The results suggest that circulating concentrations of sex hormones are associated with several of the well-established or suspected risk factors for breast cancer. The strongest associations were with BMI: concentrations of all the hormones were higher in obese women than in women with a low BMI, with the largest differences for calculated free oestradiol and free testosterone. Ovariectomy was associated with low levels of androgens, and cigarette smoking and alcohol were associated with moderate increases in all the sex hormones. Age at menarche, parity, age at first full-term pregnancy and family history of breast cancer were not strongly related to any of the hormones examined.
The large sample size provides sufficient power to detect differences in circulating hormone concentrations between categories as small as 10% for most of the analyses presented. Some of the observations replicate well-established associations, whereas others provide novel information of possible relevance to understanding the aetiology of breast cancer, as discussed in more detail below. Many previous publications on this topic are from the studies participating in this collaboration, and these publications are cited together with publications from studies not included in this collaboration. Limitations of this analysis are that the data are cross-sectional, and therefore cannot be used to attribute causality, and that some other potentially important factors such as physical activity and diet were not included in the analyses. Another potential limitation is that, with the exception of the RERF Study, the majority of the women in the contributing studies were of white European ethnic origin.
For some of the analyses, there was significant heterogeneity in the results from different studies. Inspection of the results from individual studies did not show large between-study differences in the variation of hormone levels by categories of risk factors, and suggested that the heterogeneity was largely due to differences in the magnitudes of the associations rather than qualitative differences. It is likely that the accuracy of the assays used varied; therefore, the results should be interpreted simply as an average across assays for the available data. The results suggested that the type of sex hormone assay (with or without a purification step) influenced some of the results. For example, the association of oestradiol with BMI was stronger in the studies that had used an assay with previous purification (see below); in general, hormone assays that include a purification step are both more sensitive and more specific than direct, non-extraction assays (
Stanczyk et al, 2003).
For oestradiol and testosterone, we calculated estimates of the concentration of the free hormone using the law of mass action and the concentration of SHBG, and assumed a fixed concentration of albumin. We chose not to report estimates of non-SHBG-bound oestradiol and testosterone, which are often described as ‘bioavailable' because these hormones dissociate readily from albumin. It should be noted that the calculated non-SHBG-bound oestradiol and testosterone would be perfectly correlated with free oestradiol and testosterone, respectively, and therefore that the associations reported for free oestradiol and free testosterone would be identical for the non-SHBG-bound concentrations of these hormones.
The hormones were all positively correlated with each other, presumably because they are all part of the same metabolic pathway. Whereas oestrogen and progesterone production in premenopausal women is controlled by feedback mechanisms through the anterior pituitary and the hypothalamus, the control of sex hormone production in postmenopausal women is not well understood. The oestrogens are derived from the androgens, which are secreted by the adrenal glands and by the stromal tissue of the ovaries. Adrenal androgen production may be stimulated by stress, as ACTH stimulates the adrenal cortex to synthesise cortisol and, less specifically, the adrenal androgens (
Azziz et al, 1990;
McKenna et al, 1997). Conversion of androgens to oestrogens is catalysed by the enzyme aromatase, and the major site of aromatase activity in postmenopausal women is in the adipose tissue (
Grodin et al, 1973).
Age The large decrease in DHEAS and androstenedione, the small changes in oestrogens and testosterone and the increase in SHBG with age are broadly consistent with previous reports (
Meldrum et al, 1981;
Moore et al, 1987;
Verkasalo et al, 2001;
Spencer et al, 2007). The smaller decreases in oestrogens than in two of the three androgens might suggest that the amount of substrate available only partly determines oestrogen concentrations, and that synthesis by aromatisation may become more active in older women (
Misso et al, 2005).
It is likely that the relatively high concentrations of oestradiol in women aged below 55 years are associated with the fact that many of these women have had their menopause within the last 5 years, and exclusion of women who had recently had their menopause reduced this association (see further discussion below in relation to time since menopause).
