Introduction
Endometriosis is a common gynaecological disorder, frequently associated with infertility and pelvic pain that occurs almost exclusively in menstruating women of reproductive age. Although little is known about its aetiology and pathogenesis, the theory of retrograde menstruation and metaplasia of the mesothelium or peritoneum and implantation of viable endometrial cells has been widely accepted [
1,
2]. The morphology of eutopic endometrium from women with endometriosis is similar to normal endometrium, but its physiology and biochemistry are different. Recent reports show an abnormal survival capability at the epithelial and stromal levels of the eutopic endometrium from patients with endometriosis that may result in their continuous growth [
3,
4]
In proliferative phase, the endometrium is characterized by proliferation of cells from the basal layer that respond to 17β-estradiol, reaching its maximum at ovulation time and then, falling due to the effects of progesterone with marked changes in glandular epithelium and stroma. Besides steroids, other factors are involved in these processes. In fact, after the priming of estradiol, the expression of transforming growth factor beta1 (TGF-β1) is up regulated in human endometrium coincidently with the increase of plasma progesterone concentration, being down regulated during progesterone withdrawal. Mashburn et al. (1994) [
5] reported that the highest concentration of this cytokine in the stroma was during the secretory phase and suggested that it may regulate epithelial cell proliferation and differentiation (for revision see Godkin and Dore, 1998) [
6]. In epithelial cell lines, TGF-β plays a central role in maintaining the homeostasis through limitation of growth by repression of growth-promoting transcription factors, such as c-Myc [
7]. As known, c-Myc is a transcription regulator that can both activate or inhibit gene expression in favour of cell proliferation.
It is well established that apoptosis is a temporary physiologic process by which tissues eliminate dysfunctional cells by a regulated mechanism that involves a sequence of intracellular molecular events including members of the Bcl-2 family, such as Bax, an inducer of apoptosis. In human endometrium, Bax is expressed differentially throughout the menstrual cycle, with an expression mainly in late phase according to the number of apoptotic cells detected at the end of the menstrual cycle [
8,
9].
The aim of the present study was to evaluate whether the balance between cell proliferation and apoptosis is changed in eutopic endometrium from women with endometriosis throughout the menstrual cycle. For this purpose, we evaluated: cell proliferation by the detection of Ki67 and c-myc mRNA abundance, apoptosis by DNA fragmentation detection and bax mRNA abundance, and the expression of TGF-β1 in eutopic endometrium obtained from women with and without endometriosis.
Discussion
It is known that eutopic endometrium from women with endometriosis presents physiological differences with normal endometrium. In this study we show that eutopic endometrium from these patients has a significant increase in cell survival, and alterations on DNA fragmentation and on the expression of factors involved in cell proliferation, cell differentiation and programmed cell death induction, such as Ki67, c-myc, TGF-β1 and bax.
In agreement with previous reports and in accordance with results of light microscopy [
10,
14,
15], our data show that epithelial cells from normal endometrium exhibit a higher proliferation rate than stromal cells during proliferative and early secretory phases, stages of the menstrual cycle regulated mainly by estradiol. Although eutopic endometrium from patients with endometriosis present a similar Ki67 distribution throughout the menstrual cycle, a significant augment of cells into cell cycle compared to normal endometrium was observed, in accordance with other investigators [
16‐
18]. Even though Ki67 is expressed in the cell during M, G1, S and G2 phases of cell cycle and is absent in resting cells (Go), a good correlation between Ki67 and mitotic indices has been reported in human endometrium [
19]. In contrast, other groups [
4,
20] have reported no differences between normal and eutopic endometria from women with endometriosis, probably due to the use of different techniques and the detection of proliferating cell nuclear antigen (PCNA), a nuclear protein restricted to S phase.
