Abstract
Context:
We hypothesize that endogenous sex steroids are associated with fracture risk independent of race/ethnicity.
Design and Setting:
We performed a nested case-control study within the prospective Women’s Health Initiative Observational Study. Incident nonspine fractures were identified in 381 black, 192 Hispanic, 112 Asian, and 46 Native American women over an average of 8.6 years. A random sample of 400 white women who experienced an incident fracture was chosen. One control was selected per case and matched on age, race/ethnicity, and blood draw date. Bioavailable estradiol (BioE2), bioavailable testosterone (BioT), and sex hormone–binding globulin (SHBG) were measured using baseline fasting serum. Conditional logistic regression models calculated the odds ratio (OR) and 95% confidence interval (CI) of fracture across tertiles of hormone.
Results:
In multivariable and race/ethnicity-adjusted models, higher BioE2 (>8.25 pg/mL) and higher BioT (>13.3 ng/dL) were associated with decreased risk of fracture (OR, 0.65; 95% CI, 0.50 to 0.85; P trend = 0.001 and OR, 0.76; 95% CI, 0.60 to 0.96; P trend = 0.02, respectively). The interaction term between race/ethnicity and either BioE2 or BioT was not significant. There was no association between SHBG and fracture risk. In models stratifying by race/ethnicity, higher BioE2 was associated with a lower risk of fracture in both white women (OR, 0.56; 95% CI, 0.36 to 0.87) and black women (OR, 0.61; 95% CI, 0.39 to 0.96). Higher BioT was associated with a significantly lower fracture risk in only black women (OR, 0.65; 95% CI, 0.43 to 1.00), P trend = 0.03.
Conclusions:
Serum BioE2 and BioT are associated with fracture risk in older women irrespective of race/ethnicity and independent of established risk factors for fracture.
Lower concentrations of serum bioavailable estradiol and bioavailable testosterone were associated with an increased risk of fracture irrespective of race/ethnicity.
Endogenous sex steroid hormones decline with increasing age (1), and this decline may contribute to important sequelae of aging, including osteoporotic fractures (2). Early case-control studies comparing sex steroids in women with and without a fracture were conflicting (3–6). Prospective studies support an association between higher endogenous total estradiol (E2) or bioavailable estradiol (BioE2) and lower risk of hip and vertebral fractures (7–11). Results of studies of nonvertebral and nonhip fracture are mixed, with some showing an association with E2 (12–14), whereas others do not (10, 15). In contrast, serum testosterone (T) or bioavailable testosterone (BioT) concentrations were not related to fractures in many studies (7, 9, 12, 15). In a previous study from the Women’s Health Initiative Study (WHI), which focused only on hip fracture (95% white women), high BioT (>14 ng/dL) was associated with a 25% lower risk of hip fracture compared with women with BioT (<9 ng/dL) (16). Similarly, the association with sex hormone–binding globulin (SHBG) and fracture has been inconsistent, with some studies showing an inverse association (7, 9, 10, 16, 17) and others showing no association (8, 12, 14, 15, 18).
To our knowledge, these studies have been carried out primarily in US white or European women, and little is known about sex steroids and fracture in women from other race/ethnic groups. The objective of this analysis was to test the hypothesis that sex steroids influence the risk of fractures across all race/ethnicities. We used baseline archived specimens and longitudinal fracture data in white, black, Hispanic, Asian, and Native American women enrolled in the Women’s Health Initiative Observational Study (WHI-OS).
Subjects and Methods
Study population
The study population was the WHI-OS, a prospective cohort study that enrolled 93,676 women aged 50 to 79 years, including ~15% nonwhite women from 1993 to 1998 at 40 US clinical centers. Study methods have been described in detail elsewhere (19). Briefly, women were eligible if they were postmenopausal, unlikely to move or die within 3 years, and not enrolled in the WHI clinical trials. The study was approved by the human subjects review committees at each participating institution. All participants provided written informed consent.
Information on race/ethnicity was obtained by self-report and included whites (not of Hispanic origin), blacks (not of Hispanic origin), Hispanics/Latinos (Mexican, Cuban, Puerto Rican, Central American, or South American), Asians or Pacific Islanders (Chinese, Indo-Chinese, Korean, Japanese, Pacific Islander, or Vietnamese), Native Americans (including Alaskan natives), or other.
Outcome ascertainment
Women were sent questionnaires annually to report any hospitalization and other specific outcomes, including fractures. We collected information on all fractures that occurred after study entry except those of the fingers, toes, face, skull, and sternum. Hip fractures were verified by review of medical records and confirmed by blinded central adjudicators (20). Information on other clinical fractures was obtained by self-report. In the WHI, 76% of self-reported nonhip fractures were confirmed by physician review of medical records, suggesting that the self-report of such fractures is reasonably accurate (21). For this analysis, we also excluded women with hip fractures as a result of cancer and women reporting clinical spine or coccyx fractures because of poor reliability (21).
Nested case-control design
This study is a case-control study nested within the prospective design of the WHI-OS. All self-reported cases of fracture in the WHI-OS as of August 2006 (the last date of original follow-up) were selected as potential cases. We excluded women reporting other or unknown race/ethnicity, current hormone therapy, and use of bisphosphonates or selective estrogen receptor modulators at baseline. We also excluded women with missing important covariates, such as weight, height, history of fracture, and less than two vials of unthawed serum or <0.25 mL of EDTA plasma. A total of 7116 possible cases and 37,713 controls were eligible after these exclusions. We chose all fracture cases among nonwhites, including 381 black, 192 Hispanic, 113 Asian, and 46 Native American women. A random sample of 400 fracture cases was chosen from a total of 6384 fractures among the white participants.
One control was selected for each fracture case from the risk set corresponding to the time of the case’s event. All participants (cases included) were part of the risk set until they had an event or until their last recorded visit (censored if lost to follow-up). Controls were matched to cases by age at screening (±1 year), race/ethnicity (white, black, Hispanic, Asian, or Native American), and blood draw date (±90 days). All matching factors were weighted equally during selection. Controls were chosen to have a fracture-free follow-up time at least as long as the cases’ event times. Seventeen unmatched cases during the first match attempt were matched subsequently by age at screening (±2 years), race/ethnicity, and blood draw date (±365 days).
