Properties of Hormone Parameters
The estrogen metabolite parameters during the follicular phase were generally highly correlated with each other (r = 0.71 to 0.84) and with the luteal-phase average (r = 0.66 to 0.71), but showed more moderate correlations with the baseline value (r = 0.42 to 0.71). The progesterone parameters during the luteal phase were even more highly correlated (r = 0.91 to 0.94), but less so with the baseline and the follicular-phase average (r = 0.38 to 0.54). The daily average of the estrogen metabolite during the entire cycle was highly correlated with its average during the follicular phase (r = 0.89), as well as with its peak (r = 0.83); similarly, the daily average of the progesterone metabolite throughout the cycle was highly correlated with its average during the luteal phase (r = 0.95) and with its peak (r = 0.90). The estrogen and progesterone metabolite parameters were only weakly correlated to each other (r = −0.003 to 0.13).
The variability of the hormone parameters across all cycles includes components of between- and within-woman variation (Table 2). Assay variation may also play a role, as well as time elapsed between laboratory assays. As expected, the mean variability within a woman’s cycles (Table 2, last column) is less than that between women by about one-third. Had we examined hormone parameters on the woman level, the variation would be about 20–25% less than for the overall cycle-based analysis.
Compared with average length cycles, cycles with a short follicular phase had elevations on the order of 10–13% in the estrogen baseline and follicular-phase average parameters, and a decrease in the estrogen total (Table 3). The baseline progesterone value was also elevated by 29%. Examining the minimum PdG from the 5-day moving average (intercept 0.53 ± 0.02), short follicular phases showed elevations but on the order of 16% (β = 0.09; 95% CI = 0.02 to 0.15). There was also some elevation of the luteal-phase progesterone parameters, particularly the peak. Long follicular phase was characterized by higher estrogen values, particularly the total (as might be expected given the greater number of days summed) and the luteal-phase average (Table 3). However, the daily average follicular-phase estrogen values were somewhat reduced.
Both long and short follicular phases were associated with lower progesterone parameters in the luteal phase of the previous cycle (Table 3). The hormone associations found by follicular-phase length were generally observed with short and long total cycle length as well (data not shown). This is expected because more than 84% of the variance in cycle length is attributable to follicular-phase length compared with 3% explained by luteal-phase length. 18
Cycles with short luteal phases generally had higher estrogen metabolite levels by 8–19% (Table 4). Progesterone parameters were generally decreased among cycles with short luteal phases and increased among those with long luteal phases (Table 4). The minimum PdG (alternative for the baseline) was similarly elevated in cycles with long luteal phases.
Demographic and Reproductive Covariates
Table 5 shows the adjusted mean differences for the E1C parameters by covariates; we did not include the results for the follicular-phase average in the because it was so highly correlated with the other estrogen parameters. The variables most consistently related in univariate analyses were gravidity, prior induced abortion, and older age at menarche, but the latter two were dampened after adjustment. In the adjusted model, estrogen parameters declined in a dose-response manner for number of prior pregnancies, decreasing by about 20% for three or more. In contrast, the estrogen parameters were greater among women who had prior induced (but not spontaneous) abortions. Most estrogen parameters were somewhat decreased among cycles of women with an older age at menarche. The adjusted estrogen parameters showed little association with age at first livebirth. Among women with livebirths, the estimates for history of induced abortion were greatly attenuated.
In the univariate models, Asians had somewhat lower estrogen metabolite values than whites, but less so in the multivariate models. Accordingly, they were combined with the small number of women of “other” races, who shared similar hormone values (Table 5). Except for the baseline measurement, Hispanics had estrogen parameter values 7–13% higher than whites, with a greater difference after adjustment.
The progesterone parameters were strongly related to high BMI, which had lower values of all the luteal-phase parameters (Table 6). Prior induced abortion and less education also were associated with lower progesterone values. Nonwhite, non-Hispanic women had a reduced progesterone baseline level (on the order of 15%), whereas Hispanic women did not differ much from white women by any PdG parameter. Cycles of women with an earlier or later age at menarche also had a lower baseline progesterone value. Older age at first birth (≥30 years) was strongly associated with lower adjusted luteal-phase progesterone levels (by 10–15%). Among women with livebirths, associations observed with reduced progesterone parameters were strengthened, such as those for history of induced abortion, higher BMI and lower education, and also for older age at menarche.
