Genetically determined sex hormones and functional outcome after ischemic stroke: A Mendelian randomization study

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However, the effects reported in previous studies were inconsistent, and randomized data were absent. We performed a two-sample Mendelian randomization analysis to explore the causal effects of genetically determined sex hormones on the 3-month functional outcome after IS. Methods Using genome-wide association studies, we obtained sex-specific genetic instruments for evaluating serum estradiol and bioavailable testosterone levels. The sex-specific functional outcome after IS were estimated via inverse-variance weighted Mendelian randomization analysis. Mendelian randomization-Egger regression, weighted median leave-one-SNP-out analysis, Mendelian randomization-Pleiotropy Residual Sum, and Outlier methods were used for sensitivity analyses. To validate our primary results, we also performed independent repeated and bidirectional Mendelian randomization analyses. Results Genetic predisposition to high levels of bioavailable testosterone was associated with excellent functional outcome in males (OR = 0.41, 95% CI: 0.21–0.79, P = 0.008), but worse outcome in females (OR = 2.49, 95% CI: 1.24–4.99, P = 0.009). Females with genetically predicted higher estradiol levels tend to have excellent and favorable functional outcome (excellent functional outcome, OR = 0.53; 95% CI: 0.41–0.85, P = 0.005); (favorable functional outcome, OR = 0.69; 95% CI: 0.48–0.98, P = 0.036). There was limited evidence that genetically predicted estradiol levels affect functional outcome in men (P > 0.05). Conclusions Our findings suggested that bioavailable testosterone and estradiol were promising neuroprotectants that could respectively improve the functional outcome of IS in males and females. Ischemic stroke Mendelian randomization bioavailable testosterone estradiol Figures Figure 1 Figure 2 Introduction Ischemic stroke (IS) is one of the leading causes of death and disability. 1 Although endovascular therapy is effective in treating large-vessel IS according to many recent clinical trials, the proportion of patients with good functional outcome is still relatively low. 2–4 Sex hormones have long been associated with the prognosis of IS, but their effects remain controversial. Estradiol, as the most potent estrogen, is one of the main points of interest. There is evidence from the bench suggesting that estradiol, the most potent estrogen, is a key neuroprotectant in IS. 5–8 However, some clinical trials have demonstrated otherwise. The Women's Estrogen for Stroke Trial (WEST) found that estradiol treatment did not improve mortality, stroke recurrence, or cognitive function in postmenopausal females after IS. 9, 10 Pappa et al. reported that high levels of endogenous estradiol were associated with high short-term mortality and adverse functional outcome. 11 Testosterone is the principle male sex hormone, which is also reported to have a discrepant effect on the prognosis of IS. While Ho et al. 12 demonstrated that low testosterone levels in males were associated with higher all-cause mortality after IS, two observational studies implicated a positive role for testosterone in post-stroke prognosis in the male gender. 13, 14 Current clinical findings, which predominantly come from observational studies, fail to reach consensus, probably owing to the limitations of observational studies, including residual confounding, reverse causation, and detection bias. Mendelian randomization (MR) is a novel method that uses genetic variants to determine the causality of an observational association between a risk factor and an outcome. 15 Relying on the random assortment of gene variants during meiosis, MR overcomes some core shortcomings of the observational studies mentioned above. 16 . Therefore, we performed a two-sample MR analysis to evaluate whether genetically determined sex hormone levels influence the functional outcome after IS. Methods Data availability We used summary data from publicly available original studies for our analyses. All the studies obtained ethical approval and participant consent. This study was conducted in accordance with the guidelines for Strengthening the Reporting of Observational Studies in Epidemiology-Mendelian Randomization (STROBE-MR). 17 Exposure data We retrieved publicly available sex-specific genome-wide association study (GWAS) summary-level data of the single nucleotide polymorphisms (SNPs) associated with sex hormones(Table 1 ). 18,19,20 Independent SNPs associated with bioavailable testosterone and estradiol were selected from GWAS in up to 425,097 and 311,675 white European individuals from the UK Biobank, respectively. 19,20 178,782 samples of the bioavailable testosterone levels of males (nmol/L) and 188,507 samples of females were measured by one-step competitive analysis. 147,690 samples of the Estradiol in men and 163,985 of that in women were estimated with 2-step competitive analysis on a Beckman Coulter Unicel Dxl 800. We selected SNPs associated with exposure at a genome-wide significance level (P < 5×10 − 8 ). The independence of the SNPs was assessed with stringent criteria (r 2 < 0.001; clumping window, 10 000 kb). We then harmonized the SNP alleles across the studies and removed palindromic SNPs with ambiguous allele frequencies (0.42–0.58). The data for all instrumental variables used are shown in Table S1 through S3 . Table 1 Characteristics of the Used GWAS Variable First author (year) Consortium Sample size Ancestry Used as Unit Bioavailable testosterone Ruth KS UK biobank Males: 178,782 European Exposure nmol/L Females: 188,507 Estradiol Schmitz D UK biobank Males: 147,690 European Exposure nmol/L Females: 163,985 Functional outcome after ischemic stroke Soderholm M Genetics of Ischemic Stroke Functional Outcome (GISCOME) mRS 0–1 vs 2–6: 1796 vs 2567 European Outcome NA mRS 0–2 vs 3–6: 3741 vs 2280 Outcome data Summary-level data for functional outcome after IS were obtained from the Genetics of IS Functional Outcome (GISCOME) network meta-analysis (12 studies from the US, Europe, and Australia, n = 6, 021). 18 3-month functional outcome after stroke onset was assessed by the modified Rankin Scale (mRS). The primary outcome was excellent functional outcome, defined as an mRS score of 0–1. The secondary outcome was favorable functional outcome, defined as an mRS score of 0–2. In the primary model, the analysis was adjusted for age, sex, ancestry, and baseline stroke severity, as assessed by the National Institutes of Health Stroke Scale (NIHSS) 0–10 days after stroke onset. Moreover, models without adjustment for baseline NIHSS scores were used for comparison. 