The causal impact of bioavailable testosterone levels on osteoarthritis: a bidirectional Mendelian randomized study Running Title: Causality of testosterone on OA in bidirectional MR study

The causal impact of bioavailable testosterone levels on osteoarthritis: a bidirectional Mendelian randomized study Running Title: Causality of testosterone on OA in bidirectional MR study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The causal impact of bioavailable testosterone levels on osteoarthritis: a bidirectional Mendelian randomized study Running Title: Causality of testosterone on OA in bidirectional MR study Zong Jiang, Xiaoling Yao, Yuzheng Yang, Fang Tang, Wukai Ma, Xueming Yao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3980446/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Apr, 2025 Read the published version in BMC Musculoskeletal Disorders → Version 1 posted 10 You are reading this latest preprint version Abstract Background: It has been shown that low testosterone levels are associated with the development of osteoarthritis (OA). In our study, we aimed to investigate a bidirectional causal relationship between bioavailable testosterone levels and OA using Mendelian randomization (MR) analysis. Methods: In our study, the datasets from publicly available genome-wide association study (GWAS) were adopted, including the OA-related dataset (ukb-b-14486) and the bioavailable testosterone levels-related dataset (ebi-a-GCST90012104). In total, five methods were utilized, namely MR Egger, Weighted median, Inverse variance weighted (IVW), Simple mode, and Weighted mode. Among them, IVW was the main analytical method. Additionally, the sensitivity analysis was carried out through the heterogeneity test, the horizontal pleiotropy test, and the Leave-One-Out (LOO) method. Results: The result of forward MR analysis demonstrated that bioavailable testosterone levels were considerably relevant to OA, and were a risk factor for OA (OR = 1.009, 95% CI: 1.001-1.017, P = 0.020). However, through reverse MR analysis, we did not find a causal relationship between OA and bioavailable testosterone levels. Moreover, the results of the sensitivity analysis suggested that our results were reliable. Conclusion: The results of our study supported a causal relationship between bioavailable testosterone levels and OA. Bioavailable testosterone levels osteoarthritis mendelian randomization bidirectional Figures Figure 1 Figure 2 Figure 3 Introduction Osteoarthritis (OA) is a common and complex degenerative joint disease worldwide, with chronic inflammation of joints and their appendages leading to pain, swelling, limited functional activity, and even deformities being the main clinical manifestation [ 1 – 3 ]. With the aging of the global population, the incidence rate of OA is increasing year by year, which seriously affects the quality of life of patients and brings huge economic burden to society [ 4 – 6 ]. At present, the pathogenesis of OA is not clear, but it is known that factors such as obesity, trauma, and lifestyle are all risk factors for the onset of OA [ 7 – 9 ], but even if these risk factors are avoided, the incidence rate of OA is still high. Preventing cartilage degeneration remains the key to preventing and treating OA [ 10 , 11 ]. However, there are currently no reliable biochemical biomarkers that can prevent the occurrence of OA [ 12 , 13 ]. Studies have shown that hormone replacement therapy has a clear effect on the treatment of OA and can effectively reduce joint replacement rates [ 14 ], indicating that sex steroids may play an important role in the development of OA. Therefore, exploring the causal relationship between OA and sex steroids is worth studying, so as to assess whether regulating sex steroid levels can serve as an effective therapy for preventing and treating OA . Testosterone is mainly a steroid hormone secreted by the male testes and female ovaries. It is transported to the target tissues in the body through binding of sex hormone binding globulin (SHBG) in the blood to function [ 15 ]. It is an important androgen that promotes human growth and development, and maintains normal organ function [ 16 , 17 ]. Obesity is a risk factor for OA, and studies have shown that testosterone deficiency can lead to obesity and insulin resistance, while testosterone therapy can inhibit fat deposition and reduce insulin resistance [ 18 ]. Testosterone also inhibits the endoplasmic reticulum (ER) stress mechanism and inflammatory response and cell apoptosis, and reduces the concentration of extremely low density lipoprotein and susceptibility to OA. Androgen receptors are present in human knee joint chondrocytes, synovial tissue and bone cells, and are expressed in both male and female patient chondrocytes [ 19 ], indicating a close correlation between chondrocytes and androgens [ 20 ]. There are androgen receptors in articular chondrocytes, which have the ability to synthesize androgens, and testosterone can affect the internal environment of bones by binding to receptors, which further indicates that androgens may be related to the pathogenesis of OA [ 21 ]. Mendelian Randomization (MR) is an analytical method for exploring causal relationships by introducing genome-wide association studies (GWAS) data represented by single nucleotide polymorphisms (SNPs) as mediating instrumental variables. Genetic variation is a random allocation of fixed alleles at conception, which can effectively overcome potential confounding factors and reverse the effects of causality [ 22 ]. Currently widely used in research on exposure and outcomes of various diseases, it can help us analyze and understand the causal relationship between exposure factors and diseases more conveniently and accurately. At present, a research has found a causal relationship between testosterone with hip osteoarthritis and the risk of hip replacement. However, the causal relationship between testosterone and overall OA, as well as who causes and effects OA and testosterone, is remains unclear [ 23 ]. Bidirectional MR evaluates whether there is a reverse causal relationship between exposure and outcome, that is, whether the outcome can lead to the occurrence of exposure, and conducts two double sample MR analyses to better understand the causal relationship between instrumental variables. Therefore, based on publicly available GWAS data, this study conducted a bidirectional MR analysis using bioavailable testosterone levels and OA as exposure factors or outcomes, providing a new reference for the causal relationship between bioavailable testosterone levels and OA. The overview of the study design was shown in Fig. 1 . Materials and Methods Study Design MR studies must satisfy the following three assumptions. Firstly, genetic variants selected as instrumental variables (IVs) are strongly correlated with exposure factors. Secondly, SNPs