A strength of this study is that it examines urinary steroid levels on a daily basis, through multiple cycles, in relation to demographic and menstrual cycle characteristics among a relatively large sample of premenopausal women. As the sample was not clinic-based and women had to have had a recent menstrual period to qualify, little selection by adverse health status should have occurred. Rather, as hormone parameters were examined only among ovulatory cycles, this sample probably had a lower-than-average likelihood of menstrual problems. The sample is not representative of the general population, in that subjects were willing to adhere to a labor-intensive system of urine collection and had to speak English; three-quarters of the women were white, and they tended to be highly educated and parous. This may have produced less variation in the hormone parameters within our sample than would be found in the wider population.
Although it is much more feasible to collect urine than serum for measurement of daily hormone levels, the urine levels reflect metabolism, which varies by woman depending on several intrinsic and extrinsic factors that we may not have been able to account for. Another potential limitation of the study includes the measurement of only one estrogen metabolite that may vary among women in how well it represents serum concentrations. In addition, we did not have duplicate samples for hormone assays. However, all cycles that were non-ovulatory or had an unusual day of ovulation were reassayed (24%), and the more normal and thus more conservative pattern was used for analyses. 18 Furthermore, the hormone parameters we defined were based on combining values from several days, which minimized the effect of any single extreme value.
Hormone Parameter Characteristics
Analyses were conducted with cycles as the unit of observation, accounting for repeated measures. Calculating mean hormone levels by woman would have masked the presence of within-woman variation (between cycles). Other than menstrual parameters and the amount smoked, our independent variables were primarily woman-level characteristics, but varying exposures may affect cycle level differences and should be examined in future research. As expected, the multiple parameters for each steroid metabolite were correlated and thus would not all need to be examined. The total area under the curve may be an important parameter clinically, as it represents a measure of the full “dose” or ovarian secretion of the hormone during the specified phase. This is influenced by phase length to some extent, whereas the daily average is less so. The estrogen peak does not add information over other E1C parameters, but if only limited sampling or assay is possible, measuring E1C on several days around ovulation may provide a marker of the other estrogen parameters. As the baseline levels were less correlated with the other hormone parameters and these levels are more likely to reflect non-ovarian function (eg, adrenal production), it appears useful to examine them separately. The generalized progesterone baseline is the lowest of several other time frames examined (days 3–7, 4–8 and 5–9) for typical cycles, but is influenced by short cycle length (or early ovulation). The minimum PdG determined individually for each cycle limits this problem, but requires a full cycle of data for calculation. Nevertheless, results by covariates were very similar for these two variables. In general, urinary estrogen and progesterone parameters were very weakly correlated.
Menstrual cycle characteristics presumably reflect underlying hormone patterns. However, the nature of this relation may be difficult to discern because of the variety of ways in which the hormones can be parameterized. We observed that cycles with short follicular phases had high baseline estrogen and mean estrogen during the follicular phase. Short follicular phases were also associated with higher progesterone parameters, particularly at the baseline and peak. Perhaps the hormonal feedback loop is triggered earlier in cycles with elevated baseline steroid levels, compressing time for follicle development. Women with short cycles will experience more cycles during their reproductive lifetime, thus receiving more frequent pulses of ovarian hormones and a greater daily average exposure. Cycles with a long follicular phase (which may indicate delayed follicle recruitment) had slightly lower daily average estrogen metabolite levels, but an elevation in the total follicular-phase E1C and the luteal-phase average. Another study that measured estrogen metabolites 25 similarly found higher average metabolite levels in short follicular phases and did not find appreciably lower levels in cycles with long follicular phases. In a study with daily plasma samples, 26 the follicular-phase length and cycle length were inversely related to various estradiol parameters (mean, peak and baseline), supporting our findings regarding short cycles.
We, as well as others, 26 found that luteal-phase progesterone parameters were directly associated with luteal-phase length. Therefore, measurement of luteal-phase length may be an adequate surrogate for relative PdG levels. In contrast, greater estrogen levels were observed in cycles with short luteal phases. Interestingly, a reduced progesterone response in the luteal phase was associated with alterations in the follicular-phase length of the subsequent cycle in our data. This observation is consistent with findings 27 of a relation between the follicle-stimulating hormone profile during the luteal-follicular transition and the length of the ensuing follicular phase. Thus the reduced progesterone parameters may reflect compromised corpus luteum function that has an associated perturbation of gonadotropin secretion.
