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BMJ Open: first published as 10.1136/bmjopen-2022-068053 on 11 April 2023. Downloaded from http://bmjopen.bmj.com/ on February 16, 2024 at Escola Superior de Enfermagem de
To cite: Vergauwen G, Cools P,
Denys H, et al. GRACE-­trial: a
randomised active-­controlled
trial for vulvovaginal atrophy
in patients with breast
cancer on endocrine therapy
– study protocol. BMJ Open
2023;13:e068053. doi:10.1136/
bmjopen-2022-068053
► Prepublication history for
this paper is available online.
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the journal online (http://dx.doi.​
org/10.1136/bmjopen-2022-​
068053).
Received 05 September 2022
Accepted 27 March 2023
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employer(s)) 2023. Re-­use
permitted under CC BY-­NC. No
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and permissions. Published by
BMJ.
For numbered affiliations see
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Correspondence to
Dr Glenn Vergauwen;
​glenn.​vergauwen@u​ gent.​be
GRACE-­trial: a randomised active-­
controlled trial for vulvovaginal atrophy
in patients with breast cancer on
endocrine therapy – study protocol
Glenn Vergauwen ‍ ‍,1,2 Piet Cools,3 Hannelore Denys,1,4 Tom Fiers,5
Koen Van de Vijver,1,6 Liv Veldeman,1,7 Hans Verstraelen2
ABSTRACT
Introduction Breast cancer is the most common cancer
type in women worldwide. Due to hormone receptor
positivity in the majority of the breast cancer tumours
is endocrine therapy a crucial part in the treatment
landscape of breast cancer. Endocrine therapy consists
of the use of selective oestrogen-­receptor modulators
or aromatase inhibitors. These medicines generate a
hypoestrogenic environment by reducing circulating
oestrogen or by altering the effect of oestrogen on
tissue cells by receptor blockade. As a common side
effect, vulvovaginal atrophy occurs in the majority of
patients with breast cancer using endocrine therapy.
Vulvovaginal atrophy has a significant impact on
physical and psychological well-­being due to negative
influence on quality-­of-­life, self-­esteem and sexuality.
As a consequence, adherence to endocrine therapy
for the standard duration of 5–10 years is challenging,
resulting in higher rates of therapy interruption, leading
to poorer prognosis with shorter distant disease-­free
survival. The standard treatment for vulvovaginal atrophy
in postmenopausal women is based on the use of local
hormonal treatment. However, when a patient has a history
of breast cancer, delay of treatment and undertreatment
are ubiquitous.
Methods and analysis In this first ever prospective
randomised trial patients with breast cancer on endocrine
therapy with vulvovaginal atrophy will be treated with
the available local treatment modalities with a 1:1:1:1
randomisation: oestrogen, dehydroepiandrosterone,
moisturisers and a co-­treatment of oestrogen and probiotics.
Patient-­reported outcomes measurements will be implemented
to investigate the efficacy of the implemented treatments.
Safety of the treatments will be evaluated by assessing
systemic sex hormones concentrations.
Ethics and dissemination This study was approved by
the Ethical Committee of Ghent University Hospital and
by the Federal Agency for Medicines and Health Products.
Results will be published in peer-­reviewed journals and
released in international conferences.
Trial registration number 2021-­001921-­31.
INTRODUCTION
Breast cancer is the most common cancer type
and cause of cancer-­related death in women
Vergauwen G, et al. BMJ Open 2023;13:e068053. doi:10.1136/bmjopen-2022-068053
Protocol
STRENGTHS AND LIMITATIONS OF THIS STUDY
⇒ Randomised phase IV trial.
⇒ Implementation of an active control arm instead of
placebo.
⇒ No blinding procedures in this trial, patient and phy-
sician know the treatment allocation.
⇒ Single-­
centre study with relatively small sample
size.
