Biological Sex Is Under‐Reported in Cartilage‐Related Preclinical Research: A Cross‐Sectional Analysis

ABSTRACT

Background

Intervertebral disc degeneration (IDD) and osteoarthritis (OA) share many similarities in the molecular processes involved in the onset and progression of these musculoskeletal pathologies. Biological sex is a risk factor for both conditions. Sex bias in orthopedic preclinical research affects knowledge, reproducibility, and translational aspects of basic research. This article aims to provide a comprehensive overview of how donor sex is reported in IDD and OA preclinical research using human or animal samples and in vivo models.

Methods

We performed a cross‐sectional study, searching original articles from journals with the highest impact factor in the field, to determine: (i) whether they report donor sex, and if so, whether they include this data in the analysis; and (ii) whether journals have requirements for sex reporting.

Results

Our research has four main outcomes. First, donor sex was reported in only 61.9% of the 284 cases examined. Second, among the studies where sex was reported (176), samples were predominantly from only male donors or animals (56%). Moreover, sex was rarely incorporated as a variable in outcome analysis (3.4% of cases). Finally, although 14 out of 23 journals stipulated sex reporting requirements, 37.7% of papers published in these journals failed to report donor sex.

Conclusions

Our results provide evidence for the under‐reporting of sample donor sex in OA and IDD research, which may contribute to the poor translation to clinical efficacy and the replication crisis. Our findings could guide journal policies, institutional guidelines for preclinical research, and funder requirements.

This study highlights the significant under‐reporting of donor sex in preclinical research related to intervertebral disc degeneration (IDD) and osteoarthritis (OA). We found that only 61.9% of the examined articles reported donor sex, with a predominance of male samples, and sex was rarely considered in outcome analyses. Despite some journals requiring sex reporting, a notable percentage of published papers still failed to provide this critical information. These findings underscore the need for improved reporting practices to enhance the translation of research findings to clinical applications.

Abbreviations

ARRIVE

Animal Research: Reporting of In Vivo Experiments

FAIR

Findability, Accessibility, Interoperability, and Reusability

IDD

Intervertebral Disc Degeneration

JCR

Journal Citation Report

OA

Osteoarthritis

SAGER

Sex and Gender Equity in Research

STROBE

STrengthening the Reporting of OBservational studies in Epidemiology

1. Introduction

Intervertebral disc degeneration (IDD), as a major cause of chronic low back pain, and osteoarthritis (OA) share many similarities in the molecular processes involved in the onset and progression of these pathologies [1, 2, 3, 4]. Given the lack of effective and long‐lasting treatments for IDD and OA [3, 5, 6], it is especially important to prioritize preclinical research in these areas. The main therapeutic approaches investigated are cell therapy, gene therapy, tissue engineering, and the use of growth factors and small molecules [7, 8]. Moreover, a deep comprehension of the involved molecular mechanisms is essential to make a step toward the development of new therapeutics. This implies expanding existing knowledge on the risk factors associated with the progression and pathogenesis of both IDD and OA.

Biological sex is one of these factors, as evidenced by prevalence rates of IDD and OA, where women appear to be more affected than men [9, 10, 11]. Hormonal factors play a crucial role [12, 13], and this may be supported by the increased risk of developing OA after menopause [14, 15, 16, 17]. Genetic factors are also important, with sex influencing the expression and regulation of genes involved in the pathogenesis of many noncommunicable diseases [18, 19].

Despite these facts, preliminary studies in the orthopedic field have shown that sex tends to be under‐reported in preclinical studies [20, 21]; and this can have a huge impact on new knowledge, reproducibility, and translational aspects of basic research [22, 23, 24]. Although similar studies exist in the orthopedic field, there is a lack of specificity regarding sex reporting practices within specific research areas such as OA and IDD. In addition, researchers in the field of OA and IDD have emphasized the importance of avoiding sex bias in preclinical research [25, 26].

Sex bias in preclinical research is an old and persistent problem, identified as one of the reasons for the poor translation and reproducibility of preclinical research. Furthermore, the lack of female representation in preclinical studies has led to inequalities in understanding, diagnosing, and treating diseases [22, 27, 28, 29].

