Consumption of Low-Fat Dairy Products May Delay Natural Menopause 1 3

Transcription

1 The Journal of Nutrition Nutritional Epidemiology Consumption of Low-Fat Dairy Products May Delay Natural Menopause 1 3 Jenny L. Carwile, 4 Walter C. Willett, 4 6 and Karin B. Michels 4,6,7 * Departments of 4 Epidemiology, and 5 Nutrition, Harvard School of Public Health, Boston, MA; 6 Channing Division of Network Medicine, Department of Medicine, and 7 Obstetrics and Gynecology Epidemiology Center, Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women s Hospital, Harvard Medical School, Boston, MA Abstract Later menopause is a risk factor for breast and endometrial cancer, yet few studies have investigated dietary predictors of this potentially modifiable event. In particular, dairy contains hormones and growth factors that could potentially affect menopausal timing. We therefore assessed the association between regular consumption of dairy foods and related nutrients and age at natural menopause. We conducted a prospective analysis with up to 20 y of follow-up in 46,059 participants in the NursesÕ Health Study who were premenopausal in We observed 30,816 events of natural menopause over 401,754 person-years. In the total population, the estimated mean age at natural menopause was 51.5 y for women who consumed no low-fat dairy and 51.5, 51.6, 51.7, and 51.8 y for women who consumed , , , and >3 servings of low-fat dairy daily, respectively. Premenopausal women <51 y of age consuming >3 servings of low-fat dairy per day were 14% less likely (HR: 0.86; 95% CI: 0.77, 0.96; P-trend < ) to report natural menopause in the next month relative to those consuming servings/d. Similar results were obtained for skim milk (for >6 servings/wk vs. 0 1 servings/mo: HR: 0.93; 95% CI: 0.89, 0.97; P-trend < ) but not for total high-fat dairy or whole milk. Dairy foods were not associated with age at menopause among women $51 y of age. These findings support the growing body of literature on the hormonally active nature of milk and dairy foods. J. Nutr. 143: , Introduction The mean age at natural menopause, the permanent cessation of menstrual periods, in the United States is between 50 and 51 y (1,2). Later menopause is protective against cardiovascular disease (3) and osteoporosis (4) but positively associated with development of breast (5) and endometrial cancer (6). Moreover, earlier age at menopause may be positively associated with allcause mortality (7,8). Many behavioral and socioeconomic factors appear to influence menopausal timing (1); however, despite the potential for foods and nutrients to alter the hormonal milieu, few studies have assessed dietary predictors of natural menopause. Dairy foods, especially milk, are of particular interest, because steroid hormones and growth factors are endogenous to 1 The NursesÕ Health Study is supported by grants CA87969 and HL from the National Institutes of Health (NIH). J.L.C. was supported by the Breast Cancer Research Foundation; training grant T32ES in Environmental Epidemiology from the National Institute of Environmental Health Sciences, NIH, and training grant T32HD in Reproductive, Perinatal, and Pediatric Epidemiology from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH. 2 Author disclosures: J. L. Carwile, W. C. Willett, and K. B. Michels, no conflicts of interest. 3 Supplemental Methods are available from the Online Supporting Material link in the online posting of the article and from the same link in the online table of contents at * To whom correspondence should be addressed. kmichels@research. bwh.harvard.edu. cow milk (9,10) and may retain bioactivity after ingestion (11). Furthermore, epidemiologic studies have linked milk consumption to elevated plasma concentrations of estradiol (12) and insulinlike growth factor I (IGF-I) 8 (13 16). The decline in estrogen beginning around 2 y before the final menstrual period has been well documented (17,18). Endogenous estrogen concentrations before and during this period may be affected by dairy consumption. To our knowledge, 6 studies have prospectively investigated diet and age at natural menopause (19 24), only 2 of which examined dairy consumption (22,24). In a cross-sectional analysis of the Japan NursesÕ Health Study, more frequent milk and dairy consumption was associated with a reduced likelihood of early menopause (24); however, important details about this unpublished work are unknown. In the EPIC (European Prospective Investigation into Cancer and Nutrition) cohort in Heidelberg, baseline dairy consumption was not associated with incidence of menopause, after adjusting for total energy intake and other predictors of natural menopause (22). Individual assessment of low- and high-fat dairy was not performed but may be warranted. Steroid hormones, including estrogen, are lipophilic; however, conjugated estrogens, which comprise up to 98% of estrogen 8 Abbreviations used: IGF-I, insulin-like growth factor I; NHS, NursesÕ Health Study; OC, oral contraceptive. ã 2013 American Society for Nutrition Manuscript received May 13, Initial review completed June 19, Revision accepted July 19, First published online August 14, 2013; doi: /jn

2 found in dairy products, partition into the aqueous component (9). Hence, the association between dairy consumption and natural menopause may depend on the fat content of the dairy consumed. We predicted that consumption of low-fat dairy foods may elevate circulating estrogen concentrations, supporting periodic menstrual bleeding and resulting in a later age at menopause. We examined intake of dairy foods, including total high-fat dairy, total low-fat dairy, skim and whole milk, and related nutrients, including dairy fat, dairy protein, calcium, vitamin D, and lactose, in relation to age at natural menopause in a large cohort of women with up to 20 y of follow-up. Participants and Methods Study population. The NursesÕ Health Study (NHS) is a prospective cohort study initiated in 1976, when 121,700 married, female nurses, aged 30 to 55 y, responded to a detailed baseline questionnaire on medical, lifestyle, and other health-related information. Follow-up questionnaires were mailed biennially to update information on risk factors and newly diagnosed diseases. The NHS was approved by the institutional review board of the Brigham and WomenÕs Hospital (Boston, MA). Follow-up for the present study began in 1980, when diet was first assessed, and ended in 2000, when age at menopause was ascertained for the final time. For this analysis, women were included if they had <10 total missing food items, total energy intake of 500 to 3500 kcal/d, and no diagnosis of cancer by After further restriction to women who were premenopausal at baseline and reported at least 1 status update during the study period, 46,059 women were eligible for analysis. Dietary assessment. Diet was assessed by using a self-administered semiquantitative FFQ, which was administered in 1980, 1984, 1986, 1990, 1994, and In 1980, the FFQ consisted of 61 food items. The 1984 FFQ was expanded to include 126 foods plus vitamin