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.     

The pattern of expression of Notch protein members in normal and pathological endometrium

The objective of this study was to investigate the pattern of expression and the localization of Notch-1, Notch-4 and Jagged-1 in physiological and pathological human endometrium and to evaluate the expression levels of two major regulators of the G1 checkpoint, namely cyclin D1 and p21. Sixty samples of physiological endometrium and 60 samples of pathological endometrium were used for the study. Evaluation of the expression level and the distribution of Notch pathway members and cell-cycle proteins was performed by immunohistochemistry. In the physiological endometrium we observed an increase of Notch-1 and Jagged-1 from proliferative to secretory phase and an opposite trend for Notch-4. In menopause, the level of expression of all three members of the Notch pathway decreased. We also observed a cyclin D1 increase from proliferative to secretory phase. By contrast, p21 showed a slight increase from proliferative to secretory phase. In the pathological endometrium, we observed an increase of Notch-1 expression from polyps to carcinoma and decrease for Notch-4 and Jagged-1. Moreover, we observed a higher expression of cyclin D1 in all the endometrial pathologies. By contrast, the expression level of p21 slightly increased from polyps to carcinoma. We concluded that in human endometrium Notch-4 seems to be more involved in controlling proliferation, whereas Notch-1 seems to be more involved in differentiation programming. Deregulation of these functions may induce the onset of several endometrial pathologies from polyps to cancer.     

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.     

Inhibin (10.7 kD prostatic peptide) in normal, hyperplastic, and malignant human endometria: an immunohistochemical study.

The expression of inhibin, a 10.7 kD follicle-stimulating hormone (FSH)-suppressing prostatic peptide of 94 amino acids, was investigated in normal human endometrium, endometrial hyperplasia, and adenocarcinoma, employing the avidin-biotin immunoperoxidase technique. The antiserum used was raised in rabbits against prostatic inhibin isolated from human seminal plasma. The study included 15 well differentiated, 32 moderately differentiated, and 21 poorly differentiated endometrial adenocarcinomas; 26 simple, five complex, and two complex atypical endometrial hyperplasias; and, for comparison, 25 normal proliferative and 30 normal secretory endometria. In malignant and hyperplastic endometrial tissues, inhibin was localized in the epithelial cytoplasm of endometrial glands while the stroma showed weak reactivity. On the other hand, inhibin was undetectable in the early proliferative phase, but was present on the luminal border of the glandular epithelium in the mid- and late proliferative phases. Secretory endometrium displayed strong inhibin reactivity in the cytoplasm of glandular epithelium and in the stroma. The increased inhibin reactivity in secretory endometrium as compared with the proliferative phase is indicative of a functional role for inhibin in the uterus. In addition, its localization in proliferative, hyperplastic, and malignant endometria suggests a possible regulatory role for inhibin in endometrial proliferation and growth.     

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.     

Temporal and site-specific expression of transforming growth factor- beta4 in human endometrium

We recently identified a novel member of the transforming growth factor (TGF)-beta superfamily and showed that this gene, designated as endometrial bleeding associated factor (ebaf), or TGFbeta4, has a unique expression pattern in human endometrium. By Northern blot analysis, we showed that this gene was expressed in human endometrium during the late secretory and menstrual phases and was absent in proliferative, early and mid-secretory endometria. In this report, we show by in-situ hybridization that the mRNA of the TGF-beta4 is not expressed in the proliferative endometria. On the other hand, focal expression of the TGFbeta4 mRNA first appears in some endometrial glands in the mid-secretory phase. The TGFbeta4 mRNA is strongly expressed in the endometrial stroma during the late secretory and menstrual phases of the cycle. We raised a polyclonal rabbit antiserum against a peptide at the C terminal of the protein. Western blot analysis using affinity purified antiserum shows that the TGFbeta4 precursor detected in the endometrium as well as placenta is 41 kDa. Bands in the range of 45-51 kDa are also present in human endometrium, more predominantly during the late secretory phase. Immunohistochemical staining shows a low level of immunoreactivity for TGFbeta4 in the early, mid- and late proliferative and early and mid-secretory endometria. A strong immunoreactivity for TGFbeta4 is present in the stroma and to lesser extent in the endometrial glands in late secretory and menstrual endometria. The specificity of staining was shown by neutralizing the activity of the antibody with the synthetic peptide used for raising the antibody and by omitting the antibody. The findings show that TGFbeta4, both at the mRNA and protein levels, exhibits temporal and site specific expression in human endometrium.      

