1 Institute for the Study and Treatment of Endometriosis, Oak Brook, IL 60523, 2 Rush Medical College, Chicago, IL 60612 and 3 Department of Pathology, Elmhurst Hospital, Elmhurst, IL, 60126, USA
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Abstract |
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Key words: apoptosis/endometrial glands/endometriosis/stroma
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Introduction |
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We have demonstrated previously that in women with endometriosis, proliferation of the endometrial cells in an in-vitro co-culture system is stimulated by autologous peripheral blood monocytes (Braun et al., 1994). In women without endometriosis, in the same coculture system, peripheral blood monocytes suppress proliferation of the autologous endometrial cells. This differential effect of monocytes on endometrial cells depends on the presence or absence of endometriosis, and can also be duplicated with the supernatant from the monocyte/macrophage culture, or with tumour necrosis factor alpha (TNF
), which is one of the major cytokines produced by the monocytes/macrophages (Braun and Dmowski, 1998b
; 1999
). Interesting in this respect are reports indicating that in some in-vitro cell culture systems, inflammatory cytokines, including TNF
, can stimulate through the sphingomyelin pathway, either inflammation and cell proliferation or programmed cell death (apoptosis), depending on specific conditions (Pena et al., 1997
).
Apoptosis is a fundamental physiological process responsible for maintaining homeostasis in multicellular organisms (Stellar, 1995). The orderly progression of events during apoptosis results in cell death without the leakage of protease enzymes and cellular contents from dying cells, thereby reducing the likelihood of an inflammatory response (Wyllie et al., 1980
). Accumulating evidence suggests that apoptosis is directly involved in the regulation of the menstrual cycle, through elimination of senescent cells from the functional layer of the uterine endometrium during the late-secretory and menstrual phases (Hopwood and Levison, 1976
; Tabibzadeh et al., 1994
; Kokawa et al., 1996
; Shikone et al., 1996
). This is followed by proliferation of new cells from the basal layer during the proliferative phase of the following cycle.
Recent studies from our laboratories using a cell death detection ELISA assay (Dmowski et al., 1998; Gebel et al., 1998
) demonstrated that endometrial apoptosis in the eutopic endometrium is lower in women with endometriosis than controls and is further decreased in the ectopic endometrium. However, the design of these studies did not allow identification of the apoptotic cells and the pattern of apoptosis was not studied during different phases of the cycle. The objective of the present study was to evaluate further spontaneous apoptosis in the uterine endometrium of women with and without endometriosis using a TUNEL assay and specifically: (i) to determine spontaneous apoptosis during different phases of the menstrual cycle, (ii) to determine the location of the apoptotic cells in endometrial glands and stroma, and (iii) to evaluate the degree of spontaneous apoptosis according to the stage of the disease.
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Materials and methods |
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Identification of endometrial phases and apoptosis analysis
In the pathology laboratory, endometrial specimens were dehydrated and embedded in paraffin. Two cases of normal spleen, thymus, bowel, and embryonic kidney were selected as positive controls of apoptosis. For the purpose of this study, paraffin blocks were retrieved, sectioned (5 µm), and mounted on positive charged microscopic glass slides. The slides were then coded and sent to the Pathology laboratory at another institution for a blind analysis. One set of slides was stained with haematoxylin-eosin and examined microscopically for the endometrial phase of the cycle. Endometrium was classified as early-proliferative (EP), mid-proliferative (MP), late-proliferative (LP), early-secretory (ES), mid-secretory (MS), late-secretory (LS), and menstrual (M) according to its histological appearance (Noyes et al., 1950). The distribution of the samples according to the endometrial phases and corresponding days of the menstrual cycle is presented in Table I
.
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The following criteria for apoptotic cells were applied in this study: TUNEL positive stained nucleus with nuclear morphological features of an apoptotic cell, i.e. shrinkage of the nucleus with condensed chromatin and/or densely aggregated marginal chromatin or dot-like or drop-like condensed nuclear fragments (Figure 1). TUNEL stained swollen nuclei were considered as degenerated necrotic cells and were excluded from the apoptotic cell population. Quantitative analysis of the apoptotic cells was performed with a cytometer under x400 magnification using Olympus (model BX50) microscope equipped with super-wide eyepieces. The functional layer of the endometrium in the entire tissue section was counted for the apoptotic cells. That area varied from 10 to 50 mm2 (corresponding to x1575 400 fields) depending on the size of the tissue sample. The numbers of apoptotic cells in the endometrial glandular epithelium and stroma were counted separately. The apoptotic index was defined as the number of apoptotic cells per 10 mm2 unit area.
