Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma

Adult stem cells are thought to be responsible for the high regenerative capacity of the human endometrium, and have been implicated in the pathology of endometriosis and endometrial carcinoma. The RNA-binding protein Musashi-1 is associated with maintenance and asymmetric cell division of neural and epithelial progenitor cells. We investigated expression and localization of Musashi-1 in endometrial, endometriotic and endometrial carcinoma tissue specimens of 46 patients. qPCR revealed significantly increased Musashi-1 mRNA expression in the endometrium compared to the myometrium. Musashi-1 protein expression presented as nuclear or cytoplasmic immunohistochemical staining of single cells in endometrial glands, and of single cells and cell groups in the endometrial stroma. Immunofluorescence microscopy revealed colocalization of Musashi-1 with its molecular target Notch-1 and telomerase. In proliferative endometrium, the proportion of Musashi-1-positive cells in the basalis layer was significantly increased 1.5-fold in the stroma, and three-fold in endometrial glands compared to the functionalis. The number of Musashi-1 expressing cell groups was significantly increased (four-fold) in proliferative compared to secretory endometrium. Musashi-1 expressing stromal cell and cell group numbers were significantly increased (five-fold) in both endometriotic and endometrial carcinoma tissue compared to secretory endometrium. A weak to moderate, diffuse cytoplasmic glandular staining was observed in 50% of the endometriosis cases and in 75% of the endometrioid carcinomas compared to complete absence in normal endometrial samples. Our results emphasize the role of Musashi-1-expressing endometrial progenitor cells in proliferating endometrium, endometriosis and endometrioid uterine carcinoma, and support the concept of a stem cell origin of endometriosis and endometrial carcinoma.     

子宫内膜异位症在位内膜腺上皮细胞及基质细胞的分离培养和鉴定

目的分离培养子宫内膜异位症在位内膜组织中子宫内膜腺上皮细胞及基质细胞,建立研究子宫内膜异位症的细胞模型。方法对分泌期子宫内膜异位症在位内膜组织用混合酶消化,滤网过滤和差速梯度离心的方法分离,体外培养后通过光镜观察及免疫细胞化学、免疫荧光化学方法对分离细胞鉴定。结果分离的细胞角质蛋白阳性子宫内膜腺细胞百分率约90～95%;分离的骨架蛋白形成蛋白阳性的基质细胞百分率达90%以上。结论本研究获得较高纯度的子宫内膜腺细胞和基质细胞,成功建立了研究子宫内膜异位症的细胞模型。     

子宮内膜异位症在位内膜腺上皮细胞及基质细胞的分离培养和鉴定

目的 分离培养子宫内膜异位症在位内膜组织中子宫内膜腺上皮细胞及基质细胞,建立研究子宫内膜异位症的细胞模型.方法 对分泌期子宫内膜异位症在位内膜组织用混合酶消化,滤网过滤和差速梯度离心的方法分离,体外培养后通过光镜观察及免疫细胞化学、免疫荧光化学方法对分离细胞鉴定.结果 分离的细胞角质蛋白阳性子宫内膜腺细胞百分率约90～95%;分离的骨架蛋白形成蛋白阳性的基质细胞百分率达90%以上.结论 本研究获得较高纯度的子宫内膜腺细胞和基质细胞,成功建立了研究子宫内膜异位症的细胞模型.     

Ovarian steroid receptor expression in endometriosis and in two potential parent epithelia: endometrium and peritoneal mesothelium.

Endometriosis is an oestrogen dependent condition and it is expected that the tissue of origin of endometriosis will express receptors for the ovarian steroids. Two epithelia, endometrium and peritoneal mesothelium, are the potential parent epithelium. Oestrogen and progesterone receptor expression has been studied immunohistochemically in (i) timed endometrial biopsies from 25 normal subjects and 27 patients with endometriosis, (ii) 25 endometriotic biopsies and (iii) 42 peritoneal biopsies. Endometrium but not peritoneal mesothelium expresses both oestrogen and progesterone receptors. No difference in the intensity of staining between endometria of normal subjects compared with the endometria of patients with endometriosis was noted. In paired endometrial and endometriotic biopsies, the intensity of staining for the oestrogen receptor in stromal cells and for the progesterone receptor in both glandular and stromal cells was less in the endometriotic biopsies. These data provide circumstantial evidence for an endometrial origin for endometriosis although quantitative differences exist in receptor expression between endometrium and endometriosis.     

