Maturitas 78 (2014) 205211

Contents lists available at ScienceDirect

Maturitas
journal homepage: www.elsevier.com/locate/maturitas

Effects of Glycine max (L.) Merr. soy isoflavone vaginal gel on

epithelium morphology and estrogen receptor expression in
postmenopausal women: A 12-week, randomized, double-blind,
placebo-controlled trial
Snia Maria Rolim Rosa Lima, Bianca Franco Augusto Bernardo , Silvia Saito Yamada,
Benedito Fabiano Reis, Gustavo Maximiliano Dutra da Silva,
Maria Antonieta Longo Galvo
Faculdade de Cincias Mdicas da Santa Casa de So Paulo, R. Dr. Cesrio Mota Jnior, 112, 01221-020 So Paulo, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Objective: Evaluate the effects of vaginal administration of isoflavones derived from Glycine max (L.) Merr.
Received 4 February 2014 as a treatment option for vaginal atrophy, on the morphology and expression of estrogen receptors in
Received in revised form 2 April 2014 vaginal epithelium of postmenopausal women.
Accepted 5 April 2014
Methods: The double-blind, randomized, placebo-controlled, clinical trial. Sixty women were treated for
12 weeks with isoflavone vaginal gel 4% (1 g/day) and a placebo gel. After 4 and 12 weeks, the vaginal
Keywords:
atrophy symptoms were classified at none, mild, moderate and severe and the vaginal cytology were
Isoflavones
taken to determine the maturation value. Vaginal pH was measured at the beginning and end of therapy.
Menopause
Vulvovaginal atrophy
Microbiopsies in vaginal fornix were performed before the treatment and after 12 weeks of treatment.
Intravaginal Administration Results: Isoflavone vaginal gel was effective for relief of vaginal dryness and dyspareunia symptons and
Vagina/anatomy & histology and estrogen an increase in the intermediate and superficial cells was noted. The vaginal pH in the isoflavone group
receptor was 7.1 at baseline and 5.4 after 12 weeks, whereas in the placebo group there was no significant change.
A significant increase in thickness after treatment was detected in the Isoflavone Group. The percentage
of estrogen receptor positive cells in vaginal epithelium for the Isoflavone Group ranged from 58.5% at
the beginning of treatment to 82.6% after 12 weeks. These results were superior to placebo gel.
Conclusion: Glycine max (L.) Merr. at 4% vaginal gel on a daily basis in postmenopausal women led to
improvements in vaginal atrophy symptoms, maturation values, vaginal pH, morphology and expres-
sion of estrogen receptors in vaginal epithelium. Isoflavones proved good treatment options for relief of
vulvovaginal atrophy.
2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction According to The North American Menopause Society (NAMS),

Vaginal atrophy (VA) is a common condition in postmenopausal
women associated with vaginal and/or urinary symptoms such as
vaginal dryness, itching, discomfort and dyspareunia, dysuria, uri-
nary urgency and frequency [1]. The disorder typically occurs in
postmenopausal women, but can affect women of any age who
experience a decrease in estrogenic stimulation of the urogenital
tissues [2]. Sexual function and quality of life may also be reduced
as a result of these changes [3].
Corresponding author. Tel.: +55 11991569508.
E-mail address: bfa.bernardo@gmail.com (B.F.A. Bernardo).
symptoms associated with vulvovaginal atrophy (VVA) affect
2045% of midlife and older women, but this aspect of menopause
is often overlooked and undermanaged, because only a minority
seek medical help for the problem [4]. In contrast to vasomotor
symptoms, which tend to improve over time even without treat-
ment, VVA can be progressive and is less likely to resolve without
intervention [4].
The primary goal of treating symptomatic VVA is to alleviate
symptoms. First-line therapies include nonhormonal, long-acting
vaginal moisturizers and low-dose vaginal estrogen, assuming
no contraindications [4]. Despite the effectiveness of estrogen,
concerns over side effects and safety have hindered its use by post-
menopausal women. In recent years, phytoestrogen supplements

