Plasma Estradiol and Testosterone levels and Ischemic Stroke in Postmenopausal Women

Abstract

Background and Purpose

Although exogenous hormone therapy (HT) use has been associated with increased risk of ischemic stroke in postmenopausal women, it remains unknown whether sex hormone levels contribute to ischemic stroke risk. We aimed to estimate associations between plasma sex hormone levels and ischemic stroke risk, by HT status, in a nested case-control study of postmenopausal women from the Nurses’ Health Study.

Methods

Women with confirmed incident ischemic stroke (n=419) were matched with controls (n=419) by age, HT use, and other factors. Plasma estradiol and testosterone levels were measured using liquid chromatography tandem mass spectrometry; sex hormone-binding globulin (SHBG) was assayed by electrochemiluminescence immunoassay. Associations of total and free estradiol and testosterone, the estradiol/testosterone ratio, and SHBG with ischemic stroke were estimated using conditional logistic regressions stratified by HT status with adjustment for matching and cardiovascular risk factors.

Results

Current HT users had different hormone profiles from never/past users. No clear linear trends were observed between estradiol (total or free) levels or the estradiol/testosterone ratio and ischemic stroke risk among either current users (P_trend>0.1) or never/past users (P_trend>0.6). For both current and never/past users, the associations between some of the sex hormones and ischemic stroke differed by body mass index (BMI) categories (P_interaction≤0.04). For women with a BMI<25kg/m², a higher estradiol/testosterone ratio was associated with significantly elevated ischemic stroke risk among current users (P_trend=0.01), and higher levels of total and free estradiol were significantly associated with higher ischemic stroke risk among never/past users (P_trend≤0.04). Testosterone and SHBG were not associated with ischemic stroke in either current or never/past users.

Conclusions

Our findings do not support a role of sex hormone levels in mediating ischemic stroke risk among postmenopausal women. Replications in additional larger studies are required.

Women have more stroke events than men.¹ In the Women’s Health Initiative and other clinical trials, hormone therapy (HT) was associated with a higher risk of ischemic stroke among postmenopausal women.^2-4 However, it remains unknown whether circulating sex hormone levels contribute to ischemic stroke risk in postmenopausal women, particularly in HT users. Only a few studies have investigated the relationship between sex hormone levels and ischemic stroke among postmenopausal women, and the conclusions remained inconsistent.^5-9

In this nested case-control study, we prospectively evaluated associations between sex hormone levels and ischemic stroke risk in postmenopausal women. Because of the large differences in sex hormone levels by HT use, all analyses were performed separately in current and never/past users of HT.

Methods

Because of the sensitive nature of the data collected for this study, requests to access the dataset from qualified researchers trained in human subject confidentiality protocols may be sent to the Nurses’ Health Study (NHS) at nhsaccess@channing.harvard.edu.

Study Population

Postmenopausal women from the NHS with confirmed incident ischemic stroke between blood collection (1989-1990)¹⁰ and June 2006 were included (n=419), and were matched to controls by age at blood collection, HT status, smoking, race, history of coronary heart disease, self-reported cancer, month of blood collection, and fasting. Participants who used estrogen-alone, estrogen-plus-progestogen, or other HT at blood collection were considered current HT users. Women consented to participate (Supplementary Methods). The study protocol was approved by the Institutional Review Boards of Brigham and Women’s Hospital.

Laboratory assessments

Plasma estradiol and testosterone were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS). Sex hormone-binding globulin (SHBG) was measured by electrochemiluminescence immunoassay (Supplementary Methods). Free estradiol and free testosterone were calculated by the law of mass action.¹¹

Statistical analysis

Conditional logistic regression was used to estimate ischemic stroke risk in relation to increasing quartiles of estradiol, testosterone, the estradiol/testosterone ratio, and SHBG. Multivariable models were adjusted for HT type (current users only), bilateral oophorectomy, body mass index (BMI), physical activity, alcohol intake, current aspirin use, and history of elevated cholesterol, hypertension, and diabetes. Stratified analyses by BMI, age at blood collection, and years since menopause were performed using unconditional logistic regressions with adjustment for matching factors and above covariates. Secondary and sensitivity analyses were also performed (Supplementary Methods).

Results

Women who developed ischemic stroke were more likely to have a history of hypertension and diabetes compared with controls (Table 1). Estradiol and SHBG levels were higher in current HT users than never/past users; no differences were seen by case-control status (Table I).

Table 1.

Baseline characteristics of study participants.

