| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Clinical Investigations |
Departments of Preventive Medicine and Community Health [L.-J. W. L., K. E. A., J. J. G.] and Obstetrics and Gynecology [M. N.], The University of Texas Medical Branch, Galveston, Texas 77555, and Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel [F. K.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ovarian hormone levels (19, 20, 21, 22) and related reproductive factors (e.g., age of menarche, menopause, and parity; Refs. 4 and 5 ) are known to influence breast cancer development. Increased levels of estrogens in blood and urine correlate with increased risk for breast cancer (22, 23, 24) . Serum levels of estrogens and androgens are in general lower in women who live in or have recently immigrated from low-risk areas for breast cancer, such as rural China and Japan, than in women from high-risk areas such as Britain and the United States (20 , 23 , 25 , 26) . Large cohort studies, one involving women in New York (27) and another the nationwide Nurse’s Health Study (28) , found a positive association of serum estrogens and androgens and breast cancer development. 17ß-Estradiol stimulates breast and endometrium cell proliferation (21) . Progesterone antagonizes the proliferative effect of 17ß-estradiol on the endometrium. However, the fact that breast cell proliferation increases during the luteal phase of the menstrual cycle, when progesterone concentrations are the highest, suggests that progesterone may enhance breast cell proliferation (21 , 29) .
Soya consumption may in part account for geographical variation in breast cancer incidence. Soya contains many chemopreventive components, including Bowman-Birk protease inhibitors, inositol phosphates, phytosterols, saponins, and the isoflavones genistein and daidzein (12) . Mechanisms underlying the oncoprotective effects of soya have been studied in cell cultures and laboratory animals. For example, isoflavones have been shown to have estrogen agonist and antagonist effects and can induce differentiation and inhibit cell proliferation, angiogenesis, tyrosine kinase, and topoisomerase II. Bowman-Birk protease inhibitor influences cell transformation. In a previous study, we found that circulating ovarian hormone levels were decreased by 1 month of soya consumption in six premenopausal women when measured on 3 different days during the menstrual cycle (30) . Because of the cyclic nature of ovarian hormones, to better assess their changes induced by a soya diet, we have measured circulating levels of ovarian hormones and gonadotropins in 10 women over an entire cycle once during usual home diets and once during a soya diet.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Subject Selection.
Subjects were premenopausal women who were healthy as determined by
history, physical examination, standard blood cell counts, clinical
chemistry determinations, and serum ferritin levels. Those who were
vegetarians or smokers, consumed more than two alcohol-containing
drinks/month, had experienced recent significant changes in weight or
eating habits, had taken antibiotics within the preceding 3 months, had
irregular menstrual cycles, or had taken contraceptive medications
during the preceding 6 months were excluded. Contraceptive medications
were not allowed during the study. Small doses of acetaminophen or
aspirin were permitted. One subject was taking replacement
levothyroxine, 0.1–0.25 mg/day, for hypothyroidism and was determined
to be euthyroid. Another was taking sertraline, 25 mg/day, for mild
depression before and during the study.
Baseline Study Period.
Day 1 of each cycle (the first day of menstrual bleeding) was recorded
throughout the study. After enrollment, subjects underwent baseline
studies as outpatients on the
GCRC3
for at least 3 months but no more than 6 months while consuming their
usual home diets prior to being placed on a soya diet for 1 month. Soy
products were not part of the usual diet of any subject, and all were
instructed to avoid soy products during the baseline observation
period. The major purposes of the baseline observations were to assure
that the subjects had regular cycles and to record cycle length. During
the first month of the baseline period, blood was obtained on cycle
days 5 (follicular phase), 12 (mid-cycle), and 22 (luteal phase) for
measurement of 17ß-estradiol and progesterone. Subjects were retained
in the study if luteal phase progesterone levels exceeded 4 ng/ml.
During the second month of the baseline period, blood samples were
collected on cycle days 5 and 7 and then daily from day 9 through the
second day of the subsequent cycle. After a rest period of at least one
menstrual cycle, subjects were studied as inpatients on the GCRC
beginning on cycle day 2 and discharged on cycle day 2 of the next
cycle. Intakes of energy, protein, carbohydrate, fat, and fiber during
consumption of usual home diets were estimated using Block’s Health
Habit History Questionnaire (31)
, and the values were used
for comparison with intakes during the soya diet.
Soya Diet Period.
Subjects consumed a soya-containing diet for one menstrual cycle on the
GCRC. Meals and soymilk were consumed under direct supervision. Soymilk
used for this study was an homogenized, pasteurized preparation
containing no preservatives, purchased in lots (Banyang Food Co.,
Houston, TX), stored frozen, and thawed on the day of consumption by
the subjects. The soya diet, including both soymilk and non-soya foods,
for each subject was calculated to match that needed to maintain
constant body weight, based on the Harris-Bennedict equation, with
adjustment for physical activity (32
, 33)
. The soya diet
consisted of three rotating daily menus and included a 36-ounce portion
of soymilk daily that provided 400 kilocalories, 37.9 g of
protein, 20.3 g of fat, and 16.6 g of carbohydrate (analysis
by Protein Technology Inc., St. Louis, MO). The content of protein,
carbohydrate, and fat did not vary significantly between different lots
of soymilk. Soymilk was ingested between 5 and 8 p.m. without
other foods and in place of the evening meal. The energy distribution
of the soya diet was 35.5% fat, 14.0% protein, and 50.1% from
carbohydrate daily, which is similar in macronutrient distribution to
that consumed by many residents of Western countries (34)
.