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Results |
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Discussion |
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Endometrial biopsies in our patients were obtained from the functional endometrial layer, which is primarily under the hormonal control and where cyclic events take place. All specimens in both patients and controls were carefully dated histologically and all comparisons were made according to the cycle phase. In controls, we observed a similar pattern of apoptosis during the menstrual cycle as previously reported (Tabibzadeh et al., 1994; Tabibzadeh, 1995
; Kokawa et al., 1996
; von Rango, et al., 1998
; Dahmoun, et al., 1999
; Vaskivuo, et al., 2000
). The absolute numbers of apoptotic cells in our study were lower when compared with Kokawa et al. (Kokawa et al., 1996
), who did not use morphological criteria, but similar to those of (Dahmoun et al., 1999
), who did. A recent study (Vaskivuo et al., 2000
) correlated the degree of apoptosis as determined by 3'-end labelling, DNA fragmentation analysis and expression of apoptosis-related proteins Bcl-2 and Bax with cyclic changes in serum oestradiol and progesterone concentrations. Endometrial apoptosis was negatively correlated with serum oestradiol concentrations and was most pronounced during oestradiol and progesterone withdrawal in agreement with the prior data (Koh et al., 1995
; Pecci et al., 1997
). Altogether, these studies indicate that during the late secretory phase of the cycle, functional endometrium undergoes extensive apoptosis, the peak of which appears to be associated with endometrial shedding during menstruation. A decline in oestradiol and progesterone concentrations at the end of the cycle is associated with prolonged and intense vasoconstriction of the coiled arterioles leading to endometrial ischaemia and necrosis, which coincide with the peak of apoptosis. Early during menstruation, both apoptotic and necrotic cells can be identified in the endometrial glands and stroma of the functional layer (Dahmoun et al., 1999
). This suggests that ovarian steroid-controlled endometrial cell necrosis and apoptosis may be the mechanisms involved in the regulation of the endometrial cyclicity and menstruation.
Endometrium shed during menses is typically expelled with the menstrual flow. However, endometrial cells and tissue fragments can be identified during menses in the lumen of the Fallopian tubes and in the peritoneal cavity (Ridley, 1968; Koninckx et al., 1980
; Halme et al., 1984
; Bartosik et al., 1986
; Kruitwagen et al., 1991a
). This phenomenon seems to occur with equal frequency in women with and without endometriosis (Koninckx et al., 1980
; Halme et al., 1984
; Bartosik et al., 1986
, Kruitwagen et al., 1991a
). Clinical observations and in-vitro studies further suggest that in women with endometriosis misplaced endometrial cells implant in ectopic locations giving origin to endometriotic lesions (Evers and Willebrand, 1987
; Kruitwagen et al., 1991b
; Evers, 1996
; Koks et al., 2000
), while in healthy women such implantation does not take place. The factor(s) which protect(s) healthy women from the ectopic implantation of misplaced endometrial cells and tissue fragments has been puzzling to us as well as to other investigators.
Our recent studies demonstrated that spontaneous apoptosis when measured using a cell death detection ELISA assay was significantly reduced in the uterine endometrium of women with endometriosis as compared to normal controls (Dmowski et al., 1998; Gebel et al., 1998
). These results are in agreement with the present study and suggest that in healthy women, endometrial cells and tissue fragments expelled during menses, do not survive in ectopic locations because of programmed cell death, while decreased apoptosis may lead to the ectopic survival and implantation of these cells and development of endometriosis. When paired samples of eutopic and ectopic endometria were compared, the level of apoptosis was significantly lower in the ectopic samples suggesting ectopic preselection of apoptosis-resistant cells (Gebel et al., 1998
). In the present study, we did not evaluate apoptosis in the ectopic endometrium. Apoptosis of the endometrial cells appears to be under the control of endometrial monocytes/macrophages and their secretory products. Immune cell-, and especially monocyte/macrophage-derived cytokines control proliferation versus apoptosis in the eutopic endometrial cells and may also do the same in the ectopic cells, determining thereby development of endometriosis versus normal health (Braun et al., 1994
; Tabibzadeh, et al., 1994
; Braun and Dmowski, 1998b
; Braun et al., 1999).
