Progressive rise in the expression of interleukin-6 in human endometrium during menstrual cycle is initiated during the implantation window

In order to be prepared for implantation, human endometriumundergoes a predictable series of proliferative and secretorychanges. Cytokines play an important role in regulation of thesechanges. Therefore, in this study, we immunolocalized the cytokine,interleukin-6 (IL-6), its receptor and the signal transducergp130 in human endometrium throughout the menstrual cycle. Duringthe entire menstrual cycle, the IL-6 receptor and gp130 werefound primarily in the endometrial glands and to a lesser extentin the stroma. The immunoreactivity of these proteins did notchange in endometrial cells during the entire menstrual cyclewith an exception of reduced immunoreactivity of gp130 in endometrialglands during menstrual phase. Immunostaining showed that immunoreactiveIL-6 was weakly expressed in human endometrium during the proliferativephase. Strong immunoreactivity for IL-6 appeared in endometriumduring the putative 'implantation window'. Expression was byfar most pronounced both in the glandular and surface epithelialcells. The amount of immunoreactive IL-6 in the epithelium progressivelyincreased during the secretory/menstrual phases. During thelate secretory phase, only stromal cells in the upper functionalisexhibited immunoreactivity for IL-6. Western blot analysis corroboratedthe immunohistochemical data. Human endometrial IL-6 consistedof a protein with an apparent mobility of 26 kDa. The immunoreactiveband of IL-6 was weak in the proliferative phase. The expressionof this protein increased progressively during the secretory/menstrualphases. The findings show a cell-specific pattern of distributionfor immunoreactive IL-6 in human endometrium. The menstrualcycle-dependent expression of IL-6 suggests that this cytokinemay play a role in changes in endometrium that prepare thistissue for implantation and menstrual shedding. cytokine/endometrium/implantation/interleukin/interleukin-6     

Galectin fingerprinting in human endometrium and decidua during the menstrual cycle and in early gestation

The emerging functionality of the sugar code via cell surface glycans and endogenous lectins ascribes pertinent roles in cell physiology to the carbohydrate signals of cellular glycoconjugates. To initiate monitoring of endogenous lectins in human endometrium, we focused on a family of growth/adhesion-regulatory lectins, i.e. galectins. Comprehensive fingerprinting was performed on samples throughout the menstrual cycle and in decidua. The endometrium (n = 30) and decidua (n = 7) were collected from patients undergoing hysterectomy for benign reasons and from induced abortions. Measurements by RT-PCR and then by multiprobe RNase protection assay with total endometrial and decidual tissue and with epithelial cells, stromal cells and CD45-positive cell fractions (n = 16), isolated by the use of antibody-coated magnetic beads, revealed a predominant expression of galectins-1 and -3. Protein analysis was performed by immunocytochemistry with monoclonal and polyclonal antibodies (n = 40). Galectin-1 was localized mainly in stromal cells, whereas galectin-3 was predominantly found in epithelial cells. Expression of galectin-1 increased significantly in the late secretory phase endometrium and in the decidual tissue. Expression of galectin-3 increased significantly during the secretory phase of the menstrual cycle. Cycle-dependent expression of galectin-1 in stromal cells and galectin-3 in epithelial cells suggest these lectins to be involved in the regulation of different endometrial cellular functions.     

In search of candidate genes critically expressed in the human endometrium during the window of implantation

BACKGROUND: In this prospective randomized blinded clinical trial, we examined gene expression profiles of the human endometrium during the early and mid-luteal phases of the natural cycle. METHODS: An endometrial biopsy was performed on day 16 (LH +3) or on day 21 (LH +8), followed by RNA extraction and microarray analysis using an Affymetrix HG-U95A microchip. Data analysis was carried out using pairwise multiple group comparison with the significance analysis of microarrays (SAM) software. RESULTS: With a false discovery rate of 0, the analysis revealed that 107 genes were significantly and differently expressed (> or =2-fold) during the early versus the mid-luteal phase of the cycle. Forty-five of these genes have not been previously linked to endometrial receptivity. Validation of the microarray data was accomplished using semiquantitative RT-PCR. We demonstrated the presence of estrogen and progesterone response elements (ERE and PRE) by analysis of the 5'-flanking regions of a subset of differentially regulated genes. CONCLUSIONS: Using a strict bioinformatics approach of microarray data, we demonstrated significant changes in candidate genes during the transition of the early to the mid-luteal phase of the human endometrium that may have functional significance for the opening and maintenance of the window of implantation.     

