doi:10.1093/humrep/del493
Human Reproduction Vol.22, No.4 pp. 945–952, 2007
Advance Access publication January 5, 2007
Altered apoptosis and proliferation in endometrial
stromal cells of women with adenomyosis
Jehn-Hsiahn Yang, Ming-Yih Wu, Chin-Der Chen, Mei-Jou Chen, Yu-Shih Yang
and Hong-Nerng Ho1
Department of Obstetrics and Gynecology, National Taiwan University Hospital and National Taiwan University College of Medicine,
Taipei, Taiwan
1
To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, National Taiwan University Hospital,
7 Chung-Shan South Road, 100, Taipei, Taiwan. Tel: þ 886 2 2312 3456 ext. 5161; Fax: þ886 2 2351 7459; E-mail: [email protected].
ntu.edu.tw
BACKGROUND: The eutopic endometrium in a woman suffering from adenomyosis is known to be biologically
different from that of healthy women. The aim of this study was to examine the apoptosis and proliferation of
eutopic endometrium from women with adenomyosis. METHODS: We enrolled 23 women with adenomyosis
(study group) and 21 without (control group). Eutopic endometrium was obtained and separated into single endometrial stromal cells (ESCs). ESCs were treated in vitro with hydrogen peroxide (H2O2) to examine their apoptosis
using a fluorescence-activated cell sorter. Cells were also treated with estradiol (E2), medroxyprogesterone acetate,
interleukin (IL)-6, lipopolysaccharide and interferon-g (IFN-g) to test their proliferation using a non-radioactive
cell proliferation assay. RESULTS: The percentage of annexin V (1)/7-amino-actinomycin D (1) ESCs was much
lower in women with adenomyosis after 24 h culture with and without H2O2 treatment when compared with the
control group. ESCs of adenomyosis proliferated more rapidly than those of the control group, whether they were
cultured alone or were treated with E2, MPA, IL-6 or IFN-g. The immunocytochemical Ki-67 labelling index
was much more prominent in adenomyotic ESCs than that of the control group (7.7% versus 1.1%, P < 0.001).
CONCLUSIONS: Altered apoptosis and proliferation of eutopic endometrium possibly elucidate some aspects of
the pathophysiology of adenomyosis. A high Ki-67 labelling index in immunocytochemistry might be a potential
indicator in predicting the occurrence of adenomyosis.
Key words: adenomyosis/apoptosis/endometrial stromal cell/Ki-67/proliferation
Introduction
Adenomyosis is defined as the presence of ectopic endometrial
glands and stroma within the myometrium. The pathogenic
mechanism responsible for adenomyosis is not well known as
yet. Our previous study revealed that the expression of killer
cell immunoglobulin-like receptors on natural killer (NK)
cells was decreased in eutopic endometrium in women with adenomyosis (Yang et al., 2004). It may be a compensatory effect
in which the NK cytotoxicity is activated in order to eradicate
the abnormal endometrial cells that might exit the eutopic site
of endometrium. This finding suggested that abnormal endometrial cells, but not aberrant immunological phenomenon,
account for the development of adenomyosis. Our following
studies demonstrated that interleukin (IL)-6 was abnormally
produced by endometrial stromal cells (ESCs) in women
with adenomyosis, which was either overexpressive after in
vitro co-culture with macrophage (Yang et al., 2006a) or resistant to the suppressive effect of medroxyprogesterone acetate
(MPA) and danazol (Yang et al., 2006b). These studies
strengthen the view that the eutopic endometrium of a woman
with adenomyosis is biologically different from that of a
healthy woman.
Apoptosis is a process of programmed cell death that is
involved in a diverse group of processes including reproduction
(Kokawa et al., 1996). Accumulated evidence suggests that
apoptosis is directly involved in the regulation of the menstrual
cycle through eliminating senescent cells from the functional
layer of the uterine endometrium during the late-secretory
and menstrual phases (Hopwood and Levison, 1976). In endometriosis, which is pathophysiologically similar to adenomyosis, reports demonstrated impaired apoptosis (Gebel et al.,
1998; Dmowski et al., 2001) and accelerated proliferation
(Meresman et al., 2002) of the eutopic endometrium. The
resistance to apoptosis of endometrial cells and their increased
sensitivity to proliferation suggest the possible pathogenesis of
endometriosis.
