Endocannabinoids modulate apoptosis in endometriosis and adenomyosis

Abstract

Adenomyosis that is a form of endometriosis is the growth of ectopic endometrial tissue within the muscular wall of the uterus (myometrium), which may cause dysmenorrhea and infertility. Endocannabinoid mediated apoptotic mechanisms of endometriosis and adenomyosis are not known. We hypothesized that the down regulation of endocannabinoid receptors and/or alteration in their regulatory enzymes may have a direct role in the pathogenesis of endometriosis and adenomyosis through apoptosis. Endocannabinoid receptors CB1 and CB2, their synthesizing and catabolizing enzymes (FAAH, NAPE-PLD, DAGL, MAGL) and the apoptotic indexes were immunohistochemically assessed in endometriotic and adenomyotic tissues. Findings were compared to normal endometrium and myometrium. Endometrial adenocarcinoma (Ishikawa) and ovarian endometriosis cyst wall stromal (CRL-7566) cell lines were furthermore cultured with or without cannabinoid receptor agonists. The IC50 value for CB1 and CB2 receptor agonists was quantified. Cannabinoid agonists on cell death were investigated by Annexin-V/Propidium iodide labeling with flow cytometry. CB1 and CB2 receptor levels decreased in endometriotic and adenomyotic tissues compared to the control group (p=0,001 and p=0,001). FAAH, NAPE-PLD, MAGL and DAGL enzyme levels decreased in endometriotic and adenomyotic tissues compared to control (p=0,001, p=0,001, p=0,001 and p=0,002 respectively). Apoptotic cell indexes both in endometriotic and adenomyotic tissues also decreased significantly, compared to the control group (p=0,001 and p=0,001). CB1 and CB2 receptor agonist mediated dose dependent fast anti-proliferative and pro-apoptotic effects were detected in Ishikawa and ovarian endometriosis cyst wall stromal cell lines (CRL-7566). Endocannabinoids are suggested to increase apoptosis mechanisms in endometriosis and adenomyosis. CB1 and CB2 antagonists can be considered as potential medical therapeutic agents for endometriosis and adenomyosis.

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Acta Histochemica
journal homepage: www.elsevier.com/locate/acthis
Endocannabinoids modulate apoptosis in endometriosis and adenomyosis
☆
Elif Bilgic
a
, Elif Guzel Meydanli
b
, Sevil Kose
c
, Makbule Cisel Aydin
d
, Eda Karaismailoglu
e
,
Irem Akar
c
, Alp Usubutun
d
, Petek Korkusuz
a,⁎
a
Department of Histology and Embryology, Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey
b
Department of Histology and Embryology, Istanbul University Cerrahpasa Medical Faculty, 34093 Istanbul, Turkey
c
Stem Cell Research and Application Center, Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey
d
Department of Pathology, Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey
e
Department of Biostatistics, Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey
ARTICLE INFO
Keywords:
Endocannabinoids
Endometriosis
Adenomyosis
Apoptosis
ABSTRACT
Adenomyosis that is a form of endometriosis is the growth of ectopic endometrial tissue within the muscular wall
of the uterus (myometrium), which may cause dysmenorrhea and infertility. Endocannabinoid mediated
apoptotic mechanisms of endometriosis and adenomyosis are not known. We hypothesized that the down
regulation of endocannabinoid receptors and/or alteration in their regulatory enzymes may have a direct role in
the pathogenesis of endometriosis and adenomyosis through apoptosis. Endocannabinoid receptors CB1 and
CB2, their synthesizing and catabolizing enzymes (FAAH, NAPE-PLD, DAGL, MAGL) and the apoptotic indexes
were immunohistochemically assessed in endometriotic and adenomyotic tissues. Findings were compared to
normal endometrium and myometrium. Endometrial adenocarcinoma (Ishikawa) and ovarian endometriosis cyst
wall stromal (CRL-7566) cell lines were furthermore cultured with or without cannabinoid receptor agonists.
The IC50 value for CB1 and CB2 receptor agonists was quantified. Cannabinoid agonists on cell death were
investigated by Annexin-V/Propidium iodide labeling with flow cytometry. CB1 and CB2 receptor levels
decreased in endometriotic and adenomyotic tissues compared to the control group (p = 0,001 and p = 0,001).
FAAH, NAPE-PLD, MAGL and DAGL enzyme levels decreased in endometriotic and adenomyotic tissues
compared to control (p = 0,001, p = 0,001, p = 0,001 and p = 0,002 respectively). Apoptotic cell indexes both
in endometriotic and adenomyotic tissues also decreased significantly, compared to the control group
(p = 0,001 and p = 0,001). CB1 and CB2 receptor agonist mediated dose dependent fast anti-proliferative
and pro-apoptotic effects were detected in Ishikawa and ovarian endometriosis cyst wall stromal cell lines (CRL-
7566). Endocannabinoids are suggested to increase apoptosis mechanisms in endometriosis and adenomyosis.
CB1 and CB2 antagonists can be considered as potential medical therapeutic agents for endometriosis and
adenomyosis.
1. Introduction
Endometriosis and adenomyosis are defined as the unusual location
of the endometrial tissue at ectopic sites and in the myometrium. They
can cause pelvic pain, dyspareunia, amenorrhea, dysmenorrhea and
infertility (Irving and Clement 2011; Kruse et al., 2012; Lin et al., 2014;
Lo Monte et al., 2013; Sznurkowski and Emerich, 2008; Gao et al.,
2006; Vannuccini et al., 2016; Yang et al., 2013; Yamanaka et al.,
2014). Endometriosis affects a large population and decreases quality of
life. The pathogenesis of the disease remains unclear, although it is
believed to relate with the quite aggressive behavior of endometriotic
cells at migrating ectopic locations and the resistance of these cells to
apoptosis (Agic et al., 2009; Nasu et al., 2011; Sbracia et al., 2016).
Pathogenesis of adenomyosis is similar to endometriosis; adenomyotic
cells have resistance to apoptosis as well (Yamanaka et al., 2014). The
relationship between the endocannabinoids and adenomyosis is not yet
studied.
Endocannabinoids, which are mostly located in the central nervous
system and also in other organ systems, are Cannabis ligands that
specifically act through their CB1 and CB2 receptors (Alger and Kim,
http://dx.doi.org/10.1016/j.acthis.2017.05.005
Received 24 October 2016; Received in revised form 9 May 2017; Accepted 9 May 2017
☆
Various data of this study was presented at the XII. National Congress of Histology and Embryology, Ankara, Turkey, May 27–30, 2014, and at the XIII. National Congress of
Histology and Embryology, İzmir, Turkey, April 30–May 3, 2016.
⁎
Corresponding author at: Hacettepe University Faculty of Medicine, Department of Histology and Embryology, Sihhiye 06100, Ankara, Turkey.
