Effects of Soy Milk Consumption on Estradiol Concentration in Saddleback College Males
Daniel Mosallaei and Andrew Gomez
Department of Biological Sciences
Saddleback College
Mission Viejo, CA 92692
A variety of plants contain phytoestrogens: compounds that are similar in molecular structure to
estradiol, an abundant estrogen, allowing them to exhibit estrogen-like activity. Isoflavones, the primary
phytoestrogen in soy, have the potential to cause similar effects upon ingestion. Therefore, an increase in
estradiol levels in males should result after a daily consumption of soy milk. Nine Saddleback College
males between the ages of 18-25 were chosen for this study. An initial salivary sample was taken from the
subjects; each was given a total of four quarts of soy milk to drink over a two week period. Two more
samples per subject were collected at the end of the first and second week (Week 1&2) of ingestion. Saliva
was collected in 2-mL cryovials and kept in cold storage at -4oC. Once all samples were obtained, they
were thawed, centrifuged, and decanted. The samples were analyzed according to Salimetrics EIAEstradiol protocol, and converted to optical density quantities with a micro-plate reader. The microplate
outputted optical density values, which were converted to estradiol concentrations using a 4-Paramater
Sigmoid Minus Curve. Although an increasing trend was observed, the mean concentrations of the Initial
Week (1.66 ±0.388 pg/mL), Week 1 (2.50 ±0.277 pg/mL), and Week 2 (3.45 ±1.05 pg/mL) samples are not
statistically different from one another (p =0.183, ANOVA statistical test). Thus, there is no statistical
increase in estradiol concentrations in males from drinking four quarts of soy milk over a two week
period.
Introduction
Estrogens are an important group of sex
hormones produced in the human body that are
associated with development and functioning.
Production of these hormones in the body takes
place in the testes (males) and the ovaries (females).
There are three main estrogenic steroid hormones
present in humans: estrone, estriol and, estradiol.;
estradiol is the most active estrogen and functions in
the body to aid with the sexual reproduction and
reproductive development. In addition to its role in
sexual and reproductive functioning, estradiol also
affects other parts of the body, including the cardiovascular system, the brain, and the immune system
in both males and females (McCarthy, 2008;
Balthazart, 2006). Estrogenic hormones are present
in both men and women however; estrogen levels
are significantly higher in women.
Phytoestrogens are plant-derived
compounds (phyto=plant) from a wide variety of
plants that have similar chemical structures as
estrogenic hormones. Soy contains isoflavones, a
type of phytoestrogen, which are said to exhibit
estrogen-like activity and, more recently, have been
reported to display both estrogenic and antiestrogenic effects (Davis, 1999). Soy consumption
has been suggested to exert potentially cancerpreventive effects in premenopausal women, such
as increased menstrual cycle length and sex
hormone-binding globulin levels and decreased
estrogen levels. There has been some concern that
consumption of phytoestrogens might exert adverse
effects on men’s fertility, such as lowered
testosterone levels and semen quality (Kurzer,
2002). This study investigated if there would be an
increase in estradiol levels of Saddleback males
after a daily consumption of soymilk over a twoweek period.
Materials and Methods
Nine adult males between the ages of 18-25
were used in this study. These subjects were
students attending Saddleback College, Mission
Viejo, California, enrolled in the Biology 3B course
for Fall 2013. All subjects were approached and
agreed to participate in the study; a waiver was
signed by each. Three saliva extractions were taken
between 10:30 a.m. and 12:00 p.m. on 6th, 13th, and
20th November, 2013 using labeled 2mL Salimetrics
saliva cryovials. After rinsing their mouths ten
minutes beforehand, the subjects used the provided
saliva collection aid (SCA) to deliver about 1.5 mL
(3/4ths full) saliva to the cryovials. The cryovials
were labeled with the week number and the name of
the participant, and after they were placed in a -4oC
freezer to be held until analysis.
After the first extraction (Initial Sample) on
the 6th November, 2013, participants commenced
the drinking of the daily soy milk. Each day,
subjects ingested one cup of soy milk at the same
time each day for the remaining the two weeks.
Throughout the course of this two week drinking
period, multiple emails were sent to participants as
reminders to continue drinking the milk. Otherwise,
the subjects made no changes to the rest of their diet
– as advised. At the end of each week during the
soy milk ingestion, a second and third saliva sample
was collected from each subject (Week 1 & 2
Samples). Aforementioned, the salivary extractions
were frozen at -4oC until all samples were collected
to be analyzed.