http://dx.doi.org/10.1016/j.maturitas.2014.04.007
0378-5122/ 2014 Elsevier Ireland Ltd. All rights reserved.
206 S.M.R. Rosa Lima et al. / Maturitas 78 (2014) 205211
have become attractive as safer alternatives, and their efficacy has
been investigated in experimental and clinical trials [58].
Isoflavones are the most studied of the phytoestrogens
and some trials involving its oral form for treating climacteric
symptomatology have shown no change in vaginal epithelium or
endometrium [9,10]. Similarly, topical preparations for the pre-
vention and delay of skin maturation in postmenopausal women
have shown satisfactory outcomes [11,12].
Cytologic examination of vaginal mucosa from menopausal
women shows a decreased proportion of superficial cells and an
increased proportion of parabasal cells [13]. Also, the vaginal lin-
ing thins and vaginal pH increases from the normal 3.54.0 (which
favors lactobacilli) to 6.08.0 (which favors pathogenic organisms)
[14,15].
Although cytohormonal analysis of the vaginal epithelium is
well established as a method of evaluating oestrogenic influence,
where maturation value (MV) can be calculated to express degree
of vaginal atrophy as a numeric rating [16], in cytology analysis,
only the exfoliated cells are studied. In order to gain a broader
picture of the process of maturation of the vaginal epithelium a
morphometric method should also be used.
Estrogen is a dominant regulator of vaginal physiology.
Estrogen-receptor (ER) is present in the vaginal tissues of both pre-
menopausal and postmenopausal women [17,18]. The biological
effect of estrogens is mediated by direct interaction with two ERs,
ER! and ER". The ERs belong to the superfamily of steroid nuclear
receptor transcription factors that activate binding to specific DNA
sequences, called estrogen-responsive elements, on the promoters
of the target genes [19].
Few studies investigating the effects of vaginal administration
of isoflavones on vaginal atrophy symptoms are available [7,20,21]
while no studies using the morphometric method as a means of
assessing vaginal epithelium and ER expression in vaginal cells in
postmenopausal women have been reported. The aim of the current
investigation was to evaluate the effects of vaginal administration
of isoflavones derived from Glycine max (L.) Merr. as a treatment
option for vaginal atrophy, on the morphology and expression
of estrogen receptors in vaginal epithelium of postmenopausal
women.
2. Methods
2.1. Setting
The study commenced in July 2011 and was concluded in April
2013. The clinical trial was performed in accordance with the Dec-
laration of Helsinki and International Standards of Good Clinical
Practice (ICH-E6). The study protocol and patient informed con-
sent form were approved by the Research Ethics Committee of the
Irmandade da Santa Casa de Misericrdia de So Paulo hospital,
in So Paulo Brazil. All investigations were performed at this
institution.
2.2. Study design
The double-blind, randomized, placebo-controlled, clinical trial
comprised three phases. At the first visit, written informed consent
was obtained and inclusion and exclusion criteria assessed. Eligible
participants were randomly assigned to receive Glycine max (L.)
Merr. isoflavone vaginal gel or placebo gel. Each gel was admin-
istered vaginally on a daily basis throughout the 12-week trial.
During both visits, at four and twelve weeks, the participants were
asked to rate their vaginal dryness and dyspareunia. Symptoms
were measured according to their intensity (0 null to 3 unbear-
able). Vaginal smears were taken for vaginal cytology during both
visits [22]. Vaginal pH was measured using a standardized kit (pH
Merck 014 ) at the beginning and end of therapy. Microbiopsies
in vaginal fornix were performed before the treatment and after
12 weeks of treatment. All samples were acquired by the same
investigator. Endometrial safety (endometrial thickness as mea-
sured by transvaginal ultrasonography) was evaluated at initial
screening and trial endpoint [23].
2.3. Study drug
The isoflavones of Glycine max (L.) Merr. extract 4% and
placebo gel were manufactured by Hebron Laboratory (Caruaru,
Pernambuco, Brazil). The method of extraction of the isoflavones
was not disclosed by the industrial supplier. Every 1 g of Isoflavone
gel contained 0.04 g of 10% dry soybean extract. The 10% dried
soybean extract was composed of: 3.2% Daidizin, 5.5% Genistin,
0.51% Glycitin, 0.35% Daidzein, 0.39% Genistein and 0.05% Glycitein.
The amounts of the chemical compounds in the gel varied by
10.0%. All chemical substances were characterized and quanti-
fied by HPLC/UV/DAD. The pH was 4.5 and the water proportion
was 7805%/60 g/tube. It was chosen to be like this because this for-
mulation showed the better pharmaceutics behavior and stability
in combination with dry soy extract 10%. The placebo formulation
was the same as that used in the isoflavone product. The placebo
gel consisted of carbopol, methylparaben, propylparaben, sodium
hydroxide and water. The two products were placed in similar
tubes. Treatment instructions were to administer 1 g of Isoflavone
gel or 1 g of placebo gel, vaginally at bedtime, on a daily basis.
2.4. Patients
The inclusion criteria were: non-hysterectomized, post-
menopausal (2 or more years since final menstrual cycle) women
aged 45 years or older with symptoms of vaginal dryness and/or
pruritus, pain/soreness, vulvar or vaginal burning, and dyspareu-
nia. All participants referred coital sexual activity, once or more
per week and stable partner. They were required to have serum
E2 levels less than 20 pg/mL, follicle-stimulating hormone levels
greater than 40 mIU/mL, no superficial cells on vaginal cytology, an
endometrial thickness of less than 5.0 mm as assessed by transva-
ginal ultrasonography, and a normal mammography during the 6
months leading up to study entry.
Exclusion criteria were: use of any investigational drug or
exogenous sex hormones within the 6 months leading up to study
drug initiation, or current use of corticosteroids, known or sus-
pected history of hormone-dependent tumor, breast carcinoma,
genital bleeding of unknown cause, acute thromboembolic disorder
associated with estrogen use, vaginal infection requiring treatment,
allergy to the test drug or its constituents, hot flashes, and any seri-
ous disease or chronic condition that could interfere with study
compliance.
2.5. Assessments
All participants underwent medical examination (interview,
hematology, biochemistry, urinalysis, gynecologic examination,
mammography and transvaginal ultrasonography) in order to
determine patient eligibility. During both visits at four and 12
weeks, patients reported on a questionnaire any symptoms of
vaginal dryness, pruritus, pain/soreness, vulvar or vaginal burning
and dyspareunia, which were subsequently classified as follows:
(none = 0, mild = 1, moderate = 2, severe = 3).
For vaginal cytology, vaginal smears were taken at four and 12
weeks from the upper third of the right lateral vaginal wall and
analyzed at the Department of Pathology (Santa Casa Sao Paulo Hos-
pital Sao Paulo Brazil). These samples were used to determine
 S.M.R. Rosa Lima et al. / Maturitas 78 (2014) 205211 207