In summary, there were 1132 cases and 1132 controls for a total of 2264 women. We excluded women with missing or below assay detection values for E2, BioT, or SHBG, leaving a total of 2232 (1116 pairs) participants, including 394 whites, 375 blacks, 190 Hispanics, 111 Asians, and 46 Native Americans. The average follow-up time from enrollment ± standard deviation to the last follow-up date as of August 31, 2006, was 8.60 ± 1.61 years among cases and 8.65 ± 1.61 years among controls.
Laboratory procedures
For each study participant, blood was collected at the baseline visit after at least a 12-hour fast and then stored centrally at −80°C (22). These baseline samples were shipped on dry ice to the Reproductive Endocrine Research Laboratory (University of Southern California, Los Angeles, Los Angeles, CA). Laboratory personnel were blinded to case-control status, and samples were analyzed in random order.
E2 and testosterone concentrations were quantified using radioimmunoassays (RIAs) after organic solvent extraction and celite column partition chromatography (23). For the E2 RIA, the intra-assay coefficient of variation (CV) was 7.9% at 34 pg/mL (124 pmol/L), and the interassay CVs were 8.0% and 12.0% at 16 pg/mL (58.7 pmol/L) and 27 pg/mL (99.1 pmol/L), respectively. For the T RIA, the intraassay CV was 6% at 14.3 ng/dL (0.50 nmol/L) and the interassay CV were 12.0% at 4.9 ng/dL (0.17 nmol/L), 11.0% at 14.3 ng/dL (0.50 nmol/L), and 10.0% at 47.9 ng/dL (1.66 nmol/L). Bioavailable (non-SHBG-bound) and free (non-albumin- and non-SHBG-bound) E2 (or T) concentrations were calculated using the measured total E2 (or total T) and SHBG concentrations, an assumed constant for albumin, and affinity constants of SHBG and albumin for E2 (or T) (24, 25). These calculated values are highly correlated (r ≥ 0.85) with direct measurement of BioE2 and BioT (25, 26). The sensitivities of the E2 and T assays were, respectively, 3 pg/mL (11.0 pmol/L) and 1.5 ng/dL (0.052 pmol/L), and concentrations below these values were deemed undetectable.
SHBG was quantified by a solid-phase, two-site chemiluminescent immunoassay using the Immulite Analyzer (Diagnostic Products Corp., Los Angeles, CA). The solid phase is a polystyrene bead with a monoclonal antibody specific for SHBG. The intraassay CV ranged from 4.1–7.7%, and the interassay CV ranged from 5.8–13%. The assay had a sensitivity of 0.2 nmol/L, and concentrations below these values were deemed undetectable.
Serum 25-hydroxyvitamin D [25(OH)D] was measured by RIA with DiaSorin reagents (Stillwater, MN). The sensitivity of the assay was 3.75 nM. The interassay CVs were 11.7%, 10.5%, 8.6%, and 12.5% at 14.0, 56.8, 82.5, and 1225 nM, respectively.
Other measurements
All covariates were ascertained at baseline and included education, living arrangements, personal and parental history of fracture, reproductive and medical history, alcohol intake, cigarette smoking, physical activity, general health status, diet, and falls. Current use of prescription medications was recorded by clinic interviewers by direct inspection of medicine containers brought into the clinic. Prescription names were entered into the WHI database and assigned drug codes using Medispan software (Wolters-Kluwer Health, Indianapolis, IN). Dietary intakes of calcium and vitamin D were assessed using a semiquantitative food-frequency questionnaire (27). We also collected information on the use of calcium and vitamin D supplements (i.e., amount and frequency of intake) and calculated total calcium intake.
Physical activity was classified on the basis of frequency and duration of walking and mild, moderate, and strenuous activities in the prior week. Kilocalories of energy expended in a week were calculated (metabolic equivalent score = kilocalorie h/wk/kg) (28). Physical function was measured using the 36-Item Short Form Health Survey (29). We compared women with a score >90 versus ≤90; this cutoff corresponded to the median score. Weight was measured on a balance-beam scale, and height was measured using a stadiometer. Body mass index (BMI) was calculated as weight (kg)/height (m2).
Statistical analysis
We used χ2 and t tests to compare baseline characteristics between cases and matched control participants. For continuous measures that were skewed, Wilcoxon rank-sum tests were used. In initial models, we combined all race/ethnic groups and adjusted for race/ethnicity. We also tested for interaction between sex steroid hormone and SHBG and race/ethnicity. Subsequent analyses were carried out within each race/ethnicity. To further assess confounding, we compared baseline characteristics across tertiles of BioE2, BioT, and SHBG in the combined cohort of cases and controls. A test of linear trend was performed by treating tertile as an integer value. The Cochran-Armitage test for trend across tertiles of sex hormones was used for dichotomous variables. The Jonckheere-Terpstra test for trend was used for multicategory variables.
We assessed the association between concentrations of sex steroid hormones and incident fracture in conditional logistic regression models, models appropriate for the matched case-control design. We calculated odds ratio (ORs) and 95% confidence intervals (CIs) from the conditional logistics models across tertiles. Tertiles cutoffs were determined from the distribution among control individuals in the combined multiethnic cohorts, weighted by their relative proportion in the full study. We defined the lowest tertile as the referent group.
We first examined the unadjusted association. For the multivariable (MV)-adjusted model, we adjusted for weight, height, physical activity, total calcium intake, personal history of fracture, health status, and current diabetes treatment. Backward elimination was used, and the following variables were removed from the model: alcohol, education, smoking, past hormone therapy use, glucocorticoid use, and parental history of fracture.
Because E2, testosterone, and SHBG are interrelated, we further adjusted models for the other hormones to test whether the associations were independent. We carried out our initial analyses in the total cohort with the race/ethnicity × sex steroid hormone interaction term. We then carried out models in each race/ethnic group.
Results
The average age of the subjects was 62.6 years among black women and 66.2 years among white women (Table 1). Among white women, cases were more likely to report lower physical function, a personal history of fracture, and diabetes than controls. Among black women, cases were more likely to report a positive personal fracture history than controls. The mean BMI was higher among black women than white women, but there was no difference by case-control status. A higher proportion of black women reported fair to poor health. There was no difference in smoking, alcohol, physical activity, hormone therapy, fall history, and total daily calcium intake by case-control status in either black or white women. 25(OH)D was significantly lower in white cases versus controls, but there was no difference in 25(OH)D by case-control status in women from the other ethnic groups. The characteristics of Hispanic, Asian, and Native American women by fracture case-control status are shown in Supplemental Table 1 (225.7KB, pdf) . Hispanic women with an incident fracture were more likely to report a personal history of fracture than controls. Asian women with an incident fracture were more likely to report fair/poor health status than controls. There were no relevant differences between cases and controls among Native Americans.