and mineral supplements; 1986, 1990, 1994, and 1998 questionnaires were similar to the 1984 FFQ. Participants were asked, on average, how frequently they consumed a typical portion size of specified foods over the past year. Nine possible frequency responses were available for each food, ranging from never to >6 times/d. Total dairy was calculated as the sum of the following: skim and low-fat milk, whole milk, yogurt, cottage cheese, sherbet, cream, sour cream, ice cream, cream cheese, and other cheese. The low-fat dairy food category was calculated by summing intakes of skim (nonfat) and low-fat milk, yogurt, cottage cheese, and sherbet; the high-fat dairy food category included whole milk, cream, sour cream, ice cream, cream cheese, other cheese, and butter. Red meat included beef, pork, lamb, and hamburger. If a food item was left blank, consumption was set to zero (25). Dietary nutrient intakes were calculated by multiplying the frequency of consumption of each unit of food by the nutrient content of the specified portions and summing across all foods. Nutrient composition values for each food were obtained from the USDA and other sources (26). Total nutrient intake was calculated in a similar manner; however, intake from vitamin and supplement use was also included. To calculate dairy fat and dairy protein, foods in the dairy group, as well as dairy ingredients in other foods, were considered. Total energy intake was calculated from all foods. To reduce extraneous variation in nutrient intake, nutrients were energy-adjusted by using the residuals from the regression of nutrient intake on total energy intake (27). The validity of the 61-item FFQ was assessed among a sample of 173 NHS participants by comparing intakes assessed using the FFQ to the mean intake assessed using four 1-wk diet records (28,29). Deattenuated correlation coefficients between the FFQ and the mean intake assessed by the diet records were 0.81 for skim and low-fat milk, 0.62 for whole milk, 0.94 for yogurt, 0.73 for ice cream, 0.80 for cottage cheese, and 0.57 for other (hard) cheeses (28). After a comparison of intake from the 1986 FFQ and two 1-wk diet records in 191 women, the deattenuated correlation coefficient for calcium was 0.75 (29). Assessment of menopausal status and age at menopause. Menopausal status, defined here as the permanent cessation of menses, was self-reported at baseline in 1976 and biennially thereafter. Women who reported menopause were further queried on age at menopause (y) and type of menopause (natural menopause, surgical, radiation, or chemotherapy). The midpoint of the reported age at menopause was used in analyses. For this analysis, we defined surgical menopause as unior bilateral oopherectomy and/or hysterectomy or menopause resulting from radiation/chemotherapy. To reduce misclassification, we used only the first reported age at natural menopause. Self-reported menopausal status was validated among a random sample of women in this cohort who reported surgical menopause between 1982 and 1984 by comparing self-reported hysterectomy and/or ovarian surgery with medical records (30). There was complete concordance between self-report and medical records for all but 2 out of 200 women. The reproducibility of self-reported natural menopause was assessed by comparing reported menopausal status, age at menopause, and type of menopause between the 1978 and 1980 questionnaires. More than 98% of women who reported being postmenopausal on the 1978 questionnaire also reported being postmenopausal on the 1980 questionnaire. Among women reporting natural menopause, 82% reported their age at menopause to within a year of their previous report; within-person variation increased with age since menopause. Assessment of nondietary factors. Information on nondietary predictors of age at natural menopause was collected on the 1980 questionnaire and updated whenever possible by using data from follow-up questionnaires. Age was calculated by subtracting the participantõs reported date of birth from the questionnaire return date. Age at menarche and height were assessed in Marital status was assessed in 1980, 1992, and Information on parity and age at first birth was assessed at baseline and updated every year until 1984; in 1996, women were asked to report their lifetime pregnancy history. We collected data on oral contraceptive (OC) use, which was reported until Information on smoking habits and body weight were assessed at baseline and biennially thereafter. Updated BMI (kg/m 2 ) was calculated by using weight reported at each cycle and height in Between 1980 and 1982, vigorous activity was defined as the weekly time engaged in activity sufficient to work up a sweat; for 1986 and beyond the mean weekly moderate to vigorous activity was calculated by summing the mean number of hours per week spent participating in 7 common leisuretime physical activities such as brisk walking, running, jogging, swimming, and tennis. Statistical analysis. We used Cox proportional hazards regression to estimate HRs and 95% CIs for the association between cumulative averaged dairy consumption and the event of natural menopause for women ages y. All analyses used a time scale of calendar time in months; person-months were calculated for participants from the time at the return of the 1980 questionnaire to the time at natural menopause, report of surgical menopause, death, June 2000, or, if they were lost to follow-up, the date of the last returned questionnaire, whichever came first. Women who reported menopause but who failed to report type of menopause were censored, as were women with missing menopausal status. All analyses were stratified by age in months and questionnaire cycle, making the time scale equivalent to age in months. Cumulative averaged consumption of total dairy, total low-fat dairy, total high-fat dairy, skim milk, and whole milk was calculated by using data collected between 1980 and the questionnaire before natural menopause. For example, the incidence of natural menopause from 1980 through 1984 (reported on the 1982 and 1984 questionnaires) was related to intake from the 1980 questionnaire, and the incidence of natural menopause from 1984 to 1988 (reported on the 1986 and 1988 questionnaires) was related to the mean intake from the 1980 and 1984 Dairy consumption and age at natural menopause 1643