Scientific journals are trying to encourage authors to report the sex of the samples used in their research, but this is still quite uneven across different journals. Some journals have mandatory requirements with detailed instructions; others provide external guidelines or recommendations, while some do not address the issue [30]. Although reporting samples sex is a significant step toward better science, analysis of sex as a covariate and interpretation of sex‐stratified data is also required [31].

This article aims to provide a comprehensive overview of how sample donor sex is reported in preclinical IDD and OA research. We directly searched original articles from journals with the highest impact factor in the field to determine: (i) whether they report sample sex, and if so, whether they include this data in the analysis; and (ii) whether journals have requirements for sex reporting.

2. Methods

Our protocol was prospectively registered in the open science framework (Appendix S1). We conducted a cross‐sectional study of all basic and preclinical research published between 1 January 2022 and 31 December 2022. We focused on Q1 and Q2 journals—thus including all major journals in these fields—in the following journal citation report (JCR) categories: orthopedics, rheumatology, and cell and tissue engineering, as they best represent OA and IDD publications.

2.1. Eligibility Criteria

Publications were eligible if they included any type of preclinical research related to IDD and/or OA. We included original scientific research articles in English, published in 2022 in Q1 and Q2 journals in the orthopedics, rheumatology, and cell and tissue engineering categories of the JCR, reporting basic science or preclinical studies on IDD and OA, using in vivo, ex vivo, or in vitro models. We excluded articles on clinical research or other types of articles that are not original preclinical research, such as reviews, editorials, commentaries, opinions, letters, guidelines, policy recommendations, books, book chapters, book reviews, and congress abstracts/posters.

2.2. Search Strategy and Selection Process

We identified three JCR categories that were expected to enclose a significant number of publications related to OA and IDD in preclinical research: orthopedics, rheumatology, and cell and tissue engineering. Within each category, we selected all Q1 and Q2 journals. Subsequently, we conducted a comprehensive search for all publications from the year 2022 within these selected journals.

In the screening process, we identified all articles related to IDD and OA by screening the titles and abstracts and applying the inclusion and exclusion criteria. After the title/abstract screening, the full texts were obtained and screened.

2.3. Data Extraction

We created and piloted extraction forms designed to collect information from each article of the defined cohort. These forms were used to extract details such as the type of study, the species or samples used, whether the investigators reported the sex of the subjects or animals, whether they included sex as a variable in the analysis, and if not, whether the reasons for this omission were provided. Full details of our data extraction form can be found in Appendix S3.

For each journal containing articles included in the cohort, we reviewed their author guidelines to determine if they required reporting of sex and related data for publication. The data extraction form for journals can be found in Appendix S4.

The researcher who extracted the data from the articles also reviewed the data extraction process from the journals; and vice versa.

2.4. Data Analyses and Synthesis

For the analysis stage, the results were summarized in a spreadsheet. The data were analyzed in three stages. The first consisted of a quantitative descriptive analysis of each variable in a unidimensional manner, that is, all data were summarized by column in percentages or means, as appropriate. In the second stage, the previous variables were disaggregated by subgroups based on the objectives of the study. The third and final stage consisted of synthesizing and comparing each result from stages one and two according to the available evidence.

Descriptive statistics were performed using Microsoft Excel spreadsheet and GraphPad Prism 10.1.2. We calculated the frequency distribution of the results, which were described as percentages.

2.5. Availability and Data Report

The protocol and extraction forms are available on the Open Science Framework (https://osf.io/tsqj6).

To ensure a complete and adequate report, we provide the Strengthening the Reporting of Observational studies in Epidemiology (STROBE) [32] checklist in Appendix S5.

3. Results

3.1. Journals and Publications Selection Process

Forty‐six Q1 and Q2 journals were identified in the field of “Orthopedics,” 18 in “Rheumatology” and 14 in “Cell and Tissue Engineering,” in total 78. Removal of duplications left 74 journals. From each of the 74 journals, we searched for articles published in 2022 and proceeded to title/abstract screening, resulting in 232 potentially eligible articles. After full‐text screening, we included 193 articles that met the eligibility criteria (Figure 1). These articles were published in 23 journals. The cohort of included articles and their respective journals are shown in Appendix S6.