18 Participants were of European genetic ancestry. Statistical analysis This MR approach was based on three assumptions: ( 1 ) the genetic variants associated with the exposure and ( 2 ) the instrumental variables (IVs) have no association with confounding factors, and ( 3 ) the functional outcome after IS is influenced only by the exposure, not by other pathways. 21,22 F statistics were applied to evaluate the strength of the genetic instruments. 23 We excluded the SNPs with F statistics lower than 10, which indicated that the strength of these instruments was insufficient for MR analysis 24 . The inverse-variance weighted (IVW) MR method, which assumes all genetic variants to be valid instrumental variables and produce the most accurate estimates, were performed as our primary analyses. 25 We also explored the sensitivity via the weighted median and MR-Egger regression. The weighted median approaches give more weight to the instrumental variables that are more precise, and the estimate is consistent even when up to 50% of the information comes from invalid or weak instruments. 21 The MR-Egger could detect directional pleiotropy, albeit its low precision. 25 The Cochran’s Q statistic in the IVW model were applied to assess the heterogeneity between variant-specific estimates. The MR Pleiotropy Residual Sum and Outlier (PRESSO) approaches were adopted to identify possible outliers. Finally, we performed a leave-one-SNP-out analysis, where SNPs were systematically removed to assess whether a single SNP drove the results. The association was considered significant after correction for multiple testing of the four sex hormone indices (P < 0.0125 [0.05/4]). A P value above 0.0125 but below 0.05 suggested a potential association. The results were presented as odds ratio (OR) and 95% confidence interval (CI) of functional outcome per genetically predicted unit log-transformed increase for each trait. We additionally performed analyses in the outcome database without adjusting for the baseline NIHSS score as a sensitivity evaluation. Moreover, we included a bidirectional MR analysis to test whether functional outcome of IS affected the liability to bioavailable testosterone and estradiol. All MR analyses were performed using the TwoSampleMR (version 0.5.6), Mendelian randomization (version 0.5.1), and MRPRESSO (version 1.0) packages in R (version 4.2.2). Results Genetically determined testosterone and functional outcome after ischemic stroke First, we examined the relationship between genetically determined bioavailable testosterone levels and functional outcome after IS. A total of 42 and 59 independent genetic variants were associated with bioavailable testosterone levels in males and females, respectively. The F-statistic was > 10 for all variants, indicating a low risk of weak instrument bias ( Table S1 , S2 ). Figure 1 illustrates the results of primary univariate MR analysis. The MR analyses in males showed that high testosterone levels were associated with excellent functional outcome (OR 0.41, 95% CI: 0.21–0.79, P = 0.008) for per standard deviation (approximately 3.7 nmol/L). While In females, MR analyses exhibited suggestive harmful effects of high testosterone levels on functional outcome (OR 2.49, 95% CI: 1.24–4.99, P = 0.009). Cochran Q test showed no evidence of heterogeneity. Meanwhile, none of the MR-pleiotropy Residual Sum, Outlier global test, or MR-Egger intercepts showed directional pleiotropic effects. MR analyses based on the weighted median method yielded results in the same direction as IVW but with lower precision ( Table S4 ). Leave-one-out analysis did not change the overall direction ( Figure S1 ) . Genetically determined estradiol and functional outcome after ischemic stroke Next, we examined the effects of estradiol on functional outcome after IS using MR analyses. A total of 24 variants for estradiol, including 7 for males and 17 for females, were identified ( Table S3 ). As shown in Fig. 2 , genetically determined high estradiol levels in females were associated with excellent functional outcome (OR, 0.53; 95% CI: 0.41–0.85, P = 0.005). In addition, MR analyses suggested evidence of a potential association between high estradiol levels in females and favorable functional outcome (OR, 0.69; 95% CI: 0.48–0.98, P = 0.036). However, there was no evidence of association between genetically predicted estradiol levels in men and functional outcome. The Cochran Q statistic of the IVW method indicated no horizontal pleiotropy or heterogeneity across the instrument SNP effects, and the MR-Egger analysis did not show evidence of directional pleiotropy (P > 0.05). Results of the weighted median supported the primary results ( Table S4 ). No distortion was observed in the leave-one-out plot, indicating that there was no single SNPs driving the observed effect in any of the analyses ( Figure S1 ). 18 Results of additional analysis Sensitivity analyses were conducted in models without adjustment for baseline NIHSS scores. For females, genetic predisposition to high levels of estradiol was associated with excellent and favorable functional outcome after IS (excellent functional outcome, OR = 0.68; 95% CI: 0.49–0.95, P = 0.024, favorable functional outcome, OR = 0.73; 95% CI: 0.53–0.98, P = 0.042). On the contrary, genetically predicted high bioavailable testosterone levels predicted worse functional outcome of women(OR = 2.11; 95% CI: 1.12–3.98, P = 0.021). For the male gender, additional analysis was consistent with the primary result: genetically determined high bioavailable testosterone levels suggested excellent functional outcome (OR = 0.52; 95% CI: 0.29–0.97, P = 0.042). No relationship was observed between the estradiol levels and functional outcome of men( Table S4 ). The results of the bidirectional MR analysis showed that the functional outcome after IS does not affect bioavailable testosterone and estradiol levels ( Table S4 ). Discussion In this study, we explored the potential association between sex hormone levels and functional outcome after IS using the MR method. We showed that a genetic predisposition to high levels of bioavailable testosterone is associated with excellent functional outcome in males, but worse outcome in females. Genetic predisposition to high estradiol levels is associated with excellent and favorable functional outcome in females. Bidirectional MR analysis suggested no evidence of functional outcome after IS affecting bioavailable testosterone or estradiol levels. Neither harmful nor beneficial estradiol effects on functional outcome were observed in males after IS. Previous MR studies have provided evidence of a causal relationship between sex hormones and an elevated risk of IS. 26, 27 However, these studies focused on IS risk rather than its prognosis, and the causal roles of testosterone and estradiol in post-stroke status remain largely unknown. The recently published GWAS allowed us to conduct MR analyses on this issue. 