Expression of cyclin D1 in normal, metaplastic, hyperplastic endometrium and endometrioid carcinoma suggests a role in endometrial carcinogenesis

CONTEXT: Endometrioid carcinoma is often preceded by characteristic histopathologic lesions known as endometrial hyperplasia. Estrogen appears to be involved in the development of endometrioid carcinoma. Other mechanisms of endometrial carcinogenesis include mutations in p53 and PTEN tumor suppressor genes and overexpression of cyclin D1. However, the pattern of cyclin D1 expression is not well defined in normal, hyperplastic, neoplastic, and metaplastic endometrium. DESIGN: Cyclin D1 immunohistochemical analysis was used to evaluate 108 fixed, paraffin-embedded endometrial biopsy specimens and uterine resections obtained from 108 patients. Specimens included proliferative and secretory endometria, simple and complex hyperplastic lesions, and endometrioid adenocarcinoma. Normal and metaplastic surface epithelia were also evaluated independently of glandular morphologic features. RESULTS: Cyclin D1 was significantly overexpressed in glands with complex hyperplasia and endometrioid adenocarcinoma compared with proliferative or secretory endometrium and simple hyperplasia. Significant overexpression was also noted in papillary, syncytial, and squamous metaplasias compared with normal surface epithelium or epithelium with tubal metaplasia. CONCLUSION: Overexpression of cyclin D1 increases from normal endometrium to hyperplasia and carcinoma, suggesting that it may play a role in endometrial carcinogenesis. Overexpression of cyclin D1 in endometrial glands was independent from overexpression of cyclin D1 in surface metaplastic epithelium.     

Membrane-type matrix metalloproteinases and vascularization in human endometrium during the menstrual cycle

Endometrial angiogenesis is essential for a vascularized receptive endometrium. Previously, we described that membrane type-3 metalloproteinase (MT3-MMP) is associated with endometrial angiogenesis in vitro. The association of MT-MMPs with endometrial angiogenesis in vivo is unknown. Therefore, this study analysed the presence of MT-MMPs in human endometrium and their correlation with neovascularization. RNA/protein expressions of the six MT-MMPs were determined in cultured endometrial cells. Vascularization parameters and MT-MMP expressions in vivo were evaluated by immunohistochemistry in serial endometrium sections. MT1-, MT2-, MT3- and MT4-MMP antigens were expressed in cultured endometrial endothelial cells. MT2-, MT3- and MT4-MMP were expressed by endothelium during the proliferative and secretory phase. Strikingly, these phases showed elevated vascularization, elevated total vascular surface in proliferative phases, elevated number of vessels in proliferative/late secretory phases and increased luminal surface in the proliferative phases. All MT-MMP antigens were expressed in various endometrial cell types in vivo, with decreased levels during the early secretory phase. In conclusion, all MT-MMPs are expressed in endometrium in a cycle-dependent pattern. The vascular expression of MT2-, MT3- and MT4-MMP correlated with angiogenic episodes of the cycle. Since MT2- and MT3-MMP are known to regulate tube formation, these findings support earlier in vitro data on the role of MT3-MMP in endometrial angiogenesis. Additionally, MT2-MMP appears to be associated with endometrial neovascularization also.     

Progesterone induces the fibulin-1 expression in human endometrial stromal cells

BACKGROUND: By using microarray analysis with human endometrial stromal cells (ESCs), we previously reported that the mRNA for fibulin-1, an extracellular matrix as well as a plasma glycoprotein, is up-regulated by progesterone. In the present study, we tried to clarify the spatial and temporal regulation mechanism of fibulin-1 in the human endometrium. METHODS AND RESULTS: Quantitative analysis with real-time PCR experiments on human endometrial tissues showed significantly higher fibulin-1 mRNA expressions in secretory phase endometria than in proliferative phase. Immunohistochemical studies revealed that the fibulin-1 protein is expressed in the glandular epithelium in proliferative phase endometria, and that expression switched to the stroma in secretory phase endometria. In culture experiments with ESCs, a significant increase of fibulin-1 mRNA expression was observed in cells treated with 6 alpha-methyl-17 alpha-hydroxy-progesterone acetate (MPA) or 8 bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP). MPA stimulated the fibulin-1 mRNA expression in a dose-dependent manner, and a progesterone antagonist, RU-486, inhibited the stimulatory effect almost completely. By contrast, beta-estradiol alone did not increase the fibulin-1 mRNA expression. CONCLUSIONS: These results suggest that fiblin-1 is an important molecule that mediates progesterone action in human ESC differentiation towards implantation.     