Table 1.
Baseline Characteristics of Fracture Cases and Controls in White Women and Black Women
| Characteristics |
White |
Black |
||||
|---|---|---|---|---|---|---|
| Control (n = 394) | Case (n = 394) | P Value | Control (n = 375) | Case (n = 375) | P Value | |
| Age, y | 66.2 ± 7.4 | 66.3 ± 7.3 | 0.96 | 62.6 ± 7.4 | 62.6 ± 7.6 | 0.99 |
| Education, less than high school | 1.5 | 1.3 | 0.77 | 3.8 | 4.6 | 0.71 |
| Smoking status | 0.39 | 0.08 | ||||
| Never | 51.5 | 55.5 | 54.9 | 47.6 | ||
| Past | 42.9 | 38.1 | 33.7 | 41.6 | ||
| Current | 5.6 | 6.4 | 11.4 | 10.9 | ||
| Alcohol consumption | 0.54 | 0.27 | ||||
| Never | 10.9 | 13.3 | 22 | 16.7 | ||
| Former | 18 | 20.4 | 31.6 | 36.2 | ||
| <7 drinks per wk | 58.8 | 55.6 | 42.2 | 42.7 | ||
| >7 drinks per wk | 12.2 | 10.7 | 4 | 4.4 | ||
| Health status | 0.19 | 0.64 | ||||
| Excellent to very good | 64.8 | 58.8 | 36.7 | 33.6 | ||
| Good | 29.6 | 33.5 | 41.3 | 42.3 | ||
| Fair to poor | 5.6 | 7.7 | 22 | 24.1 | ||
| SF-36 physical functioning score >90 | 40.7 | 33.4 | 0.035 | 28.1 | 28.5 | 0.91 |
| Physical activity, median (IQR), METs/wk | 9 (2.9–20.8) | 8.5 (2.5–18.1) | 0.34 | 6.2 (1.5–15.4) | 5 (1.3–15.4) | 0.40 |
| Treated diabetes mellitus | 2.8 | 6.1 | 0.02 | 11.5 | 15.5 | 0.11 |
| Broke bone, ever | 37 | 56 | <0.0001 | 22.3 | 33.4 | 0.0008 |
| Hormone therapy use | 0.57 | 0.57 | ||||
| Past | 25.9 | 27.7 | 19.2 | 17.6 | ||
| Never | 74.1 | 72.3 | 80.8 | 82.4 | ||
| Oral glucocorticoid use, daily | 0.8 | 2 | 0.13 | 1.9 | 3.2 | 0.25 |
| Falls in last 12 mo | 0.52 | 0.20 | ||||
| None | 69 | 65.1 | 70.8 | 64.6 | ||
| 1 | 18 | 20.5 | 16.4 | 20 | ||
| ≥2 | 12.9 | 14.4 | 12.8 | 15.4 | ||
| Height, cm | 161.3 ± 6.8 | 161.3 ± 6.4 | 0.98 | 162.1 ± 7.1 | 162.7 ± 6.8 | 0.28 |
| Weight, kg | 70.9 ± 15.5 | 71.9 ± 14.8 | 0.36 | 80.4 ± 18.3 | 81 ± 17.7 | 0.59 |
| BMI, kg/m2 | 27.2 ± 5.7 | 27.6 ± 5.5 | 0.34 | 30.5 ± 6.8 | 30.5 ± 6.4 | 0.97 |
| Total calcium intake, median (IQR), mg/d | 1125.5 (703.5–1557.4) | 1037.5 (700–1577.6) | 0.42 | 598.4 (366.8–981.5) | 649.9 (379.8–1083.7) | 0.24 |
| 25(OH)D level, median (IQR), ng/mL | 24.5 (18.4–30.7) | 22.8 (17.5–29.2) | 0.03 | 15.8 (11.2–21.5) | 16 (10.9–22.8) | 0.49 |
| BioE2, median (IQR), pg/mL | 6.4 (4–9.5) | 5.8 (3.8–9) | 0.14 | 8.1 (5.3–12.2) | 7.4 (4.6–12.1) | 0.15 |
| BioT, median (IQR), ng/dL | 10.3 (7.1–14.5) | 9.7 (7–13.8) | 0.23 | 12.2 (8.7–17.4) | 11.5 (8–16.6) | 0.09 |
| SHBG, median (IQR), nmol/mL | 48.2 (35.5–69.6) | 45.5 (31.8–64.8) | 0.08 | 38.1 (29.4–55) | 43.8 (28.5–61.3) | 0.027 |
Values are mean ± SD, percentages, or as otherwise indicated.
Abbreviations: IQR, interquartile range; MET, metabolic equivalent; SF-36, 36-Item Short Form Health Survey.
Characteristics of women across tertiles of BioE2
Among white women, higher BioE2 was associated with younger age. The percentage of those with fair or poor health, poor physical function, and diabetes increased with increasing BioE2. The median physical activity was lower in women with the highest BioE2. The mean body weight and BMI was greater with increasing BioE2. Use of daily oral corticosteroids was uncommon and did not differ significantly by BioE2. Median BioT increased and median SHBG and 25(OH)D decreased with increasing BioE2. These associations likely reflected the positive association between body weight and BioE2.
Among black women, the patterns across BioE2 were similar to white women for age, health status, physical functioning, physical activity, prevalence of diabetes, body weight, BMI, and 25(OH)D (Table 2). The percentage differences in median BioT and SHBG across tertiles of BioE2 were greater in black versus white women.
Table 2.