Subjects continued their usual daily activities, including work, study, and exercise. Basal body temperatures were recorded daily to determine the time of ovulation. Fasting blood samples were obtained between 7 and 9 a.m. on cycle days 5, 7, and 9 and then daily until the second day of the next cycle for measurement of 17ß-estradiol, progesterone, gonadotropins, daidzein, and genistein. Two 12-h urine samples were collected daily starting on cycle day 2 and continuing until cycle day 2 of the next cycle for analysis of urinary excretion of daidzein and genistein. Sera were separated and stored immediately at -80°C until analysis. Urine samples were refrigerated during collection and then stored at -20°C until analyzed.
Blood cell counts, liver function tests, serum calcium, phosphorous, creatinine, cholesterol, HDL, LDL, triglycerides, and ferritin were determined on cycle days 3 and 15, as well as on day 2 of the next cycle during the baseline and the soya diet periods. If the serum ferritin fell below 10 ng/ml during the study, an oral iron supplement was provided.
Hormone Analysis.
17ß-Estradiol concentrations in the serum, after extraction with
hexane and ethyl acetate (v/v, 3:2), were measured by a specific RIA as
described previously (35)
. Progesterone levels were
measured by direct RIA using commercial kits (Diagnostic Laboratory
Inc., Webster, TX; Ref. 30
). Blank and control sera were
run with each assay. Assays were performed in duplicate. Levels of LH
and FSH were measured by immunoradiometric assay using commercial kits
(Diagnostic Laboratory, Inc.). The intra-assay coefficients of
variation were 4–8%, and inter-assay variation was 5–9%. All of the
samples from each subject, i.e., baseline and treatment
samples, were analyzed together in a single batch.
Analysis of Soya Isoflavones Genistein and Daidzein in Soymilk
and in Urine.
Isoflavone content in soymilk and urine was analyzed by a gas
chromatography flame ionization detection method as described
previously (36)
. Amounts of isoflavones in soymilk or
urine were expressed as amounts of the aglycone forms.
Serum Levels of Daidzein and Genistein.
Serum levels of daidzein and genistein were analyzed by competitive
enzyme-linked immunoassays, using monoclonal antibodies generated
against daidzein and genistein, and horseradish peroxidase conjugates
of daidzein and genistein as tracers, as described previously
(37
, 38) . The detection limit of the assay is 0.5 ng/well.
Sera were obtained 
15 h after soymilk ingestion, and daily blood
samples of each study cycle were individually measured for daidzein and
genistein. Mean values for the entire cycle were calculated.
Data Analysis.
The outcome measures were serum concentrations of 17ß-estradiol,
progesterone, LH, FSH, and menstrual cycle length. Because of the
cyclic nature of these hormones, summary measures of the data were used
in the data analysis (39)
. The hormone data were expressed
as AUCs, mean daily levels, peak levels, and rates, as appropriate for
assessing changes in hormones over two entire cycles under the two
different dietary conditions.
Serum levels of hormones, obtained once every other day initially and daily after day 9 until day 2 of the next cycle, were used to calculate area under the serum concentration versus time curves (AUC, concentration x time) using WinNonlin software (Scientific Consulting, Inc., Cary, NC). AUC represents the integrated or cumulative exposure to these cyclic hormones during each dietary period. AUC was divided by the number of days to obtain the mean daily level. Peak levels represent the maximum levels recorded during the cycle and during the follicular and luteal phases. The rates of increase in 17ß-estradiol level leading up to the follicular (or mid-cycle) peak were calculated for each subject by solving for the parameter b in the second order polynomial regression equation: Y = intercept + bX + cX2, where Y is the serum 17ß-estradiol level at day X of the cycle, using the day of the LH surge as the reference point. The statistical analysis compared the b estimates from before and during the soya diet. AUCs and mean daily levels were calculated for the entire cycle and for the follicular and luteal phases using the day of the serum LH surge as a reference point. The LH surge represents the end of the follicular phase and the beginning of the luteal phase. For graphical analysis of the time courses of hormone levels, values were plotted using the day of serum LH surge as a reference point.
Each outcome summary measure, i.e., cycle length, AUC, mean daily levels, peak levels, and rates of increase, was analyzed across the entire cycle and then also within the follicular and the luteal phases. Most comparisons were for within-subject changes and used paired t tests or Wilcoxon signed rank tests.
Secondary analyses by multiple regression were carried out to further
explore the possible mechanisms of the hormone changes between home
diet and soya diet periods as related to study-associated variables.
