Our study demonstrated that the E/T ratio was independently associated with both AIS and poor early functional outcomes. In this study, the highest tertile of the E/T ratio was significantly associated with AIS. Our findings suggest that the E/T ratio may be a possible independent biological marker of AIS. Furthermore, an elevated E/T ratio may be used to predict early unfavorable functional outcomes. To the best of our knowledge, the current study is the first to investigate the inter-relationship between the ratio of sex hormones and early functional neurological outcomes in patients with AIS.
The presence of sexual dimorphism and their function has now been supported by evidence from the various fields of genetics, epigenetics, immunology, cellular physiology, and neurosciences. These mechanisms include apoptotic signaling cascades in neurons and glia, resident microglial activation, neuroglial response to ionic imbalance, autophagy, and mitochondrial toxicity . In addition, the different effects of sexual hormone levels on the disease in males and females should be considered.
Clinical trials have demonstrated that estrogen increase the stroke incidence and severity in elderly women . There is also evidence that estrogen therapy may increase thrombotic conditions and the risk of stroke. This suggests that hormones affect not only the brain, but also the coagulation system [15, 16]. Similar trends were also observed in experimental stroke models. When administered to aged female rodents, estrogen paradoxically increased infarct volume after ischemic stroke [17, 18]. These neurotoxic effects may be associated with a decrease in the utilization of insulin-like growth factor 1, a neuroprotective agent that decreases with age and is downregulated by estrogen treatment . However, these studies were conducted only in women. Furthermore, the effects of estrogen therapy duration or dose on the experimental results were unclear .
Estradiol is the most potent endogenous estrogen. Estradiol is the main female sex hormone and plays an important role in the promotion of secondary sexual characteristics, and development of the reproductive system and breast tissue [10, 21]. One study obtained blood samples from 302 post-menopausal women hospitalized with AIS and found an association between higher estradiol levels and stroke severity, 1-month mortality, and poor functional outcomes . The study reported that estradiol levels remained independently associated with the aforementioned parameters, even after correction of confounders. The study concluded that stroke itself may elevate estradiol levels as an endogenous protective mechanism. Another study reported that higher circulating estradiol levels in late post-menopausal women were associated with higher mortality, independent of age and multiple confounders . This study also suggested that the pro-thrombotic effects of estradiol may explain the high rate of thrombotic events observed in post-menopausal women. Lee et al. suggested that post-menopausal women with high endogenous free estradiol levels had 2.3-fold greater odds of stroke, independent of age . However, these prospective studies can only reveal associations, not causality [22-24]. Nevertheless, higher levels of estradiol may be a marker of cerebrovascular disease and could help in identification of patients with AIS who are at risk of worsening unfavorable functional outcomes and mortality [22, 23].
Related studies of testosterone levels provide conflicting data on the true effects of the hormone on visceral adiposity, independent of other sex hormones. High serum testosterone is associated with reduced subcutaneous and increased abdominal adiposity in women, while low testosterone is associated with the same effect in men [25-27]. In older men, sex hormone levels do not decrease as rapidly as in menopausal women however, testosterone levels decline steadily with age .These declining testosterone levels have been reported to be associated with elevated visceral adiposity in older men . This may play a role in increased risk of cardiovascular diseases in aging men. Low testosterone levels are associated with increased systemic inflammation  and endothelial dysfunction , both of which contribute to atherosclerosis. Epidemiological studies have shown that low testosterone levels independently predict the atherosclerosis development [32, 33]. Some meta-analyses have demonstrated that low testosterone levels increase the risk of major cardiovascular diseases as well as mortality [34, 35]. While many studies have reported the association between mortality in men and low circulating levels of testosterone , few have investigated the association between testosterone and the incidence of AIS. Levels of testosterone fluctuate throughout life in males. Increased levels observed in young men and decreased levels of the hormone in older men have been shown to increase the risk of ischemic stroke [37-39]. It has also been hypothesized that aged men have reduced functional outcomes after AIS as a result of the progressive decreased testosterone, in contrast to the neuroprotective effect observed in the female brain . Although the effects of estradiol and testosterone vary greatly depending on age and sex, it is reported that in elderly men, as total testosterone levels decrease, the incidence of stroke increases and functional outcomes may be poor even after adjusting for existing cardiovascular risk factors [41-43]. This is probably due to the anti-inflammatory and anti-oxidant effects of testosterone itself on brain endothelial cells and vascular smooth muscle cells in the pathologic state, but suggests that the balance between estradiol and testosterone may also be important [44-46]. Based on these results, in order to determine the impact of testosterone on the outcomes of ischemic stroke, a more robust study should be designed that considers the levels of endogenous testosterone and other sex hormones.