There have been few studies investigating the apoptosis
and proliferation of the eutopic endometrium of adenomyosis.
# The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
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J.-H.Yang et al.
An immunohistochemical study found rare apoptosis in eutopic
endometrium from women with adenomyosis as well as from the
healthy controls (Jones et al., 1998). This report demonstrated
that the B cell lymphoma/leukemia-2 (bcl-2) expression in
stromal cells, which provided resistance to apoptosis, was less
prominent in the ectopic endometrium of adenomyosis than the
paired eutopic endometrium. Currently, there are no studies
comparing the apoptosis and cell proliferation of eutopic endometrium between women with and without adenomyosis.
Fluorescence-activated cell sorter (FACS) unlike immunohistochemistry, is useful in assessing changes in the intensity
of expression of surface markers and, therefore, can be used
to quantify their up- or down-regulation. There has been a
study comparing immunohistochemistry and FACS results in
gastric carcinoma using a monoclonal antibody against the
carcinoembryonic antigen. Comparable results were achieved
with these two methods. However, quantitative evaluation of
the immunoreactive cell populations was available only with
the use of FACS (Tamai et al., 1993). In addition, FACS
facilitates the evaluation of larger numbers of cells than
tissue sections in small specimens. Studies have proven the
efficacy of FACS in the apoptotic analysis of endometrial
cells (Stackievicz et al., 2001; Wang et al., 2005).
In this study, ESCs were obtained from women with and
without adenomyosis. We investigated the apoptosis of ESCs
with FACS and determined the proliferation of ESCs using
non-radioactive assay kits and immunocytochemistry, in an
attempt to find possible deviations in apoptosis and proliferation in the eutopic endometrium of adenomyosis.
Materials and methods
Subjects and specimens
This study consisted of 23 women who suffered from adenomyosis (study group) and 21 women in whom cervical intraepithelial
neoplasia or leiomyoma was found (control group). These women
underwent hysterectomy, either via abdominal or vaginal route, at
our hospital. All the participating women were at premenopausal
status. As apoptosis and cell proliferation might vary in different
phases of the menstrual cycle (Kokawa et al., 1996; Jones et al.,
1998; Matsumoto et al., 1999), only women in the early- to midsecretory phase were included in this study. The histology of the
early- to mid-secretory phase endometrium was characterized by tortuous glands, subnuclear vacuoles in the glandular epithelium, prominent glandular secretion, stromal edema and inconspicuous spiral
arteries. The diagnosis of adenomyosis was made by histopathologic
examination when there is ingrowth of endometrium below the endometrial –myometrial interface more than 2.5 mm. Informed consent
was obtained from each woman before surgery, and this study protocol
was approved by the institutional review board at our hospital.
Purification of ESCs
Endometrium was obtained immediately after the uterus was removed
from the women, and was placed in an ice-cold 1 : 1 mixture of
Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F-12 for
transport to the laboratory. The tissue was gently dissected into
small pieces (1–2 mm3) and washed by centrifugation (400 g) in
a fresh medium to remove any debris or excess blood cells. The
tissue was then incubated for 2 h at 378C in a shaking water bath in
946
DMEM/F-12 containing 0.5% collagenase and 0.02% DNase. The
dispersed cells were filtered through a 70-mm nylon mesh to remove
the undigested tissue pieces containing the glandular epithelium.
The filtered fraction was separated further from epithelial cell
clumps by differential sedimentation at unit gravity, as follows. The
cells were resuspended in 2 ml of culture medium and layered
slowly over 10 ml of the medium in a centrifuge tube. Sealed tubes
were placed in an upright position at 378C in air with 5% CO2 for
30 min. After sedimentation, the top 8 ml of medium was collected.
The medium containing stromal cells was filtered through a 40-mm
nylon mesh. Final purification was achieved by allowing stromal
cells to adhere selectively to culture dishes for 30 min at 378C in
5% CO2 in air. Non-adhering epithelial cells were removed. Cell
purity was assessed by immunocytochemistry using antibodies
against cytokeratin (Dako Corp., Carpenteria, CA, USA), vimentin
(Dako) and Factor VIII (Dako). The purity of the stromal cell preparations used in this study was more than 98%.
