Our study shows that aromatase levels are significantly higher in post-menopausal women after acute ischemic stroke when measured within 24 hours from symptom onset. Testosterone levels significantly decrease in men at this time point. No significant change in estradiol levels was seen within 24 hours post ischemic stroke in either sex, although there was a trend for higher estradiol levels in women with stroke. Interestingly, there was a significant positive correlation between estradiol levels and change in mRS (functional outcome measure), regardless of sex.
In intrauterine life, sexual differentiation of the brain occurs by aromatization of androgens to estrogens (9). It has been shown that neurotoxic insult to the brain (induced by systemic administration of kainic acid), leads to increased aromatase expression in astrocytes (17). In spontaneously hypertensive rats, aromatase was elevated at 24 h and at 8 days after focal cerebral ischemia in the penumbra, specifically in the astrocytic processes (11). The expression of aromatase has also been known to be sex specific. Aromatase activity and expression was found to be greater in female than in male primary cultured cortical astrocytes. Arimidex, an aromatase inhibitor, abolished sex differences in astrocytic cell death induced by oxygen glucose deprivation (OGD), while addition of estradiol protected both sexes. This implied that local estradiol production by aromatase is protective in both sexes (18). Studies in aromatase knock out mice (ArKO) further clarified the role of aromatase. Female ArKO mice subjected to reversible middle cerebral artery occlusion had larger infarcts compared to wild type mice. Similarly, wild type female mice treated chronically with the aromatase inhibitor, fadrozole, had more ischemic damage when compared with ovariectomized females, suggesting that extragonadal estradiol is also important for neuroprotection (12). The role of aromatase in cardiovascular disease has also been highlighted by studies in breast cancer patients who were administered aromatase inhibitors (19). A recent study demonstrated increased risks of heart failure and cardiovascular mortality in patients taking aromatase inhibitors as compared with patients taking tamoxifen (20). Administration of the specific aromatase inhibitor, fadrozole, in male rats enhanced the neurodegenerative effects of kainic acid and this was reversed by administration of estradiol, confirming that neuroprotective effects of aromatase are mediated by estradiol (21). Consistent with these reports in preclinical literature, our clinical study suggests that the aromatization pathway may become active in the event of brain injury, as evidenced by the increased levels of serum aromatase seen after stroke in women. This may be an early endogenous protective mechanism of the brain activated by transcription factors like hypoxia inducible factor or cytokines(11). Future studies on the dynamics of these transcription factors, cytokines and their associations with sex hormone levels may assist in further deciphering the role of these sex hormones in ischemic stroke.
Although no significant change in estradiol levels was seen after stroke, we found a significant positive correlation between estradiol levels and change in mRS, regardless of sex, which suggests that a higher peripheral estradiol level may be associated with a higher mRS, suggestive of worse outcomes. This has been shown in previous studies in elderly women (22-24). This correlation highlights the complex and the long debated role of estradiol in stroke. Boys are known to have higher stroke incidence compared to girls. In fact, it has been seen that for each 1 nmol/liter increase in testosterone in young boys, there was a 1.3 fold increase in risk of stroke (25). This epidemiology reverses with advanced age, and elderly women have higher stroke incidence and worse outcomes, which is often attributed to the loss of estrogen at menopause. Preclinical studies in stroke models have shown a robust neuroprotective effect of estrogen (5), but this was not recapitulated in early clinical trials. Both the WHI and WEST trials of estrogen supplementation led to increased mortality in post-menopausal women (6, 7), although issues with trial design, including the dose, type and timing of estrogen supplementation were raised. The Kronos Early Estrogen Prevention Study (KEEPS) study found that neither oral nor transdermal estrogens affected the progression of atherosclerosis (measured as CIMT, carotid-artery intima–media thickness), when given to recently postmenopausal women (26). On the other hand, Early versus Late Intervention Trial with Estradiol (ELITE) found that oral estradiol therapy (when initiated within 6 years of menopause) was associated with less progression of subclinical atherosclerosis (measured as CIMT) (27). It appears that the type, timing of therapy and the dose response effect of estradiol on the vasculature may be important in determining the benefits of exogenous estrogen for stroke prevention. We speculate that higher post stroke injury (as gauged by worse outcomes, in this case mRS) causes increased aromatase expression and that in turn attempts to increase estradiol levels to protect the brain. However, these results should be interpreted with caution, as causality cannot be determined with a correlation. We did not see any significant change in estradiol levels with stroke, there was only an incremental trend. This may be due to a few reasons. It is possible that estradiol levels are not elevated in the hyperacute phase after stroke and increase later than the 24 hour time period. This can be ascertained in future studies as we did not perform a longitudinal analysis. Another possible explanation may be that the peripheral endogenous levels of estradiol in the serum are not an accurate representation of the sex steroid milieu in the brain after an injury. Moreover, a low patient sample size to study differences in estradiol levels was one of the limitations of this study.
We also found decreased testosterone levels in men after stroke. This has been reported previously in preclinical models of stroke (28) and also in men (29), which may be due to an acute stress response leading to lower testosterone levels. However, it is possible that decreasing androgens is a protective mechanism in men. The use of testosterone therapy has been associated with increased risk of adverse cardiovascular outcomes in some studies (30), while others have shown benefit of testosterone replacement therapy, therefore making the role of testosterone in stroke and cardiovascular disease unclear (31). Our study did not find any correlation of testosterone levels with mRS but again, causality is difficult to establish at one time point after stroke.
This study had some limitations which should be considered. Due to limited number of patients meeting the inclusion and exclusion criteria, our sample size was relatively small, especially for multivariate analysis. Obesity is a known common co-morbidity with cerebrovascular diseases, and adipose tissue is a significant source of aromatase (32). Our database did not record body mass index for most patients and therefore obesity was not accounted for in the multivariate regression model comparing hormone levels. In addition, only total hormone levels were measured by ELISA. It would be interesting to see the distribution between free, total and sex hormone binding globulin bound fractions in future studies. Unlike preclinical studies, it is difficult to obtain pre-stroke serum samples in human stroke biorepositories. Therefore, although the control population does make an adequate comparative group, the pre-stroke differences in hormone levels in this patient population could not be ascertained in this study. It should also be realized that the levels of peripheral endogenous hormones may not simulate the endogenous levels of sex steroids in the injured brain. Finally, this study was performed on serum samples at only 24 hour time point after stroke. Future studies assessing the hormone levels longitudinally would help us in understanding the complex dynamics of sex steroids after stroke.