18 To our knowledge, this study is the first MR analysis exploring the association between sex hormones and functional outcome after IS. Androgens’s effects on the cerebral vasculature are highly complexed, which differ based on sex, dose, and age. 28 Our study exhibited protective effect of genetic predisposition to high levels of bioavailable testosterone on functional outcome in males. Testosterone may have protective anti-inflammatory effects in pathological conditions, such as hypoxia, endotoxin-induced inflammation, or ischemia. 28 Yi Pan et al. demonstrated an accelerated functional recovery in castrate rats treated with testerone 29 . They found less GFAP expression and reactive astrocyte hypertrophy around the infarct area in testosterone-treated rats, suggesting that the beneficial effects of testosterone on post-stroke status is presumably related to the function of microlia and astrocyte. Fanaei et al. reported testosterone treatment in castrated male rats to reduce infarct volume and improve sensorimotor recovery following transient focal ischemia, which is probably mediated by promoting antioxidant defenses, BDNF levels and neurogenesis 30 . However, few studies have been done to explore the underline mechanism of testosterone’s effect on post-stroke female. More studies need to be conducted regarding this question in the future. Our study showed that estradiol could exert a protective effect on the post-stroke functional outcome of female, which is probably related to the neuroprotective function of estradiol. Estradiol is reported to reduce the infarct size in ovariectomized rats whether its administered before or after experimental stroke 31 . Several mechanisms have been identified for estrogen-mediated neuroprotection following cerebral ischemia. Firstly, the genomic effects of estrogen are modulated by the activation of the estrogen response element (ERE), which enhances the transcription of genes containing ERE, such as bcl-2 and seladin‐1 . 32–34 Once activated, these genes participate in cell proliferation, differentiation 35 , and neuroprotection 36 . In vitro studies revealed that astrocytes actively participate in the neuroprotective effects of estrogen by modulating inflammatory responses via the downregulation of pro-inflammatory transcription factor NF-κB and the major pro-inflammatory cytokine interleukin-6 after cerebral ischemia. 37, 38 In addition, estrogen promotes neurogenesis and angiogenesis via selective estrogen receptor modulators (SERMs) 39 , which are critical in poststroke plasticity. The efficacy of estrogens after stroke seems to be age-sensitive in both sexes; 40 however, our study failed to confirm the association between estradiol levels in males and functional outcome after IS because the limited number of SNPs in this study might not provide sufficient statistical power. The results of this study have some limitations. First, we used genetic variants derived from a study with a relatively large sample size strongly associated with BP to avoid weak instrument problems; however, weak instrument bias may still affect our findings. Second, we were limited by the fact that the MR explores the effects of lifelong exposure, whereas sex hormones typically have much shorter periods of action and may have sex-, dose-, model-, and age-dependent effects. The effect sizes that we estimated do not represent the associations between critical periods of exposure and outcome. 41 Therefore, MR studies can only provide additional evidence, and further randomized controlled trials are warranted to investigate the effects of sex hormones on functional outcome after IS. Third, we failed to explore the effects of estradiol on functional outcome in men after IS. This null result did not indicate that there were no protective or deleterious effects of estradiol in males, only that the limited number of included SNPs did not offer sufficient statistical power to perform meaningful analyses. It is also worth mentioning that gender itself may affect the stroke prognosis. Females are reported to be more likely to recognize the warning signs of stroke, which may predict favorable outcomes 42, 43 . Meanwhile, male gender has been associated with less engagement, attentiveness, and receptiveness to health-related knowledge, exerting a negative effect on their recovery 44, 45 . Finally, as all the participants were of European ancestry, the results of this study are not necessarily valid for other ethnic groups. Abbreviations IS: ischemic stroke; MR: mendelian randomization; OR: SNPs: single nucleotide polymorphisms; mRS: modified Rankin Scale; GWAS: genome-wide association study; NIHSS: National Institutes of Health Stroke Scale; IVW: inverse-variance weighted; OR: odds ratio; CI: confidence interval. Conclusion In this MR analysis, a genetic predisposition to high levels of bioavailable testosterone was associated with excellent functional outcome after IS in males, but with worse outcomes after IS in females. On the contrary, a genetic predisposition to high estradiol levels was associated with excellent and favorable functional outcome after IS in females. Further studies are needed to determine the effect of estradiol on functional outcome after IS in males. Declarations Ethics approval and consent to participate All the studies involved in our work obtained ethical approval and participant consent. This study was conducted in accordance with the guidelines for Strengthening the Reporting of Observational Studies in Epidemiology-Mendelian Randomization (STROBE-MR). Consent for Publication Not applicable. Competing interests The authors declare no competing interests. Funding This work was supported by the Shanghai Changhai Hospital (Grant No. GH145-05). Author Contribution Tianxiang Gao: participated in study design, analyzed data, prepared figures, wrote manuscript. Hongjian Zhang and Hanchen Liu: participated in study design, analyzed data, wrote manuscript. Manyue Ge: wrote manuscript. Jianmin Liu and Shaojun Mo: participated in study design, interpreted data, wrote manuscript. 