Co-expression of CXCR4 and CXCR7 in human endometrial stromal cells is modulated by steroid hormones

Endometrium modulated by estrogen (E) and progesterone (P) is important for implantation and pregnancy. The present study compared the expression of chemokine CXCL12 and chemokine receptor CXCR4 and CXCR7 between human cycling and early pregnant endometria by immunohistochemistry (IHC). Then the modulation of E and P on expression of CXCL12, CXCR4 and CXCR7 in human endometrial stromal cells (ESCs) was explored at both mRNA and protein level. The result of IHC showed that human ESCs of the menstrual period did not express CXCL12, CXCR4 or CXCR7 protein, however, the expression of CXCR4 and CXCR7 but not CXCL12 in ESCs increased in the proliferative and secretory phase, and the expression intensity for CXCR4 and CXCR7 in ESCs was the highest in the first trimester. Moreover, E and P were able to up-regulate the mRNA and protein expression of CXCR4 and protein expression of CXCR7 in ESCs (P<0.01). Thus, ESCs spatiotemporally co-express CXCR4 and CXCR7 rather than CXCL12, and E and P are able to regulate the expression of CXCR4 and CXCR7 in ESCs, suggesting the modulation of steroid hormones on chemokine receptor expression in ESCs.     

Secretory endometrium highly expresses urocortin messenger RNA and peptide: possible role in the decidualization process

BACKGROUND: Urocortin (UCN) gene expression and synthesis have been reported in epithelial and stromal cells of the human endometrium. In this study we evaluated (i) UCN messenger RNA (mRNA) expression and peptide production in uterine specimens collected throughout the endometrial cycle, (ii) UCN secretion after decidualization of cultured human endometrial stromal cells (HESCs) and (iii) the effect of UCN on endometrial decidualization. METHODS: HESCs were isolated from samples of human endometrium collected from healthy patients with normal menstrual cycle and cultured in presence of cAMP, 17-beta-estradiol (E(2)) + medroxyprogesterone acetate (MPA) and UCN. UCN levels were measured in endometrial extracts by an enzyme immunoassay, and changes of endometrial UCN mRNA expression were measured by RT-PCR analysis. RESULTS: UCN peptide concentrations and mRNA expression were highest in the secretory phase of the menstrual cycle (P < 0.001, late secretory versus early and late proliferative phase) and higher in the late than the early secretory phase (P < 0.01). After decidualization of HESC with cAMP or E(2) + MPA, UCN levels rose in parallel with prolactin concentrations by days 6 (P < 0.01, for all). Finally, the addition of UCN to HESCs, with or without E(2) + MPA, induced the release of prolactin. CONCLUSIONS: The evidence that (i) UCN is highly expressed in the secretory phase of the endometrial cycle; (ii) cAMP and E(2) + MPA modulate secretion of UCN and (iii) UCN induces HESCs decidualization together suggest a possible role for UCN in endometrial physiology.     

P-glycoprotein expression is increased in human secretory and gestational endometrium.

To determine the expression, distribution, and intracellular localization of the multi-drug resistance gene product P-glycoprotein (Pgp) in the human menstrual cycle and in early gestational endometrium, we retrospectively studied 36 endometrial samples utilizing 3 murine monoclonal antibodies (MAbs), MAb C219, MAb C494, and MAb JSB-1, which recognize spatially distinct cytoplasmic epitopes of Pgp. Formalin-fixed, paraffin-embedded endometrial samples obtained from 36 women of reproductive age with normal menstrual cycles were assigned morphologic menstrual dates: proliferative (N = 10), secretory (N = 19), menstrual (N = 1), and gestational endometrium (N = 6). The cellular localization, staining intensity, and percentage of Pgp immunoreactive cells varied with the phase of the menstrual cycle. Early proliferative endometria revealed no Pgp immunoreactivity for all three MAbs. Mid-proliferative endometria showed weak immunostaining in less than 15% of the glandular epithelia. Late proliferative endometria showed a strong apical paranuclear/Golgi staining pattern. Early secretory endometria showed strong luminal membranous, subnuclear vacuolar membranous, and supranuclear vacuolar membranous immunostaining to all 3 MAbs in greater than 80% of the glandular epithelia. Apical paranuclear/Golgi and membranous staining were present in nonvacuolated mid-secretory glands. Immunoreactivity diminished in the late secretory phase with mild to moderate staining in less than 35% of the endometrial glands. Menstrual endometria showed weak, focal staining. All gestational endometria showed marked cytoplasmic, membranous, and apical/Golgi immunostaining both in the hypersecretory (Arias-Stella) endometrial glands as well as in the decidua. In general, the intensity of MAb C494 immunostaining was weaker than that of MAb C219 or JSB-1. These results suggest the following: Pgp expression parallels that of nuclear progesterone receptor expression in the normal human endometrial cycle and early gestational endometrium; Pgp expression corresponds to rising plasma and tissue levels of progesterone as well as to morphologic changes in the endometrial glandular epithelium associated with the marked development of the secretory apparatus; Pgp expression is hormonally regulated and may be involved in uteroplacental transport of substrates important in the implantation process and in early embryo-endometrial interactions; and Pgp may be involved in the transport of progesterone across the uterine epithelium during pregnancy.     

