Endogenous TWEAK is critical for regulating the function of mouse uterine natural killer cells in an immunological model of pregnancy loss

Uterine natural killer (uNK) cells are the most abundant lymphocyte population in the feto–maternal interface during early gestation, and uNK cells play a significant role in the establishment and maintenance of pregnancy‐related vascularization, as well as in tolerance to the fetus. Tumour necrosis factor‐like weak inducer of apoptosis (TWEAK) and its receptor, fibroblast growth factor‐inducible molecule (Fn14), are involved in preventing local cytotoxicity and counterbalancing the cytotoxic function of uNK cells. Here, we studied the regulation of TWEAK/Fn14‐mediated innate immunity in the uterus using a lipopolysaccharide (LPS)‐induced model of abortion in pregnant mice. Specifically, we detected the expression of TWEAK and Fn14 in the uterus and in uNK cells following LPS treatment. Our results revealed that TWEAK and Fn14 are expressed by uNK cells in pregnant mice; in particular, it appears that the cytokine TWEAK is primarily derived from uNK cells. Interestingly, the down‐regulation of TWEAK in uNK cells and the up‐regulation of the Fn14 receptor in the uterus in LPS‐treated mice may contribute to the disruption of decidual homeostasis by altering uNK cell cytotoxicity – ultimately leading to fetal rejection. In conclusion, the present study strongly suggests that the TWEAK–Fn14 axis in uNK cells is involved in maintaining the tolerance necessary for successful pregnancy.     

Modulation of NK Cell Lytic Function by Endometrial Secretory Factors: Potential Role in Endometriosis

PROBLEM: Development and progression of endometriosis have been suggested to be related to a defect of NK cells in the ability to eliminate endometrial fragments regurgitated with menstrual debris in ectopic sites. However, it has not been clarified yet whether the origin of this defect has to be attributed to an intrinsic alteration of NK cell population or to a consequence of the environmental system. The present study was designed to evaluate whether soluble factors of endometrial origin might directly interfere with NK cell lytic function. METHOD: Media conditioned by 20 endometrial stromal cultures (10 from endometriosis patients and 10 from women without the disease) were examined to test their effects on NK cell-mediated cytotoxicity directed against endometrial stromal targets. NK cells were purified from peripheral blood samples by a two steps panning technique and incubated overnight with and without endometrial supernatants. Thereafter, the percentage of lysis against endometrial cells was detected in a ⁵¹Cr cytotoxicity assay. RESULTS: Media conditioned by the overall endometrial stromal cultures exerted a significant suppressive effect (P < 0.001) on NK cell function when compared to control NK fractions (mean percentage of cytotoxicity ± SEM 17.46 ± 2.8 and 29.41 ± 1.49, respectively). No inhibitory effect was detect with conditioned media obtained from peritoneal cells and keratinocytes. Moreover, NK cell-mediated cytotoxicity resulted significantly more inhibited by the addition of endometrial supernatants obtained from endometriosis patients (mean percentage cytotoxicity ± SEM 13.55 ± 2.3, P < 0.05) when compared to those from women without the disease (mean percentage cytotoxicity ± SEM 21.38 ± 4.9). CONCLUSION: The results of this study indicate the existence of soluble non-specific factor(s) released by human endometrial cells able to interfere with NK cell killing. These factors could participate in the local regulation of the immunological mechanisms suggested to be involved in the development of endometriosis.     

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.     

Paracrine effects of uterine leucocytes on gene expression of human uterine stromal fibroblasts

The endometrium contains a distinct population of immune cellsthat undergo cyclic changes during the menstrual cycle and implantation.The majority of these leucocytes are uterine NK (uNK) cells,however how these cells interact with uterine stromal fibroblastsremains unclear. We therefore investigated the paracrine effectof medium conditioned by uterine decidual leucocytes (whichare enriched for uNK cells) on the gene expression profile ofendometrial stromal fibroblasts in vitro using a cDNA microarray.Our results, verified by real-time PCR, ELISA and FACS analysis,reveal that soluble factors from uterine leucocytes substantiallyalter endometrial stromal fibroblast gene expression. The largestgroup of up-regulated genes found was chemokines and cytokines.These include IL-8, CCL8 and CXCL1, which have also been shownto be stimulated by contact of stromal fibroblasts with trophoblast,suggesting that uNK cells work synergistically to support trophoblastmigration during implantation. The decidual leucocytes alsoup-regulated IL-15 and IL-15R in stromal fibroblasts which couldproduce a niche for uNK cells allowing proliferation withinand recruitment into the uterus, as seen in bone marrow. Overallthis study demonstrates, for the first time, the paracrine communicationbetween uterine leucocytes and uterine stromal fibroblasts,and adds to the understanding of how the uterine immune systemcontributes to the changes seen within the cycling endometrium.     