E-mail addresses: elifbilgic8@gmail.com (E. Bilgic), elifguzelctf@yahoo.com (E.G. Meydanli), sevil.arslan@hacettepe.edu.tr (S. Kose), ciselaydin@gmail.com (M.C. Aydin),
edaozturk1982@yahoo.com (E. Karaismailoglu), iremakar@yahoo.com (I. Akar), alpusubutun@yahoo.com (A. Usubutun), petek@hacettepe.edu.tr (P. Korkusuz).
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2011; Coskun and Bolkent, 2014; Mercati et al., 2012; Muccioli, 2010;
Scotchie et al., 2015; Yazulla, 2008). The most well-known endocan-
nabinoids are anandamide (AEA) and di-arachidonoylglycerol (2-AG)
(Alger and Kim, 2011; Muccioli, 2010; Scotchie et al., 2015; Yazulla,
2008). AEA is known to act more over the CB1 receptor in the female
genital system, while 2-AG displays its effects usually through the CB2
receptor (Maccarrone, 2009; Taylor et al., 2010). In the female genital
system, endocannabinoids and their receptors are generally located in
the endometrium, the myometrium, the ovarian cortex and the medulla
and the uterine tubes; they have critical roles in menstrual cycle,
ovarian maturation, embryo transplantation and implantation
(Brighton et al., 2011; El-Talatini et al., 2009, 2010; Karasu et al.,
2011; Maccarrone, 2009; Scotchie et al., 2015; Sun et al., 2009; Taylor
et al., 2010).
Recent researches suggested that endocannabinoids are involved in
the pathophysiology of endometriosis in a variety of ways.
Endocannabinoid agonists have anti-proliferative and analgesic effects
on endometriotic cells or patients. Endometriosis-associated pain is
shown to decrease by WIN 55212-2, CB1 and CB2 receptor agonist in
experimental studies or by palmitoylethanolamide in patients with
endometriosis (Cobellis et al., 2011; Dmitrieva et al., 2010; Giugliano
et al., 2013; Indraccolo and Barbieri, 2010; Lo Monte et al., 2013). Cell
proliferation in deep infiltrating endometriosis decreased with WIN-
55212-2, both in vitro and in vivo (Leconte et al., 2010). In vitro
stimulatory effect of endocannabinoid agonists on cell migration
moreover was presented (Gentilini et al., 2010; McHugh et al., 2012).
According to this, enhanced endometrial stromal cell migration via CB1
and GPR18 receptor with use of methanandamide, which is another
endocannabinoid agonist, or N-arachidonyl glycine, which is an
endogenous metabolite of anandamide, were shown through the
activation of PI3K/Akt, ERK1/2 or MAPK pathways (Gentilini et al.,
2010; McHugh et al., 2012). Although some activities of endocannabi-
noids are defined, we still do not know the effects of endocannabinoids
on apoptosis in endometriosis. Anandamide leaded to apoptosis
through CB1 receptor and p38 pathway on decidual cells (Almada
et al., 2015; Fonseca et al., 2009).
Given the apoptotic and anti-proliferative effects of endocannabi-
noids, we hypothesized that the down regulation of endocannabinoid
receptors and/or alteration in their regulatory enzymes may have a
direct role in the pathogenesis of endometriosis and adenomyosis
through apoptosis. We aimed to define the potential apoptosis related
classical receptor mediated effects of endocannabinoids on endome-
triosis and adenomyosis. We investigated the differences of immune
labelings of CB1 and CB2 receptors, AEA and 2-AG catabolizing and
synthesizing enzymes, as well as apoptotic index between the endome-
triotic and adenomyotic patients and age-matched controls. Depending
on the supposedly pro-apoptotic effects of endocannabinoids (Almada
et al., 2015; Siegmund et al., 2016), endometrial adenocarcinoma cell
line (Ishikawa) and ovarian endometriosis cyst wall stromal cell line
(CRL 7566) were cultured with or without cannabinoid classical
receptor agonists. The xCELLIgence cell impedance based system was
used to calculate the IC50 value for CB1 and CB2 receptor agonists.
Cannabinoid agonists’effect on cell death was investigated with flow
cytometry by Annexin-V/propidium iodide labeling. Outcomes of these
experiments may explain endocannabinoid effects of cell survival and
death mechanisms in endometriosis.
2. Materials and methods
2.1. Design
A double blind randomized experimental study was designed. We
received endometrial archive samples belonging to patients having
been diagnosed as endometriotic and adenomyotic from January 2010
to July 2012. Age-matched paraffin endometrial tissue blocks of 20
endometriosis, 17 adenomyosis patients and 19 normal controls
between 24 and 52 years were obtained from Hacettepe University
Pathology Department (Table 1). Control tissues were obtained from
patients undergoing dilatation and curettage surgery for benign gyne-
cological conditions other than endometrial disease. Control endome-
trial tissues were sub-grouped according to the phase of menstrual cycle
as proliferative (n = 12) and secretory phases (n = 7). The endome-
triotic tissues were also sub-grouped as cystic (n = 9) and non-cystic
(n = 11). The use of endometriotic cells and the paraffin blocks of
endometrial tissue was approved by the Hacettepe University Non-
invasive Clinical Researches Ethical Committee (TBK 12/05-08), An-
kara, Turkey.
2.1.1. CB1 and CB2 receptors and FAAH, NAPE-PLD, MAGL, DAGL
enzymes immune labeling
5–6μm Thick paraffin sections were deparaffinized. Endogenous
peroxidase activity was blocked by 3% hydrogen peroxide (cat#
216763, Sigma-Aldrich, St. Louis, USA) after antigen retrieval.
Nonspecific binding was blocked with 5% normal mouse serum
(cat#M5905, Sigma-Aldrich, St. Louis, USA) for 30 min. Slides were
incubated with following primary antibodies overnight at 4 °C:
CB1(cat#C2866, rabbit polyclonal;1/100 dilution; Sigma-Aldrich, St.
Louis, USA), CB2 (cat#HPA028718, rabbit polyclonal; 1/100 dilution;
Sigma-Aldrich, St. Louis, USA), FAAH (cat#HPA007425, rabbit poly-
clonal, 1/50 dilution; Sigma-Aldrich, St. Louis, USA), MAGL
(cat#100035, rabbit polyclonal, 1/100 dilution, Cayman, Michigan,
USA), DAGL (cat#ab106979, rabbit polyclonal, 1/100 dilution, Abcam,
Cambridge, USA). Incubation with NAPE-PLD (cat#HPA019832, rabbit
polyclonal, 1/100 dilution, Sigma-Aldrich, St. Louis, USA) was per-
formed overnight at RT. The secondary antibody incubation
(cat#EXTRA3, mouse monoclonal, Sigma-Aldrich, St. Louis, USA) was
performed for 30 min at RT at 1/20 dilution. After washing slides and
incubating with DAB (cat#D3939, Sigma-Aldrich, St. Louis, USA) we
used haematoxylin for counterstaining. Digital images were analyzed
and captured using the Leica DM6000B microscope equipped with a
DFC480 digital camera.