Mismatch repair system in endometriotic tissue and eutopic endometrium of unaffected women

Objective: To test the immunohistochemical staining pattern of some mismatch repair (MMR) system proteins in endometriotic tissue (ET) and eutopic endometrium. Methods: This was a retrospective study conducted at the Pathology and Obstetrics and Gynecology Departments of the Udine University Hospital. We analyzed 528 samples obtained from 246 patients affected by endometriosis and 71 samples from 71 patients with normal endometrium. A tissue microarray model was used to analyze the immunohistochemical expression of MMR system proteins. Results: Significant loss of MMR proteins was found in the stromal component of ETs. We found MSH2 to be expressed at a higher level than any other MMR system proteins in eutopic endometrium and ETs, to be significantly correlated to Ki-67 expression in both stromal and glandular components of ETs, and to be expressed at a significantly higher level in ETs than in eutopic endometrium. When considering the subgroup of endometriosis with high recurrence rate and glandular cytoplasmic staining for aurora A kinase, we found MMR proteins expressed at a significantly higher level in these ETs than in other ETs and eutopic endometrium of unaffected women. Conclusions: We found significant loss of MMR proteins (known to be associated with microsatellite instability) in the stromal component of ETs. The group of ETs with glandular cytoplasmic staining for aurora A kinase had higher MMR protein expression, suggesting an increased activity of this system. Our result suggests a novel role of increased MSH2 expression in cellular proliferation of endometriosis.     

Immunohistochemical localization of androgen receptor in the human endometrium, decidua, placenta and pathological conditions of the endometrium.

The immunohistochemical localization of the androgen receptor in the human endometrium at various stages of the menstrual cycle and post-menopausal period, in decidua and placenta of early pregnancy, and in several pathological conditions of the endometrium has been investigated. At any phase of the menstrual cycle, both endometrial glandular cells and endometrial stromal cells showed positive nuclear staining. Endometrial stromal cells of the functional layer showed stronger staining than those of the basal layer, but endometrial glandular cells of both layers showed the same staining intensity. There was little staining in myometrium. Even after menopause, endometrial glandular and stromal cells showed the same staining pattern as the basal layer of pre-menopausal endometrium and the staining intensity of endometrial stromal cells was weak. In decidua and placenta of early pregnancy, decidual and trophoblastic cells showed positive staining and there was no staining in the stromal cells of placenta. The expression of the androgen receptor was also detected in adenomyosis, endometriosis and endometrial carcinoma. Although the proliferation and differentiation of endometrium are mediated mainly by oestrogen and progesterone receptors, the androgen receptor may play some role in modulating these changes. These results suggest that it may be involved in both physiological and pathological changes of the endometrium.     

Epidermal growth factor in human endometrium: proliferative effects in culture and immunocytochemical localization in normal and endometriotic tissues.

Endometrial proliferation is stimulated by oestradiol. The precise mechanism is poorly understood, but may be mediated by epidermal growth factor (EGF). The aim of the present study was to assess the effects of oestradiol and EGF on glandular and stromal proliferation in human endometrial cell cultures, and to determine the localization of EGF-like immunoreactivity (EGF-IR) using immunocytochemistry in normal and endometriotic tissue. Endometrium was obtained from women undergoing curettage or hysterectomy for benign disease, or laparoscopy for endometriosis. For tissue culture experiments, enriched glandular and stromal cells were prepared by digestion with collagenase and DNAase, and cultured for 4 days with oestradiol or EGF, both alone and in combination. Immunocytochemical studies were performed using sheep primary antibody against EGF, with binding visualized using the unlabelled antibody--enzyme method. In combination, oestradiol and EGF increased mean cell counts from 1.15 +/- 0.06 and 1.36 +/- 0.05 x 10(5)/ml to 1.68 +/- 0.1 (+46%) and 1.94 +/- 0.16 (+43%) x 10(5)/ml, in proliferative and secretory gland preparations respectively (n = 10, P less than 0.01). No effect was seen in stromal cell preparations; however the stimulation in gland preparations was further augmented after the addition of stromal-conditioned medium. EGF-IR was detected in endometrium from normal women, and in normal and ectopic endometrium from women with endometriosis. EGF-IR was seen in glands and stroma and was not related to the phase of the menstrual cycle. EGF may play a role in the oestrogen-stimulated proliferation of normal and endometriotic endometrium.     