Once the three sets of samples were
obtained on the 20th November, 2013, they were
taken out of cold storage and thawed. The bags
holding the cryovials were placed in warm areas to
increase the rate of melting. After, the samples were
transferred to smaller tubes and stirred using a
micro centrifuge, separating the solid and liquid in
the bacteria. The supernatant liquid was decanted
from the salivary precipitate.
An analysis – using Salimetrics Enzyme
Immunoassay (EIA) kit reagents – was performed
to quantify the concentration of estrogen within
each supernatant sample. The wells on the
microplate were mapped out for each subject and
their respective saliva samples. The experimenters
used the kit to fully analyze the samples according
to Salimetrics Salivary 17β-Estradiol EIA Protocol.
The microtitre plate is already coated with estradiol
antibodies, to which the estradiol from the saliva
and the standards compete with the estradiolperoxidase for a spot on the antibodies. Therefore,
the greater the estradiol concentration in the sample,
the less estradiol-peroxidase will be able to bind to
the coated antibodies. The tetramethylbenzidine
(TMB) combined with the stop solution (2M
sulfuric acid) detects the amount of estradiolperoxidase and exerts a level of opaqueness, or
optical density. When this optical density becomes
available, the micro-plate was read by a Fischer
Scientific Multiskan micro-plate reader at 450 ƞm,
giving assorted quantitative data of optic density for
each well. Since the value outputted by the
microplate reader is proportional to the amount of
estradiol-peroxidase, this value will be the inverse
of the actual estradiol concentration. These values
were transferred to MS Excel (Microsoft
Corporation, Redmond, Washington) where further
statistical manipulations and processing were
performed.
The sample and standard values were
inputted into a MS Excel Estradiol spreadsheet
created by Yousef Azzazy. Based on the standard
estradiol concentrations, the spreadsheet produced a
fitted 4-Parameter Sigmoid Curve embedding the
samples’ concentrations. To output the most
accurate data, the experimenters adjusted the four
parameters of the Sigmoid Curve to obtain a best−.59
fit: 𝐹(𝑥) = 0.7 + 𝑥 −1.45 . Aforementioned, the
1+( )
4
actual estradiol levels will be the inverse of the data
outputted by the microplate; therefore, the inverse
4-parameter sigmoid curve was used to obtain the
concentrations. These values – containing three
estradiol concentrations for each of the nine
subjects – were transferred into another MS Excel
spreadsheet. The mean estradiol concentration of
each week was obtained, and compared using an
ANOVA test. The differences in concentration
between the three weeks was also compared and
analyzed with a paired T-test.
Results
As mentioned above, the data was run
through Yousef’s excel program to output the 4Parameter Sigmoid Minus Curve. The curve,
𝐴−𝐷
represented by the equation 𝐹(𝑥) = 𝐷 + 𝑥 𝐵 was
1+( )
𝐶
adjusted to fit along the plot points created by the
standards as best as possible. The minimum
asymptote (A) was set at 0.11; the steepness (B)
was changed to -1.45; the inflection point (C) fit the
scatterplot best at 4, and the maximum asymptote
(D) was set to 0.7. This graph is shown in Figure
One. The original function created by the sigmoid
curve outputs the log of concentration; therefore, to
obtain the estradiol concentrations the inverse of the
function was taken. Based on the inverse sigmoid
function, the lower the ratio of optical density of
Bound (B) to Unbound (Bo) estradiol-peroxidase of
the saliva sample, the higher the estradiol
concentration. Aforementioned, this is because the
concentration of estradiol is inversely proportional
to the level of estradiol-peroxidase.
The mean estradiol concentrations for
Initial, Week 1, and Week 2 samples were
calculated. For the Initial sample, the average
estradiol concentration was 1.66 ± 0.388 pg/mL (±
SEM, C.L. ±0.894, N=9). At the end of Week 1, the
mean concentration was calculated to be 2.50 ±
0.277 pg/mL (± SEM, C.L. ±0.638, N=9). For
Week 2, the mean estradiol concentration was 3.45
± 1.05 pg/mL (±SEM, C.L. ±2.41, N=9). These data
are shown in Figure Two.