2.1.2. Image analysis
Two pathologists according to pathological criteria for the diseases
selected endometriotic and adenomyotic foci under the microscope (Fu
et al., 2013; Yu et al., 2015).
Table 1
Age-matched control and experimental groups are presented with mean ± standard deviation/median (min–max) of their immune reactivities and apoptotic indexes.
Control n AGE
mean ± SD
CB1
mean ± SD
CB2
mean ± SD
FAAH
mean ± SD
NAPE-PLD
mean ± SD
MAGL
mean ± SD
DAGL
median (min-max)
TUNEL
median (min-max)
Proliferative 12 37,5 ± 1,27 0,80 ± 0,11 0,79 ± 0,10 0,79 ± 0,07 0,73 ± 0,15 0,69 ± 0,09 0,64 (0,49–0,79) 0,63 (0,0–0,85)
Secretory 7 41,4 ± 2,5 0,80 ± 0,11 0,76 ± 0,10 0,83 ± 0,11 0,71 ± 0,11 0,76 ± 0,10 0,72 (050–0,82) 0,65 (0,4–0,91)
Endometriosis
Non-cystic 11 40,4 ± 2,07 0,35 ± 0,16 0,35 ± 0,21 0,37 ± 0,26 0,27 ± 0,23 0,22 ± 0,29 0,60 (0,0–0,66) 0,0 (0,0–0,70)
Cystic 9 35,6 ± 2,5 0,39 ± 0,20 0,40 ± 0,25 0,34 ± 0,34 0,32 ± 0,29 0,38 ± 0,24 0,52 (0,48–0,74) 0,11 (0,0–0,66)
Adenomyosis 17 42,5 ± 0,9 0,37 ± 0,19 0,34 ± 0,32 0,41 ± 0,21 0,43 ± 0,25 0,29 ± 0,26 0,48 (0,0–0,81) 0,05 (0,0–0,69)
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Ten endometriotic foci or equal amount of glands have been
selected at non-overlapping fields of each endometrial, endometriotic
and adenomyotic sections by the motorized stage module of a Leica
DM6000B microscope (Lin et al., 2014). Photomicrographs of each
focus were generated by the microscope (Leica DM6000B) attached
computerized digital camera (DFC 480, Leica Westlar Germany) and
captured as TIFF at 200×magnification. The bright-field images were
analyzed quantitatively by image processing program (LAS 3.8 Leica
Inc., Westlar Germany version 3.8). Areas of interest (ROI) consisting of
endometrial, adenomyotic or normal glandular (for control group) foci
have been chosen at the x and y stages at the binary mode; and the total
ROI was calculated for 10 foci. The measurements were done at
minimum 45,876 um
2
- maximum 125,214 um
2
for each endometriotic,
adenomyotic or glandular focus (Hey-Cunningham et al., 2013). Brown
stained particles (immune labeled cells) were counted in the binary
defined area, at counting mode of LAS. Haematoxylin was extracted
from DAB by RGB level of the software. The blue threshold value was
106,49 px for the nuclei, and the brown threshold value was 65,22 px
for peroxidase labeling. The number of total immune reactive cell
percentage was expressed as the ratio of immune positive particles
(both the glandular epithelial and stromal cells) to total ROI.
2.1.3. TUNEL analysis for apoptosis
Slides were rinsed after de-waxing and dehydrating. Endogenous
peroxidase activity was blocked in 3% hydrogen peroxide for 10 min.
Sections were treated with permeabilization solution (0.1% Triton X-
100 in 0.1% sodium citrate) for 8 min at room temperature. We used
the in-situ cell death detection kit (Roche, Indianapolis, IN, USA) for
detecting the DNA fragments. Digital images were analyzed and
captured using the Leica DM6000B microscope equipped with the
DC500 digital camera after DAB incubation and haematoxylin staining.
The apoptotic index was expressed as a percentage of apoptotic
glandular and stromal cells over total glandular and stromal cells at
200×magnification. Average of 4 analyzed non-overlapping fields was
reported (Shen et al., 2010) in every specimen.
2.1.4. Cell culture
Ishikawa cell line (99040201, Sigma, Germany) was used to
simulate normal endometrial glandular cells with their well-known
phenotypic similarity and their response to steroids, resembling the
physiological conditions (Tamm-Rosenstein et al., 2013). Ishikawa cell
line is provided at passage 15 and authenticated by the manufacturer
(European Collection of Authenticated Cell Cultures, London, UK).
Endometriosis cyst wall stromal cell line (CRL-7566, ATCC, USA) was
used at passage 15, to analyze the cannabinoid agonistic effect on
endometriosis model. CRL-7566 is provided at passage 15 and,
authenticated by DNA-based method. Although stated as mycoplasma
free by the providers, the cell lines were tested by EZ-PCR Mycoplasma
test kit (cat#20-700-10, Biological Industries, Kibbutz Beit Haemek,
Israel) before use. Ishikawa cells were incubated in DMEM F-12 with
10% FBS and 2% L-glutamine and 1% pen-strep solution at 37 °C and
5% CO
2.
CRL-7566 cells were incubated in DMEM with % 20 FBS and %
2L-glutamine and % 1 pen-strep solution at 37 °C and 10% CO
2.
Cells
were used for the experiments at passage 18.
2.1.5. Impedance-based real-time cell proliferation analysis
Real-time cell proliferation was assessed with the xCELLIgence
system (Roche Applied Science, Mannheim, Germany; ACEA
Biosciences, San Diego, CA). Disposable 96-well e-plates were coated
with 10 μgr/ml fibronectin, cells were seeded and incubated at 37 °C, %
5 CO
2
until cell index was 1 at 22 h (Lowin et al., 2012).
Ishikawa cell line was used to detect half maximal inhibitory
concentration (IC
50
) of selective CB1 and CB2 agonists ACPA (1318,
Tocris Bioscience, Bristol, UK), respectively. ACPA (100 nM, 1 μM,
10 μM, 100 μM) and CB 65 (1 μM, 10 μM, 100 μM) were applied at
different concentrations and the IC
50
was calculated accordingly (to
determine the of cannabinoids RTCA software was used) (Fig. 5A and
C). Because our experimental procedure took about 146 h; IC50
concentrations were calculated both at 126th and 46th hours (Fig. 5B
and D). After detecting IC50 concentrations, Ishikawa and CRL-7566
cells were exposed to the determined IC
50
concentration of ACPA
(9.3 ×10
−6
M) and CB65 (1.9 ×10
−4
M) for 46 h and monitored at
every 15 min for 146 h (Fig. 6).
