449

RBMO VOLUME 37 ISSUE 4 2018

ARTICLE

hsa-miRNA-154-5p expression in plasma of

endometriosis patients is a potential diagnostic
marker for the disease
BIOGRAPHY
Iveta Yotova received her PhD in endocrinology from Sofia University, Bulgaria, in
2001. After postdoctoral training in epigenetic research at the Austrian Academy of
Science, she joined the Medical University of Vienna. Since 2006 her research has
focused on molecular mechanisms in endometriosis, including epigenetic aspects of
the disease.

Petra Pateisky1, Dietmar Pils2, Ladislaus Szabo1, Lorenz Kuessel1,

Heinrich Husslein1, Arndt Schmitz3, René Wenzl1, Iveta Yotova1,*

KEY MESSAGE
Alterations in level of expression of hsa-miR-154-5p in plasma of endometriosis patients alone or in combination with age
and BMI and differentially expressed hsa-miR-196b-5p, hsa-miR-378a-3p and hsa-miR-33a-5p are potentially new diagnostic
markers for the disease. This might allow earlier identification, a more timely diagnosis and potentially in the future an
optimized treatment for patients suffering from endometriosis.

ABSTRACT
Research question: As microRNA (miRNA) are stable in circulation, this study tested whether they could serve as putative non-
invasive biomarkers for endometriosis, and their expression differences between endometriosis patients and controls. It also
addressed whether the combination of differently expressed miRNA together with clinical parameters in a statistical model could
distinguish between endometriosis patients and controls.

Design: This prospective cohort study explored the possibility of using changes in extracellular miRNA spectra in plasma of
51 patients with endometriosis compared with 41 controls combined with clinical data as non-invasive biomarkers for the disease.
The project was divided into three different phases for biomarker screening, discovery and validation. The differences in expression
levels of plasma miRNA obtained from women with and without endometriosis were analysed with quantitative PCR-based
microarrays. The diagnostic performance of the selected individual and/or combined differentially expressed miRNA candidates and
clinical parameters was assessed using in silico bioinformatics modelling and receiver operating characteristic curve analysis.

Results: Data showed that a specific plasma miRNA signature is associated with endometriosis and that hsa-miR-154-5p, which
alone or in combination with hsa-miR-196b-5p, hsa-miR-378a-3p, and hsa-miR-33a-5p and the clinical parameters of body mass
index and age, are potentially applicable for non-invasive diagnosis of the disease. Changes in the levels of expression of certain
circulating plasma miRNA also occurred within the phases of the menstrual cycle.

Conclusions: miRNA seem to be promising candidates for the non-invasive diagnosis of endometriosis. Further, other clinical
parameters may help in distinguishing women suffering from endometriosis from healthy individuals.

KEYWORDS
1  Department of Obstetrics and Gynecology, Medical University of Vienna, Waehringer Guertel 18–20, Vienna A-1090,
Austria
2  Center for Medical Statistics, Informatics and Intelligent Systems, Institute of Clinical Biometrics, Medical University of
Endometriosis
miRNA
Vienna, Spitalgasse 23, Vienna A-1090, Austria
3  Bayer AG, Pharmaceuticals Division Drug Discovery, Muellerstraße 178, Berlin 13353, Germany Non-invasive biomarkers
Subfertility
© 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. E-mail address: iveta.yotova@meduniwien.ac.at (I Yotova). https://doi.org/10.1016/j.
rbmo.2018.05.007 1472-6483/© 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Declaration: The authors report no financial or commercial conflicts of interest.

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450 RBMO VOLUME 37 ISSUE 4 2018
INTRODUCTION
E
ndometriosis is a
hormone-driven gynaecological
disease characterized by the
presence of endometrial
tissue outside of the uterine cavity
and affecting 2–10% of women of
reproductive age. The symptoms of
the disease may vary, but are most
often associated with pain and/
or unexplained infertility (Eskenazi
and Warner, 1997). The diagnosis
relies on surgical assessment and
visualization by laparoscopy. Non-
invasive imaging by ultrasound or
magnetic resonance is applicable
to a subset of symptomatic patients
with deep infiltrating and ovarian
endometriosis, but not to patients with
superficial endometriosis (Hsu et al.,
2010). As a result, the diagnosis and
intervention in the disease are often
delayed due to lack of symptoms
and sensitive biomarkers. Recent
studies have focused on proteomic,
transcriptomic, metabolomic and
methylome approaches for discovering
new non-invasive endometriosis
biomarkers (Fassbender et al., 2015;
Kuessel et al., 2017; Rizner, 2014). In
the last few years, microRNA (miRNA)
have emerged as novel and promising
candidates mainly based on their
function as important epigenetic
regulators of gene expression in diverse
physiological (Chen et al., 2008;
Zhao et al., 2012b) and pathological
processes, including inflammation, cell
growth and proliferation, extracellular
matrix remodelling and angiogenesis
(for review see Teague et al., 2010).
In cancer, the pattern of miRNA
expression has been shown to be
correlated with aberrant expression
of tumour suppressor genes and
oncogenes, defining their high
diagnostic and prognostic values (Zhang
et al., 2008). Accumulating data also
indicate that miRNA might be involved
in the development of endometriosis.
Several studies have reported an
altered pattern of miRNA expression in
eutopic endometrium of patients with
endometriosis compared with healthy
endometrium as well as ectopic lesions.
They are known to regulate pathways
involved in proliferation, inflammation
and angiogenesis, thereby potentially
influencing the disease pathogenesis
and/or progression (Burney et al.,
2009; Filigheddu et al., 2010; Hawkins
et al., 2011).
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Being secreted by exosomes and stable
in the circulation in association with
RNA-binding proteins such as the
Ago2 component of the RNA-induced
silencing complex (RISC) (Turchinovich
et al., 2011) and nucleoplasmin (NPM1)
(Wang et al., 2010), miRNA show
disease-associated differences (Wang
et al., 2013), raising the prospect
of using them as biomarkers and
therapeutic tools. Interestingly, Arroyo
et al. (2011) suggested that Ago2-
associated miRNA could be involved
in the regulation of gene expression in
recipient cells. Based on these reports,
some authors proposed a hormone-
like mechanism of action of the
circulating miRNA (Cortez et al., 2011)
and suggested their role in cell-to-cell
communication (Bang et al., 2014;
Kosaka et al., 2013). The functional
analysis of differentially expressed (DE)
circulating miRNA and their use as
biomarkers for early prediction and
diagnosis of endometriosis are just
starting to emerge. Some circulating
miRNA biomarkers for endometriosis
have already been proposed (Cho
et al., 2015; Cosar et al., 2016; Hsu
et al., 2014; Jia et al., 2013; Rekker
et al., 2015; Suryawanshi et al., 2013;
Wang et al., 2013, 2016a). However,
the discrepancy in study design,
normalization strategies and statistical
evaluation of the data among the
reports resulted in high inconsistency
of the nominated miRNA candidates.
For example, in the study by Jia et al.
(2013), the study cohort only consisted
of patients with endometriosis stage
III–IV and the expression analysis was
performed using quantitative PCR
(q-PCR)-based microarray with 1205
candidates. The normalization to hsa-
miR-16 resulted in the identification of
three putative biomarkers, hsa-miR-
17–5p, hsa-miR-20a and hsa-miR-22,
which did not show association with
disease stage and/or lesion type.
In contrast, although the study by
Wang et al. (2013) used the same
experimental technique with a lower
target number (n = 765), the study
cohort consisted of endometriosis
patients in all disease stages. The
normalization of the data was
performed to the level of expression of
U6. This resulted in the identification
of six putative biomarkers. From these,
only two (hsa-miR-199a and hsa-
miR-122) showed association with the
disease stage. Thus, the identification
of new putative candidates, together
with the independent validation of
already suggested miRNA biomarkers
for diagnosis of the disease, will allow
better selection of the best miRNA
candidates for conducting large-scale
validation studies.
The aim of the current study was
to further explore the possibility of
using changes in extracellular miRNA
spectra in plasma of patients with
endometriosis combined with clinical
data to identify putative non-invasive
biomarkers for the disease. Statistical
modelling approaches were used, which
include a stringent cross-validation
based method to narrow down the
number of targets, for discrimination of
women with and without endometriosis.
Our findings confirm some of the
previously reported DE miRNA in the
circulation of women with the disease
and highlight additional examples, which
together with clinical parameters, may
serve as new diagnostic tools for the
disease.
MATERIALS AND METHODS
Study design
This study was designed in three
phases (summarized in Supplementary
FIGURE S1). In Phase 1 (screening phase)
a sample pool-based q-PCR analysis
on a subset of the study cohort
was performed in order to screen
for disease-associated differences.
The pools were separated by cycle
phase to account for changes due
to the cycle phases (Cho et al.,
2015). Each pool comprised a total
of five samples (taking 500 µl of each
plasma sample) from: (i) control
women in the proliferative phase
of the menstrual cycle; (ii) control
women in the secretory phase of the
menstrual cycle; (iii) endometriosis
patients in the proliferative phase
of the menstrual cycle; and (iv)
endometriosis patients in the secretory
phase of the menstrual cycle. In this
phase, only women without hormonal
therapy treatment prior to diagnostic
surgery were included to exclude
eventual therapy-related effects on
miRNA secretion. miRNA candidate
identification was performed using
two different statistical approaches,
namely GenEx Software provided
by Exiqon and R statistical software
(https://www.r-project.org). In order to
validate and narrow down the number
of putative miRNA marker candidates,