the Maturation Index, which describes the proportion of parabasal, Table 1

Patient baseline characteristics (mean SD* ).
intermediate and superficial cells. The Maturation Value (MV), or
Frost Index, was calculated according to the formula: MV = (0 % Characteristic Isoflavone (n = 29) Placebo (n = 26) p*
parabasal cells) + (0.5 % intermediate cells) + (1.0 % superficial Age (years) 59.2 4.8 59.9 2.9 0.490
cells) [22]. Maturation of the vaginal epithelium (a positive treat- Age at menopause (years) 49.4 4.7 47.3 4.3 0.080
ment effect) is evidenced by a decrease in parabasal cells and an Time since menopause (years) 9.7 4.4 11.8 4.9 0.100
increase in the proportion of superficial cells. Weight (kg) 64.7 11.7 66.8 12.8 0.530
Body mass index (kg/m2 ) 26.7 5.2 27.8 4.3 0.420
The pH indicator paper (014 Merck ) was placed in direct con-
tact with the lower third of the right vaginal wall for at least 60 s. SD: standard deviation.
*
Readings were taken in a comparative manner, in accordance with p < 0.05 (Student t-test).

previously established standards according to the Manufacturer.

The biopsy specimens originating from the vaginal mucosa were
fixed in 10% buffered formaldehyde solution for a period of 24 h and
study material processed at the Department of Pathology of the
Irmandade da Santa Casa de Misericrdia de So Paulo hospital.
The sections were dehydrated in ethanol, cleared by xylene and
embedded in paraffin for preparation of blocks. The blocks were cut
by microtome blade calibrated to produce 4 #m-thick sections. The
sections were stained with hematoxylin and eosin (HE) and then
examined under a standard optical microscope [24]. For anatomical
and pathological determination, the criteria established by Kurman
and Solomon [25] were adopted, with the diagnosis confirmed by
two pathologists.
Each slide was evaluated by light microscopy (Axioscop 40
Zeiss) with a lens providing 400 times magnification and a micro
adapted for use as the monitor (LG Flatron 14 inches). The cal-
culation of area in square millimeters for high power fields was
performed with the assistance of a graduated Neubauer chamber,
consisting of a thick glass slide, rectangular in shape, with a retic-
ulated central area [26].
As proposed by Campaner and Galvo, the thickness of the vagi-
nal epithelium was determined on an optical microscope with the
aid of the AxioVision 3.0 software, ZEIZZ, which allows the creation
of a metric scale area. These measurements were made from the
base of the basal cell layer to the apex of the superficial epithelial
cells [26].
For immunohistochemistry, representative areas of the vaginal
mucosa paraffin blocks were selected and sent to the Pathology
Department of the Santa Casa de Misericordia de So Paulo hos-
pital, where all immunohistochemical reactions were performed
together in order to minimize technical errors. To demonstrate the
presence of ER, mouse monoclonal antibodies M7047 clone 1D5
field kits manufactured by a Californian laboratory (DAKO Corpo-
ration, Carpinteria, CA).
Serum FSH levels were analyzed using the chemiluminescence
system ACS-180, Chiron DL = 0.30 mIU/L and serum estra-
diol levels were analyzed using chemiluminescence/Chiron
LD = 10.0 pg/mL. The postmenopausal reference ranges used were
serum FSH levels from 23.0 to 116.3 mIU/mL and estradiol lev-
els from 0 to 19 pg/mL. The assay detection limits were 5 pg/mL
for estradiol and 0.3 mIU/mL for FSH. Limits of quantitation were
500 pg/mL for estradiol and 200 mIU/mL for FSH. Blood for hormone
analysis was collected at baseline and after 90 days of the study.
After collection, blood was allowed to stand for approximately
30 min at ambient temperature and then centrifuged at >1200 g
(3500 rpm) to separate blood cells from serum. The serum was care-
fully transferred to a another serum container by means of pipette
and analyzed for estradiol and FSH.
2.6. Blinding of drugs
The bottles were identified using four-digit numbers separated
by a slash, as follows: 01/01, where the first number indicates
the patient and the second indicates the month. Example: patient
01 should always receive samples identified with these numbers:
01/01; 01/02; 01/03.
2.7. Randomization
The women included in the study (n = 60) were randomized into
two groups by using specialized site [27]. Sample blinding codes
were disclosed after completion of treatment for all patients.
2.8. Statistical analyses
Without information on the vaginal effect of the isoflavone
Glycine max (L.) Merr. a sample size of 60 patients was required.
This sample size allowed treatment effect to be determined at
= 0.05 with a statistical power of 90%. All data reported at week
0 and week 12 are from the intent-to-treat analyses, with missing
values for each individual computed using the last observation car-
ried forward approach. Statistical analyses (Student t-test) were
performed by comparing variables for each group: patient base-
line characteristics, FSH, E2, pH, vaginal epithelium thickness,
percentage of positive ER cells and endometrial thickness. The
MannWhitney test was used to compare the atrophic symptoms
and MV of each group at baseline and after 4 and 12 weeks of treat-
ment. Data were expressed as mean or median and significance
level was set at less than 0.05. The analyses were performed on
change in scores between baseline and weeks 4 and 12. Treatment
differences (isoflavones and placebo) were expressed as means
(standard error), and 95% confidence intervals (CIs) accompany p
values for primary endpoints [28].
2.9. Registration
The trial was registered in REBEC (www.ensaiosclinicos.gov.br).
The registration number of the trial was RBR-88vgp6 and the name
of the trial registry was Efeitos das isoflavonas do Glycine max
(L.) Merr. no epitelio vaginal e no endometrio em mulheres apos
a menopausa.
3. Results
Of the 117 participants enrolled, 60 subjects remained after
applying the inclusion and exclusion criteria. A total of 30 women
received 1 gram of isoflavone vaginal gel 4% daily for 12 weeks
and 30 women received 1 gram of placebo vaginal gel for the same
period. In the placebo group, two women discontinued treatment
due to lack of improvement and two subjects were lost to follow up.
In the isoflavone group, one woman discontinued due to leucorrhea
(Fig. 1). Patient baseline characteristics are shown in Table 1. No sig-
nificant differences were observed among the two groups regarding
age, age at menopause, and time since menopause, weight or BMI.
The post-menopausal genital complaints reported were vagi-
nal dryness in 100% for both groups, dyspareunia in 93.1% for
the isoflavone group and in 92.4% for the placebo group, pruri-
tus in 48.2% of the isoflavone group and 64.4% in the placebo
group, pain/soreness in 61.1% for the isoflavone group and 42.3%
in the placebo group, vulvar and/or vaginal burning in 44.8% of the
isoflavone group and 42.3% for the placebo group and presence of
secretion in 24.1% for the isoflavone group and 15.3% in the placebo
208 S.M.R. Rosa Lima et al. / Maturitas 78 (2014) 205211