FIGURE 1.

Flow diagram of the journal and article selection process. This figure illustrates the multi‐step process used to identify eligible studies published in 2022. The text describes the journal screening steps and article selection process. Articles were selected from Q1 and Q2 journals in three journal citation report (JCR) categories: orthopedics, rheumatology, and cell and tissue engineering. The final screening resulted in 193 included articles published across 23 journals.

3.2. Characteristics of Publications and Journals

Of the 193 articles analyzed, 73.6% (n = 142) were targeting OA and 26.4% (n = 51) were targeting IDD. The funding of the articles was reported as follows: 72.1% (n = 139) from public sources, 11.9% (n = 23) from mixed sources (public and private), 4.1% (n = 8) from private institutions, and 11.9% (n = 23) did not report funding or stated that they were not funded. The four methods that were most present included gene therapy, which represented 16% (n = 31) of articles; 13.5% (n = 26) reported on molecular therapies, 7.3% (n = 14) on tissue engineering, and 6.2% (n = 12) on cell‐based therapies, while 57% (n = 110) addressed other types of approaches. In terms of study type (i.e., in vivo, in vitro, ex vivo), some articles reported more than one study type per article. Figure 2 provides a summary of the main study results. In total, we found 284 different study types in the eligible publications, of which 48.9% (n = 139) were in vitro studies, 40.5% (n = 115) were in vivo studies, and 10.5% (n = 30) were ex vivo studies. In vitro studies involve all the experiments conducted on isolated cells, while ex vivo studies refer to experiments on tissue explants or organs from an organism in an external environment. In vivo studies include experiments conducted within a living organism, in this case, with animal models.

FIGURE 2.

Summary of key findings on sex reporting practices in included studies. This figure provides an overview of sex reporting across the 284 study types identified within the 193 included articles. It summarizes: (i) whether the sex of sample donors was reported, (ii) the distribution of donor sex (male, female, both), (iii) whether sex was considered in the analysis (as a covariate or in the discussion), and (iv) the distribution of study models (in vivo, in vitro). Percentages refer to proportions out of the total number of study types. This summary highlights that sex was not reported in 38.1% of study types and was rarely considered analytically or acknowledged as a limitation.

Of the 23 journals analyzed, 43.5% (n = 10) were Q1 and 56.5% (n = 13) were Q2. The publishers were Springer Nature (26.1%), Wiley (17.4%), Elsevier (13.1%), Sage Journals (13.1%), Oxford Academic (8.7%), Taylor & Francis, Hindawi, Lippincott Williams & Wilkins, Forum Multimedia Publishing, and own publisher 4.3% each. Of all the journals included, 30.4% (n = 7) were from an academic society. Full details can be found in Appendix S7.

3.3. Sex Report

The 193 included articles reported 284 different study types (in vitro, in vivo, ex vivo), as some articles included more than one study type. Of these, 61.9% (n = 176) reported the sex of the samples used in the research. Of the 176 that reported the sex of the samples, 56.3% (n = 99) were male samples, 26.1% (n = 46) were both sexes, and 17.6% (n = 31) were female samples.

Of note, of the 176 study types that reported sample sex, only 6 (3.4%) included sex as a covariate in the analysis of results. In addition, only 19 studies (10.8%) included sex in the discussion. Of the studies that reported sample sex, 57.4% (n = 101) were in vivo studies (Figure 3).

FIGURE 3.

Was the sex of the samples reported? Numbers of each study type (in vivo, in vitro, ex vivo) based on whether biological sex was reported or not.

The 38.0% (n = 108) of study types that did not report sample sex did not mention this as a limitation or any other comment in the discussion. Of the 108 studies that did not report sample sex, 75.9% (n = 82) were in vitro studies (Figure 3).