in Phase 2 (discovery phase) the 20
individual samples from patients and
controls (same samples as in the pools)
were used, which were subjected to
q-PCR array-based analysis. Further,
statistical model building was applied
to search for possible combinations of
miRNA signatures and clinical patient
data, revealing the best discrimination
between endometriosis patients
and controls. In Phase 3 (validation
phase) the intention was to reassess
the miRNA candidates and clinical
features with an independent larger
cohort of 72 individual patients (n
= 41 endometriosis patients and n
= 31 controls). In addition, single
significant differences in miRNA
expression were tested for between
endometriosis patients and controls in
the validation cohort of n = 72 samples.
In order to evaluate the cumulative
prediction power for endometriosis
of the interesting single factors, their
combination was tested on the total
patient cohort (patients from Phase 1/2,
n = 19–20 individual samples minus one
strong outlier in the control group plus
patients from Phase 3 after exclusion of
samples based on quality criteria, n =
64, resulting in a total cohort of n = 83
patients).
Study population
The present prospective cohort
study, Endometriosis Marker Austria
(EMMA), is being conducted at the
tertiary referral certified Endometriosis
Centre of the Medical University of
Vienna and has been going on since
2010. From all women undergoing
laparoscopic surgery for different
benign medical indications, between
December 2010 and December
2012, 92 premenopausal women with
complete available data sets were
selected from the EMMA protocol into
this study. Women eligible for study
participation were premenopausal
undergoing laparoscopic surgery
because of suspected endometriosis,
pelvic pain of unknown origin, adnexal
cysts, infertility work-up or uterine
fibroids. Patients with a history of any
malignant disease, acute inflammatory
process or infection and systemic
autoimmune disorders such as systemic
lupus erythematosus or rheumatoid
arthritis were excluded from study
participation. Patients were between 18
and 50 years old with regular menstrual
cycles. For each patient, height and
weight measurements were used for
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body mass index (BMI) calculation.
Blood was collected preoperatively in a
fasting state from the patients directly
on the day of surgery. Furthermore,
all patients were asked to fill out a
detailed questionnaire in order to
evaluate pain symptoms potentially
associated with endometriosis (e.g.
visual analogue scale [VAS] for severity
of dysmenorrhoea, 0 = no pain;
10 = excessive pain) and a detailed
anamnesis sheet, resulting in a very well
characterized patient cohort. In order
to build a cohort representing the
target population in the clinical routine
setting, patients under hormonal
treatment due to pain symptoms were
not excluded from the study.
The presence or absence of
endometriosis was confirmed visually
by laparoscopy and additional
histopathological analysis of the
samples. The different stages of
endometriotic disease were classified
according to the revised American
Society for Reproductive Medicine
(1997). Patients who did not show
any macroscopic or histological
endometriotic lesions were taken as
controls. The menstrual cycle phase
was evaluated by histological analysis of
endometrial biopsy taken from every
patient via diagnostic dilatation and
curettage (D and C) supplemented by
hormonal blood analysis. For women
under hormonal treatment, no cycle
phase was assigned.
The study was approved by the
institutional ethics committee of
the Medical University of Vienna
on 6 July 2010 (reference number
545/2010). All patients gave their
written informed consent prior to study
inclusion.
Sample preparation, RNA isolation and
cDNA synthesis
All samples were collected between
07:00 h and 11:00 h prior to the
surgical intervention. The collected
blood was centrifuged according to a
standard protocol at 3000g for 10 min
at 4°C, 30 min to 1 h after sampling.
The sediment-free serum and plasma
samples were frozen in aliquots at
−80°C until further analysis. The total
RNA was extracted from 500 µl of
plasma using the QIAGEN miRNeasy
Serum/Plasma Kit according to the
manufacturer's protocol. The RNA
quality was ensured using haemolysis
RBMO VOLUME 37 ISSUE 4 2018 451
tests as previously described by Blondal
et al. (2013). For all experimental
phases, the cDNA was synthesized
from 500 ng of total RNA using the
Exiqon miRCURY LNA™ Universal
RT microRNA PCR, polyadenylation
and cDNA synthesis kit II according
to the manufacturer's protocol. For
internal quality control of cDNA
synthesis, RNA spike-in controls were
used as recommended by Exiqon. A
10 µl sample of each cDNA was added
to 45 µl nuclease-free water and an
equal volume of Universal PCR Master
Mix and amplification was performed
according to the manufacturer's
protocol.
Expression profiling and validation
using q-PCR-based arrays
To screen for and validate the
differences in the levels of miRNA
expression in plasma samples
of patients and controls either
ready-to-use or customer-based
q-PCR-based expression arrays from
Exiqon (MA, USA) were used (Ready-
to-Use Exiqon Human microRNA PCR
Panel I, V3.M with 372 spotted LNA™
primer). All arrays were run on an ABI
Prism 7900HT sequence detection
system (Applied Biosystems, Life
Technologies).
Expression arrays data normalization
(pre-processing)
Prior to data normalization, the raw
expression values were first filtered
by excluding samples with cycle
threshold (Ct) values higher than
37 cycles. Except for Phase 1, where
two different software programs were
used (i.e. R and GenEx6 software)
to identify differences in the levels
of expression of the miRNA plasma
secretome, normalization of the data
in Phases 2 and 3 was achieved by
subtracting the geometric mean of the
housekeeping miRNA from the mean
expression value of each miRNA. The
housekeeping miRNA were selected
using geNorm software (R-package,
‘NormqPCR’ v.1.24) and defined as
the most stably expressed miRNA
in this study population. The main
differences in data pre-processing with
the two independent approaches in
Phase 1 included differences in data
normalization. The GenEx6 software
normalization was performed by a two-
step approach and included inter-plate
normalization to the mean value of the
UniSp3 and subsequent normalization
452 RBMO VOLUME 37 ISSUE 4 2018
to the mean of six housekeeping genes
identified by Normfinder. Normalization
with the statistical program R was
performed by subtracting the
geometric mean of the six most
stably expressed miRNA identified by
geNorm software. It is important to
note that the two approaches used for
housekeeper identification revealed
slightly different results (Supplementary
FIGURE S2A). In this study, miRNA-199a,
being recently described as DE in
serum and showing either up-regulation
(Wang et al., 2013) or down-regulation
(Hsu et al., 2014) in endometriosis
patients versus controls, was classified
by both normalization procedures
(Normfinder and geNorm) as a stably
expressed miRNA. Therefore, it was
used as a ‘housekeeping’ normalization
control in all phases of the project.
After normalization and exclusion of
miRNA candidates showing Ct values
above 37 in more than half (i.e. ten)
of the samples, in every phase of the
study the remaining missing values were
imputed with the highest Ct values of
the corresponding miRNA plus one.
Finally, all normalized and filtered
expression values were log2 transformed
to get near-normal distributions and to
allow parametric statistical testing. The
samples showing missing values for ≥10
of all analysed miRNA per sample were
excluded from further analysis. The
residual missing values were imputed
with the highest Ct values of the
corresponding miRNA plus 1.
Statistical analysis and data
presentation
Statistical analysis of the normalized
and log2-transformed expression data
was performed with R (v3.1.2). The
expression levels of plasma miRNA
between the groups were compared
using linear models fitted for each
miRNA using a design matrix including
information for phase and disease
status, and differentially abundant
miRNA called using an empirical
Bayes statistic (eBayes function, limma
R-package) (Ritchie et al., 2015). miRNA
were considered DE between both
groups, endometriosis patients vs
controls or proliferative vs secretory
cycle phase, when showing at least
an absolute log2 fold change equal
or greater than 1 (abs (log2FC) ≥ 1).
For Phase 3, a statistical cut-off at
FDR (false discovery rate) of 20% for
differences in the miRNA abundance
was set. Principle component analysis
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(PCA) of centred and scaled expression
data was used to identify and visualize
the associations between miRNA,
endometriosis and menstrual cycle
phase.
In silico model building strategies
for disease prediction and diagnostic
performance of DE miRNA
Complete data sets were necessary
for model building. Therefore, the
missing values of each miRNA were
imputed by the maximal obtained Ct
of the corresponding miRNA plus 1
(cycle threshold + 1), meaning half
the abundance of the smallest value
across all samples. Secretory menstrual
cycle status, age, dysmenorrhoea
and BMI were always included during
model building. Three different model
building strategies were applied: (i)
the penalized support vector machine
(pSVM) approach from the R-package
‘penalizedSVM’ was used to perform
internal cross-validated SVM model
building and feature selection (Becker
et al., 2009); (ii) internal cross-validated
Lasso (least absolute shrinkage and
selection operator)/elastic net model-
generation using R-package ‘Glmnet’
was used as a second method for
feature selection (Friedman et al.,
2010); (iii) finally, feature selection was
performed by randomized generalized
linear models as implemented in
R-package ‘randomGLM’ (Song et al.,
2013). Within the model building
process all samples were from either
the discovery phase (n = 20) or the
validation phase (n = 64); from the
total cohort of 72 patients in Phase 3,
eight patients had to be excluded from
further analysis based on our quality
criteria for miRNA expression, which
excludes samples with missing values
for ≥10 of all analysed miRNA per
sample). The samples were randomly
partitioned into k subsets and with k–1
subsets a predictive model was built
and tested on the remaining subset,
each k times, so that every subset was
used once as a validation set. This was
performed for each tuning parameter
value and the misclassifications for
all k model building and testing
procedures were averaged and finally
the tuning parameter value with the
lowest averaged misclassification value
was chosen (Burman, 1989). Those
randomly built training and validation
cohorts are different by their mean
from the study cohorts used in the
discovery Phase 2 and the validation
Phase 3. In the study outlined here, the
entire patient cohort (n = 20) in the
discovery phase was used to evaluate
which of the DE miRNA from Phase 1
are the most suitable for endometriosis
prediction in a model. The final patient
cohort in Phase 3 (n = 64) was used
to validate the prediction power of
the selected parameters in Phase 2
on an independent set of patients and
controls.
The diagnostic performance of the
models was assessed using receiver
operating characteristic (ROC)
curves to plot the test sensitivity
versus its false positive rate. The
quality of a diagnostic model can
be illustrated by the area under the
ROC curve (AUC). An AUC of 0.5
indicates classifications assigned by
chance. To select optimal cut-points
from the diagnostic tests an R-package
based method known as ‘MaxSpSe’ was
used.
Identification of miRNA gene targets
and functional enrichment analysis
To identify putative miRNA gene targets
for the DE miRNA in women with and
without endometriosis, MirTarget2 was
used, a freely available software (http://
mirdb.org). This software identifies
common features associated with
miRNA target binding, which is further
used to predict miRNA targets with
machine-learning methods (Wang,
2016; Wong and Wang, 2015). To test
whether the miRNA gene targets are
associated with specific biological
functions, gene ontology enrichment
analysis was performed using DAVID
(https://david-d.ncifcrf.gov/) (Huang
Da et al., 2009). This analysis used
predicted gene targets showing a
target score in the range of 80–100 by
miRTarget2 identification software. In
the DAVID annotation system, Fisher's
exact test is adopted to measure the
gene enrichment in annotation terms.
Gene ontology (GO) terms showing
a Fisher's exact P-value <0.05 and
fold enrichment >1.5 were considered
significantly enriched among miRNA
gene targets.
RESULTS
Demographic data
Overall, 92 participants were included
in the study, consisting of 51 (55%)
endometriosis patients and 41 (45%)
controls. In Phases 1 and 2 of the
 RBMO VOLUME 37 ISSUE 4 2018 453

