Associations between Reproductive and Menstrual Factors and Postmenopausal Sex Hormone Concentrations

Introduction

Hormone-related cancers in postmenopausal women are associated with certain menstrual and reproductive events. Early age at menarche, late age at menopause, late age at first full-term pregnancy, and low parity are putative risk factors for breast cancer (1-5), whereas breast-feeding is thought to have a protective effect (4, 6, 7). Nulliparity and late age at menopause are also established risk factors for endometrial cancer (8). Parity and past use of oral contraceptives have been associated with reduced risk of ovarian cancer in postmenopausal women (9, 10).

These menstrual/reproductive characteristics may modulate cancer risk by influencing lifetime or acute exposure to estrogens and other sex hormones (1, 5, 7, 9, 11, 12). We examined the associations between menstrual and reproductive history and serum hormone concentrations in postmenopausal, overweight, sedentary women. This information will be valuable in understanding possible mechanisms through which reproductive and menstrual factors may affect the risk of hormone-related cancers in postmenopausal women.

Methods

Overview of the Study

Subjects were from the Physical Activity for Total Health study, which is described in detail elsewhere (13). The trial was designed to investigate the effects of a yearlong moderate intensity exercise intervention versus stretching control in 173 postmenopausal women on hormone end points and, secondarily, on changes in body mass index, fat distribution, and immune function. Women were ages 50 to 75 years, sedentary (<60 minutes per week of moderate-to-vigorous intensity exercise), overweight or obese (body mass index ≥25.0 or between 24.0 and 24.9 and percentage body fat >33%), and resided in the greater Seattle, WA area. All study procedures, including a written informed consent, were approved by the Fred Hutchinson Cancer Research Center Institutional Review Board (Seattle, WA).

We identified potentially eligible women primarily via mass mailings and media advertisements (14). Interested women were screened for eligibility by telephone interview and clinic visit. Major ineligibility criteria included using hormone therapy in the past 6 months; being too physically active; having medical conditions contraindicating moderate-to-vigorous intensity exercise; having a clinical diagnosis of diabetes; and currently using tobacco.

Baseline Data Collection

Women were considered postmenopausal if they had not had a menstrual period during the previous 12 months and, for women ages 50 to 54 years, had a serum follicle stimulating hormone (FSH) concentration of >30 mIU/mL. We collected demographic information, medical history, reproductive history, hormone use history, and diet (15) via self-administered questionnaires. We assessed total kilograms of body fat using a dual-energy X-ray absorptiometry whole-body scanner (Hologic QDR 1500, Hologic Inc., Waltham, MA). A 12-hour fasting blood was processed within 1 hour of collection, aliquoted into 1.8 mL tubes, and stored at −70°C.

Hormone Assays

Serum hormone assays were done at the Reproductive Endocrine Research Laboratory (University of Southern California, Los Angeles, CA) directed by one of the authors (F.Z.S.). Estradiol and estrone were quantified in baseline samples by sensitive and specific RIA following organic solvent extraction and Celite column partition chromatography (16, 17). Chromatographic separation of the steroids was achieved using different concentrations of toluene in isooctane and ethyl acetate in isooctane. Sex hormone binding globulin (SHBG) and FSH were quantified via an immunometric assay both using the Immulite Analyzer (Diagnostic Products Corporation, Los Angeles, CA). Free estradiol was calculated using the measured estradiol concentrations, SHBG concentrations, and an assumed constant for albumin (18), which is valid compared with direct measurement (19).

Samples were batched such that, within each batch, subject randomization dates were similar, the number of intervention and control subjects was approximately equal, and the sample order was random. Two quality control pooled samples were placed in each batch. Laboratory personnel were blinded to subject and quality control sample identity. The intraassay coefficients of variation (CV) were <10%, and the interassay CVs were <12% for all assays, except estrone (intraassay CV 12.4%, interassay CV 17.6%) and estradiol (intraassay CV 12.4%, interassay CV 15.8%).

Statistical Analysis

We assessed the associations between sex hormone concentrations and the following variables: age at menopause (≤44, 45 to 49.9, 50 to 54.9, or ≥55 years); time since menopause (≤4, 5 to 9, 10 to 14, 15 to 19, or ≥20 years); age at menarche (≤11, 12, 13, or ≥14 years); regularity of menstrual periods (always, sometimes, or never); years of oral contraceptive use (none, ≤4, 5 to 9, or ≥10); years since last oral contraceptive use (never used, ≥30, 25 to 29, or ≤24); reason for menopause (natural, surgery, or other); years of hormone therapy (none, ≤5, or >5); years since last hormone therapy use (never, ≤5, or >5); past herbal therapy use (yes or no); had a pregnancy lasting ≥6 months (yes or no); age at first pregnancy lasting ≥6 months (<20, 20 to 34, 25 to 29, or ≥30 years); number of pregnancies ≥6 months (1, 2, 3, or ≥4); and months spent breast-feeding (<1, 1 to 12, or ≥13).

For each characteristic, we determined adjusted geometric means and 95% confidence intervals using linear regression of log-transformed concentrations of estradiol, estrone, SHBG, and free estradiol. For FSH, we computed arithmetic means and confidence intervals using linear regression. All analyses were adjusted for age (linear), kilograms of body fat from dual-energy X-ray absorptiometry (linear), oophorectomy status (no ovaries, at least part of one ovary, or unknown), race (non-Hispanic white or other), marital status (never married, divorced/separated, widowed, or married/living with partner), and alcohol consumption (g/d). We also tried adjusting for body mass index (continuous) instead of body fat from dual-energy X-ray absorptiometry. Secondarily, we assessed time since menopause and age at menopause together in one model and age at first pregnancy, number of pregnancies, and breast-feeding together in another model, which did not include age. Tests for trend were done using the ranks of the categorizations listed above.