Baseline Characteristics Across BioE2 Tertiles in White Women and Black Women
| Characteristics |
White |
Black |
||||||
|---|---|---|---|---|---|---|---|---|
| ≤4.89 pg/mL (n = 303) | >4.89–8.25 pg/mL (n = 254) | >8.25 pg/mL (n = 231) | P Trend | ≤4.89 pg/mL (n = 186) | >4.89–8.25 pg/mL (n = 215) | >8.25 pg/mL (n = 349) | P Trend | |
| Age, y | 67 ± 7.2 | 66.7 ± 7.1 | 64.9 ± 7.7 | 0.0011 | 64 ± 7.8 | 63.3 ± 7.8 | 61.4 ± 7.2 | 0.0001 |
| Education, less than high school | 2 | 0.8 | 1.3 | 0.45 | 4.9 | 2.8 | 4.6 | 0.95 |
| Smoking status | 0.26 | 0.74 | ||||||
| Never | 56.7 | 51.6 | 51.5 | 51.4 | 54 | 49.4 | ||
| Past | 35.7 | 41.3 | 45.9 | 37 | 33.2 | 40.7 | ||
| Current | 7.7 | 7 | 2.6 | 11.6 | 12.8 | 9.9 | ||
| Alcohol | 0.67 | 0.75 | ||||||
| Never | 14.3 | 12.2 | 9.1 | 19.9 | 20.3 | 18.6 | ||
| Former | 17.9 | 17.7 | 22.6 | 34.8 | 26.9 | 37.8 | ||
| <7 drinks per wk | 55.8 | 59.5 | 56.5 | 41.4 | 49.5 | 38.6 | ||
| >7 drinks per wk | 12 | 10.6 | 11.7 | 3.9 | 3.3 | 5 | ||
| Health status | 0.053 | 0.003 | ||||||
| Excellent to very good | 65.1 | 63.1 | 56.1 | 38.6 | 44.6 | 27.5 | ||
| Good | 28.6 | 30.2 | 37 | 36.9 | 39.9 | 45.5 | ||
| Fair to poor | 6.3 | 6.8 | 7 | 24.6 | 15.5 | 27 | ||
| SF-36 physical functioning score >90 | 46.8 | 33.5 | 28.4 | <0.0001 | 35 | 32.9 | 22 | 0.001 |
| Physical activity, median (IQR), METs/wk | 10.5 (3.5–21.1) | 8.8 (3.3–18.8) | 7.5 (1.5–17.5) | 0.016 | 6.4 (1.5–14.7) | 9 (3–19.8) | 3.8 (0.8–12.3) | 0.001 |
| Treated diabetes mellitus | 2 | 3.5 | 8.7 | 0.0003 | 8.7 | 8.4 | 19.2 | 0.001 |
| Broke bone, ever | 48.8 | 43.8 | 46.5 | 0.53 | 30.9 | 25.4 | 27.7 | 0.54 |
| Hormone therapy use | 0.94 | 0.82 | ||||||
| Past | 27.7 | 24.8 | 27.7 | 19.9 | 16.7 | 18.6 | ||
| Never | 72.3 | 75.2 | 72.3 | 80.1 | 83.3 | 81.4 | ||
| Oral glucocorticoid use, daily | 2.6 | 0.4 | 0.9 | 0.06 | 3.8 | 2.3 | 2 | 0.24 |
| Falls, last 12 mo | 0.36 | 0.79 | ||||||
| None | 68 | 69.3 | 63.3 | 67.2 | 69.1 | 67.1 | ||
| 1 | 18.3 | 17.9 | 22.1 | 19.4 | 17.4 | 18.1 | ||
| ≥2 | 13.7 | 12.8 | 14.6 | 13.3 | 13.5 | 14.9 | ||
| Height, cm | 161.2 ± 6.5 | 161.2 ± 6.8 | 161.7 ± 6.4 | 0.37 | 163 ± 6.9 | 161.7 ± 7.3 | 162.5 ± 6.7 | 0.58 |
| Weight, kg | 64.8 ± 11.5 | 71.6 ± 13.7 | 79.9 ± 16.6 | <0.0001 | 71.3 ± 13.8 | 76.8 ± 14.2 | 88.2 ± 18.9 | <0.0001 |
| BMI, kg/m2 | 24.9 ± 4.1 | 27.6 ± 5.1 | 30.6 ± 6.2 | <0.0001 | 26.9 ± 5.1 | 29.3 ± 5.4 | 33.3 ± 6.8 | <0.0001 |
| Total calcium intake, median (IQR), mg/d | 1089.8 (684.8–1577.8) | 1063 (703.4–1589.3) | 1064.5 (653–1546.4) | 0.62 | 757.4 (413.6–1091.1) | 566.4 (357.8–977.5) | 602.6 (370.8–1007.3) | 0.12 |
| 25(OH)D level, median (IQR), ng/mL | 25.1 (18.6–32.3) | 23.3 (18.4–28.5) | 22.0 (17.0–28.0) | 0.0004 | 17.3 (12.7–23.3) | 16.4 (11.6–23.0) | 14.5 (9.9–21.5) | 0.0003 |
| BioT, median (IQR), ng/dL | 7.8 (5.6–10.2) | 11.4 (8.7–15.3) | 12.8 (9.1–18.2) | <0.0001 | 8.1 (5.6–11.6) | 10.7 (8.4–14.5) | 14.8 (11.1–19.9) | <0.0001 |
| SHBG, median (IQR), nmol/mL | 57.8 (43.9–76) | 42.5 (32.5–56.1) | 37.6 (25.2–54.2) | <0.0001 | 58.6 (45.7–76.3) | 44.7 (33.1–60.2) | 32.3 (24.2–43.4) | <0.0001 |
Values are mean ± SD, percentages, or as otherwise indicated.
Abbreviations: IQR, interquartile range; MET, metabolic equivalent; SF-36, 36-Item Short Form Health Survey.
We observed similar patterns in Hispanic women among those with highest BioE2, with a higher percentage of women reporting fair to poor health status and diabetes (Supplemental Table 2 (225.7KB, pdf) ). Mean BioE2 increased with increasing weight and BMI. There was no association between BioE2 and 25(OH)D among Hispanic women. The percentage difference in median BioT and SHBG across tertiles of BioE2 was similar to white women.
Asian women with higher BioE2 were younger, better educated, less likely to drink ≥7 drinks per week, more likely to have diabetes, and had a higher body weight and BMI (Supplemental Table 2 (225.7KB, pdf) ). The mean BioT was 56% higher and mean SHBG was 57% lower in Asian women with the highest BioE2 compared with the lowest.
Among Native American women, the patterns across BioE2 were generally similar to women in the other race/ethnic groups, but given the smaller number of Native American women, they did not consistently reach statistically significance (Supplemental Table 2 (225.7KB, pdf) ).