TABLE 1  – SUMMARY OF THE CLINICAL CHARACTERISTICS OF THE STUDY COHORTS ANALYSED IN EACH PHASE
OF THE STUDY

Baseline characteristics Phase 1 and 2, n = 20 Phase 3, n = 72

Type EM Controls P-value EM Controls P-value

Number 10 10 NA 41 (56.9%) 31 (43.1%) NS
Age 36.2 ± 6.4 34.0 ± 5.0 NS 33.3 ± 7.8 37.6 ± 8.2 0.025
BMI 19.9 ± 3.1 26.7 ± 6.3 0.02 22.2 ± 3.6 23.9 ± 4.0 NS
Cycle phase NS NS
Proliferative 5 5 17 (41.5%) 18 (58.1%) NS
Secretory 5 5 14 (34.1%) 6 (19.4%) NS
Hormonal intake 0 0 NA 10 (24.4%) 7 (22.6%) NS
Dysmenorrhoea VAS 5.9 ± 3.9 6.0 ± 4.1 NS 5.85 ± 2.8 3.8 ± 3.7 0.015
Overall cyclic pain 6.6 ± 3.0 4.7 ± 2.8 NS 5.90 ± 2.9 4.4 ± 3.1 0.031
rASRM stage NA NA
  Minimal to mild 4 (40%) NA 16 (39%) NA
  Moderate to severe 6 (60%) NA 25 (61%) NA
EM lesion from total EM NA NA
  Ovarian plus other lesions 3 (30%) NA 12 (29.3%) NA
  Peritoneal plus other lesions 10 (100%) NA 23 (56.1%) NA
  Only ovarian lesion 0 NA 6 (14.6%) NA
  Only peritoneal lesions 3 NA 12 (29.3%) NA
DIE status from total EM NA NA
a
  With DIE 6 (60%) NA 17 (41.5%) NA
  Without DIE 4 (40%) NA 24 (58.5%) NA
  Only DIE 0 NA 0 NA
BMI = body mass index; DIE = deep infiltrating endometriosis; EM = endometriosis; NA = not applicable; NS = not significant; rASRM = revised American Society for
Reproductive Medicine, Endometriosis classification; VAS = visual analogue scale.
a
  Irrespective of other lesion types.