Coimbra. Protected by copyright.
worldwide.1 Tumour growth and develop-
ment in breast cancer tends to be most often
hormone-­dependent, with up to 80% of all
diagnosed breast cancer tumours displaying
hormone receptor positivity.2 Accordingly, a
cornerstone of hormone-­ receptor positive
breast cancer therapy is endocrine therapy
with selective oestrogen-­ receptor modula-
tors (SERM) and aromatase inhibitors (AI)
for a treatment duration of 5–10 years being
the most commonly used. The primary goal
of endocrine therapy in patients with breast
cancer is to improve long-­ term prognosis
through a reduction of oestrogen-­effect on
tissues by either reducing the circulating
oestrogen (AI) or blocking the oestrogen-­
receptor (SERM).3 4
Vulvovaginal atrophy (VVA) is one of the
most common yet upmost undertreated and
underdiagnosed side effects of endocrine
therapy in patients with breast cancer. Patients
suffering from VVA will typically experience
symptoms of vaginal dryness, irritation,
pruritus and dyspareunia.5 VVA has its impact
on quality-­of-­life, self-­
esteem and sexuality
and therefore has an important role in the
overall well-­being of the patient.5 6 Vasomotor
symptoms, such as hot flushes and night
sweats, are about equally common, yet by far
more discussed and treated than VVA. While
vasomotor symptoms generally diminish over
time, VVA will persist (lifelong), stressing
1
Open access
the importance of diagnosing and discussing symptoms
of VVA in patients with breast cancer and in extent with
all postmenopausal patients. The majority of patients
with breast cancer on endocrine therapy will experience
symptoms of VVA during endocrine therapy.7 Due to the
occurrence of these symptoms, adherence to endocrine
therapy in patients with breast cancer is challenged and
high rates of interruption or termination of endocrine
therapy are reported, impeding long-­term prognosis of
the patient.8
The standard treatment for VVA in postmenopausal
women is based on the use of local hormonal treatment.
Facing a history of breast cancer, patients will experi-
ence delay of treatment and undertreatment for VVA.
Hesitance for discussing vaginal health and sexuality is
a facilitator for the deprivation of VVA treatment along
with reluctance of initiation of local hormonal treatment
in breast cancer survivors.9 Besides moisturisers and local
hormonal treatments, recent evidence points towards
targeting the vaginal microbiome in order to restore
vaginal health.10–12 Supplying probiotics can contribute
in restoring and maintaining a healthy vaginal homoeo-
stasis.13 14
At present, there is lack of large prospective randomised
data on local treatments for VVA in patients with breast
cancer, yet the available literature showed no increased
recurrence risk for breast cancer and their use is
supported by official guideline statements from organisa-
tions such as the American College of Obstetricians and
Gynecologists.9 15
This randomised trial will assess prospectively four
different treatment strategies for VVA, including
Figure 1 Study overview.
2 Vergauwen G, et al. BMJ Open 2023;13:e068053. doi:10.1136/bmjopen-2022-068053
BMJ Open: first published as 10.1136/bmjopen-2022-068053 on 11 April 2023. Downloaded from http://bmjopen.bmj.com/ on February 16, 2024 at Escola Superior de Enfermagem de
moisturiser, local oestrogen, local oestrogen with probi-
otics and dehydroepiandrosterone (DHEA).
MATERIALS AND METHODS
Study design and objectives
GRACE-­trial is a single-­centre, prospective, randomised
active-­controlled clinical trial for the assessment of effi-
cacy and safety of different available local treatment
modalities in patients with breast cancer on endocrine
therapy with VVA. Four different treatment modalities
will be assessed in this clinical trial: oestrogen, DHEA,
moisturisers and a co-­treatment of oestrogens and probi-
otics. A study overview can be found in figure 1.
Ethics and dissemination
This study was reviewed and approved by the Ethical
Committee of Ghent University Hospital (study number
BC-­09638, protocol V.1.0, approval on 22 October 2021)
and by the Federal Agency for Medicines and Health
Products (approval on 25 October 2021). Study results
will be published in peer-­reviewed journals and further
disseminated at international conferences.
Coimbra. Protected by copyright.
Study population and eligibility criteria
Patients with breast cancer on endocrine therapy (SERM
or AI) with symptoms of VVA are the primary target popu-
lation for this study.
A total number of 160 patients will be enrolled in this
study (40 patients in each treatment group).