Fluorescence-activated cell sorter
The ESCs were divided into three portions. The first portion was
processed immediately for FACS analysis. The second portion was
cultured for 24 h in T25 flasks containing DMEM/F-12 supplemented
with 100 IU ml21 penicillin G, 50 mg ml21 of streptomycin and
2.5 mg ml21 of amphotericin B at 378C in air with 5% CO2. The third
portion was also cultured for 24 h as was done in the second portion,
except that 200 mM of hydrogen peroxide (H2O2) was added to the
media. The second and third portions were processed for FACS analysis
after culture.
The method of FACS analysis has been described in detail previously
(Yang et al., 2000, 2004). Briefly, after washing twice with cold
phosphate-buffered saline (PBS), the ESCs were resuspended in 1
binding buffer at a concentration of 1106 cells ml21. About 100 ml
of ESCs were incubated with 5 ml of phycoerythrin (PE)-conjugated
annexin V (BD Biosciences, San Jose, CA, USA) and 5 mL of 7-amino-actinomycin D (7-AAD, BD Biosciences) for 15 min at room
temperature (RT) in the dark. After that, 400 ml of 1binding buffer
was added to the ESCs.
For staining with caspase-3, ESCs were resuspended in 0.5 ml of
Cytofix/Cytoperm (BD Biosciences) solution for 20 min. After being
washed twice with Perm/Wash buffer (BD Biosciences), the ESCs
were incubated with PE-conjugated anti-caspase-3 (BD Biosciences)
for 30 min in the dark at RT. For staining with bcl-2, ESCs were
treated with 0.5 ml of lysing solution for 10 min, and were cultured
with 0.5 ml of permeabilizing solution for 10 min. The ESCs were
washed with 0.5% bovine serum albumin (BSA) in 1 PBS and 0.1%
sodium azide, and then incubated with 20 ml of PE-conjugated
anti-bcl-2 (BD Biosciences) for 30 min in the dark at RT.
FACS analysis was done within 1 h using a FACScan cytofluorimeter (BD) with computer interface to software (Hewlett-Packard
Consort 32, BD) for full-list mode data storage, recovery and analysis.
Samples of ESCs were subjected to FACS, and background noise in
the clinical samples was excluded by establishing a window in
which only the signals derived from ESCs appeared. Electrical compensation was performed to eliminate the interference of fluorescence
during simultaneous two-colour measurement. The compensation procedure was performed before each measurement. In each cell suspension, a minimum of 30 000 events acquired for gated ESCs were
measured (Figure 1).
Proliferation assay
ESC proliferation was determined by CellTiter 96 AQueous NonRadioactive Cell Proliferation Assay kit (Promega, Madison, WI,
USA). This kit is a colorimetric assay for determining the number of
Altered apoptosis and proliferation in adenomyosis
Figure 1. (A) This shows the 2D histogram of forward scatter (FSC, x-axis) and side scatter (SSC, y-axis) on linear scales. The endometrial
stromal cells (ESCs) were located in the round area, and the signals of cell debris, red blood cells and lymphocytes were seen in the lower
left region. We made a window setting for ESCs in this round area and measured the fluorescence intensity of the signal in this area. (B) In
the annexin V assay, viable ESCs are annexin V and 7-amino-actinomycin D (7-AAD) negative. ESCs that are in early apoptosis are annexin
V positive and 7-AAD negative, and those in late apoptosis or already dead are both annexin V and 7-AAD positive. After ESCs were permeabilized, fixed and stained with phycoerythrin-conjugated monoclonal antibodies, cells positive for bcl-2 (C) and caspase-3 (D) were detected.
viable cells as a surrogate measure of proliferation. It is composed of solutions of a novel tetrazolium compound [3-(4,5-dimethylthiazol2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium,
inner salt (MTS)] and an electron coupling reagent phenazine methosulfate (PMS). MTS is bioreduced by cells to a formazan product
that is soluble in tissue culture medium. The absorbance of formazan
at 490 nm can be measured directly in 96-well assay plates without
additional processing. The conversion of MTS into aqueous, soluble
formazan was accomplished by dehydrogenase enzymes found in
metabolically active cells. The quantity of formazan product, as
measured by the amount of 490 nm absorbance, was directly proportional to the number of living cells in culture. Experiments have
demonstrated a high correlation between this assay and [3H]thymidine
incorporation assay (Bounous et al., 1992; Wagner et al., 1999). Its
advantages over the [3H]thymidine assay are that it does not deal with
radioactivity, it is less costly and not very labour intensive (Ahmed
et al., 1994).