project 20 samples (10 endometriosis
cases and 10 controls) from the
total study cohort were analysed
after selection, as explained in the
Materials and methods section (Study
population). Women under hormonal
treatment were excluded from the
study population in Phases 1 and 2. In
terms of baseline characteristics the
10 endometriosis patients showed a
significantly lower BMI in comparison
to the 10 controls (19.9 ± 3.1 versus
26.7 ± 6.3, P = 0.020) (TABLE 1). Initially,
for analysis in Phase 3 the 72 other
patients (endometriosis group, n =
41, 56.9% and control group, n = 31,
43.1%) were taken for validation of
the results. Between the two groups,
age, dysmenorrhoea and overall cyclic
pain (expressed as VAS – points) were
significantly different (P = 0.025, 0.015
and 0.031, respectively) (TABLE 1). Finally,
from the whole validation cohort, 64
patients and controls were subjected
to ROC analysis after exclusion of a
total of 8 samples not meeting the
predefined quality criteria. Overall,
from the 64 patients in the validation
cohort, 25% were under hormonal
treatment less than 3 months prior to
the diagnostic surgery.
Phase 1 (screening phase) –
identification of secreted miRNA
expression profiles for endometriosis/
controls and menstrual cycle phase
To identify a subset of miRNA with
potential clinical application for diagnosis
of endometriosis, the expression spectra
of 372 miRNA targets in plasma pools
of cases and controls using q-PCR-
based array were initially profiled.
The q-PCR-based array contains the
majority of already published putative
diagnostic miRNA candidates detectable
in the circulation. After normalization
(exclusion of miRNA candidates showing
Ct values higher than 37 in more than
50% of samples) two different statistical
approaches were applied to identify DE
miRNA in disease and/or in different
menstrual cycle phases. PCA of the
normalized expression data showed
that miRNA expression profiles are
disease and menstrual cycle phase-
dependent and independent of the
type of data normalization procedure
(Supplementary FIGURE S2B). Using
R-software a total of 49 DE (abs(log2 FC)
≥ 1) miRNA were identified (FIGURE 1A).
From these, 12 DE miRNA candidates
were identified as cycle phase-specific
(FIGURE 1A and Supplementary TABLE S1B)
and 27 as disease-associated (FIGURE 1A
and Supplementary TABLE S1A). An
additional 10 miRNA were differentially
regulated in both cell cycle and disease
state (FIGURE 1A). Supervised hierarchical
clustering analysis highlighted that
both increase and decrease of miRNA
expression levels occur in plasma of
patients versus controls and in cycle
phases (FIGURE 1B). Independent data
analysis using GenEx6 software resulted
in a list of 78 DE (abs(log2 FC) ≥ 1)
miRNA. From these, 39 DE miRNA
were specific for the disease state,
20 for the cycle phase and 19 miRNA

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454 RBMO VOLUME 37 ISSUE 4 2018

FIGURE 1  – A subset of microRNA (miRNA) is differentially expressed (DE) in plasma of patients with endometriosis compared with controls. (A)
Distribution of DE miRNA in plasma of patients with endometriosis. The Venn diagram represents the comparison between DE miRNA candidates
based on cycle phase and presence/absence of endometriosis. Total number 12 DE candidates were identified as cycle phase-specific and 27 as
disease specific. Ten candidates were differentially regulated in both cell cycle and disease state. Proliferative versus secretory phase; controls
versus endometriosis. (B) Supervised hierarchical clustering of 49 DE (log2 fold change >1) miRNA between analysed groups is shown. Biological
samples are on the x-axis and DE miRNA are on the y-axis with relative levels of expression indicated by colour scale. Red colour represents an
expression level above the mean across all samples, black represents mean expression and green represents expression lower than the mean.
(C) Venn diagram depicting the overlap between DE miRNA identified by R-software and the GeneEx6 program. CyPh = cycle phase-dependent
expression. The percentage of overlapped or uniquely expressed miRNA between the compared groups is given in brackets under each number.

were differentially regulated in both
disease and cycle phases, respectively
(Supplementary FIGURE S2C). Eight
miRNA showed unique and reciprocal
expression patterns using both statistical
approaches (Supplementary TABLE S1C).
The final list of n = 75 selected miRNA
(TABLE 2) for screening and validation
was conducted by including: (i) most
but not all of the overlapping DE
miRNA candidates identified by the
two independent statistical approaches
(FIGURE 1C); (ii) the reciprocally expressed
disease-associated candidates; (iii) the
geNorm housekeeping gene list; and (iv)
additional five not DE miRNA candidates
as putative new housekeeping genes for
the validation phase. The study included
100% of the miRNA from the following
major overlapping groups: (i) DE
candidates based on disease status and
(ii) DE candidates based on menstrual
cycle phase distribution (FIGURE 1C).
From the remaining overlapping groups
and based on a systematic literature
search, the top ranked DE miRNA
candidates with putative functions in
female reproductive physiology and/or
pathology, including endometriosis, were
selected (TABLE 2).
Phase 2 (discovery phase) – test for
reproducibility and reliability of DE
miRNA and discovery of diagnostically
relevant miRNA targets
To test for reproducibility and reliability
of selected miRNA candidates in
Phase 2 of the study, an expression
array analysis was performed for the
69 miRNA candidates (75 miRNA
minus the 6 most stably expressed
miRNA across the total study cohort
used henceforth as housekeeper
miRNAs; see TABLE 2) on individual
samples, previously used for pool-
based analysis in Phase 1. After initial
data normalization and filtering, a
total of 67 reliably expressed miRNA
were obtained for further analysis.
Statistically, the differences between
the groups were assessed with logistic
regression modelling, correcting for
either secretory phase or disease
status, respectively. Based on this
analysis the differences in the levels of
expression of 25 of the preselected DE
miRNA representing 37.3% of the initial
67 DE candidates were confirmed.
From these miRNA, 19 showed both
disease and cell cycle aberration
in their secretion (Supplementary
TABLES S2A and S2B).

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 RBMO VOLUME 37 ISSUE 4 2018 455

TABLE 2  – LIST OF DE AND MOST STABLY EXPRESSED (HOUSEKEEPING) MIRNA SELECTED AT THE END OF
THE SCREENING STUDY PHASE 1

miRNA Accession number Regulation in Regulation by cycle phase Referencesa(reproduction)

endometriosis (contr. (PPh versus SPh)
versus EM)
hsa-miR-128 MI0000447 ND down Yanokura et al., 2015
hsa-miR-532-5p MIMAT0002888 ND down NF
hsa-miR-140-3p MIMAT0004597 ND down Hawkins et al., 2011
hsa-miR-663a MIMAT0003326 ND down NF
hsa-miR-362-5p MIMAT0000705 ND down Suryawanshi et al., 2013
hsa-miR-152 MI0000462 ND down Widodo et al., 2016
hsa-miR-487b MI0003530 ND up Wang et al., 2016a
hsa-miR-409-3p MIMAT0001639 ND up Hiroki et al., 2010
hsa-miR-30d-5p MIMAT0000245 ND up Vilella et al., 2015
hsa-miR-34a-5p MIMAT0000255 ND up Schirmer et al., 2014
hsa-miR-136-5p MIMAT0000448 ND up Yoneyama et al., 2015
hsa-miR-132-3p MIMAT0000426 ND down Leinders et al., 2016a
hsa-miR-181a-5p MIMAT0000256 up ND He et al., 2015a
hsa-miR-32-5p MIMAT0000090 up ND Yoneyama et al., 2015a
hsa-miR-99b-5p MIMAT0000689 up ND Yanokura et al., 2015a
hsa-miR-144-3p MIMAT0000436 up ND Pan and Chegini, 2008a
hsa-miR-331-3p MIMAT0000760 up ND Xue et al., 2015a
hsa-miR-378a-3p MIMAT0000732 up ND Bidarimath et al., 2014a
hsa-miR-127-3p MIMAT0000446 up up Pan and Chegini, 2008a
hsa-miR-339-5p MIMAT0000764 up up Wessels et al., 2013a
hsa-miR-934 MIMAT0004977 up down Gu et al., 2013a
hsa-miR-485-3p MIMAT0002176 up up Wang et al., 2016aa
hsa-miR-296-5p MIMAT0000690 down down Widodo et al., 2016a
hsa-miR-22-5p MIMAT0004495 down down Jia et al., 2013a
hsa-miR-421 MIMAT0003339 down up Salilew-Wondim et al., 2016a
hsa-miR-181d MI0003139 down up Widodo et al., 2016a
hsa-miR-370 MI0000778 up up Widodo et al., 2016a
hsa-miR-629-5p MIMAT0004810 up up Laudanski et al., 2013a
hsa-miR-33a-5p MIMAT0000091 down ND NF
hsa-miR-196b-5p MIMAT0001080 down ND Shi et al., 2017
hsa-miR-625-3p MIMAT0004808 down ND Wang et al., 2016a
hsa-miR-326 MIMAT0000756 down ND NF
hsa-miR-361-3p MIMAT0004682 down ND NF
hsa-miR-433 MI0001723 down ND NF
hsa-miR-324-5p MIMAT0000761 down ND Wang et al., 2016a
hsa-miR-17-5p MIMAT0000070 down ND Jia et al., 2013
hsa-miR-98-5p MIMAT0000096 down ND NF
hsa-miR-193a-5p MIMAT0004614 down ND Pu et al., 2016
hsa-let-7d-3p MIMAT0004484 down ND Wang et al., 2016b
hsa-miR-140-5p MIMAT0000431 up ND NF
hsa-miR-431-5p MIMAT0001625 up ND Wang et al., 2016a
hsa-miR-133a MI0000450 up ND Wang et al., 2012
hsa-miR-451a MIMAT0001631 up ND Li et al., 2011