Erythropoietin and erythropoietin receptor expression in human endometrium throughout the menstrual cycle

Erythropoietin (Epo) is an important regulator of erythropoiesis. Recent studies have demonstrated non-classical sites of Epo and Epo-receptor (Epo-R) expression, suggesting new physiological roles unrelated to erythropoiesis. Other studies have shown that the mouse uterus expresses Epo and its receptor, and produces Epo protein in an estrogen-dependent manner. We therefore hypothesized that Epo is one of the growth factors involved in cyclic endometrial changes. We determined Epo and Epo-R mRNA expression in isolated endometrial epithelial and stromal cells using RT-PCR. While both Epo and Epo-R were detected in all samples of isolated epithelial cells analysed throughout the menstrual cycle, neither one was detected in isolated stromal cells. In addition, using quantitative real-time RT-PCR with the TaqMan detection system, we showed that isolated epithelial cells had higher Epo mRNA levels in the secretory phase than in the proliferative phase. Immunohistochemical analyses revealed that Epo and Epo-R protein expression in glandular epithelial cells was increased during the mid-proliferative phase and was maintained during the late proliferative and the early, mid- and late secretory phases. These findings suggest that Epo may be involved in cyclic proliferation and differentiation of endometrial glandular epithelial cells, acting in an autocrine manner. In addition, we also hypothesize that ovarian steroids may stimulate Epo production in human endometrial glandular epithelial cells.     

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.     

Dipeptidyl peptidase IV as a differentiation marker of the human endometrial glandular cells.

To investigate the involvement of membrane-bound peptidases in the human endometrial function, we examined the expression of dipeptidyl peptidase (DPP) IV and its enzyme activity. Immunohistological studies revealed that DPP IV was detected on human endometrial glandular cells and endometrial surface epithelium, but not on endometrial stromal cells or decidual cells in the first trimester of pregnancy. DPP IV expression on glandular cells and surface epithelium was weak in the proliferative phase, began to increase gradually in the early secretory phase, and was strong in mid-to late secretory phase and in the first trimester of pregnancy. DPP IV enzyme activity was detected histochemically in glandular cells and surface epithelium in the mid-secretory phase, and became stronger in the late secretory phase, but was rarely detected in the proliferative phase and early secretory phase. During the first trimester of pregnancy DPP IV enzyme activity in glandular cells and surface epithelium was slightly weaker than in the late secretory phase. Endometrial stromal cells and decidual cells, however, had no detectable DPP IV enzyme activity at any time throughout the menstrual cycle or during the first trimester of pregnancy. These findings indicate that DPP IV is a differentiation marker for glandular cells and surface epithelium and that active DPP IV is present in both areas during the peri-implantation period and thereafter.     

LMO2 promotes angiogenesis probably by up-regulation of bFGF in endothelial cells: an implication of its pathophysiological role in infantile haemangioma

Sun Z‐J, Cai Y, Chen G, Wang R, Jia J, Chen X‐M, Zheng L‐W & Zhao Y‐F
(2010) Histopathology 57 , 622–632
LMO2 promotes angiogenesis probably by up‐regulation of bFGF in endothelial cells: an implication of its pathophysiological role in infantile haemangioma Aims: Infantile haemangiomas (IHs) are common benign vascular tumours distinctive for their perinatal presentation, rapid growth during the first year of life and subsequent slow involution. Recent research has indicated that endothelial cells of haemangiomas express LIM‐only protein 2 (LMO2). The aim of this study was to investigate the role of LMO2 in the pathogenesis of IHs was investigated. Methods and results: Immunoreactivity of LMO2 was assessed in specimens of 19 IH. Stable transfection of LMO2 into human endothelial cell lines (EAhy926) was performed to evaluate the role of LMO2 in terms of the change in cell proliferation, cell cycle and cell migration as well as the expression level of angiogenic factors. Immunoreactivity for LMO2 was detected in all IH specimens, specifically in the nucleus of the endothelial cells. The intensity of LMO2 immunostaining decreased significantly from proliferative to involuting stages. Furthermore, the overexpression of LMO2 enhanced the proliferation and migration of the endothelial cells and promoted G0/G1–S‐phase transition in vitro, together with an up‐regulation of bFGF expression. Conclusions: LMO2 probably promotes angiogenesis by up‐regulation of bFGF expression and thereby consequently influences progression of IH.     