The eligibility criteria can be found in figure 2.

Figure 2 Eligibility criteria. FSH: follicle-­stimulating hormone, GnRH: gonadotropin-­releasing hormone.
Recruitment and randomisation
Recruitment of patients will take place at Ghent Univer-
sity Hospital during routine follow-­ up appointments
at one of the participating departments (Gynaecology,
Medical Oncology and Radiotherapy). Initial screening
will be performed by the treating physician. If a patient
with breast cancer on endocrine therapy with symptoms
of VVA is seen on a routine follow-­up breast cancer consul-
tation, the study will be introduced and the patients inter-
ested in the study will be referred to the study team for
evaluation of inclusion and exclusion criteria.
Randomisation will be performed electronically
through a computer-­driven randomisation list. No strati-
fication factors will be implemented. There is no blinding
of treatment for participant or investigator. Withdrawn
subjects will be replaced until the predefined goal of
160 subjects have completed the study. If the following
data and samples are available, the subject will not be
replaced: at least two patient-­reported outcome measure-
ments (PROMs) assessments, at least two serum samples
and two vaginal microbiome samples.
Trial status
Recruitment is ongoing and started in March 2022 and is
expected to be completed in March 2026.
Allocation and initial assessment
The study team will contact the patient and a prescreening
with control of eligibility criteria will be performed. When
a patient is eligible, a first study visit will be planned.
Initial assessment will be performed at the first study visit
after agreement and signing the informed consent file.
Patients will be allocated to a treatment group based on
the result of the randomisation process.
Data management and monitoring
Registered study data will be stored digitally, using
Research Electronic Data Capture (REDCap, Nashville,
Vergauwen G, et al. BMJ Open 2023;13:e068053. doi:10.1136/bmjopen-2022-068053
Open access
BMJ Open: first published as 10.1136/bmjopen-2022-068053 on 11 April 2023. Downloaded from http://bmjopen.bmj.com/ on February 16, 2024 at Escola Superior de Enfermagem de
USA). Data will be not be publicly available and stored
securely at the study centre. Adverse events and serious
adverse events will be reported. All serious adverse events
Coimbra. Protected by copyright.
(initial and follow-­up information) occurring during this
study will be reported within 24 hours to the Clinical
Trial Unit of Ghent University Hospital. The latter will be
responsible for data monitoring.
Primary objectives
Three primary objectives have been determined. The effi-
cacy of the different treatment modalities will be assessed
by means of the first two primary objectives. The first
primary objective is the assessment based on PROMs.
The second primary objective is the clinical evaluation by
means of vaginal pH and the Vaginal Maturation Index,
the latter being a direct microscopical evaluation of the
vaginal epithelium using a vaginal smear sample.
Lastly, the third primary objective is the evaluation of
systemic sex hormone concentrations. This objective is
frequently used to address safety in regard to sustained
systemic elevations of these hormones.
Secondary objectives
The secondary objective in this study is the character-
isation of vaginal microbial alterations after treatment
initiation. Identification of these alterations can help in
further understanding of the pathophysiology of VVA
and potentially create opportunities for new treatment
strategies towards VVA in patients with breast cancer on
endocrine therapy.
Study treatment
Patients will be allocated to one of four treatment arms,
with 40 patients to be enrolled in each arm. All treatment
groups will receive a topical (vaginal) treatment for VVA
for 12 weeks. An overview of treatment regimens can be
found in figure 1.
3
Open access
Group A will receive 0.03 mg oestriol (Oekolp, Dr
KADE Pharmazeutische Fabrik, Berlin, Germany) in
a regime of one vaginal ovule daily for three consecu-
tive weeks, followed by one vaginal ovule two times per
week. Group B will receive 6.5 mg prasterone (DHEA)
(Intrarosa, Basic Pharma Manufacturing, Geleen, The
Netherlands) in a regime of one vaginal ovule daily.
