healthmedicinet

Patients and tissue samples

Samples were collected between 2010 and 2014 and were analyzed under protocols approved
by the institutional review board of the Medical University of Vienna (6th July 2010,
reference number 545/2010). Signed informed consent was obtained from each participant
of this study.

For qRT-PCR, tissue samples were obtained from 121 premenopausal women (mean age 32.3?±?5.9 years)
who underwent laparoscopic surgery at the certified Endometriosis Centre at the university-affiliated
General Hospital of Vienna between 2010 and 2014 due to the suspicion of endometriosis
with or without infertility. The 121 cases consisted of 74 patients with endometriosis
and 47 control patients who also underwent hysteroscopy, including dilation and curettage,
due to unexplained infertility. Among the 74 cases with endometriosis, we obtained
matched samples of ectopic and eutopic endometrium in 30 cases, exclusively eutopic
endometrium in 12 cases, and exclusively ectopic endometrium in 32 cases. The matched
sample tissues were collected during the same surgical procedure. Endometriosis was
diagnosed histologically in 62 patients and by visual inspection in 12 patients. Staging
was performed according to the revised American Fertility Society (rAFS) classification
guidelines (I, n?=?10; II, n?=?9; III, n?=?25; IV, n?=?30) 32]. Patients with malignant diseases of the ovaries or the endometrium were excluded.
Ectopic lesions consisted of ovarian lesions (n?=?40), peritoneal lesions (n?=?13),
and deep infiltrating lesions (n?=?9). Characteristics of the study populations are
provided in Additional file 1: Table S1.

For IHC, tissue samples were collected under the same conditions as for the qRT-PCR
samples. The 160 cases consisted of 110 patients with endometriosis and 50 control
patients who underwent dilation and curettage for benign indications. Among the 110
cases with endometriosis, we obtained matched samples of ectopic and eutopic endometrium
in 49 cases, exclusively eutopic endometrium in 20 cases, and exclusively ectopic
endometrium in 41 cases. Staging was performed according to the revised American Fertility
Society (rAFS) classification guidelines (I, n?=?17; II, n?=?23; III, n?=?22; IV,
n?=?25) 32]. Characteristics of the study populations are provided in Additional file 2: Table S2.

Quantitative Real-Time PCR (qRT-PCR)

Briefly, total RNA was isolated from fresh frozen tissues with the Absolutely RNA
miRNA Kit (Agilent) and reverse-transcribed with the SuperScript First-Strand Kit
(Invitrogen) according to the manufacturers’ instructions. Each sample was analyzed
by real-time PCR on an Applied Biosystems 7500 fast instrument, using gene-specific
primers and fluorescent probes obtained from Applied Biosystems: CDH1, Hs_01023894_m1;
TWIST1, Hs_01675818_m1; SNAIL, Hs_00195591_m1; SLUG, Hs_00950344_m1; GAPDH, Hs_99999905_m1
(control), and ACTB (control), Hs_99999903_m1. The mRNA levels of CDH1, TWIST1, SNAIL
and SLUG were normalized to those of ACTB and GAPDH in each sample by subtracting
the mean Ct (threshold cycle) values of the controls from the Ct value of CDH1, TWIST1,
SNAIL and SLUG as described previously 33]. For binary analysis, the cutoff was set at 0.162 for CDH1 expression, at 0.031 for
TWIST1 expression, at 0.0075 for SNAIL expression and at 0.156 for SLUG expression.

Immunohistochemistry (IHC)

TWIST1

Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissues.
Three-micrometer-thick sections were cut and placed on glass slides. Heat antigen
retrieval was performed in 10 mM Sodium Citrate Buffer pH6. Nonspecific background
staining was blocked by incubation in H
₂
O
₂
and with Ultra V Block (Thermo Scientific, Ultra Vision LP Kit, TL-060-HL) according
to the protocol. The rabbit polyclonal IgG to humanTWIST amino acids 12–27 (Abcam,
ab50581) was applied at a dilution of 1:1200 with Antibody Diluent with Background
Reducing Components (Dako, S3022) and incubated overnight at 4 °C. The Ultra Vision
LP Kit was used according to the protocol (Thermo Scientific, Ultra Vision LP Kit,
TL-060-HL). Finally, all slides were incubated with DAB-Substrate (Dako, K346811)
and counterstained in hematoxylin before they were dehydrated and mounted.