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.      

Mid-luteal serum inhibin-A concentration as a marker of endometrial differentiation.

BACKGROUND: Recent studies have indicated that the corpus luteum is a major source of circulating inhibin-A and serum concentrations of inhibin-A may reflect the human luteal function. The present prospective study was undertaken to determine the usefulness of mid-luteal serum concentrations of inhibin-A as markers of endometrial receptivity (as assessed by histological dating and alphavbeta3 integrin expression) and whether they are better predictors of endometrial function than serum progesterone. METHODS: Consecutive infertile women (experimental group, n = 50) with regular menstrual cycles, and fertile women who were requesting contraception and had regular menstrual patterns and normal secretory endometria (control group, n = 10) were included. In all women basal body temperature, luteal serum concentrations of oestradiol, progesterone, prolactin, and inhibin-A, and endometrial biopsies were used in the same cycle to assess luteal function. RESULTS: Out-of-phase mid-secretory endometria were detected in 17 of the 50 infertile women. Lack of alphavbeta3 integrin expression was detected in 27 of the 50 mid-luteal endometrial biopsies. Thus, hormonal concentrations were compared in the mid-luteal phase between the following eight groups of women: group 1 (n = 10), control fertile women; group 2 (n = 50), infertile women (all); subdivided into group 3 (n = 33), with in-phase biopsies; group 4 (n = 17), with out-of-phase endometria; group 5 (n = 23), expressing alphavbeta3 integrin in endometria; group 6 (n = 27), whose endometria did not express alphavbeta3 integrin; group 7 (n = 18), with both in-phase endometrial biopsy and alphavbeta3 integrin expression; and finally group 8 (n = 12), whose endometria were out-of-phase and did not express alphavbeta3 integrin. Mid-luteal serum concentrations of oestradiol, progesterone, prolactin, and inhibin-A of the seven infertile groups were similar to those of the control group of fertile women. No statistically significant difference between the infertile groups was observed for any hormonal parameter considered. CONCLUSION: Mid-luteal serum inhibin-A determination does not accurately reflect endometrial function/maturation and it is not a better indicator of endometrial luteal phase dysfunction than mid-luteal serum progesterone.     

Expression of endometrial glycogen synthase kinase-3beta protein throughout the menstrual cycle and its regulation by progesterone

Glycogen synthase kinase-3beta (GSK-3beta) is a serine/threonine kinase that plays a role in glycogen synthesis by inhibiting glycogen synthase (GS) through phosphorylation. We hypothesized that GSK-3beta by virtue of its role in glycogen synthesis through the inhibition of GS will play a role in the preparation of the endometrium for blastocyst implantation. Immunohistochemical (IHC) analysis and Western blot analysis (WBA) detected GSK-3beta in the endometrium, myometrium, Fallopian tube and ovary. WBA showed more than 5-fold higher endometrial expression of the phosphorylated GSK-3beta (pGSK-3beta) isoform (inactive) in the secretory phase as compared with the proliferative phase (P < 0.001), whereas no differences in total GSK-3beta expression were detected. IHC analysis confirmed the WBA and showed marked expression of pGSK-3beta predominantly in glandular epithelial cells in early and mid secretory endometrium with scant expression during the proliferative phase. In in vitro experiments using human endometrial-derived epithelial cell line (HES), progesterone did not alter total GSK mRNA or protein expression. However, progesterone induced a dose-dependent increase in the expression of pGSK-3beta, which could be blocked by RU486. Cyclic expression of GSK-3beta's active and inactive forms in the endometrium suggests that sex hormones regulate the expression of this enzyme. In vitro experiments demonstrate that progesterone through receptor-mediated mechanisms induces phosphorylation of endometrial GSK-3beta.     

