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\begin{document}

\title{Vaginal changes after ovariectomy in ewes -- a large animal model for
Genitourinary Syndrome of Menopause}



\author[1]{Eva Vodegel}%
\author[1]{Zeliha Guler}%
\author[2]{Lamees Ras}%
\author[3]{Katerina Mackova}%
\author[1]{Anne Groeneveld}%
\author[2]{Deon Bezuidenhout}%
\author[4]{Jan Deprest}%
\author[2]{Stephen Jeffrey}%
\author[1]{Jan-Paul Roovers}%
\affil[1]{Amsterdam UMC Location AMC}%
\affil[2]{University of Cape Town}%
\affil[3]{KU Leuven}%
\affil[4]{KU Leuven Department of Development and Regeneration}%


\vspace{-1em}



  
  \date{May 20, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Objective: To evaluate the effect of iatrogenic menopause on the
physiology of the vagina of the ewe, and to evaluate if vaginal changes
in ewes can be translated to women with the genitourinary syndrome of
menopause (GSM). Design: Animal study, randomised design for ovariectomy
or control, blinded for allocation and outcome assessment. Setting:
University of Cape Town, South Africa. Population or Sample: Twenty-five
Dohne Merino ewes. Methods: Iatrogenic menopause was induced in 20
animals by bilateral ovariectomy. Five animals served as a control group
(no intervention). Differences between groups were determined by linear
regression analyses (adjusted for baseline scores) at 5 months after
ovariectomy. Main Outcome Measures: Vaginal epithelial thickness, pH,
vaginal maturation value, vaginal maturation index, epithelial glycogen
accumulation, content of elastin fibres, collagen, and vascularity.
Results: Ovariectomised ewes showed epithelial thinning of the vaginal
wall from 146 \selectlanguage{greek}µ\selectlanguage{english}m to 47 \selectlanguage{greek}µ\selectlanguage{english}m (mean, p \textless{}0.001 ). In addition,
epithelial glycogen accumulation (43\%) and the vascularity (23\%) of
the vaginal wall significantly decreased as compared to the control
group. No differences were found for vaginal pH, vaginal cytology
outcomes, elastin fibres and collagen content. Conclusions: This study
established the ewe as a suitable large animal model for GSM.
Furthermore, the similar relevant outcomes in humans and ewes hold great
value for future translational research for the evaluation and
optimisation of different treatment modalities for GSM. Funding: None.
Keywords: Atrophy, ewe, genitourinary syndrome of menopause, model,
ovariectomy.%
\end{abstract}%



\sloppy


\textbf{Vaginal changes after ovariectomy in ewes -- a large animal
model for Genitourinary Syndrome of Menopause}

\textbf{Authors:} Eva V. VODEGEL (MD)\textsuperscript{1,2}, Zeliha GULER
(PhD)\textsuperscript{1,2}, Lamees RAS (MD)\textsuperscript{3}, Katerina
MACKOVA (MD, PhD)\textsuperscript{4,5}, Anne C.H.M.
GROENEVELD\textsuperscript{1,2}, Deon BEZUIDENHOUT
(PhD)\textsuperscript{6}, Jan DEPREST (MD, PhD)\textsuperscript{4,7},
Stephen T. JEFFERY (MD)\textsuperscript{3}, Jan-Paul W.R. ROOVERS (MD,
PhD)\textsuperscript{1,2}

\begin{enumerate}
\tightlist
\item
  Amsterdam UMC location University of Amsterdam, Dept. of Obstetrics
  and Gynaecology, Meibergdreef 9, Amsterdam, The Netherlands.
\item
  Amsterdam Reproduction and Development research institute, Amsterdam,
  the Netherlands.
\item
  Department of Obstetrics and Gynaecology, Groote Schuur Hospital,
  University of Cape Town, Cape Town, South Africa.
\item
  Department of Development and Regeneration, KU Leuven, Leuven,
  Belgium.
\item
  Institute for the Care of Mother and Child, Third Faculty of Medicine,
  Charles University, Prague, Czech Republic.
\item
  Cardiovascular Research Unit, Cape Hearth Centre, University of Cape
  Town, Cape Town, South Africa.
\item
  Pelvic Floor Unit, University Hospitals KU Leuven, Leuven, Belgium.
\end{enumerate}