Distribution of cyclooxygenase-2 in eutopic and ectopic endometrium in endometriosis and adenomyosis

The objective of this study was to determine the distribution of cyclooxygenase-2 (COX-2) in eutopic and ectopic endometria in endometriosis and adenomyosis. The subjects were 35 patients with endometriosis diagnosed by laparoscopy, 33 patients with histologically confirmed adenomyosis and 50 female controls with normal fecundity. Expression of COX-2 was immunohistochemically investigated in tissues from eutopic endometrium and myometrium and ectopic endometrium of the wall of ovarian chocolate cysts using polyclonal antibody. Surface epithelial cells, endometrial glandular epithelial cells or stromal cells were assessed. Cells were semi-quantitatively assessed on a scale of 1 to 5 using a nomogram created from positive cell count and the degree of staining. COX-2 expression in surface and glandular epithelia of the control group varied markedly during the menstrual cycle. It was lowest in the early proliferative phase and gradually increased thereafter. It remained high throughout the secretory phase. However, in patients with endometriosis, expression of COX-2 in glandular epithelium was higher than that in the control group, though it varied throughout the menstrual cycle. On the other hand, there was no variation in expression of COX-2 in the adenomyosis group during the menstrual cycle, and it was lower than that in the endometriosis group in all phases. Pronounced COX-2 expression was observed in glandular cells from ectopic endometrial tissue of ovarian chocolate cyst walls in all cases regardless of the menstrual phase. In summary, increased COX-2 expression in eutopic and ectopic endometria was believed to be strongly correlated with pathological abnormalities in these disorders.     

Expression of glycodelin and cyclooxygenase-2 in human endometrial tissue following three-dimensional culture

PROBLEM: Our previous study showed that in vitro culture of human endometrial tissue in a three-dimensional (3D) fibrin matrix could mimic the early stages of endometriosis with invasion, gland and stroma formation and sprouting of new vessels. The objective of the present study was to evaluate the expression of glycodelin (Gd) and cyclooxygenase-2 (COX-2), two angiogenic factors, to further validate the 3D culture model of endometriosis. METHOD OF STUDY: Human endometrial fragments were obtained from endometrial biopsies and placed in a 3D fibrin matrix culture. Immunohistochemistry with specific antibodies to Gd and COX-2 was used to examine endometrial epithelium and blood vessels, and 4, 6-diamidino-2-phenylindole staining was used for nuclear identification. RESULTS: Three-dimensional culture of human endometrial tissue in the fibrin matrix resulted in the proliferation of endometrial stromal cells, glandular epithelium and angiogenesis. Gd positive glandular epithelium was seen in 85% of wells with developing endometrial glands and COX-2 positive new vessels were seen in 80% of wells with angiogenesis-like structures after 4 weeks of culture. CONCLUSION: Our findings confirm that angiogenesis occurs following the culture of endometrial tissue in the 3D fibrin matrix, and suggests that Gd and COX-2 might play important roles in promoting neovascularization and cell proliferation in the establishment of endometriosis.     

The role of peritoneal washings in the diagnosis of endometriosis

Endometriosis, the presence of endometrial tissue outside the uterine corpus, is a common finding in reproductive age women. It is classically diagnosed based on the presence of at least two of the following elements: endometrial glands, endometrial stroma, and hemosiderin‐laden macrophages (HLMs). Although a common finding in surgical pathology specimens at the time of gynecologic surgery, there is little literature on the role of pelvic washings in diagnosing endometriosis. Our study aimed to examine the characteristics of endometriosis in pelvic washings at the time of gynecologic surgery. We report nine cases of endometriosis diagnosed on pelvic washing. Two had a reported history of endometriosis. Four had endometriosis on the concurrent surgical pathology specimen. Liquid‐based cytology was diagnostic of endometriosis in seven patients, including five with glandular cells and HLMs and two with glandular cells, HLMs, and endometrial stromal cells. Cell block was diagnostic of endometriosis in eight patients, including four cases with intact fragments of endometrial glands and stroma. Three cases showed glandular cells and HLMs, while one showed separate fragments of glandular cells and stromal cells. Pelvic washings increased the diagnostic yield for endometriosis at the time of gynecologic surgery, as only four out of nine cases had endometriosis diagnosed on surgical pathology. Cell block in particular aids in the diagnosis, since intact glandular and stromal fragments frequently can be identified.     

Immunohistochemical analysis of oestrogen and progesterone receptors of eutopic and ectopic endometrium in the rabbit model of endometriosis: the effect of pregnancy.