N= 117 women
Inclusion and
exclusion criteria

N= 60

Group 1 Group 2
(Isoflavones) (Placebo)
1g/ daily/ 90 days 1g/ daily/ 90 days
N= 30 N= 30

Discon!nued treatment
Discon!nued due to due to lack of
leucorrhea (1) improvement (2)
Lost to follow up (2)

N= 29 N=26
Fig. 1. Study flowchart illustrating enrollment, number of women in the intent-to-treat population, randomization into treatment groups, and follow-up of study participants.

Table 2 Estradiol (pg/mL): No statistically significant differences were

Symptoms and frequency of vaginal atrophy at study baseline.
found for mean sera estradiol concentrations between the two
Symptoms Isoflavones n (%) Placebo n (%) p* value treatment groups (p = 0.776 and p = 0.638, respectively) Table 4.
Vaginal dryness 29 (100) 26 (100) 0.285 Endometrial thickness (mm): Analysis of endometrial echo before
Dyspareunia 27 (93.1) 24 (92.4) 0.191 and after 12 weeks treatment between the groups revealed no sta-
Pruritus 14 (48.2) 17(64.4) 0.032 tistically significant difference (p = 0.202 and 0.241, respectively).
Pain/soreness 18 (61.1) 11 (42.3) 0.113 Endometrial thickness was < 5 mm in all cases (Table 4).
Burning 13 (44.8) 11 (42.3) 0.643
Vaginal pH: The average pH in the isoflavone group was 7.1 at
Secretion 7 (24.1) 4 (15.3) 0.295
baseline and 5.4 after 12 weeks, whereas in the placebo group there
*
p < 0.05 (Chi-square test).
was no significant change in the average vaginal pH after 12 weeks
of treatment (Table 4).
Maturation Value (MV): In the Isoflavone Group, MV had
increased from 0 to 70 after 4 weeks and remained at 70 after 12
group. The two most prevalent complaints were investigated in this weeks of treatment. An increase in the intermediate and superficial
study, namely, vaginal dryness and dyspareunia (Table 2). cells was noted. Statistically significant differences in median MV
Vaginal dryness: In the Isoflavone Group, the median score for values were found in the Isoflavone Group among the time points
vaginal dryness complaints at baseline was 3. At four weeks treat- (p < 0.000). Differences among medians between baseline and four
ment, the median score was 2 and after 12 weeks the score was 1. weeks, as well as between baseline and 12 weeks, reached statis-
Comparing with the Placebo Group, the median score for vaginal tical significance. However, no statistically significant difference in
dryness complaints was 2 at baseline, and remained the same after MV was found between four and 12 weeks. In the Placebo Group,
four weeks and 12 weeks (Table 3). MV was 5, 16.2 and 32.5 for the 3 time points, respectively. An
Dyspareunia: In the Isoflavone Group, the median score of dys- increase in intermediate and superficial cells was noted. In this
pareunia complaints at baseline was 3. At four weeks treatment, group, statistically significant differences in median MV among the
the median score was 2 and after 12 weeks, was 0. Comparing with 3 time points (p < 0.001) were found. However, the MV attained
the Placebo Group, the median score for dyspareunia complaints after 12 weeks of treatment in the isoflavone group was higher
was 2 at baseline and 1 after four and 12 weeks (Table 3). than that obtained with placebo (Table 5).
FSH (mUI/mL): No statistically significant differences were found Vaginal epithelium thickness: In the Isoflavone Group, a signif-
for mean FSH concentration at baseline and at 12 weeks between icant increase in thickness after treatment was detected. Vaginal
the two treatment groups (p = 0.850 and 0.912, respectively) epithelium thickness was 153.5 at baseline and 259.8 at 12 weeks.
Table 4. In the Placebo Group, vaginal epithelium thickness was 145.3 at
 S.M.R. Rosa Lima et al. / Maturitas 78 (2014) 205211 209