MYC

MYC IHC was performed with a professional staining system (AutostainerLink48, DAKO,
Glostrup, Denmark) at the Department of Pathology in the Wilhelminen Hospital. Briefly,
antigen retrieval was performed by boiling the slides in EnVision FLEX Target Retrieval
Solution at high pH (Dako Kit, K8000) for 15 min at 97 °C. The blocking procedure
was performed according to the protocol (Dako, K8000). The rabbit monoclonal IgG to
human c-MYC [Y69] (Biocare, CME415AK, CK) was applied at a dilution of 1:100 with
Renoir Red Diluent (Biocare) and incubated for 20 min at room temperature. The slides
were incubated with polymer according to the protocol (Dako, K8000). Finally, all
slides were incubated with DAB-Substrate (Dako, K8000) and counterstained in hematoxylin
(Dako real hematoxylin, S2020) before they were dehydrated and mounted.

Scoring and immunohistochemical analysis

Prior to immunohistochemistry, endometriotic lesions consisting of well-defined glandular
epithelial and stromal cells were identified in hematoxylin-eosin-stained sections
by a pathologist. Serial sections were cut from the chosen samples. A semiquantitative
subjective scoring system to evaluate the localization, quantity and intensity of
immunoreactivity was employed using light microscopy (200 × magnification). In each
sample, the staining of glandular epithelial cells and stromal cells was scored separately.
The intensity of the staining was scored using a four-point scoring scale (0, negative
staining; 1, weak staining; 2 moderate staining, 3, strong staining). The percentage
of positively stained cells was again scored using a four-point scoring scale (0,
negative staining; 1, 1-35 % positive cells; 2, 36-70 % positive cells; 3, 67 % positive
cells). The two scores were combined by multiplication to derive a final IHC score
(0–9). For epithelial or stromal TWIST1 and epithelial MYC expression, a final score
of ?4 was regarded as positive, and for stromal MYC expression, a final score of ?3
was regarded as positive (Fig. 1). Evaluations were performed by two blinded investigators. The outcomes analyzed
by two experienced investigators showed statistical significance for the same results.
An automatic quantitative analysis system was not robust/adequate for the analysis
of our probes and was therefore not used. Positive and negative (without primary antibody)
controls were run concurrently. The MYC protein was expressed in the nucleus of the
epithelial and the stromal cells of eutopic and ectopic endometrium. TWIST expression
was observed in the cytoplasm and the nucleus of epithelial and stromal cells. However,
as a transcription factor, activated TWIST exerts its main function in the nucleus.
Thus, for both factors, only the nuclear staining of epithelial and stromal cells
was evaluated.

Fig. 1. Immunohistochemical staining of MYC and TWIST1 in eutopic and ectopic endometrial
tissue. Anti-MYC antibody was applied at a dilution of 1:100 and yielded negative
(a, b) or positive (c, d) nuclear staining in eutopic (a, c) and ectopic (b, d) tissue. Anti-TWIST1 antibody was applied at a dilution of 1:1200 and yielded cytoplasmatic
and nuclear staining. For evaluation, only nuclear staining was analyzed. Anti-TWIST1
antibody yielded negative (e, f) or positive (g, h) nuclear staining in eutopic (e, g) and ectopic (f, h) tissue. Magnification?=?200x

Statistical analysis

Data were analyzed using SPSS (17.0). For association analyses, chi-squared tests
were used. Wilcoxon and Mann Whitney U tests were used to compare the two groups.
For correlation analyses, Spearman tests were used. For paired statistics, the McNemar
Test and Wilcoxon Signed Ranks Test were used. A linear regression model was computed
to describe associations between MYC, TWIST1, and cycle phase. We considered the subgroup
analyses as exploratory and hence did not adjust for multiple testing, as recommended
by Bender and Lange 34]. Statistical significance was defined as p??0.05.