ESCs (2 104 per well) were seeded in 96-well culture plates. The
first column of each 96-well plate did not contain any cells and was
used as a blank. The remaining columns were ESCs without treatment,
followed by ESCs treated with estradiol (E2) 1028 M, E2 1027 M,
MPA 1028 M, MPA 1027 M, E2 1028 M þ MPA 1027 M, E2
1027 M þ MPA 1027 M, IL-6 10 ng ml21, IL-6 50 ng ml21,
lipopolysaccharide (LPS) 10 ng ml21 and interferon-g (IFN-g)
100 ng ml21. Cell proliferation was evaluated after 48 h of treatment
when 20 ml of MTS/PMS solution was added to all wells, and plates
were incubated at 378C for 4 h. At the end of the incubation period,
plates were read with an enzyme-linked immunosorbent assay plate
reader (SpectraMax, Molecular Devices, Menlo Park, CA, USA).
Data were expressed in optical density (OD) units. Experiments
were conducted in triplicate.
Immunocytochemistry for Ki-67
ESCs were plated in four-well chamber slides (Nunc, Naperville, IL,
USA). After 24 h of incubation, the medium was removed and cells
were fixed for 5 min with 4% paraformaldehyde in PBS. After
washing with PBS, cells were permeabilized with 0.1% Triton
X-100 in PBS for 10 min at RT. ESCs were blocked by incubating
at RT in 1% BSA in PBS for 1 h, followed by 3% H2O2 for 5 min.
Cells were then incubated with a 1 : 50 dilution of mouse monoclonal
anti-human Ki-67 antibody (BD, Franklin Lakes, NJ, USA) as a
primary antibody for 40 min at RT. Next, cells were washed in PBS,
incubated with biotinylated mouse anti-human antibody (BD) for
40 min, and washed again in PBS. Cells were then incubated with
3,30 diaminobenzidine chromogen (Dako) for 10 min, and were
947
J.-H.Yang et al.
washed again in PBS. Finally, cells were counterstained with hematoxylin and slides were mounted with water-soluble mounting
medium. Experiments with the omission of primary antibodies were
used as negative controls.
Ki-67 protein is a nuclear and nucleolar protein that is strictly
associated with cell proliferation. The Ki-67 labelling index was calculated manually by counting the number of Ki-67 positive cells per
1000 total cells by two trained observers blinded to the experiment.
Two consecutive, randomly selected 100 microscopic fields were
selected for each specimen processed.
Statistical analysis
All values are expressed as mean + SE. The one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was employed to
compare between treated and untreated ESCs, and between the study
and control groups in cell proliferation. The independent samples’
t-test was used to compare the difference between the study and
control groups in cell apoptosis and immunocytochemistry. A
P-value ,0.05 was considered statistically significant.
Results
The mean age was 44.4 + 0.8 years for women with adenomyosis and 44.6 + 1.2 years for those without adenomyosis.
The body mass indices were similar between the two groups
(mean, 24.8 versus 23.6). All the participating women were
in the early- to mid-secretory phase which was confirmed by
histopathologic examination.
ESC apoptosis
In Figure 1B, viable ESCs are negative for both annexin V and
7-AAD. ESCs in early apoptosis are annexin V positive and
7-AAD negative, and those in late apoptosis or already dead
are positive for both annexin V and 7-AAD. In ESC suspensions tested on the day of specimen collection (day 0), both
the percentages of annexin V (þ)/7-AAD (þ) and annexin
V (þ)/7-AAD (2) were similar between women with and
without adenomyosis. After 24 h culture, however, the percentage of annexin V (þ)/7-AAD (þ) ESCs was much lower in
women with adenomyosis than in those without (4.9 + 1.2%
versus 14 + 3.5%, respectively, P ¼ 0.021), but the percentages of annexin V (þ)/7-AAD (2) ESCs remained similar
between the two groups. In ESCs treated with H2O2 for 24 h,
the percentages of annexin V (þ)/7-AAD (þ) ESCs increased
even further. These percentages were also lower in women with
adenomyosis than in women without (8 + 2.3% versus
20.5 + 4.6%, respectively, P ¼ 0.019) (Figure 2).