\textbf{Corresponding author:} Eva V. Vodegel, MD Amsterdam UMC,
University of Amsterdam, location AMC Dept. of Obstetrics and
Gynaecology, room H4-222 Meibergdreef 9, 1105 AZ Amsterdam, the
Netherlands

Telephone: +31(0)20-5666429

E-mail: e.v.vodegel@amsterdamumc.nl

\textbf{Running title:} Vaginal changes after ovariectomy in ewes

\textbf{Study conducted in:} South Africa

\textbf{ABSTRACT}

\textbf{Objective:} To evaluate the effect of iatrogenic menopause on
the physiology of the vagina of the ewe, and to evaluate if vaginal
changes in ewes can be translated to women with the genitourinary
syndrome of menopause (GSM).

\textbf{Design:} Animal study, randomised design for ovariectomy or
control, blinded for allocation and outcome assessment.

\textbf{Setting:} University of Cape Town, South Africa.

\textbf{Population or Sample:} Twenty-five Dohne Merino ewes.

\textbf{Methods:} Iatrogenic menopause was induced in 20 animals by
bilateral ovariectomy. Five animals served as a control group (no
intervention). Differences between groups were determined by linear
regression analyses (adjusted for baseline scores) at 5 months after
ovariectomy.

\textbf{Main Outcome Measures:} Vaginal epithelial thickness, pH,
vaginal maturation value, vaginal maturation index, epithelial glycogen
accumulation, content of elastin fibres, collagen, and vascularity.

\textbf{Results:} Ovariectomised ewes showed epithelial thinning of the
vaginal wall from 146 \selectlanguage{greek}µ\selectlanguage{english}m to 47 \selectlanguage{greek}µ\selectlanguage{english}m (mean, \emph{p} \textless{}0.001 ). In
addition, epithelial glycogen accumulation (43\%) and the vascularity
(23\%) of the vaginal wall significantly decreased as compared to the
control group. No differences were found for vaginal pH, vaginal
cytology outcomes, elastin fibres and collagen content.

\textbf{Conclusions:} This study established the ewe as a suitable large
animal model for GSM. Furthermore, the similar relevant outcomes in
humans and ewes hold great value for future translational research for
the evaluation and optimisation of different treatment modalities for
GSM.\textbf{Funding:} None.

\textbf{Keywords:} Atrophy, ewe, genitourinary syndrome of menopause,
model, ovariectomy.

\textbf{Tweetable abstract:} Iatrogenic menopause in ewes mimic the
physiologic vaginal changes in postmenopausal women encountering
genitourinary syndrome of menopause (GSM). This animal model is vital to
improve treatment for GSM.

\textbf{1 \textbar{} INTRODUCTION}

The genitourinary syndrome of menopause (GSM), formerly known as
vulvovaginal atrophy, represents a variety of genitourinary symptoms and
signs associated with menopause and is directly related to decreased
circulating oestrogen levels.\textsuperscript{1} Clinical symptoms
include vaginal dryness, pruritus, burning, irritation, dyspareunia and
urinary symptoms of urgency, dysuria and recurrent urinary tract
infections. Physiologic changes result in epithelial thinning, increased
vaginal pH, and reduced vaginal blood flow, collagen content, vaginal
secretions, and elasticity.\textsuperscript{2-4}

GSM has a negative impact on women's quality of life and their sexual
health.\textsuperscript{3} Although the true prevalence of GSM is
difficult to determine, it is estimated that up to 50\% of
postmenopausal women are affected.\textsuperscript{5} These symptoms can
be chronic and progressive.

With the increasing life expectancy, women will spend one-third of their
life in the postmenopausal period.\textsuperscript{6} This emphasises
the great extent of the problem and growing interest in the development
and improvement of existing and future therapies for GSM. Current
treatments are vaginal moisturizers, lubricants and vaginal oestrogen
therapy. Alternative treatments could be vaginal dehydroepiandrosterone
(DHEA), ospemifene or vaginal laser therapy. However, more data is
needed to attain proof of concept if new therapies are safe and
efficacious in the treatment of GSM. For approval for clinical use, the
Food and Drug Administration (FDA) requires preclinical evidence of
safety and efficacy of any novel treatments. It is for this reason that
the establishment of a suitable animal model is crucial. The
preferential use of an animal model over in-human studies is critical to
initially establish safety and efficacy. Some of the reasons for this
are the potential safety concerns when evaluating treatment dosing and
timing (for example with vaginal laser therapy settings), concerns with
the invasiveness of multiple vaginal biopsies in women when these
measurements are required, and the high cost of clinical trials.