(continued on next page)

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456 RBMO VOLUME 37 ISSUE 4 2018

Table 2 – (continued)

miRNA Accession number Regulation in Regulation by cycle phase Referencesa(reproduction)

endometriosis (contr. (PPh versus SPh)
versus EM)
hsa-miR-381-3p MIMAT0000736 up ND Wang et al., 2016a
hsa-miR-101-3p MIMAT0000099 up ND Pan and Chegini, 2008
hsa-miR-154-5p MIMAT0000452 up ND Estella et al., 2012
hsa-miR-134 MI0000474 up ND NF
hsa-miR-143-3p MIMAT0000435 up ND Cosar et al., 2016
hsa-miR-15b-5p MIMAT0000417 up ND Wang et al., 2016a
hsa-miR-375 MIMAT0000728 up ND Filigheddu et al., 2010
hsa-miR-215 MI0000291 down ND Wang et al., 2013a
hsa-miR-192-5p MIMAT0000222 down ND Myatt et al., 2010a
hsa-miR-133b MIMAT0000770 down ND Zhao et al., 2012aa
hsa-miR-376b-3p MIMAT0002172 down ND Georgieva et al., 2012a
hsa-miR-199b-5p MIMAT0000263 down ND Wang et al., 2015a
hsa-miR-744-5p MIMAT0004945 down ND Wang et al., 2016aa
hsa-miR-330-3p MIMAT0000751 upb ND Wu et al., 2009
b
hsa-miR-365a-3p MIMAT0000710 down ND Xu et al., 2015
hsa-miR-497-5p MIMAT0002820 downb ND Laudanski et al., 2013
hsa-miR-590-5p MIMAT0003258 downb ND Xu et al., 2015
hsa-miR-7-5p MIMAT0000252 downb ND Xu et al., 2015
hsa-miR-1 MIMAT0000416 downb ND Hawkins et al., 2011
hsa-miR-183-5p MIMAT0000261 downb ND Shi et al., 2014
hsa-miR-185-3p MIMAT0004611 upb ND Laudanski et al., 2013
hsa-miR-106a-5p MIMAT0000103 housekeeper housekeeper NA
hsa-miR-150-5p MIMAT0000451 housekeeper housekeeper NA
hsa-miR-425-5p MIMAT0003393 housekeeper housekeeper NA
hsa-miR-125a-5p MIMAT0000443 housekeeper housekeeper NA
hsa-miR-199a-3p MIMAT0000232 housekeeper housekeeper NA
hsa-miR-30e-5p MIMAT0000692 housekeeper housekeeper NA
hsa-miR-301a-3p MIMAT0000688 NAc NAc NA
hsa-miR-337-5p MIMAT0004695 NAc NAc NA
hsa-miR-542-5p MIMAT0003340 NAc NAc NA
hsa-miR-551b-3p MIMAT0003233 NAc NAc NA
hsa-miR-877-5p MIMAT0004949 NAc NAc NA
The six miRNA labelled as ‘housekeepers’ in the table were chosen for normalization based on their most stable expression pattern across the study population.
DE = differentially expressed; EM = endometriosis; miRNA = microRNA; NA = not applicable; ND = no differential expression; NF = not found for indicated search
category; PPh = proliferative phase; SPh = secretory phase.
a  References for miRNA included in the final validation list for Phase 2 of the study based on systematic literature search.
b  Reciprocal expression pattern.
c  Additional not differentially expressed candidates – putative normalization (housekeeping) targets.

Clinical parameters, such as age, BMI,
VAS score (visual analogue scale for
pain measurement) of dysmenorrhoea,
and the cycle phase are known to
be associated with the occurrence
of endometriosis (Fauconnier and
Chapron, 2005; Hediger et al., 2005;
Sinaii et al., 2008). Therefore, these
four clinical confounders and the 67
DE miRNA from Phase 1 were used
to develop discriminative models
for disease prediction. Overall,
three principally different machine-
learning approaches with included
cross-validated feature selection
approaches were employed: Lasso,
penalized support vector machine
(pSVM) and random generalized linear
model (rGLM). The last of these is an
ensemble predictor method, which
selects predictors from subsets of
candidates in each round of model
building to avoid selecting only models
with candidates with the strongest
discriminative power. Two rounds of
modelling were performed (TABLE 3),
with the first round including all 20