Expression of Telomerase Activity in Human Endometrium Is Localized to Epithelial Glandular Cells and Regulated in a Menstrual Phase-Dependent Manner Correlated with Cell Proliferation

Telomerase activity is observed in most malignant tumors and germ cells, whereas normal somatic cells usually do not express it. Human endometrium is composed of glandular and stromal components and exhibits dramatic changes in proliferative activity during the menstrual cycle, which is exquisitely regulated by estrogen function. We previously reported that normal human endometrium expresses telomerase activity. However, it remains unclear which of the above components are the major sources of telomerase activity and how levels of telomerase activity are regulated over the menstrual cycle. Quantitative analysis of telomerase activity revealed that it changes dramatically over the course of the menstrual cycle and is strictly regulated in a menstrual-phase-dependent manner. Maximal activity equivalent to that in endometrial cancer was present in late proliferative phase, and minimal activity in late secretory phase. Postmenopausal endometrium and endometrium treated with anti-estrogen drugs exhibited decreased telomerase activity. Testing isolated epithelial glandular cells and stromal cells, we found that telomerase activity was localized to epithelial glandular cells. In situ RNA hybridization analysis also revealed epithelial-specific expression of human telomerase RNA. In vitro analysis of cultured epithelial cells demonstrated that telomerase activity is correlated with epithelial proliferation but not affected by estrogen treatment. These findings suggest that expression of telomerase activity is specific to epithelial cells and linked to cell proliferative status. The involvement of estrogen in telomerase regulation remains to be elucidated.     

Distinct subsets of stromal cells confined to unique microenvironments in human endometrium throughout the menstrual cycle.

Human endometrial stroma exhibits rather uniform morphology throughout the endometrium. However, predecidualization develops characteristically around vessels and subsequently around glands and under surface epithelium, demonstrating existence of regional differences among stromal cells. Immunoreactivity of stromal cells in endometrial tissues from various phases of the menstrual cycle, as elucidated by employing monoclonal antibodies to cytokeratin, vimentin, very late antigen-1 (VLA-1), Ber-EP4, and HLA-DR, revealed presence of phenotypically distinct subsets of stromal cells confined to unique microenvironments throughout the menstrual cycle. All stromal cells strongly expressed vimentin and weakly expressed cytokeratin. However, Ber-EP4 positive stromal cells were distinctly confined around glands and to the subluminal regions of the surface epithelium. The intervening stromal cells were Ber-EP4 negative. The HLA-DR positive stromal cells were characteristically present in three different locations: around glands and under surface epithelium, around blood vessels and around HLA-DR positive lymphoid cells. From all antigens studied, only expression of VLA-1 in the stromal cells showed a characteristic change throughout the menstrual cycle. Stromal cells in the proliferative and early secretory phases were VLA-1 negative. However, VLA-1 characteristically developed initially in the HLA-DR positive cells around vessels and then in HLA-DR/Ber-EP4 positive cells around glands and under surface epithelium. Eventually, all stromal cells in the upper functionalis expressed VLA-1 in the late secretory phase of the menstrual cycle. These data underscore a heterogeneity in stromal cells not exemplified by their morphology. Also, they provide a basis for understanding the differences that the stroma exhibits in morphologic and functional differentiation throughout the menstrual cycle.     