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Mendelian randomization in cardiometabolic disease: challenges in evaluating causality. Nat Reviews Cardiol. 2017;14. 10.1038/nrcardio.2017.78 . Madsen TE, Baird KA, Silver B, et al. Analysis of Gender Differences in Knowledge of Stroke Warning Signs. J Stroke Cerebrovasc Dis. 2015;24:1540–7. 10.1016/j.jstrokecerebrovasdis.2015.03.017 . 2015/04/23. Focht KL, Gogue AM, White BM et al. Gender differences in stroke recognition among stroke survivors. J Neurosci Nurs 2014; 46: 18–22; quiz 22, E11-12. 2014 /01/09 . DOI: 10.1097/JNN.0000000000000026. Ek S. Gender differences in health information behaviour: a Finnish population-based survey. Health Promot Int. 2015;30:736–45. 10.1093/heapro/dat063 . 2013/08/30. Eriksson-Backa K, Ek S, Niemela R, et al. Health information literacy in everyday life: a study of Finns aged 65–79 years. Health Inf J. 2012;18:83–94. 2012/06/27. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-3918724","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":271963339,"identity":"06d19b6e-d270-4be9-b807-a7c70a0a66ae","order_by":0,"name":"Tianxiang Gao","email":"","orcid":"","institution":"University of Shanghai for Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tianxiang","middleName":"","lastName":"Gao","suffix":""},{"id":271963341,"identity":"52e69ba3-acc3-4528-89f1-08fe03c14543","order_by":1,"name":"Hongjian Zhang","email":"","orcid":"","institution":"Naval Medical University Changhai hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongjian","middleName":"","lastName":"Zhang","suffix":""},{"id":271963342,"identity":"7e3bc324-5315-4e50-9500-15ec53d3b500","order_by":2,"name":"Hanchen Liu","email":"","orcid":"","institution":"Naval Medical University Changhai hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hanchen","middleName":"","lastName":"Liu","suffix":""},{"id":271963343,"identity":"3418b675-e84b-4a98-8727-a92b445a6c80","order_by":3,"name":"Manyue Ge","email":"","orcid":"","institution":"Naval Medical University Changhai hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Manyue","middleName":"","lastName":"Ge","suffix":""},{"id":271963344,"identity":"5e86d7a1-4f70-422c-9e7d-45aef278ac4c","order_by":4,"name":"Shaojun Mo","email":"","orcid":"","institution":"Naval Medical University Changhai hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shaojun","middleName":"","lastName":"Mo","suffix":""},{"id":271963345,"identity":"f827fede-10bc-40a1-8b8d-ab81dc5bdc60","order_by":5,"name":"Lei Zhang","email":"","orcid":"","institution":"Naval Medical University Changhai hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Zhang","suffix":""},{"id":271963346,"identity":"854b085b-151c-41fb-b24d-0bc7a9a9f3c5","order_by":6,"name":"Pengfei Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYDACCQjFz8/MfPgBSVokZ7azpRmQpmXDeR4FCaJ08M/uMfxc8OuOhPFhHgYDhhqbaMKW3DljLD2z75mE2WHeAw8YjqXlNhDSYiCRYyDN23O4zuwwX4IBY8NhorQY/wZqkTBu5jGQIFaLmTTPj8MSBszEapG4kVZmzdvwTELiMDCQE4jxC/+M5M23ef7ckeDvP3z4wYcaG8JaGBg4DBgY2w5A2AmElYMA+wMGhj8HiFM7CkbBKBgFIxMAAPkLPq9/fwdtAAAAAElFTkSuQmCC","orcid":"","institution":"Naval Medical University Changhai hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Pengfei","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-02-01 21:00:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3918724/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3918724/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51007786,"identity":"162c70fc-e99d-4a8b-a322-c0a78723e4fd","added_by":"auto","created_at":"2024-02-12 15:43:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":196484,"visible":true,"origin":"","legend":"\u003cp\u003eGenetically predicted Bioavailable Testosterone (BioT) with functional outcome after ischemic stroke (IS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, Inverse-variance weighted estimates for the association between a genetically determined unit increase in exposure on the functional outcome after IS. \u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e, Scatter plots of individual single-nucleotide polymorphism (SNP) effects and estimates from different Mendelian randomization (MR) methods for the effect of (\u003cstrong\u003eB\u003c/strong\u003e) the BioT of males on excellent functional outcome and (\u003cstrong\u003eC\u003c/strong\u003e) the BioT of females on excellent functional outcome. P-het is the P-value belonging to the Q statistic for heterogeneity and P-pleio is the P-value for pleiotropic effects. IVW indicates inverse-variance weighted; and OR, odds ratio.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3918724/v1/d87ce7f44b10bb4468dd44d9.png"},{"id":51007789,"identity":"b35b125b-0886-470a-a1dc-ca24851e8632","added_by":"auto","created_at":"2024-02-12 15:43:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":190975,"visible":true,"origin":"","legend":"\u003cp\u003eGenetically predicted estradiol with functional outcome after ischemic stroke (IS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e, Inverse-variance weighted estimates for the association between a genetically determined unit increase in exposure on the functional outcome after IS. \u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e, Scatter plots of individual single-nucleotide polymorphism (SNP) effects and estimates from different Mendelian randomization (MR) methods for the effect of (\u003cstrong\u003eB\u003c/strong\u003e) the estradiol of females on excellent functional outcome and (\u003cstrong\u003eC\u003c/strong\u003e) the estradiol of females on favorable functional outcome. P-het is the P-value belonging to the Q statistic for heterogeneity and P-pleio is the P-value for pleiotropic effects. IVW indicates inverse-variance weighted; and OR, odds ratio.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3918724/v1/a81a2fef06596b6b17a9262f.png"},{"id":66569936,"identity":"a3886919-4a8e-4d44-aaf2-2a2cc83ff101","added_by":"auto","created_at":"2024-10-14 11:32:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":895521,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3918724/v1/4e7422ce-63ef-49cd-bf86-414c7adff9a7.pdf"},{"id":51007790,"identity":"64e274c8-89c5-45a4-873c-6683e8648381","added_by":"auto","created_at":"2024-02-12 15:43:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4249231,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3918724/v1/e01fddc9854b90f22344ed42.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genetically determined sex hormones and functional outcome after ischemic stroke: A Mendelian randomization study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIschemic stroke (IS) is one of the leading causes of death and disability.