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.     

Human milk-fat globule membrane antigens (Mam-3 group) in normal cycling endometrium and endometrial carcinomas--an immunohistochemical study. A preliminary report

The expression of Mam-3 antigens in normal human endometrium and endometrial carcinomas was investigated employing the monoclonal antibodies 67D11 (anti Mam-3a), 115H10 (anti Mam-3b), and 115C2 (anti Mam-3c). Dewaxed sections of formalin-fixed, paraffin-embedded curettings of 9 proliferative-, 6 interval-, 13 secretory endometria, as well as 16 endometrial carcinomas were used. In normal cycling endometrium the Mam-3a, -3b and -3c antigens were detected at the apical membrane of surface and/or glandular epithelium only, but to a different degree. The Mam-3a antigen was only detected in the surface epithelium of 66% of proliferative and 33% of interval endometrium. The Mam-3b antigen was detected in surface and glandular epithelium of all normal endometria, but was most widespread in proliferative endometrium. The Mam-3c antigen was detected in surface epithelium of proliferative, and interval endometrium especially, and in glandular epithelium in 22% of endometria irrespective of the phase. Increased expression of Mam-3a and -3c antigens was recorded in most carcinomas. A loss of polarity of staining was seen in grade III carcinomas especially, staining of the cytoplasma was recorded in half of the carcinomas. The expression of Mam-3c antigen in carcinomas seemed to decline with increasing grade of anaplasia. All carcinomas expressed the Mam-3b antigen. The present results indicate that the Mam-3a and -3c antigens are tumor-associated antigens of endometrial carcinomas and that they might be useful as diagnostic tools in endometrial pathology.     

Disparate actions of mifepristone (RU 486) on glands and stroma in the primate endometrium.

Besides being an antiprogestin, mifepristone (RU 486) was recently shown to antagonize oestrogen-dependent growth in the endometrium. To explore the molecular mechanisms for this phenomenon, we investigated whether or not the morphological effects of mifepristone are mediated by the progesterone receptor (PR) and whether mifepristone has disparate effects on the glandular epithelium and stroma. Six groups of hypogonadal, oestrogen-primed cynomolgus monkeys were treated for 2 weeks with: vehicle only (group I); mifepristone (group II); mifepristone plus progesterone at 0.2 mg/kg (group III), 1.0 mg/kg (group IV) or 5.0 mg/kg (group V); and progesterone only (5.0 mg/kg) (group VI). Histomorphological evaluation showed strikingly compacted stroma in the mifepristone-exposed endometria (group II), which was partially reversible by additional progesterone treatment (groups III-V). Glandular proliferation (pseudostratification, glandular mitoses) in mifepristone-treated monkeys was not significantly different from that in vehicle (oestradiol)-treated monkeys, but was inhibited by progesterone-only treatment. Cells containing vacuoles were scarce in the mifepristone-exposed endometrium, but detected frequently in progesterone-exposed endometria, indicating the strong antisecretory effect of mifepristone on glands. We conclude that oestrogen-dependent oedema in the stroma is antagonized by mifepristone. The reversal of this effect by progesterone suggests a PR-mediated mechanism. In glands, mifepristone is antiprogestogenic, but not antioestrogenic. Thus, stromal cells may be the target of antiprogestin-induced inhibition of oedema and endometrial growth.     

The effects of levonorgestrel implants on vascular endothelial growth factor expression in the endometrium

Vascular endothelial growth factor (VEGF) expression and the microvascular density of the endometrium were studied in Norplant users and normal controls, using immunohistochemistry on formalin-fixed paraffin-embedded endometrial sections. The VEGF staining index was quantified using computerized image analysis. The VEGF staining index between stages of the menstrual cycle and between normal and Norplant endometria were compared. Norplant VEGF staining index was analysed for correlation with microvascular density, duration of Norplant use, the number of bleeding/spotting days in the reference period up to 90 days prior to biopsy, and the length of time since the last bleeding/spotting episode. The results showed that immunoreactive VEGF was detected predominantly in endometrial glands but weakly expressed in the stroma throughout the menstrual cycle, and also in Norplant users. Large variation in the VEGF staining index between individuals was observed and no significant difference in the VEGF staining index was detected between stages of the menstrual cycle for the glands and stroma. The glandular and stromal VEGF staining indices were significantly higher in Norplant than in normal endometrium (P<1x10(-4)). No correlation was found between the Norplant VEGF staining index and endometrial microvascular density, duration of Norplant use, the number of bleeding/spotting days in the reference period, and the length of time since the last bleeding/spotting episode. The VEGF staining index was higher in glands than stroma for both normal and Norplant endometrium. The results suggest a differential control of endometrial glandular versus stromal VEGF expression, and possible positive effects of levonorgestrel on VEGF expression.     