The three mean estradiol concentrations
were run through an ANOVA statistical test,
revealing p=0.183. Furthermore, the mean increase
in estrogen concentrations between the first and
second week, the second and third week, and the
first and third week were calculated and found to be
0.833 pg/mL, 0.95652 pg/mL, and 1.71 pg/mL
respectively. An ANOVA test was also run for
these increases, outputting a p value of 0.533. A
one-tailed paired equal variance t-test was run to
test for statistical difference in the sequential
increases between the three weeks, revealing p =
0.4273. Finally, a one-tailed paired equal variance ttest was run between the mean concentrations of the
first and final week (Initial and Week 2), outputting
p = 0.072.
Figure 2. A bar graph comparing the Week the
Sample was taken with Estrogen Concentration
(n=9). Initial sample mean concentration was 1.66
± 0.388 pg/mL. Week 1 mean concentration was
2.50 ± 0.277 pg/ mL. Week 2 mean estradiol
concentration was 3.45 ± 1.05 pg/mL. There is no
statistical difference between the three weeks (? =
0.183, ANOVA statistical test). Error Bars are mean
± SEM.
Ratio of optical density (B/Bo)
0.7
0.6
𝐹 𝑥 =𝐷+
0.5
𝐴−𝐷
𝑥
1 + ( )𝐵
𝐶
0.4
0.3
0.2
0.1
0
1
10
Log of Estradiol Concentration (pg/ml)
100
Figure 1. Four Parameter Sigmoid Minus Curve
relating the Percent Bound of Standards and
Unknowns with the Log of Testosterone
Concentration (n=9). As the log of concentration
increases, the ratio of bound to unbound decreases.
Estrogen Concentration
(pg/mL)
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Initial Mean
Week 1 Mean
Week 2 Mean
Week of Sample
Discussion
Based off initial observations of Figure
Three, an increasing trend can be seen between the
mean estradiol concentrations between the three
separate samples – which would confirm the
hypothesis. However, the statistical tests proved
otherwise. When the microplate reader scanned the
microplate, it generated a quantitative value based
on optical density of each well; these optical
densities acted as the raw data. The raw data was
converted to estradiol concentration values using
the Inverse Sigmoid Minus Curve function that was
generated with the high/low concentration standards
and four parameters.
Even though an increasing trend in estradiol
concentrations with soy milk consumption was
observed, statistical tests are needed for
confirmation. An ANOVA test comparing the
means of the three weeks was inconclusive,
outputting 0.183. Furthermore, a second ANOVA
test was run comparing the differences between
each week, which outputted a p value of 0.533 –
another statistically insignificant result.
Furthermore, a one tailed, paired T-test comparing
the linear/sequential increase between the weeks
(Initial and Week 1, Week 1 and Week 2) came out
undesirable, with a p value of 0.4273. Thus far, the
trend observed is not statistically backed up,
meaning there is no significant difference between
the weeks of soy milk ingestion and the week
without it. A final one-tailed paired t-test was run
comparing the first week (Initial) – before any soy
milk was ingested – and the final week (Week 2) –
after two weeks of soy milk was ingested, revealing
p = 0.072. Relative to the others, this is the lowest p
value; yet since it is greater than 0.05, there is no
statistical one-direction difference of the estradiol
concentrations between the first week (without soy
milk) and final week (after two weeks of soy milk
ingestion), rejecting the proposed hypothesis.
This study was performed over a three-week
period; a continuation of this study could be
achieved by performing the experiment over a
longer duration of time to examine any greater
change in estrogen levels. This would include an
increase in the period of time for soymilk ingestion.
A study performed by Jorge E. Chavarro in 2008
suggested that a higher intake of soy foods and soy
isoflavones had a relation with lower sperm
concentration among men. Furthermore, an
increased amount of soymilk intake for each subject
to consume per week could be observed. Increased
soymilk consumption could have a greater effect on
the estradiol levels of males in this experiment. R.C.
Griggs experimented on the effect of testosterone
on body size and concluded that testosterone
increases the muscle mass of men by increasing
muscle protein synthesis; therefore, body size could
have an effect on the amount of soymilk needed for
ingestion. Perhaps, individuals of bigger size
require higher dosage of soy to obtain a change in
estradiol levels. A study done in 1978 by Martin M.
Pierre on phytoestrogens reported that
phytoestrogens had the ability to interact with
estrogen receptors in human breast cancer cells,
suggesting that phytoestrogens could potentially
inhibit the production of breast cancer cells.
Phytoestrogens are still a great area of study for
scientists to this day.
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