Regulated expression of cytokines in human endometrium throughout the menstrual cycle: dysregulation in habitual abortion

It is widely assumed that, after ovulation, the human endometrium undergoes specific changes and becomes receptive to the implantation of embryo during the mid-secretory phase. When implantation does not take place, further changes occur which eventually result in the shedding of human endometrium. The present study was carried out to examine whether there are changes in the cytokine gene expression in human endometrium which are correlated with endometrial function in various phases of the menstrual cycle. The RNase protection assay was performed on carefully dated endometria from normal subjects to characterize the expression of cytokines which potentially contribute to endometrial function. These included: tumour necrosis factor (TNF), interleukin (IL)-1beta, IL-6, IL-8, leukaemia inhibitory factor (LIF), transforming growth factor beta1 (TGF-beta1), macrophage colony stimulating factor (MCSF or colony stimulating factor-1), and vascular endothelial growth factor (VEGF) mRNAs. A low level of expression of these cytokine mRNAs was found during the proliferative and early secretory phase. Expression of cytokine mRNA increased during the mid-secretory phase and rose to a peak in the late secretory phase. The level of cytokine mRNA expression during gestation was most akin to that observed during the mid-secretory phase. Individuals with habitual abortion presented with an abnormal expression of IL-1beta and IL-6 mRNA in endometrium, during the mid-secretory phase. Taken together, these findings are consistent with a progressive rise in the expression of cytokines in human endometrium during the secretory phase in natural cycles. Furthermore, the findings show that habitual abortion is associated with the abnormal expression of IL-1beta and IL-6 in the mid-secretory phase.     

Progestin-dependent effect of forskolin on human endometrial aromatase acitivity

We have previously demonstrated that the aromatase activityin human endometrial stromal cells is stimulated by progestinand enhanced by oestrogen. In this study, we have investigatedthe effect of forskolin (Fit), an agent that stimulates thehormone-sensitive adenylate cydase in mammalian cells, on theintracellular cAMP content and aromatase activity in endometrialstromal cells in primary culture. Stromal cells were isolatedfrom proliferative and secretory endometria and were individuallycultured in nutrient medium or medium supplemented with medroxyprogesteroneacetate (MPA), oestradiol (E2) and Fk, separately or in combination.The intracellular cAMP content of stromal cells was increasedafter incubation with Fk. Stromal cells treated with Fk aloneor FK and MPA for 1-3 days sustained the elevated intracellularcAMP content but 90% of this nuckotide was released to the medium.Aromatase activity was either not affected or was increasedup to 5-fold over the control by Fk alone. Forskolin exerteda synergistic effect upon aromatase activity in the presenceof progestin or progestin and oestradiol. Sequential incubationof the stromal cells with MPA and then Fk indicated that theadditional increase in aromatase activity caused by Fk occurredafter 24 h of incubation. These results demonstrate that intracellularcAMP exerts a stimulatory effect on aromatase activity in progestin-conditionedstromal cells. We also investigated whether the endometrialaromatase activity changes in vivo during the reproductive cycleby measuring the activities in endometrial specimens obtainedfrom women during their reproductive age. The mean values ofaromatase activities (fmol/h x mg protein) in proliferative,secretory endometria and decidua from first trimester of thegestation were 1.6 ? 1.0 (mean ? SD, n = 10), 3.0 ? 3.4 (n =13) and 1500 ? 900 (n = 20), respectively. These results indicatethat stimulation of endometrial aromatase activity occurs afterconception under physiological conditions. These observationsopen possibilities for the study of the role of cAMP in theexpression of regulatory processes dependent upon steroid hormonesin endometrial cells     

Steroid receptors and proliferative activity in non-neoplastic and neoplastic endometria