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.     

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.     

The effect of various doses of mifepristone on endometrial leukaemia inhibitory factor expression in the midluteal phase--an immunohistochemical study

Leukaemia inhibitory factor (LIF) is a cytokine which plays an obligatory role in mouse blastocyst implantation. In human endometrium, LIF expression is significantly increased in the mid-luteal phase indicating that LIF may also play an important role in the human. We have previously shown that a single dose of 200 mg of mifepristone immediately post-ovulation is an effective contraceptive method, probably due to inhibition of endometrial development and function. The purpose of this study was to investigate the effect of various doses of mifepristone on endometrial LIF expression. A total of 22 fertile, regularly-menstruating women were studied during control and treatment cycles. The subjects were divided into four groups: group I received a single dose of 200 mg of mifepristone on cycle day LH + 2 (n = 7). The subjects in groups II and III were treated with either 5 mg (n = 5) or 2.5 mg (n = 5) once a week for 2 months. Group IV subjects received 0.5 mg per day (n = 5) of mifepristone for 3 months. LIF was measured immunohistochemically in endometrial tissue specimens taken on the corresponding day (cycle day LH + 6 to LH + 8) in hormonally- characterized control and treatment cycles. LIF immunostaining was observed in all controls and located to the cytoplasm of the luminal and glandular epithelial cells and stromal cells. In the treatment cycles the staining of luminal epithelium and stroma was similar to controls, while the glandular staining was reduced in all treatment groups. This study reveals that early luteal phase treatment as well as intermittent or daily low dose treatment with mifepristone reduces endometrial glandular LIF expression at the expected time of implantation. The results further support the contraceptive potential of mifepristone in low doses.      

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.     

Ubiquitous expression of TNF-alpha/cachectin immunoreactivity in human endometrium.

Emerging evidence suggests that cytokines play significant roles as intercellular communication signals in human endometrium. In the present report, an immunohistochemical staining method and tumor necrosis factor-alpha (TNF-alpha) specific polyclonal antibody was used to identify potential in vivo sources of this cytokine in human endometrium. During the proliferative and early secretory phases of the menstrual cycle, glandular epithelium was distinctly free of any immunoreactive product. With the emergence of mid- and particularly late-secretory phases, however, some glandular epithelial cells started to express TNF-alpha. In the late secretory phase, glands exhibiting strong staining were adjacent to those that were negative for TNF-alpha or only sparingly contained positively immunostained cells. Remarkably, the glandular epithelium constituted a major source of enhanced expression of TNF-alpha during gestation. In the proliferative phase, virtually all stromal cells strongly expressed TNF-alpha, and in the secretory phase, some variability of staining could be observed among various stromal cells. In gestational endometria, decidual cells and in all endometria, endothelial cells strongly expressed immunoreactivity for TNF-alpha. These data demonstrate that various constituents of endometria are constitutively primed to express TNF-alpha and that this expression is distinctly associated with the changes in endometrium that are compelled by hormonal stimuli.     

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.     

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.     

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.     