Thus far, the ovariectomised ewe has been used in a few studies as a
large animal model for GSM.\textsuperscript{12-14} However, the validity
of these studies is questionable as there were a few flaws in the study
design. These include a lack of premenopausal control groups, baseline
measurements, long term follow-up, comparative analyses between the
study groups at the same point in time and methods of statistical
analyses. Hence, this study is needed to objectively examine the effect
of (iatrogenic) menopause on the physiologic changes in the ewe's vagina
using specific points of measurement that are affected in women with
GSM, thereby validating the animal model for GSM. Such a large animal
model is vital for future development and optimisation of new treatment
interventions for women suffering with GSM.

\textbf{2 \textbar{} METHODS 2.1 \textbar{} Animals}

Twenty six (26) multiparous Dohne Merino ewes (6 years old) of similar
origin were recruited for this study. However, the study was performed
in 25 ewes as one animal died due to age related health issues before
the study started. During the study the flock was held on the field with
free access to food and water (ad libitum). The animals were
acclimatised one week before the study started. During the study, the
animals were monitored daily and weighed weekly, conforming with the
care and husbandry guidelines of the National Health and Medical
Research Council of South Africa for animals in research. This study was
approved by the Animal Ethics Committee of the University of Cape Town
(Protocol number: 018\_005).

\textbf{2.2 \textbar{} Study design \& procedures}

\emph{2.2.1 \textbar{} Cycle synchronisation}

Ewes have cyclical changes in the vaginal epithelium with epithelial
thickening during oestrus and a decrease in thickness during the
pro-oestrus and dioestrus period.\textsuperscript{15, 16} Cycle
synchronisation was performed in all animals using progestogen
impregnated sponges (flugestone acetate, 40mg/sponge, Ovakron\selectlanguage{ngerman}®, Ani
Pharm, Bloemfontein, South Africa). A sponge was inserted intravaginally
for 14 days. In ewes, oestrus occurs approximately 48 hours after sponge
removal.\textsuperscript{17}

\emph{2.2.2 \textbar{} Allocation, randomisation and blinding}

After acclimatisation and cycle synchronisation, all the ewes were
allocated with a unique study number using an ear clip. Twenty animals
were randomly allocated to the (iatrogenic) menopause intervention arm,
and had bilateral ovariectomy (OVX) performed on them. The remaining
five ewes were allocated to the premenopausal control arm and therefore
recieved no intervention (see Supplementary material Appendix S1 for the
power calculation, including an explanatory note on the difference in
group size). During the study, researchers and caregivers were blinded
for allocation (including the identifying code), except at time of the
ovariectomy when the animals were clipped, and the abdominal scar was
still visible. All animals were operated and examined in a random order
during the entire study. Also, the histology and cytology images were
blinded for allocation and time points (using Blind Analysis tools,
ImageJ; 1.50i), and were assessed in a random order by two independent
researchers.

\emph{2.2.3 \textbar{} Bilateral ovariectomy -- study intervention}

48-72 hours after sponge removal, bilateral ovariectomy was performed
via a small laparotomy (6-8 cm midline incision) under general
anaesthesia. Surgery details are presented in the Supplementary
material, Appendix S2. For the study design, including the intervention
and assessments, see Table S1 of the Supplementary material.

\textbf{2.3 \textbar{} Outcome measures}

\emph{2.3.1 \textbar{} Vaginal pH \& cytology}

The vaginal pH was assessed monthly using litmus strips (VWR
International bv, Leuven, Belgium, range 2-9; resolution 0.5), which was
placed in the vagina for 4 seconds.

Vaginal cytology assessment was performed monthly using a cytology
brush, which was placed in liquid-based fixative after swabbing the
vagina. A Papanicolaou (PAP) test was performed (SA Path - Cape Town
Laboratory) and slides were digitalised (bright field digital slide
scanner; NOCTN44, Philips). The percentage of superficial, intermediate
and parabasal cells were counted out of 100 cells using a Cell counter
Plugin tool (ImageJ, 1.5i). The vaginal maturation value (VMV) was
calculated (1 × superficial cells + 0.6 × intermediate cells + 0.2 ×
parabasal cells).\textsuperscript{18}

\emph{2.3.2 \textbar{} Biopsies}

Monthly biopsies were taken in all ewes at the distal vaginal anterior
wall 6 consecutive times (see also the Supplementary material, Table
S1). Biopsies were performed under sedation, (ketamine, 5-10mg/kg,
intramuscular) with the animal being restrained in a dorsal recumbent
position on a surgical trolley. Biopsy size was ± 5 mm and they were
taken at various locations of the distal anterior wall, 1 cm proximal
from the hymen (at 11, 3, 9, 2, 10, and 1 o'clock respectively -- where
the anterior midline is assumed to be 12 o'clock), to prevent
involvement of scar tissue.