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.     

Patterns of expression of integrin molecules in human endometrium throughout the menstrual cycle.

A heterogeneous group of cells interact with each other and with the surrounding matrix to form the complex structure of human endometrium. Since the integrin superfamily of molecules is involved in the cell-cell and cell-matrix interactions, this study was designed to screen, in situ, the cellular distribution of CDW49a-f molecules in human endometrium throughout the menstrual cycle. The integrin molecules were localized by immunohistochemistry using monoclonal antibodies. Glandular epithelium expressed all integrin molecules. With the exception of CDW49d (alpha 4 beta 1), surface epithelium also expressed all these molecules. Endothelial cells were positive for all integrin molecules except CDW49a (alpha 1 beta 1). Endometrial lymphoid cells were positively immunostained for CDW49a, d and e (alpha 5 beta 1) and were negative for CDW49b (alpha 2 beta 1), CDW49c (alpha 3 beta 1) and CDW49f (alpha 6 beta 1). Regional differences in the expression of integrin molecules were observed. As compared to the functionalis epithelium, basalis epithelium characteristically exhibited higher expression of CDW49a, d and e. Two integrins in endometrium, CDW49a and d exhibited changes related to the menstrual cycle. CDW49a, which was not expressed in glandular epithelial cells in the proliferative phase, was strongly expressed in these cells after ovulation and its expression was diminished in the late secretory phase. This molecule was not expressed in the stromal cells, however, predecidual cells characteristically expressed this molecule in the late secretory phase. CDW49d was only expressed in the glandular epithelial cells in the mid-proliferative to mid-secretory phases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Implantation and early pregnancy: Endometrial Th2 cytokine expression throughout the menstrual cycle and early pregnancy

Embryonic implantation and maintenance of pregnancy is influencedby the maternal immunological response. Type 2 T-helper (Th2)cells secrete interleukins 4, 5, 6 and 10 and are associatedwith suppression of cell-mediated immunity. Temporal changesin the expression of these cytokines were detectable by immunohistochemistrythroughout the menstrual cycle. In pregnancy, 10-fold or greaterincreases in interleukin 6 and 10 secretion were detectableby enzyme-linked immunoassay compared with the non-pregnantstate. The pattern of expression of Th2 cytokines suggests thatprogesterone may Influence endo metrial cytokine secretion.During pregnancy, Th2 expression paralleled that of viinentin,a stromal cell marker, suggesting that these cells may be aprincipal source of these cytokines in the gravid uterus. Increasedsecretion of Th2 cytokines may be a mechanism of suppressingcell- mediated immunity in the endometriuin, which may in turnenhance embryonic implantation and maintenance of pregnancy.     

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.     

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.     

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.     

Changes in basement membrane thickness in the human endometrium during the luteal phase of the menstrual cycle

We have examined aspects of the fine structure of the basal laminae associated with the luminal and glandular epithelium and small blood vessels in the human endometrium. Four short studies are presented and reviewed. Study 1 examined biopsies from 20 fertile women taken on days after the luteinizing hormone surge (LH): LH +2, 4, 6, 8 and 10. The basal lamina (both lamina densa and lucida) increased in thickness over the period studied. Study 2 again studied the glandular epithelium and examined the effect of RU486 (a progesterone receptor blocker) administered on day LH +3 and biopsied on day LH +6. The basal laminae were found to be the same as LH +2 control group but thinner than LH +6 control. Study 3 documented increased thickness of the basal laminae between LH +6, 8 and 13 in the luminal epithelium. The within-group coefficient of variation was 16% and 27% for LH +6 and LH +13 groups but only 2 % for LH +8. Study 4 demonstrated an increase in basal lamina thickness associated with small blood vessels between LH +6 and LH +10 in normal fertile women. The basal lamina provides the interface between epithelial and mesenchymal environments; changes in its structure can alter the phenotypic expression of the epithelia. It is one of the maternal barriers that must be transgressed by the trophoblast during implantation. Together, these combined studies provide quantitative baseline structural information on the electron microscopical appearance of the basal lamina during the luteal phase of the menstrual cycle.     