3 questionnaires. Questionnaires ascertained typical diet and attainment of natural menopause over the previous year; thus, relating menopause to diet reported on the previous questionnaire was necessary to minimize reverse causation. To reduce the impact of outliers and to avoid misclassification of dairy consumption, cumulative averaged consumption was categorized into 5 groups and modeled by using indicator variables. To aid interpretation, consumption of total and individual dairy foods was reported in units of servings per day or week. For tests of linear trend across categories of dairy consumption, we assigned the median value to each category and modeled the values as a continuous variable. The proportional hazards assumption was tested by using a likelihood ratio test comparing a model with cross-product terms for continuous age in months and the indicator variables for the exposure to the model containing the main effects only. The proportional hazards assumption was violated in the total cohort; therefore, we stratified all models by age at questionnaire return ($51 and <51 y). This cutoff was chosen because it represents the median age at natural menopause in the cohort. To explore whether dietary calcium explained any of the association between dairy consumption and natural menopause, we examined the association between dairy and age at natural menopause after adjustment for calcium. Similar models were constructed for other nutrients of interest. We conducted sensitivity analyses in which the exposure was classified in 2 additional ways: 1) baseline (1980) diet and 2) diet from 1980 to 1998 using simple updates. Similar results were obtained using each approach; only results for cumulative averaged diet are presented, as this method reflects long-term dietary intake and reduces measurement error due to within-person variation over time (31). OC use during the perimenopausal period may prolong menstrual bleeding, resulting in misclassification of menopausal status; therefore, we repeated analyses after excluding women who reported OC use at any point during the study period. Some studies (20,32), but not all (33), suggest that heavier women experience later natural menopause, so we also investigated whether associations differed by BMI (<25, , $30 kg/m 2 ) at the time of questionnaire return. Skim milk and low-fat dairy were similarly correlated with individual nutrients: skim milk and dairy protein, r = 0.63; low-fat dairy and dairy protein, r = 0.73; skim milk and dairy fat, r = 0.06; low-fat dairy and dairy fat, r = 0.10; skim milk and dietary calcium, r = 0.71; low-fat dairy and dietary calcium, r = 0.73; skim milk and dietary vitamin D, r = 0.68; low-fat dairy and dietary vitamin D, r = 0.63; skim milk and lactose, r = 0.69; and low-fat dairy and lactose, r = The following variables were chosen for inclusion in multivariate models on the basis of their potential associations with natural menopause: age at menarche (<10, , , $15 y), age at first birth and parity (nulliparous, <25 y and 1 or 2 children, <25 y and >2 children, $25 y and 1 or 2 children, $25 y and >2 children), hours per week of moderate to vigorous activity (quintiles), height (quintiles), BMI (<21, , , , $30 kg/m 2 ), OC use (never; current user; former user, <5 y; former user, $5 y), smoking (never; former; current, 1 14 cigarettes/d; current, cigarettes/d; and current, $25 cigarettes/d), marital status (married or not married), total energy (kcal/d, continuous), red meat consumption (0, 0.1 1, 1.1 2, 2.1 3, >3 servings/d), and egg consumption (0 1/mo, 1.1/mo 2/wk, 2.1 4/wk, 4.1 6/wk, >6/wk). The missing indicator method was used for covariates with missing data. Additionally, we estimated multivariable-adjusted mean age at natural menopause in the interval between 33.5 and 60.5 y by using baseline survival probabilities (Supplemental Methods). Statistical tests were 2-sided and performed at the 0.05 level of significance. We used SAS (version 9.2; SAS Institute) for all analyses. Results Of the 121,700 women enrolled in the NHS, 46,059 were premenopausal at baseline and met other eligibility criteria. Over a total of 401,754 person-years of follow-up, two-thirds (n = 30,816) of women reported their age at natural menopause, 13% (n = 6069) reported their age at surgical menopause, 2% (n = 1051) remained premenopausal at the end of follow-up, 1% (n = 447) died during the study period, and the remaining 17% was censored for reasons including incomplete data on age at or type of menopause (n = 7676). Women reporting natural menopause were followed for a mean of 8.5 y and had a median unadjusted age at natural menopause of 51.5 y. Among women who remained premenopausal in 1990, those consuming >3 servings of total dairy per day were more likely to have children and, among parous women, had more children relative to women consuming no dairy (Table 1). Women consuming greater amounts of dairy foods also reported a higher energy intake, were less likely to be smokers, and engaged in more physical activity than those reporting the lowest intake of dairy foods. Overall cohort. In the overall cohort, higher intake of low-fat dairy, skim milk, dairy protein, dairy fat, vitamin D, and lactose predicted a later age at natural menopause. For example, women consuming >3 servings of low-fat dairy daily reported reaching natural menopause 3.6 mo later than those consuming no lowfat dairy (estimated mean age at natural menopause: 51.8 vs y) (Table 2). However, the proportional hazards assumption was violated for several exposures. Therefore, all additional analyses were stratified by age at questionnaire return (<51 or $51 y). Women aged <51 y at the time of questionnaire return. Among women <51 y of age (accruing 16,557 events over 357,498 person-years), more frequent consumption of low-fat dairy and skim milk, but not high-fat dairy or whole milk, predicted a later age at natural menopause (i.e., a lower rate of natural menopause). Specifically, premenopausal women who consumed >3 servings/d of low-fat dairy were 18% less likely (HR: 0.82; 95% CI: 0.73, 0.91; P-trend < ) to report natural menopause in the next month relative to those consuming servings/d of low-fat dairy, holding total energy constant (Table 3). Multivariable analyses yielded similar effect estimates (HR: 0.86; 95% CI: 0.77, 0.96; P-trend < ). Frequency of skim milk consumption also predicted a later age at natural menopause (for 0 1 servings/mo of skim milk vs. >6 servings/wk HR: 0.93; 95% CI: 0.89, 0.97; P-trend < ). Intake of dairy protein, dairy fat, and lactose, but not total calcium or vitamin D, also predicted a marginally later age at natural menopause (i.e., a lower rate of natural menopause) (Table 4). Relative to those in the lowest quintile of dairy protein intake, premenopausal women in the highest quintile of intake were 9% less likely (HR: 0.91; 95% CI: 0.86, 0.95; P-trend < ) to reach natural menopause in the next month, holding covariates constant (adjusted for total energy only HR: 0.88; 95% CI: 0.84, 0.93). Corresponding figures were 0.94 for dairy fat (95% CI: 0.89, 0.99; P-trend: 0.02) and 0.92 for lactose (95% CI: 0.87, 0.97; P-trend: ) (adjusted for total energy only HR for dairy fat: 0.95; 95% CI: 0.90, 1.00; HR for lactose: 0.91; 95% CI: 0.86, 0.95). Women aged $51 y at the time of questionnaire return. Among women $51 y (accruing 14,259 events over 44,256 person-years), dairy foods and nutrients did not predict age at natural menopause. Sensitivity analyses. To explore which, if any, component of dairy might explain the association between low-fat dairy and natural menopause observed among women <51 y of age, nutrients were individually added to the original fully adjusted model (model 2). For models including lactose, dairy fat, or 1644 Carwile et al.