	Current HT users			Never/past users of HT
Mean (SD) or No. (%)	Cases (n=198)	Controls (n=198)	P	Cases (n=221)	Controls (n=221)	P
Age, year†	61.8 (5.1)	61.8 (5.1)	0.86	63.1 (4.4)	63.1 (4.4)	0.51
BMI, kg/m2†	25.2 (4.0)	24.7 (4.1)	0.18	26.2 (5.2)	26.1 (5.3)	0.85
Bilateral oophorectomy	64 (32.3%)	70 (35.4%)	0.52	37 (16.7%)	31 (14.0%)	0.43
History of hypertension	87 (43.9%)	65 (32.8%)	0.02	120 (54.3%)	81 (36.7%)	0.0002
History of diabetes	21 (10.6%)	6 (3.0%)	0.003	30 (13.6%)	19 (8.6%)	0.10
History of elevated cholesterol	93 (47.0%)	90 (45.5%)	0.76	112 (50.7%)	110 (49.8%)	0.85
History of coronary heart disease†	12 (6.1%)	14 (7.1%)	0.68	14 (6.3%)	19 (8.6%)	0.37
Family history of MI prior to age 60	89 (45.0%)	80 (40.4%)	0.36	93 (42.1%)	88 (39.8%)	0.63
Aspirin use >1 tabl/wk	97 (49.0%)	90 (45.5%)	0.48	99 (44.8%)	122 (55.2%)	0.03
Physically activity, MET-hour/wk	15.6 (21.3)	17.5 (20.7)	0.36	15.1 (17.7)	15.3 (16.3)	0.92
Alcohol consumption, g/m2	6.6 (12.1)	5.9 (10.0)	0.51	5.1 (9.4)	4.8 (10.5)	0.75
Smoking status†			0.94			0.93
Never smokers	80 (40.4%)	79 (39.9%)		93 (42.1%)	93 (42.1%)
Past smokers	86 (43.4%)	89 (45.0%)		82 (37.1%)	85 (38.5%)
Current smokers	32 (16.2%)	30 (15.2%)		46 (20.8%)	43 (19.5%)
HT type			0.59
Estrogen alone	108 (54.6%)	114 (57.6%)
Estrogen plus progestin	62 (31.3%)	55 (27.8%)
Other hormones	23 (11.6%)	20 (10.1%)
Unknown	5 (2.5%)	9 (4.6%)

^†Matching factors.

Estradiol, testosterone, the estradiol/testosterone ratio, and SHBG were not associated with ischemic stroke in current or never/past users of HT (multivariable P_trend≥0.4; Figure 1). The results remained unchanged after additionally adjusting for other biomarkers (Table II), excluding women whose HT status changed after blood collection (Table III), or restricting the analysis to current HT users who took the most common estrogen preparation and dose.

Figure 1. Associations of sex hormone and SHBG levels with ischemic stroke.

Numbers of case/control were shown by quartiles of each marker.

We observed significant interactions of BMI and total and free estradiol with ischemic stroke in both current and never/past users of HT (P_interaction≤0.04), and the interaction for the estradiol/testosterone ratio was also significant in current HT users (P_interaction=0.02) (Figure 2). Among current HT users, associations between the estradiol/testosterone ratio and stroke differed significantly by years since menopause (P_interaction=0.01; Table IV). Associations between sex hormone biomarkers and ischemic stroke did not vary by age (P_interaction≥0.13; Table V). All interaction P-values were not significant after Bonferroni correction.

Figure 2. Adjusted associations of sex hormones and SHBG with ischemic stroke by BMI.

Numbers of case/control were shown by quartiles of each marker.

Discussion

In this nested case-control study with a large number of ischemic stroke cases, we did not observe significant associations between estradiol levels and ischemic stroke in current or never/past users of HT. Associations of estradiol levels (both current and never/past users) and the estradiol/testosterone ratio (current users only) with ischemic stroke may vary across BMI categories. No association for testosterone or SHBG with ischemic stroke was observed.

Our findings are consistent with previous studies that did not observed significant associations, but prior studies only focused on postmenopausal women who did not use HT.^6-8 Only one study found that detectable total estradiol (vs. non-detectable by immunoassay) were associated with higher stroke risk among women who had established carotid atherosclerosis.⁵

We utilized the highly sensitive LC-MS/MS technique – the gold standard for assessing testosterone and estradiol levels in postmenopausal women,¹² which ensured the accuracy and reliability of the assessment. Other strengths include the prospective study design and detailed covariate information in the NHS. Additionally, this study had the largest number of ischemic stroke cases in women thus far, but the statistical power was still limited for HT groups. Another limitation is that sex hormones and SHBG were only measured at one timepoint. We did not have information on atrial fibrillation, a known risk factor for ischemic stroke that has been associated with HT, at or prior to the blood collection.¹³ We cannot rule out residual confounding by unmeasured confounders.

Conclusions

Our data do not confirm an association of plasma sex hormone and SHBG levels with ischemic stroke risk in postmenopausal women who are either current or never/past HT users. Future studies with a larger sample size are needed.