Summary In the glands of cyclic endometria, proliferative activity (PA), as revealed by expression of the Ki-67 antigen, is highest in the proliferative phase (P) and early secretory phase (S1). The PA decreases in the middle secretory phase (S2). In the stroma the PA is low during the whole cycle. In P and S1, the oestrogen receptor (ER) and the progesterone receptor (PR) are strongly expressed in glands and stroma. The number of positive cells and the staining intensity decreases in S2, particularly in the glands. In atrophic endometria, fibro-glandular polyps and in endometria with arrested secretion the PA is low in both glands and stroma. ER and PR can be detected in glands and stroma. The PA in atypical hyperplasias is only slightly higher than in cyclic endometria and endometria with simple hyperplasia. The ER and PR levels are comaparable to those in proliferative endometria. The PA of endometrial adenocarcinomas is positively and the ER and PR negatively correlated with the degree of de-differentiation. No ER-negative carcinoma displays the PR. Immunohistologically, nonneoplastic receptor positive tissue can be seen in many ER- and PR-negative carcinomas. These structures may falsify the biochemical receptor analysis. Prof. Dr. Curt Froboese dedicated to his⁹⁹th birthday     

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.     

Large scale validation of human N-myc downstream-regulated gene (NDRG)-1 expression in endometrium during the menstrual cycle

A major challenge in the comprehension of the endometrial transformations leading to the completion of each menstrual cycle in humans is in the identification of specific molecular pathways underlying these monthly turnovers. Towards this goal we compared, by the differential display technique, the relative expression of mRNA in endometrial biopsies harvested in individuals (n = 48) either at the proliferative or the secretory phase of the menstrual cycle. We isolated a cDNA fragment homologous to NDRG1 (N-myc Downstream-Regulated Gene-1) that is present in markedly higher amounts in the secretory phase. Northern blot analysis and quantitative real time PCR experiments confirmed this result in distinct cohorts of individuals (44 and 560 respectively). A closer examination of data showed that the highest mRNA levels were found during the range of 25-28 days of the uterine cycle. Consistent with the mRNA data, the temporal profile of the NDRG1 protein showed a 15-fold increase during the secretory phase, as demonstrated by using semi-quantitative dot blot analyses (n = 92). Immunohistochemical localization revealed that NDRG1 was expressed both in epithelial and stromal cells. This large scale validation of the NDRG1 mRNA and protein increase in endometrium during the secretory phase is consistent with its differentiation-related function described in other tissues and its potential involvement in the window of implantation of the human endometrium, as suggested by previous chip-based evidence.     

Assessment of the proliferative status of epithelial cell types in the endometrium of young and menopausal transition women

BACKGROUND: We determined protein and mRNA expressions of markers of normalhuman endometrial proliferation and hypothesized that dysregulationof the endometrial response to estradiol (E₂) and progesteronewould be observed in the older menopausal transition (MT) womencompared with mid-reproductive age (MRA) controls. METHODS: Endometrial biopsies were prospectively obtained from MRA andMT non-randomized healthy volunteers during proliferative (±exogenous E₂) and secretory (MRA only) menstrual cycle phases.mRNA and/or nuclear protein expressions of proliferative markers(MKI67, PCNA and MCM2), cell-cycle regulators (cyclins A1, E1and D1 and cyclin dependant kinase Inhibitor B; CCNA1, CCNE1,CCND1 and CDKN1B) and sex-steroid receptors [estrogen receptor(ER) and progesterone receptor (PR)] were assessed in endometriallumen, gland and stroma. RESULTS: MRA women had significantly higher proliferative than secretoryexpression of MKI67, PCNA, MCM2, CCNA1, CCNE1, ESR1 and PGRin lumen and gland (minimal stromal changes), whereas CDKN1Bprotein expression was higher during the secretory phase. E₂-treatmentof MT women led to relatively less MKI67 glandular protein expressioncompared with MRA women; no other age-related differences wereobserved. CONCLUSION: Although the MT does not appear to alter the proliferative cellphenotype of endometrial epithelium and stroma, the data suggestthat prior to the MT, age is associated with a decrease in someproliferative markers and steroid receptor expression statuswithin different endometrial cell types.     

