovascular research is to ensure that authors consider the potential impact of sex and gender on results, interpretations, and conclusions. Complete and unbiased data sets are more reliable, and rigorous study design includes randomization, data collection, and analysis conducted across sexes and genders in a blinded manner. When reporting collective data, demarcation to allow sex or gender to be distinguished in individuals within groups will provide visual clarity for our readers. The American Physiological Society (APS) provides an APS Journals Rigor and Reproducibility Checklist (https://journals.physiology.org/author-info.rigor-and-reproducibility-checklist) to help our authors, and the Sex and Gender Equity in Research (SAGER) Guidelines were specifically developed to help authors and journal editors incorporate sex and gender reporting in publications (8–10). Further support for designing experiments and reporting results is available (11, 12).

One assumption often raised as a potential issue is the sample size. We would like to state clearly to authors that there is no need to automatically double your sample size. Working with a statistician will help to ensure that your study is adequately powered to address the question(s) evaluated. If power may be an issue, Bonapersona et al. (13) recently developed an alternative solution to use historical data to increase power. Using Bayesian priors from historical control data, they capitalized on the observation that control groups generally are expected to be similar. When they performed a simulation study to test this idea, the inclusion of data from control groups from previous studies reduced by 50% the minimum sample size required to attain 80% power. They further validated the approach on a data set based upon seven independent rodent studies and provided an open-source tool, RePAIR, to use this approach (https://utrecht-university.shinyapps.io/repair/). The bottom line is this: do not assume that there will be an issue with statistical power for studies using all sexes and genders. Spending time to develop the best experimental design will save effort later.

Because the expectations regarding considering sex as a biological variable will be applied to new research-type article submissions beginning January 2023, the editors anticipate that there will be several months of transition in the published literature in AJP-Heart and Circ. For any questions you have, the editors are ready to assist authors in meeting the expectations outlined in Table 1 (you can reach us at ajphearteditor@gmail.com). This editorial also serves as a reminder that we remain interested in submissions that focus on improving rigor and reproducibility within the cardiovascular research community, particularly regarding sex and gender use in research. If you have ideas for contributions that move us toward that goal, please reach out to us for discussion. In summary, we thank you for your commitment to ensuring the cardiovascular research field is the strongest it can be. As we continue to evolve our journal toward this goal, Vincent Van Gogh comes to mind: “Great things are done by a series of small things brought together.”

GRANTS

We acknowledge funding from National Institutes of Health Grants AA024892 (to J.R.C.), AG053585 (to A.J.L.), HL111600 (to C.M.R.), HL136737 (to J.A.K.), and HL147570 (to J.A.K.); Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development Grant 5I01BX000505 (to M.L.L.); Department of Defense Grants W81XWH-19-RTRP-IDA and W81XWH-13-2-0057 (to A.J.L.); Gheen’s Foundation (to A.J.L.); Natural Sciences Engineering Research Council (to K.R.B.); Canadian Institutes of Health Research (to Z.K. and K.R.B.); Canadian Foundation for Innovation (to K.R.B.); Heart and Stroke Foundation of Canada (to Z.K. and K.R.B.); Heart and Stroke Foundation of New Brunswick (to K.R.B.); and New Brunswick Health Research and Innovation Foundations (to K.R.B.).

DISCLAIMERS

The content is solely the responsibility of the authors and does not necessarily represent the official views of any of the funding agencies or APS. All authors have reviewed and approved the article.

DISCLOSURES

The authors hold the position as the executive editorial board of AJP-Heart and Circ and were blinded from reviewing or making decisions for the manuscript. The authors have no other conflicts of interest to disclose.

AUTHOR CONTRIBUTIONS

M.L.L. drafted manuscript; M.L.L., J.R.C., C.M.R., Z.K., K.H.K., K.R.B., J.A.K., P.K., and A.J.L. edited and revised manuscript; M.L.L., J.R.C., C.M.R., Z.K., K.H.K., K.R.B., J.A.K., P.K., and A.J.L. approved final version of manuscript.