Cell kinetic study of the endometrium by nonisotopic in situ hybridization for histone H3 messenger RNA and immunohistochemistry for Ki‐67 and for estrogen and progesterone receptors

We investigated the cell kinetics of the endometrium in hysterectomy specimens taken for leiomyoma from 22 women with regular ovulatory menstrual cycles. Formalin‐fixed, paraffin‐embedded tissue sections were examined for proliferating activity using histone H3 messenger RNA in situ hybridization (H3 mRNA‐ISH) and immunostaining for the Ki‐67 antigen. The relationship of the proliferative activity of endometrial cells to the immunohistochemical expression of the estrogen receptor (ER) and the progesterone receptor (PR) was also examined. During the menstrual cycle, H3 mRNA expression was observed in both the epithelial cells and the stromal cells of the endometrium. In the functional layer, the labeling indices for H3 mRNA (H3 mRNA‐LIs) in the epithelial cells peaked in the late proliferative phase, decreased sharply in the early secretory phase, and remained unchanged thereafter. On the other hand, H3 mRNA‐LIs of stromal cells displayed two peaks: one in the midproliferative phase and the other in the late secretory phase, the former peak being the greater. In the basal layer, epithelial cells and stromal cells showed low H3 mRNA‐LIs and no significant variation throughout the menstrual cycle. The H3 mRNA‐LIs correlated well with the Ki‐67‐LIs and were lower than the corresponding Ki‐67‐LIs. The regression coefficient (H3 mRNA‐LIs against the Ki‐67‐LIs) was 0.33 for epithelial cells and 0.49 for stromal cells, suggesting that the cell cycle time was longer for epithelial cells than for stromal cells. The proliferative activity of endometrial cells showed close relationships with the expressions of ER and PR in the endometrium. When used in combination with other proliferative markers in paraffin‐embedded tissue sections, H3 mRNA‐ISH could open broader perspectives on the cell kinetics of the endometrium. Anat Rec 266:234–240, 2002. © 2002 Wiley‐Liss, Inc.     

Molecular aspects of implantation: Variation in the expression of cellular retinoid binding proteins in human endometrium throughout the menstrual cycle

Human endometrium is a glandular epithelial tissue with a substantialunderlying stroma. Under the influence of ovarian steroids,endometrium undergoes a cyclical pattern of proliferation followedby secretory differentiation. Since retinoids promote the differentiationof many epithelia to secretory phenotypes they may be involvedin controlling the secretory differentiation of human endometrialepithelium. Cytosolic binding proteins for retinol (cellularretinol binding protein) and retinoic acid (cellular retinoicacid binding protein) may play an important part in regulatingthe availability of retinoic acid to its nuclear receptors andwe have therefore asked whether expression of mRNA for theseproteins varies in relation to endometrial differentiation.In a series of 54 endometrial biopsies, both endometrial epithelialand stromal cells expressed mRNA for cellular retinol bindingprotein type I at a constant level throughout the menstrualcycle. Cellular retinoic acid binding protein type II was alsoexpressed but the level of expression varied dramatically, beingelevated in the proliferative phase and depressed during thesecretory phase of the menstrual cycle in both epithelial andstromal cells. These data suggest that cytosolic binding proteinsmodulate the supply of retinoic acid to the nuclei of endometrialcells during the menstrual cycle and that retinoic acid is involvedin the cyclical control of endometrial differentiation.     

Differential localization and expression of urokinase plasminogen activator (uPA), its receptor (uPAR), and its inhibitor (PAI-1) mRNA and protein in endometrial tissue during the menstrual cycle

Normal endometrium is a highly dynamic tissue, which responds to ovarian steroids with cyclic proliferation, differentiation (secretion), and degradation (menstruation). The urokinase plasminogen activator (uPA)-dependent proteolytic cascade as well as ligand activation of the uPA receptor (uPAR) is critically involved in physiological as well as pathophysiological aspects of tissue expansion and remodelling. Cyclic variation and distribution of uPA, uPAR and plasminogen activator inhibitor 1 (PAI-1) mRNA were examined by in situ hybridization, real-time PCR and northern blot in normal endometrium. Their corresponding proteins were localized with immunohistochemistry. uPA mRNA is exclusively expressed by stromal cells, whereas uPA protein is present in both epithelial and stromal cells. Immunostaining for uPA protein is reduced or undetectable at midcycle, thus coinciding with peak concentration of uPA in the uterine fluid. uPAR mRNA is expressed by epithelial cells in the proliferative phase and by stromal cells in the secretory phase. However, epithelial cells stain for uPAR protein throughout the cycle, suggesting that uPAR may detach from stromal cells and then bind to epithelial cells in the secretory phase. PAI-1 mRNA is located in vessel walls. The late secretory phase has greatly increased expression of all three mRNA and their proteins, mainly in pre-decidual cells in the superficial stroma. Discordant localization of the mRNA and proteins suggest that uPA is produced by stromal cells, released and bound to epithelial cells in both the proliferative and secretory phases, whereas uPAR is released from the stroma and bound to epithelial cells in the secretory phase. Also, the present data together with earlier reports suggest that uPA is released from the epithelial cells to the uterine fluid.     