\u003csup\u003e1\u003c/sup\u003e Although endovascular therapy is effective in treating large-vessel IS according to many recent clinical trials, the proportion of patients with good functional outcome is still relatively low.\u003csup\u003e2\u0026ndash;4\u003c/sup\u003e Sex hormones have long been associated with the prognosis of IS, but their effects remain controversial.\u003c/p\u003e \u003cp\u003eEstradiol, as the most potent estrogen, is one of the main points of interest. There is evidence from the bench suggesting that estradiol, the most potent estrogen, is a key neuroprotectant in IS.\u003csup\u003e5\u0026ndash;8\u003c/sup\u003e However, some clinical trials have demonstrated otherwise. The Women's Estrogen for Stroke Trial (WEST) found that estradiol treatment did not improve mortality, stroke recurrence, or cognitive function in postmenopausal females after IS.\u003csup\u003e9, 10\u003c/sup\u003e Pappa et al. reported that high levels of endogenous estradiol were associated with high short-term mortality and adverse functional outcome.\u003csup\u003e11\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTestosterone is the principle male sex hormone, which is also reported to have a discrepant effect on the prognosis of IS. While Ho et al. \u003csup\u003e12\u003c/sup\u003edemonstrated that low testosterone levels in males were associated with higher all-cause mortality after IS, two observational studies implicated a positive role for testosterone in post-stroke prognosis in the male gender.\u003csup\u003e13, 14\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eCurrent clinical findings, which predominantly come from observational studies, fail to reach consensus, probably owing to the limitations of observational studies, including residual confounding, reverse causation, and detection bias. Mendelian randomization (MR) is a novel method that uses genetic variants to determine the causality of an observational association between a risk factor and an outcome.\u003csup\u003e15\u003c/sup\u003e Relying on the random assortment of gene variants during meiosis, MR overcomes some core shortcomings of the observational studies mentioned above.\u003csup\u003e16\u003c/sup\u003e. Therefore, we performed a two-sample MR analysis to evaluate whether genetically determined sex hormone levels influence the functional outcome after IS.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eWe used summary data from publicly available original studies for our analyses. All the studies obtained ethical approval and participant consent. This study was conducted in accordance with the guidelines for Strengthening the Reporting of Observational Studies in Epidemiology-Mendelian Randomization (STROBE-MR).\u003csup\u003e17\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExposure data\u003c/h2\u003e \u003cp\u003eWe retrieved publicly available sex-specific genome-wide association study (GWAS) summary-level data of the single nucleotide polymorphisms (SNPs) associated with sex hormones(Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003csup\u003e18,19,20\u003c/sup\u003e Independent SNPs associated with bioavailable testosterone and estradiol were selected from GWAS in up to 425,097 and 311,675 white European individuals from the UK Biobank, respectively.\u003csup\u003e19,20\u003c/sup\u003e 178,782 samples of the bioavailable testosterone levels of males (nmol/L) and 188,507 samples of females were measured by one-step competitive analysis. 147,690 samples of the Estradiol in men and 163,985 of that in women were estimated with 2-step competitive analysis on a Beckman Coulter Unicel Dxl 800. We selected SNPs associated with exposure at a genome-wide significance level (P\u0026thinsp;\u0026lt;\u0026thinsp;5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e). The independence of the SNPs was assessed with stringent criteria (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; clumping window, 10 000 kb). We then harmonized the SNP alleles across the studies and removed palindromic SNPs with ambiguous allele frequencies (0.42\u0026ndash;0.58). The data for all instrumental variables used are shown in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e through S3\u003c/b\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\u003eCharacteristics of the Used GWAS\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFirst author (year)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConsortium\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSample size\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAncestry\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUsed as\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eUnit\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBioavailable testosterone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRuth KS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eUK biobank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMales: 178,782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eExposure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003enmol/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFemales: 188,507\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEstradiol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSchmitz D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eUK biobank\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMales: 147,690\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eExposure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003enmol/L\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFemales: 163,985\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFunctional outcome after ischemic stroke\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSoderholm M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGenetics of Ischemic Stroke Functional Outcome (GISCOME)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003emRS 0\u0026ndash;1 vs 2\u0026ndash;6: 1796 vs 2567\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEuropean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOutcome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003emRS 0\u0026ndash;2 vs 3\u0026ndash;6: 3741 vs 2280\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 \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eOutcome data\u003c/h2\u003e \u003cp\u003eSummary-level data for functional outcome after IS were obtained from the Genetics of IS Functional Outcome (GISCOME) network meta-analysis (12 studies from the US, Europe, and Australia, n\u0026thinsp;=\u0026thinsp;6, 021).