Group C will receive a moisturiser treatment, based on
hyaluronic acid (Premeno Duo, DeltaMed, Friedberg,
Germany) in a regime of one vaginal ovule daily for 10
consecutive days, followed by one vaginal ovule two times
per week. Group D will receive a combination treatment
of 0.03 mg oestriol and Lactobacillus acidophilus (Gynoflor,
Gideon Richter, Budapest, Hungary) in a regime of one
vaginal ovule daily for 12 consecutive days, followed by
one vaginal ovule two times per week.
Interventions
Enrolled patients will have three study visits. Study visits
will be organised at baseline, after 6 weeks (±14 days)
and after 12 weeks (±14 days, end of study). On these
study visits the following interventions will be performed
(figure 1).
Vaginal assessments
These will include vaginal pH measurement using a pH
indicator strip (art. 662–0201, Macherey-­Nagel, Düren,
Germany) after insertion of a non-­lubricated strip against
the mid-­vaginal lateral wall for 10 s. After measuring the
vaginal pH, a vaginal sampling brush (Viba-­Brush, Rovers
Medical Devices, Oss, The Netherlands) will be used to
gently scrape the upper third of the vaginal wall to obtain
exfoliating squamous cells to assess the Vaginal Matura-
tion Index.16 The Vaginal Maturation Index represents
the proportion of parabasal, intermediate and superfi-
cial squamous cells. Using a predefined formula (0.2 ×
% parabasal cells + 0.6 × % intermediate cells + 1.0 × %
superficial cells), a qualitative indicator for oestrogenic
effect on the vaginal epithelium can be obtained.
Venous blood sampling
Due to the low concentrations, general laboratory tests
of sex steroids are inadequate for the evaluation of
possible alterations in these concentrations, except for
dehydroepiandrosterone sulphate (DHEA-­ S) which is
abundantly present in serum. Previous work already indi-
cated the strength and importance of measurements by
mass spectrometry (liquid chromatography with tandem
mass spectrometry (LC-­ MS/MS)) in order to achieve
competent assessment of concentration variations. The
following sex steroid concentrations will be determined:
oestrone, oestradiol, DHEA, DHEA-­S, testosterone and
dihydrotestosterone.
Mid-vaginal swab
This will be used for microbial analysis. After collecting the
swab, the sample will be stored at −80°C until processed.
Bacterial DNA will be extracted and the microbiome will
4 Vergauwen G, et al. BMJ Open 2023;13:e068053. doi:10.1136/bmjopen-2022-068053
BMJ Open: first published as 10.1136/bmjopen-2022-068053 on 11 April 2023. Downloaded from http://bmjopen.bmj.com/ on February 16, 2024 at Escola Superior de Enfermagem de
be characterised using cpn60 sequencing metagenomics
as previously described.17
PROMs
Apart from these clinical interventions, patients will also
be required to fill in questionnaires for PROM assess-
ment, using EQ-­5D-­questionnaire18 and the FACT-­ ES
questionnaire.19
Data analysis
Sample size
Given the lack of direct comparative studies between the
included treatments, we based our sample size calculation
on the only available retrospective review that compares
DHEA and oestrogens with placebo, although not in a
direct comparison but through retrospective interstudy
comparison.20 In this review, the difference of effect of
DHEA and oestrogen to placebo in previous studies was
compared using severity scores for vaginal dryness.21 22
DHEA improved the severity score in three studies with
a mean of 0.33 compared with placebo, while this was
0.40 for oestrogen. Using these numbers, aiming for a
power of 90%, a sample size of 12 for each group would
be needed. However, due to lack of prospective compara-
tive data, we will increase our sample size to 40 patients in
Coimbra. Protected by copyright.
each group to overcome potential lower differences, yet
we acknowledge that this is an arbitrary chosen increase.
Statistics
Descriptive statistics will be implemented (number of
observations, mean, SEM). Comparison of treatments
will be based on t-­tests, analysis of variance and, for sex
steroid concentrations, analysis of covariance with the
baseline concentration as covariate.
Patient and public involvement
Patients were involved in the design of this research,
where the research question, implemented question-
naires and outcome measures were discussed.
