Apoptosis is a process of programmed cell death by which the numbers of cells in a variety of tissues, including those in the human uterus, are physiologically regulated and the process differs from events related to necrosis.1 Microscopically, apoptosis is associated with cell shrinkage; the condensation of chromatin; and the formation of small, spherical, membrane-bound organelles, referred to as apoptotic bodies,1 and DNA fragmentation is evident.2
The human endometrium undergoes characteristic proliferative, secretory, and menstrual phases as a result of cycle-related changes in accordance with levels of steroid hormones. Apoptosis is found primarily in glandular cells during the secretory phase, whereas it is less frequent in proliferative endometrium,3,4 thereby suggesting an important role for apoptosis in the menstrual cycle. The protein encoded by the bcl-2 gene was detected using a monoclonal antibody and validated immunohistochemical techniques.5 This protein has been reported to play an important role as a cell death repressor.6,7 There is an inverse correlation of bcl-2 protein expression with the induction of apoptosis in the normal human uterine endometrium.4,8,9 There are reports on the induction of apoptosis in endometriotic lesions; however, there is no consensus on the mode of appearance of apoptosis, compared with the eutopic endometrium.10,11 Less is known about apoptosis and bcl-2 expression in the ectopic endometrium of adenomyotic lesions. A proliferative marker, Ki-67, is expressed only in the nucleus of cycling proliferative but not in resting cells.12 Expression of Ki-67 is comparatively greater in the functionalis than the basalis endometrium,13 and immunoreactivity of Ki-67 in endometrial stromal cells is weak during the proliferative phase but increased in secretory phase endometrium. There have been two reports on proliferative activity in endometriotic lesions; however, the results are conflicting. Li et al14 noted the higher proliferative activity in endometriotic lesions compared with eutopic endometrium, whereas Jones et al15 found a lower proliferative activity in these lesions. We find no reports on details of Ki-67 expression in adenomyotic lesions.
Our preliminary data on detection of apoptosis in adenomyotic lesions indicated that induction of apoptosis in these lesions is more frequent than in the corresponding eutopic endometrium, which suggests that the ectopic endometrium in adenomyotic lesions might have properties that differ from those in the eutopic endometrium. There are various opinions as to the origin of the endometrium found in adenomyotic lesions.16 Cullen17 suggested that it has its source in the endometrium lining of the uterine cavity, and it is now universally accepted that the endometrium of the uterine cavity penetrates interspaces of the uterine wall. On the other hand, von Recklinghausen (Von Recklinghausen F. Adenomyoma and cystadenomas of the wall of the uterus and tube: Their origin as remnants of the Wolffian body. Wien Klin Wochenschr 1896;8:530) proposed that the ectopic endometrium is the result of embryonic cell rests. The theory of origin of müllerian rests has been supported by investigators who suggested the possibility that the histopathogenesis of adenomyotic lesions is related to metaplasia of müllerian remnants.18,19
The present study was performed to determine whether the adenomyotic lesion has its origin in the endometrial lining of the uterine cavity, especially in the basal layer. For this purpose, the dynamics of induction of apoptosis, bcl-2 gene expression, and Ki-67 expression were examined in adenomyotic lesions and the findings were compared with those in the corresponding eutopic endometrium.
Materials and Methods
Human endometrial and myometrial tissues were obtained from 23 Japanese patients, 28–51 years of age (mean 42.9 ± 5.4 years), all with regular menstrual cycles. All patients underwent abdominal hysterectomy between January 1992 and December 1993 at Saga Medical School Hospital because of dysmenorrhea, hypermenorrhea, or lower abdominal pain. Preoperative diagnoses were myoma uteri in 13, myoma uteri with ovarian tumor in two, endometriosis in five, and adenomyosis in three. Histopathology confirmed adenomyotic lesions in all patients. Eight patients had adenomyosis, four had a concomitant pelvic endometriosis, 10 were complicated with myoma uteri, and two were complicated with ovarian mucinous cystadenoma. Patients with an irregular menstrual cycle and those who received hormonal therapy, including hormonally based contraception, were not included in this study. The endometria, dated according to the method of Noyes et al,20 were separated into five phases as follows: the menstrual phase (n = 3), the early proliferative phase (n = 8), the late proliferative phase (n = 3), the early secretory phase (days 15–21; n = 3), and the mid- to late secretory phase (days 22–28; n = 6), with day 1 being the first day of the last menstrual period.