\u003csup\u003e18\u003c/sup\u003e 3-month functional outcome after stroke onset was assessed by the modified Rankin Scale (mRS). The primary outcome was excellent functional outcome, defined as an mRS score of 0\u0026ndash;1. The secondary outcome was favorable functional outcome, defined as an mRS score of 0\u0026ndash;2. In the primary model, the analysis was adjusted for age, sex, ancestry, and baseline stroke severity, as assessed by the National Institutes of Health Stroke Scale (NIHSS) 0\u0026ndash;10 days after stroke onset. Moreover, models without adjustment for baseline NIHSS scores were used for comparison.\u003csup\u003e18\u003c/sup\u003e Participants were of European genetic ancestry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThis MR approach was based on three assumptions: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) the genetic variants associated with the exposure and (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) the instrumental variables (IVs) have no association with confounding factors, and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) the functional outcome after IS is influenced only by the exposure, not by other pathways.\u003csup\u003e21,22\u003c/sup\u003e F statistics were applied to evaluate the strength of the genetic instruments.\u003csup\u003e23\u003c/sup\u003e We excluded the SNPs with F statistics lower than 10, which indicated that the strength of these instruments was insufficient for MR analysis\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe inverse-variance weighted (IVW) MR method, which assumes all genetic variants to be valid instrumental variables and produce the most accurate estimates, were performed as our primary analyses.\u003csup\u003e25\u003c/sup\u003e We also explored the sensitivity via the weighted median and MR-Egger regression. The weighted median approaches give more weight to the instrumental variables that are more precise, and the estimate is consistent even when up to 50% of the information comes from invalid or weak instruments.\u003csup\u003e21\u003c/sup\u003e The MR-Egger could detect directional pleiotropy, albeit its low precision.\u003csup\u003e25\u003c/sup\u003e The Cochran\u0026rsquo;s Q statistic in the IVW model were applied to assess the heterogeneity between variant-specific estimates. The MR Pleiotropy Residual Sum and Outlier (PRESSO) approaches were adopted to identify possible outliers. Finally, we performed a leave-one-SNP-out analysis, where SNPs were systematically removed to assess whether a single SNP drove the results. The association was considered significant after correction for multiple testing of the four sex hormone indices (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0125 [0.05/4]). A P value above 0.0125 but below 0.05 suggested a potential association. The results were presented as odds ratio (OR) and 95% confidence interval (CI) of functional outcome per genetically predicted unit log-transformed increase for each trait.\u003c/p\u003e \u003cp\u003eWe additionally performed analyses in the outcome database without adjusting for the baseline NIHSS score as a sensitivity evaluation. Moreover, we included a bidirectional MR analysis to test whether functional outcome of IS affected the liability to bioavailable testosterone and estradiol.\u003c/p\u003e \u003cp\u003eAll MR analyses were performed using the TwoSampleMR (version 0.5.6), Mendelian randomization (version 0.5.1), and MRPRESSO (version 1.0) packages in R (version 4.2.2).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGenetically determined testosterone and functional outcome after ischemic stroke\u003c/h2\u003e \u003cp\u003eFirst, we examined the relationship between genetically determined bioavailable testosterone levels and functional outcome after IS. A total of 42 and 59 independent genetic variants were associated with bioavailable testosterone levels in males and females, respectively. The F-statistic was \u0026gt;\u0026thinsp;10 for all variants, indicating a low risk of weak instrument bias (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, S2\u003c/b\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the results of primary univariate MR analysis. The MR analyses in males showed that high testosterone levels were associated with excellent functional outcome (OR 0.41, 95% CI: 0.21\u0026ndash;0.79, P\u0026thinsp;=\u0026thinsp;0.008) for per standard deviation (approximately 3.7 nmol/L). While In females, MR analyses exhibited suggestive harmful effects of high testosterone levels on functional outcome (OR 2.49, 95% CI: 1.24\u0026ndash;4.99, P\u0026thinsp;=\u0026thinsp;0.009).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCochran Q test showed no evidence of heterogeneity. Meanwhile, none of the MR-pleiotropy Residual Sum, Outlier global test, or MR-Egger intercepts showed directional pleiotropic effects. MR analyses based on the weighted median method yielded results in the same direction as IVW but with lower precision (\u003cb\u003eTable S4\u003c/b\u003e). Leave-one-out analysis did not change the overall direction (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGenetically determined estradiol and functional outcome after ischemic stroke\u003c/h2\u003e \u003cp\u003eNext, we examined the effects of estradiol on functional outcome after IS using MR analyses. A total of 24 variants for estradiol, including 7 for males and 17 for females, were identified (\u003cb\u003eTable S3\u003c/b\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, genetically determined high estradiol levels in females were associated with excellent functional outcome (OR, 0.53; 95% CI: 0.41\u0026ndash;0.85, P\u0026thinsp;=\u0026thinsp;0.005). In addition, MR analyses suggested evidence of a potential association between high estradiol levels in females and favorable functional outcome (OR, 0.69; 95% CI: 0.48\u0026ndash;0.98, P\u0026thinsp;=\u0026thinsp;0.036). However, there was no evidence of association between genetically predicted estradiol levels in men and functional outcome.