The equation for the regression analysis for changes in hormone,
Y, is
 |
where ß0 is the intercept and
ß1 is the parameter for one of the predictor
variables of isoflavone levels, i.e., intakes, plasma
levels, or urinary levels of daidzein, genistein, or the sum of
daidzein and genistein. The second variable in the model associated
with parameter ß2 can be viewed as a covariate
adjustment using one of the five possible variables for nutrients
(i.e., home - soya values for: energy, protein, fat,
carbohydrate, or fiber), age, or BMI. The final term is the interaction
of the two main effects, X1 and
X2. The residual error is denoted as
. Each isoflavone level predictor variable was fitted separately
with changes in hormone levels expressed as AUCs, mean daily levels,
and peak values. Each of these models was then adjusted for age, BMI
(kg/m2), or nutrient intake variables. Because of
the small sample size, it was necessary to make each adjustment
separately. To identify highly correlated variables for the regression
models (R > 0.5), pair-wise correlations
were computed. Because hormonal profiles are different in the
follicular and luteal phases, the models were obtained separately for
the total cycle and for the follicular and luteal phases. Not
surprisingly, AUC, mean daily, and peak level were influenced in the
same manner by all factors examined, and therefore, model results only
for mean daily levels are presented. Likewise, because results for the
total cycle were generally consistent with the results of separate
analyses for the follicular and luteal phase, only the latter two are
presented. For brevity of presentation, groups of related regression
models are reported as a range of R2
values plus the maximum P (e.g., all
P 
0.05).
All statistical analyses were performed using SAS (SAS Institute, Inc., Cary, NC). All results were expressed as mean ± SE, and all Ps are from two-tailed tests.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. They were (137 ± 7)% (mean ± SE) of
ideal body weight and had regular cycles (CV in cycle length during the
3-month baseline of 5.7–11%). Seven were Caucasians, three were
African-Americans, and nine were nulliparous. (Table 1)
. The
demographic variables of the dropouts were in general very similar to
those who completed the study regarding age (29.7 ± 7.7
years; P = 0.31, Student’s t
test), body weight (69.9 ± 13.9 kg;
P = 0.77), height (1.7 ± 0.7 m; P = 0.55), and BMI
(25.1 ± 5.1 kg/m2;
P = 0.40) except race (3 African-Americans, 1
Asian, 3 Hispanics, and 9 Caucasians). The dropouts all occurred during
the baseline observation period. The main reason for the dropouts was
frequency of blood drawing.
|
.
For the group, average energy and fat provided by home diets and the
soya diet did not differ significantly. The soya diet on average
provided more energy from carbohydrate than did the home diets (50.1
versus 45.3% of calories; P = 0.02), less energy from protein (14 versus 17%;
P = 0.01), and less fiber (6
versus 18.3%; P = 0.001; Table 1
). There was individual variation in these dietary differences.
Intakes of Soya Isoflavones.
The soymilk used in this and previous studies (36)
contained the isoflavones daidzein and genistein, mostly as conjugates
(85 mol-% glucoside conjugates and 15 mol-% free). Because
soybean isoflavone content varies with season of harvesting and storage
(40)
, there was variation in isoflavone content between
lots. Because different subjects ingested soymilk from different lots,
isoflavone intake varied among the subjects. However, each subject
ingested the same lot throughout the soya-feeding period.
Table 2
shows that subjects ingested 68.8 ± 3.7 mg/day
(n = 10) of daidzein and 85.2 ± 5.4 mg/day of genistein. The daily total isoflavone intakes
varied from 113 to 207 mg/day (154.0 ± 8.4 mg/day).
Because of interindividual variation in cycle length as well as
isoflavone content of the soymilk lots, isoflavone intake over entire
cycles varied from 2712 to 6624 mg/cycle (4129.5 ± 329.6 mg/cycle). The individual intakes were used to determine the
association of isoflavones and observed changes in various hormones
(see below).
|
). There was also
considerable intraindividual variation in isoflavone excretion (CV,
26% for daidzein and 28% for genistein). Subjects excreted more
daidzein (35.1 ± 4.2%; range, 19.6–58.0%) than
genistein (11.3 ± 2.3%; range, 1.6 to 21.3%; Table 2
and Fig. 1
) as reported in previous studies (41
, 42)
. Average daily
levels of daidzein and genistein (sum of free and conjugated forms)
over a cycle in serum were 2.95 µg/ml (range, 0.45–6.17 µg/ml) and
0.85 µg/ml (range, 0.14–2.16 µg/ml), respectively, with
substantial interindividual variation (Table 2)
.
|
A, urinary excretion
of ingested isoflavones decreased progressively during the menstrual
cycle such that isoflavone excretion was greater during the follicular
phase than the luteal phase.
This was examined further by calculating average daily excretion during
the follicular and luteal phases for each subject. This confirmed that
the excretion of daidzein (in all 10 subjects) and genistein (in 8 of
10 subjects) were both greater during the follicular phase than the
luteal phase (daidzein excretion, 25.72 ± 3.58 mg/day
and 23.71 ± 3.35 mg/day during the follicular and
luteal phases, respectively; P = 0.006;
genistein excretion, 9.97 ± 1.99 mg/day and
8.62 ± 1.73 mg/day during follicular and luteal phases,
respectively; P = 0.03;
n = 10; Fig. 1B
).
Effects on Serum Lipids.