Table 3
Symptoms of vaginal atrophy at basal 4 weeks and 12 weeks (study endpoint) in isoflavone group and placebo group (median and maximumminimum values).

Symptoms Isoflavone Placebo Isoflavones compared with

placebo- p* value

Basal 4 weeks 12 weeks p value Basal 4 weeks 12 weeks p value

Dryness 3 (31) 2 (30) 1 (20) 0.000 2 (31) 2 (30) 2 (30) 0.002 0.049
Dyspareunia 3 (30) 2 (30) 0 (30) 0.006 2 (30) 1 (30) 1 (30) 0.255 0.028
*
p < 0.05 (MannWhitney test).

Table 4
FSH, estradiol and endometrial thickness at study baseline and endpoint in isoflavone group and placebo group (mean SD).

Parameters Isoflavones Placebo

Basal 12 weeks p value Basal 12 weeks p value

FSH (mIU/mL) 59.2 22.6 58 25.9 0.850 71.6 29.9 70.6 32.5 0.912
Estradiol (pg/mL) 40.3 26.1 38.3 26.9 0.776 30.3 9.6 31.5 9.2 0.638
Endometrial echo (mm) 3.3 1.3 3.8 1.6 0.202 3.4 1.0 3.8 0.8 0.241
Vaginal pH 7.1 0.9 5.4 0.8 0.000* 7.4 0.8 7.1 0.8 0.172

SD: standard deviation.

*
p < 0.05 (Student t-test).

Table 5
Comparison of maturation value at baseline, four weeks and 12 weeks in isoflavone (Iso) group and placebo (Plc) group (median and maximumminimum values).

Groups Median p value Iso compared with

plc p value

Basal 4 weeks 12 weeks Basal-4 weeks 412 weeks Basal-12 weeks

Isoflavones 0 (330) 70 (090) 70 (973) 0.000 0.091 0.000* 0.000*

Placebo 5(300) 16.2(050) 32.5(080) 0.001 0.026* 0.000*
*
p < 0.05 (MannWhitney test).

Table 6
Comparison of vaginal epithelium thickness (#m) and estrogen receptor (ER) positive cells (%) at baseline and 12 weeks in isoflavone (Iso) group and placebo (Plc) group
(mean SD).

Parameters Group Mean Iso compared with plc p value

Basal 12 weeks p value

Thickness
Isoflavones 153.5 66.1 259.8 56.9 0.329 0.001*
Placebo 145.3 60.5 191.9 83.7
ER positive
Isoflavones 58.5 33.9 82.6 17.4 0.071 0.824
Placebo 73.4 24.5 83.7 8.8

SD: standard deviation.

*
p < 0.05 (Student t-test).