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TABLE 3  – SUMMARY OF THE PERFORMANCE OF THE DIFFERENT CLASS PREDICTION IN SILICO STATISTICAL METH-
ODS IN THE DISCOVERY PHASE (PHASE 2) OF THE PROJECT ON COHORTS OF N = 20 (10 ENDOMETRIOSIS PATIENTS
AND 10 CONTROLS) AND N = 19 (10 ENDOMETRIOSIS PATIENTS AND 9 CONTROLS – ONE STRONG OUTLIER WITH NO
CLINICAL OR OTHER IDENTIFIABLE REASON WAS EXCLUDED FROM FURTHER ANALYSIS), RESPECTIVELY
Number of samples (10 patients/10 controls)
miRNA input
Clinical parameters input
miRNA output
Clinical parameters output
Mean % of correct identification
Number of samples (10 patients/9 controls)
miRNA input
Clinical parameters input
miRNA output
Clinical parameters output
Mean % of correct identification
miRNA = microRNA; ND = not determined.
individual samples and the second
round excluding one patient classified
as an outlier by topological group
distribution analysis of the study
population using Isomap (data not
shown). By building the three different
models mentioned above, an attempt
was made to perform a stringent
selection of the miRNA candidates
by narrowing down the number
of DE miRNA candidates to those
showing putative diagnostic power.
As expected and even with stringent
internal cross-validation (k-fold cross-
validation method) during feature
selection, all models revealed 100%
correct classifications of the samples
into disease and controls (TABLE 3). This
is presumably due to overfitting of the
models in a setting of more predictors
than samples. In none of the models
did the menstrual cycle phase turn
out to be an influencing factor for
discriminating patients from controls.
Thus, for validation of the data on an
independent patient cohort, from the
selected miRNA and clinical parameters
those called by at least one of the
three models (n = 42 miRNA and age,
BMI and dysmenorrhoea) were chosen
(TABLE 4, Supplementary TABLE S3).
Phase 3 (validation phase) – in
silico modelling approach did not
discriminate cases from controls
Here, the prediction power of two
of the discovery phase models was
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Lasso Penalized support vector machines
(SVM)
10/10 10/10
67 67
4 4
36 9 ND
2 1
100 100
10/9 10/9
67 67
4 4
10 24
1 1
100 100
tested, on an independent and
larger patient cohort consisting of 72
patients. For validation, only patients
having n ≤ 10 missing values for all
tested miRNA targets per sample were
included (Supplementary TABLE S4).
As in Phase 2, the remaining missing
values in each individual sample were
imputed with the highest Ct values
of the corresponding miRNA plus 1.
This resulted in a list of 64 patients
distributed in 28 controls and 36
patients with endometriosis. The
validation was performed building the
final Lasso model based on the data
from the cohort used in Phase 2 and
including all 42 preselected miRNA
and the clinical parameters age, BMI
and dysmenorrhoea (Supplementary
TABLE S5A). As seen in FIGURE 2A, the
model can discriminate patients from
controls perfectly in the cohort from
the discovery phase, but not at all in
the validation cohort (AUC = 0.514).
Similar results were obtained by using
an ensemble random GLM predictor
including all possible factors (miRNA,
age, BMI and VAS dysmenorrhoea)
(FIGURE 2B and Supplementary TABLE S5B).
A subset of miRNA is significantly
altered in plasma of patients with
endometriosis and shows disease
and/or menstrual cycle-dependent
regulation
The models based on the characteristics
chosen in Phase 2 could not differentiate
between endometriosis patients and
RBMO VOLUME 37 ISSUE 4 2018 457
Random generalized
linear model (rGLM)
ND
ND
ND
ND
ND
10/9
67
4
4
1
100
controls in our validation cohort.
Therefore, the study further investigated
whether any single differences between
patients and controls could serve
alone or in combination with clinical
parameters as putative biomarkers for
the disease. Applying logistic regression
modelling to the data correcting for
cycle phase and hormonal intake, single
significant differences (FDR < 0.2) in
miRNA expression levels between cases
and controls in the validation cohort (n
= 64) were looked at (Supplementary
TABLES S6A and S6B). It was found that hsa-
miRNA-154-5p was significantly down-
regulated in endometriosis (P = 0.008)
and the difference in its expression
levels was independent of cycle phase
and hormonal intake (FIGURE 3A and
Supplementary TABLES S6B and S6C). The
levels of expression of hsa-miRNA-196b-
5p, hsa-miRNA-378a-3p and hsa-miRNA-
33a-5p were disease- and menstrual
cycle-dependent and were either
down-regulated (hsa-miRNA-196b-5p
and hsa-miRNA-378a-3p, FIGURE 3B and
3C) or up-regulated (hsa-miRNA-33a-5p,
FIGURE 3D) in patients with endometriosis
(see also Supplementary TABLE S6). Two
miRNA, hsa-miRNA-326 and hsa-
miRNA-485-3p, showed menstrual cycle
specific regulation in healthy individuals
and were lower in the secretory phase.
This regulation was lost in patients
with endometriosis (FIGURE 3E and 3F;
Supplementary TABLES S6A and SB). The
age and BMI were significantly lower
in patients compared with controls
458 RBMO VOLUME 37 ISSUE 4 2018

TABLE 4  – LIST OF 42 DE MIRNA BETWEEN WOMEN WITH AND WITHOUT ENDOMETRIOSIS CHOSEN FOR VALIDATION
IN PHASE 3

miRNA Accession number

hsa-miR-337-5p MIMAT0004695
hsa-miR-877-5p MIMAT0004949
hsa-miR-132-3p MIMAT0000426
hsa-miR-30d-5p MIMAT0000245
hsa-miR-34a-5p MIMAT0000255
hsa-miR-362-5p MIMAT0000705
hsa-miR-663a MIMAT0003326
hsa-miR-22-5p MIMAT0004495
hsa-miR-421 MIMAT0003339
hsa-miR-629-5p MIMAT0004810
hsa-miR-127-3p MIMAT0000446
hsa-miR-133a MI0000450
hsa-miR-181a-5p MIMAT0000256
hsa-miR-32-5p MIMAT0000090
hsa-miR-485-3p MIMAT0002176
hsa-let-7d-3p MIMAT0004484
hsa-miR-1 MIMAT0000416
hsa-miR-183-5p MIMAT0000261
hsa-miR-185-3p MIMAT0004560
hsa-miR-133b MIMAT0000770
hsa-miR-199b-5p MIMAT0000263
hsa-miR-215 MI0000291
hsa-miR-378a-3p MIMAT0000732
hsa-miR-744-5p MIMAT0004945
hsa-miR-144-3p MIMAT0000436
hsa-miR-99b-5p MIMAT0000689
hsa-miR-134 MI0000474
hsa-miR-140-5p MIMAT0000431
hsa-miR-154-5p MIMAT0000452
hsa-miR-15b-5p MIMAT0000417
hsa-miR-17-5p MIMAT0000070
hsa-miR-196b-5p MIMAT0001080
hsa-miR-326 MI0000808
hsa-miR-33a-5p MIMAT0000091
hsa-miR-361-3p MIMAT0004682
hsa-miR-375 MI0000783
hsa-miR-381-3p MIMAT0000736
hsa-miR-451a MI0001729
hsa-miR-625-3p MIMAT0004808
hsa-miR-98-5p MIMAT0000096
hsa-miR-365a-3p MIMAT0000710
hsa-miR-7-5p MIMAT0000252
miRNA = microRNA.

(P = 0.026 and 0.024, respectively) were significantly higher (P = 0.023) FIGURE S3C).In these experimental settings,
(Supplementary FIGURES S3A and S3B) in the secretory phase of the cycle in the hormonal therapy did not significantly
and VAS scores of dysmenorrhoea endometriosis patients (Supplementary affect either miRNA expression levels or

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 RBMO VOLUME 37 ISSUE 4 2018 459

FIGURE 2  – Discovery (Phase 2) and validation (Phase 3) of the Lasso (A) and rGLM (B) models for disease prediction. ROC curves for disease
prediction on discovery (n = 19, left) and validation (n = 64, right) cohorts, using normalized log2 Ct values of n = 42 miRNA and clinical
parameters (age, BMI and dysmenorrhoea) are shown. Specific 100% prediction power is seen in the discovery phase (AUC = 1 and 0.94,
respectively) and random in the validation phase (AUC = 0.514 and 0.66, respectively) for both statistical models.

differences in clinical parameters found
by disease and/or cycle phase (FIGURE 3
and Supplementary TABLE S6C).
To further evaluate the relationship
between DE miRNA and disease stage,
the levels of expression of hsa-miRNA-
154-5p, hsa-miRNA-378a-3p, hsa-miRNA-
33a-5p and hsa-miRNA-196b-5p were
compared in patients with rAFS I and
II (n = 15) and rAFS III and IV (n = 21)
endometriosis. As shown in FIGURE 4A
and Supplementary FIGURE S4A, there was
no significant difference in the levels of
expression of any of the miRNA between
the disease stages. Similarly, there was
no significant difference in the levels
of expression of hsa-miRNA-154-5p,
hsa-miRNA-378a-3p, hsa-miRNA-33a-5p
and hsa-miRNA-196b-5p between the
different lesion entities (FIGURE 4B and data
not shown).
hsa-miRNA-154-5p is an assumed
biomarker for endometriosis
A final question was whether these DE
miRNA, either alone or in combination,
might serve as markers for non-invasive
diagnosis of the disease. ROC curve
analysis, performed on the validation
cohort (n = 64 cases and controls),
showed that DE hsa-miRNA-154-5p has a
possible diagnostic potential (FIGURE 4C).
The AUC was 0.72 (95% confidence
interval [CI]: 0.587, 0.851, P = 0.001)
and the sensitivity and specificity were
67% and 68% at the cut-off value of
5.24 (log2 expression value relative to the
housekeepers). ROC curve analysis for
hsa-miRNA-378a-3p, hsa-miRNA-33a-5p
and hsa-miRNA-196b-5p in the validation
cohort showed that these miRNA are
not predictive markers for the disease
(Supplementary FIGURE S4B). The ROC
curve analysis for the interesting clinical
factors of age and BMI showed that they
only have low predictive power (both
AUC = 0.67, P = 0.01) for the disease
(Supplementary FIGURE S4C).
Combining the above-mentioned miRNA
(hsa-miRNA-154-5p, hsa-miRNA-378a-3p,
hsa-miRNA-33a-5p and hsa-miRNA-196b-
5p) in the logistic regression model on
a larger cohort (n = 83, representing
the total study population), the AUC
value for differentiating women with and
without endometriosis was 0.72 (95% CI:
0.604, 0.830; P = 0.0003) (FIGURE 4D).
When the clinical parameters of age
and BMI were taken into account the
AUC value did not improve (AUC =
0.72, 95% CI: 0.607, 0.832, P = 0.0002)
(FIGURE 4D). However, in comparison to
the combination of the ROC analysis of
the four interesting miRNA, the addition