Characteristics of women across tertiles of BioT are shown in Supplemental Table 3 (225.7KB, pdf) and across tertiles of SHBG in Supplemental Table 4 (225.7KB, pdf) . In whites, blacks, and Asians, 25(OH)D decreased with increasing BioT. The most consistent finding across all ethnic groups was higher BMI and body weight with increasing BioT, with the opposite trends with increasing SHBG. 25(OH)D increased with increasing SHBG in whites, blacks, Asians, and Native Americans.
BioE2 and fracture risk
The median BioE2 tended to be lower among fracture cases but did not differ significantly by case-control status in any race/ethnic groups (Table 1; Supplemental Table 1 (225.7KB, pdf) ). The association between BioE2 and fracture is shown in Table 3.
Table 3.
OR (95% CI) of Fracture Across Tertiles of BioE2
| Race/Ethnic Group |
BioE2 Tertiles |
|||
|---|---|---|---|---|
| ≤4.89 pg/mL | >4.89–8.25 pg/mL | >8.25 pg/mL | P Trend | |
| MV adjusteda | Ref | 0.80 (0.63–1.02) | 0.65 (0.50–0.85) | 0.001 |
| MV adjusteda plus 25(OH)D | Ref | 0.79 (0.63–1.014) | 0.65 (0.50–0.84) | 0.001 |
| MV adjusteda plus BioT | Ref | 0.82 (0.64–1.05) | 0.68 (0.52–0.90) | 0.007 |
| MV adjusteda plus SHBG | Ref | 0.79 (0.62–1.01) | 0.64 (0.49–0.84) | 0.001 |
| White | (n = 303) | (n = 254) | (n = 231) | |
| Unadjusted | Ref | 0.72 (0.50–1.02) | 0.71 (0.49–1.03) | 0.06 |
| MV adjustedb | Ref | 0.72 (0.48–1.06) | 0.56 (0.36–0.87) | 0.01 |
| MV adjustedb plus 25(OH)D | Ref | 0.71 (0.48–1.05) | 0.55 (0.35–0.86) | 0.009 |
| MV adjustedb plus BioT | Ref | 0.72 (0.48–1.08) | 0.56 (0.35–0.89) | 0.015 |
| MV adjustedb plus SHBG | Ref | 0.68 (0.45–1.01) | 0.54 (0.34–0.85) | 0.007 |
| Black | (n = 186) | (n = 215) | (n = 349) | |
| Unadjusted | Ref | 0.68 (0.44–1.03) | 0.67 (0.45–0.99) | 0.06 |
| MV adjustedb | Ref | 0.74 (0.47–1.16) | 0.61 (0.39–0.96) | 0.04 |
| MV adjustedb plus 25(OH)D | Ref | 0.74 (0.47–1.16) | 0.61 (0.38–0.96) | 0.03 |
| MV adjustedb plus BioT | Ref | 0.76 (0.48–1.20) | 0.67 (0.41–1.08) | 0.11 |
| MV adjustedb plus SHBG | Ref | 0.80 (0.50–1.28) | 0.69 (0.43–1.12) | 0.13 |
| Hispanic | (n = 126) | (n = 120) | (n = 134) | |
| Unadjusted | Ref | 1.07 (0.63–1.81) | 1.15 (0.69–1.91) | 0.60 |
| MV adjustedb | Ref | 1.18 (0.62–2.25) | 1.04 (0.52–2.09) | 0.90 |
| MV adjustedb plus 25(OH)D | Ref | 1.22 (0.64–2.36) | 1.01(0.50–2.04) | 0.95 |
| MV adjustedb plus BioT | Ref | 1.07 (0.55–2.09) | 0.89 (0.42–1.88) | 0.76 |
| MV adjustedb plus SHBG | Ref | 1.17 (0.60–2.30) | 1.04 (0.51–2.10) | 0.92 |
| Asian/Pacific Islander | (n = 101) | (n = 69) | (n = 52) | |
| Unadjusted | Ref | 0.87 (0.47–1.61) | 1.15 (0.56–2.35) | 0.78 |
| MV adjustedb | Ref | 0.69 (0.34–1.38) | 1.03 (0.45–2.37) | 0.92 |
| MV adjustedb plus 25(OH)D | Ref | 0.71 (0.35–1.43) | 1.06 (0.46–2.46) | 0.99 |
| MV adjustedb plus BioT | Ref | 0.77 (0.37–1.60) | 1.16 (0.49–2.77) | 0.80 |
| MV adjustedb plus SHBG | Ref | 0.65 (0.32–1.33) | 1.01 (0.44–2.34) | 0.90 |
| Native American | (n = 26) | (n = 26) | (n = 40) | |
| Unadjusted | Ref | 1.37 (0.41–4.58) | 0.56 (0.19–1.65) | 0.30 |
| MV adjustedb | Ref | 0.74 (0.15–3.65) | 0.44 (0.10–2.00) | 0.29 |
| MV adjustedb plus 25(OH)D | Ref | 0.62 (0.12–3.19) | 0.28 (0.05–1.56) | 0.15 |
| MV adjustedb plus BioT | Ref | 1.21 (0.21–7.01) | 0.68 (0.13–3.45) | 0.63 |
| MV adjustedb plus SHBG | Ref | 0.69 (0.14–3.49) | 0.41 (0.09–1.87) | 0.25 |
Values are OR (95% CI) or as otherwise indicated. Matching factors included age, race/ethnicity, and blood draw date.
Abbreviation: Ref, reference.
MV model adjusted for weight, height, physical activity, total calcium intake, personal history of fracture, health status, diabetes treatment, and race/ethnicity.
MV model adjusted for weight, height, physical activity, total calcium intake, personal history of fracture, health status, and diabetes treatment.
In the total population, adjusting models for race/ethnicity × BioE2 interaction, there was a significant trend whereby increasing BioE2 was associated with a decreased risk of fracture (P trend = 0.001) (Table 3). Women with BioE2 in the second tertile (>4.89 to 8.25 pg/mL) had a 20% (OR, 0.80; 95% CI, 0.63 to 1.02) and women in the third tertile (>8.25 pg/mL) a 35% (OR, 0.65; 95% CI, 0.50 to 0.85) lower risk of fracture compared with women with the lowest BioE2. There was no significant interaction between race/ethnicity and BioE2 in either the unadjusted model (P = 0.19) or MV model (P = 0.23). Further adjustment for BioT and SHBG had no effect on our results.