baseline and 191.9 after 12 weeks of treatment, representing a
statistically significant increase. However, the result obtained in
the Isoflavone Group was higher than that of the Placebo Group
(Table 6).
Estrogen receptor (ER) positive cells: The percentage of ER positive
cells in vaginal epithelium for the Isoflavone Group ranged from
58.5% at the beginning of treatment to 82.6% after 12 weeks, rep-
resenting a statistically significant increase. In the Placebo Group,
values ranged from 73.4% at the beginning of treatment to 83.7%
after 12 weeks, although this increase was not statistically signifi-
cant.
4. Discussion
The effect of isoflavones derived from Glycine max (L.) Merr. in
the treatment of vaginal atrophy by topical gel compared to placebo
was evaluated. Systemic hormone therapy with natural estrogens is
used for the relief of menopausal symptoms. However, a substantial
proportion of women with urogenital complaints and changes in
sexuality have no clinical improvement from systemic hormone
treatment, where the combination of local treatment for relief of
symptoms is required in this group [29].
Studies on the vaginal use of isoflavones after menopause are
scant [20,21,8] and no publications evaluating their effect on the
morphology of the vaginal epithelium and the expression of estro-
gen receptors are available, thus prompting the present study.
Clinical improvement was found after 4 weeks of treatment
with isoflavone gel for vaginal dryness and dyspareunia, while the
same was not observed in the placebo group, in which only an
improvement in dryness after 12 weeks occurred. Furthermore,
when comparing the effect of isoflavones versus placebo for vagi-
nal dryness after 12weeks, the former treatment proved superior
to the latter. This finding was similar to that described by Lima et al.
who reported significant improvement in these symptoms [8].
The placebo group also showed improvement in vaginal dryness,
a finding previously reported with the use of vaginal lubricants (Le
Donne et al. [20]). The degree of this improvement however, was
lower than that found in the Isoflavone group.
210 S.M.R. Rosa Lima et al. / Maturitas 78 (2014) 205211
The placebo group used a vaginal gel containing no thera-
peutic substances and products known to be inert and harmless
with the aim of comparing the placebo effect on the treatment of
vaginal symptoms, thereby avoiding possible therapeutic results
not attributed to the treatments. A number of the product com-
ponents in the placebo vaginal gel may improve some vaginal
symptoms immediately after use, such as moisturizing products.
Nevertheless, cellular changes and vaginal symptoms have been
found not to persist in the longer term [30].
Tedeschi and Benvenuti [21] compared the symptoms of vagi-
nal atrophy in women treated with isoflavone vaginal gel versus
women given no vaginal treatment. The authors observed a signif-
icant improvement in dryness and dyspareunia after four weeks
in the group of women who used the vaginal gel compared to the
untreated group. In the study, the women used an isoflavone gel
that differed from the gel used in the present study, in that it con-
tained aglycone isoflavones (10 mg) associated with Lactobacillus
sporogenes, Calendula officinalis and lactic acid.
Serum concentrations of FSH and estradiol before and after the
use of each product remained unchanged in both groups. This find-
ing is especially relevant since the systemic absorption of vaginal
estrogen is a major concern regarding the safety of the treatment.
Systemic hormonal absorption may occur depending on the
duration of use and dose applied [31,32]. On the other hand, it has
also been demonstrated that the level of absorption can decrease
with time [33,34]. However, this effect is more relevant for women
with a history of breast cancer and other hormone-dependent can-
cers [35].
In the present study, an endometrial investigation was carried
out in order to ensure the safety of the proposed therapy. It is
known that, in the presence of hormone therapy, episodes of spot-
ting and endometrial thickening may occur. In this study however,
there were no reports of genital bleeding and sonographic findings
confirmed no increase in endometrial thickness in either group, as
evidenced by measurements below five millimeters and no change
in thickness of endometrial echoes between the beginning and end
of treatment in both groups.
We evaluated vaginal pH at baseline and again at 90 days of
treatment and observed that only the group of women who used
the gel with isoflavones had a significant reduction in this mea-
sure. The acidification of vaginal pH observed in participants was
similar to that typically seen in premenopausal women. Studies
with isoflavones administered orally remain inconclusive. Kaari
et al. compared the use of isoflavones and conjugated equine estro-
gens administered orally for six months and noted no change in
vaginal pH in the isoflavone group [36]. Manonai et al. obtained
similar results when evaluating the vaginal pH of 36 women in
perimenopause and after menopause for twelve weeks who con-
sumed a soy-rich diet (50 mg isoflavones/day) compared to those
on a control diet (isocaloric and low in soy) [37].
The administration of isoflavones by the vaginal route exerts
a greater effect on pH. In a study conducted by Tedeschi and Ben-
venuti, a non-significant reduction in vaginal pH from 5.9 to 4.9 was
observed after four weeks of treatment with vaginal gel isoflavones
[21].
In the present study, we found a significant increase in Meisels
Index after 30 and 90 days of treatment in both groups, with a
comparative analysis between groups demonstrating the superi-
ority of isoflavones over placebo. Regarding the activity of orally