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Cochran Q statistic of the IVW method indicated no horizontal pleiotropy or heterogeneity across the instrument SNP effects, and the MR-Egger analysis did not show evidence of directional pleiotropy (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Results of the weighted median\u003c/p\u003e \u003cp\u003esupported the primary results (\u003cb\u003eTable S4\u003c/b\u003e). No distortion was observed in the leave-one-out plot, indicating that there was no single SNPs driving the observed effect in any of the analyses (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). \u003csup\u003e18\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eResults of additional analysis\u003c/h2\u003e \u003cp\u003eSensitivity analyses were conducted in models without adjustment for baseline NIHSS scores. For females, genetic predisposition to high levels of estradiol was associated with excellent and favorable functional outcome after IS (excellent functional outcome, OR\u0026thinsp;=\u0026thinsp;0.68; 95% CI: 0.49\u0026ndash;0.95, P\u0026thinsp;=\u0026thinsp;0.024, favorable functional outcome, OR\u0026thinsp;=\u0026thinsp;0.73; 95% CI: 0.53\u0026ndash;0.98, P\u0026thinsp;=\u0026thinsp;0.042). On the contrary, genetically predicted high bioavailable testosterone levels predicted worse functional outcome of women(OR\u0026thinsp;=\u0026thinsp;2.11; 95% CI: 1.12\u0026ndash;3.98, P\u0026thinsp;=\u0026thinsp;0.021). For the male gender, additional analysis was consistent with the primary result: genetically determined high bioavailable testosterone levels suggested excellent functional outcome (OR\u0026thinsp;=\u0026thinsp;0.52; 95% CI: 0.29\u0026ndash;0.97, P\u0026thinsp;=\u0026thinsp;0.042). No relationship was observed between the estradiol levels and functional outcome of men(\u003cb\u003eTable S4\u003c/b\u003e). The results of the bidirectional MR analysis showed that the functional outcome after IS does not affect bioavailable testosterone and estradiol levels (\u003cb\u003eTable S4\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we explored the potential association between sex hormone levels and functional outcome after IS using the MR method. We showed that a genetic predisposition to high levels of bioavailable testosterone is associated with excellent functional outcome in males, but worse outcome in females. Genetic predisposition to high estradiol levels is associated with excellent and favorable functional outcome in females. Bidirectional MR analysis suggested no evidence of functional outcome after IS affecting bioavailable testosterone or estradiol levels. Neither harmful nor beneficial estradiol effects on functional outcome were observed in males after IS.\u003c/p\u003e \u003cp\u003ePrevious MR studies have provided evidence of a causal relationship between sex hormones and an elevated risk of IS.\u003csup\u003e26, 27\u003c/sup\u003e However, these studies focused on IS risk rather than its prognosis, and the causal roles of testosterone and estradiol in post-stroke status remain largely unknown. The recently published GWAS allowed us to conduct MR analyses on this issue.\u003csup\u003e18\u003c/sup\u003e To our knowledge, this study is the first MR analysis exploring the association between sex hormones and functional outcome after IS.\u003c/p\u003e \u003cp\u003eAndrogens\u0026rsquo;s effects on the cerebral vasculature are highly complexed, which differ based on sex, dose, and age.\u003csup\u003e28\u003c/sup\u003e Our study exhibited protective effect of genetic predisposition to high levels of bioavailable testosterone on functional outcome in males. Testosterone may have protective anti-inflammatory effects in pathological conditions, such as hypoxia, endotoxin-induced inflammation, or ischemia.\u003csup\u003e28\u003c/sup\u003e Yi Pan et al. demonstrated an accelerated functional recovery in castrate rats treated with testerone\u003csup\u003e\u003cem\u003e29\u003c/em\u003e\u003c/sup\u003e. They found less GFAP expression and reactive astrocyte hypertrophy around the infarct area in testosterone-treated rats, suggesting that the beneficial effects of testosterone on post-stroke status is presumably related to the function of microlia and astrocyte. Fanaei et al. reported testosterone treatment in castrated male rats to reduce infarct volume and improve sensorimotor recovery following transient focal ischemia, which is probably mediated by promoting antioxidant defenses, BDNF levels and neurogenesis\u003csup\u003e30\u003c/sup\u003e. However, few studies have been done to explore the underline mechanism of testosterone\u0026rsquo;s effect on post-stroke female. More studies need to be conducted regarding this question in the future.\u003c/p\u003e \u003cp\u003eOur study showed that estradiol could exert a protective effect on the post-stroke functional outcome of female, which is probably related to the neuroprotective function of estradiol. Estradiol is reported to reduce the infarct size in ovariectomized rats whether its administered before or after experimental stroke\u003csup\u003e31\u003c/sup\u003e. Several mechanisms have been identified for estrogen-mediated neuroprotection following cerebral ischemia. Firstly, the genomic effects of estrogen are modulated by the activation of the estrogen response element (ERE), which enhances the transcription of genes containing ERE, such as \u003cem\u003ebcl-2\u003c/em\u003e and \u003cem\u003eseladin‐1\u003c/em\u003e.\u003csup\u003e32\u0026ndash;34\u003c/sup\u003e Once activated, these genes participate in cell proliferation, differentiation\u003csup\u003e35\u003c/sup\u003e, and neuroprotection\u003csup\u003e36\u003c/sup\u003e. \u003cem\u003eIn vitro\u003c/em\u003e studies revealed that astrocytes actively participate in the neuroprotective effects of estrogen by modulating inflammatory responses via the downregulation of pro-inflammatory transcription factor NF-κB and the major pro-inflammatory cytokine interleukin-6 after cerebral ischemia.\u003csup\u003e37, 38\u003c/sup\u003e In addition, estrogen promotes neurogenesis and angiogenesis via selective estrogen receptor modulators (SERMs)\u003csup\u003e39\u003c/sup\u003e, which are critical in poststroke plasticity.\u003c/p\u003e \u003cp\u003eThe efficacy of estrogens after stroke seems to be age-sensitive in both sexes;\u003csup\u003e40\u003c/sup\u003e however, our study failed to confirm the association between estradiol levels in males and functional outcome after IS because the limited number of SNPs in this study might not provide sufficient statistical power.\u003c/p\u003e \u003cp\u003eThe results of this study have some limitations. First, we used genetic variants derived from a study with a relatively large sample size strongly associated with BP to avoid weak instrument problems; however, weak instrument bias may still affect our findings. Second, we were limited by the fact that the MR explores the effects of lifelong exposure, whereas sex hormones typically have much shorter periods of action and may have sex-, dose-, model-, and age-dependent effects. The effect sizes that we estimated do not represent the associations between critical periods of exposure and outcome.