in genetic variants are independent of confounding factors that are related to exposure and outcome. Thirdly, genetic variants affect outcomes only through exposure and not through other biological pathways [ 24 ]. The study used data from published public databases, and therefore, this study did not require any additional ethical approval. Data Source and Pre-processing The IEU OpenGWAS database ( https://gwas.mrcieu.ac.uk/ ) was utilized to download OA-related dataset (ukb-b-14486) and bioavailable testosterone levels-related dataset (ebi-a-GCST90012104). The ukb-b-14486 contains 38,472 OA samples, 424,461 control samples, and 9,851,867 SNP samples. The ebi-a-GCST90012104 includes 382,988 samples and 16,137,327 SNP samples [ 25 ]. Afterwards, SNPs selected as IVs were screened via “TwoSampleMR” R package (version 0.5.6) with P < 5 × 10 − 8, and linkage disequilibrium analysis (LDA) was performed to ensure independence (r2 = 0.001 and kb = 10,000). Statistical analyses After the IVs were filtered, MR analyses were performed by combining the MR function and five methods, as follows MR Egger, Weighted median, Inverse variance weighted (IVW), Simple mode, and Weighted mode. Since the IVW method provides more precise causation and its results are unbiased, the results are mainly referred to the IVM. Then, odds ratios (ORs) were calculated, with an OR equal to 1 indicating no correlation between exposure and outcome; an OR greater than 1 indicating that exposure promotes the outcome; and an OR less than 1 indicating that exposure inhibits the occurrence of the outcome event. The results were presented using scatter plots, forest plots, and funnel plots. To determine the reliability of the results of the analysis, a sensitivity analysis was conducted via heterogeneity test, the Horizontal pleiotropy test, and the Leave-One-Out (LOO) method. When performing the heterogeneity test, a Q value greater than 0.05 indicates that there is no heterogeneity. A P value is greater than 0.05 demonstrating that there is no horizontal pleiotropy in Horizontal pleiotropy test. The LOO is used to see if there are outliers in the effect of each SNP. Results Causal effect of bioavailable testosterone levels on OA After screening, 105 SNPs were obtained that were strongly relevant to bioavailable testosterone levels but not associated with OA. As shown in Table 1 , there was a causal relationship between bioavailable testosterone levels and OA ( P = 0.020), and bioavailable testosterone levels were a risk factor for OA (OR = 1.009). The results of the scatter plot showed that the slopes of the lines were positive, further validating bioavailable testosterone levels as a risk factor for OA ( Fig. 2 A ) . Of the forest plot results, the point of IVM was on the right, which supported the view that bioavailable testosterone levels increase risk of OA ( Fig. 2 B ) . The funnel plot showed that MR conformed to Mendel's second law of random grouping ( Fig. 2 C ) . Table 1 MR analysis results of bioavailable testosterone levels for OA Exposure Outcome Method Nsnp P-value OR(95% CI) Bioavailable testosterone levels Osteoarthritis MR Egger 98 0.757 1.002(0.987–1.018) Weighted median 98 0.027 1.012(1.001–1.024) Inverse variance weighted 98 0.020 1.009(1.001–1.017) Simple mode 98 0.561 1.007(0.984–1.031) Weighted mode 98 0.185 1.009(0.996–1.023) Evaluation of the reliability of the results, we then did a sensitivity analysis. Firstly, the Q value of IVM was less than 0.05, suggesting that there was heterogeneity (Supplementary Table 1) . However, the P value was less than 0.05 for IVM, demonstrating that heterogeneity did not cause a significant effect on the results. Then, according to the horizontal pleiotropy test, there were no confounding factors in this study (P = 0.328) (Supplementary Table 2) . Thereafter, the LOO method suggested that there were no points of deviation ( Fig. 2 D ) . In conclusion, the results that we obtained were reliable, and there was a causal relationship between bioavailable testosterone levels and OA. Causal effect of OA on bioavailable testosterone levels To illustrate the exact causal relationship between bioavailable testosterone levels and OA, we performed a reverse MR analysis, with OA as the exposing factor and bioavailable testosterone levels as the outcome. A total of six independent SNPs were obtained with the same screening criteria. Under the IVW model, OA was not significantly associated with bioavailable testosterone levels (P = 0.779) ( Table 2 ) . Moreover, the remaining four models demonstrated the same results. Furthermore, the sensitivity analysis results manifested that our results were reliable ( Fig. 3 ; Supplementary Table 3–4) . Table 2 MR analysis results of OA on bioavailable testosterone levels (Reverse MR). Exposure Outcome Method Nsnp P-vauel Beta Osteoarthritis Bioavailable testosterone levels MR Egger 6 0.518 -0.482 Weighted median 6 0.986 -0.005 Inverse variance weighted 6 0.779 -0.076 Simple mode 6 0.856 -0.078 Weighted mode 6 0.932 0.032 Discussion There have been reports on the relationship between bioavailable testosterone levels and OA, but the causal relationship between them is still unclear. In this study, we conducted a bidirectional two sample MR analysis using the maximum GWAS data of genetic variation to evaluate the causal relationship between testosterone and OA, demonstrating strong genetic evidence. We found a positive causal relationship between bioavailable testosterone levels and the risk of OA. Research on OA mainly focuses on estrogen, while research on androgens is relatively scarce. Research on OA mainly focuses on estrogen, while research on androgens is relatively scarce. Testosterone is mainly dissociated by binding to sex hormone binding protein (SHBG) and albumin, resulting in free testosterone, which is an important bioavailable testosterone in the human body and plays an important role in human growth and lipid metabolism [ 26 , 27 ]. The lack of testosterone leads to disorders in lipid metabolism, protein metabolism, and glucose metabolism, resulting in a series of metabolic syndromes, obesity, and other conditions [ 28 ]. Biologically available testosterone is one of the most important androgens in the human body, and its concentration varies in a peak line with age [ 29 , 30 ]. After the age of 40, there is a linear decline in testosterone levels in males and postmenopausal females [ 31 ]. Under normal circumstances, testosterone binds to specific receptors in the cell to form a testosterone receptor complex, and the active testosterone receptor complex binds to specific androgen response sheets (ARE) on the target gene to regulate gene expression. Studies have shown that both estrogen and androgen receptors exist in osteoblasts [ 32 , 33 ]. In addition to its direct effects on bone and cartilage, testosterone not only binds to