DNA fragmentation associated with apoptosis was detected using a modified terminal deoxynucleotidyl-transferase-biotin nick end-labeling (TUNEL) method,21 for all 23 cases. The 4-μm–thick sections from paraffin-embedded blocks were deparaffinized, immersed in 0.3% H2O2 to block endogenous peroxidase, and then treated with 20 μg/mL proteinase K for 15 minutes and incubated at 37C for 1 hour in a solution including terminal deoxynucleotidyltransferase (TdT) and digoxigenin-labeled deoxyuridinetriphosphate. After a wash, they were treated with a peroxidase-labeled anti-digoxigenin antibody for 30 minutes and stained with 3,3′-diaminobenzidine containing 0.01% H2O2, as the chromogen. The slides were counterstained with hematoxylin. A normal thymus of a fetus at 20 weeks' gestation served as the positive control, and omission of the enzyme TdT as the negative control.
Serial sections were deparaffinized followed by immunostaining with bcl-2 antibody (bcl-2 124; Dako Ltd, Glostrup, Denmark) or with Ki-67 monoclonal antibody (Dakopatts, Copenhagen, Denmark); the avidinbiotin peroxidase complex method was used. We compared bcl-2 immunoreactivity and the Ki-67 labeling index in eutopic and ectopic endometria. For negative control the primary antibody was replaced by phosphate-buffered saline.
The quantitation of apoptosis, bcl-2 expression, and Ki-67 expression was carried out in a blind fashion by two independent observers. Each observer counted at least 1000 cells in more than 10 randomly chosen fields per case, and the number of positive cells per 1000 cells was designated as the apoptotic index on TUNEL-stained sections. The extent of the staining in the case of bcl-2 expression was scored as follows: strongly positive, 3; definite positive, 2; weakly positive, 1; and negative, 0. The Ki-67 labeling index was determined by counting at least 1000 cells in more than ten different areas of each section and was expressed in percentage. Interobserver agreement was 82%, 85%, and 91% for the analysis of apoptosis, bcl-2 expression, and Ki-67 expression, respectively.
For statistical analysis, Kruskal-Wallis test was used for multiple comparisons among all groups, and Wilcoxon's paired test was used for comparison of each pair of groups. A level of P < .05 was considered to have statistical significance.
To assess hormonal responsiveness of the ectopic endometrium in adenomyotic lesions, endometrial dating was performed for both eutopic and ectopic endometrium, in the same specimen. Comparative results are shown in Table 1. Various phases of the menstrual cycle were evident in the normal endometrium, whereas only the proliferative phases, with or without hemorrhagic degeneration, were seen in most cases of the ectopic endometrium, although only partial secretory change was seen in one third of cases at the secretory phase. This means that the ectopic endometrium in adenomyosis is less sensitive to the cyclic change of endogenous sex steroids, especially to progesterone.
Using the TUNEL method, the induction of apoptotic cells was examined in eutopic and ectopic tissues obtained from the 23 patients with adenomyosis. In Table 2, the mean apoptotic labeling indices were compared by phases of the menstrual cycle. In the eutopic endometrium, apoptotic cells were most frequently detected in the mid- to late secretory phase, especially in glandular cells (Figure 1A) and were also frequently detected in the menstrual phase. The rate of detection of apoptotic cells was low in the early proliferative phase and was very low during the late proliferative phase through early secretory phases (Figure 1B). Quantitative analysis revealed a significant increase in the apoptotic index in epithelial cells during mid- to late secretory phases (6.42 ± 4.09) compared with findings during the early proliferative (0.83 ± 0.85) (P < .01), late proliferative (0.00) (P < .01), and early secretory (0.33 ± 0.58) (P < .01) phases. A similar tendency was noted in stromal cells; however, the difference was not significant (Table 2).
In the ectopic endometrium of the adenomyotic lesions, apoptotic cells were induced in all the menstrual phases, and measured apoptotic indices showed no cyclic change, either in glandular or in stromal cells (Table 2, Figure 1C). Significantly higher apoptotic indices were noted in stromal cells of the ectopic endometrium in menstrual (37.40 ± 4.47) (P < .05) and proliferative (22.39 ± 8.84) (P < .01) phases compared with findings in the functional layer of the corresponding eutopic endometrium (Table 3). The apoptotic index in stromal cells of the ectopic endometrium during the secretory phase (23.86 ± 19.63) was also significantly higher than that in the basal layer (6.16 ± 7.15) (P < .01) and was higher than that in the functional layer (7.70 ± 7.48), although the difference was not significant. These findings strongly suggest that biologic properties of the ectopic endometrium in adenomyotic lesions differ from findings in the eutopic endometrium, in both functional and basal layers.
