In this study, we explored the genetic associations and causality of SHBG, TT, and BT with VTE, PE, and DVT using a combination of two-sample MR, MRPRESSO, and LDSC. In the primary MR analyses, we found that elevated serum SHBG levels increased the risk of VTE and PE, whereas elevated serum TT levels increased the risk of VTE and DVT. Furthermore, the LDSC and MR sensitivity analyses supported the causal effect of SHBG on VTE. However, the causal effects of testosterone levels have not been verified.

Previous studies have shown that the use of exogenous oestrogen, androgens, and endogenous oestrogen increases the risk of VTE. However, the association between endogenous androgens such as SHBG and VTE remains controversial.

Numerous investigations have substantiated the independent prognostic value of SHBG in assessing VTE risk subsequent to the use of hormonal contraceptives [19,20,21]. A recent case-control study involving women aged ≤ 45 years revealed that the occurrence risk of VTE exhibits a dose-response association with SHBG levels, indicating an OR of up to 2.0 for individuals in the highest quartile of SHBG levels compared to those in the lowest quartile, suggesting the pathophysiological effects of SHBG VTE in young women not using contraceptives [22]. In this study, primary IVW before BH correction and MR-PRESSO suggested a causal effect of SHBG on VTE. The replicated MR analyses in the largest VTE database currently knownand MVMR also supported this result, combined with the results of LDSC, which supports the genetic correlation between SHBG and VTE. We suggest that SHBG is causally associated with VTE, consistent with the conclusions of previous studies. Simultaneously, owing to the differences in sex hormones and SHBG between genders, we used the largest GWAS data available on female VTE to confirm the causal relationship between SHBG and VTE in females. Unfortunately, there are currently no available male VTE data that do not significantly overlap with our exposure data (VTE data for both males and females can be obtained in UKBB, but our previous LDSC analysis suggests a high overlap with exposure data). In addition, it must be emphasized that in the original GWAS study of the exposure data, the authors analysed and pointed out that the genetic structure of SHBG levels is highly consistent between males and females [16]; therefore, the results of the MR analysis using population-wide SHBG genetic IVs are credible, we concluded that a causal relationship exists between SHBG and VTE. Among the subtypes of VTE, SHBG was causally associated with PE in the primary MR and MVMR analyses, which is consistent with the genetic correlation of LDSC. However, we recognize that DVT and PE are considered two clinical presentations of VTE, with approximately 50–70% of PE patients concurrently experiencing DVT and 50% of DVT patients having an associated asymptomatic PE noted on lung scans [35]. The conclusion that SHBG is causally associated with PE but not DVT requires further validation. The transition from DVT to PE involves intricate physiological regulation, and several risk factors which predict the occurrence of PE in DVT patients, such as hypertension, diabetes, long lying state, glucocorticoid therapy, and D-dimer levels, have been reported [36, 37]. Therefore, our findings may allow us to speculate that SHBG could potentially be associated with a higher risk of PE development in DVT patients within the context of VTE formation. However, clinical research on the high-risk factors and underlying mechanisms for the occurrence of PE in DVT patients are limited. Our speculation in this regard necessitates further validation through subsequent research endeavours.

However, the mechanisms underlying the regulation of VTE by SHBG remain unclear. Among women using hormonal contraceptives, investigators have considered SHBG a marker of the ‘oestrogenicity’ of contraceptives, and oestrogen is an established risk factor for VTE [19]. Some researchers have also hypothesized that hepatically metabolized hormonal contraceptives may influence the production of SHBG and coagulation factors, ultimately contributing to VTE [20]. In addition, SHBG can be regarded as a potent amplifier of steroid activity, and it is the main transporter of hydrophobic androgens in hydrophilic blood, but perhaps less so for oestrogens, which can also undergo glucuronidation and sulfation to facilitate their transportation to target organs [38]. Therefore, their effects may also be via testosterone [39].Furthermore, the interaction between SHBG and megalin might act as an additional autocrine-controlled mechanism, actively transporting steroids into cells and potentially modulating hormone signalling pathways [38]. It is also believed that SHBG may be implicated in the modulation of chronic inflammation, cellular proliferation, and lipid metabolism, and these processes can potentially impact the development of VTE independent of sex hormones [17, 40]. Regardless of the underlying mechanism, SHBG may be a potential clinical biomarker of VTE. Follow-up studies should focus on exploring the pathophysiological mechanisms of SHBG in VTE to look forward to identifying novel therapeutic targets.