Oestrogen receptors (ER) and progesterone receptors (PR) were measured in the rabbit model of endometriosis in eutopic and ectopic endometrium of pregnant animals (n = 7) and controls (n = 7). Immunostaining of cryostat sections of ectopic and eutopic endometrium was performed using monoclonal antibodies against ER and PR. Levels of ER and PR were 'evaluated' in a semi-quantitative manner using a modified histoscore. The ER and PR content in stromal and glandular cells was not different in eutopic and ectopic endometrium in either pregnant or non-pregnant control animals. There was a significant difference between the PR content of the glandular epithelium of pregnant animals and controls for both eutopic (P less than 0.02) and ectopic (P less than 0.001) endometrium. The disappearance of glandular PR and the persistence of stromal PR suggest that the function of the glandular endometrium is mediated by the paracrine and autocrine action of stromal cells. The decidual cells are likely to produce substances that may be of importance in embryo implantation and early pregnancy.     

Suppression of Natural Killer Cell Function and Production of Soluble ICAM-1: Endometrial Stroma Versus Melanoma

PROBLEM: It has been suggested that specific mechanisms commonly used by different cellular systems to evade immunologic recognition are involved in the development of endometriosis. To gain insight into this aspect, we looked at the relationship between two of these mechanisms in endometrial stroma and the melanoma system for which the ability to create an environment of immune privilege has been well established. METHOD OF STUDY: Media conditioned by endometrial stromal cultures and malignant melanoma A375 were examined to test their effects on peripheral blood mononuclear cell-mediated cytotoxicity directed against K562 target. Moreover, these media were tested for the concentration of the soluble form of intercellular adhesion molecule-1 (sICAM-1), which has been suggested as a marker for spreading potential. RESULTS: Media conditioned by endometrial stromal cultures exerted a significant suppressive effect on cell cytotoxicity when compared with those derived from malignant melanoma Moreover, the constitutive release of sICAM-1 was significantly higher in supernatants from endometrial stromal than in melanoma cells. CONCLUSIONS: These results indicate that two specific properties suggested to be involved in the ability of tumor cells to evade the immune system are more pronounced in the endometrium than in a malignant melanoma. Since the properties evaluated have been previously demonstrated to be even more notable in endometrial samples derived from endometriosis patients, a role of these mechanisms in the development of the disease may be hypothesized.     

Deficient expression of tumor necrosis factor receptor type 2 in the endometrium of women with endometriosis

PROBLEM: Tumor necrosis factor-alpha (TNF-alpha) is secreted mainly during the menstrual phase and has been suggested to play a role in induction of apoptosis in endometrial cells and menstrual shedding. TNF-alpha receptor type 2 (TNF-RII) is believed to play a central role in TNFalpha-mediated cytotoxic, mitogenic, anti-proliferative and apoptotic effects. The aim of this study was to assess whether TNF-RII maybe expressed differentially in the endometrium of women with different degrees of endometriosis. METHOD OF STUDY: TNF-RII expression in the endometrial tissue of women with and without endometriosis was investigated by immunohistochemical techniques and in situ hybridization. RESULTS: In histological sections, we observed TNF-RII mRNA and the corresponding protein localized mainly in endometrial glandular cells, with only very faint immunostaining in the surrounding stromal cells. Statistical analysis of our data showed a significant decrease in protein and mRNA expression of TNF-RII in endometrial glandular cells of patients with stages I and II endometriosis compared to normal subjects. TNF-RII expression was also found to decrease significantly in the secretory phase of the menstrual cycle in women with early endometriosis stages (I and II). CONCLUSIONS: In view of the relevant role of TNF-RII in the modulation of the inflammatory and the proapoptotic effects of TNFalpha, deficient expression of TNF-RII mRNA in the endometrium of women at the earliest stages of endometriosis may play a significant role in the pathophysiology of this disease.     

Papanicolaou tests associated with cervical mucosal endometriosis: An analysis of cellular features and comparison to endocervical adenocarcinoma in situ

Endometrium directly sampled from endocervical mucosal endometriosis can mimic endocervical adenocarcinoma in situ (AIS) in Papanicolaou (Pap) tests. We analyzed a series of Pap tests to investigate the cellular features of mucosal endometriosis and to assess the utility of stroma and apoptotic bodies in the differential diagnosis with AIS. Pap test samples from patients known to have endocervical mucosal endometriosis were compared with samples containing AIS. Pap tests from patients with mucosal endometriosis had lesional cells in 13 (62%) cases which includes glandular and stromal cells (10 cases), stroma only (two cases), and glandular cells only (one case). Three (23%) cases had gland‐stromal aggregates. Three (23%) cases had mitotic figures and two (15%) had apoptotic bodies. By comparison, only one (8%) AIS case had endometrial‐type stroma. Seven (58%) AIS cases had apoptotic bodies and three (25%) had mitotic figures. We conclude that Pap tests from patients with mucosal endometriosis usually (62%) have lesional cells. These lesional cells almost always include stroma, which is useful in the differential diagnosis with AIS. We identified stroma significantly more often in endometriosis cases (92%) than in AIS cases (8%). Pathologists should look for endometrial stroma when considering an interpretation of directly sampled endometrium. In the absence of stroma, AIS should be considered. Diagn. Cytopathol. 2010;38:551–554. 2009 Wiley‐Liss, Inc.     