The percentages of ESCs positive for bcl-2 staining were
similar between women with and without adenomyosis on
day 0 (9 + 2.7% versus 6.6 + 1.2%) as well as after 24 h
culture with H2O2 treatment (7.6 + 2.7% versus 5.6 + 0.9%)
or without H2O2 treatment (7.5 + 2.5% versus 5.2 + 1.2%).
The percentages of ESCs positive for caspase-3 staining were
also not different between these two groups (Figure 3).
ESC proliferation
With regard to the effects of various agents on ESC proliferation, the OD units were not different (P . 0.98 by ANOVA)
between ESCs cultured alone and ESCs treated with various
948
Figure 2. The annexin V assay in ESCs of women with adenomyosis
(black bars, n ¼ 23) and without adenomyosis (white bars, n ¼ 21).
(A) Both the percentages of annexin V (þ)/7-AAD (þ) and
annexin V (þ)/7-AAD (2) ESCs were similar between the two
groups on day 0. (B) After 24 h culture either with or without hydrogen peroxide (H2O2), the percentage of annexin V (þ)/7-AAD (þ)
ESCs was much lower in women with adenomyosis than in those
without adenomyosis. *P ¼ 0.021 by independent-samples’ t-test.
**P ¼ 0.019 by independent-samples’ t-test.
agents including E2, MPA, E2 þ MPA, IL-6, LPS and IFN-g,
either in women with or without adenomyosis. However, the
OD unit of ESCs cultured alone for 48 h in women with adenomyosis was significantly higher than that in the control group
(0.42 + 0.05 versus 0.24 + 0.03 respectively, P , 0.05).
After treatment with E2 1028 M (P , 0.05), E2 1027 M (P ,
0.01), MPA 1028 M (P , 0.05), MPA 1027 M (P , 0.05), E2
1028 M þ MPA 1027 M (P , 0.05), E2 1027 M þ MPA
1027 M (P , 0.05), IL-6 10 ng ml21 (P , 0.05), IL-6
50 ng ml21 (P , 0.05) and IFN-g 100 ng ml21 (P , 0.05)
for 48 h, the OD units were also much higher in women with
adenomyosis than those in women without (Figure 4).
Ki-67 labelling index of ESCs
The immunocytochemical staining of ESCs was shown in
Figure 5. A high density of immunopositive nuclei is a
feature of proliferating ESCs. Ki-67 positive cells were
observed in 7.7 + 1.2% of ESCs in women with adenomyosis,
Altered apoptosis and proliferation in adenomyosis
Figure 4. ESC proliferation determined by CellTiter 96 AQueous
Non-Radioactive Cell Proliferation Assay kit. The optic density
units were much higher in the ESCs of adenomyosis (black bars,
n ¼ 23) than in those of the control group (white bars, n ¼ 21),
whether ESCs were cultured alone or were treated with estradiol
(E2), medroxyprogesterone acetate (MPA), E2 þ MPA, interleukin
(IL)-6 or interferon-g. *P , 0.05 by one-way ANOVA followed by
Tukey’s post hoc test. **P , 0.01 by one-way ANOVA followed
by Tukey’s post hoc test.
Figure 3. The expression of bcl-2 (A) and caspase-3 (B) in ESCs of
women with adenomyosis (black bars, n ¼ 23) and without adenomyosis (white bars, n ¼ 21). All the percentages of bcl-2 (þ) and
caspase-3 (þ) ESCs were similar between the two groups on day 0,
or after 24 h in culture either with or without H2O2.
which was significantly higher than that in the control group
(1.1 + 0.2%, P , 0.001) (Figure 6).