4 TABLE 1 Age-standardized characteristics of the 17,238 women in the NursesÕ Health Study who remained premenopausal in 1990, across categories of 1990 milk consumption 1 Dairy consumption 2 #1.0 serving/d (n = 2875) servings/d (n = 6957) servings/d (n = 4474).3 servings/d (n = 2932) Age 3,4, y Age at menarche, y Nulliparous, % Children (among parous women), n Age at first birth, y Oral contraceptive use, % Never user Former user,5 y $5 y Total energy intake, kcal/d Total meat, servings/d Red meat, servings/d Eggs, servings/d BMI, kg/m Height, cm Cigarette smoking 5, % Never Former Current 1 14 cigarettes/d cigarettes/d $25 cigarettes/d Moderate to vigorous activity, h/wk Married, % Values are means 6 SDs or percentages and are standardized to the age distribution of the study population. 2 Total dairy consumption was calculated from reported intake of skim and low-fat milk, whole milk, yogurt, cottage cheese, sherbet, cream, sour cream, ice cream, and cream cheese. 3 Values were not age-adjusted. 4 For this table, data on demographic and dietary variables were obtained between 1980 and Total does not sum to 100 due to rounding. dairy protein, the association between low-fat dairy and natural menopause was attenuated but generally retained significance (P-trend: 0.007, <0.0001, and 0.03, respectively), suggesting that the observed association was attributable to a component of milk other than these nutrients. When the association between skim milk and natural menopause was examined in a corresponding fashion, HRs were similarly attenuated (P-trend: 0.01, <0.0001, and 0.08, respectively). We also assessed whether findings persisted after restriction to the 40,684 women who did not report menopause during their first year in the study or the 45,973 women who never reported using OCs during follow-up. Results were not materially different in either of these subpopulations (data not shown). Effect estimates were materially unchanged after consideration of crude fiber intake (data not shown). Finally, results were not modified by overweight or obesity status (data not shown). Discussion In this prospective study of dairy consumption and age at natural menopause, more frequent consumption of total low-fat dairy and skim milk, but not high-fat dairy or whole milk, predicted a modest delay in natural menopause among women aged <51 y. One potential biological rationale for these findings is that consumption of low-fat dairy may increase circulating estrogen concentrations, delaying natural menopause. However, although we collected data on and adjusted for many potential confounders, observed effect estimates were modest and compatible with residual confounding. Biological mechanism. Cow milk contains various estrogen metabolites, including 17b-estradiol, estrone, and estriol (9), ~80 98% of which are present in their conjugated form (9,34). Estrone sulfate and other conjugated estrogens are hydrophilic (unlike lipophilic free estrogens) and might therefore be expected to be present in the highest concentrations in low-fat dairy products, including skim milk (9). However, studies relating estrogen concentrations and dairy fat content in dairy foods have yielded mixed results (9,34,35). Conjugated estrogens bypass first-pass hepatic metabolism, and are therefore considered to be the most biologically relevant in the context of oral administration (36). The bioactivity of orally administered conjugated estrogens is demonstrated by the effectiveness of OCs and hormone replacement therapy. Limited evidence supports the hypothesis that frequent consumption of milk increases circulating estrogen concentrations. In rats, plasma estrone sulfate concentrations were 29% higher in animals fed low-fat milk compared with those fed a macronutrient milk substitute (37). Similarly, dairy consump- Dairy consumption and age at natural menopause 1645

5 TABLE 2 Estimated mean age at natural menopause among 46,059 women in the NursesÕ Health Study, Estimated mean age at natural menopause 2 y Low-fat dairy 3 0 servings/d serving/d servings/d servings/d servings/d 51.8 High-fat dairy 4 0 servings/d serving/d servings/d servings/d servings/d 51.6 Skim milk 0 1 serving/mo serving/mo 2 servings/wk servings/wk servings/wk servings/wk 51.6 Whole milk 0 1 serving/mo serving/mo 2 servings/wk servings/wk servings/wk servings/wk Diet was assessed in 1980, 1984, 1986, 1990, 1994, and Food intakes were cumulatively updated over follow-up. 2 Values of questionnaire cycle and risk factors quantified by using quantiles were set to the median category, other categorical risk factors were set to the observed mode in the study population, and continuous variables were set to their observed mean in the study population. 3 Total low-fat dairy intake was calculated from reported intake of skim and low-fat milk, yogurt, cottage cheese, and sherbet. 