Isolation and purification of human endometrial stromal and glandular cells using immunomagnetic microspheres.

Isolation of pure preparations of the different cell populations of human endometrium is a prerequisite for studies of in-vitro function. Sieving of dispersed endometrial cells, followed by adsorption onto immunomagnetic microspheres coated with antibody to Thy-1 was used to separate glandular and stromal cells. The purity of these cell populations was checked with antibodies to cytokeratin and Thy-1. The stromal cells were 98% pure and 90% viable, gland cells were 82% pure with 76% viability. The purified cells were able to proliferate in vitro as shown by thymidine incorporation.     

Expression of Intercellular Adhesion Molecule-1 (ICAM-1) on Cultured Human Endometrial Stromal Cells and Its Role in the Interaction With Natural Killers

PROBLEM: Recent evidence emphasizes the role of natural killer cells (NKs) as potential effectors of peritoneal immune surveillance directed against the outgrowth of endometrial cells, refluxed with menstrual debris, in ectopic sites. This NK-mediated cytotoxicity toward autologous endometrial antigens seems to be significantly decreased in endometriosis patients. METHOD: We set up experiments to clarify which molecules are involved in NK-endome-trial cell interaction. In particular, we evaluated the surface expression and functional activity of intercellular adhesion molecule-1 (ICAM-1), a cell surface glycoprotein that has been identified as one of the ligands for lymphocyte function-associated antigen-1 (LFA-1), present on almost all leucocyte cell types. Immunofluorescence flow cytometry was used to assess ICAM-1 expression on resting and IL 1β-activated endometrial stromal cells in culture. Dermal fibroblasts were used as control cells. Cytotoxicity and binding assays by ⁵¹Cr release in presence and absence of a specific monoclonal antibody (mAb) against ICAM-1 were then performed in order to determine the effect of this molecule on NK-mediated cytotoxic and binding activity toward endometrial stromal cells. RESULTS: The results of this study indicated that ICAM-1 expression on endometrial stromal cells seems to be constitutively higher than on dermal fibroblasts and can be up-regulated upon exposure to IL 1β. Furthermore, a mAb against ICAM-1 strongly inhibits the binding but not the cytotoxicity of NKs toward endometrial cells. No difference in the expression of this molecule was observed throughout the cycle. CONCLUSIONS: The presence of ICAM-1 on human endometrium might relate to the action of the immunocompetent cells in human specific reproductive events.     

Characterization of Lymphocyte Subpopulations and T Cell Activation In Endometriosis

PROBLEM: Numerous studies have characterized the lymphocyte subpopulations in normal eutopic endometrium and suggested a role for the cytokine secretory products of these lymphocytes in regulating endometrial cell proliferation and differentiation. Recent studies have shown that ectopic endometrium contains a greater concentration of scattered stromal lymphocytes than does eutopic endometrium. However, the lymphocyte subpopulations and their activation status have not been characterized in ectopic endometrium. METHODS: We performed immunohistochemical studies on serial sections of proliferative and secretory phase eutopic endometrium and ectopic endometrium obtained during the proliferative phase using monoclonal antibodies to CD4 (T helper-inducer cells), CD8 (T cytolytic-suppressor cells), CD22 (B-cells), CD56 (natural killer cells), and VLA-1 (T-cell activation marker). RESULTS: Ectopic endometrium contained significantly more scattered stromal CD4, CD8, and activated T cells than did proliferative and secretory eutopic endometrium. There were more activated T-cells in proliferative than in secretory eutopic endometrium. Ectopic endometrium contained significantly fewer NK cells than proliferative and secretory endometrium. CONCLUSIONS: These results demonstrate that (1) the increased lymphocyte population in ectopic endometrium is due to increased numbers of CD4 and CD8 cells, and (2) a greater number of activated T cells are present in ectopic endometrium as compared to eutopic endometrium. Increased concentration of stromal T cells and enhanced VLA-1 expression in ectopic endometrium suggest that cytokine products of the activated T-cells may be involved in regulating cellular processes of endometriosis tissue.