2.1.6. Flow cytometry analysis
We incubated cells with the determined IC
50
values of ACPA
(9,3 ×10
−6
M) and CB65 (1,9 ×10
−4
M) for 46 h after expansion.
Cells with and without cannabinoid agonists were analyzed by
Annexin-V/propidium iodide labeling in FACS Aria flow cytometer
(Becton, Dickinson Biosciences, USA) at 46th hour. Cells were classified
as live (Annexin V−, PI−), necrotic (Annexin V−, PI+), early
apoptotic (Annexin V+, PI−), and late apoptotic (Annexin V+, PI+)
cells. The acquired data was analyzed by using BD FACSDiva software
v6.1.2 (Becton Dickinson Biosciences, USA) (Fig. 7). The ratio of
apoptosis was reported as early apoptotic percentage plus late apoptotic
percentage in the text.
2.2. Statistics
Distribution of normality of immune labeling was evaluated by the
Shapiro-Wilk test. Age and CB1, CB2, FAAH, NAPE-PLD, MAGL immune
labelings of stromal and glandular cell variables were evaluated by one-
way ANOVA followed by post hoc Tukey testing. DAGL immune
labeling in stromal and glandular cells was not normally distributed.
Therefore, they were evaluated by the Kruskal-Wallis and the Mann-
Whitney U-tests with Bonferroni correction. Correlation analysis was
performed using the Pearson’s (for parametric data) or the Spearman
correlation (for nonparametric data) tests. Parametric data were
presented as the mean ± standard error of mean, while others were
presented as minimum, median, and maximum values. Confidence
interval was 95% and statistical significance was defined as p < 0.05.
The SPSS (15.0 version) program and the NCSS-PASS 2007 software
were used for analysis.
3. Results
3.1. CB1 and CB2 receptor immune labeling
CB1 and CB2 receptor immune labeling was cytoplasmic and intense
in both endometrial glandular and stromal cells in the control group
(Fig. 1A–F). Immune labeling percentages for CB1 and CB2 receptors in
the experimental groups were significantly (p = 0,001) lower than that
of the control group (Fig. 1G). The CB1 and CB2 receptor immune
labeling was similar in the endometriosis and the adenomyosis groups.
Endometrial glandular and stromal cells in proliferative and secretory
phases of the control group exhibited similar CB1 receptor immune
labeling. The CB2 receptor labeling of the glandular cells was sig-
nificantly (p = 0,020) higher in the proliferative phase than the
secretory phase however it remained unchanged in the stromal cells
between proliferative and secretory menstrual phases in this group. CB2
immune labeling for glandular epithelial and stromal cells decreased
with age (r = −0,612, p = 0,012; r = −0,53, p = 0,033) in the
adenomyosis group.
3.2. FAAH and NAPE-PLD immune labeling
FAAH and NAPE-PLD enzymes presented a compatible pattern of
immune labeling with CB1 receptor (Fig. 2A–F). Immune labeling
analysis indicated that FAAH and NAPE-PLD enzyme immune labeling
were significantly lower (p = 0,001) in glandular and stromal cells in
the endometriosis and the adenomyosis groups when compared to the
control group (Fig. 2G). Although FAAH enzyme levels in the glandular
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cells was significantly lower in the proliferative phase when compared
to the secretory phase (p = 0,004), FAAH enzyme immune labeling did
not show any difference in stromal cells between menstrual phases of
the control group. The control group furthermore showed similar
NAPE-PLD enzyme expression in endometrial glandular and stromal
cells in proliferative and secretory phases.
3.3. MAGL and DAGL enzyme immune labeling
MAGL and DAGL showed a compatible immune labeling pattern
with the CB2 receptor (Fig. 3A–F). Immune labeling analysis indicated
that immune labeling of MAGL (p = 0,001 both for glandular and
stromal cells) and DAGL (p = 0,002 for glandular cells) in the experi-
mental groups compared to the control group (Fig. 3G–I).
3.4. TUNEL assay for apoptosis
Lower TUNEL positivity was detected in the endometriosis and the
adenomyosis groups compared to the control group in glandular and
stromal cells (p = 0,001) (Fig. 4A–E). Apoptotic index revealed no
difference in the glandular and stromal cells among the phases of the
cycle in the control group and between the endometriosis and the
adenomyosis groups as well as cystic and solid subgroups of endome-
triotic patients.
3.5. Impedance-based real-time cell proliferation analysis
Optimal anti-proliferative effect of ACPA and CB65 at IC50 con-
centrations was at the 46th hour (Fig. 5B and D). The IC
50
concentra-
Fig. 1. A–F are endometrial micrographs exhibiting cytoplasmic CB1 (A–C) and CB2 (D–F) receptor immune labeling on glandular epithelial (*) and stromal cells (**), Haematoxylin
400×. (G) Immune labelings for CB1 and CB2 receptor distribution in control and experimental groups are shown. (*) p = 0,001. Note the significantly decreased immune labeling in
adenomyosis (C and F) and endometriosis groups (B and E) comparing to control (A and D) in the micrographs and the graphic (n = 19 for control; n = 20 for endometriosis and n = 17
for adenomyosis).
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tions of CB1 and CB2 agonists were detected as 9.3 ×10
−6
M for ACPA
and 1.9 ×10
−4
M for CB 65 on Ishikawa cells (Fig. 5A and C).
Ishikawa and CRL-7566 endometriotic cells exhibited decreasing
cell indices immediately after application of the IC50 concentration of
ACPA and CB 65. Cell proliferation index for CRL-7566 cells decreased
76% with ACPA and 86% with CB65 (Fig. 6A and B). Cell proliferation
index for Ishikawa cells decreased 95% with ACPA and 81% with CB65
(Fig. 6C and D).
3.6. Flow cytometry analysis
Annexin-V/propidium iodide labeled total (early and late) apoptotic
Ishikawa and CRL-7566 cell numbers increased with ACPA and CB65
exposure compared to the untreated control (Fig. 7). The 71,7% of
Ishikawa cells and 81,7% of CRL-7566 cells were apoptotic (early and
late) with ACPA (Fig. 7A and D). The 80,5% of Ishikawa cells and
78,3% of CRL-7566 cells were apoptotic (early and late) (Fig. 7B and E)
with CB65. In the untreated control group, only 0,8% of Ishikawa cells,
but 76,9% of CRL-7566 cells were apoptotic (early plus late) (Fig. 7C
and F).