4 Total high-fat dairy intake was calculated from reported intake of whole milk, cream, sour cream, ice cream, cream cheese, other cheese, and butter. tion has been positively related to total and free estradiol concentrations in postmenopausal women (12). In a crosssectional analysis of NHS participants, total and free estradiol concentrations were positively associated with the Western pattern, a dietary pattern characterized by consumption of highfat dairy and red and processed meats, as well as refined grains, sweets, and desserts (38). Serum estradiol concentrations did not differ by treatment status in an 18-mo intervention in which 12- y-old girls were randomly assigned to receive 568 ml (1 pint) of whole or reduced-fat milk per day or a usual diet (39). However, ovarian production of estrogens is relatively high in this group, and circulating estrogen concentrations would therefore be more tightly regulated than in a population of older females, in whom peripheral estrogen production is more abundant. Although these studies suggest that, in certain populations, milk consumption may increase circulating estrogen concentrations, whether the magnitude of this increase is sufficient to stimulate menstruation is uncertain. On the basis of this evidence, we expected consumption of dairy, and, in particular, low-fat dairy foods, to increase estrogen concentrations, postponing menopause. Associations between low-fat dairy, skim milk, and natural menopause could not be fully explained by intake of dairy protein, dairy fat, or lactose. This is compatible with the hypothesis that a nonnutrient component of dairy foods accounts for the observed associations with age at natural menopause. The observed association between skim milk, low-fat dairy, and natural menopause was restricted to women aged <51 y at the time of questionnaire return. This suggests that only younger women may be susceptible to dairy consumption, possibly because menopause has already been initiated to an irreversible degree in older women. Dairy contains other hormones and nutrients that may influence timing of natural menopause, and we considered several alternative hypotheses related to these possible mechanisms. We hypothesized that dairy consumption may predict earlier natural menopause because galactose, a lactose metabolite, has been posited as an ovarian toxin. Women with galactosemia experience early menopause (40) and women with low activity of galactose-1-phosphate uridyl transferase, an enzyme responsible for galactose metabolism, may reach menopause earlier than women with normal enzyme activity (41). However, we observed only positive associations between dairy foods and related nutrients, including lactose, and age at natural menopause. Frequent dairy consumption is also associated with a 10 20% increase in circulating IGF-I concentrations (13 16), which could possibly stimulate the ovaries, postponing natural menopause. Circulating IGF-I concentrations decrease with age and may be involved in the menopausal transition, as suggested by studies in rats, in which decreased IGF-I signaling leads to disrupted surges in luteinizing hormone similar to that observed during reproductive senescence (42). However, the relation between IGF-I and natural menopause is still unclear. Additionally, intake of an unidentified component of low-fat dairy may increase the frequency of anovulatory menstrual cycles (43), a hypothesized predictor of later age at natural menopause (44). Last, our results could be explained by a dietary pattern characterized by frequent consumption of low-fat dairy. Strengths and limitations. Major strengths of this study include the prospective design as well as detailed collection of dietary information using previously validated questionnaires. Dietary recall on FFQs is subject to both within- and betweenperson errors. Calculation of cumulative averages can reduce measurement error due to within-person variation (29), but findings were materially unchanged when we instead examined baseline or recent diet. We observed large variation in total energy, which likely resulted in part from the under- or overreporting of dietary intake by some women. We accounted for this by adjusting for total energy intake (29); however, systematic under- or overreporting of dairy foods may remain. We related natural menopause to diet averaged over the preceding questionnaire cycles, making it unlikely that menopausal status influenced dietary reporting. Exposure misclassification unrelated to age at menopause likely resulted in slightly attenuated findings. Misclassification of the outcome was minimized by using only the first reported age at menopause and by collecting data every 2 y. Thus, reporting errors in age of menopause are expected to be nondifferential with respect to dairy consumption. NHS participants are not a random sample of the general United States population, but our results are likely widely generalizable because the biological mechanisms of interest are unlikely to differ between our population and other women in the United States or elsewhere. Moreover, circulating estrogen concentrations are likely to vary with body size (45), activity (46), dietary composition (38), and other factors (47). The women in our study reported considerable variability in these and other 1646 Carwile et al.