White women with the highest BioE2 (>8.25 pg/mL) had approximately a 45% lower risk of fracture (P trend = 0.01) compared with white women with lowest BioE2 (≤4.89 pg/mL). This association was independent of many confounding factors, including body weight. Among black women, a similar pattern was observed whereby black women with the highest BioE2 had a MV-adjusted 39% lower risk of fracture than women with the lowest BioE2 (P trend = 0.04). Additional adjustment for 25(OH)D had no effect on our results. These trends in black women were slightly attenuated after adjusting for BioT and SHBG. There was no association between BioE2 and fracture risk in Hispanics, Asians, or Native Americans (Table 3).
BioT and fracture risk
Median BioT was similar in white women cases and controls (Table 1). In models adjusting for race/ethnicity × BioE2 interaction, women in the top tertile of BioT (>13.3 ng/dL) had approximately a 25% lower risk of fracture (P trend = 0.02) that was unaffected by adjusting for BioE2 or SHBG. There was no race/ethnicity × BioT interaction (unadjusted model, P = 0.94; MV-adjusted model, P = 0.90). In race/ethnic-specific models, white women with BioT (>13.3 ng/dL) had approximately a 20% lower risk of fracture, but it was not statistically significant (Table 4). Median BioT was lower among black women cases compared with controls, but it was not significant (P = 0.09). Results showed a significant dose response between increasing BioT and reduced fracture risk in black women (Table 4). In MV-adjusted models, black women with BioT >13.3 ng/dL had a 35% lower risk of fracture compared with women with BioT ≤8.33 ng/dL (P trend = 0.03). There was no association between BioT and fracture risk in Hispanics or Asians. Among Native Americans, there was a suggestion of an association between BioT and lower fracture rates, especially among women with BioT >13.3 ng/dL, but CIs were wide and trends were not significant. Additional adjustment for 25(OH)D had no effect on our results.
Table 4.
OR (95% CI) of Fracture Across Tertiles of BioT
| Race/Ethnic Group |
BioT Tertiles |
|||
|---|---|---|---|---|
| ≤8.33 ng/dL | >8.33–13.3 ng/dL | >13.3 ng/dL | P Trend | |
| MV adjusteda | Ref | 1.04 (0.84–1.30) | 0.76 (0.60–0.96) | 0.02 |
| MV adjusteda plus 25(OH)D | Ref | 1.05 (0.84–1.31) | 0.76 (0.61–0.96) | 0.02 |
| MV adjusteda plus BioE2 | Ref | 1.04 (0.84–1.30) | 0.76 (0.60–0.96) | 0.02 |
| MV adjusteda plus SHBG | Ref | 1.02 (0.81–1.28) | 0.74 (0.58–0.95) | 0.01 |
| White | (n = 286) | (n = 271) | (n = 231) | |
| Unadjusted | Ref | 1.00 (0.72–1.38) | 0.78 (0.55–1.11) | 0.20 |
| MV adjustedb | Ref | 1.08 (0.75–1.54) | 0.80 (0.54–1.18) | 0.30 |
| MV adjustedb plus 25(OH)D | Ref | 1.06 (0.74–1.52) | 0.79 (0.53–1.16) | 0.26 |
| MV adjustedb plus BioE2 | Ref | 1.07 (0.75–1.54) | 0.82 (0.55–1.21) | 0.35 |
| MV adjustedb plus SHBG | Ref | 0.97 (0.67–1.41) | 0.69 (0.46–1.04) | 0.09 |
| Black | (n = 185) | (n = 255) | (n = 310) | |
| Unadjusted | Ref | 0.99 (0.67–1.46) | 0.72 (0.49–1.06) | 0.06 |
| MV adjustedb | Ref | 0.95 (0.62–1.45) | 0.65 (0.43–1.00) | 0.03 |
| MV adjustedb plus 25(OH)D | Ref | 0.93 (0.61–1.43) | 0.64 (0.42–0.98) | 0.02 |
| MV adjustedb plus BioE2 | Ref | 0.92 (0.60–1.41) | 0.61 (0.39–0.94) | 0.02 |
| MV adjustedb plus SHBG | Ref | 1.11 (0.70–1.75) | 0.75 (0.48–1.18) | 0.97 |
| Hispanic | (n = 119) | (n = 141) | (n = 120) | |
| Unadjusted | Ref | 1.27 (0.79–2.06) | 1.08 (0.66–1.77) | 0.75 |
| MV adjustedb | Ref | 1.22 (0.68–2.17) | 1.21 (0.66–2.19) | 0.53 |
| MV adjustedb plus 25(OH)D | Ref | 1.24 (0.70–2.22) | 1.22 (0.67–2.23) | 0.51 |
| MV adjustedb plus BioE2 | Ref | 1.24 (0.69–2.22) | 1.15 (0.63–2.11) | 0.62 |
| MV adjustedb plus SHBG | Ref | 1.22 (0.68–2.19) | 1.20 (0.64–2.25) | 0.56 |
| Asian/Pacific Islander | (n = 82) | (n = 87) | (n = 53) | |
| Unadjusted | Ref | 0.97 (0.55–1.72) | 0.84 (0.41–1.72) | 0.65 |
| MV adjustedb | Ref | 0.84 (0.44–1.61) | 0.61 (0.26–1.44) | 0.27 |
| MV adjustedb plus 25(OH)D | Ref | 0.86 (0.45–1.64) | 0.63 (0.26–1.51) | 0.32 |
| MV adjustedb plus BioE2 | Ref | 0.83 (0.44–1.60) | 0.62 (0.26–1.46) | 0.28 |
| MV adjustedb plus SHBG | Ref | 0.82 (0.42–1.58) | 0.58 (0.24–1.39) | 0.23 |
| Native American | (n = 28) | (n = 35) | (n = 29) | |
| Unadjusted | Ref | 0.45 (0.14–1.49) | 0.34 (0.11–1.05) | 0.07 |
| MV adjustedb | Ref | 0.70 (0.19–2.76) | 0.37 (0.10–1.41) | 0.13 |
| MV adjustedb plus 25(OH)D | Ref | 0.76 (0.18–3.17) | 0.33 (0.08–1.42) | 0.12 |
| MV adjustedb plus BioE2 | Ref | 0.67 (0.17–2.73) | 0.38 (0.10–1.48) | 0.15 |
| MV adjustedb plus SHBG | Ref | 0.37 (0.07–1.95) | 0.17 (0.03–1.07) | 0.06 |
Values are OR (95% CI) or as otherwise indicated. Matching factors included age, race/ethnicity, and blood draw date.