\emph{2.3.3 \textbar{} Histology and histological examination}

All details about histology sample preparation and staining procedures
can be found in the Supplementary material, Appendix S3. Vaginal
biopsies were stained with haematoxylin and eosin (H\&E) to evaluate the
epithelial thickness. The presence of epithelial glycogen accumulation
was demonstrated by Periodic acid-Schiff (PAS) staining. Van Gieson's
and Masson's Trichrome stains were used to detect elastin fibres and to
quantify collagen and vascularity in the specimens respectively.

For the primary outcome epithelial thickness, a representative part of
the biopsy was selected by two blinded researchers (at 100×
magnification). The average epithelial thickness was assessed using a
computer-assisted measurement program (ImagePro, v.10), taking the
variability of the dermal papillae into
consideration.\textsuperscript{19} The upper border of the epithelium
and the wave-like border of the epithelial basal layer were marked with
a minimum length of 200 \selectlanguage{greek}μ\selectlanguage{english}m and a maximum length of 3000 \selectlanguage{greek}μ\selectlanguage{english}m. The
epithelial thickness between these two lines was automatically
calculated on a resolution of 1 \selectlanguage{greek}μ\selectlanguage{english}m and was averaged to determine a
representative and standardised epithelial thickness of each specimen.
In case of unresolved discrepancies or a questionable location of the
basal membrane, another researcher was consulted.

For the assessment of the epithelial glycogen accumulation, elastin
fibres, collagen, and vascularity; five images were randomly taken (at
400\selectlanguage{ngerman}× magnification) from each specimen by a blinded researcher. Each
image was scored for abundance on a 4-point grading scale, from 0 till
3, by two other independent blinded researchers, providing an average
score (between 0-3) calculated from 10 scores per biopsy. Details are
given in the Supplementary material Appendix S4. In case of
discrepancies, the scoring was adjudicated by a third reviewer.

\textbf{2.4 \textbar{} Data analyses}

Data was analysed using computer software IBM SPSS Statistics for
Windows (Version 28.0. Armonk, NY: IBM Corp). Data and residuals from
models were assessed for normality by histograms and Q-Q plots. At 5
months after OVX, differences between groups (OVX and non-OVX) were
determined by linear regression analyses, adjusting for baseline scores.
Data is reported as mean ± standard deviation (SD). Data analyses was
performed in consultation with a statistician -- Dr. van Eekelen.

\textbf{3 \textbar{} RESULTS}

At baseline (before OVX), 25 Dohne Merino ewes were all aged six years
old with a mean weight of 68.0 kg (SD 7.5), and all being multiparous
(all ewes had a parity of four). At baseline, the two groups were
similar for all outcomes (Table 1).

\textbf{3.1 \textbar{} Vaginal pH \& cytology}

At five months after OVX, the vaginal pH levels were not different
between the OVX and the control group (OVX 8.1 ±0.31 SD; control 8.0
±0.35 SD). For both the VMV (OVX 57 ±8 SD; control 62 ±13 SD) and the
percentage of superficial cells (OVX 7\% ±8 SD; control 32\% ±25 SD)
there were lower values in the OVX group when compared to the
premenopausal control group, but these were not statistically
significant. See also Table 1 and Figure 1.

\textbf{3.2 \textbar{} Histology}

At five months after OVX, there is a statistically significant
(\emph{p}\textless{}0.001) decrease in epithelial thickness of in the
OVX group (47 \selectlanguage{greek}µ\selectlanguage{english}m \selectlanguage{ngerman}±34 SD) when compared to the premenopausal control
group (146 \selectlanguage{greek}µ\selectlanguage{english}m \selectlanguage{ngerman}±53 SD). Further analyses showed statistically significant
(\emph{p} =0.02) lower levels of glycogen accumulation in the OVX group
(score 1.36 ±0.77 SD) when compared to control (score 2.40 ±0.60 SD).
There was also a statically significant lower amount of blood vessels
(\emph{p} \textless{}0.01) in the OVX group (score 2.07 ±0.49 SD) when
compared to control (score 2.68 ±0.28 SD). No differences were found for
elastin fibres and collagen content when the two groups were compared at
five months after OVX (See also Table 1, Figure 1, 2 and 3).