androgen receptors, but also to estrogen receptors to affect bone calcium metabolism balance [ 21 ]. In addition, testosterone can aromatise and convert into estradiol, which then binds to estrogen receptors and participates in the physiological regulation of bone and cartilage [ 34 ]. The decrease in testosterone levels affect cartilage metabolism through androgen receptors and ion channels, as well as leading to decreased in estradiol conversion rate, which leading to cartilage degeneration and the formation of OA [ 35 ]. When testosterone is at normal levels in men but there is an aromatase deficiency in the body, most of the androgens cannot be converted into estrogen, which often results in lower levels of estrogen in the body, which leads to joint cartilage degeneration and the development of OA [ 36 ]. This indicates a significant correlation between androgens, especially testosterone, and OA. In a study of male calf knee joint cartilage, it was found that testosterone increased the content of glycosaminoglycans in the extracellular matrix of chondrocytes, promote the coverage of type II collagen on the cartilage surface and the growth of cartilage fiber structure in joint cartilage [ 37 ]. Currently, clinical studies on the correlation between testosterone levels and OA are mainly case reports. A study of the correlation between hormone levels and hand OA in 573 premenopausal women found a significant correlation between lower levels of serum testosterone and the prevalence of hand OA [ 38 ]. In another study targeting men, serum testosterone levels were found to be positively correlated with cartilage thickness [ 39 ]. Testosterone can increase male muscle strength and is often recommended for the treatment of male musculoskeletal pain, its also can effective to reduce fat content and inhibit inflammatory reactions [ 40 , 41 ]. In the study of serum testosterone levels and OA symptoms, it was found that higher levels of serum testosterone can reduce the joint osteoarthritis index (WOMAC) [ 42 ]. These studies have shown a significant correlation between serum testosterone levels and OA, but the causal relationship remains unclear. Our study suggests a causal relationship between bioavailable testosterone levels and OA from a genetic perspective, providing indicative evidence for the prevention and treatment of OA. In this study, we had sufficient samples for MR analysis to explore the causal relationship between bioavailable testosterone levels and OA, and found a causal relationship between bioavailable testosterone levels and OA. This study has several advantages. Firstly, the data is sourced from the GWAS database, which can exclude the interference of confounding factors. Secondly, we use bidirectional MR analysis to study the impact of causal relationships on causal inference. We also used sensitivity analysis using multiple methods to exclude bias caused by related and unrelated pleiotropy. In summary, we demonstrate a potential causal relationship between bioavailable testosterone levels and OA, whereas OA is the cause and bioavailable testosterone levels are the result, and there is no causal relationship between the two. However, our research still has certain limitations. Firstly, the study population is of European ancestry, and the scope of the study is relatively limited. Secondly, due to the lack of raw data from the WGAS database, subgroup analysis was not conducted. Finally, we only found a causal relationship between bioavailable testosterone levels and OA from a genetic perspective, and the mechanism of its occurrence is still unclear. Nevertheless, this study provides new insights into the causal relationship between bioavailable testosterone levels and OA from a genetic perspective, thereby providing new insights into the study of OA. Conclusion Our research suggests a positive causal relationship between testosterone levels and OA, which may provide effective biomarkers for the prevention and treatment of OA. Abbreviations OA: osteoarthritis; MR: Mendelian randomization; GWAS: genome-wide association study; IVW: Inverse variance weighted; SNP: single nucleotide polymorphism: IV: instrumental variable; OR: odds ratio. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The data analyzed in this research was downloaded from IEU database (https://gwas.mrcieu.ac.uk/). Competing interests The authors declare that they have no competing interests. Funding This research was funded by Supported by Guizhou Provincial Basic Research Program(Natural Science)(Fundamentals of Qian Kehe-ZK[2023] General 436); Guizhou Provincial Department of Education Youth Science and Technology Talent Growth Project (Qian Jiaohe-KY Word[2022] No. 262); Guizhou University of Traditional Chinese Medicine Graduate Education Innovation Program Project(YCXZRB202201); Guizhou Province College Student Innovation and Entrepreneurship Training Program Project(S202310662064); Key Laboratory of Integrated Traditional Chinese and Western Medicine in the Prevention and Treatment of Disease Transformation in Higher Education Institutions in Guizhou Province (Qian Jiao Ji [2023] No. 017). Author Contributions Conceptualization, Z.J. and X.L.; methodology, Y.Z.; software, W.Y.; validation, Z.J., L.X. and Y.Z.; formal analysis, X.M.; investigation, F.T.; resources, W.K.; data curation, F.T.; writing—original draft preparation, Z.J.; writing—review and editing, F.T.; visualization, X.M.; supervision, W.K.; project administration, F.T.; funding acquisition, W.K. All authors have read and agreed to the published version of the manuscript.ding acquisition, F.T. All authors have read and agreed to the published version of the manuscript. Acknowledgments Thank you to all participants for their selfless dedication. References Cho Y, Jeong S, Kim H, Kang D, Lee J, Kang SB, Kim JH. Disease-modifying therapeutic strategies in osteoarthritis: current status and future directions. Exp Mol Med. 2021; 53(11):1689-1696. Favero M, Belluzzi E, Ortolan A, Lorenzin M, Oliviero F, Doria A, Scanzello CR, Ramonda R. Erosive hand osteoarthritis: latest findings and outlook. 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Total Serum Testosterone and Western Ontario and McMaster Universities Osteoarthritis Index Pain and Function Among Older Men and Women With Severe Knee Osteoarthritis. Arthrit Care Res. 2020; 72(11):1511-1518. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials.doc Supplementary information Supplementary Table 1: Heterogeneity test(Forward MR); Supplementary Table 2: Horizontal pleiotropy test (Forward MR); Supplementary Table 3: Heterogeneity test (Reverse MR); Supplementary Table 4: Horizontal pleiotropy test (Reverse MR). Cite Share Download PDF Status: Published Journal Publication published 21 Apr, 2025 Read the published version in BMC Musculoskeletal Disorders → Version 1 posted Editorial decision: Revision requested 11 Dec, 2024 Reviews received at journal 05 Dec, 2024 Reviewers agreed at journal 29 Nov, 2024 Reviews received at journal 11 Aug, 2024 Reviewers agreed at journal 10 Aug, 2024 Reviewers invited by journal 30 May, 2024 Editor assigned by journal 22 May, 2024 Editor invited by journal 29 Feb, 2024 Submission checks completed at journal 29 Feb, 2024 First submitted to journal 22 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3980446","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":276038579,"identity":"f1da0705-3945-48a9-aca5-ec1f5fc9123c","order_by":0,"name":"Zong Jiang","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zong","middleName":"","lastName":"Jiang","suffix":""},{"id":276038580,"identity":"32e181d4-d9b4-480d-8934-b75fedd8378b","order_by":1,"name":"Xiaoling Yao","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"Yao","suffix":""},{"id":276038581,"identity":"4c643374-a044-4841-b6c7-e7bb85ddc277","order_by":2,"name":"Yuzheng Yang","email":"","orcid":"","institution":"Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuzheng","middleName":"","lastName":"Yang","suffix":""},{"id":276038582,"identity":"c9a49072-fd54-489a-a588-b7fce6896c8f","order_by":3,"name":"Fang Tang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIie2PP0vEMBiHWwLtEsmagB8iUKgeHM1XeUvhpuLcwSFyEDdde1/E+S0Bp6jrgQ6dxOGG3uKfRcwtOpm7UTAPvL/h5ffwJkkSifxVOj+MkGEEOaeM6XCb7sL5EZemkWO3OBY9HqhI50oxOjuXGsKKyu/sC54/VXoNktfmgcoE02nbBq7Qs8Up3j43Fz2ArM0jPSGaiNVN6GFtWUyZbQgHhJ0y05iRo5DCNqXET9tkvNZYm3sqEfYovC3GwdiKUptqcHiAst6UyXBlgeeGJNA1VPTDMviX/LotJny1Sln29vEuK8XYcpi2AcWTcR+1/lmk+pfmN2Tyofa1IpFI5B/zBehLXBOKEhHbAAAAAElFTkSuQmCC","orcid":"","institution":"The Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Fang","middleName":"","lastName":"Tang","suffix":""},{"id":276038583,"identity":"7a52e754-cfe1-4761-bd77-8e85668f4b9c","order_by":4,"name":"Wukai Ma","email":"","orcid":"","institution":"The Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wukai","middleName":"","lastName":"Ma","suffix":""},{"id":276038584,"identity":"af3c758b-6a29-469b-8714-5c32b4b06dd6","order_by":5,"name":"Xueming Yao","email":"","orcid":"","institution":"The Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xueming","middleName":"","lastName":"Yao","suffix":""},{"id":276038585,"identity":"420707c3-260e-4d76-a4ff-91000fd37630","order_by":6,"name":"Weiya Lan","email":"","orcid":"","institution":"The Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Weiya","middleName":"","lastName":"Lan","suffix":""}],"badges":[],"createdAt":"2024-02-23 03:00:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3980446/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3980446/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12891-025-08626-8","type":"published","date":"2025-04-21T15:57:47+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51933323,"identity":"90a6bd94-9872-4ef6-a519-4c56aee17b32","added_by":"auto","created_at":"2024-03-04 06:16:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":438778,"visible":true,"origin":"","legend":"\u003cp\u003eStudy design and workflow of this study.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3980446/v1/150eb8efbdaff1411efa505f.png"},{"id":51933321,"identity":"2ce5c9d9-0dbd-42cc-906f-e2066cf07ff6","added_by":"auto","created_at":"2024-03-04 06:16:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1692847,"visible":true,"origin":"","legend":"\u003cp\u003eForward mendelian randomization (MR) analysis analysis. (A) The scatter plot of MR analysis. The vertical axis represents the causal effect of instrumental variables on outcomes, while the horizontal axis represents the causal effect of instrumental variables on exposure. (B) Forest map of MR Analysis. Each horizontal solid line in the forest map reflects the estimated results of a single SNP using the Wald ratio method. The red line in the figure reflects that exposure factors can increase the risk of disease under the IVW method. (C) Funnel plot of MR analysis. (D) Leave-One-Out for MR Analysis.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3980446/v1/2021a02c4bf095bb82267753.png"},{"id":51933322,"identity":"823d49b5-e46a-4832-ae9b-ab3b9c6ce07f","added_by":"auto","created_at":"2024-03-04 06:16:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":203132,"visible":true,"origin":"","legend":"\u003cp\u003eLeave-One-Out for MR Analysis (Reverse MR).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3980446/v1/daff680549dafb1c71afb136.png"},{"id":81570595,"identity":"6558f4d1-e253-43b0-8747-2408660654ff","added_by":"auto","created_at":"2025-04-28 16:13:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3421336,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3980446/v1/3f7b1f61-4001-4ebf-830d-421420abdad8.pdf"},{"id":51933320,"identity":"390b3666-75c0-4b30-a870-dfdf81f70d6a","added_by":"auto","created_at":"2024-03-04 06:16:30","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":38400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary Table 1: Heterogeneity test(Forward MR);\u003c/p\u003e\n\u003cp\u003eSupplementary Table 2: Horizontal pleiotropy test (Forward MR);\u003c/p\u003e\n\u003cp\u003eSupplementary Table 3: Heterogeneity test (Reverse MR);\u003c/p\u003e\n\u003cp\u003eSupplementary Table 4: Horizontal pleiotropy test (Reverse MR).\u003c/p\u003e","description":"","filename":"SupplementaryMaterials.doc","url":"https://assets-eu.researchsquare.com/files/rs-3980446/v1/d9fbb8b7e63070baae56ef07.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"The causal impact of bioavailable testosterone levels on osteoarthritis: a bidirectional Mendelian randomized study Running Title: Causality of testosterone on OA in bidirectional MR study","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOsteoarthritis (OA) is a common and complex degenerative joint disease worldwide, with chronic inflammation of joints and their appendages leading to pain, swelling, limited functional activity, and even deformities being the main clinical manifestation [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. With the aging of the global population, the incidence rate of OA is increasing year by year, which seriously affects the quality of life of patients and brings huge economic burden to society [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. At present, the pathogenesis of OA is not clear, but it is known that factors such as obesity, trauma, and lifestyle are all risk factors for the onset of OA [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], but even if these risk factors are avoided, the incidence rate of OA is still high. Preventing cartilage degeneration remains the key to preventing and treating OA [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, there are currently no reliable biochemical biomarkers that can prevent the occurrence of OA [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Studies have shown that hormone replacement therapy has a clear effect on the treatment of OA and can effectively reduce joint replacement rates [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], indicating that sex steroids may play an important role in the development of OA. Therefore, exploring the causal relationship between OA and sex steroids is worth studying, so as to assess whether regulating sex steroid levels can serve as an effective therapy for preventing and treating OA .