Differential Expression of IGF-I and IGF-II in Eutopic and Ectopic Endometria of Women With Endometriosis and in Women Without Endometriosis

PROBLEM: Factors regulating the development, growth, and differentiation of endometrial cells of endometriotic lesions are poorly understood. To investigate the paracrine-autocrine regulation of ectopic endometrial cell growth, the expression of IGF-I and IGF-II were studied. METHOD: Tissue specimens of eutopic and ectopic endometria were obtained from eight patients with endometriosis at laparoscopy and from the endometria of 14 women without endometriosis as controls. They were tested for the expression of IGF-I and IGF-II by immunohistochemical analysis. RESULTS: Immunohistochemical study for IGF-I in controls showed a more intense staining during the proliferative phase both in stromal and epithelial cells. In eutopic endometria of women with endometriosis a reduction in the staining was observed, whereas in epithelial cells of fibrotic peritoneal adhesions an intense immunostaining for IGF-I was observed. Immunohistochemical study of IGF-II in controls showed a more intense staining during secretory phase both in stromal and epithelial cells. In eutopic endometria of women with endometriosis, a reduction in the staining was observed, whereas in epithelial cells of fibrotic peritoneal adhesions an intense immunostaining for IGF-I was observed. Immunohistochemical study of IGF-II in controls showed a more intense staining during secretory phase both in stromal and epithelial cells. In eutopic endometria of women with endometriosis, a reduction in the staining was observed, whereas in epithelial cells of ovarian lesions and fibrotic peritoneal adhesions, no immunostaining for IGF-II was observed. CONCLUSIONS: In endometriosis there is an alteration of mechanisms regulating cell proliferation and differentiation.     

Proliferative activity in endometrial stromal granulocytes throughout menstrual cycle and early pregnancy.

The proliferative activity in endometrial stromal granulocytes was studied using two approaches. Firstly, mitotic activity was studied in paraffin wax embedded sections of normal non-pregnant endometrium and early pregnancy decidua stained with phloxine-tartrazine. Secondly, the monoclonal antibody Ki67 was applied to cryostat sections of similar tissues. Endometrial stromal granulocytes were identified by their labelling with NKH1, Dako T11, UCHL1 or Dako-LC. The percentage of cases in which endometrial stromal granulocytes showed mitosis was 25%, 75%, 86%, and 93%, respectively in proliferative, early secretory, mid secretory, and late secretory phases, and 14% in early decidua. There were at most one or two endometrial stromal granulocytes in mitosis per 10 high power fields. Double labelled cells were present in small numbers in proliferative endometrium and in moderate numbers in secretory endometrium. Only a few cells in early decidua double labelled with Ki67/T11; moderate numbers of cells double labelled with Ki67/Dako-LC. It is concluded that proliferative activity does occur in endometrial stromal granulocytes and is particularly prominent in the late secretory phase.     

Lack of correlation between vascular endothelial growth factor production and endothelial cell proliferation in the human endometrium.

The role of vascular endothelial growth factor (VEGF) in endometrial angiogenesis was examined by measuring its production in human endometrial tissues from different stages of the menstrual cycle and relating these data to endothelial cell proliferation in the same tissues. Conditioned medium was collected from explant, and separated glandular epithelial and stromal cells cultured from 24 normal human endometrial biopsies and VEGF measured by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was also used to assess VEGF and the percentage of proliferating microvessels in the samples. Wide variation in results between individual endometrial samples at each stage of the menstrual cycle was observed for all parameters measured. There was no significant difference in VEGF secretion by explant, glandular epithelial or stromal cell cultures across the menstrual cycle, or in the percentage of proliferating vessels. VEGF immunostaining in the stroma was elevated during the early proliferative stage (P = 0.03). Epithelial cells secreted more VEGF than stromal cells (1.76 +/- 0.46 versus 0.46 +/- 0.06 ng per 10(5) cells; P = 0.002). There was no correlation between VEGF secreted by cultured explants, epithelial or stromal cells, VEGF immunostaining and the proportion of proliferating microvessels. These results show that the majority of endometrial VEGF is produced by glands, but neither total glandular nor stromal VEGF is correlated with endometrial endothelial cell proliferation. There is still no clear understanding on the regulation of human endometrial angiogenesis.     