administered isoflavones on vaginal epithelium, conclusions are
controversial [3842]. However, a study by Le Donne et al. analyz-
ing the effect of isoflavones administered to the vagina showed an
increase in the Meisels Index for both groups [20], results mirrored
by the present study. Lima et al. compared isoflavone vaginal gel
with a cream containing conjugated equine estrogens or placebo for
three months and also detected an increase in Meisels Index over
time in the groups, noting no significant difference when compar-
ing isoflavones with conjugated equine estrogens [8].
We believe that treatment of vaginal atrophy by topical applica-
tion of isoflavone gel influenced the epithelium maturation process,
promoting an increase in the number of cells and layers of the vagi-
nal epithelium. This corroborates the results reported by Nilsson
et al. who studied five women who received 1.25 mg of conjugated
equine estrogens orally for 30 days and underwent vaginal biop-
sies before and after treatment. An increase in the thickness of the
vaginal epithelium and the total number of cells was observed [43].
Carbonel et al. found a similar effect upon studying the vaginal
epithelium of rats fed soy by gavage for 21 days and comparing
them to a group of rats in use of conjugated equine estrogens and
control animals. A proliferative effect was observed with thicken-
ing of the epithelium and mucosa and increased collagen fibers in
the groups fed soy at a concentration of 120 mg/kg per day and also
in the group treated with conjugated equine estrogens. This same
finding was observed in the placebo group.
We attribute the increased thickness of vaginal epithelium in the
Placebo Group to hydration of the hydrophilic substances contained
in the gel applied. This increase in cell volume represented only
a transient effect however, and it is noteworthy that the average
thickness of vaginal mucosa in the Placebo Group was lower than
that observed in the Isoflavone Group at the end of the study.
Our literature search we found no other studies evaluating the
morphology of the vaginal epithelium in women using isoflavone
orally and vaginally.
ERs are members of the superfamily of nuclear receptor ligand-
dependent transcription factors. It has been found that an absence
of ligand ERs located in target cells is associated with the com-
plex of heat shock proteins and chaperone complex [44]. Using the
vaginal gel, isoflavones can express their activities after interaction
with ERs causing its activation. A significant increase in ER expres-
sion was observed in the immunohistochemical study of vaginal
epithelium samples in the Isoflavone group at 90 days. Notably,
this effect was not found in women in placebo group, since the gel
contained no substances capable of interacting with ER.
In summary, our results showed isoflavone vaginal gel to be
effective for the treatment and management of symptoms of
vaginal estrogen deficiency-induced vaginal atrophy and for the
proliferative effect and increased expression of ER in the epithe-
lium of the vagina. The gel exhibited no systemic absorption and
can therefore be used in women who do not want to use hormone
therapy or that have contraindications. Further long-term studies
involving women with genital atrophy symptoms are needed to
confirm the efficacy of this treatment and the endometrial protec-
tion conferred.
Contributors
They were responsible for the clinical history, physical examina-
tion and treatment of the study participants, and also contributed
to the writing of the paper, statistical tests and conclusions
Maria Antonieta L. Galvo da Silva (PhD) contributed to the cyto-
logical, morphological and immunohistochemical study.
Competing interests
None of the authors involved in this study have any form of
conflict of interest.
Funding information
Funding for the entire study was conducted by researchers.
 S.M.R. Rosa Lima et al. / Maturitas 78 (2014) 205211
References
[1] Pastore LM, Carter LA, Hulka BS, et al. Self-reported urogenital symp-
toms in postmenopausal women: womens health initiative. Maturitas
2004;49:292303.
[2] North American Menopause Society. The 2012 Hormone Therapy Posi-
tion Statement of The North American Menopause Society. Menopause
2012;19(3):25171.
[3] Archer DF. Efficacy and tolerability of local estrogen therapy for urogenital
atrophy. Menopause 2010;17(1):194203.
[4] North American Menopause Society. Management of symptomatic vulvovagi-
nal atrophy: 2013 position statement of The North American Menopause
Society. Menopause 2013;20(September (9)):888902.
[5] Drapier FE, Chantre P, Mares P. Effects of a standardized soy extract on hot
flushes: a multicenter, double blind, randomized, placebo controlled study.
Menopause 2002;9:32934.
[6] Nikander E, Rutanen EM, Nieminen P. Lack of effect of isoflavonoids
on the vagina and endometrium in postmenopausal women. Fertil Steril
2005;83:13742.
[7] Alves DL, Lima SM, da Silva RR, et al. Effects of Tripholium pratense and Cimifuga
racemosa on the endometrium of wistar rats. Maturitas 2008;56:23842.
[8] Lima SMRR, Yamada SS, Reis BF, Postigo S, Silva MALG, Aoki T. Effec-
tive treatment of vaginal atrophy with isoflavone vaginal gel. Maturitas
2013;74(3):2528.
[9] Upmalis DH, Lobo R, Bradley L, Warren M, Cone FL, Lamia CA. Vasomo-
tor symptom relief by soy isoflavone extract tablets in post-menopausal
women: a multi-center, randomized, placebo-controled study. Menopause
2000;7(4):23642.
[10] DAnna R, Cannata ML, Atteritano M, et al. Effects of the phytoestrogen
genistein on hot flushes, endometrium and vaginal epithelium in post-
menopausal women: a 2-year randomized, double-blind, placebo-controlled
study. Menopause 2009;16(2):3016.
[11] Moraes ARB. The effects of topical isoflavones on postmenopausal skin: double-