Endometrial expression of epithelial neutrophil-activating peptide-78 during the menstrual cycle or in progestin-only contraceptive users with breakthrough bleeding and the influence of doxycycline therapy

BACKGROUND: Endometrial breakthrough bleeding is characterized by an inflammatory reaction and increased production of proinflammatory mediators, one of which may be epithelial neutrophil-activating peptide-78 (ENA-78), a chemokine with neutrophil-activating properties. METHODS AND RESULTS: We therefore investigated the endometrial expression of ENA-78 in Norplant users as progestin-only contraceptive with various bleeding patterns (n=35) as compared with non-users with a normal menstrual cycle (n=55). The endometrial stromal cells (ESCs) were the major site of ENA-78 expression with the highest levels found during the secretory phase. The expression of ENA-78 was increased in Norplant users with irregular bleeding as compared with those with regular cycles and amenorrhoea. The levels of ENA-78 detected in uterine washes and sera after the use of Norplant for 3-6 months (n=25) increased compared with baseline (P < 0.05). These levels did not significantly change in Norplant users who received doxycycline (Dox) therapy (25 mg/twice daily for 6 months) when measured midway through or at the conclusion of study when compared with the baseline (n=25). Treatments with medroxyprogesterone acetate (MPA) and tumour necrosis factor-alpha (TNF-alpha) (25 ng/ml), but not 17beta-estradiol (E2) or E2 + MPA (10(-8) M), representing endometrium exposed to contraceptive and inflammatory conditions, respectively, increased the levels of ENA-78 production by ESCs, and this was reduced by co-treatments with Dox (25 microg/ml) (P < 0.05). CONCLUSIONS: The endometrial production of ENA-78 is altered in progestin-only contraceptive users experiencing breakthrough bleeding and is regulated by MPA and TNF-alpha in ESCs. Although Dox therapy did not alter uterine ENA-78 secretion, its suppression in ESCs suggests that Dox, acting site-specifically and through an anti-inflammatory mechanism, may influence the outcome of breakthrough bleeding in contraceptive users.     

Involvement of cyclin-dependent kinase inhibitor p27Kip1 in growth inhibition of endometrium in the secretory phase and of hyperplastic endometrium treated with progesterone

A cyclin-dependent kinase (cdk) inhibitor, p27Kip1 (p27), binds to the cyclin E-cdk2 complex and functions as a suppressor of cell cycle promotion. Here, the involvement of p27 in the growth of normal human endometrium was immunohistochemically studied, and the findings were compared with those of Ki-67, cyclin E and cdk2. In addition, to elucidate the effect of progesterone on the expression of p27, tissues from patients with endometrial hyperplasia were examined before and after the administration of medroxyprogesterone acetate (MPA) for the treatment of this disease. In the glandular cells of the normal endometrium, p27 was negligible during the proliferative phase, whereas it was markedly increased in the secretory phase. The staining pattern of Ki-67 was the reverse. Cyclin E/cdk2-positive cells were observed throughout the menstrual cycle. In the secretory phase, the cyclin E/cdk2-positive cells were also positive for p27, suggesting an interaction between these molecules. Stromal cells, especially in the basalis, showed a consistent expression of p27 throughout the menstrual cycle. The expression of p27 in hyperplastic epithelia before the MPA treatment was negligible, whereas it was greatly increased after the treatment. The Ki-67 positivity decreased after the treatment. These findings suggest that p27 is involved in the progesterone-induced growth suppression of normal and hyperplastic endometria.      

Modulation of Expression of Toll-like receptors in the Human Endometrium

Problem  The purpose of the study was to investigate the different expressions of toll-like receptors (TLRs) in the proliferative and the secretory phase of endometrial tissue.
Method of study  Eight infertile women were included in this study. The endometrial tissues of proliferative and secretory phase were obtained from each woman. The tissues were evaluated for the expression of mRNA for TLR1–10 by reverse transcribed polymerase chain reaction (RT-PCR). The proteins of TLR2, 3, 4, and 9 were evaluated by Western blot. The mRNA and protein level of proliferative and secretory endometrial tissue from the same woman was compared. The data were analysed with SPSS15.0.
Results  TLR1–7, 9, and 10 mRNA were expressed throughout the menstrual cycle, but in the same woman, the expression of TLR2–6, 9, and 10 mRNA was higher during the secretory phase than that in the proliferative phase. The Western blot also showed that the protein expression of TLR2, 3, 4, and 9 was stronger in the secretory phase than that in the proliferative phase in the same woman.
Conclusion  The expression of TLRs is cycle dependent in human endometrial tissue. The expressions of TLRs were higher in the secretory phase than that in the proliferative phase: this indicated that TLRs may be regulated by sex hormones throughout the menstrual cycle.     