We have suggested a unique concept in this study, namely the E/T ratio, and investigated the association between this ratio, AIS, and unfavorable functional outcomes. The novelty of this work is that it presents a new form of biomarker by combining estradiol and testosterone to quantitatively identify the influence of acute ischemic stroke. In this study, the E/T values showed clear differences between the two groups, but the values of estradiol and testosterone did not show statistical differences. This is probably considered a problem of diagnostic values. Although not statistically significant, there were an increase in estradiol and a decrease in testosterone level in AIS group, compared with control. The estradiol and testosterone levels were affected by age and sex. This study is also meaningful in comparing estradiol and testosterone respectively in patients with AIS, but more focused suggests that using the E/T ratio can increase the diagnostic value. This study suggests that estradiol and testosterone may have clinical significance in AIS patients, but lower testosterone level compared to estradiol level is more correlated with AIS and early unfavorable functional outcome. E/T ratio was concluded to be a useful biomarker for AIS and early unfavorable functional outcomes. The results of our study are consistent with some previously reported observations. In a study on middle-aged men, wherein testosterone or estradiol was measured by immunoassay, higher estradiol levels were associated with cardiovascular disease . In a study on elderly men, higher total testosterone using mass spectrometry was associated with a lower incidence of cardiovascular events, with similar hazard ratio in men with and without pre-existing cardiovascular disease . Another study in aged men using mass spectrometry for hormonal analysis found no association between testosterone and cardiovascular events, defined as myocardial infarction (MI), stroke, or cardiovascular disease-related death . A recent study on elderly men reported that higher plasma testosterone is an associated biomarker for reduced risk of stroke, but not MI . Another recent study reported that extremely low plasma testosterone levels were associated with a high risk of ischemic stroke in men, and that this was caused by being overweight, and HTN . Testosterone was found to independently induce endothelial relaxation through secretion of endothelial nitric oxide synthase, which causes vasodilation . In obese men, the reduction in these neuroprotective effects may be related to development of AIS, due to increase in blood pressure. Furthermore, the conversion of testosterone to estradiol arises in adipose tissue by aromatization, and excessive adipose tissue may reduce plasma testosterone concentrations [50, 52]. Therefore, the applicability of the E/T ratio, and the association between higher E/T ratio and prevalence of AIS should be considered.
The present study has several limitations. First, this was a single-center study conducted on a single race. The sample size was small for the results may not be generalizable to other demographic groups. A large-scale, multi-center external validation of the current study is necessary. Second, we primarily considered cross-sectional observational data, which may reduce accuracy. Particularly, female were excluded from this study because plasma sex hormones vary widely depending on menstruation or menopause. Such variations are also observed in men. However, not to the extent seen in women. It would be interesting to conduct a study that distinguishes the causal relationship between sex hormones and stroke in pre- and post-menopausal women. Furthermore, it would be also interesting to conduct a large-scale study comparing reproductive and elderly age for various sex hormones in stroke. Third, we did not serially measure plasma sex hormone levels, which prevented us from determining the cause and effect relationship between the hormones and AIS or other functional outcomes. We did not illustrate the longitudinal relationship between sex hormones and AIS. Fourth, the E/T ratio may be related to inflammation and short-term outcomes, which may be related to the infarct volume, but could not be measured in this study. MRI volumetry should be used to provide more accurate and reliable information on infarct volume. A large-scale study using this technique is warranted. Fifth, access to the proven gold standard mass spectrometry estradiol measurement technique for routine clinical use is limited. Therefore, immunoassay, as used in this study, may have resulted in inaccurate measurement of low serum concentrations of estradiol in men, which could lead to overestimation of estradiol . Finally, since early functional outcomes were assessed in this study, it is necessary to verify the association with long-term neurological outcomes. Despite these limitations, the results suggest that the E/T ratio may be of diagnostic and prognostic value for AIS.