Multiple observational studies have indicated a potential elevation in short-term VTE risk among men receiving testosterone therapy [6, 7, 41]. A recent case-crossover study demonstrated that testosterone therapy was associated with an elevated risk of VTE within 12 months of follow-up in men with and without hypogonadism, with an OR of 2.32 (95% CI, 1.97−2.74) and 2.02 (95% CI, 1.47−2.77), respectively [42]. The mechanism underlying this effect might be attributed to the elevation of Hct levels in men following testosterone treatment, which is significantly associated with VTE [7, 43]. The current observational findings do not support an association between endogenous testosterone levels and the risk of VTE in men or women [10, 11, 22, 44]. However, a recent MR study observed a positive association between endogenous testosterone, genetically predicted by mutations in the JMJD1C gene region, and thromboembolism (VTE, arterial embolism, and thrombosis) (OR: 2.09, 95% CI: 1.27−3.46) in men, whereas no such association was found in women, and no association was observed between endogenous testosterone, genetically predicted by mutations in the SHBG gene region, and thromboembolism [45]. However, similar to this study, the correlation between testosterone and VTE failed to obtain consistent results in validation studies. Coupled with the small sample size (3225 men), we believe that their conclusions need to be further verified. In this study, the results of both IVW and MR-PRESSO suggested a causal relationship between TT but not BT and VTE. However, the replicated MR analyses in both sexes and females did not support this result, nor did the MVMR data in either sex or LDSC. Additionally, in the original GWAS of exposure data, the authors highlighted that the genetic contribution to the variability in circulating testosterone levels varies considerably between males and females, with several variants having genome-wide significance for testosterone in opposite directions, thus emphasising that sex-disaggregated data are best used in genetic association analyses of testosterone [16]. Additionally, in our MVMR analyses of female data, TT and BT had a significant causal effect on VTE. Therefore, we cannot completely exclude the correlation between testosterone levels and VTE when conducting sex-specific analyses. In conclusion, although this study had a larger sample size than previous MR studies, given the negative results of the LDSC and replicated analyses and the lack of better available sex-disaggregated data, we considered whether there was a causal association between testosterone and VTE needs the support of further large-sample sex-disaggregated MR analysis results.

We conducted the first MR study to confirm the genetically predicted causal relationship between SHBG and VTE; however, this study had a few limitations. First, because the three assumptions of MR studies cannot be precisely satisfied, corresponding bias cannot be eliminated. In terms of instrument relevance, we adopted a stricter P-value (P < 5 × 10− 8), calculated the F value of each SNP, and deleted SNPs with F ≤ 10. The R2 of the IVs used in all MR analyses ranged from 1.9 to 10.5%, thereby reducing the bias introduced by weak IVs. Horizontal pleiotropy was the main reason for the bias in instrument validity. We used MR-PRESSO to exclude outliers that may have pleiotropic effects and then performed a subsequent MR analysis. Although IVW was utilized as the principal methodology to ascertain causal associations, we ensured the consistency of direction using various MR analysis methods. In addition, the leave-one-out analyses showed no significant horizontal pleiotropy, and the MVMR analyses showed robust results, consistent with the main MR analysis of the overall results, which were mainly used to rule out bias owing to the correlation between sex hormones. Additionally, we performed replicated MR analyses using an independent outcome database and LDSC to confirm the genetic correlation and reliability of primary MR analyses. Reverse causality was ruled out using MR Steiger. Second, heterogeneity tests revealed some heterogeneity in this study, which may reflect the different responses of different populations or environments to research effects. In response, a random-effects model was applied for IVW analyses. Third, this study was conducted in a European population; therefore, generalising the results to other races requires further verification. Fourth, sex hormones differ between sexes, and the genetic IVs of testosterone also show large differences. However, owing to the lack of sex-specific outcome data that did not overlap with our exposure data, our sex-disaggregated data were only for women. Therefore, the correlation between testosterone levels and VTE was used as the reference in this study. However, because the genetic IVs of SHBG described by the original author were not significantly different between the sexes, the results of this study on SHBG remain credible. Further, large-sample sex-disaggregated MR analyses of sex hormones, especially testosterone, in VTE are necessary to determine their causal relationship. Therefore, SHBG may be a potential clinical biomarker for VTE, and follow-up studies should focus on exploring the pathophysiological mechanisms of SHBG in VTE to look forward to developing novel therapeutic targets.