TGF-beta 1 and IGF-1 expression in atrophic post-menopausal endometrium

OBJECTIVES: Endometrial cells may synthetize cytokines and growth factors which may modulate some of the molecular mechanisms of endometrial proliferation and differentiation. PATIENTS AND METHODS: We investigated the role of transforming growth factor beta-1 (TGF-beta 1), insulin-like growth factor-1 (IGF-1) and relative receptors in five tissue samples from atrophic post-menopausal endometria. The control group was represented by proliferative and secretory endometria from 10 healthy, normally-menstratued women. TGF-beta 1 and IGF-1 m-RNA expression was evaluated by Northern hybridization analysis, while TGF-beta 1 and IGF-1 receptors distribution was studied by immunohistochemistry. RESULTS: In atrophic endometria Northern hybridization analysis showed a significant decrease of IGF-1 expression, and an increase of TGF-beta 1 expression compared to proliferative and secretory endometria. By immunohistochemistry it was demonstrated that TGF-beta 1 and IGF-1 receptors were both localized in cell cytoplasm, mainly in the stromal compartment. CONCLUSIONS: The results of our study would suggest a possible role of IGF-1 and TGF-beta 1 in maintaining the quiescent differentiative state of atrophic post-menopausal endometrium. The persistence of IGF-1 and TGF-beta 1 receptors in epithelial compartment could play a key role in proliferative response of atrophic endometrium to exogenous hormone replacement therapy (HRT) or endogenous intervening high estrogens levels.     

Cyr61, a deregulated gene in endometriosis

Gene expression profiling was performed to identify genes involved in the development of endometriosis. In the secretory phase of the menstrual cycle, several estrogen-regulated genes were up-regulated in endometria of women with endometriosis. The most consistent regulation with one of the highest factors was observed for the Cyr61 gene, which codes for a secreted, cysteine-rich, heparin-binding protein that promotes cell adhesion, migration, and neovascularization. Estrogen responsiveness of endometrial Cyr61 expression was suggested by the higher expression during the proliferative phase and the reduction observed in human endometrial fragments grafted into nude mice subsequently treated with an anti-estrogen. The expression level of Cyr61 was found to be further increased in ectopic endometriotic lesions, as compared to eutopic endometria. In these lesions, an imbalance in expression of the estrogen-converting enzymes 17beta-hydroxysteroid dehydrogenase type 1 and 2 was found, which might explain the elevated Cyr61 level. However, Cyr61 expression was not altered in endometriotic lesions of women treated with a GnRH agonist. These results suggest that Cyr61 may represent a gene characteristic for endometriosis and also play an important role in the development and persistence of endometriotic lesions.     

Expression of c-kit (CD117) in benign and malignant human endometrial epithelium

BACKGROUND: The proto-oncogene c-kit encodes a tyrosine kinase receptor (CD117) with a molecular weight of 145 kd. Previous studies, predominantly utilizing immunohistochemistry, have led to contradictory findings regarding the expression of CD117 in the endometrium. To help resolve this issue, we analyzed a series of benign and malignant endometrial tissues using both immunohistochemistry and Western blot analysis. OBJECTIVE: To examine the expression of CD117 in benign and malignant human endometrial tissues. METHODS: The expression of CD117 in 35 benign endometrial tissues (7 hyperplastic, 14 proliferative, 14 secretory) and 10 endometrioid carcinomas was investigated by immunohistochemistry (clone K45 monoclonal antibody). Immunoprecipitation (clone K69 monoclonal antibody) followed by Western blotting (clone K45 monoclonal antibody and clone 1.D9.3D6 monoclonal antibody) was performed to confirm CD117 expression. RESULTS: Fifty-seven percent of the hyperplasias, 93% of proliferative endometria, and 79% of secretory endometria immunostained positively for CD117. In benign endometria, epithelial staining tended to be more intense in the hyperplastic and proliferative endometria as compared to the secretory endometria, whereas endometrial stromal cells were not immunoreactive. Of the 10 frozen endometrial tissues analyzed by immunohistochemistry, 4 of 9 endometrioid carcinomas and a single case of an endometrioid polyp developing in association with a carcinoma expressed CD117. Immunoprecipitation followed by Western blot analysis confirmed expression of full-length CD117 in an endometrial polyp and carcinoma, and revealed a correlation between levels of immunoprecipitated CD117 and immunohistochemical staining intensity. CONCLUSIONS: Benign and malignant endometrial tissues express CD117. Our data suggest (a) a possible relationship between estrogen and CD117 expression in benign endometrium and (b) potential involvement of this growth factor receptor in endometrial carcinogenesis.