The effects of 1 month of soya consumption on serum lipid levels in
this group of premenopausal women are shown in Fig. 2
. Cholesterol, triglycerides, LDL, HDL, and the ratios of cholesterol to
HDL were measured three times per cycle during the baseline and during
soya diet periods, generally at cycle onset, mid-cycle, and at the end
of the cycle. Averages during both dietary periods were calculated,
with exclusion of the first measurement in the soya diet period,
because this was taken after only one day of this diet. As shown in
Fig. 2
, during the soya diet period, the average serum cholesterol
decreased by 6% (P = 0.07, paired
t test), triglyceride by 17% (P = 0.08), HDL by 6% (P = 0.23), and LDL by 4%
(P = 0.32). The ratios of cholesterol to HDL
did not change significantly (not shown).
|
, consumption of soymilk containing 113–207 mg of isoflavones/day for 1
month did not significantly alter menstrual cycle length (1.2%
decrease; P = 0.51) or follicular phase
length (2.7% increase; P = 0.67) in this
group of women but was associated with a decrease in average length of
the luteal phase that approached statistical significance (6%;
P = 0.07). By multiple regression analysis,
decreases in luteal cycle lengths among individuals were positively and
significantly associated with the amounts of daidzein and genistein
ingested or excreted and with the decrease in protein intake during
soya feeding, and there was some evidence of an interaction between
these two variables (R2, 0.51–0.54;
all P < 0.05).
|
), as were AUC (Fig. 4B
), mean daily level
(AUC/cycle length), and peak levels (Fig. 4C
) of
17ß-estradiol in most subjects. These decreases in circulating
17ß-estradiol levels were evident when analyzed over the entire
cycle, over the follicular phase, and over the luteal phase. For the
total cycle, follicular phase, and luteal phase AUCs (pg/ml x day), 17ß-estradiol decreased by 24% during soya diet
feeding (all P < 0.04; Fig. 4B
).
Average daily levels (pg/ml) of 17ß-estradiol were decreased by
19–23% during the follicular and the luteal phases of the cycle
(P 0.05; Fig. 4C
). Maximal
levels of 17ß-estradiol during the follicular phase were suppressed
to a similar degree during the soya diet (28%;
P = 0.004; Fig. 4C
).
|
The effects of intakes and levels of isoflavones, macronutrient
intakes, age, and BMI on soy-induced decreases in 17ß-estradiol were
examined by multiple regression analysis as described in "Materials
and Methods." The results showed that during the soya diet, the
decreases of both follicular and luteal phase 17ß-estradiol levels
were positively associated with subject’s age and with the urinary
levels of daidzein, genistein, or the sum of daidzein and genistein,
and with plasma levels of genistein, but were inversely related to the
interaction of age and isoflavone levels
(R2 = 0.60–0.80;
P < 0.05 for follicular phase levels; and
R2 = 0.61–0.68;
P < 0.05 for luteal phase levels). The
decrease in 17ß-estradiol was also inversely associated with change
in protein intake (R2 = 0.53–0.64; P < 0.05 for follicular phase
levels; and R2 = 0.81–0.83;
P < 0.001 for luteal phase levels), either
with or without adjustment for age or urinary isoflavone levels. This
suggests that a greater decrease in energy intake from protein during
the soya diet may blunt the inhibitory effect of isoflavones on
17ß-estradiol levels. This influence of protein intake on
17ß-estradiol levels was greater during the luteal phase than during
the follicular phase. Changes in 17ß-estradiol were not predicted by
variables such as BMI, fat intake, carbohydrate intake, or fiber
intake. None of these independent variables singularly explained the
outliers in Fig. 4
. Group mean changes did not differ between
Caucasians and African-Americans.
Effects on Progesterone Levels.
Average daily levels of progesterone for the group of subjects during
the luteal phase decreased during the soya diet (Fig. 5A
). The luteal phase AUC (Fig. 5B
;
P = 0.002), mean daily level, and mean peak
level (Fig. 5C
; P 0.004) of
progesterone were lower in all 10 subjects during the soya diet than
during the home diets. Progesterone levels were 45% lower over the
total cycle during the soya diet period than during the home diet
period. Progesterone levels during the follicular phase were generally
very low, as expected, and the difference during the two dietary
periods was not statistically significant (P = 0.84).
|
0.002), with or without
adjustment for age and intakes of protein, carbohydrate, or fat.
Intakes of energy and fiber modified the influence of isoflavone and
daidzein dose (R2 = 0.87–0.94; all P 0.05), but not genistein
dose, on progesterone level change induced by the soya diet. Urinary or
serum levels of daidzein and genistein and the sum of these isoflavones
were not significantly related to changes in progesterone. When urinary
or serum isoflavone levels were included in the regression analysis,
energy, protein, carbohydrate, fat, and fiber intakes became
significant predictors for changes in serum progesterone levels
(R2 = 0.46–0.87;
P < 0.05). By this analysis, providing more
energy during the soya diet compared with the home diet, for example,
led to a greater soya-induced decrease in progesterone level. Other
study-associated variables, such as fat intake, carbohydrate intake,
and age, were not predictors for progesterone changes. Qualitatively,
group mean changes for African-Americans (n = 3) were greater than those of Caucasians (n = 7) but without statistical significance.
Effects on Gonadotropin Levels.