\u003csup\u003e41\u003c/sup\u003e Therefore, MR studies can only provide additional evidence, and further randomized controlled trials are warranted to investigate the effects of sex hormones on functional outcome after IS. Third, we failed to explore the effects of estradiol on functional outcome in men after IS. This null result did not indicate that there were no protective or deleterious effects of estradiol in males, only that the limited number of included SNPs did not offer sufficient statistical power to perform meaningful analyses. It is also worth mentioning that gender itself may affect the stroke prognosis. Females are reported to be more likely to recognize the warning signs of stroke, which may predict favorable outcomes\u003csup\u003e42, 43\u003c/sup\u003e. Meanwhile, male gender has been associated with less engagement, attentiveness, and receptiveness to health-related knowledge, exerting a negative effect on their recovery\u003csup\u003e44, 45\u003c/sup\u003e. Finally, as all the participants were of European ancestry, the results of this study are not necessarily valid for other ethnic groups.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eIS: ischemic stroke; MR: mendelian randomization; OR: SNPs: single nucleotide polymorphisms; mRS: modified Rankin Scale; GWAS: genome-wide association study; NIHSS: National Institutes of Health Stroke Scale; IVW: inverse-variance weighted; OR: odds ratio; CI: confidence interval.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this MR analysis, a genetic predisposition to high levels of bioavailable testosterone was associated with excellent functional outcome after IS in males, but with worse outcomes after IS in females. On the contrary, a genetic predisposition to high estradiol levels was associated with excellent and favorable functional outcome after IS in females. Further studies are needed to determine the effect of estradiol on functional outcome after IS in males.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eAll the studies involved in our work obtained ethical approval and participant consent. This study was conducted in accordance with the guidelines for Strengthening the Reporting of Observational Studies in Epidemiology-Mendelian Randomization (STROBE-MR).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for Publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Shanghai Changhai Hospital (Grant No. GH145-05).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eTianxiang Gao: participated in study design, analyzed data, prepared figures, wrote manuscript. Hongjian Zhang and Hanchen Liu: participated in study design, analyzed data, wrote manuscript. Manyue Ge: wrote manuscript. Jianmin Liu and Shaojun Mo: participated in study design, interpreted data, wrote manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. All data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSaini V, Guada L, Yavagal DR. Global Epidemiology of Stroke and Access to Acute Ischemic Stroke Interventions. Neurology. 2021;97:6\u0026ndash;S16. 2021/11/18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshimura S, Sakai N, Yamagami H, et al. Endovascular Therapy for Acute Stroke with a Large Ischemic Region. 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Health Inf J. 2012;18:83\u0026ndash;94. 2012/06/27.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ischemic stroke, Mendelian randomization, bioavailable testosterone, estradiol","lastPublishedDoi":"10.21203/rs.3.rs-3918724/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3918724/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and purpose\u003c/h2\u003e \u003cp\u003eSex hormones may affect functional outcome after ischemic stroke (IS). However, the effects reported in previous studies were inconsistent, and randomized data were absent. We performed a two-sample Mendelian randomization analysis to explore the causal effects of genetically determined sex hormones on the 3-month functional outcome after IS.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eUsing genome-wide association studies, we obtained sex-specific genetic instruments for evaluating serum estradiol and bioavailable testosterone levels. The sex-specific functional outcome after IS were estimated via inverse-variance weighted Mendelian randomization analysis. Mendelian randomization-Egger regression, weighted median leave-one-SNP-out analysis, Mendelian randomization-Pleiotropy Residual Sum, and Outlier methods were used for sensitivity analyses. To validate our primary results, we also performed independent repeated and bidirectional Mendelian randomization analyses.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eGenetic predisposition to high levels of bioavailable testosterone was associated with excellent functional outcome in males (OR\u0026thinsp;=\u0026thinsp;0.41, 95% CI: 0.21\u0026ndash;0.79, P\u0026thinsp;=\u0026thinsp;0.008), but worse outcome in females (OR\u0026thinsp;=\u0026thinsp;2.49, 95% CI: 1.24\u0026ndash;4.99, P\u0026thinsp;=\u0026thinsp;0.009). Females with genetically predicted higher estradiol levels tend to have excellent and favorable functional outcome (excellent functional outcome, OR\u0026thinsp;=\u0026thinsp;0.53; 95% CI: 0.41\u0026ndash;0.85, P\u0026thinsp;=\u0026thinsp;0.005); (favorable functional outcome, OR\u0026thinsp;=\u0026thinsp;0.69; 95% CI: 0.48\u0026ndash;0.98, P\u0026thinsp;=\u0026thinsp;0.036). There was limited evidence that genetically predicted estradiol levels affect functional outcome in men (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur findings suggested that bioavailable testosterone and estradiol were promising neuroprotectants that could respectively improve the functional outcome of IS in males and females.\u003c/p\u003e","manuscriptTitle":"Genetically determined sex hormones and functional outcome after ischemic stroke: A Mendelian randomization study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-12 15:43:10","doi":"10.21203/rs.3.rs-3918724/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1bc3fe05-057b-44ad-bb72-14131f39e0fe","owner":[],"postedDate":"February 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-14T11:23:55+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-12 15:43:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3918724","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3918724","identity":"rs-3918724","version":["v1"]},"buildId":"zQwnuV7TCBrMSSSToR1PI","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}