Discussion
There have been several reports describing the cyclic apoptosis
of endometrial cells in the regulation of menstrual cycles
(Kokawa et al., 1996; Tao et al., 1997). A decreased sensitivity
of endometrial cells to apoptosis was found in the ectopic
locations (Beliard et al., 2004; Nishida et al., 2005, Izawa
et al., 2006) as well as in the eutopic endometrium (Dmowski
et al., 1998; Gebel et al., 1998) of endometriosis when compared with the fertile controls. For the first time, the present
study demonstrates reduced apoptosis and increased proliferation of the eutopic endometrium in women with adenomyosis.
Our results revealed that ESCs of adenomyosis were resistant to late apoptosis (both annexin V-PE and 7-AAD positive)
after 24 h incubation when compared with the control group.
This differential susceptibility of ESCs to apoptosis between
women with and without adenomyosis was also found when
ESCs were treated with H2O2. H2O2 is an apoptosis inducing
agent that injures various kinds of cells by both apoptosis
and necrosis (Wagner et al., 2000; Nakamura et al., 2006).
Our findings suggest that the apoptosis-resistant ESCs might
escape from programmed cell death and lead to implantation
in the ectopic sites and the development of adenomyosis.
Bcl-2 is a regulator of the apoptotic process. Elevated levels
of bcl-2 can provide resistance to cell death (DiGiuseppe et al.,
1996; Reed 1997). Bcl-2 has been considered to inhibit apoptosis in the human endometrium during the proliferative
phase (Otsuki et al., 1994). Meresman et al. (2000) reported
an increased expression of bcl-2 protein in the proliferative
eutopic endometrium from women with endometriosis when
compared with the normal controls. In this study, however,
Figure 5. Immunocytochemical staining of Ki-67 in ESCs from
women without adenomyosis (A) and with adenomyosis (B).
Paraformaldehyde-fixed ESCs were incubated with a 1:50 dilution
of mouse monoclonal anti-human Ki-67 antibody. ESCs were then
incubated with 3,30 diaminobenzidine chromogen and counterstained
by hematoxylin. A high density of immunopositive nuclei is the
feature of proliferating ESCs. Magnification, 100.
949
J.-H.Yang et al.
Figure 6. Ki-67 labelling index in women with adenomyosis (†,
n ¼ 23) and without (W, n ¼ 21) adenomyosis. This index was calculated by counting the number of Ki-67 positive cells per 1000 total
ESCs. Two consecutive, randomly selected 100 microscopic fields
were selected for each specimen processed. The mean value of the
Ki-67 labelling index was 7.7% in women with adenomyosis and
1.1% in women without adenomyosis. The P-value was ,0.001 by
independent-samples’ t test.
the bcl-2 expression on ESCs was not different between women
with and without adenomyosis. This result implies that the
decreased apoptosis of ESCs in adenomyosis is regulated by
mediators other than bcl-2. It might also be due to the fact
that we studied endometrium in the early- to mid-secretory
phase, whereas Meresman et al. (2000) investigated endometrium in the proliferative phase.
Caspase-3 is a key protease that is activated during the early
stage of apoptosis. In this study, we did not demonstrate different expression of caspase-3 in ESCs between women with and
without adenomyosis. The reason may be that the membrane
phospholipid phosphatidylserine externalization, which has a
high affinity for annexin V, may not be directly mediated by
caspase-3 (Johnson et al., 2000). It is compatible with a previous report that caspase-independent pathways of apoptosis
are also involved in the programmed cell death (Bouscary
et al., 2000).
Proliferative disorders (hyperplasia and cancer) of the
human endometrium have been linked to hyperestrogenic
states and to unopposed estrogen replacement therapy (Rose,
1996). In this study, there was a trend that the ESCs treated
with E2 proliferated more rapidly than the untreated ESCs
did, and the proliferation was limited after the addition of
MPA to E2. This is compatible with previous reports that E2
augments endometrial cell proliferation, but MPA arrests
their growth (Harada et al., 2004; Blauer et al., 2005).
IL-6 has been reported to promote endometrial cell proliferation (Khan et al., 2005). IL-6 achieves its effects through the
ligand-specific IL-6 receptor. IL-6 receptor has been found to
950
be expressed on ESCs during in vitro culture (Zarmakoupis
et al., 1995), and that the expression level of IL-6 receptor
did not differ between the proliferative and the secretory
phases (Yoshioka et al., 1999). However, our results did not
demonstrate increased endometrial cell proliferation after
stimulation with IL-6.