Demographic and Reproductive Characteristics
Our strongest findings were with age at menarche, pregnancy history variables, BMI and (to a lesser extent) race. We found generally lower estrogen parameter values among cycles of women with older age at menarche, supporting an inverse relation noted by some serum studies, 28–30 but not all. 31,32 Progesterone values were also lower with older age at menarche among parous women. Consistent with our findings of reduced estrogen metabolite parameters with higher gravidity, Bernstein et al.33 found that parous women had a 20% lower follicular-phase estrogen level (day 11) in serum and urine than nulliparous women. In our study, age at first livebirth was not related to any estrogen parameter, but was inversely associated with progesterone parameters. Two studies 16,33 support these findings with estrogen, whereas another study 32 found an inverse association. The only study 16 that examined progesterone did not find an association. We found history of induced abortion associated with reduced progesterone, but with elevated estrogen. These associations with reproductive history were also observed in models limited to average-length cycles. Thus, these patterns do not appear to reflect variation in cycle characteristics, as confirmed by our previous analysis 18 in which we did not find differences in cycle or follicular-phase length with these variables.
Our observation that women with a high BMI had cycles with lower mean luteal-phase progesterone metabolite levels is consistent with other recent findings. 16,34 Gold et al.15 reported a higher peak PdG in women with low BMI; although we did not observe this, their finding supports an inverse association. BMI was not strongly related to estrogen parameters in our study, nor in several other studies of similar-aged, primarily parous women. 16,30,35
By ethnic group, we found that nonwhite, non-Hispanic women (primarily Asians) tended to have lower mean estrogen and progesterone metabolite levels than white women, but the associations were generally reduced after adjustment. Other studies 36,37 have found that Asians had lower estrogen levels than European women; one of these studies suggested different routes of estradiol metabolism. 37 In our study, Hispanics had higher estrogen metabolite levels than whites. However, the magnitude of this difference was reduced by about half among average length cycles, indicating that some of the hormonal differences may reflect the longer mean cycle length we found in Hispanics. 18 A study of parous women 16 did not report strong associations with race, but it was based on small numbers. In that study, Hispanics also tended to have higher urinary estrogen (12%) in the mid-luteal phase than whites. We are not aware of any study of follicular-phase estrogen levels in Hispanics. In addition to biological differences, variation by ethnicity may reflect life-style differences. Although we have information on a number of covariates, there are some, such as diet, for which we have no data.
We found little association between age (within 18–39 years old) and the estrogen parameters. Other study results are inconsistent with respect to age and luteal-phase estrogen values, 16,30,32 but several report elevated follicular-phase serum estradiol levels as age increases, within a similar reproductive age range as our study. 30,32,33,35
It is commonly accepted that ovarian hormones play a critical role in breast cancer risk, and there have been several hypotheses proposed for the mechanism. 2,4 Therefore, we examined various risk factors associated with breast cancer, by their steroid metabolite levels. Factors considered protective for breast cancer such as parity, older age at menarche, and Asian ethnicity were associated with lower estrogen parameter values. This supports the hypothesis that these factors affect breast cancer risk through an estrogen mechanism. Similarly, we observed higher estrogen values with history of induced abortion, a suspected risk factor for breast cancer. Increasing weight in premenopausal women is not associated with an increased risk of breast cancer, 2 and so the lack of an association of BMI with urinary estrogen is consistent. However, these findings do not preclude a possible relation in older women. Examining progesterone with respect to breast cancer risk factors, we found slightly lower progesterone parameter values with the protective factors of older age at menarche and nonwhite, non-Hispanic race/ethnicity. However, progesterone levels were also reduced with purported risk factors such as older age at first birth and induced abortion. 1 Short cycle length has been associated with an increased risk of breast cancer 1,38; we found that with cycles short follicular phases had higher baseline levels of both steroids, higher follicular-phase daily estrogen levels, and generally higher luteal-phase progesterone levels.
In conclusion, specific cycle characteristics appear to be representative of consistent alterations in steroid metabolite patterns, providing a crude surrogate of hormonal milieu. In particular, luteal-phase length is directly related to progesterone metabolite levels. Also, short follicular phases (and thus short cycles) had elevated baseline steroid and average follicular-phase estrogen metabolite levels, preceded by reduced progesterone levels in the previous luteal phase. Several reproductive characteristics were associated with variation in urinary steroid levels. It appears that these would not be revealed by a study of menstrual cycle characteristics alone, 18 as these variations occurred in average-length cycles as well.
We acknowledge the contributions of Robert Hiatt and Catherine Schaeffer of Kaiser Permanente’s Division of Research in the original design and conduct of the study. We thank David Epstein for managing the fieldwork, Ceciley Wilder and David Paniagua for manuscript preparation, and Sioban Harlow and Peggy Reynolds for valuable comments on earlier drafts.
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Keywords:© 2002 Lippincott Williams & Wilkins, Inc.
hormones; estrogen; menstrual cycle; progesterone; breast cancer; women’s health