\textbf{4 \textbar{} DISCUSSION}

\textbf{4.1 \textbar{} Main findings}

This study shows that ovariectomy in ewes induces epithelial thinning of
the vaginal wall, and decreases the glycogen accumulation and the
vascularity in the vaginal wall. The effects of ovariectomy in ewes seem
to reflect the physiologic changes of the vagina in women encountering
GSM.

\textbf{4.2 \textbar{} Strengths and limitations}

Our findings are obtained from a well-designed and conducted study
including randomisation, appropriate sample size, similar group
characteristics at baseline, allocation concealment, blind outcome
assessment, and the longitudinal design including repeated
measurements.\textsuperscript{20, 21} Also, the use of a premenopausal
control group made it possible to perform between-group analyses and
therefore eliminate confounders due to time-related and environmental
effects. This includes factors like fluctuating seasonal oestrogen
levels (the ewe is a short-day breeder), or seasonal related intake of
red clover (containing high levels of phyto-oestrogen). However, a
limitation of between-group analyses is the individual difference
variable. To minimise this confounder, we adjusted for baseline scores
when performing linear regression analyses.

Furthermore, our research group has extensive experience with the
evaluation of physiologic changes in the vagina and pelvic floor,
including a variety of outcome measures and innovative
technologies.\textsuperscript{22-27} Also for this study, we devised an
innovative way to determine the mean epithelial thickness of the vaginal
wall, which provided a solid and representative value for our primary
outcome. In addition, the 4-point grading scale, used for histology
assessment, was determined from the data available from this study. As a
result, the full range of abundancy was optimally used which enabled us
to capture the effect of ovariectomy on these outcome measures the best
possible. Lastly, the longitudinal design allowed us to see when a
plateau phase of epithelial thinning was reached.

We are aware that our research has some limitations as well. The quality
of the PAP stained cytology slides varied from poor to excellent,
leading to difficulties in reading and interpretating with subsequent
missing data. Therefore, analysis was done in a smaller sample size than
originally planned. Furthermore, due to the small introitus of the ewe,
we decided to not include gynaecological examination to evaluate the
clinical signs of GSM. According to the principles of humane animal
research (replacement, reduction and refinement), we aimed to minimise
pain and distress. Last, due to an unexpected loss of one animal just
before the study started, the control group consisted of five instead of
six animals. However, the statistical power was not affected since a
10\% loss of animals was taken into account.

\textbf{4.3 \textbar{} Interpretation}

GSM comprises a variety of changes in the genitourinary tissues in
response to the loss of oestrogen with menopause. In this study we
obtained iatrogenic menopause in ewes by performing bilateral
ovariectomy. As a result, there was a significant decrease in epithelial
thickness, glycogen accumulation and vascularity of the vaginal wall.
Similar changes are seen in women affected by GSM following menopause.
When taking a closer look to the epithelial thickness as the primary
outcome measure for GSM, an approximately 3 fold decrease after
menopause in both women (220 \selectlanguage{greek}µ\selectlanguage{english}m to 80-125 \selectlanguage{greek}µ\selectlanguage{english}m.\textsuperscript{27, 28})
and ewes (146 \selectlanguage{greek}µ\selectlanguage{english}m to 47 \selectlanguage{greek}µ\selectlanguage{english}m) were seen, even though women have a thicker
vaginal wall than ewes.