4. Discussion
Endometriosis and adenomyosis that co-exist with endometriosis
(Garavaglia et al., 2015), affecting nearly 10–15% of the female
population (Jeung et al., 2016) increase the risk of gynecological
malignancies (Krawczyk et al., 2016). Adenomyosis was furthermore
recognized in 16–34% of endometrial carcinoma hysterectomy speci-
mens (Gizzo et al., 2016). Endometriotic cells tend to have cancer cell
like aggressive features for migrating to ectopic locations and they are
resistant to apoptosis (Agic et al., 2009; Nasu et al., 2011; Sbracia et al.,
2016). Adenomyotic cells have resistance to apoptosis as well
(Yamanaka et al., 2014).
Cannabinoid receptors (CB1, CB2) and the NAPE-PLD, FAAH,
DAGL, MAGL enzymes exhibited different regulation in the glandular
epithelial cells and stromal cells of the normal endometrial, the
Fig. 2. A–F are endometrial micrographs showing cytoplasmic AEA catabolizing FAAH (A–C) and synthesizing NAPE-PLD (D–F) enzyme immune labeling on glandular epithelial (*) and
stromal cells (**), Haematoxylin 400×. (G) Graphic shows the immune labeling for FAAH and NAPE-PLD in control and experimental groups. (*) p = 0,001. Note that both enzymes
significantly decreased in endometriosis (B and E) and adenomyosis (C and F) groups comparing to control (A and D). (n = 19 for control; n = 20 for endometriosis and n = 17 for
adenomyosis).
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endometriotic and the adenomyotic tissues in this study. Immune
labeling values for CB1 and CB2 receptors in glandular and stromal
cells of endometriosis and adenomyosis groups were lower than that of
the control group. Our findings for CB1 immune labeling were similar
with the results of Resuehr et al. (2012) who showed that CB1 immune
reactivity decreased in endometriosis compared to the control group,
however they were different than that of Leconte et al. (2010) who
suggested no difference for the CB1 receptor expression level between
endometriosis and controls (Leconte et al., 2010; Resuehr et al., 2012).
CB1 and CB2 receptor distribution in the endometriosis and
adenomyosis groups were not significantly different in our study.
Findings of this study demonstrated lower immune labeling for
cannabinoid receptors in adenomyosis and endometriosis compared
to the control group.
Labeling for NAPE-PLD and FAAH, synthesizing and catabolizing
enzymes of AEA, decreased in glandular epithelial cells and stromal
cells in both endometriosis and adenomyosis compared to the control.
Taylor et al. (2010) showed the existence of the receptors of AEA (CB1)
together with its synthesizing and catabolizing enzymes in the endome-
trium at different stages of the menstrual cycle and in the ovary by
immunohistochemistry (Taylor et al., 2010). It is known that AEA is
more common in endometrium than 2-AG at physiological conditions
(Maccarrone, 2009; Taylor et al., 2010). Although Sanchez et al. (2016)
detected increased systemic levels of AEA, 2-AG and OEA in patient
derived serum, lower expressions of CB1 mRNA was detected in the
same cases’endometriotic cells compared to controls at secretory phase
of menstruation (Sanchez et al., 2016). Our study is the first that
searched for the synthesizing and catabolizing enzymes of AEA in
endometriosis and adenomyosis patients. Immune reactivity of synthe-
sizing and catabolizing enzymes were detected to be similar in
endometriosis and adenomyosis. Since the expression of both NAPE-
PLD and FAAH decreased in endometriosis and adenomyosis groups in
line with receptor immune reactivity, we suggest that synthesis and
degrading of AEA get slower together in both epithelial and stromal
cells during the pathogenesis of the disease.
Tissues from the proliferative and secretory phases of the normal
endometrium exhibited the same immune labeling pattern for CB1 in
this study. This finding revealed that CB1 receptor immune reactivity is
not menstrual cycle dependent. This finding correlated well with the
data of Taylor et al. (2010) and colleagues (Taylor et al., 2010).
Fig. 3. A–F are endometrial micrographs showing cytoplasmic 2-AG synthesizing MAGL (A–C) and 2-AG catabolizing DAGL (D–F) enzyme immune labeling on glandular epithelial (*)
and stromal cells (**), Haematoxylin 400×. (G) Graphic shows the immune labeling percentages for MAGL enzyme distribution in control and experimental groups. (*) p = 0,001. (H)
(boxplot graph) shows nonparametric distributed immune labeling scores for DAGL on glandular epithelial cells of all groups respectively. (**) p = 0,002. (n = 19 for control; n = 20 for
endometriosis and n = 17 for adenomyosis).
E. Bilgic et al. $FWD+LVWRFKHPLFD[[[[[[[[[[²[[[


Resuehr et al. (2012) however reported that secretory phase of the
normal endometrium exhibits increased CB1 immune reactivity
(Resuehr et al., 2012). The strength of our study is the larger number
of patients and also larger panel of labeling comparing to previous
reports.
FAAH immune labeling was higher in the secretory compared to the
proliferative phase in glandular epithelial cells in this study. This
revealed that glandular epithelial cells at secretory phase were inde-
pendent from CB1 receptor activity. We found that immune labeling for
NAPE-PLD was similar at different phases of the control group.
Fig. 4. From A to C are endometrial micrographs from control and experimental groups exhibiting apoptotic stromal (**) and epithelial (*) cells undergoing apoptosis with their nuclei
labeled in dark brown. Haematoxylin 400×. D and E show boxplot graphs of apoptotic indices for glandular epithelial and stromal cells respectively (*) p < 0,05. Note that control
group exhibits significantly higher apoptotic rate comparing to both adenomyosis and endometriosis groups. (n = 19 for control; n = 20 for endometriosis and n = 17 for adenomyosis).
Fig. 5. (A and C) Real time cell proliferation curves of Ishikawa cells following application of different doses of ACPA (100 nM, 1 μM, 10 μM, 100 μM) and CB65 (1 μM, 10 μM, 100 μM)
are shown. (B) and (D) are logarithmic graphics representing the calculated value for IC
50
concentration. The anti-proliferative effect of ACPA (9.3 ×10
−6
M) and CB65 (1.9 ×10
−4
M)
at IC50 concentrations was observed from 46th hour. All plots were generated using the RTCA Software 1.1.
E. Bilgic et al. $FWD+LVWRFKHPLFD[[[[[[[[[[²[[[


According to Taylor et al. (2010), FAAH enzyme level is higher at late
secretory phase and lower at early proliferative phase of the menstrual
cycle, while NAPE-PLD immune reaction is higher at late secretory and
early proliferative phases than late proliferative and early secretory
phases in glandular epithelial cells of normal endometrium (Taylor
et al., 2010). Our findings for phase distribution of FAAH are consistent
with the data of Taylor et al. (Taylor et al., 2010). We suggest that the
FAAH enzyme rather than the CB1 receptor or the NAPE-PLD enzyme
regulates endocannabinoid activity. The limitation of our study was
using archive blocks but not fresh tissue samples. Working on archived
paraffin blocks is a well-established method for evaluating homogenous
patient groups.