When analyzing sex reporting according to the type of experiments (Figure 4), we observed that in in vitro studies, sex was not reported in 59% (n = 82) of the cases, while cells from both sexes were used in 22.3% (n = 31) of cases. Cells from only male donors were used in 12% (n = 17) of cases, and from only female donors in 6.5% (n = 9) of cases. In ex vivo studies, sex was not reported in 40% (n = 12) of cases. Samples from both male and female were used in 20% (n = 6) of cases, exclusively from male donors in 30% (n = 9) of cases, and exclusively from female donors in 10% (n = 3) of cases. Lastly, in in vivo studies, sex was not reported in 12.2% (n = 14) of cases. Male animals were used exclusively in 66.5% (n = 73) of cases, female animals in 16.5% (n = 19) of cases, and animals from both sexes in 7.8% (n = 9) of cases.

FIGURE 4.

Sex reporting per type of study and species. Total sex reporting (a) and sex reporting per type of study (b). Percentages of species in the studies (c) and sex reporting per species (d).

Within the 284 studies, various species were used, including humans, rats, mice, rabbits, bovines, pigs, sheep, dogs, horses, goats, and monkeys (Figure 4). Specifically, experiments involved human samples in 94 cases (33.1%), rat samples in 74 cases (26.1%), mouse samples in 67 cases (23.6%), and smaller percentages of other species like rabbit (4.6%) and bovine (4.6%), among others. Sex reporting remains low for species used mostly for in vitro and ex vivo tests, such as human (45.7%) and bovine (23.1%) while the percentage of reporting increases for species used mostly for in vivo studies, such as mouse (73.1%), rat (73.0%) and rabbit (92.3%).

In the 284 experiments, the numbers and sex of all sample donors or animals were fully reported in 50.4% of cases, while in 16.6% numbers per sex were not specified. In 28.5% of cases, the number of sample donors or animals used was not clear. In the remaining 4.6% (13 experiments) full number reporting was not applicable since the authors used cell lines.

3.4. Obligation to Report Sex Data According to Journals

Of the journals included, 60.9% (n = 14) require the sex of samples to be reported. The requirement appears in the authors' guidelines; although in most cases, they refer to available external guidelines and provide a link to them. Three journals refer to the SAGER (The Sex and Gender Equity in Research) guideline and the rest to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guideline. Four of the journals, in addition to mentioning ARRIVE, have a paragraph explaining in detail why it is important to report sex, the differences between sex and gender, and other important and clarifying information. Some journals (21.7%, n = 5) recommended reporting the sex of samples or using the ARRIVE guidelines for reporting results. Finally, others (17.4%, n = 4) made no recommendation or reference to guidelines for reporting sex. Further details can be found in Appendix S7.

The ARRIVE guideline has a section where animal characteristics should be reported, including the sex of the animal. In our cohort of in vivo studies (n = 115), 24.3% (n = 28) of the publications mentioned the ARRIVE guideline in the text of the article. Of these, only 32.1% (n = 9) included the guideline checklist that should be included when mentioning ARRIVE as a tool for reporting in vivo results. Furthermore, of the nine that mentioned the guideline and included the checklist, 2 did not report the sex of the animals.

3.5. Relationship Between Articles Reporting Sex and Journals Requiring Sex Reporting

A total of 138 articles were published in the 14 journals where sex reporting was mandatory. Among these publications, 62.3% (n = 86) effectively reported sex, while 37.7% (n = 52) did not.

A visual summary of all results can be found in Appendix S2.

4. Discussion

Although the importance of using and reporting samples of both sexes in basic science was highlighted in the 1990s [33] and evidence of lack of sex reporting is growing, there is still a huge gap in the consideration of sex as a variable in research analysis.