Epithelial thinning results in less exfoliation of cells into the
vagina, meaning less available glycogen, which is broken down into
lactic acid by the action of lactobacilli. Therefore, vaginal pH rises
after menopause and is associated with overgrowth of anaerobic bacteria
causing opportunistic vaginal infections in women.\textsuperscript{2,
29} Although we observed a significant decrease of epithelial glycogen
accumulation in ewes following ovariectomy, interestingly the vaginal pH
remained unchanged. This is explained by Miller et al. by the fact that
lactobacilli dominance appears to be unique to the human vaginal
microbiome, where the abundance of lactobacilli is \textgreater{}70\%,
compared to \textless{}1\% in other mammals.\textsuperscript{29}

Beyond outcome measures as epithelial thickness, glycogen levels,
vaginal pH and vascularity, it is also recommended to evaluate vaginal
cytology for objective assessment of GSM in women.\textsuperscript{18}In
this study, although not statistically significant, we observed lower
levels of the VMV and the VMI following ovariectomy in ewes. On the
other hand, it could be hypothesised that the distribution of
superficial, intermediate and parabasal cells is less pronounced due to
the fact that ewes have thinner vaginal walls.

Furthermore, a hypo-oestrogenic state results in loss of dermal
collagen, elastin fibres, and blood vessels in the lamina
propria.\textsuperscript{30} Although a significant decrease in
vascularity was observed, no significant difference in elastin and
collagen content between ovariectomised ewes and the control group was
found. Elastin is a very stable protein, where the metabolic turnover of
mature elastin fibres is relatively slow.\textsuperscript{31,
32}Although elastin likely turns over in the vulvovaginal tissues after
menopause, the exact mechanism remains still unknown. It could be
hypothesised that the fragmentation of elastin fibres in ewes was not
yet completed at 5 months after ovariectomy. Regarding collagen content,
the vaginal collagen levels might already be affected by the
reproductive state. For instance, Ulrich et al. showed that the total
collagen levels in parous ewes are different from both virgin and
pregnant ewes.\textsuperscript{33} The unchanged collagen levels after
ovariectomy have been described previously.\textsuperscript{13} In
addition, there are studies reporting decreased collagen I/III ratio in
women with GSM, whereas this has not been observed in
ewes.\textsuperscript{33, 34}

\textbf{5 \textbar{} CONCLUSION}

Previously, adequate evaluation of physiologic vaginal changes in ewes
following ovariectomy was lacking. This study used rigorous and novel
assessment tools to evaluate the effect of iatrogenic menopause in ewes
on multiple outcome measures for GSM. These effects seem to reflect the
physiologic changes of the vagina in women encountering GSM. In
addition, this study shows that relevant outcome measures used in women,
are also applicable in the evaluation of GSM in ewes. Hence, this large
animal model is of great value for future translational research for the
evaluation and optimisation of different treatment modalities for GSM.
In a successive study, we evaluated different interventions in the
treatment of GSM in the ovariectomised ewe.

\textbf{Abbreviations}

DHEA Dehydroepiandrosterone

FDA Food and Drug Administration

GSM Genitourinary syndrome of menopause

H\&E Haematoxylin and eosin

OVX Ovariectomy

PAP Papanicolaou

PAS Periodic acid-Schiff

SD Standard deviation

VMI Vaginal maturation index

VMV Vaginal maturation value

\textbf{Declarations}

\textbf{Acknowledgement}

We like to thank Mr. Woudstra, Mr. Morris and the animal caretakers for
the use of the research facility and taking proper care of the animals.
We like to thank Mr. Mogotlane (MD), Ms. van Wilsem and Mr. Atigan (MD)
for their contributions in the tissue processing. We also like to thank
statistician Dr. van Eekelen for statistical advice and support.

\textbf{Disclosure of interests}

The authors report no conflict of interest.

\textbf{Contributions to authorship}

JPR, ZG, EVV and LR were responsible for the conception and design of
this study. EVV, ZG, LR, ACHMG and STJ were responsible for the data
collection and data management. EVV was responsible for the conduct
analyses. All authors were involved in the interpretation of the data.
EVV drafted the initial manuscript, which was reviewed and critically
revised by all authors. All authors approved the final manuscript as
submitted.

\textbf{Details of ethics approval}

This study was approved by de Animal Ethics Committee of the University
of Cape Town (Protocol number: 018\_005).

\textbf{Funding}

No funding is available for this study.

\textbf{SUPPLEMENTARY MATERIAL}

Additional supporting information may be found online in the Supporting
Information section at the end of the article.

\begin{itemize}
\tightlist
\item
  Supplementary Table S1. Study design: interventions and assessments
\item
  Supplementary Table S2. Regression table
\item
  Supplementary Appendix S1. Power calculation
\item
  Supplementary Appendix S2. Bilateral ovariectomy
\item
  Supplementary Appendix S3. Histology and immunohistochemistry
\item
  Supplementary Appendix S4. Histological examination
\end{itemize}

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\textbf{FIGURE LEGENDS}

\textbf{Figure 1:} Vaginal pH, VMV, VMI (\% superficial cells),
epithelial glycogen accumulation, elastin fibres, collagen, and
vascularity in ewes. Blue represents the control (premenopausal) group,
orange represents the OVX (postmenopausal) group. Measurements are taken
five months after OVX. Data presented as mean ± SD, * = statistical
significant difference between groups.