6 TABLE 3 HRs and 95% CIs for associations between dairy and milk consumption and natural menopause among 46,059 women in the NursesÕ Health Study, Dairy consumption P-trend 2 Low-fat dairy 3, servings/d Events/person-year 1137/29,909 10,080/206, /75, /36, /9227 Model (0.94, 1.07) 1.00 (ref) 0.94 (0.90, 0.97) 0.87 (0.82, 0.92) 0.82 (0.73, 0.91), Model (0.94, 1.07) 1.00 (ref) 0.96 (0.92, 1.00) 0.90 (0.85, 0.95) 0.86 (0.77, 0.96), Events/person-year 585/ /25, /11, / /991 Model (0.79, 0.94) 1.00 (ref) 1.01 (0.97, 1.05) 0.98 (0.92, 1.04) 0.91 (0.81, 1.03) 0.74 Model (0.87, 1.04) 1.00 (ref) 0.99 (0.95, 1.04) 0.98 (0.92, 1.04) 0.95 (0.84, 1.07) 0.41 High-fat dairy 4, servings/d Events/person-year 52/ /166, /105, /42, /41,341 Model (0.63, 1.09) 1.00 (ref) 1.01 (0.97, 1.05) 1.01 (0.96, 1.06) 1.02 (0.96, 1.07) 0.46 Model (0.64, 1.12) 1.00 (ref) 1.00 (0.96, 1.04) 0.99 (0.94, 1.04) 0.98 (0.92, 1.03) 0.39 Events/person-year 49/ /20, /14, / /4342 Model (0.78, 1.40) 1.00 (ref) 1.00 (0.96, 1.04) 1.00 (0.95, 1.06) 0.99 (0.93, 1.05) 0.79 Model (0.88, 1.58) 1.00 (ref) 1.00 (0.96, 1.04) 1.00 (0.95, 1.06) 0.99 (0.93,1.06) 0.81 Skim milk, servings 0 1/mo 1.1/mo 2/wk 2.1 4/wk 4.1 6/wk.6/wk Events/person-year 4676/106, /58, /51, /28, /112,471 Model (ref) 1.01 (0.97, 1.06) 0.95 ( ) 1.00 (0.94, 1.06) 0.89 ( ), Model (ref) 1.02 (0.97, 1.07) 0.96 ( ) 1.02 (0.96, 1.08) 0.93 ( ), Events/person-year 2805/ / / / /14,033 Model (ref) 1.12 (1.06, 1.18) 1.09 (1.03, 1.15) 1.08 ( ) 1.08 ( ) 0.23 Model (ref) 1.04 (0.98, 1.10) 1.02 (0.97, 1.09) 1.01 ( ) 1.03 ( ) 0.69 Whole milk, servings 0 1/mo 1.1/mo 2/wk 2.1 4/wk 4.1 6/wk.6/wk Events/person-year 8843/201, /72, /32, /11, /39,083 Model (ref) 1.04 (1.00, 1.08) 1.08 (1.02, 1.14) 1.12 (1.03, 1.22) 1.04 (0.98, 1.10) 0.01 Model (ref) 1.01 (0.97, 1.05) 1.05 (0.99, 1.10) 1.07 (0.98, 1.16) 1.00 (0.95, 1.06) 0.34 Events/person-year 7381/23, /12, / / /3381 Model (ref) 1.03 (0.99, 1.08) 1.00 (0.94, 1.07) 1.04 (0.94, 1.13) 1.00 (0.93, 1.07) 0.95 Model (ref) 1.01 (0.97, 1.05) 1.00 (0.94, 1.06) 1.00 (0.91, 1.09) 1.02 (0.96, 1.09) Diet was assessed in 1980, 1984, 1986, 1990, 1994, and Food intakes were cumulatively updated over follow-up. Model 1 is a Cox proportional hazards model adjusted for total energy intake (continuous) and stratified by age in months and period. Model 2 is a Cox proportional hazards model adjusted as for model 1 covariates as well as age at menarche (,10, , , $15 y), age at first birth and parity (nulliparous,,25 y and 1 or 2 children,,25 y and.2 children, $25 y and 1 or 2 children, $25 y and.2 children), moderate to vigorous activity (quintiles), 1980 height (quintiles), BMI (,21, , , , $30 kg/m 2 ), oral contraceptive use (never; current user; former user,,5 y; former user, $5 y), smoking (never; former; current, 1 14 cigarettes/d; current, cigarettes/d; current, $25 cigarettes/d), marital status (married or not married), red meat consumption (0, 0.1 1, 1.1 2, 2.1 3,.3 servings/d), and egg consumption (0 1/mo, 1.1/mo 2/wk, 2.1 4/wk, 4.1 6/wk,.6/wk). ref, reference. 2 Calculated by using the median of each category as a continuous term. 3 Total low-fat dairy consumption was calculated from reported intake of skim and low-fat milk, yogurt, cottage cheese, and sherbet. 4 Total high-fat dairy intake was calculated from reported intake of whole milk, cream, sour cream, ice cream, cream cheese, other cheese, and butter. characteristics, supporting the generalizability of our findings to women with a range of circulating estrogen concentrations. The unweighted median age at natural menopause was 51.5 y, which is later than those calculated in other populations, especially after accounting for differences in definition of menopause (1). The baseline exclusion of women who had already experienced menopause likely resulted in a slight upward bias in reported age at natural menopause; however, this would not affect the validity of our reported effect estimates. In conclusion, our findings suggest that women <51 y of age who frequently consume low-fat dairy and skim milk experience a slightly later natural menopause relative to women consuming these items less frequently. A 1-y increase in age at natural menopause has been found to predict a 2.5% increase in risk of breast cancer (48); therefore, the modest observed effect estimates we report are unlikely to be of clinical significance. However, our results are consistent with a growing body of literature supporting the hormonally active nature of milk and other dairy foods (49). Continued research on the health consequences of dairy consumption is especially critical in light of the current Dietary Guidelines for Americans recommendation of $3 servings of low-fat dairy per day (50). Dairy consumption and age at natural menopause 1647