The hypothesis that decreased apoptosis in the endometrial or immune cells of the reproductive system may contribute to the pathogenesis of endometriosis has been considered and studied by several investigators with mixed results (Harada et al., 1996; McLaren et al., 1997
; Suganuma et al., 1997
; Dmowski et al., 1998
; Gebel et al., 1998
; Jones et al., 1998
; Matsumoto et al., 1999
; Meresman et al., 2000
). In agreement with this report, apoptotic cells were observed in adenomyotic and ovarian endometriotic tissues, without apparent cyclic pattern and without correlation between the intensity of apoptosis and the phase of the menstrual cycle (Harada et al., 1996
; Suganuma et al., 1997
; Matsumoto et al., 1999
). The assays used in these studies were semiquantitative and there was no comparative evaluation of normal healthy controls. Jones et al., who also used a semiquantitative TUNEL assay, reported only rare apoptotic stromal or epithelial cells without apparent difference between normal, eutopic, ectopic, or adenomyotic endometrium (Jones et al., 1998
). These authors, `only rarely' identified apoptosis in the normal endometrium and it is unclear how carefully did they match endometrial phases between patients and controls. Only one recent study compared the frequency of endometrial apoptosis in normal controls and women with endometriosis according to the phase of the cycle (Meresman et al., 2000
). The authors used a similar patient population as in our study, had a similar study design, and evaluated apoptosis with the TUNEL assay. Although the number of subjects was smaller than in our study, and only two endometrial phases were compared, the results were similar.
It has been suggested that ovarian steroids may control endometrial apoptosis by up and down regulation of Bcl-2 and Bax expression (Rotello et al., 1992; Koh et al., 1995
; Tabibzadeh, 1995
). Meresman et al. noted increased Bcl-2 and absent Bax expression in the late proliferative eutopic as compared to normal endometrium (Meresman et al., 2000
). In the late secretory eutopic endometrium there was a significant decrease in Bax expression. Decreased apoptosis was found in Bcl-2 immunopositive and Bax-immunonegative tissues. The authors suggested that in women with endometriosis increased Bcl-2 and decreased Bax expression are the anti-apoptotic factors. However, McLaren et al. reported essentially similar patterns of Bcl-2 and Bax expression in the glandular cells of normal, eutopic, and ectopic endometrium during the proliferative and secretory phases (McLaren et al., 1997
). These authors also reported an increased percentage of Bcl-2+ macrophages in the peritoneal fluid from women with endometriosis, and concluded that Bcl-2+ macrophages may predispose endometriotic cells to resist apoptosis. Interestingly, Jones et al. noted a significant Bcl-2 expression in the endometrial stroma in normal and eutopic endometrium with further increase during the late secretory phase (Jones et al., 1998
). Using double labelling, these authors demonstrated that most Bcl-2+ cells were leukocytes. The ectopic stroma contained significantly higher numbers of Bcl-2+ cells than eutopic, only some of which were leukocytic. Altogether, these reports indicate that endometrial apoptosis in both eutopic and ectopic endometrium in women with endometriosis may be quantitatively different than in normal healthy women and that abnormal expression of apoptosis controlling proteins Bcl-2 and Bax in the endometrial cells, as well as leukocytes, may play a role in this phenomenon.
In the present study, the apoptotic index was significantly lower in women with endometriosis than in controls. The difference was caused primarily by a significant decrease in apoptosis during the late secretory/menstrual and early proliferative phases. Interestingly, the decrease in the apoptotic index between endometriosis and controls was much higher in the glandular epithelium than in the stroma, indicating that these two cell types may not contribute equally to the subsequent development of the disease. The above seems to be consistent with the histological appearance of endometriotic lesions.
If the decrease in programmed cell death facilitates ectopic survival and implantation of the endometrial cells, one might expect an inverse relationship between the level of apoptosis and the severity of the disease. To test this hypothesis, we analysed our data according to the stage of endometriosis. Disappointingly, we were unable to demonstrate statistically significant differences in apoptosis between different stages, although the trend was apparent. It is quite likely that apoptosis is only one of the mechanisms that control development and progression of endometriosis. Growth autonomy of endometriotic cells and immune inflammatory reaction within the peritoneal cavity are some of the other factors involved (Braun and Dmowski, 1998a). Local and systemic immune response may lead to spontaneous resorption of old endometriotic lesions, while retrograde dissemination to the development of new ones. The balance between these two events may determine the progression or spontaneous regression of the disease and may explain the existence of microscopic endometriosis. Future studies should correlate the stage of endometriosis with the intensity of eutopic and ectopic endometrial apoptosis, taking into consideration the local and systemic immune response, as well as the effect of the cycle and serum hormone concentrations.
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Notes |
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References |
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Submitted on December 29, 2000; accepted on June 7, 2001.