Telomerase activity is found in the epithelial cells but not in the stromal cells in human endometrial cell culture

Telomerase activity is associated with the proliferative activity of cells. In the endometrium, telomerase activity is higher in the proliferative phase than in the secretory phase of the menstrual cycle, suggesting that telomerase activity may occur primarily in the glandular epithelial cells. To test this, a dissociated cell culture of the endometrium was performed, and the telomerase activity in each cell fraction was analysed. Telomerase activity was found in all 10 endometrial tissues of the proliferative phase of the menstrual cycle. Both the fragments of epithelial glands and single cells, which were prepared by enzymatic dissociation, showed telomerase activity. In the 7 day cell culture, it was found in nine out of 10 epithelial cell enriched fractions, but in none of the stromal cell enriched fractions. Flow cytometric analysis showed that the epithelial enriched fraction was contaminated with a predominant number of stromal cells, while the stromal cell enriched fraction was comprised mostly of stromal cells with apparent proliferative activity. Our results suggest that telomerase activity of the endometrium occurs primarily in the epithelial cells in the endometrium and that the stromal cells do not express telomerase activity regardless of their potent proliferative activity.      

Expression of the epidermal growth factor system in human endometrium during the menstrual cycle

The epidermal growth factor (EGF) system is ubiquitous in humans and plays fundamental roles in embryogenesis, development, proliferation and differentiation. As the endometrium of fertile women is characterized by proliferation and differentiation, we hypothesize a role for the EGF system. Fourteen premenopausal women had endometrial samples removed on day 6 +/- 1 and day 6 +/- 1 and 12 +/- 1 after ovulation during one menstrual cycle. RNA was extracted and analysed by real-time PCR, and immunohistochemistry was performed to localize the components of the EGF system. Human EGF Receptor 1 (HER1) showed highest expression during the proliferative phase, HER2 and HER4 during the early and HER3 during the late secretory phase. Amphiregulin (AR) and transforming growth factor alpha (TGFalpha) expression is highest in proliferative phase. Heparin binding (HB)-EGF and betacellulin (BCL) show no variation. Epiregulin (EP) is detectable in some samples. EGF is undetectable. HER1, HER2, HER3 and HER4 were localized to the epithelium and glands HER3 and HER4 solely in the secretory phase. Amphiregulin was seen in leucocytes and stromal cells, TGFalpha and betacellulin in the epithelial lining, epiregulin in stromal cells whereas HB-EGF and EGF are undetectable. In conclusions, we observed cyclical expression of the four EGF receptors and two ligands and localized all four receptors and four ligands in endometrial biopsies. This suggests a role for the EGF system in growth of the endometrium.     

Cytomorphologic appearances of normal endometrial cells during different phases of the menstrual cycle: A cytologic approach to endometrial dating

From 1988 to 1992, 156 endometrial cytologic preparations were procured by directly brushing hysterectomy specimens from premenopausal women with normal endometrium and regular menstruation. These brushing smears were obtained by using the cyto-brush and/or the endometrial brush. The cytologic findings were correlated with histologic endometrial dating. Of these, 56 cases were classified as proliferative phase endometrium; 89, secretory phase; and 11, menstrual phase. The cytomorphologic features of endometrial glandular and stromal cells at different stages of the menstrual cycle are summarized and compared. On the basis of the different cytomorphologic features of glandular and stromal cells seen during various phases of the cycle, cytologic differentiation between proliferative phase and secretory phase endometria can be readily established. It appears possible to date the endometrium by direct endometrial brushing technique. It is also possible to determine if the glandular and/or stromal cells seen in the endocervical smears procured by the cytobrush are normal endometrial cells from cytobrush extraction, and are consistent with the date of the menstrual cycle, which should be made clear in the cytology report. © 1995 Wiley-Liss, Inc.