Levels of synthesizing and catabolizing enzymes (DAGL and MAGL
respectively) of 2-AG were correlated with CB2 receptor immune
labeling in all groups. DAGL enzyme activities in glandular and stromal
cells increased with age in solid subgroup of endometriosis, suggesting
that 2-AG synthesis decreases with aging at disease. Immune labeling
for both enzymes of 2-AG decreased in glandular and stromal cells in
the experimental groups compared to that of the control group. Our
findings regarding synthesizing and catabolizing enzymes of 2-AG and
its receptor are original since their immune labeling pattern has not
been studied in the normal endometrium in comparison with endome-
triosis and adenomyosis until now. It is likely that 2-AG might be a
molecule playing an important role in the pathogenesis of endome-
triosis and adenomyosis.
CB2 receptor reactivity was higher in the proliferative phase than
the secretory phase in the control group. This finding was consistent
with the results of Taylor et al. (2010), while the enzyme reactivity
score was similar in both phases (Taylor et al., 2010). These findings
reveal that the enzymes do not regulate the effect of 2-AG through CB2
receptors in the modulation of the menstrual cycle and these receptors
do not mediate the effects of other endocannabinoids. We suggest 2-AG
activity might be taking an active role in endometrium progressing to
late phases of reproductive ages as we detected increased MAGL
enzyme activity in the proliferative phase on glandular cells with
increasing age. We suggest that the pathophysiological mechanism of
endometriosis could be different than adenomyosis. Aging may play
role on dysregulation of endocannabinoids via CB2 receptors, taking
into consideration the decreased CB2 receptor reactivity with increas-
ing age in adenomyosis.
Our data from TUNEL analysis was in line with the literature and
correlated with our immune labeling results. The proliferation capacity
of glandular epithelial and stromal cells of endometrium is higher in
endometriosis than normal endometrium (Agic et al., 2009; Nasu et al.,
2011; Sanchez et al., 2012).
The IC
50
value was confirmed as 9.3 ×10
−6
M for ACPA and
1.9 ×10
−4
M for CB 65 on Ishikawa cells. ACPA exhibited stronger
anti-proliferative effect on Ishikawa cells and, CB65 caused stronger
anti-proliferation on CRL-7566 respectively. Our study is the first to
examine the real time direct and dose dependent anti-proliferative
effect of cannabinoid agonists in both control and endometriotic cells.
We report CB65 exhibits stronger pro-apoptotic effect on Ishikawa cells
and, ACPA causes stronger pro-apoptosis on CRL-7566 respectively.
Truthfully, there is more than one cell death mechanism (Galluzzi et al.,
Fig. 6. (A–D) Real time cell proliferation curves and bar graphics are shown following application of ACPA (9.3 ×10
−6
M) and CB65 (1.9 ×10
−4
M) at IC50 concentrations on control
(Ishikawa) and endometriotic (CRL-7566) cells. Note that the cell proliferation indices of CRL-7566 (A and B) and Ishikawa (C and D) cell lines decreased through hours comparing to
untreated controls. All plots were generated using the RTCA Software 1.1.
E. Bilgic et al. $FWD+LVWRFKHPLFD[[[[[[[[[[²[[[


2012). The deviation of our results may be because of the different
apoptotic pathways, which play role on endometrial cell death.
Cannabinoid agonists and their receptors have been shown in endo-
metrial cancers (Ayakannu et al., 2013, 2015; Guida et al., 2010). AEA
and 2-AG synthesis and degradation pathways are known in cancer
angiogenesis and overall gene expression levels were reported for
endometrial carcinoma (Ayakannu et al., 2015). Although both syn-
thetic CB1 and CB2 agonists increased the apoptotic cell percentage
compared to control group and decreased the cell proliferation indexes
in our study, the molecular pathways of cannabinoid-dependent cell
mechanisms need to be searched. We suggest that cannabinoid agonists
can potentially inhibit endometriotic cell proliferation. Palmitoyletha-
nolamide was recently used to reduce chronic pelvic pain in endome-
triotic patients (Angioni, 2015). Based upon this finding, we suggest
that cannabinoid agonists can be assessed for their molecular mechan-
isms on endometriotic cell proliferation regression and apoptosis and,
can be a potential therapeutic agent.
In conclusion, endocannabinoids and their receptor distribution on
endometriotic and adenomyotic tissue samples were compared with
healthy endometrial tissue samples. We presented that expression of
endocannabinoid receptors and synthesizing and catabolizing enzymes
and apoptotic cell ratio decrease in endometriosis and adenomyosis,
compared to normal endometrium. Cannabinoid agonist presented anti-
proliferative and apoptotic effect on cell culture.
Conflict of interest
The authors listed above have no financial interest with any
company or organization in the subject matter or materials discussed
in this manuscript.
Acknowledgements
This study was supported by the Scientific and Technological
Research Council of Turkey (TUBITAK, # 112S217).
References
Agic, A., Djalali, S., Diedrich, K., Hornung, D., 2009. Apoptosis in endometriosis. Gynecol.
Obstet. Invest. 68 (4), 217–223.
Alger, B.E., Kim, J., 2011. Supply and demand for endocannabinoids. Trends Neurosci. 34
(6), 304–315.
Almada, M., Piscitelli, F., Fonseca, B.M., Di Marzo, V., Correia-da-Silva, G., Teixeira, N.,
2015. Anandamide and decidual remodelling: COX-2 oxidative metabolism as a key
regulator. Biochim. Biophys. Acta 1851 (11), 1473–1481.
Ayakannu, T., Taylor, A.H., Marczylo, T.H., Willets, J.M., Konje, J.C., 2013. The
endocannabinoid system and sex steroid hormone-dependent cancers. Int. J.
Endocrinol. 2013, 259676.
Ayakannu, T., Taylor, A.H., Willets, J.M., Konje, J.C., 2015. The evolving role of the
endocannabinoid system in gynaecological cancer. Hum. Reprod. Update 21 (4),
517–535.
Brighton, P.J., Marczylo, T.H., Rana, S., Konje, J.C., Willets, J.M., 2011. Characterization
of the endocannabinoid system, CB(1) receptor signalling and desensitization in
human myometrium. Br. J. Pharmacol. 164 (5), 1479–1494.
Cobellis, L., Castaldi, M.A., Giordano, V., Trabucco, E., De Franciscis, P., Torella, M.,
Colacurci, N., 2011. Effectiveness of the association micronized N-
Palmitoylethanolamine (PEA)-transpolydatin in the treatment of chronic pelvic pain
related to endometriosis after laparoscopic assessment: a pilot study. Eur. J. Obstet.
Gynecol. Reprod. Biol. 158 (1), 82–86.