Circulating levels of gonadotropins LH and FSH were measured during
both dietary periods to determine whether the soya diet might influence
17ß-estradiol and progesterone levels (Figs. 4
5
) by decreasing
the levels of these gonadotropins, perhaps by the interaction of the
weakly estrogenic isoflavones genistein and daidzein with receptors in
the pituitary and hypothalamus. As shown in Fig. 6
, consumption of the soya diet for 1 month did not influence the total
cycle, mean daily, or peak levels of LH and FSH.
|
5
6
, of our study subjects are typical of those found in
premenopausal women (44, 45, 46)
. These figures indicate that
intracycle variation is substantial for all individuals. To assess the
within-subject correlation during one cycle of home diet measurements,
autocorrelations for each subject were plotted for progesterone and
17ß-estradiol (Fig. 7
). These correlations give information about how much a single
measurement correlates with previous measurements. For 17ß-estradiol
(Fig. 7A
), measurements within a few days (e.g.,
<2 days) are highly correlated, but for >4 days apart the
correlations diminish. For progesterone (Fig. 7B
),
measurements within 3 days or >10 days apart are highly correlated,
whereas the correlations drop off for measurements 3–9 days apart. The
negative correlations for measurements >5 days apart reflect the
cyclic nature of these two ovarian hormones. The similarity of the
shapes and closeness of the lines for progesterone show that subjects
follow a similar cyclical pattern, whereas the more scattered pattern
for 17ß-estradiol shows higher between-subject variability.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
5
) and that this hormonal effect occurs
throughout the menstrual cycle. This expands the result of a prior
study during which subjects ate unrestricted while consuming soymilk as
a supplement (30)
. Furthermore, we have shown that soya
feeding has no effects on gonadotropins (Fig. 6
). The soya diet also
marginally shortened luteal phase duration (Fig. 3
) and reduced serum
lipids (Fig. 2
). Decreases in serum 17ß-estradiol were observed
during both the follicular and the luteal phases of the menstrual cycle
(Fig. 4
), whereas decreases in progesterone were observed only during
the luteal phase, when progesterone synthesis in ovaries is more active
(Fig. 5
). The results suggest that the synthesis and/or metabolism of
these two ovarian steroids may be affected by a soymilk-containing
diet.
Secondary analysis by multiple regression, the use of which was made
possible by individual differences among subjects in intakes of
isoflavones (ranging from 113 to 207 mg/day; Table 2
) and other
nutrients and in metabolism and disposition of ingested isoflavones
(Fig. 1
) provided insight into other factors in the soya diet in
addition to isoflavones that may influence these ovarian hormones.
After adjustment for these variations, isoflavone intake and plasma and
urinary levels of isoflavones were found to be associated with
soya-induced changes in ovarian hormone levels.
The isoflavones daidzein and genistein in soybeans are weak estrogens
and may bind to estrogen receptors (47)
in the pituitary
and hypothalamus and regulate ovarian hormone synthesis by influencing
production of gonadotropins. However, despite the high daily intakes of
isoflavones in this study (113–207 mg/day; Table 2
), the levels of LH
and FSH (Fig. 6
) were not significantly different between the soya and
the home dietary periods, even after controlling for individual
differences in intakes and urinary levels of isoflavones. These data
suggest that the inhibitory effects of the soya diet on ovarian steroid
hormone levels may not be mediated by gonadotropins. Two other studies
in which women ingested 45–60 mg of isoflavones/day found a
suppressive effect on serum LH and FSH concentrations (48
, 49)
. In the present study providing isoflavone doses >100
mg/day, no effects on serum LH and FSH were observed compared with
basal non-soya diet (Fig. 6
). This is consistent with the results of
Duncan et al. (49)
. In fact, in the present
study peak LH levels were higher during soya feeding in 8 of the 10
women. Comparisons of results from these different studies suggest the
possibility that low doses of isoflavones may act as estrogen agonists
in the hypothalamus and pituitary, whereas high doses do not.
Additional studies in humans over a wide range of isoflavone dosages
are needed to fully characterize whether gonadotropins mediate the
effects of soya isoflavones on ovarian steroids. Alternatively, soya
isoflavones may directly inhibit steroid synthesis enzymes in ovaries,
as has been suggested by in vitro studies
(50, 51, 52, 53)
.
Multiple regression analysis has shown that individual decreases in
17ß-estradiol level are not associated with individual differences in
isoflavone intake but are positively associated with individual plasma
and urinary levels of daidzein and genistein, and this association is
influenced by age. This result suggests that isoflavones play a role in
influencing 17ß-estradiol levels. The lack of correlation between
isoflavone intakes and changes in 17ß-estradiol is attributable to
large interindividual differences in plasma and urinary levels of
isoflavones (Fig. 1
and Refs. 41
, 42,
and
54
). We showed previously that urinary recoveries
of ingested isoflavones varied from 3 to 80% (41
, 42 , 54)
. Isoflavones in soy exist mostly as glucosides (glycones),
and the hydrolysis of these glycones to aglycones (free forms) by
intestinal flora is thought to be necessary prior to systemic
absorption (55)
. The large interindividual variability in
urinary recovery of ingested isoflavones is attributable possibly to
interindividual differences in composition of intestinal flora as
discussed previously (41)
. Thus, plasma or urinary levels
of isoflavones may be better biomarkers of soya exposure than
isoflavone intake per se.