Several genes involved in cell proliferation, such as the proto-oncogene
c-myc are up-regulated by estradiol in different cell types that include human breast cancer cell line MCF-7 [
21,
22], rat hepatocyte [
23], and pituitary and somatolactotrophic cell line GH3 [
24]. Little information is available of the proto-oncogen
c-myc in the endometrium. The presence of the protein c-Myc in nuclei and cytoplasm of endometrial glands and nuclei stroma from eutopic and ectopic endometria has been reported [
25], even though its role in the endometrium is not clear. In the present investigation, the
c-myc mRNA abundance was not modified throughout the menstrual cycle in both types of endometria studied, although it was augmented in eutopic endometrium from women with endometriosis compared to normal women. Several findings suggest that in endometriosis, ectopic endometrium grows and regresses in an estrogen-dependent manner. Even more, eutopic endometrium of women with endometriosis contains not only estradiol receptors, but also P
450Arom, the enzyme that catalyzes the conversion of androgen to estrogen, provoking an estrogenic micro-environment in this tissue that may act through intracrine pathways [
26‐
28]. Therefore, the estrogenic microenvironment could induce the expression of the transcription factor
c-myc and facilitates cell proliferation in the eutopic endometria in this disease. This effect may be improved by the reduction of TGF-β1 detected in the endometrium of these patients, as probable consequence of the loss of the negative regulation on P
450AROM described in choriocarcinoma cell line (JEG-3) [
29].
On the other hand, TGF-βs are likely candidates to partially mediate the effects of progesterone on the expression of other proteins, which have been suggested to be involved in the regulation of endometrial cell proliferation and differentiation [
5,
6]. In addition, the cyclic expression of TGF-β1, mainly observed in stromal cells during the secretory phase as reported in this study, is in accord to progesterone receptor location [
30]. In several cell types, TGF-β1 plays a role in maintaining tissue homeostasis by modulating genes involved in the arresting of cell cycle including down-regulation of c-Myc [
7,
31]. Even more, in rabbit uterine epithelial cells, a coordinated but inverse regulation of cell proliferation and apoptosis by this cytokine was described [
32]. The fact that the cyclicity of TGF-β1 observed in normal endometrium was absent in eutopic endometrium from women with endometriosis indicates an impaired local production in these patients that may favors the cell proliferation. More studies are required to understand the complex and strict interactions between these molecules involved in the deregulation on cell proliferation in the eutopic endometrium of these women.
Proliferation and apoptosis are important biologic processes in the endometrium remodeling with cell cycle changes that take place during the menstrual cycle [
14,
33]. In the late secretory phase, and related with a decrease of endometrial progesterone receptor and plasma progesterone concentration, cell death by apoptosis increases in the functional endometrial layer, and at the time of menstruation it becomes necrotic and hypoxic and is shed. In the present investigation, the expected increase of apoptotic cells during late secretory phase was not observed in eutopic endometrium from women with endometriosis. Concomitantly to the absence of apoptosis in those endometria, the pro-apoptotic gene
bax mRNA abundance was unchanged during the menstrual cycle, at difference at the increase exhibited in normal endometrium, suggesting that the reduction of bax expression may be a mechanism that could explain the decreased incidence of apoptosis in endometriosis. In agreement with these results, recent publications [
3,
34,
35] also reported the absence of apoptotic cells and the increase of Bcl-2 and the reduction of Bax expression in eutopic endometrium from women with endometriosis, although Béliard et al. (2004) [
4] were unable to find these differences between patients with endometriosis and normal women. Curiously, we detected that both
bax mRNA abundance and apoptotic cells were increased in early secretory phase in eutopic endometrium from women with endometriosis but not from normal women. The implication of this augmented cell death in eutopic endometrium from the patients needs more studies.
It is well known that menstrual fragments are composed of both necrotic and living cells, which do not survive in ectopic locations because of its programmed cell death [
36,
37]. The loss of cell death by apoptosis in late secretory phase in these patients seems to be consistent with cell ectopic survival and implantation of endometrial cells in the peritoneal cavity, effect that is independent of the disease stages as it was reported by Dmowski et al. (2001) [
34]. The altered expression of
c-myc, TGF-β1 and
bax observed in eutopic endometrium from women with endometriosis, suggests their participation in the deregulation of the cell survival in this tissue. It is known that the deregulation of apoptotic signaling can play a primary or secondary role in various diseases; in fact, an insufficient apoptosis degree contributes to pathogenic processes including cancer. The present investigation shows that cell viability is augmented in eutopic endometrium from women with endometriosis compared to normal endometrium, primarily due to a reduction in cell death by apoptosis, and also, an increase in cell proliferation. These data strongly suggest that this condition may facilitate the invasive character of the endometrium in these patients. Until now, it is not clear why misplaced endometrial cells from healthy women do not implant and do not develop into endometriotic lesions as occurs in women that develop endometriosis, indicating that some factor(s) which facilitate(s) their survival and implantation may be involved. However, we cannot rule out the possible presence of endometrium cells that belong to basal layer, aspect not studied in the present work.