7 TABLE 4 HRs and 95% CIs for associations between nutrients and natural menopause among 46,059 women in the NursesÕ Health Study, Q1 Q2 Q3 Q4 Q5 P-trend 2 Dairy fat Events/person-year 3072/68, /71, /72, /72, /72,432 Model (ref) 0.98 (0.93, 1.03) 0.97 (0.92, 1.01) 0.97 (0.92, 1.02) 0.95 (0.90, 1.00) 0.06 Model (ref) 0.98 (0.93, 1.03) 0.96 (0.92, 1.01) 0.97 (0.92, 1.02) 0.94 (0.89, 0.99) 0.02 Events/person-year 2346/ / / / /9564 Model (ref) 1.00 (0.95, 1.06) 1.03 (0.97, 1.09) 1.01 (0.96, 1.07) 0.99 (0.94, 1.05) 0.72 Model (ref) 0.97 (0.91, 1.02) 0.99 (0.94, 1.05) 0.98 (0.93, 1.04) 0.98 (0.92, 1.03) 0.60 Dairy protein Events/person-year 3415/70,787 3,606/74, /73, /72, /66,265 Model (ref) 0.98 (0.94, 1.03) 0.96 (0.91, 1.00) 0.91 (0.87, 0.96) 0.88 (0.84, 0.93), Model (ref) 0.98 (0.94, 1.03) 0.97 (0.92, 1.02) 0.92 (0.88, 0.97) 0.91 (0.86, 0.95), Events/person-year 2571/ / / / /8506 Model (ref) 1.04 (0.99, 1.10) 1.03 (0.97, 1.09) 1.04 (0.98, 1.10) 1.00 (0.94, 1.05) 0.69 Model (ref) 1.00 (0.95, 1.06) 0.99 (0.94, 1.05) 1.01 (0.95, 1.06) 0.98 (0.92, 1.04) 0.49 Calcium 3 Events/person-year 3618/76, /77, /74, /69, /58,844 Model (ref) 0.99 (0.94, 1.03) 0.99 (0.94, 1.03) 0.98 (0.93, 1.03) 0.97 (0.92, 1.02) 0.21 Model (ref) 0.98 (0.94, 1.03) 0.99 (0.94, 1.04) 0.98 (0.93, 1.03) 0.97 (0.92, 1.03) 0.35 Events/person-year 2754/ / / / /6967 Model (ref) 1.03 (0.97, 1.08) 1.03 (0.98, 1.09) 1.05 (1.00, 1.11) 1.05 (0.99, 1.11) 0.05 Model (ref) 0.99 (0.94, 1.04) 0.98 (0.93, 1.03) 1.00 (0.95, 1.06) 0.99 (0.93, 1.05) 0.95 Vitamin D 3 Events/person-year 3697/76, /74, /73, /71, /61,695 Model (ref) 0.98 (0.93, 1.02) 0.99 (0.94, 1.03) 0.95 (0.90, 0.99) 0.94 (0.89, 0.99) Model (ref) 0.99 (0.95, 1.04) 1.00 (0.95, 1.05) 0.96 (0.92, 1.01) 0.96 (0.91, 1.01) 0.07 Events/person-year 2830/ / / / /7849 Model (ref) 1.03 (0.97, 1.08) 1.04 (0.99, 1.09) 1.01 (0.96, 1.07) 0.97 (0.92, 1.03) 0.10 Model (ref) 1.00 (0.95, 1.06) 1.01 (0.96, 1.06) 0.99 (0.93, 1.04) 0.95 (0.90, 1.01) 0.06 Lactose Events/person-year 3468/67, /73, /72, /73, /70,604 Model (ref) 1.00 (0.95, 1.05) 0.97 (0.92, 1.02) 0.96 (0.91, 1.01) 0.91 (0.86, 0.95), Model (ref) 1.00 (0.95, 1.05) 0.99 (0.94, 1.04) 0.98 (0.93, 1.02) 0.92 (0.87, 0.97) Events/person-year 2784/ / / / /8387 Model (ref) 1.04 (0.99, 1.10) 1.02 (0.96, 1.07) 1.05 (0.99, 1.10) 1.00 (0.95, 1.06) 0.88 Model (ref) 1.01 (0.96, 1.07) 0.99 (0.94, 1.05) 1.02 (0.96, 1.07) 0.99 (0.93, 1.04) Diet was assessed in 1980, 1984, 1986, 1990, 1994, and Nutrient intakes were cumulatively updated over follow-up. Model 1 is a Cox proportional hazards model adjusted for total energy intake (continuous) and stratified by age in months and period. Model 2 is a Cox proportional hazards model adjusted as for model 1 covariates as well as age at menarche (,10, , , $15 y), age at first birth and parity (nulliparous,,25 y and 1 or 2 children,,25 y and.2 children, $25 y and 1 or 2 children, $25 y and.2 children), moderate to vigorous activity (quintiles), 1980 height (quintiles), BMI (,21, , , , $30 kg/m 2 ), oral contraceptive use (never; current user; former user,,5 y; former user, $5 y), smoking (never; former; current, 1 14 cigarettes/d; current, cigarettes/d; current, $25 cigarettes/d), marital status (married or not married), red meat consumption (0, 0.1 1, 1.1 2, 2.1 3,.3 servings/d), and egg consumption (0 1/mo, 1.1/mo 2/wk, 2.1 4/wk, 4.1 6/wk,.6/wk). Q, quintile; ref, reference. 2 Calculated by using the median of each category as a continuous term. 3 Total calcium and vitamin D include intake from food and supplements Carwile et al.

8 Acknowledgments The authors thank Donna Spiegelman, Ellen Hertzmark, and Molin Wang of the Harvard School of Public Health for their advice on statistical methods. J.L.C., W.C.W., and K.B.M. designed the research; J.L.C. analyzed the data and wrote the manuscript; W.C.W. and K.B.M. provided important consultation; and J.L.C. has primary responsibility for the final content. All authors read and approved the final manuscript. Literature Cited 1. Gold EB, Bromberger J, Crawford S, Samuels S, Greendale GA, Harlow SD, Skurnick J. Factors associated with age at natural menopause in a multiethnic sample of midlife women. Am J Epidemiol. 2001;153: McKinlay SM, Brambilla DJ, Posner JG. The normal menopause transition. Maturitas. 2008;61: Atsma F, Bartelink ML, Grobbee DE, van der Schouw YT. Postmenopausal status and early menopause as independent risk factors for cardiovascular disease: a meta-analysis. Menopause. 2006;13: Kritz-Silverstein D, Barrett-Connor E. Early menopause, number of reproductive years, and bone mineral density in postmenopausal women. Am J Public Health. 1993;83: Kelsey JL, Gammon MD, John EM. Reproductive factors and breast cancer. Epidemiol Rev. 1993;15: Karageorgi S, Hankinson SE, Kraft P, De Vivo I. Reproductive factors and postmenopausal hormone use in relation to endometrial cancer risk in the Nurses Health Study cohort Int J Cancer. 2010;126: Cooper GS, Sandler DP. Age at natural menopause and mortality. Ann Epidemiol. 1998;8: Jacobsen BK, Heuch I, Kvale G. Age at natural menopause and all-cause mortality: a 37-year follow-up of 19,731 Norwegian women. Am J Epidemiol. 2003;157: Farlow DW, Xu X, Veenstra TD. Quantitative measurement of endogenous estrogen metabolites, risk-factors for development of breast cancer, in commercial milk products by LC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci. 2009;877: Vicini J, Etherton T, Kris-Etherton P, Ballam J, Denham S, Staub R, Goldstein D, Cady R, McGrath M, Lucy M. Survey of retail milk composition as affected by label claims regarding farm-management practices. J Am Diet Assoc. 2008;108: Andersson AM, Skakkebaek NE. Exposure to exogenous estrogens in food: possible impact on human development and health. Eur J Endocrinol. 