Our research shows four main concerns:

1. There is low reporting of sex in the samples; in most cases, no justification for this is given.

2. When sex is reported, the samples are predominantly male.

3. Even when sex is reported, it is not included as a variable in the outcome analysis in almost any of the studies.

4. Most journals require the sex of samples to be reported through external guidelines, mostly ARRIVE guidelines.

4.1. Low Reporting of Sex

Low reporting of sample sex might not be a scientific problem if some justification was given. As in many studies in other areas of basic science [27, 34, 35] our study shows that the lack of sex reporting is rarely accompanied by adequate justification and not even mentioned as a limitation of the study. Moreover, in the few cases where it was justified, the argument was based on commercial issues (e.g., availability of only male animals in the market) [36]. In other studies, the justification given was “to avoid experimental variability,” which is a questionable research practice [33, 35]. Another observation to note is that in 13 cases, authors utilized cell lines without providing information regarding the original donors. Despite the accessibility of such information, we included these 13 cases in the statistic of not reported sex, as we support that sex information is fundamental and should not be omitted from any publications.

4.2. Male Samples Are Predominant

As basic research underpins clinical research, differences in male and female physiology and pathophysiology cannot be ignored at this early stage. The contributions of sex to the prevalence, presentation, and progression of many diseases are profound. However, it has been a common and preferred practice to use male subjects in basic and preclinical research [36]. Our study shows the same situation in OA and IDD research, which is highly problematic as both diseases affect women more than men [9, 10, 11]. Preference for male samples may occur for many reasons, as mentioned, to reduce experimental variability due to a lack of hormone cycle dependency, because they are cheaper to purchase in some countries, confound exposure in case of undetected pregnancy, among others [29]. Using only males is then based on the false assumption that there are no major differences between the sexes outside of reproductive functions [29, 33, 37, 38].

4.3. Sex as Variable

Reporting sex is not enough. Sex of sample donors or subjects needs to be reported, but not as another characteristic of the samples. Sex should be reported and included as a variable in the outcome analysis [27, 33, 34, 35]. In our study, we found that only a small fraction (less than 3.4%) of the articles that reported sample sex included sex as a covariate in the results. Several prescription medicines have been withdrawn from the market following post‐marketing reports of greater adverse effects in women than in men [39]. In reviewing their approval, all of the preclinical and clinical studies supporting the evidence for the drug were conducted on predominantly male samples and participants [36]. This is particularly evident in cardiovascular research, where the prevalence of certain diseases is higher in women, yet they are under‐represented in cardiovascular clinical trials [40]. The case of digoxin, which was later found to increase the risk of death in women, is an example in the cardiovascular field of the consequences of a lack of diversity in preclinical and clinical research [41].

4.4. Sex Reporting as Journal Requirement for Publication

The journals where it is mandatory to report the sex of sample donors or subjects mostly request to do so by external guidelines, mostly ARRIVE guidelines. This could be problematic as the ARRIVE guidelines only cover animal studies, that is in vivo studies. This means that journals that only rely on ARRIVE guidelines to report the sex of samples do not include any strategies, recommendations, or obligations to include the sex of samples in in vitro or ex vivo studies.

Only 3 journals, from the same publisher, mention the SAGER guideline, a guideline specific for gender and sex reporting. These journals also offer a full section entitled ‘Reporting sex and gender‐based analyse’, which defines sex, gender, and the importance of reporting, among other things.

Surprisingly, we identified 7 articles that used the term “gender” instead of “sex,” confusing these concepts. Scientific research, and therefore scientific writing such as articles published in academic journals, should demonstrate a clear understanding of the terminology employed. “Sex” is defined as “a set of biological characteristics in humans and animals, primarily associated with physical and physiological traits, including chromosomes, gene expression, hormone levels and function, and reproductive/sexual anatomy” [38]. “Gender” is defined as “the socially constructed roles, behaviors, expressions and identities of girls, women, boys, men and gender diverse people” [38]. Therefore, “sex” and “gender” are different concepts and should be used appropriately. However, misuse of the concepts was found in articles about mice, horses, dogs, and human cells, all published in journals in the Orthopedic category of the JCR. This discrepancy shows a need for increased awareness and training among authors, editors and reviewers within the academic community.

Although our study examined a limited sample of journals, it is interesting to note that among the journals where sex reporting was mandatory (n = 14), the majority belonged to the Q2 category (71.4%, n = 10). This suggests that sex reporting is still a marginal issue in those journals that are typically associated with higher editorial standards.