\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eTestosterone is mainly a steroid hormone secreted by the male testes and female ovaries. It is transported to the target tissues in the body through binding of sex hormone binding globulin (SHBG) in the blood to function [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It is an important androgen that promotes human growth and development, and maintains normal organ function [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Obesity is a risk factor for OA, and studies have shown that testosterone deficiency can lead to obesity and insulin resistance, while testosterone therapy can inhibit fat deposition and reduce insulin resistance [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Testosterone also inhibits the endoplasmic reticulum (ER) stress mechanism and inflammatory response and cell apoptosis, and reduces the concentration of extremely low density lipoprotein and susceptibility to OA. Androgen receptors are present in human knee joint chondrocytes, synovial tissue and bone cells, and are expressed in both male and female patient chondrocytes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], indicating a close correlation between chondrocytes and androgens [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. There are androgen receptors in articular chondrocytes, which have the ability to synthesize androgens, and testosterone can affect the internal environment of bones by binding to receptors, which further indicates that androgens may be related to the pathogenesis of OA [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMendelian Randomization (MR) is an analytical method for exploring causal relationships by introducing genome-wide association studies (GWAS) data represented by single nucleotide polymorphisms (SNPs) as mediating instrumental variables. Genetic variation is a random allocation of fixed alleles at conception, which can effectively overcome potential confounding factors and reverse the effects of causality [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Currently widely used in research on exposure and outcomes of various diseases, it can help us analyze and understand the causal relationship between exposure factors and diseases more conveniently and accurately. At present, a research has found a causal relationship between testosterone with hip osteoarthritis and the risk of hip replacement. However, the causal relationship between testosterone and overall OA, as well as who causes and effects OA and testosterone, is remains unclear [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Bidirectional MR evaluates whether there is a reverse causal relationship between exposure and outcome, that is, whether the outcome can lead to the occurrence of exposure, and conducts two double sample MR analyses to better understand the causal relationship between instrumental variables.\u003c/p\u003e\u003cp\u003eTherefore, based on publicly available GWAS data, this study conducted a bidirectional MR analysis using bioavailable testosterone levels and OA as exposure factors or outcomes, providing a new reference for the causal relationship between bioavailable testosterone levels and OA. The overview of the study design was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMR studies must satisfy the following three assumptions. Firstly, genetic variants selected as instrumental variables (IVs) are strongly correlated with exposure factors. Secondly, SNPs in genetic variants are independent of confounding factors that are related to exposure and outcome. Thirdly, genetic variants affect outcomes only through exposure and not through other biological pathways [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The study used data from published public databases, and therefore, this study did not require any additional ethical approval.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eData Source and Pre-processing\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe IEU OpenGWAS database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gwas.mrcieu.ac.uk/\u003c/span\u003e\u003cspan address=\"https://gwas.mrcieu.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized to download OA-related dataset (ukb-b-14486) and bioavailable testosterone levels-related dataset (ebi-a-GCST90012104). The ukb-b-14486 contains 38,472 OA samples, 424,461 control samples, and 9,851,867 SNP samples. The ebi-a-GCST90012104 includes 382,988 samples and 16,137,327 SNP samples [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Afterwards, SNPs selected as IVs were screened via \u0026ldquo;TwoSampleMR\u0026rdquo; R package (version 0.5.6) with P\u0026thinsp;\u0026lt;\u0026thinsp;5 \u0026times; 10\u0026thinsp;\u0026minus;\u0026thinsp;8, and linkage disequilibrium analysis (LDA) was performed to ensure independence (r2\u0026thinsp;=\u0026thinsp;0.001 and kb\u0026thinsp;=\u0026thinsp;10,000).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAfter the IVs were filtered, MR analyses were performed by combining the MR function and five methods, as follows MR Egger, Weighted median, Inverse variance weighted (IVW), Simple mode, and Weighted mode. Since the IVW method provides more precise causation and its results are unbiased, the results are mainly referred to the IVM. Then, odds ratios (ORs) were calculated, with an OR equal to 1 indicating no correlation between exposure and outcome; an OR greater than 1 indicating that exposure promotes the outcome; and an OR less than 1 indicating that exposure inhibits the occurrence of the outcome event. The results were presented using scatter plots, forest plots, and funnel plots.\u003c/p\u003e \u003cp\u003eTo determine the reliability of the results of the analysis, a sensitivity analysis was conducted via heterogeneity test, the Horizontal pleiotropy test, and the Leave-One-Out (LOO) method. When performing the heterogeneity test, a Q value greater than 0.05 indicates that there is no heterogeneity. A P value is greater than 0.05 demonstrating that there is no horizontal pleiotropy in Horizontal pleiotropy test. The LOO is used to see if there are outliers in the effect of each SNP.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCausal effect of bioavailable testosterone levels on OA\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAfter screening, 105 SNPs were obtained that were strongly relevant to bioavailable testosterone levels but not associated with OA. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, there was a causal relationship between bioavailable testosterone levels and OA (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.020), and bioavailable testosterone levels were a risk factor for OA (OR\u0026thinsp;=\u0026thinsp;1.009). The results of the scatter plot showed that the slopes of the lines were positive, further validating bioavailable testosterone levels as a risk factor for OA \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Of the forest plot results, the point of IVM was on the right, which supported the view that bioavailable testosterone levels increase risk of OA \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. The funnel plot showed that MR conformed to Mendel's second law of random grouping \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMR analysis results of bioavailable testosterone levels for OA\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExposure\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOutcome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMethod\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNsnp\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOR(95% CI)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eBioavailable\u003c/p\u003e \u003cp\u003etestosterone levels\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eOsteoarthritis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMR Egger\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.757\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.002(0.987\u0026ndash;1.018)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWeighted median\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.012(1.001\u0026ndash;1.024)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInverse variance weighted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.009(1.001\u0026ndash;1.017)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSimple mode\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.561\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.007(0.984\u0026ndash;1.031)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWeighted mode\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.009(0.996\u0026ndash;1.023)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eEvaluation of the reliability of the results, we then did a sensitivity analysis. Firstly, the Q value of IVM was less than 0.05, suggesting that there was heterogeneity \u003cb\u003e(Supplementary Table\u0026nbsp;1)\u003c/b\u003e. However, the P value was less than 0.05 for IVM, demonstrating that heterogeneity did not cause a significant effect on the results. Then, according to the horizontal pleiotropy test, there were no confounding factors in this study (P\u0026thinsp;=\u0026thinsp;0.328) \u003cb\u003e(Supplementary Table\u0026nbsp;2)\u003c/b\u003e. Thereafter, the LOO method suggested that there were no points of deviation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. In conclusion, the results that we obtained were reliable, and there was a causal relationship between bioavailable testosterone levels and OA.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCausal effect of OA on bioavailable testosterone levels\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo illustrate the exact causal relationship between bioavailable testosterone levels and OA, we performed a reverse MR analysis, with OA as the exposing factor and bioavailable testosterone levels as the outcome. A total of six independent SNPs were obtained with the same screening criteria. Under the IVW model, OA was not significantly associated with bioavailable testosterone levels (P\u0026thinsp;=\u0026thinsp;0.779) \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Moreover, the remaining four models demonstrated the same results. Furthermore, the sensitivity analysis results manifested that our results were reliable \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cb\u003eSupplementary Table\u0026nbsp;3\u0026ndash;4)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMR analysis results of OA on bioavailable testosterone levels (Reverse MR).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExposure\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOutcome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMethod\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNsnp\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP-vauel\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBeta\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eOsteoarthritis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eBioavailable\u003c/p\u003e \u003cp\u003etestosterone levels\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMR Egger\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.518\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.482\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWeighted median\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.986\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInverse variance weighted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.779\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.076\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSimple mode\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.856\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.078\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWeighted mode\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.932\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.032\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThere have been reports on the relationship between bioavailable testosterone levels and OA, but the causal relationship between them is still unclear. In this study, we conducted a bidirectional two sample MR analysis using the maximum GWAS data of genetic variation to evaluate the causal relationship between testosterone and OA, demonstrating strong genetic evidence. We found a positive causal relationship between bioavailable testosterone levels and the risk of OA.