Metabolic syndrome (MS) increases the risk of cardiovascular disease (CVD) in both sexes and is associated with multiple diseases including sleep apnea, liver disease, polycystic ovary syndrome (PCOS), and hormone-sensitive cancers (1–3). High atherosclerotic CVD accounts for 33-40% of all mortality in the United States and the European Union (4). Accumulative evidence indicates that MS-associated CVD risk is highly linked with sex and sex hormones over the lifetime (5). Although men are more likely to be at risk for CVD than women, the risk of developing CVD increases drastically after menopause in women, particularly in those with menstrual dysfunction. It has been postulated that changes in sex hormones owing to menopause transition or menstrual dysfunction may be contributory factors for adverse CVD risk in postmenopausal women (6–8). However, MS is often comorbid with systemic low-grade inflammation, making a worse prognosis (9). Emerging evidence suggests that chronic low-grade inflammation may serve as an independent marker for atherosclerotic CVD. In addition, sex-specific differences have also been observed in MS-associated inflammation (10). The current prevalence of MS is 34.7% in the United States. A notable increase in MS has become a major global health concern, especially among women, young adults, and some ethnic groups (11). Since imbalances in the sex hormones and sex chromosomes are associated with the development of metabolic abnormalities and inflammatory diseases, sex hormones may serve as global markers for MS and inflammation-associated CVD outcomes (12).

Multiple studies have investigated the association between sex hormones and MS in women with PCOS, pre- or postmenopausal women, or men (5, 13). A large meta-analysis confirmed the sex-dependent association of total testosterone (TT) and free testosterone with MS while a sex-independent association between sex hormone-binding globulin (SHBG) and MS (14). Recently, some studies highlighted a balance between TT to estradiol (E) is critical for CVD risk evaluation in men or women (15–22). Limited studies also evaluated a ratio of TT to SHBG or E to SHBG as a marker for CVD risk (23–27). However, these studies produced contradictory findings. Although several studies highlight the influence of sex hormones on inflammatory and immune cell functions, limited studies evaluated the role of a ratio of sex hormones on inflammatory markers (28–30). Several studies demonstrated that C-reactive protein (CRP) and MS are associated with carotid atherosclerosis differently in different sexes indicating different sex hormones might be associated with MS and CRP in different sexes (31). Despite the potential influence of MS and inflammation on CVD risk, the associations of ratios of sex hormones with metabolic and inflammatory markers are not evaluated according to sex and age groups. A direct comparison of the ratios of sex hormones in association with MS and inflammatory markers may provide a valuable global marker for risk stratification of MS and inflammatory-related diseases.

A total of 9,167 subjects including 4,360 males and 4,807 females were included in the analysis. The study contains subjects with an average age (SD) of 45 (17.1) years, mostly non-Hispanics (84.7%) and married (61.3%). Relatively, male participants were younger, smokers, drinkers, physically active, and overweight or obese than females ( Table 1 ). The prevalence of MS and high CRP was 42%, 25.1% in males, and 43.6% and 37.8% in females, respectively. The median E concentration was markedly lower in women with age≥50 years (22.0 vs. 241.2) while the median SHBG (48.8 vs. 31.7) was significantly higher in males with age≥50 years without much difference in TT ( Supplementary Table 1 ). Most sex hormone ratios had an interaction with the age group (<50 years vs. ≥50 years) on both MS and high CRP outcomes ( Supplementary Table 2 ). Accordingly, the association of sex hormones with outcomes was reported separately for each age group.

In our study, the FEI was found to be associated with MS and CRP outcomes to a greater extent than EAI or FAI in males across age groups. In addition, the FEI remained greatly associated with MS and CRP outcomes in females with age≥50 years. However, the FAI was positively associated with MS and CRP in females with age<50 years. These findings were confirmed in multiple sensitivity analyses including after additionally adjusting for each sex hormone concentration. These findings suggest a regular evaluation of sex hormone ratios, particularly FEI in both sexes and FAI in younger women. These evaluations may help physicians to stratify individuals at risk for CVD and develop potential strategies for early management of MS and CRP through maintaining sex hormone ratios.

We observed FEI as the strongest factor associated with MS and high CRP in younger or older men. To the best of our knowledge, there is no study directly evaluating the association between FEI with MS or CRP in young men. In elderly men, only one study reported associations between FEI and MS and inflammatory markers. This study on older Italian men found a strong association between free estradiol and MS supporting our study findings (36). Our findings that a higher FEI consistently associates with adverse metabolic and inflammatory profiles compared to EAI or FAI in older women have also been supported in emerging studies (24, 37, 38). Exogenous E treatment appears to be associated with the increased risk of stroke in postmenopausal women suggesting that reducing free E levels could potentially reduce the risk of MS and inflammation and subsequently CVD events (6, 24). As confirmed in multiple studies (30, 39, 40) and meta-analyses (14, 41), SHBG and TT were also inversely associated with MS and high CRP in our study. Although the association between E and MS has been conflicting among men and women (29, 36, 37, 42, 43), the FEI was positively associated with MS and high CRP even after adjusting for TT, E, and SHBG concentrations indicating the usefulness of FEI in the context of MS and CRP-related diseases.

In addition to FEI, we also observed an inverse association between EAI and MS and high CRP in men. Consistent with our findings, multiple studies reported the protective effects of a high EAI for MS and its components, inflammation, and cardiac events in men (16, 18, 19, 28). Contrary to our study findings, a few studies showed that a higher EAI is adversely associated with some metabolic abnormalities (44) and cerebrovascular disease (21) in elderly men. These contradictory associations particularly in elderly men may be due to a positive association between a higher TT and LDL cholesterol as observed in our study and other studies (45). In our healthy women sample, EAI was not consistently associated with MS and CRP outcomes. In contrast, multiple studies reported that a higher EAI is associated with incident MS (46), cardiometabolic diseases in women with menopause transition (22), development of CVD in postmenopausal women (17), and cardiometabolic markers in women with CHD (15). The role of the EAI in older men and women needs to be further explored after adjusting for SHBG and E levels.