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FIGURE 3  – A subset of miRNA is altered in plasma of patients with endometriosis and show disease- and/or cycle phase-specific regulation. Box
plots representing the absolute log2 fold change difference in the levels of expression of hsa-miR-154-5p (A), difference in the expression levels of
hsa-miR-196b-5p (B), difference in the expression levels of hsa-miR-378a-3p (C) and hsa-miR-33a-5p (D) and difference in the expression levels of
hsa-miR-326 (E) and hsa-miR-485–3p (F) in cases and controls assessed with logistic regression modelling, corrected for cycle phase, hormonal
intake and disease status in the validation cohort of n = 64 cases and controls are shown. For each significant comparison, the P-values are
indicated below the graphs. P = proliferative phase, S = secretory phase, H = hormonal intake, Co = control group, EM = endometriosis group.

of age and BMI showed substantial
improvement of the prediction validity
represented by the P-value. As seen in
Supplementary TABLE S6D the probability
factor (Pr) in the logistic regression
model for the combined clinical
parameters BMI, age and DE miRNA
was significant (Pr(>|t|) < 0.05) only
for miR-378a-3p (Pr = 0.0468) and
miR-33a-5p (Pr = 0.0217), respectively.
Therefore, these two miRNA can
function independently from each other
in order to predict endometriosis by the
logistic regression model. In contrast,
age, BMI, hsa-miR-154-5p and hsa-miR-
196b-5p had Pr(>|t|) > 0.05. This result
suggests that these factors can't function
as independent predictors for the disease
and their prediction power for the
disease is dependent on the interaction
between all variables in the model.

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 RBMO VOLUME 37 ISSUE 4 2018 461

FIGURE 4  – The hsa-miRNA-154-5p is a putative non-invasive biomarker for endometriosis. (A) Graphical representation of the influence of rASRM
stage on the levels of plasma hsa-miRNA-154-5p is given. The Mann–Whitney test was used and the P-value of the group comparisons was P >
0.05, indicated on the top of the graph as not significant (NS). The number of samples in each group are indicated in the brackets. (B) Graphical
representation of the influence of ectopic lesion location on the levels of hsa-miRNA-154-5p expression is plotted. The mean values of miRNA
expression levels are indicated on each box plot. Statistical multiple group analysis of the data (Kruskal-Wallis and Dunn multiple comparison
tests) showed that the differences in the levels of circulating plasma miRNA between the groups are random (adjusted P-value, P > 0.05) and
not associated with the type of the lesions. NS = not significant. (C) Diagnostic value of hsa-miRNA-154-5p for endometriosis expressed by ROC
curve analysis (left) of the log2 transformed expression data (right) obtained in a cohort of n = 64 cases and controls. The AUC and P-values
are indicated in the graph. (D) Diagnostic performance of the combined four DE miRNA in a cohort of n = 83 when analysed together (left) or
in combination with the clinical parameters age and BMI (right) is graphically presented. The AUC and corresponding P-values for each test are
indicated in the graphs.

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462 RBMO VOLUME 37 ISSUE 4 2018
FIGURE 5  – The predicted gene targets for the four altered miRNA in endometriosis are enriched in regulatory pathways associated with
endometriosis. Graphical visualization of the gene ontology terms (y-axis) showing fold enrichment >1.5 and P-value <0.05 (x-axis).
The supposed gene targets of hsa-miRNA-
154-5p, hsa-miRNA-378a-3p, hsa-miRNA-
33a-5p and hsa-miRNA-196b-5p are
involved in pathways with known or
suspected role for endometriosis
To gain insight into the biological role of
DE hsa-miR-154-5p, hsa-miRNA-378a-3p,
hsa-miRNA-33a-5p and hsa-miRNA-
196b-5p, gene target prediction and
GO annotation analysis was carried
out using MiRTarget2 (http://mirdb.org)
(Supplementary TABLE S7A) and DAVID
(https://david.ncifcrf.gov) (Supplementary
TABLE S7). This approach revealed
significant enrichment in miRNA gene
targets for those DE miRNA that control
cell motion, proliferation, differentiation,
adhesion, neovascularization and
innervation (FIGURE 5). This result suggests
that the pathogenesis of endometriosis
may involve miRNA-mediated modulation
of gene regulatory programmes at
the site of ectopic lesion formation,
implantation and growth.
DISCUSSION
Endometriosis is a disease that still
poses challenges in diagnosis, optimal
treatment and follow-up. In recent
years, a growing body of evidence
suggests an involvement of miRNA in
endometriosis pathogenesis and lesion
formation. In several diseases, including
endometriosis, DE miRNA in the
circulation have already been identified
and become interesting targets in the
search for non-invasive biomarkers
(Cosar et al., 2016; Reid et al., 2011).
In the current study, a q-PCR-based
profiling array of plasma miRNA
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was used to search for expression
differences between endometriosis
patients and controls to nominate
putative biomarkers for the disease.
For many diseases, a single biomarker
can be informative on a population
level, but not at the level of the
individual patient. This inadequacy has
already shifted attention to the use of
multiple biomarker discovery sets and
measurements (Fu et al., 2010). A panel
of several biomarkers could comprise
different entities of the same type of
variable, or alternatively, could contain
a combination of disparate types of
features, such as, in this case, a collection
of clinical data and miRNA. Therefore,
in the screening and discovery Phases
1 and 2 of our study, a combination of
in silico disease-predictive models and
individual group comparisons were
applied to detect miRNA candidates and
clinical parameters with a high potential
to identify patients with endometriosis.
It was found that models based on 42
DE miRNA candidates and the clinical
parameters BMI, age and dysmenorrhoea
can discriminate endometriosis patients
and controls with high specificity and
sensitivity when applied to a small
sample set, but are classified as random
in an independent larger study cohort.
Nevertheless, when using logistic
regression modelling for individual group
comparisons, corrected either for cycle
phase, hormonal treatment or disease
status, it was shown that the relative
expression levels of hsa-miR-154-5p,
hsa-miR-196b-5p, hsa-miR-378a-3p are
significantly lower and hsa-miR-33a-5p
significantly higher in plasma of patients
with endometriosis versus controls. It
was also demonstrated that hsa-miR-
154-5p has a quite acceptable sensitivity
and specificity in discriminating patients
with endometriosis from controls. When
combining the four DE miRNA with the
clinical parameters age and BMI, the
AUC value for endometriosis prediction
did not change, but the prediction
validity improved substantially. This result
adds to an evolving picture showing
an advantage of using combined DE
serum or plasma miRNA targets for
endometriosis prediction (Cosar et al.,
2016; Wang et al., 2013). However,
the outlined test model showed only a
fair disease prediction power (AUC =
0.72) and so its clinical value should be
considered as moderate. Nevertheless, to
be able to make a final conclusion on the
practical applicability of the outlined test
model, the clinical value of the model
needs to be further validated on a larger
study population.
Another interesting finding of our study
was that some circulating miRNA seem
to show, apart from endometriosis-
related changes, significant menstrual
phase-dependent differences. It was
shown that the expression levels of
hsa-miR-196b-5p, hsa-miR-378a-3p and
hsa-miR-33a-5p are both disease- and
menstrual cycle-dependent. It was also
found that hsa-miR-326 and hsa-miR-
485-3p display unequivocally cycle
phase-dependent regulation and are
down-regulated in plasma samples of
control women in the secretory versus
the proliferative phase. Comparing these