Thioredoxin expression in the human endometrium during the menstrual cycle

Human thioredoxin, a cellular disulphide reducing protein, is known to be secreted by some types of cells and to display unique extracellular activities including modulation of cytokine actions and protection of the cell against damage from oxidative stress. This study has been undertaken to investigate the pattern of expression and tissue distribution of thioredoxin in human endometrium during the menstrual cycle. Immunohistochemical studies showed increased thioredoxin immunoreactivity in the glands of the secretory phase compared to those of the proliferative phase. Although the staining of thioredoxin was relatively intense in predecidual stromal cells, the most prominent staining of thioredoxin was present in both glands and stroma of the endometrium in the early secretory phase of the menstrual cycle. Northern hybridization analyses revealed that expression of thioredoxin mRNA in the endometrium of the early secretory phase was approximately 3-fold compared to the other phases of the menstrual cycle, consistent with the results of the immunohistochemical studies. These results suggest that both protein and gene expression of thioredoxin in the endometrium are menstrual cycle phase-specific and highly active in the phase of endometrial differentiation which occurs in preparation for implantation (early secretory phase of the menstrual cycle). Thioredoxin expressed in the early secretory phase of the menstrual cycle may be advantageous for blastocyst implantation.      

Variable expression of Ia antigens in human endometrium and in chronic endometritis

Recent studies suggest that Ia antigens may be expressed in epithelial cells and that their expression may be under hormonal control. Therefore, the distribution of these antigens was studied in frozen sections of 37 human endometria with two monoclonal antibodies (Mab) to monomorphic determinants of Ia antigens using an avidin-biotin-complex (ABC) method. Five early proliferative, 9 midproliferative, 3 late proliferative, and 12 secretory endometria were examined. Two gestational endometria and six endometria with chronic endometritis were also used. Four consecutive sections from each case were stained for Ia, OKT8, Leu-3a, and B1 antigens. Throughout the cycle, the endothelial cells, many lymphocytes, and various monocytic-macrophagic cells in endometrial stroma were Ia positive. Furthermore, Ia antigens were localized to the normal endometrial epithelium. The intensity and the pattern of Ia expression, however, varied in different phases of the cycle. Ia antigens were stained weakly in endometrial glands and surface epithelium in early proliferative phase, and strongly in surface epithelium and glandular cells of the basalis and to a lesser extent of the functionalis in midproliferative and late proliferative phases. The expression of Ia antigens in epithelium was absent or focal during the secretory phase and in gestational endometria. Throughout the cycle and in gestational endometria, glandular cells in intimate association with lymphocytic aggregates were Ia positive. In chronic endometritis, the increased number of Ia positive stromal lymphoid cells was associated with a strong display of Ia antigens in epithelium. The findings indicate that, in addition to endothelial and lymphoid cells, Ia antigens are expressed in endometrial glandular and surface epithelial cells. This expression may be influenced by lymphoid cells in endometrium and by hormones.     

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.     

Thymidine phosphorylase expression in normal and hyperplastic endometrium

AIMS: To investigate the expression of thymidine phosphorylase (TP), a known angiogenic factor for endothelial cells, in normally cycling endometrium and various forms of endometrial hyperplasia. METHODS: TP expression was assessed with the P-GF.44C monoclonal antibody, using the alkaline phosphatase anti-alkaline phosphatase method. Ninety two normal and hyperplastic endometria were studied. RESULTS: In normal proliferative endometrium, TP is found exclusively in the basal layer and the inner third of the functionalis; expression is cytoplasmic in glandular epithelium and nuclear in stromal cells. It is invariably patchy. This immunohistochemical picture remains almost unaltered during the early and mid secretory phase of the normal menstrual cycle but, most impressively, TP is expressed uniformly in the epithelium of all endometrial glands towards the end of the cycle. At this stage, expression is mixed nuclear/cytoplasmic and there is very little stromal nuclear staining. In simple endometrial hyperplasia, the staining pattern for TP is identical to normal proliferative endometrium, with a distribution that is usually limited to a few rather weakly proliferating glands and to the adjacent periglandular stroma of the deep endometrium. The distribution is more extensive in complex and atypical endometrial hyperplasias, where a mixed nuclear/cytoplasmic pattern usually prevails over the pure cytoplasmic reaction. CONCLUSIONS: TP is expressed consistently in normal and hyperplastic endometrium, suggesting a role in physiological and pathological angiogenesis. In normal endometrium, TP has a definite pattern of distribution, which is dependent on the phase of the menstrual cycle, whereas in all forms of endometrial hyperplasia the enzyme is randomly distributed and lacks an orderly pattern.     