The most intensive staining of bcl-2 gene expression was observed in the proliferative phase of the eutopic endometrium. The staining was also positive in the early secretory phase and was very weak during mid-to late secretory and menstrual phases (Table 2). This observation inversely correlates with the induction of apoptosis. In contrast, no cyclic change of bcl-2 expression was observed in the ectopic endometrium (Table 2).
Ki-67–positive cells were observed in 5.82% of epithelial cells of the functional layer of the eutopic endometrium during the proliferative phase, and the number was significantly higher than that in the secretory phase (1.21%, P < .01) and that in the menstrual phase (1.02%, P < .01), respectively (Table 4, Figure 2A and B). On the other hand, Ki-67 expression was constantly high in epithelial cells of the ectopic endometrium, irrespective of the phases of the menstrual cycle (Figure 2C). Labeling indices in the ectopic endometrium during secretory and menstrual phases were significantly higher than those in the eutopic endometrium of the functional layer (P < .01 and P < .05, respectively) (Table 4). The labeling index in adenomyotic lesions during the secretory phase was also significantly higher than that in the eutopic glandular epithelium of the basal layer (P < .01) (Table 4). Proliferative activity in stromal cells of the ectopic endometrium during the secretory phase was higher than that in the basal layer of the eutopic endometrium; however, the difference was not significant (Table 4).
In 1975, Hopwood and Levison22 were the first to report on apoptosis in the human endometrium, and they identified characteristic morphological changes of apoptosis in glandular epithelial cells during the menstrual cycle. There are now several reports on apoptosis during menstrual cycles.8–10,23,24 Gompel et al8 investigated bcl-2 expression in the endometrium throughout the menstrual cycle and found that bcl-2 staining predominated in glandular cells, peaked at the end of the follicular phase, and disappeared at the onset of secretory activity. Otsuki et al9 also found cyclic change of bcl-2 expression in the endometrium during the menstrual cycle, and they suggested that this change was associated with distribution of estrogen and progesterone receptors. All the other reports are in good agreement with these studies. Nevertheless, the role of apoptosis remains unknown.
In the present study, we found cyclic change of induction of apoptotic cells and the inverse correlation of bcl-2 expression in normal human endometrium during the menstrual cycle, consistent with data in previous reports.10,23,24 We also noted no cyclic change in apoptosis and bcl-2 expression in the ectopic endometrium in adenomyosis. We found only one report that referred to apoptosis and bcl-2 expression in adenomyotic lesions. Suganuma et al11 analyzed 12 adenomyotic lesions and stated that apoptotic cells were rarely found in adenomyotic lesions or in the eutopic endometrium, findings incompatible with previous observations and with our data. They reported that bcl-2 staining was positive in all adenomyotic tissues in the proliferative phase but in none of those in the secretory phase. This would suggest that bcl-2 expression in the adenomyotic endometrium shows a cyclic change, which is also incompatible with our observations.
In the analysis of proliferative activity, Ki-67 expression was high in the proliferative phase of the functional eutopic endometrium but was low in the secretory and menstrual phases. A similar result was found in the secretory phase of the basal eutopic endometrium. Li et al14 examined the eutopic endometrium and endometriotic lesions when immunostaining for proliferating cell nuclear antigen and noted that staining in the glandular epithelium reached a maximum in the proliferative phase and decreased in the secretory phase in both functional and basal layers of the eutopic endometrium, which is consistent with our observation. We found that Ki-67 expression in adenomyotic lesions was constantly high, irrespective of aspects of the menstrual cycle, findings differing from the eutopic endometrium in the basal layer. We observed that induction of apoptosis was remarkably high in stromal cells in adenomyotic lesions, compared with findings in the basal layer of the eutopic endometrium. The difference would reflect the constant proliferation of the ectopic endometrium among the narrow spaces of the myometrium. This mechanism can be explained by Moulton's observation that increasing the density of stromal cell cultures increases levels of apoptosis.25
On the basis of findings of apoptosis, bcl-2 expression, and Ki-67 staining in adenomyotic lesions, we conclude that adenomyotic lesions do have a constant proliferative activity that is rarely influenced by endogeneous sex steroids, especially by progesterone. As to the pathogenesis of adenomyosis, the direct extension theory was proposed by Cullen,17 and has been widely accepted. However, evidence that the adenomyotic endometrium has different biologic or proliferative activities from the eutopic endometrium in the basal layer does support the metaplasia theory of müllerian remnants, as emphasized by Dougherty and Anderson18 and recently by Nisolle and Donnez for the pathogenesis of adenomyotic lesions.19 If one explains this evidence according to the direct extension theory, it would be essential to presuppose that only a selected cell population in the basal layer that is highly proliferative and less responsive to progesterone invades diffusely and deeply into the myometrium.
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