Background: Concentrations of estrogen and progesterone within the breast could provide a better reflection of breast cancer risk than levels in the circulation. We developed highly sensitive immunoassays for multiple steroid hormones and proteins in the nipple aspirate fluid (NAF), which can be obtained noninvasively with a simple suction device. Previous studies showed that NAF hormone levels are strongly correlated between breasts and within a single breast over time and are predictably related to hormone replacement therapy or use of oral contraceptives. This study evaluates the relationship of NAF estrogen and progesterone levels to those in serum and saliva, the relationship of NAF estradiol to androgenic and estrogenic precursors in NAF, and the relationship of NAF hormone levels to those of response proteins such as cathepsin D and epidermal growth factor (EGF). Methods: Normal premenopausal women collected saliva daily and donated blood and NAF in the midluteal phases of menstrual cycles at intervals of 0, 4, 12, and 15 months. Analytes were measured by immunoassays after solvent fractionation. Log-transformed values were fit to repeated measures analysis of covariance models to ascertain associations between analytes. Results: Small nonsignificant associations were found between NAF and serum or salivary estradiol. However, progesterone in NAF was significantly associated with progesterone in serum and saliva (R = 0.18 and 0.32, respectively). Within NAF, the estradiol precursors estrone sulfate, androstenedione, and dehydroepiandrosterone were significantly associated with estradiol concentration (P < 0.06), and a multiprecursor model explained the majority of variance in NAF estradiol (model R2 = 0.83). Cathepsin D and EGF in NAF could not be predicted from serum or salivary steroid measurements; however, both could be predicted from estradiol and its precursors in NAF (model R2 = 0.70 and 0.93, respectively). Conclusions: By showing consistent associations between estradiol and its precursors and response proteins, these data provide support for the biological validity of NAF hormone measurements and for the importance of steroid interconversion by aromatase and sulfatase within the breast. The low correlation between estrogen levels in NAF and those in serum or saliva suggests that the degree of association between estrogen or its androgen precursor levels and risk of breast cancer observed in epidemiologic studies using serum estimates might be highly attenuated.
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