Hysterectomy and ovariectomy Previous analyses of data from studies contributing to this collaboration (
Laughlin et al, 2000) and others (
Judd et al, 1974a;
Vermeulen, 1976;
Davison et al, 2005;
Cappola et al, 2007;
Fogle et al, 2007;
McTiernan et al, 2008) have shown that postmenopausal women who have had a bilateral ovariectomy have lower circulating androgen concentrations than postmenopausal women with intact ovaries. Furthermore, clinical studies have shown that androgen concentrations are higher in blood samples from the ovarian veins than in the peripheral circulation, demonstrating that the ovaries secrete androgens (
Grodin et al, 1973;
Judd et al, 1974b). Our analyses replicated these relationships; women who had undergone a bilateral ovariectomy had testosterone concentrations about 30% lower than those of naturally postmenopausal women, together with smaller reductions in androstenedione and DHEAS, but no significant differences for total oestradiol or oestrone. Women who had undergone a hysterectomy without bilateral ovariectomy had circulating androgen concentrations intermediate between those of women who had a natural menopause and women who had undergone a bilateral ovariectomy; this intermediate effect might be due to misclassification of some women who had undergone bilateral ovariectomy into the hysterectomy category and/or damage to the ovarian blood supply during hysterectomy (
Laughlin et al, 2000); alternatively, it is possible that long-standing differences in circulating androgens somehow influence the likelihood of hysterectomy.
Body mass index Levels of the oestrogens were higher in women with higher BMI, probably because women with more adipose tissue have more aromatase (in their adipose tissue) and therefore increased total aromatase activity (
Grodin et al, 1973), and the high oestrogen levels of obese postmenopausal women probably explain their increased risk for breast cancer (
Endogenous Hormones and Breast Cancer Collaborative Group, 2003a). This relationship has been observed in many previous studies that are not included in this collaboration (
Meldrum et al, 1981;
Kaye et al, 1991;
Madigan et al, 1998;
Nagata et al, 2000;
Verkasalo et al, 2001;
Boyapati et al, 2004;
McTiernan et al, 2006;
Chavez-MacGregor et al, 2008). Androstenedione and testosterone also increased with increasing BMI, whereas DHEAS was not clearly related to BMI. These associations have been observed previously (
Cappola et al, 2007), but the reason why androgens increase with BMI is not clear. Obesity is associated with increases in insulin, which may stimulate androgen production in the ovarian stroma (
Barber et al, 2006), whereas adrenal androgen synthesis is not known to be stimulated by insulin; however, our results suggested that androgens also increased with BMI in women who had undergone a bilateral ovariectomy and in whom most androgen synthesis must be in the adrenals. Sex hormone-binding globulin also decreased with increasing BMI, as has been consistently observed in many studies (e.g.,
Baglietto et al, 2009;
Moore et al, 1987; and
Newcomb et al, 1995); this decrease may be due to raised insulin concentrations inhibiting SHBG synthesis in the liver (
Crave et al, 1995).
Overall, total oestradiol was 47% higher and calculated free oestradiol was 89% higher in obese women than in thin women. There was significant heterogeneity between studies in these estimates, and some of this heterogeneity was explained by the oestradiol assay method, with increases of total oestradiol between thin and obese women of 82% for studies that used assays with previous purification but only 30% for studies that used direct non-extraction assays. A similar difference has been noted previously, and it is likely that the larger estimate of 82% is more accurate because assays with previous purification for oestradiol are more sensitive and more specific than direct assays (
Lee et al, 2006).
Smoking We observed higher concentrations of oestrogens and androgens in current heavy cigarette smokers (at least 15 cigarettes per day) than in non-smokers, whereas SHBG did not vary significantly according to smoking status. Cigarette smokers were on average thinner than non-smokers, and the differences in oestrogens were accentuated by the adjustment for BMI. Previous studies have not shown clear associations of smoking with circulating oestrogens, but have generally observed that at least some of the androgens were higher in smokers than in non-smokers (
Khaw et al, 1988;
Cassidenti et al, 1992;
Baron et al, 1995;
Manjer et al, 2005). The mechanism for these associations is unknown but may involve more general effects on the hypothalamic–pituitary–adrenal axis (
Kapoor and Jones, 2005).
Most epidemiological studies have suggested that smoking has little or no effect on breast cancer risk (
Collaborative Group on Hormonal Factors in Breast Cancer, 2002;
International Agency for Research on Cancer, 2004), but some recent large cohort studies have suggested that a long duration of smoking does have a small positive association with breast cancer risk in postmenopausal women, particularly in association with starting to smoke at a young age (
Cui et al, 2006;
Luo et al, 2011) and perhaps because of the chemical carcinogens in tobacco smoke (
Secretan et al, 2009). The moderate differences between heavy smokers and non-smokers in circulating hormones might affect breast cancer risk, although the total effect of smoking on lifetime exposure to hormones also includes the relatively early menopause of smokers (
Midgette and Baron, 1990) and possibly changes in oestrogen metabolism (
Michnovicz et al, 1986).
Alcohol Alcohol intake was positively associated with all the sex hormones, with the strongest association for DHEAS, whereas SHBG was inversely associated with alcohol consumption.