LPS was reported to promote (Iba et al., 2004), and IFN-g to
suppress (Clayton et al., 2004; Nishida et al., 2005) endometrial cell proliferation. The action of LPS on ESCs was
mediated through toll-like receptor 4 (Hirata et al., 2005),
and that of IFN-g on ESCs was through IFN-g receptor 1
(Nishida et al., 2005). However, we did not achieve similar
results, in which neither the ESCs with adenomyosis nor
those of the control group proliferated differently after treatment with LPS and IFN-g when compared with untreated
ESCs. Our method of purifying ESCs from uterine endometrium is consistent with what has been used previously
(Osteen et al., 1989) and yields cells of very good purity,
.98% based on vimentin, cytokeratin and Factor VIII staining.
However, the isolation procedure that relies on a combination
of enzymatic digestion, unit gravity sedimentation, filtration
and adherence is not likely to remove macrophages from the
ESC cultures. As we attempted to stimulate ESCs with
various agents including those that are expected to activate
macrophages (e.g. LPS, IFN-g, E2), but did not find any
effect of LPS or IFN-g on proliferation of ESCs, this would
suggest that our ESC cultures are not contaminated with
macrophages, which is in contrast to those seen by others
who have suggested stimulation of proliferation by LPS (Iba
et al., 2004) and suppression by IFN-g (Clayton et al., 2004;
Nishida et al., 2005). In addition, previous reports studied
ESCs from women with endometriosis, whereas we investigated ESCs from women with adenomyosis. ESCs between
these two disease entities might respond differently to the cytokine stimulation. Furthermore, cells that proliferated prominently after the stimulation with LPS are ESCs (Iba et al.,
2004) which might be biologically different from the eutopic
ESCs in this study.
Using immunocytochemistry, we demonstrated a higher
Ki-67 labelling index in ESCs with adenomyosis than in the
control group. Ki-67 protein is a nuclear and nucleolar
protein that is strictly associated with cell proliferation
(Verheijen et al., 1989). A detailed cell cycle analysis revealed
that the Ki-67 antigen was present in the nuclei of cells in the
G1, S and G2 phases of the cell division cycle as well as in
mitosis. Quiescent or resting cells in the G0 phase did not
express the Ki-67 antigen (Gerdes et al., 1984).
The identification of an individual indicator that predicts the
occurrence of adenomyosis is still several years away. Our
results revealed a mean immunocytochemical Ki-67 index of
7.7% in adenomyosis, whereas the fraction of Ki-67-positive
cells in the control group is generally ,2%. Although it is
unclear whether the Ki-67 protein expression remains constant
before and after the development of adenomyosis, the much
higher Ki-67 protein expression of ESCs in adenomyosis might
be a potential indicator of adenomyosis. Further studies are
required to establish which values of Ki-67 labelling index
might be used to predict the occurrence of adenomyosis.
Altered apoptosis and proliferation in adenomyosis
In this study, we used uterine samples from patients with cervical intraepithelial neoplasia and leiomyoma as controls.
Although it is certainly true that they do not have adenomyosis,
they would not be considered normal in the strictest sense of
the word. Cervical intraepithelial neoplasia, in particular, is a
dysplastic condition that precedes an invasive neoplastic
process in which substantial field cancerization can be present
in the surrounding tissues. The expression of bcl-2 in affected
tissues would be predicted to be higher when compared with
truly normal cells.
In conclusion, we report for the first time the differential
expression of ESC apoptosis and proliferation between
women with and without adenomyosis. The altered apoptosis
and proliferation of the eutopic endometrium possibly reveal
some aspects of the pathophysiology of adenomyosis. A high
Ki-67 labelling index in immunocytochemistry might be a
potential indicator in predicting the occurrence of adenomyosis.
Acknowledgements
This study was supported by grant NSC93-2314-B002-159 from the
National Science Council of the Republic of China and the research
grants NTUH95-S357 and NTUH96-S573 from the National Taiwan
University Hospital.
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Submitted on August 13, 2006; resubmitted on October 19, 2006; resubmitted
on November 27, 2006; accepted on December 1, 2006