In contrast to the positive association between individual levels of isoflavones and individual decreases of 17ß-estradiol, the inhibitory effect of soya on progesterone levels is inversely related to individual intakes of genistein with or without controlling for age and intake changes in macronutrients: protein, fat, and carbohydrate. The influence of daidzein intake on progesterone changes is affected by intakes of energy and fiber. It was surprising that despite this strong inverse relationship between isoflavone doses and decreases in progesterone, there was no correlation between urinary and serum levels of genistein and daidzein and progesterone changes. When serum and urinary levels of both isoflavones were included in the regression analysis, all macronutrient intakes became significant predictors of progesterone level changes. These data suggest that the macronutrient components of the soya diet also influence progesterone levels. Dosage of genistein provided in soymilk in this study ranged from 63–100 mg/day. It remains to be determined whether genistein intakes lower than 63 mg/day might have a different dose-response relationship with progesterone level changes. Dose-response relationships for genistein are frequently biphasic or U-shaped in many biological systems. For example, the effects of genistein on progesterone synthesis (50) , cell proliferation (56 , 57) , pituitary responsiveness to the stimulation of gonadotropin-releasing hormone (58) , and bone loss (59) can be stimulatory or inhibitory, depending upon genistein dose. Additional studies with lower doses of soya isoflavones are needed to fully characterize human responses to soya isoflavone effects.
BMI and fat intake, each of which may influence hormone levels and
cancer risk (60
, 61)
, do not explain the observed
soya-induced changes in ovarian steroid hormone levels, because none of
our study subjects gained weight during the month of soya feeding. This
is not surprising because the soya diet was eucaloric compared with the
home diets for the group. Total fat intakes for the group were similar
during both dietary periods (Table 1)
and approximated the amounts of
fat (e.g., 35%) commonly consumed by populations in the
United States (34)
. Despite the high fat content, fat
intake change is not a predictor for hormonal effects. The study
subjects consumed more energy from carbohydrate by the soya diet than
by the home diets (Table 1)
, but this change in carbohydrate intake was
not a predictor of soya-induced inhibition of ovarian hormone levels.
Protein intake was somewhat lower during the soya diet than during the
home diets of this group of study subjects (14% versus 17%
of total energy, respectively; Table 1
). This change in energy intake
from protein appears to have modified soya-induced changes in
17ß-estradiol but not progesterone. The regression data suggest that
increased energy intake from protein during the soya diet enhanced
soya-induced inhibition of 17ß-estradiol levels and modified the
influence of isoflavone levels on luteal phase lengths. Altered
fat:carbohydrate (60)
and protein:carbohydrate ratios
(62)
, where energy intake remains constant, have been
shown to affect sex hormone levels. In this study, a decrease in
dietary protein appears to have lessened the isoflavone-related
reduction in 17ß-estradiol. More dietary intervention studies are
needed to determine the role of protein and other specific nutrients on
sex hormone levels in humans.
In the present study, in which the lengths of the follicular and luteal phases were estimated based on daily measurement of serum LH, a significant decrease in luteal phase length was observed after controlling for the isoflavone levels and change in protein intake. Effects of soya feeding on menstrual cycle lengths have been examined in five other studies (30 , 48 , 49 , 63 , 64) , four of which showed an increase or a trend toward an increase in the lengths of the follicular phase or total cycle during soya ingestion (30 , 48 , 63 , 64) . By accurately measuring the occurrence of serum LH surge day, we showed that soya feeding significantly reduced luteal phase lengths after controlling for isoflavone levels. Because breast cells are more proliferative during the luteal phase than the follicular phase, a shortening of the luteal phase may reduce the length of time of proliferation of breast cells, and thereby reduce the probability of neoplastic transformation and breast cancer development.
In a prior study, in which women were provided the same amount of
soymilk as in this study for 1 month but began soymilk ingestion 3–6
days after the onset of menstrual bleeding rather than on cycle day 2
as in this study, and consumed self-selected hospital diets rather than
a controlled diet as in this study, soya feeding was found to reduce
ovarian hormone levels when measured on cycle days 5, 12, and 22
(30)
. Therefore, two different study designs involving two
different groups of premenopausal women both have shown that
consumption of a soya diet can lower circulating ovarian steroid
hormone levels (30)
. The sample size of our study is
small, and this makes inference to a larger population difficult.
However, as shown in Fig. 5
, all 10 subjects had decreases in
progesterone levels during the soy diet. On the basis of a binomial
event, the lower bound of the 95% confidence limits for the
probability of success for 10 successes in 10 trials is 0.69. This
implies that our results can be generalized to at least 70% of the
population randomly sampled. Despite a small sample size, our results
are consistent with the observation in Japanese women of a 25%
reduction of 17ß-estradiol level after consumption of soymilk
containing 109 mg isoflavones daily and also with the result of a cross
sectional study showing an inverse relationship between soya intake and
17ß-estradiol levels (64
, 65)
.