胎儿及育龄期子宫内膜P63表达及其意义

目的观察P63蛋白在胎儿及育龄期子宫内膜上皮的表达。方法12～39周龄的胎儿和育龄期子宫内膜标本各20例。采用免疫组织化学SP法检测子宫内膜P63蛋白的表达情况。结果胎儿子宫内膜自12周起可检测到P63蛋白阳性细胞,主要定位于表面上皮,随孕龄增加表达逐渐减弱;育龄期子宫内膜仅在1例增生期子宫内膜检测到P63阳性细胞,主要定位于腺上皮散在的零星细胞,表达强度低于胎儿子宫内膜组织。结论P63蛋白与子宫内膜上皮细胞的成熟程度、分化潜能与增殖有关。     

The effect of mifepristone on the expression of insulin-like growth factor binding protein-1, prolactin and progesterone receptor mRNA and protein during the implantation phase in human endometrium

Insulin-like growth factor binding protein-1 (IGFBP-1) and prolactin are recognized as crucial signals for the initiation and maintainance of decidualization. The purpose of the study was to investigate the effect of mifepristone on the expression of IGFBP-1, prolactin and progesterone receptors (PR) during the implantation phase in human endometrium. Eight fertile women were studied during control and treatment cycles. Treatment with 200 mg of mifepristone was administered on day LH +2. Endometrial samples were collected on day LH +6 to +8. Expression of IGFBP-1, prolactin and PR was identified using immunohistochemistry, and mRNA levels were determined with RT-PCR. In control specimens, IGFBP-1 and prolactin were localized to the cytoplasm of the endometrial glandular and to a lesser extent in stromal cells. In the same samples, PR immunoreactivity was detected in the nucleus of the endometrial stromal cells, and was absent from the glandular cells. After mifepristone treatment, there was a significant increase in the immunostaining and mRNA expression for IGFBP-1 and PR. Prolactin expression increased only slightly after treatment. These results support the view that administration of mifepristone in the early luteal phase does not simply retard endometrial development. Our findings provide further insight into the regulation of IGFBP-1 and prolactin by PR in the human endometrium in vivo.     

Angiogenic factors in normal endometrium and endometrial adenocarcinoma

In the endometrium, angiogenesis plays important roles not only in tumor growth but also in the menstrual cycle. The purpose of the present paper was to investigate immunohistochemically the correlation between angiogenic factor expression and angiogenic score in normal and neoplastic endometrium. Immunohistochemical staining for vascular endothelial growth factor (VEGF), angiopoietin (Ang)-1, Ang2, Tie2, CD34 and CD105 was performed on formalin-fixed and paraffin-embedded tissues from 31 normal endometrium and 85 endometrial adenocarcinoma. VEGF, Ang1, Ang2 and Tie2 expression was localized in the cytoplasm of glandular and tumor cells. The levels of each angiogenic factor were different in the phases of the menstrual cycle and each layer of normal endometrium. In general, VEGF and Tie2 expression was higher in adenocarcinoma than in normal epithelial cells. Conversely, Ang1 and Ang2 expression was higher in normal epithelium than in adenocarcinoma. The angiogenic score (CD105/CD34) tended to be higher in the adenocarcinoma than in the normal epithelium. It is suggested that the angiogenic pathway and the role of these factors seem to differ between normal tissue and carcinoma of the endometrium.     

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.      

Effects of mifepristone on proliferation and apoptosis of human endometrium in new users of medroxyprogesterone acetate

BACKGROUND: Mifepristone has been demonstrated to decrease breakthrough bleeding (BTB) in users of progestin-only contraceptives. METHODS: Endometrial biopsies were collected from 50 normal cycling women who were new users of depot medroxyprogesterone acetate (DMPA) randomized to receive either mifepristone or placebo before, during and after treatment. Proliferation, apoptosis and sex steroid receptors were evaluated by either immunohistochemistry or TUNEL assay. RESULTS: Administration of mifepristone to DMPA-exposed endometrium for 1 week significantly increased endometrial expression of Ki-67 (MKI67), estrogen receptor (ER)alpha and progesterone receptors A and B (PRAB) and decreased the number of TUNEL-positive and caspase-3 (CASP3)-active cells in the endometrial stroma. However, after 10 weeks of mifepristone treatment, no significant difference in proliferation, apoptosis and the expression of ERalpha or PRAB could be detected between the endometrium treated with DMPA alone and endometrium treated with mifepristone and DMPA. CONCLUSIONS: Administration of mifepristone to DMPA users significantly increases endometrial proliferation and decreases endometrial stromal apoptosis in the short term. Prolonged exposure to mifepristone does not counteract the inhibitory effects of progestin therapy on endometrial proliferation. Estrogen and progesterone receptors may play an important role in these effects.