1999;140: Brinkman MT, Baglietto L, Krishnan K, English DR, Severi G, Morris HA, Hopper JL, Giles GG. Consumption of animal products, their nutrient components and postmenopausal circulating steroid hormone concentrations. Eur J Clin Nutr. 2010;64: Giovannucci E, Pollak M, Liu Y, Platz EA, Majeed N, Rimm EB, Willett WC. Nutritional predictors of insulin-like growth factor I and their relationships to cancer in men. Cancer Epidemiol Biomarkers Prev. 2003;12: Heaney RP, McCarron DA, Dawson-Hughes B, Oparil S, Berga SL, Stern JS, Barr SI, Rosen CJ. Dietary changes favorably affect bone remodeling in older adults. J Am Diet Assoc. 1999;99: Holmes MD, Pollak MN, Willett WC, Hankinson SE. Dietary correlates of plasma insulin-like growth factor I and insulin-like growth factor binding protein 3 concentrations. Cancer Epidemiol Biomarkers Prev. 2002;11: Ma J, Giovannucci E, Pollak M, Chan JM, Gaziano JM, Willett W, Stampfer MJ. Milk intake, circulating levels of insulin-like growth factor-i, and risk of colorectal cancer in men. J Natl Cancer Inst. 2001;93: Randolph JF Jr, Zheng H, Sowers MR, Crandall C, Crawford S, Gold EB, Vuga M. Change in follicle-stimulating hormone and estradiol across the menopausal transition: effect of age at the final menstrual period. J Clin Endocrinol Metab. 2011;96: Sowers MR, Zheng H, McConnell D, Nan B, Harlow SD, Randolph JF Jr. Estradiol rates of change in relation to the final menstrual period in a population-based cohort of women. J Clin Endocrinol Metab. 2008;93: Bromberger JT, Matthews KA, Kuller LH, Wing RR, Meilahn EN, Plantinga P. Prospective study of the determinants of age at menopause. Am J Epidemiol. 1997;145: Dorjgochoo T, Kallianpur A, Gao YT, Cai H, Yang G, Li H, Zheng W, Shu XO. Dietary and lifestyle predictors of age at natural menopause and reproductive span in the Shanghai Women s Health Study. Menopause. 2008;15: Nagata C, Takatsuka N, Kawakami N, Shimizu H. Association of diet with the onset of menopause in Japanese women. Am J Epidemiol. 2000;152: Nagel G, Altenburg HP, Nieters A, Boffetta P, Linseisen J. Reproductive and dietary determinants of the age at menopause in EPIC-Heidelberg. Maturitas. 2005;52: Torgerson DJ, Thomas RE, Campbell MK, Reid DM. Alcohol consumption and age of maternal menopause are associated with menopause onset. Maturitas. 1997;26: Yasui T. Endometriosis as a risk factor for early menopause. Climacteric. 2008;II: Suppl 2:SM Michels KB, Willett WC. Self-administered semiquantitative food frequency questionnaires: patterns, predictors, and interpretation of omitted items. Epidemiology. 2009;20: Feskanich D, Willett WC, Colditz GA. Calcium, vitamin D, milk consumption, and hip fractures: a prospective study among postmenopausal women. Am J Clin Nutr. 2003;77: Willett WC, Howe GR, Kushi LH. Adjustment for total energy intake in epidemiologic studies. Am J Clin Nutr. 1997;65 Suppl:1220S 8S; discussion 9S 31S. 28. Salvini S, Hunter DJ, Sampson L, Stampfer MJ, Colditz GA, Rosner B, Willett WC. Food-based validation of a dietary questionnaire: the effects of week-to-week variation in food consumption. Int J Epidemiol. 1989;18: Willett W. Nutritional epidemiology. New York: Oxford University Press; Colditz GA, Stampfer MJ, Willett WC, Stason WB, Rosner B, Hennekens CH, Speizer FE. Reproducibility and validity of self-reported menopausal status in a prospective cohort study. Am J Epidemiol. 1987; 126: Hu FB, Stampfer MJ, Rimm E, Ascherio A, Rosner BA, Spiegelman D, Willett WC. Dietary fat and coronary heart disease: a comparison of approaches for adjusting for total energy intake and modeling repeated dietary measurements. Am J Epidemiol. 1999;149: Kato I, Toniolo P, Akhmedkhanov A, Koenig KL, Shore R, Zeleniuch- Jacquotte A. Prospective study of factors influencing the onset of natural menopause. J Clin Epidemiol. 1998;51: Willett W, Stampfer MJ, Bain C, Lipnick R, Speizer FE, Rosner B, Cramer D, Hennekens CH. Cigarette smoking, relative weight, and menopause. Am J Epidemiol. 1983;117: Courant F, Antignac JP, Laille J, Monteau F, Andre F, Le Bizec B. Exposure assessment of prepubertal children to steroid endocrine disruptors. 2. Determination of steroid hormones in milk, egg, and meat samples. J Agric Food Chem. 2008;56: Pape-Zambito DA, Roberts RF, Kensinger RS. Estrone and 17betaestradiol concentrations in pasteurized-homogenized milk and commercial dairy products. J Dairy Sci. 2010;93: Remesar X, Tang V, Ferrer E, Torregrosa C, Virgili J, Masanes RM, Fernandez-Lopez JA, Alemany M. Estrone in food: a factor influencing the development of obesity? Eur J Nutr. 1999;38: Qin LQ, Xu JY, Wang PY, Ganmaa D, Li J, Wang J, Kaneko T, Hoshi K, Shirai T, Sato A. Low-fat milk promotes the development of 7,12- dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats. Int J Cancer. 2004;110: Fung TT, Hu FB, Barbieri RL, Willett WC, Hankinson SE. Dietary patterns, the Alternate Healthy Eating Index and plasma sex hormone concentrations in postmenopausal women. Int J Cancer. 2007; 121: Cadogan J, Eastell R, Jones N, Barker ME. Milk intake and bone mineral acquisition in adolescent girls: randomised, controlled intervention trial. BMJ. 1997;315: Kaufman FR, Kogut MD, Donnell GN, Goebelsmann U, March C, Koch R. Hypergonadotropic hypogonadism in female patients with galactosemia. N Engl J Med. 1981;304: Dairy consumption and age at natural menopause 1649