We excluded one woman who was using vaginal estrogen cream at her blood draw, resulting in a premenopausal estradiol concentration; one woman with an extremely high SHBG concentration (191 nmol/L); and one woman missing information on race/ethnicity (a variable used to adjust all models). Thus, 170 women were included. Analyses of time since and age at menopause excluded 5 women who were missing age at menopause and 18 women who had had a hysterectomy without bilateral oophorectomy before menopause. All analyses were done using Stata 8 (StataCorp, College Station, TX).

Results

Subjects were, on average, 61 years old and had 38.4 kg of body fat (Table 1). Most participants were non-Hispanic white, had a natural menopause, and had a history of regular menses. On average, study participants began menstruating at age 13 years and stopped at age 50 years. Parous women (83%) had an average of 2.1 pregnancies. The first of these pregnancies occurred at an average age of 24 years; 70% of parous women breast-fed.

Women ≥20 years past menopause had 23% lower total estradiol and 30% lower free estradiol concentrations than women within 4 years of menopause (P for trend = 0.04 and 0.02, respectively; Table 2). Age at menopause was positively associated with total and free estradiol concentrations; however, adjustment for time since menopause substantially attenuated the associations (data not shown). Women reporting a history of irregular menses had 25% lower FSH concentrations than women reporting regular menses (P < 0.01).

Nulliparous women had 19% higher FSH concentrations than parous women (P = 0.02; Table 3). Parous women with ≥4 births had 20% lower free estradiol and 38% higher SHBG concentrations compared with those with one birth (P for trend = 0.02 and 0.01, respectively). Breast-feeding was modestly associated with lower FSH concentrations (P for trend = 0.07). Results were similar when age at first pregnancy, number of pregnancies, and breast-feeding were included in the same model.

Ever versus never use of herbal hormones was associated with 28% higher FSH concentrations (P = 0.02; Table 4). We did not detect statistically significant associations between other hormone use variables and sex hormone concentrations. Adjusting for body mass index instead of body fat did not change results meaningfully.

Discussion

Our study suggests that parity and time since menopause may be related to postmenopausal sex hormone concentrations. Time since menopause was inversely associated with concentrations of total and free estradiol in our study. Similarly, in study of 125 postmenopausal women, bioavailable estradiol was lower and SHBG was higher in the later postmenopausal years (20). Another study suggested that estradiol and estrone concentrations were lower 8 years versus 1 year after menopause in 159 women (21). However, time since menopause was not associated with urinary estrogen concentrations in another study of 220 women (22). We found no association between age at menopause and sex hormone concentrations after adjusting for time since menopause, which is consistent with most (20, 23-25) but not all (21) previous data. Overall, these studies suggest a modest decline in estrogen concentrations with increasing time since menopause.

Only FSH differed significantly between parous and nulliparous women. Previous studies have reported that women with high versus low basal FSH concentrations have lower pregnancy rates (26). Gonadotropin stimulation (e.g., high FSH or luteinizing hormone) has been associated with an increased risk of ovarian cancer in some (10, 26-28) but not all (29) studies. The null results for estrogen and SHBG concentrations are consistent with most studies (20, 21, 23, 25). However, one study found that postmenopausal parous women had nonsignificantly higher urinary estrogens than nonparous women (22).

Among parous women, parity was inversely associated with free estradiol and positively associated with serum SHBG concentrations. Hankinson et al. (11) reported a similar relationship with plasma estrone, estrone sulfate, estradiol, and free estradiol in 216 postmenopausal women; however, only the association with estrone sulfate was statistically significant. Our observations are consistent with an established association of parity with lower breast cancer risk. Pregnancy may cause an enduring change in a woman's hormone profile (e.g., lowering her estrogen concentrations). Alternatively, a woman's genetic and environmental profile, or her premenopausal hormone levels, may influence both her ability to have children and her postmenopausal sex hormone concentrations.

We were unable to detect an association between postmenopausal sex hormone concentrations and age at first pregnancy lasting ≥6 months, which is consistent with most (20, 21, 25) but not all (11) studies. FSH concentrations and time spent breast-feeding were modestly inversely associated. To our knowledge, there have been no previous studies of this relationship in postmenopausal women. There is greater biological plausibility for the observed relationship between history of menstrual regularity and FSH concentration, as this hormone is involved in cycle regulation; again, an underlying genetic and environmental profile may be important for both premenopausal and postmenopausal hormone concentrations. The reason for the observed relationship between herbal therapies and FSH is less clear. Either of these associations with FSH concentrations could have been due to small sample sizes, as only 12 women reported ever using herbal hormones and only 8 reported always having had irregular menses.

Our study has several limitations. Because we did not know the participants' premenopausal hormone concentrations, we could not examine whether a woman's underlying hormonal profile influenced both her reproductive capabilities and her postmenopausal sex hormone concentrations. Second, the participants had to meet the stringent eligibility criteria of our exercise intervention trial to be included in the present study (e.g., sedentary and overweight/obese). Therefore, caution must be used in generalizing our results to all postmenopausal women, especially those taking hormone therapy. Like our study population, however, the majority of American women in the age range we studied are overweight or obese (30).

Our results suggest that time since menopause and parity are related to postmenopausal sex hormone concentrations. We do not have evidence to suggest that age at menarche, first pregnancy, or menopause is independently associated with postmenopausal sex hormone concentrations. Our study may, however, help elucidate biological mechanisms through which reproductive and menstrual characteristics modulate risk of postmenopausal hormone-related cancers.