Coskun, Z.M., Bolkent, S., 2014. Biochemical and immunohistochemical changes in delta-
9-tetrahydrocannabinol-treated type 2 diabetic rats. Acta Histochem. 116 (1),
112–116.
Dmitrieva, N., Nagabukuro, H., Resuehr, D., Zhang, G., McAllister, S.L., McGinty, K.A.,
Mackie, K., Berkley, K.J., 2010. Endocannabinoid involvement in endometriosis. Pain
151 (3), 703–710.
El-Talatini, M.R., Taylor, A.H., Elson, J.C., Brown, L., Davidson, A.C., Konje, J.C., 2009.
Localisation and function of the endocannabinoid system in the human ovary. PLoS
Fig. 7. From A to F are representative flow cytometry analysis of CRL-7566 and Ishikawa, cells that were labeled with Annexin-V/PI. The percentage of apoptotic cells is equal to Q
2
+Q
4
(early apoptotic and late apoptotic cells
)
areas. Both o cell lines were exhibited apoptosis with cannabinoid agonists. CB65’seffect was more prominent on Ishikawa and, ACPA’s was more
prominent on CRL-7566 cells. All the quadrants arranged according to the isotyping control values. All analyses were done using BD FACSDiva software v6.1.2 (Becton Dickinson
Biosciences, USA).
E. Bilgic et al. $FWD+LVWRFKHPLFD[[[[[[[[[[²[[[


One 4 (2), e4579.
El-Talatini, M.R., Taylor, A.H., Konje, J.C., 2010. The relationship between plasma levels
of the endocannabinoid, anandamide, sex steroids, and gonadotrophins during the
menstrual cycle. Fertil. Steril. 93 (6), 1989–1996.
Fonseca, B.M., Correia-da-Silva, G., Teixeira, N.A., 2009. Anandamide-induced cell death:
dual effects in primary rat decidual cell cultures. Placenta 30 (8), 686–692.
Fu, G., Che, X., Sun, Y., Huang, X., Xu, H., Zhou, C., Zhang, X., 2013. Pigment epithelial-
derived factor expression in endometriotic lesions in a rat model of endometriosis.
Acta Histochem. 115 (4), 301–307.
Galluzzi, L., Vitale, I., Abrams, J.M., Alnemri, E.S., Baehrecke, E.H., Blagosklonny, M.V.,
Dawson, T.M., Dawson, V.L., El-Deiry, W.S., Fulda, S., Gottlieb, E., Green, D.R.,
Hengartner, M.O., Kepp, O., Knight, R.A., Kumar, S., Lipton, S.A., Lu, X., Madeo, F.,
Malorni, W., Mehlen, P., Nunez, G., Peter, M.E., Piacentini, M., Rubinsztein, D.C., Shi,
Y., Simon, H.U., Vandenabeele, P., White, E., Yuan, J., Zhivotovsky, B., Melino, G.,
Kroemer, G., 2012. Molecular definitions of cell death subroutines: recommendations
of the Nomenclature Committee on Cell Death 2012. Cell Death Differ. 19 (1),
107–120.
Gao, X., Outley, J., Botteman, M., Spalding, J., Simon, J.A., Pashos, C.L., 2006. Economic
burden of endometriosis. Fertil. Steril. 86 (6), 1561–1572.
Gentilini, D., Besana, A., Vigano, P., Dalino, P., Vignali, M., Melandri, M., Busacca, M., Di
Blasio, A.M., 2010. Endocannabinoid system regulates migration of endometrial
stromal cells via cannabinoid receptor 1 through the activation of PI3K and ERK1/2
pathways. Fertil. Steril. 93 (8), 2588–2593.
Giugliano, E., Cagnazzo, E., Soave, I., Lo Monte, G., Wenger, J.M., Marci, R., 2013. The
adjuvant use of N-palmitoylethanolamine and transpolydatin in the treatment of
endometriotic pain. Eur. J. Obstet. Gynecol. Reprod. Biol. 168 (2), 209–213.
Gizzo, S., Patrelli, T.S., Dall’asta, A., Di Gangi, S., Giordano, G., Migliavacca, C., Monica,
M., Merisio, C., Nardelli, G.B., Quaranta, M., Noventa, M., Berretta, R., 2016.
Coexistence of adenomyosis and endometrioid endometrial cancer: role in surgical
guidance and prognosis estimation. Oncol. Lett. 11 (2), 1213–1219.
Guida, M., Ligresti, A., De Filippis, D., D'Amico, A., Petrosino, S., Cipriano, M., Bifulco, G.,
Simonetti, S., Orlando, P., Insabato, L., Nappi, C., Di Spiezio Sardo, A., Di Marzo, V.,
Iuvone, T., 2010. The levels of the endocannabinoid receptor CB2 and its ligand 2-
arachidonoylglycerol are elevated in endometrial carcinoma. Endocrinology 151 (3),
921–928.
Hey-Cunningham, A.J., Markham, R., Fraser, I.S., Berbic, M., 2013. Dysregulation of
vascular endothelial growth factors and their neuropilin receptors in the eutopic
endometrium of women with endometriosis. Reprod. Sci. 20 (11), 1382–1389.
Indraccolo, U., Barbieri, F., 2010. Effect of palmitoylethanolamide-polydatin combination
on chronic pelvic pain associated with endometriosis: preliminary observations. Eur.
J. Obstet. Gynecol. Reprod. Biol. 150 (1), 76–79.
Jeung, I., Cheon, K., Kim, M.R., 2016. Decreased cytotoxicity of peripheral and peritoneal
natural killer cell in endometriosis. Biomed. Res. Int. 2016, 2916070.
Karasu, T., Marczylo, T.H., Maccarrone, M., Konje, J.C., 2011. The role of sex steroid
hormones, cytokines and the endocannabinoid system in female fertility. Hum.
Reprod. Update 17 (3), 347–361.
Krawczyk, N., Banys-Paluchowski, M., Schmidt, D., Ulrich, U., Fehm, T., 2016.
Endometriosis-associated malignancy. Geburtshilfe Frauenheilkd 76 (2), 176–181.
Kruse, C., Seyer-Hansen, M., Forman, A., 2012. Diagnosis and treatment of rectovaginal
endometriosis: an overview. Acta Obstet. Gynecol. Scand. 91 (6), 648–657.
Leconte, M., Nicco, C., Ngo, C., Arkwright, S., Chereau, C., Guibourdenche, J., Weill, B.,
Chapron, C., Dousset, B., Batteux, F., 2010. Antiproliferative effects of cannabinoid
agonists on deep infiltrating endometriosis. Am. J. Pathol. 177 (6), 2963–2970.