results to previous profiling studies in
bio-fluids, it was found that hsa-miR-326
and hsa-miR-485-3p have been reported
as DE and down-regulated in serum
of patients with endometriosis (Wang
et al., 2016a). It is important to note
that the analysis of the data in the work
by Wang et al. (2016a) was conducted
without selective calculation for
identification of cycle phase-dependent
miRNA differences. In the cited study,
the cycle phase distributions were
tested in terms of statistically significant
differences between endometriosis
patients and controls. Consequently,
as this was not the case, this factor was
not further taken into consideration for
miRNA expression analysis. In contrast,
in this study, the cycle phase was
incorporated as a possible confounder
of miRNA expression when testing for
single significant differences between
women with and without endometriosis.
This approach was intended to ensure
that any feasible interference of the
cycle phase is controlled for when
calculating differences arising from
endometriosis. It should be noted that,
as the analysis was limited to 372 known
miRNA, it does not have the genome-
wide analytical power of the recently
published deep-sequencing based
profiling data from Wang et al. (2016a).
Thus, the discrepancy of this data with
the study mentioned above might reflect
differences in research approaches
and does not exclude study protocol,
functional or cohort differences. Besides,
although this study does not show
associations between the DE miRNA and
rAFS stages or lesion types, evidence
exists that some of the proposed miRNA
biomarkers might indicate disease
severity with progression of the rAFS
score (Wang et al., 2013).
Up to now, little has been known about
the existence of concurrently DE
miRNA in circulation and tissue and
the mechanisms of their dysregulation
in endometriosis. For this reason,
this study examined whether some
of the DE candidates in plasma were
previously found to be de-regulated
in tissue samples of women with
endometriosis and controls. The
hsa-miR-154-5p is located at the DLK1-
MEG3 imprinted genomic locus which
hosts 54 miRNA and is the largest one
currently known in the human genome
(Benetatos et al., 2013). Up-regulation
of hsa-miR-154-5p is found in different
human cancers including endometrial
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carcinosarcomas, where it is a good
marker for distinguishing epithelial-to-
mesenchymal transition (Castilla et al.,
2011). Furthermore, increased expression
of this miRNA has been associated with
endometriomas (Kiba et al., 2016) and
endometrial stroma decidualization
(Estella et al., 2012). These observations,
together with the data from this study,
showing down-regulation of circulating
plasma hsa-miR-154-5p levels in
endometriosis, point to a more complex
and assorted biological function of the
transcript. In contrast to hsa-miR-154-
5p, hsa-miR-196b is consistently down-
regulated in plasma and tissue samples
of women with endometriosis at ectopic
sites of lesion implantation (Wei et al.,
2015). These data suggest that hsa-miR-
196b might act as an opposing factor in
the process of ectopic lesion formation.
Experimental evidence supporting this
idea is provided by the findings of Abe
et al. (2013), showing that overexpression
of hsa-miR-196b in endometrial and
endometriosis stroma cells in culture
leads to suppression of cell proliferation
and induction of apoptosis via a
mechanism including c-myc and Bcl-2
repression. Additionally, the hsa-miR-196b
was characterized as an epigenetically
regulated, hypermethylated and
down-regulated gene in endometriosis
compared with control endometrial
stroma cells. The hsa-miR33a-5p was
identified as down-regulated in tissue
samples of patients with endometrial
serous adenocarcinoma (Hiroki et al.,
2010) and hsa-miR-378a as up-regulated
in tissue of patients with ovarian cancer
(Chan et al., 2014). Recently, hsa-
378a was shown to regulate tumour
angiogenesis (Lee et al., 2007), epithelial-
to-mesenchymal transition (Zaravinos et
al., 2012) and to promote vascularization
under hypoxia (Xing et al., 2014). All
of these processes are also known to
be associated with the development of
endometriosis. Thus, as this study has
shown that these miRNA are DE and are
potentially good markers for diagnosis
of the disease, it might be interesting to
further evaluate their biological relevance
for endometriosis.
An overall strength of this study is the
design, with the stepwise approach for
marker detection using independent
patient cohorts for marker discovery
and validation. The normalization
method used slightly differed from
previous miRNA studies (Cosar et al.,
2016; Hsu et al., 2014; Jia et al., 2013;
RBMO VOLUME 37 ISSUE 4 2018 463
Suryawanshi et al., 2013; Wang et al.,
2013, 2016a), where the normalization of
expression levels was performed using
either single stably expressed miRNA or
spike-in controls. Here, the geometric
mean of the most stably expressed
miRNA within all experimental phases
was used. This method of normalization
particularly enables exploration of the
biological relevance of small expression
differences (Vandesompele et al., 2002).
Additional strengths of the study include
the confirmation or exclusion of the
disease by surgery and pathology, the
homogeneity of the study population
with respect to cycle phase and disease
stage type and group distribution and
the equal time-frame of blood collection
for cases and controls. Further, the
inclusion of potential confounders such
as dysmenorrhoea, age, BMI and cycle
phase into the disease prediction models
represents an internal cross-validation
approach that might be applicable in
future studies.
Although this study adds important
information to the field of miRNA
research in endometriosis, there are
several limitations that need to be
addressed. A general limitation in all
published data, including ours, seems
to be the moderate number of samples
in the discovery and validation phases.
Albeit well characterized, the control
population in this study included women
with other gynaecological diseases, which
may have impacted the levels of plasma
miRNA, e.g. benign ovarian cysts, uterine
fibroids or unexplained infertility. Thus,
the results of our analysis have to be
interpreted carefully and in the context
of the current experimental settings. One
way to overcome these limitations in the
future would be collaboration between
different study centres to conduct
studies based on larger cohorts. As
proposed by the World Endometriosis
Research Foundation (Fassbender et al.,
2013; Rogers et al., 2013), cooperative
strategies for choosing adequate
control cohorts and data normalization
methodology are also needed to support
the establishment of a consensus
on miRNA analysis for non-invasive
marker prediction and validation for
endometriosis.
As some authors proposed, a
hormone-like mechanism of action of
circulating miRNA, a role in cell-to-
cell communication (Bang et al., 2014;
Kosaka et al., 2013) and intracellular
464 RBMO VOLUME 37 ISSUE 4 2018
transcription in recipient cells (Arroyo
et al., 2011), MiRTarget2 and DAVID
enrichment analysis was used to identify
hsa-miR-154-5p, hsa-miR-196b-5p,
hsa-miR-378a-3p and hsa-miR-33a-
5p target gene sets. The study found
enrichment in genes controlling cell
motion, proliferation, cell differentiation,
neovascularization and innervation.
Thus, these miRNA seem to affect
genes with known or suspected roles
in endometriosis development, making
them interesting putative biomarkers for
the disease.
In conclusion, hsa-miR-154-5p, alone or
in combination with hsa-miR-196b-5p,
hsa-miR-378a-3p and hsa-miR-33a-5p
and the clinical parameters BMI and age,
is potentially applicable for non-invasive
diagnosis of the disease. Future studies
in different and larger patient cohorts are
needed to investigate the clinical value of
the described model.
ACKNOWLEDGEMENT
This study was supported by Bayer's
Grants4Targets (appl. no. 2012-08-0735).
Arndt Schmitz is employed by and
holds stocks in Bayer AG. Dr Schmitz
supported the team in study design and
critically reviewed the manuscript on
behalf of Grants4Targets. There are no
other conflicts of interests to declare. All
authors approved the final version of the
manuscript. The authors want to thank all
the participants and health professionals
involved in the present study for their
enthusiasm and commitment, and also
the study nurse of the Endometriosis
Centre, Manuela Gstöttner, for her
diligent work, constant assistance and
time flexibility whenever needed.
APPENDIX SUPPLEMENTARY
MATERIAL
Supplementary data to this article
can be found online at doi:10.1016/j.
rbmo.2018.05.007.
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For personal use only. No other uses without permission. Copyright ©2019. Elsevier Inc. All rights reserved.
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Received 19 July 2017; refereed 2 May 2018;
accepted 4 May 2018.