Abbreviation: Ref, reference.
MV model adjusted for weight, height, physical activity, total calcium intake, personal history of fracture, health status, diabetes treatment, and race/ethnicity.
MV model adjusted for weight, height, physical activity, total calcium intake, personal history of fracture, health status, and diabetes treatment.
SHBG and fracture risk
In the total population adjusting for race/ethnicity, there was no association between SHBG and fracture. The interaction between race/ethnicity and SHBG was also not statistically significant.
In race/ethnic-specific models, white women with the highest SHBG (>58 nmol/mL) had a 31% lower risk of fracture compared with women with SHBG (≤37.6 nmol/mL, P trend = 0.06) (Table 5). In contrast, black women with the highest SHBG (>58 nmol/mL) had a 70% increased risk of fracture (P trend = 0.007). Additional adjustment for 25(OH)D had no effect on our results. There was no association between SHBG and fracture risk in women from the other race/ethnic groups.
Table 5.
OR (95% CI) of Fracture Across Tertiles of SHBG Levels
| Race/Ethnic Group |
SHBG Tertiles |
|||
|---|---|---|---|---|
| ≤37.6 nmol/mL | >37.6–58 nmol/mL | >58 nmol/mL | P Trend | |
| MV adjusteda | Ref | 1.20 (0.96–1.49) | 1.01 (0.80–1.28) | 0.83 |
| MV adjusteda plus 25(OH)D | Ref | 1.20 (0.96–1.50) | 1.02 (0.8–1.29) | 0.80 |
| MV adjusteda plus BioE2 | Ref | 1.20 (0.96–1.49) | 1.01 (0.80–1.28) | 0.83 |
| MV adjusteda plus BioT | Ref | 1.16 (0.93–1.45) | 0.96 (0.76–1.23) | 0.84 |
| White | (n = 260) | (n = 268) | (n = 260) | |
| Unadjusted | Ref | 0.85 (0.61–1.19) | 0.76 (0.54–1.06) | 0.10 |
| MV adjustedb | Ref | 0.85 (0.58–1.23) | 0.69 (0.46–1.02) | 0.06 |
| MV adjustedb plus 25(OH)D | Ref | 0.86 (0.59–1.26) | 0.71 (0.48–1.06) | 0.08 |
| MV adjustedb plus BioE2 | Ref | 0.83 (0.57–1.20) | 0.69 (0.46–1.02) | 0.06 |
| MV adjustedb plus BioT | Ref | 0.81 (0.55–1.18) | 0.64 (0.42–0.96) | 0.03 |
| Black | (n = 328) | (n = 237) | (n = 185) | |
| Unadjusted | Ref | 1.59 (1.12–2.24) | 1.66 (1.15–2.39) | 0.005 |
| MV adjustedb | Ref | 1.62 (1.12–2.35) | 1.70 (1.13–2.58) | 0.007 |
| MV adjustedb plus 25(OH)D | Ref | 1.62 (1.12–2.35) | 1.68 (1.11–2.55) | 0.008 |
| MV adjustedb plus BioE2 | Ref | 1.64 (1.13–2.39) | 1.80 (1.18–2.74) | 0.004 |
| MV adjustedb plus BioT | Ref | 1.59 (1.09–2.31) | 1.63 (1.07–2.48) | 0.01 |
| Hispanic | (n = 161) | (n = 124) | (n = 95) | |
| Unadjusted | Ref | 0.67 (0.41–1.07) | 0.73 (0.43–1.2) | 0.16 |
| MV adjustedb | Ref | 0.87 (0.49–1.54) | 0.84 (0.45–1.60) | 0.56 |
| MV adjustedb plus 25(OH)D | Ref | 0.89 (0.49–1.59) | 0.83 (0.44–1.58) | 0.56 |
| MV adjustedb plus BioE2 | Ref | 0.87 (0.49–1.56) | 0.83 (0.44–1.58) | 0.55 |
| MV adjustedb plus BioT | Ref | 0.92 (0.51–1.65) | 0.93 (0.48–1.82) | 0.80 |
| Asian/Pacific Islander | (n = 83) | (n = 75) | (n = 64) | |
| Unadjusted | Ref | 1.92 (0.89–4.18) | 0.93 (0.44–1.96) | 0.78 |
| MV adjustedb | Ref | 2.45 (0.99–6.02) | 0.86 (0.37–2.03) | 0.69 |
| MV adjustedb plus 25(OH)D | Ref | 2.43 (0.98–6.01) | 0.84 (0.36–1.99) | 0.66 |
| MV adjustedb plus BioE2 | Ref | 2.39 (0.97–5.91) | 0.82 (0.34–1.97) | 0.63 |
| MV adjustedb plus BioT | Ref | 2.43 (0.97–6.09) | 0.81 (0.34–1.93) | 0.58 |
| Native American | (n = 43) | (n = 28) | (n = 21) | |
| Unadjusted | Ref | 0.85 (0.31–2.34) | 0.65 (0.23–1.88) | 0.43 |
| MV adjustedb | Ref | 1.16 (0.32–4.28) | 0.47 (0.11–2.00) | 0.35 |
| MV adjustedb plus 25(OH)D | Ref | 1.29 (0.34–4.88) | 0.49 (0.11–2.15) | 0.39 |
| MV adjustedb plus BioE2 | Ref | 1.46 (0.35–6.05) | 0.45 (0.10–2.06) | 0.36 |
| MV adjustedb plus BioT | Ref | 0.72 (0.16–3.22) | 0.18 (0.03–1.19) | 0.86 |
Values are OR (95% CI) or as otherwise indicated. Matching factors included age, race/ethnicity, and blood draw date.
Abbreviation: Ref, reference.
MV model adjusted for weight, height, physical activity, total calcium intake, personal history of fracture, health status, diabetes treatment, and race/ethnicity.
MV model adjusted for weight, height, physical activity, total calcium intake, personal history of fracture, health status, and diabetes treatment.