Lin, K., Zhan, H., Ma, J., Xu, K., Wu, R., Zhou, C., Lin, J., 2014. Increased steroid receptor
RNA activator protein (SRAP) accompanied by decreased estrogen receptor-beta (ER-
beta) levels during the malignant transformation of endometriosis associated ovarian
clear cell carcinoma. Acta Histochem. 116 (5), 878–882.
Lo Monte, G., Soave, I., Marci, R., 2013. Administration of micronized
palmitoylethanolamide (PEA)-transpolydatin in the treatment of chronic pelvic pain
in women affected by endometriosis: preliminary results. Minerva Ginecol. 65 (4),
453–463.
Lowin, T., Zhu, W., Dettmer-Wilde, K., Straub, R.H., 2012. Cortisol-mediated adhesion of
synovial fibroblasts is dependent on the degradation of anandamide and activation of
the endocannabinoid system. Arthritis Rheum. 64 (12), 3867–3876.
Maccarrone, M., 2009. Endocannabinoids: friends and foes of reproduction. Prog. Lipid
Res. 48 (6), 344–354.
McHugh, D., Page, J., Dunn, E., Bradshaw, H.B., 2012. Delta(9) –tetrahydrocannabinol
and N-arachidonyl glycine are full agonists at GPR18 receptors and induce migration
in human endometrial HEC-1B cells. Br. J. Pharmacol. 165 (8), 2414–2424.
Mercati, F., Dall’Aglio, C., Pascucci, L., Boiti, C., Ceccarelli, P., 2012. Identification of
cannabinoid type 1 receptor in dog hair follicles. Acta Histochem. 114 (1), 68–71.
Muccioli, G.G., 2010. Endocannabinoid biosynthesis and inactivation, from simple to
complex. Drug Discov. Today 15 (11–12), 474–483.
Nasu, K., Nishida, M., Kawano, Y., Tsuno, A., Abe, W., Yuge, A., Takai, N., Narahara, H.,
2011. Aberrant expression of apoptosis-related molecules in endometriosis: a possible
mechanism underlying the pathogenesis of endometriosis. Reprod. Sci. 18 (3),
206–218.
Resuehr, D., Glore, D.R., Taylor, H.S., Bruner-Tran, K.L., Osteen, K.G., 2012.
Progesterone-dependent regulation of endometrial cannabinoid receptor type 1 (CB1-
R) expression is disrupted in women with endometriosis and in isolated stromal cells
exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Fertil. Steril. 98 (4),
948–956 (e941).
Sanchez, A.M., Vigano, P., Mugione, A., Panina-Bordignon, P., Candiani, M., 2012. The
molecular connections between the cannabinoid system and endometriosis. Mol.
Hum. Reprod. 18 (12), 563–571.
Sanchez, A.M., Cioffi, R., Vigano, P., Candiani, M., Verde, R., Piscitelli, F., Di Marzo, V.,
Garavaglia, E., Panina-Bordignon, P., 2016. Elevated systemic levels of
endocannabinoids and related mediators across the menstrual cycle in women with
endometriosis. Reprod. Sci. 23 (8), 1071–1079.
Sbracia, M., Valeri, C., Antonini, G., Biagiotti, G., Pacchiarotti, A., Pacchiarotti, A., 2016.
Fas and fas-ligand in eutopic and ectopic endometrium of women with endometriosis:
the possible immune privilege of ectopic endometrium. Reprod. Sci. 23 (1), 81–86.
Scotchie, J.G., Savaris, R.F., Martin, C.E., Young, S.L., 2015. Endocannabinoid regulation
in human endometrium across the menstrual cycle. Reprod. Sci. 22 (1), 113–123.
Shen, Z., Zhang, S., Zou, C.C., Gu, W.Z., Shang, S.Q., 2010. DNA microarray analysis of
the gene expression profile of kidney tissue in a type 2 diabetic rat model. Mol. Med.
Rep. 3 (6), 947–952.
Siegmund, S.V., Wojtalla, A., Schlosser, M., Schildberg, F.A., Knolle, P.A., Nusing, R.M.,
Zimmer, A., Strassburg, C.P., Singer, M.V., 2016. Cyclooxygenase-2 contributes to the
selective induction of cell death by the endocannabinoid 2-arachidonoyl glycerol in
hepatic stellate cells. Biochem. Biophys. Res. Commun. 470 (3), 678–684.
Sun, X., Wang, H., Okabe, M., Mackie, K., Kingsley, P.J., Marnett, L.J., Cravatt, B.F., Dey,
S.K., 2009. Genetic loss of Faah compromises male fertility in mice. Biol. Reprod. 80
(2), 235–242.
Sznurkowski, J.J., Emerich, J., 2008. Endometriomas are more frequent on the left side.
Acta Obstet. Gynecol. Scand. 87 (1), 104–106.
Tamm-Rosenstein, K., Simm, J., Suhorutshenko, M., Salumets, A., Metsis, M., 2013.
Changes in the transcriptome of the human endometrial Ishikawa cancer cell line
induced by estrogen, progesterone, tamoxifen, and mifepristone (RU486) as detected
by RNA-sequencing. PLoS One 8 (7), e68907.
Taylor, A.H., Abbas, M.S., Habiba, M.A., Konje, J.C., 2010. Histomorphometric evaluation
of cannabinoid receptor and anandamide modulating enzyme expression in the
human endometrium through the menstrual cycle. Histochem. Cell Biol. 133 (5),
557–565.
Vannuccini, S., Lazzeri, L., Orlandini, C., Tosti, C., Clifton, V.L., Petraglia, F., 2016.
Potential influence of in utero and early neonatal exposures on the later development
of endometriosis. Fertil. Steril. 105 (4), 997–1002.
Yamanaka, A., Kimura, F., Kishi, Y., Takahashi, K., Suginami, H., Shimizu, Y., Murakami,
T., 2014. Progesterone and synthetic progestin, dienogest, induce apoptosis of human
primary cultures of adenomyotic stromal cells. Eur. J. Obstet. Gynecol. Reprod. Biol.
179, 170–174.
Yang, Y., Zhang, X., Zhou, C., Huang, X., Lin, J., Xu, H., 2013. Elevated immunoreactivity
of RANTES and CCR1 correlate with the severity of stages and dysmenorrhea in
women with deep infiltrating endometriosis. Acta Histochem. 115 (5), 434–439.
Yazulla, S., 2008. Endocannabinoids in the retina: from marijuana to neuroprotection.
Prog. Retin. Eye Res. 27 (5), 501–526.
Yu, J., Zhao, L., Zhang, D., Zhai, D., Shen, W., Bai, L., Liu, Y., Cai, Z., Li, J., Yu, C., 2015.
The effects and possible mechanisms of puerarin to treat endometriosis model rats.
Evid. Based Complement. Altern. Med. 2015, 269138.
E. Bilgic et al. $FWD+LVWRFKHPLFD[[[[[[[[[[²[[[
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