The following additional studies showed effects of soya feedings on ovarian steroids that differ from data presented here. With lower doses of isoflavones (<65 mg/day) an increase (48 , 66) or no effect (49 , 63) of soya feedings on serum 17ß-estradiol levels were found. Our present and previous studies (30) have noted an effect of soya with isoflavones on progesterone levels. Others have not noted such an effect. The studies of Duncan et al. (49) and Petrakis et al. (66) , which did not control for energy intake, as was done in the present study, and provided soya as a supplement, found no effects on 17ß-estradiol levels. Our regression analysis indicates that endocrine effects of soya feeding are complex and may relate to multiple dietary components, including doses of isoflavones as discussed above and characteristics of the subjects such as age, which may explain the differing endocrine effects of studies of soya feeding.
As shown in Fig. 7
, single measurements of blood ovarian hormone levels
have limited ability to project the actual cycle levels of ovarian
hormones in cycling women. Daily blood samples may have enhanced the
capacity of this study to detect changes in circulating ovarian
hormones. Differences in results among reported studies might also be
attributable in part to differences in soya preparations. The soya
preparation used in this study was a homogenate prepared from whole
soybeans and contained compounds other than isoflavones and might
differ from the preparations used by other investigators. The other
chemopreventive components of soya are Bowman-Birk protease inhibitor,
which was detected in our soya preparation, inositol phosphates,
phytosterols, and saponins, and these have established biological
effects in laboratory animals and in cell cultures (reviewed in Refs.
12
, 67 ). Whether the latter compounds can influence
ovarian steroids remains to be determined.
Serum concentrations of cholesterol and other lipids tended to decrease
during soya feeding (Fig. 2
). These effects approached statistical
significance (P = 0.07–0.08, in the two
tailed t test; Fig. 2
). Small decreases in serum lipids have
also been observed in other studies in subjects with normal lipid
levels (48
, 68)
. Soya has been reported to be quite
effective in lowering serum lipids in hypercholesterolemic patients
(69
, 70) . Given the role of fat in cancer risk
(61)
, a small decrease in serum lipids if it occurred over
an extended period might be significant for cancer risk reduction.
In summary, a diet maintaining energy intake and containing soya and weakly estrogenic isoflavones is effective in reducing circulating levels of ovarian hormones in premenopausal women without apparent effect on the levels of gonadotropins. Analysis of data suggests that multiple components of the soya diet including isoflavones may modulate ovarian hormone levels directly and the effect is not mediated by gonadotropins. Because decreased levels of ovarian hormones may reduce breast cell proliferation and breast cancer risk, the results of this study have implications for breast cancer prevention by dietary intervention.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by USPHS, NIH Grants CA65628, CA56273,
and CA45181, and National Center for Research Resources GCRC Grants
MO1-RR-00073 and AICR95B119. 
2 To whom requests for reprints should be
addressed, at Department of Preventive Medicine and Community Health,
The University of Texas Medical Branch, 700 Harborside Drive,
Galveston, TX 77555-1110. Phone: (409) 772-1730; Fax: (409) 772-9108;
E-mail: LLu{at}utmb.edu
3 The abbreviations used are: GCRC, General
Clinical Research Center; LH, luteinizing hormone; FSH,
follicle-stimulating hormone; AUC, area under the concentration time
curve; BMI, body mass index; CV, coefficient of variation; LDL, low
density lipoprotein; HDL, high-density lipoprotein.
Received 1/14/00. Accepted 5/25/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-induced protein tyrosine kinase. Endocrinology, 129: 2790-2792, 1991.This article has been cited by other articles:
|
|
 |
|
  S.-A. Lee, X.-O. Shu, H. Li, G. Yang, H. Cai, W. Wen, B.-T. Ji, J. Gao, Y.-T. Gao, and W. Zheng Adolescent and adult soy food intake and breast cancer risk: results from the Shanghai Women's Health Study Am. J. Clinical Nutrition, June 1, 2009; 89(6): 1920 - 1926. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Caserta, L. Maranghi, A. Mantovani, R. Marci, F. Maranghi, and M. Moscarini Impact of endocrine disruptor chemicals in gynaecology Hum. Reprod. Update, January 1, 2008; 14(1): 59 - 72. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. Wood, T. C. Register, and J.M. Cline Soy isoflavonoid effects on endogenous estrogen metabolism in postmenopausal female monkeys Carcinogenesis, April 1, 2007; 28(4): 801 - 808. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Cotterchio, B. A. Boucher, M. Manno, S. Gallinger, A. Okey, and P. Harper Dietary Phytoestrogen Intake Is Associated with Reduced Colorectal Cancer Risk J. Nutr., December 1, 2006; 136(12): 3046 - 3053. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Atkinson, J. W Lampe, D. Scholes, C. Chen, K. Wahala, and S. M Schwartz Lignan and isoflavone excretion in relation to uterine fibroids: a case-control study of young to middle-aged women in the United States. Am. J. Clinical Nutrition, September 1, 2006; 84(3): 587 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. O. Moore, Y. Wang, W. G. Stebbins, D. Gao, X. Zhou, R. Phelps, M. Lebwohl, and H. Wei Photoprotective effect of isoflavone genistein on ultraviolet B-induced pyrimidine dimer formation and PCNA expression in human reconstituted skin and its implications in dermatology and prevention of cutaneous carcinogenesis Carcinogenesis, August 1, 2006; 27(8): 1627 - 1635. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. Wood, T. C. Register, A. A. Franke, M. S. Anthony, and J. M. Cline Dietary Soy Isoflavones Inhibit Estrogen Effects in the Postmenopausal Breast Cancer Res., January 15, 2006; 66(2): 1241 - 1249. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Dave, R. R. Eason, S.R. Till, Y. Geng, M. C. Velarde, T. M. Badger, and R. C.M. Simmen The soy isoflavone genistein promotes apoptosis in mammary epithelial cells by inducing the tumor suppressor PTEN Carcinogenesis, October 1, 2005; 26(10): 1793 - 1803. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Huang, S. Cao, M. Nagamani, K. E. Anderson, J. J. Grady, and L.-J. W. Lu Decreased Circulating Levels of Tumor Necrosis Factor-{alpha} in Postmenopausal Women during Consumption of Soy-Containing Isoflavones J. Clin. Endocrinol. Metab., July 1, 2005; 90(7): 3956 - 3962. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. F. Skibola, J. D. Curry, C. VandeVoort, A. Conley, and M. T. Smith Brown Kelp Modulates Endocrine Hormones in Female Sprague-Dawley Rats and in Human Luteinized Granulosa Cells J. Nutr., February 1, 2005; 135(2): 296 - 300. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. R. Eason, M. C. Velarde, L. Chatman Jr, S. R. Till, Y. Geng, M. Ferguson, T. M. Badger, and R. C. M. Simmen Dietary Exposure to Whey Proteins Alters Rat Mammary Gland Proliferation, Apoptosis, and Gene Expression during Postnatal Development J. Nutr., December 1, 2004; 134(12): 3370 - 3377. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Maskarinec, A. A. Franke, A. E. Williams, S. Hebshi, C. Oshiro, S. Murphy, and F. Z. Stanczyk Effects of a 2-Year Randomized Soy Intervention on Sex Hormone Levels in Premenopausal Women Cancer Epidemiol. Biomarkers Prev., November 1, 2004; 13(11): 1736 - 1744. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. H. Choi, M. H. Lee, A. E. Bale, D. Carter, and B. G. Haffty Incidence of BRCA1 and BRCA2 Mutations in Young Korean Breast Cancer Patients J. Clin. Oncol., May 1, 2004; 22(9): 1638 - 1645. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D Brooks, W. E Ward, J. E Lewis, J. Hilditch, L. Nickell, E. Wong, and L. U Thompson Supplementation with flaxseed alters estrogen metabolism in postmenopausal women to a greater extent than does supplementation with an equal amount of soy Am. J. Clinical Nutrition, February 1, 2004; 79(2): 318 - 325. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. L. Horn-Ross, E. M. John, A. J. Canchola, S. L. Stewart, and M. M. Lee Phytoestrogen Intake and Endometrial Cancer Risk J Natl Cancer Inst, August 6, 2003; 95(15): 1158 - 1164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Yamamoto, T. Sobue, M. Kobayashi, S. Sasaki, and S. Tsugane Soy, Isoflavones, and Breast Cancer Risk in Japan J Natl Cancer Inst, June 18, 2003; 95(12): 906 - 913. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Hilakivi-Clarke, E. Cho, A. Cabanes, S. DeAssis, S. Olivo, W. Helferich, M. E. Lippman, and R. Clarke Dietary Modulation of Pregnancy Estrogen Levels and Breast Cancer Risk among Female Rat Offspring Clin. Cancer Res., November 1, 2002; 8(11): 3601 - 3610. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Dai, A. A. Franke, F. Jin, X.-O. Shu, J. R. Hebert, L. J. Custer, J. Cheng, Y.-T. Gao, and W. Zheng Urinary Excretion of Phytoestrogens and Risk of Breast Cancer among Chinese Women in Shanghai Cancer Epidemiol. Biomarkers Prev., September 1, 2002; 11(9): 815 - 821. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. de Lemos, C. L. Van Patten, K. A. Gelmon, and I. A. Olivotto Safety Issues of Soy Phytoestrogens in Breast Cancer Patients J. Clin. Oncol., July 1, 2002; 20(13): 3040 - 3042. [Full Text] [PDF]
|
|
|
|
|
|
M. S. Kurzer Hormonal Effects of Soy in Premenopausal Women and Men J. Nutr., March 1, 2002; 132(3): 570S - 573. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. H. Ju, C. D. Allred, K. F. Allred, K. L. Karko, D. R. Doerge, and W. G. Helferich Physiological Concentrations of Dietary Genistein Dose-Dependently Stimulate Growth of Estrogen-Dependent Human Breast Cancer (MCF-7) Tumors Implanted in Athymic Nude Mice J. Nutr., November 1, 2001; 131(11): 2957 - 2962. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Messina and C. L. Loprinzi Soy for Breast Cancer Survivors: A Critical Review of the Literature J. Nutr., November 1, 2001; 131(11): 3095S - 3108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. D. Allred, K. F. Allred, Y. H. Ju, S. M. Virant, and W. G. Helferich Soy Diets Containing Varying Amounts of Genistein Stimulate Growth of Estrogen-dependent (MCF-7) Tumors in a Dose-dependent Manner Cancer Res., July 1, 2001; 61(13): 5045 - 5050. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