DISCUSSION
At present, patients with breast cancer with VVA are inad-
equately treated for this common side effect of endocrine
therapy. A direct head-­to-­head comparison for efficacy
is lacking, therefore it was chosen as one of our primary
objectives. With this comparison we aim to clearly distinct
the magnitude of treatment efficacy between the different
modalities. In close relationship with efficacy resides the
question of safety with regard to breast cancer recur-
rence. Systemic alterations of sex steroid concentrations
have previously been described where the magnitude of
these alterations depend on the implemented treatment
modality. No increased recurrence has been described
after initiation of local treatment for VVA.15 16 With this
direct comparison we aim to objectify the relative magni-
tude of alterations of sex steroid concentrations. This
new information may contribute to the increase of aware-
ness and may help clinical decision-­ making, possibly

generating a lower threshold for clinicians to start treat-
ment for VVA in patients with breast cancer. This in turn
may improve adherence to endocrine therapy which is a
prerequisite for its success. Non-­adherence to endocrine
therapy is driven by the side effects of this treatment.
The vaginal microbiome fulfils a crucial role in the
vaginal homoeostasis. Previously, differences in the
vaginal microbiome of postmenopausal women with and
without VVA have been identified.14 Microbial changes
induced by treatment will be evaluated in this study.
Furthermore, we implement probiotics in combination
with oestrogen in the vaginal treatment of VVA. Previous
work demonstrated the safety and efficacy of this combi-
nation treatment in patients with breast cancer in a phase
1 trial.13 However, the evaluation of the additive effect
of vaginal probiotics in an oestrogen-­based vaginal treat-
ment is lacking. With this study we also aim to investigate
the effect of probiotics in comparison to oestrogen-­only
and the other treatments for VVA.
Trial status
Recruitment for the study was commenced in March
2022. At time of manuscript submission, patient recruit-
ment was ongoing.
Author affiliations
1 12 Cruickshank R, Sharman A. The biology of the vagina in the human
Cancer Research Institute Ghent, Ghent University, Gent, Belgium
2
Department of Obstetrics - Gynaecology, University Hospital Ghent, Gent, Belgium
3 age-­periods and their relations to glycogen in the vaginal epitehlial.
Department of Diagnostic Sciences, Ghent University, Gent, Belgium
4 BJOG An Int J Obstet Gynaecol 1934;41:190–207.
Department of Medical Oncology, University Hospital Ghent, Gent, Belgium
5
Department of Medical Microbiology, University Hospital Ghent, Gent, Belgium
6
Department of Pathology, University Hospital Ghent, Gent, Belgium
7
Department of Radiation Oncology, University Hospital Ghent, Gent, Belgium
Contributors Study conception: GV, HV. Initial study design: GV, HV. Revision of
study design and protocol: GV, HV, PC, HD, TF, KVdV, LV. Study coordination: GV.
Patient recruitment: GV, HD, LV. Laboratory analysis: TF, PC. Cytological analysis
(VMI): GV, KVdV. Drafting the manuscript: GV. All authors read and approved the final
manuscript.
Funding This trial was funded by Kom Op Tegen Kanker.
Competing interests None declared.
Patient and public involvement Patients and/or the public were involved in the
design, or conduct, or reporting, or dissemination plans of this research. Refer to
the Methods section for further details.
Patient consent for publication Consent obtained directly from patient(s).
Provenance and peer review Not commissioned; externally peer reviewed.
Open access This is an open access article distributed in accordance with the
Creative Commons Attribution Non Commercial (CC BY-­NC 4.0) license, which
permits others to distribute, remix, adapt, build upon this work non-­commercially,
and license their derivative works on different terms, provided the original work is
properly cited, appropriate credit is given, any changes made indicated, and the use
is non-­commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.
ORCID iD
Glenn Vergauwen http://orcid.org/0000-0002-2213-163X
Vergauwen G, et al. BMJ Open 2023;13:e068053. doi:10.1136/bmjopen-2022-068053
Open access
BMJ Open: first published as 10.1136/bmjopen-2022-068053 on 11 April 2023. Downloaded from http://bmjopen.bmj.com/ on February 16, 2024 at Escola Superior de Enfermagem de
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