In younger women, FAI was found to be the most critical factor for MS and CRP in our study. Hyperandrogenic profile as measured with FAI has been observed as a typical feature of MS in premenopausal women (26). A recent study further attested a link between androgenicity and the risk of developing MS before menopause using a Coronary Artery Risk Development in Young Adults (CARDIA) study (47). In addition to the FEI, we also observed a higher FAI associated with MS and CRP in women with age ≥50 years. Several studies confirmed our findings related to FAI for most metabolic and inflammatory markers in postmenopausal women (8, 23, 29, 48, 49). Rexrode et al. (25) reported a direct comparison of all combinations of sex hormones with CVD events among postmenopausal women without hormonal therapy and demonstrated that higher FAI levels are associated with CVD events. Moreover, Phillips et al. (42) showed that elevated free testosterone but not SHBG or E is associated with an increased degree of coronary artery disease. These findings confirm the potential utility of the FAI for atherosclerotic CVD risk stratification in women. In our study, the direction of associations of sex hormone ratios with MS and high CRP was generally consistent except in women of ages <50 years. A high FEI was positively associated with MS while inversely associated with high CRP in younger females. In addition, a high FAI was only associated with MS but not with high CRP in younger males while a high EAI was only associated with high CRP but not with MS in younger females. Chen et al. (31) demonstrated that CRP and MS differently predict carotid atherosclerosis in men and women. They reported CRP levels are associated with extracranial carotid artery plaques in men while MS is associated with common carotid artery intima-media thickness in women. These findings explain the potential sex hormones and sex differences related to MS and CRP-induced diseases as discussed in several studies (5, 50, 51).

Although it is unclear how a higher FEI can influence MS and CRP in men and postmenopausal women, a higher FEI above a threshold likely induces hypoandrogenemia in males and hyperandrogenism in postmenopausal females, which is associated with insulin resistance and obesity leading to MS and systematic inflammation (5, 12). Since E is primarily produced through the aromatization of TT to E in males, higher free E levels can be detrimental to cardiometabolic outcomes in aging or TT-deficient men. Owing to numerous factors, especially inflammation, insulin resistance, and obesity, the level of biologically active E (FEI) is affected in menopausal women and excess FEI can affect cardiometabolic health as confirmed in several studies (52). TT and E both are involved in oxidative phosphorylation in the mitochondria affecting adenosine triphosphate production leading to mitochondrial dysfunction, MS, and inflammation (53). Moreover, deficiency in the functions of androgen receptors and estrogen receptors has the potential to affect metabolic health. Our study also identified SHBG as a consistent marker inversely associated with MS and CRP in both sexes across age groups. In addition to FAI, lower SHBG concentrations emerged to be a robust marker for MS and CRP in our study of younger females. Since SHBG is associated with reduced E to TT ratio levels in older men (54) and TT in postmenopausal women (55), lower SHBG can influence free TT and E levels affecting cardiometabolic health in both sexes. Multiple studies confirm that SHBG has a direct effect on metabolic health and diabetes through SHBG polymorphisms and apolipoprotein B (56, 57). Recent studies discovered SHBG in the human heart produced by cardiomyocytes in dilated cardiomyopathy patients, indicating that SHBG transports sex steroids into the heart and may have a direct effect on heart condition (58). Lifestyle, dietary changes, and treatments can be used to enhance SHBG expressions and functions (59). In older subjects, estrogen blockers may be used to reduce biologically active E levels. Furthermore, prevention strategies such as minimizing environmental exposures and enhancing immunity may be universally adopted across all populations (60, 61).

In our study, increases in FEI appeared to be strongly associated with metabolic and inflammatory markers in males across age groups. In addition, FEI was also markedly associated with a high prevalence of MS and high CRP in females of age≥ 50 years. In younger females, a higher FAI was associated with metabolic and inflammatory markers. In addition, lower SHBG concentrations were also associated with MS and high CRP in both sexes across age groups. These findings confirm the potential role of sex hormone ratios in metabolic and inflammation processes in both sexes. A regular evaluation of sex hormone ratios specific to sex is critical across age groups for CVD risk stratification and early prevention. A longitudinal study is required to validate the findings obtained in our study.

Publicly available datasets were analyzed in this study. This data can be found here: https://www.cdc.gov/nchs/nhanes/index.htm.

Ethical approval was not required for the studies involving humans because the study dataset is a publicly available database without PHI. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements because This is a publicly available database (NHANES) without PHI. The NHANES collects data after obtaining approval from the National Center for Health Statistics Research Ethics Review Board, Centers for Disease Control and Prevention, and receiving written consent from the participants.

PD: Conceptualization, Data curation, Validation, Visualization, Writing – original draft, Writing – review & editing, Resources. VS: Data curation, Visualization, Writing – review & editing, Formal analysis, Methodology, Software. NV: Data curation, Visualization, Writing – review & editing, Project administration, Resources. MT: Data curation, Project administration, Resources, Visualization, Writing – review & editing. SR: Resources, Visualization, Writing – review & editing, Conceptualization, Supervision, Validation. RL: Conceptualization, Supervision, Validation, Visualization, Writing – review & editing. AD: Conceptualization, Supervision, Validation, Visualization, Writing – review & editing, Data curation, Investigation, Methodology, Project administration, Writing – original draft.