Decreased level of the cell cycle regulator p27 and increased level of its ubiquitin ligase Skp2 in endometrial carcinoma but not in normal secretory or in hyperstimulated endometrium

p27 is a cyclin-dependent kinase (CDK) inhibitor whose specific late G(1) destruction allows progression of the cell across the G(1)/S boundary. The protein is ubiquitinated by S-phase kinase-interacting protein-2 (Skp2) following its specific phosphorylation, and is subsequently degraded by the 26s proteasome. There is a direct relationship between low level of p27 and rapid proliferation occurring in several benign states and in many malignancies. In the glandular cells of the normal endometrium, the level of p27 is exceedingly low during the proliferative phase, whereas it is markedly increased during the secretory phase. The expression of p27 in endometrial carcinoma is very low but has been found to increase following treatment with progesterone. However, estrogen exposure is considered as a major risk factor in developing endometrial cancer. The implications of the high dose of estrogen and progesterone induced during IVF treatment are still unknown. We have examined the expression of p27 and Skp2 as well as of Ki67 proliferation marker by using endometrial extracts and cells from normal endometrium, from ovarian hyperstimulated patients, and from endometrial carcinoma patients. The expression of p27, Skp2 and Ki67 was found to be similar in both normal secretory endometrium and endometrium from ovarian hyperstimulated patients. In striking contrast, p27 is significantly lower while Skp2 and Ki67 are significantly higher in the endometrial carcinoma and in endometrium from the proliferative phase compared with their normal secretory counterpart tissue.     

The progressive rise in the expression of alpha crystallin B chain in human endometrium is initiated during the implantation window: modulation of gene expression by steroid hormones

Human endometrium undergoes sequential changes during the menstrual cycle and becomes receptive to implantation during a defined period in the secretory phase. We attempted to identify the genes expressed during this period by representational difference analysis (RDA). When the cDNAs of a proliferative endometrium were used as the driver and the cDNAs of a post-ovulatory day 5 endometrium were used as the tester, a number of bands were identified by RDA. DNA of the cloned RDA products revealed that the majority of the clones contained a fragment of a cDNA identical to that of a crystallin B chain. Northern blot analysis showed that the expression of the alpha crystallin B chain mRNA was absent during the proliferative phase. The expression of the mRNA of alpha crystallin B chain first appeared in the secretory phase, progressively increased during this phase and peaked in the late secretory endometria. The pattern of expression of alpha crystallin B chain mRNA in the endometrium of mature cycling baboons (Papio anubis) was similar to that seen in human endometrium. As revealed by Western blot analysis, the expression of the alpha crystallin B chain protein in human endometrium followed a pattern of expression similar to its mRNA. At the cellular level, the immunoreactive protein first appeared in the surface epithelial cells of human endometrium within the implantation window without significant immunoreactivity in the underlying glandular cells. During the mid- and late secretory phases, the intensity of staining in the epithelial cells was enhanced and an intense immunoreactivity was developed in the glandular epithelium, alpha crystallin B chain was virtually an epithelial product and no immunoreactivity for this protein was detectable in the stromal cells, endothelial cells or lymphoid cells. The expression of alpha crystallin B chain could be regulated, by medroxy progesterone acetate as well as by oestrogen withdrawal, in human endometrial carcinoma cells (EnCa- 101), transplanted to nude mice. Based on the data presented here, the known function of alpha crystallin B chain and its distinct pattern of expression in human endometrium, we suggest that this protein is an important factor within the molecular repertoire that makes endometrium receptive to implantation.