Discussion
In our prospective nested case-control study, women with the highest BioE2 (>13.3 pg/mL) had a 35% lower risk of nonspine fracture compared with women with lowest BioE2 (≤4.89 pg/mL), independent of race/ethnicity and many other factors associated with fracture risk, including body weight. These findings extend previous findings in primarily US white women and European women to women of all race/ethnicities. Similar results were observed for BioT where women with the highest BioT (>13.3 ng/dL) had a 24% lower risk of nonspine fracture compared with women with the lowest BioT (≤8.33 ng/dL), irrespective of race/ethnicity. Both the associations of BioE2 and BioT with fracture were independent of each other. The association between BioT and fracture in the literature is less consistent than BioE2, but our results are in accord with a previous WHI study of hip fracture in which 95% of cases and controls were white (16).
One of our objectives was to examine the association between sex steroid hormones and fracture in individual race/ethnic groups. We found that both white and black women with the highest BioE2 had a lower risk of fracture. The overall trend between BioE2 and lower fracture risk was significant in both groups, with no evidence of a threshold. The association of BioT with fracture was not significant in white women. In contract, black women with the highest BioT had a 35% lower risk of having an incident fracture compared with black women with the lowest BioT. BioT levels tended to be higher in black women compared with white women: 45% of black women were in the highest BioT tertile compared with 25% of white women. Thus, it is possible that we did not observe this association between BioT and fracture in whites because fewer of them had a high BioT level. The high BioT in black women may reflect their higher BMI compared with white women.
Overall, SHBG was unrelated to risk of nonspine fracture. These results conflict with a previous WHI study of hip fracture (16) where higher SHBG was associated with an increased risk of subsequent hip fracture. The difference with our results may reflect the heterogeneity of fracture types in our study. We had few hip fractures, especially in nonwhites. Examination of the association between SHBG and fracture in individual race/ethnic groups showed the expected positive relationship between SHBG and fractures in black women. However, in whites, we found the opposite: Higher SHBG was associated with a lower risk of fracture. This finding may be spurious because it does not reflect the underlying biology of SHBG and free hormones. For example, among both black and white women, the median BioE2 and BioT decreased with increasing SHBG. Thus, these results in white women should be interpreted with caution.
A major clinical question is whether measurement of BioE2 and BioT could be used to identify individual women at high risk of fracture. We used a state-of-the-art reproductive endocrine research laboratory. On the other hand, E2 and testosterone measured in the clinical setting is problematic. As noted in a recent position statement of the council of the Endocrine Society (30), routine measurements of E2 are difficult for a number of reasons, including their low concentrations in postmenopausal women, cross-reactivity with other conjugated and unconjugated metabolites, precision and reliability issues, and poor accuracy across multiple laboratories. Thus, although E2 and testosterone are clearly in the biologic pathway to fractures, clinical use of these laboratory tests is limited until the laboratory issues can be resolved.
Lower 25(OH)D levels have been linked to an increased risk of fracture (31, 32), and 25(OH)D levels were lower among the white cases versus controls. However, 25(OH)D did not differ between women who fractured versus controls in the other race/ethnic groups. Additional adjustment for 25(OH)D had no effect on our results.
There are a number of strengths to our study. This study examined the association between fracture and sex steroid hormones in multiethnic groups of women. All assays were carried out blindly to case-control status at a state-of-the-art reproductive hormone laboratory, and we adjusted for many confounding factors in our analyses.
There are, however, several limitations to our study. We had limited power to detect an association between sex steroids and fracture in Hispanic, Asian, and Native American women. Bone mineral density was not broadly available, and we were unable to test whether associations between BioE2 and BioT and fracture were mediated by bone mineral density. Finally, hormone levels were measured at a single time point, and fractures occurred up to 8 years after the baseline measures.
In conclusion, higher circulating levels of BioE2 and BioT were associated with a decreased risk of nonspine clinical fractures. Our results were independent of race/ethnicity, extending previous finding in primarily US white and European women to include a diverse group of postmenopausal women.
Acknowledgments
We thank members of the program office (National Heart, Lung, and Blood Institute, Bethesda, Maryland): Jacques Rossouw, Shari Ludlam, Joan McGowan, Leslie Ford, and Nancy Geller. We also thank members of the clinical coordinating center (Fred Hutchinson Cancer Research Center, Seattle, Washington): Garnet Anderson, Ross Prentice, Andrea LaCroix, and Charles Kooperberg. We thank the following investigators and academic centers: (Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts) JoAnn E. Manson, (MedStar Health Research Institute/Howard University, Washington, DC) Barbara V. Howard, (Stanford Prevention Research Center, Stanford, California) Marcia L. Stefanick, (The Ohio State University, Columbus, Ohio) Rebecca Jackson, (University of Arizona, Tucson/Phoenix, Arizona) Cynthia A. Thomson, (University at Buffalo, Buffalo, New York) Jean Wactawski-Wende, (University of Florida, Gainesville/Jacksonville, Florida) Marian Limacher, (University of Iowa, Iowa City/Davenport, Iowa) Jennifer Robinson, (University of Pittsburgh, Pittsburgh, Pennsylvania) Lewis Kuller, (Wake Forest University School of Medicine, Winston-Salem, North Carolina) Sally Shumaker, (University of Nevada, Reno, Nevada) Robert Brunner, and (University of Minnesota, Minneapolis, Minnesota) Karen L. Margolis.
Acknowledgments
The Women’s Health Initiative Study is funded by the National Heart, Lung, and Blood Institute, National Institutes of Health, US Department of Health and Human Services through Contracts HHSN268201100046C, HHSN268201100001C, HHSN268201100002C, HHSN268201100003C, HHSN268201100004C, and HHSN271201100004C.
Disclosure Summary: The authors have nothing to disclose.
Footnotes
- 25(OH)D
- serum 25-hydroxyvitamin D
- BioE2
- bioavailable estradiol
- BioT
- bioavailable testosterone
- BMI
- body mass index
- CI
- confidence intervals
- CV
- coefficient of variation
- E2
- estradiol
- MV
- multivariable
- OR
- odds ratio
- RIA
- radioimmunoassay
- SHBG
- sex hormone–binding globulin
- T
- testosterone
- WHI
- Women’s Health Initiative Study
- WHI-OS
- Women’s Health Initiative Observational Study.
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