\u003c/p\u003e \u003cp\u003eResearch on OA mainly focuses on estrogen, while research on androgens is relatively scarce. Research on OA mainly focuses on estrogen, while research on androgens is relatively scarce. Testosterone is mainly dissociated by binding to sex hormone binding protein (SHBG) and albumin, resulting in free testosterone, which is an important bioavailable testosterone in the human body and plays an important role in human growth and lipid metabolism [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The lack of testosterone leads to disorders in lipid metabolism, protein metabolism, and glucose metabolism, resulting in a series of metabolic syndromes, obesity, and other conditions [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Biologically available testosterone is one of the most important androgens in the human body, and its concentration varies in a peak line with age [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. After the age of 40, there is a linear decline in testosterone levels in males and postmenopausal females [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Under normal circumstances, testosterone binds to specific receptors in the cell to form a testosterone receptor complex, and the active testosterone receptor complex binds to specific androgen response sheets (ARE) on the target gene to regulate gene expression. Studies have shown that both estrogen and androgen receptors exist in osteoblasts [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In addition to its direct effects on bone and cartilage, testosterone not only binds to androgen receptors, but also to estrogen receptors to affect bone calcium metabolism balance [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In addition, testosterone can aromatise and convert into estradiol, which then binds to estrogen receptors and participates in the physiological regulation of bone and cartilage [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The decrease in testosterone levels affect cartilage metabolism through androgen receptors and ion channels, as well as leading to decreased in estradiol conversion rate, which leading to cartilage degeneration and the formation of OA [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. When testosterone is at normal levels in men but there is an aromatase deficiency in the body, most of the androgens cannot be converted into estrogen, which often results in lower levels of estrogen in the body, which leads to joint cartilage degeneration and the development of OA [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This indicates a significant correlation between androgens, especially testosterone, and OA. In a study of male calf knee joint cartilage, it was found that testosterone increased the content of glycosaminoglycans in the extracellular matrix of chondrocytes, promote the coverage of type II collagen on the cartilage surface and the growth of cartilage fiber structure in joint cartilage [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Currently, clinical studies on the correlation between testosterone levels and OA are mainly case reports. A study of the correlation between hormone levels and hand OA in 573 premenopausal women found a significant correlation between lower levels of serum testosterone and the prevalence of hand OA [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In another study targeting men, serum testosterone levels were found to be positively correlated with cartilage thickness [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Testosterone can increase male muscle strength and is often recommended for the treatment of male musculoskeletal pain, its also can effective to reduce fat content and inhibit inflammatory reactions [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the study of serum testosterone levels and OA symptoms, it was found that higher levels of serum testosterone can reduce the joint osteoarthritis index (WOMAC) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. These studies have shown a significant correlation between serum testosterone levels and OA, but the causal relationship remains unclear. Our study suggests a causal relationship between bioavailable testosterone levels and OA from a genetic perspective, providing indicative evidence for the prevention and treatment of OA.\u003c/p\u003e \u003cp\u003eIn this study, we had sufficient samples for MR analysis to explore the causal relationship between bioavailable testosterone levels and OA, and found a causal relationship between bioavailable testosterone levels and OA. This study has several advantages. Firstly, the data is sourced from the GWAS database, which can exclude the interference of confounding factors. Secondly, we use bidirectional MR analysis to study the impact of causal relationships on causal inference. We also used sensitivity analysis using multiple methods to exclude bias caused by related and unrelated pleiotropy.\u003c/p\u003e \u003cp\u003eIn summary, we demonstrate a potential causal relationship between bioavailable testosterone levels and OA, whereas OA is the cause and bioavailable testosterone levels are the result, and there is no causal relationship between the two. However, our research still has certain limitations. Firstly, the study population is of European ancestry, and the scope of the study is relatively limited. Secondly, due to the lack of raw data from the WGAS database, subgroup analysis was not conducted. Finally, we only found a causal relationship between bioavailable testosterone levels and OA from a genetic perspective, and the mechanism of its occurrence is still unclear. Nevertheless, this study provides new insights into the causal relationship between bioavailable testosterone levels and OA from a genetic perspective, thereby providing new insights into the study of OA.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOur research suggests a positive causal relationship between testosterone levels and OA, which may provide effective biomarkers for the prevention and treatment of OA.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eOA: osteoarthritis; MR: Mendelian randomization; GWAS: genome-wide association study; IVW: Inverse variance weighted; SNP: single nucleotide polymorphism: IV: instrumental variable; OR: odds ratio.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data analyzed in this research was downloaded from IEU database (https://gwas.mrcieu.ac.uk/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Supported by Guizhou Provincial Basic Research Program(Natural Science)(Fundamentals of Qian Kehe-ZK[2023] General 436); Guizhou Provincial Department of Education Youth Science and Technology Talent Growth Project (Qian Jiaohe-KY Word[2022] No. 262); Guizhou University of Traditional Chinese Medicine Graduate Education Innovation Program Project(YCXZRB202201); Guizhou Province College Student Innovation and Entrepreneurship Training Program Project(S202310662064); Key Laboratory of Integrated Traditional Chinese and Western Medicine in the Prevention and Treatment of Disease Transformation in Higher Education Institutions in Guizhou Province (Qian Jiao Ji [2023] No. 017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Z.J. and X.L.; methodology, Y.Z.; software, W.Y.; validation, Z.J., L.X. and Y.Z.; formal analysis, X.M.; investigation, F.T.; resources, W.K.; data curation, F.T.; writing\u0026mdash;original draft preparation, Z.J.; writing\u0026mdash;review and editing, F.T.; visualization, X.M.; supervision, W.K.; project administration, F.T.; funding acquisition, W.K. All authors have read and agreed to the published version of the manuscript.ding acquisition,\u0026nbsp;F.T. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThank you to all participants for their selfless dedication.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCho Y, Jeong S, Kim H, Kang D, Lee J, Kang SB, Kim JH. Disease-modifying therapeutic strategies in osteoarthritis: current status and future directions. Exp Mol Med. 2021; 53(11):1689-1696.\u003c/li\u003e\n\u003cli\u003eFavero M, Belluzzi E, Ortolan A, Lorenzin M, Oliviero F, Doria A, Scanzello CR, Ramonda R. Erosive hand osteoarthritis: latest findings and outlook. Nat Rev Rheumatol. 2022; 18(3):171-183.\u003c/li\u003e\n\u003cli\u003eYin B, Ni J, Witherel CE, Yang M, Burdick JA, Wen C, Wong S. 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Arthritis Rheum. 2008; 58(6):1696-1700.\u003c/li\u003e\n\u003cli\u003eEnglert C, Blunk T, Fierlbeck J, Kaiser J, Stosiek W, Angele P, Hammer J, Straub RH. Steroid hormones strongly support bovine articular cartilage integration in the absence of interleukin-1beta. Arthritis Rheum. 2006; 54(12):3890-3897.\u003c/li\u003e\n\u003cli\u003eSowers MF, Hochberg M, Crabbe JP, Muhich A, Crutchfield M, Updike S. Association of bone mineral density and sex hormone levels with osteoarthritis of the hand and knee in premenopausal women. Am J Epidemiol. 1996; 143(1):38-47.\u003c/li\u003e\n\u003cli\u003eCicuttini FM, Wluka A, Bailey M, O\u0026apos;Sullivan R, Poon C, Yeung S, Ebeling PR. Factors affecting knee cartilage volume in healthy men. Rheumatology. 2003; 42(2):258-262.\u003c/li\u003e\n\u003cli\u003eKolind MI, Christensen LL, Caserotti P, Andersen MS, Glintborg D. Muscle function following testosterone replacement in men on opioid therapy for chronic non-cancer pain: A randomized controlled trial. Andrology-Us. 2022; 10(3):551-559.\u003c/li\u003e\n\u003cli\u003eAndrade S, Mucida YM, Xavier J, Fernandes LN, Silva RO, Bandeira F. Bone mineral density, trabecular bone score and muscle strength in transgender men receiving testosterone therapy versus cisgender men. Steroids. 2022; 178:108951.\u003c/li\u003e\n\u003cli\u003eFreystaetter G, Fischer K, Orav EJ, Egli A, Theiler R, Munzer T, Felson DT, Bischoff-Ferrari HA. Total Serum Testosterone and Western Ontario and McMaster Universities Osteoarthritis Index Pain and Function Among Older Men and Women With Severe Knee Osteoarthritis. Arthrit Care Res. 2020; 72(11):1511-1518.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-musculoskeletal-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmsd","sideBox":"Learn more about [BMC Musculoskeletal Disorders](http://bmcmusculoskeletdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://author-welcome.nature.com/12891","title":"BMC Musculoskeletal Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bioavailable testosterone levels, osteoarthritis, mendelian randomization, bidirectional","lastPublishedDoi":"10.21203/rs.3.rs-3980446/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3980446/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eIt has been shown that low testosterone levels are associated with the development of osteoarthritis (OA). In our study, we aimed to investigate a bidirectional causal relationship between bioavailable testosterone levels and OA using Mendelian randomization (MR) analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e In our study, the datasets from publicly available genome-wide association study (GWAS) were adopted, including the OA-related dataset (ukb-b-14486) and the bioavailable testosterone levels-related dataset (ebi-a-GCST90012104). In total, five methods were utilized, namely MR Egger, Weighted median, Inverse variance weighted (IVW), Simple mode, and Weighted mode. Among them, IVW was the main analytical method. Additionally, the sensitivity analysis was carried out through the heterogeneity test, the horizontal pleiotropy test, and the Leave-One-Out (LOO) method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The result of forward MR analysis demonstrated that bioavailable testosterone levels were considerably relevant to OA, and were a risk factor for OA (OR = 1.009, 95% CI: 1.001-1.017, \u003cem\u003eP\u003c/em\u003e = 0.020). However, through reverse MR analysis, we did not find a causal relationship between OA and bioavailable testosterone levels. Moreover, the results of the sensitivity analysis suggested that our results were reliable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e The results of our study supported a causal relationship between bioavailable testosterone levels and OA.\u003c/p\u003e","manuscriptTitle":"The causal impact of bioavailable testosterone levels on osteoarthritis: a bidirectional Mendelian randomized study Running Title: Causality of testosterone on OA in bidirectional MR study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-04 06:16:17","doi":"10.21203/rs.3.rs-3980446/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-11T11:10:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-05T16:45:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180587170332839588641944006782516036976","date":"2024-11-29T05:32:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-11T04:37:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"103364091074271922429281631633079597222","date":"2024-08-11T02:00:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-30T17:25:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-22T12:37:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-02-29T10:38:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-29T10:35:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Musculoskeletal Disorders","date":"2024-02-23T02:57:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-musculoskeletal-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmsd","sideBox":"Learn more about [BMC Musculoskeletal Disorders](http://bmcmusculoskeletdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://author-welcome.nature.com/12891","title":"BMC Musculoskeletal Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6356d7db-649c-4ccb-b2d0-6343f6819406","owner":[],"postedDate":"March 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-28T16:12:05+00:00","versionOfRecord":{"articleIdentity":"rs-3980446","link":"https://doi.org/10.1186/s12891-025-08626-8","journal":{"identity":"bmc-musculoskeletal-disorders","isVorOnly":false,"title":"BMC Musculoskeletal Disorders"},"publishedOn":"2025-04-21 15:57:47","publishedOnDateReadable":"April 21st, 2025"},"versionCreatedAt":"2024-03-04 06:16:17","video":"","vorDoi":"10.1186/s12891-025-08626-8","vorDoiUrl":"https://doi.org/10.1186/s12891-025-08626-8","workflowStages":[]},"version":"v1","identity":"rs-3980446","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3980446","identity":"rs-3980446","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}