High alcohol consumers were on average thinner than non-drinkers, and the differences in oestrogens and in SHBG were accentuated by the adjustment for BMI. These findings are broadly consistent with those of previous observational studies (
Onland-Moret et al, 2005). Furthermore, a randomised trial in 51 postmenopausal women showed that DHEAS increased by 5.1% in women consuming 15
g of alcohol per day and by 7.5% in women consuming 30
g of alcohol per day; alcohol consumption also increased circulating concentrations of oestrone sulphate (
Dorgan et al, 2001). In another randomised crossover trial in nine postmenopausal women, 30
g of alcohol per day increased circulating DHEAS by 18% but had no detectable effect on oestradiol or testosterone (
Sierksma et al, 2004). The mechanism for these associations is unknown.
Alcohol is mostly consumed in the evening, whereas blood samples are usually collected during the day. As the half-life of oestradiol is ~3
h (
Ginsburg et al, 1998), it is possible that there is a marked increase of oestradiol shortly after alcohol consumption in the evening, which cannot be assessed in typical epidemiological studies. In a study of the acute effects of 40
g of alcohol in premenopausal women, circulating oestradiol concentrations increased and reached a peak 25
min after alcohol ingestion (
Mendelson et al, 1988). The half-life of DHEAS is longer (~14
h), and this might explain why this sulphated hormone has the strongest association with alcohol consumption (
Dorgan et al, 2001).
The increases in sex hormone concentrations associated with alcohol consumption might contribute to the increase in breast cancer risk with alcohol consumption (
Baan et al, 2007), although other factors not related to circulating hormone concentrations might mediate the effect (
Seitz and Stickel, 2007).
Other risk factors Early research suggested that young age at menarche is positively associated with oestrogen levels in young women (
MacMahon et al, 1982), but previous studies have not demonstrated clear associations of age at menarche with circulating hormones in postmenopausal women (
Verkasalo et al, 2001). We found that, after adjusting for BMI, mean concentrations of oestradiol were 6% lower in postmenopausal women who had had a late menarche than in those who had an early menarche, but age at menarche was not clearly associated with the other hormones or SHBG.
The mean concentration of DHEAS varied significantly with parity, but without a clear pattern. Previous studies have reported that parity was not associated with sex hormones in postmenopausal women (
Ness et al, 2000) and that DHEAS was not significantly related to parity in postmenopausal women (
Key et al, 1990). Age at first full-term pregnancy in parous women was not associated with any of the sex hormones or SHBG.
Age at menarche, parity and age at first full-term pregnancy are risk factors for breast cancer (
Kelsey et al, 1993). Apart from the very weak association of age at menarche with circulating oestradiol, the current findings that these risk factors are not associated with circulating sex hormone concentrations in postmenopausal women suggest that their effects on breast cancer risk are not likely to be mediated through postmenopausal hormone levels. These risk factors may operate through long-term effects on sex hormone levels in premenopausal women (
MacMahon et al, 1982), through changes in the duration rather than the level of exposure to premenopausal sex hormones (
Key and Pike, 1988), by hormonally mediated long-lasting changes in breast structure (
Russo et al, 2005) or by other mechanisms.
Mean concentrations of oestradiol, free oestradiol and DHEAS were significantly higher in women who had had their menopause within 5 years of blood collection than in other women, but the other sex hormones and SHBG did not differ significantly between these groups. The higher levels of oestradiol and free oestradiol were observed primarily up to about 1 year after menopause, and this is compatible with results from a longitudinal study in which oestradiol levels declined from about 2 years before the final menstrual period until ~2 years after (
Sowers et al, 2008). In another report from the same longitudinal study (
Crawford et al, 2009), there was a small increase in circulating concentrations of DHEAS during the menopausal transition, followed by a decline (more than 2 years since last menstrual period).
Previous use of exogenous sex hormones was significantly associated with several endogenous hormones, although the magnitude of these associations was small. Previous use of oral contraceptives was associated with higher concentrations of oestradiol and free oestradiol and lower concentrations of androstenedione; previous use of hormonal therapy for menopause was associated with lower concentrations of oestrone, androstenedione, testosterone and free testosterone. In another study (
Chan et al, 2008), previous use of oral contraceptives was associated with lower concentrations of oestradiol, oestrone, androstenedione, testosterone and SHBG, and previous use of hormonal therapy for the menopause was associated with lower concentrations of testosterone and 17
α-hydroxyprogesterone. It is possible that the associations with hormonal therapy for menopause may be partly because women with relatively low endogenous hormone levels may have more symptoms, and therefore be more likely to be prescribed hormonal therapy.
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