blind and randomized clinical trial of efficacy. Eur J Obstet Gynecol Reprod Biol
2009;146(2):18892.
[12] Patriarca MT, Barbosa de Moraes AR, Nader HB, et al. Hyaluronic acid con-
centration in postmenopausal facial skin after topical estradiol and genistein
treatment: a double-blind, randomized clinical trial of efficacy. Menopause
2013;20(March (3)):33641.
[13] Bachmann GA, Nevadunsky NS. Diagnosis and treatment of atrophic vaginitis.
Am Fam Phys 2000;61:30906.
[14] Semmens JP, Wagner G. Estrogen deprivation and vaginal function in post-
menopausal women. J Am Med Assoc 1982;248:4458.
[15] Pandit L, Ouslander JG. Postmenopausal vaginal atrophy and atrophic vaginitis.
Am J Med Sci 1997;314:22831.
[16] Hammond D. Cytological assessment of climacteric patients. Clin Obstet
Gynecol 1977;4:4970.
[17] Chen GD, Oliver RH, Leung BS, Lin LY, Yeh J. Estrogen receptor alpha and beta
expression in the vaginal walls and uterosacral ligaments of premenopausal
and postmenopausal women. Fertil Steril 1999;71:1099102.
[18] Gebhart JB, Rickard DJ, Barrett TJ, et al. Expression of estrogen receptor iso-
forms alpha and beta messenger RNA in vaginal tissue of premenopausal and
postmenopausal women. Am J Obstet Gynecol 2001;185:132530.
[19] Cavallini A, Dinaro E, Giocolano A, et al. Estrogen receptor (ER) and ER-
related receptor expression in normal and atrophic human vagina. Maturitas
2008;59:21925.
[20] Le Donne M, Caruso C, Mancuso A, et al. The effect of vaginally administered
genistein in comparison with hyaluronic acido on atrophic epithelium in post-
menopause. Arch Gynecol Obstet 2011;283:131923.
[21] Tedeschi C, Benvenuti C. Comparison of vaginal gel isoflavones no topical treat-
ment in vaginal dystrophy: results of a preliminary prospective study. Gynecol
Endocrinol 2012;28(August (8)):6524.
211
[22] Meisels A. The maturation value. Acta Cytol 1967;11:24955.
[23] McGurgan P, Taylor LJ, Duffy SR, ODonovan PJ. An immunohistochemical com-
parison of endometrial polyps from postmenopausal women exposed and not
exposed to HRT. Maturitas 2006;53:45461.
[24] Brancroft JD, Stevens A. Theory and Practice of Histological Techniques. New
York: Churchill Livingstone; 1977, 436 pp.
[25] Kurman RJ, Solomon DO. Sistema Bethesda para o relato de diagnstico
citolgico crvicovaginal. Rio de Janeiro: Revinter; 1997.
[26] Campaner AB, Galvo MAL. Application of na easy and useful morphomet-
ric technique for immunohistochemistry counting. Gynecol Oncol 2009;112:
282.
[27] Urbaniak GC, Plous S. Research Randomizer (Version 4.0) [Computer software];
2013. Available from: http://www.randomizer.org. [22.06.13].
[28] Long CY, Liu CM, Hsu SC, Wu CH, Wang CL, Tsai EM. A randomized comparative
study of the effects of oral and topical estrogen therapy on the vaginal vas-
cularization and sexual function in hysterectomized postmenopausal women.
Menopause 2006;13(5):73743.
[29] Santos I, Clissold S. Urogenital disorders associated with oestrogen deficiency:
the role of promestriene as topical oestrogen therapy. Gynecol Endocrinol
2010;April.
[30] Marques-Teixeira J. Placebo effect. Int J Psychiatry Clin Pract 2006 [Editorial].
[31] Weisberg E, Ayton R, Darling G, et al. Endometrial and vaginal effects of
low-dose estradiol delivered by vaginal ring or vaginal tablet. Climacteric
2005;8:8392.
[32] Freedman M, Kaunitz AM, Reape KZ, Hait H, Shu H. Twice-weekly synthetic
conjugated vaginal cream for the treatment of vaginal atrophy. Menopause
2009;16:73541.
[33] Pitkin J, Rees MC, Gray S, Lumsden MA, Stevenson J, Williamson J, et al. Manag-
ing the menopause British Menopause Society Council consensus statement
on hormone replacement therapy. J Br Menopause Soc 2003;9:12931.
[34] Labrie F, Cusan L, Gomez JL, Ct I, Brub R, Blanger P, et al. Effect of one-
week treatment with vaginal estrogen preparations on serum estrogen levels
in postmenopausal women. Menopause 2009;16:306.
[35] Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML,
et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal
women: principal results from the Womens Health Initiative randomized con-
trolled trial. JAMA 2002;288:32133.
[36] Kaari C, Haidar MA, Jnior JM, Nunes MG, Quadros LG, Kemp C, et al. Random-
ized clinical trial comparing conjugated equine estrogens and isoflavones in
postmenopausal women: a pilot study. Maturitas 2006;53(January (1)):4958
[Epub 2005 Oct 27].
[37] Manonai J, Songchitsomboon S, Chanda K, Hong JH, Komindr S. The effect of a
soy-rich diet on urogenital atrophy: a randomized, cross-over trial. Maturitas
2006;54(May (2)):13540.
[38] Wilcox G, Wahlqvist ML, Burger HG, Medley G. Oestrogenic effects of plant food
in postmenopausal women. BMJ 1990;301:9056.
[39] Uesugi T, Toda T, Okuhira T, Chen JT. Evidence of estrogenic effect by the three-
month-intervention of isoflavone on vaginal maturation and bone metabolism
in early postmenopausal women. Endocr J 2003;50(October (5)):6139.
[40] Yin DK, Baracat EC, Han KK, Giro MJBC, Panizzi MCC. Efeitos da isoflavona na
sndrome do climatrio. Rev Bras Med 2000;57:214.
[41] Galhardo CL, Soares Jr JM, Simes RS, Haidar MA, Rodrigues de Lima G,
Baracat EC. Estrogen effects on the vaginal pH, flora and cytology in late post-
menopause after a long period without hormone therapy. Clin Exp Obstet
Gynecol 2006;33:859.
[42] Nahas PE, Nahs NJ, De Luca L, Traiman P, Pontes A, Dalben I. Benefits of soy germ
isoflavones in postmenopausal women with contraindication for conventional
hormone replacement therapy. Maturitas 2004;48(August (4)):37280.
[43] Nilsson K, Risberg B, Heimer G. The vaginal epithelium in the postmenopause
cytology, histology and pH as methods of assessment. Maturitas 1995;21:516.
[44] Parker MG. Mortyn Jones Memorial Lecture. Structure and function of the
oestrogen receptor. J Neuroendocrinol 1993;5:2238.