\textbf{Figure 2:} Longitudinal measurements of the vaginal epithelial
thickness (\selectlanguage{greek}µ\selectlanguage{english}m) in ovariectomised ewes (orange) compared to the control
group (blue); Data presented as mean \selectlanguage{ngerman}± SD. X-axis shown in months after
OVX.

\textbf{Figure 3:} Representative figures of vaginal changes before (A)
and 5 months after (B) ovariectomy in ewes. \textbf{1} : Epithelial
thickness; Haematoxylin and Eosin stain, magnification ×200. \textbf{2}
: Glycogen accumulation (marked with yellow arrows); Periodic
acid-Schiff stain, magnification ×200. \textbf{3} : Vascularity, (marked
with yellow arrows); Masson's Trichrome stain, magnification ×100.
\textbf{4} : Elastin fibres (black lines, marked with yellow arrows);
Van Gieson's stain, magnification ×400. \textbf{5} : Collagen (stained
blue); Masson's Trichrome stain, magnification ×400.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
Outcome & Baseline (before OVX) & Baseline (before OVX) & 5 months post
OVX & 5 months post OVX & 5 months post OVX\tabularnewline
\midrule
\endhead
& Premeno-pausal (n=5) & Postmeno-pausal (n=20) & Premeno-pausal (n=5) &
Postmeno-pausal (n=20) & \emph{p} value\tabularnewline
Epithelial thickness (\selectlanguage{greek}µ\selectlanguage{english}m) & 110 (45) & 116 (39) & 146 (53) & 47 (34) &
\textbf{\textless{} 0,001*}\tabularnewline
Vaginal pH (2-9) & 8,0 (0,35) & 8,1 (0,46) & 8,0 (0,35) & 8,1 (0,31) &
0,509\tabularnewline
Vaginal cytology (VMV) & 57 (19) \selectlanguage{greek}\textsuperscript{\textbf{β1}}\selectlanguage{english} & 52 (9)
\selectlanguage{greek}\textsuperscript{\textbf{β2}}\selectlanguage{english} & 65 (13) & 57 (8)
\selectlanguage{greek}\textsuperscript{\textbf{β3}}\selectlanguage{english} & 0,493\tabularnewline
Superficial cells (VMI \%) & 24,0 (29,7) \selectlanguage{greek}\textsuperscript{\textbf{β1}}\selectlanguage{english} &
12,7 (15,3) \selectlanguage{greek}\textsuperscript{\textbf{β2}}\selectlanguage{english} & 32,4 (24,9) & 7,4 (7,9)
\selectlanguage{greek}\textsuperscript{\textbf{β3}}\selectlanguage{english} & 0,223\tabularnewline
Glycogen accumulation (0-3) & 2,56 (0,36) & 2,37 (0,47) & 2,40 (0,60) &
1,36 (0,77) & \textbf{0,02*}\tabularnewline
Elastin fibres (0-3) & 2,10 (0,83) & 1,27 (0,80) & 1,72 (0,70) & 1,63
(0,60) & 0,637\tabularnewline
Collagen (0-3) & 1,38 (0,37) & 1,53 (0,48) & 1,82 (0,46) & 1,76 (0,49) &
0,869\tabularnewline
Vascularity (0-3) & 2,12 (0,56) & 1,92 (0,57) & 2,68 (0,28) & 2,07
(0,49) & \textbf{\textless{}0,01*}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 1. Outcomes at baseline and 5 months post OVX.} Values
presented as mean (SD). At baseline, there were no significant
differences for all outcomes between groups. \selectlanguage{greek}β\selectlanguage{english}1: n=4. \selectlanguage{greek}β\selectlanguage{english}2: n=9. \selectlanguage{greek}β\selectlanguage{english}3: n=5.
* = significant difference between groups.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure1/Figure1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure2/Figure2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure3/Figure3}

\end{center}
\end{figure}

\selectlanguage{english}
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