Another aspect to consider is that 72.1% of the publications included in this work were funded from public sources. This highlights the importance of enhancing the quality of design and reporting in publications within the field to ensure the responsible use of public funds and mitigate the repetition of studies without clear purpose.

In this context, it could be beneficial to reanalyze previously obtained data when applicable, incorporating sex as a covariate or including sex‐specific analyses. This can offer the potential to identify new insights without the need to conduct additional experiments. To enable such analyses, it is essential to adhere to the FAIR (findability, accessibility, interoperability, and reusability) guiding principles [42]. These standards aim to maximize the value and impact of scientific data to facilitate the advancement of scientific discovery and innovation.

This study has some limitations that should be considered. We focused on basic and preclinical research published in Q1 and Q2 journals in the orthopedics, rheumatology, and cell and tissue engineering categories of the JCR for the year 2022. Consequently, this work does not allow us to derive information regarding trends over different time periods or the possibility of an increasing awareness of sex reporting in the field. The methodological approach employed, involving manual screening of publications one by one, may have introduced some imprecisions in the extraction of data. Moreover, the extraction of article data was performed by one researcher, while the extraction of journal data was carried out by another researcher. Although it is a limitation that data extraction was not done by two researchers independently, we want to emphasize that one researcher appraised the data extraction process of the other.

Biological sex plays a significant role as a risk factor for both OA and IVDD, emphasizing the importance of analyzing and reporting data separately based on sex [43, 44]. This approach can enhance our understanding of the pathophysiology and improve the potential for translational application of research findings. Additionally, sex differences in pain [45, 46], drug response, and treatment side effects should be considered in basic and preclinical research [20]. A lack of sex‐specific reporting neglects the chance to develop effective treatments for these diseases, which involve high morbidity burdens. Therefore, we need to adopt more strict guidelines for sex reporting. We also recommend that institutions and funding agencies adopt policies that support sex reporting in preclinical research to ensure that this bias will be reduced. Addressing this, we can improve the quality and reproducibility of preclinical research and eventually improve treatments for patients with IDD and OA.

5. Conclusion

Our results provide evidence of the under‐reporting of sample sex in OA and IDD research, which has been identified as one of the problems of poor translation to clinical trials and the replication crisis. Our findings could guide journal policies [47], institutional guidelines for preclinical research, as well as the requirements of funders.

Author Contributions

Daniele Zuncheddu: conceptualization, methodology, data curation, investigation, visualization, formal analysis, writing – original draft. Paola Buedo: conceptualization, methodology, data curation, investigation, writing – review and editing, writing – original draft. Martin J. Stoddart: conceptualization, funding acquisition, project administration, writing – review and editing. Laura B. Creemers: conceptualization, funding acquisition, supervision, project administration, writing – review and editing. Sibylle Grad: conceptualization, supervision, funding acquisition, project administration, resources, writing – review and editing, methodology. Marcin Waligora: conceptualization, methodology, supervision, funding acquisition, project administration, resources, writing – review and editing.

Conflicts of Interest

Sibylle Grad is an Editorial Board member of JOR Spine and is the corresponding author of this article. To minimize bias, she was excluded from all editorial decision‐making related to the acceptance of this article for publication.

Supporting information

Appendix S1: Study protocol.

Appendix S2: Summary of the results.

Appendix S3: Extraction data form for articles.

Appendix S4: Extraction data form for journal author guidelines.

Appendix S5: STROBE checklist.

Appendix S6: Cohort of included articles.

Appendix S7: Summary of the journals.

Zuncheddu D., Buedo P., Stoddart M. J., Creemers L. B., Grad S., and Waligora M., “Biological Sex Is Under‐Reported in Cartilage‐Related Preclinical Research: A Cross‐Sectional Analysis,” JOR Spine 8, no. 3 (2025): e70104, 10.1002/jsp2.70104.

Data Availability Statement

All data generated or analyzed during this study are included in this published article (and its [Link], [Link], [Link], [Link], [Link], [Link], [Link] files).