Immunohistochemical staining of von Willebrand factor in human endometrium during normal menstrual cycle

Endometrial biopsies were obtained from 73 normal women throughoutthe menstrual cycle. Using a polyclonal antibody and a streptavidin–biotin–peroxidasemethod, formalinfixed paraffin sections of the tissue were stainedfor von Willebrand factor (vWF). Both subjective scoring andobjective quantitative colour image analysis were used to assessthe staining intensity, and the results obtained by the twomethods were in concordance with each other. Positive stainingwas observed at all stages of the menstrual cycle. Specificstaining was confined to the vascular endothelium and showedcyclical changes. The staining intensity was the weakest duringthe menstrual phase and was significantly (P < 0.02) reducedfrom all other stages of the cycle, except late secretory phase.This was followed by a rapid increase in the early proliferativephase to reach a peak in the mid cycle before gradually fallingoff towards the end of the cycle. The staining intensity inthe late secretory phase was significantly reduced (P < 0.05)from other stages except menstrual, early proliferative andmid secretory phase. Vascular staining for vWF was heterogeneouswith some vessels devoid of any positive staining.     

Heat shock proteins in human endometrium throughout the menstrual cycle

Human endometrium is a steroid-sensitive tissue and there isevidence that supports the viewpoint that heat shock proteins(HSP) are implicated in the regulation of steroid function.Therefore, in this study we examined the expression of variousmembers of the heat shock family of proteins in the steroid-responsivehuman endometrium. Western blot analysis revealed that the expressionof HSP90 showed minimal changes throughout the menstrual cycle.When normalized to the amount of HSP90, the expression of HSP27,HSP60 and the constitutive form of heat shock protein 70 (HSC70)increased progressively during the late proliferative and earlysecretory phases, and diminished in the mid- to late secretoryand menstrual phases. In contrast, the inducible form of heatshock protein 70 (HSP70) did not undergo these changes. Thecellular and subcellular localizations of these proteins wereexamined in human endometria by immunohistochemical staining.With the exception of HSP70, which was found primarily in theepithelial cells, the immunoreactivity for other heat shockproteins was found in both the stroraa and the epithelium. Immunoreactivityfor HSP27 was found in the lymphoid aggregates within endometrialstroma, and both HSP27 and HSP90 were found in endothelial cells.The immunoreactive heat shock proteins were found in the nucleiand/or cytoplasm of cells. However, no consistent nuclear versuscytoplasmic staining emerged, and such localization was irrespectiveof the site, the cell type or the phase of the menstrual cycle.Our findings show that endometrium has a full complement ofheat shock proteins. The menstrual cycle-dependent changes inthe amounts of heat shock protein suggest regulation by steroidhormones.     

Uterus and endometrium: Inhibition of human endometrial stromal cell proliferation by interleukin 6

We investigated the ability of interleukin 6 (IL-6) to modulatehuman endometrial stromal cell growth in vitro Stromal cellproliferation in response to treatment with varying concentrationsof IL-6 was determined. Endometrial tissue was obtained from10 normally cycling women during the secretory phase of theirmenstrual cycle. Treatment with IL-6 resulted in a dose- andcell-density-dependent inhibition of endometrial stromal cellproliferation in vitro. The maximal inhibition was observedwith 200 pg/ml of IL-6 and at a concentration of 10⁵ cells/well.During in-vitro culture, stromal cells produced low amountsof IL-6 and demonstrated the presence of IL-6 receptor. Thesedata demonstrate that IL-6 acts as a growth-regulatory signalfor human endometrial stromal cells. We postulate that IL-6may contribute to the maintenance of homeostasis in normal endometriumand that perturbation of IL-6 mediated responses may play arole in disorders of the endometrium such as endometrial cancerand endometriosis.     

Expression of gap junction connexins in the human endometrium throughout the menstrual cycle

Expression of connexins, the proteins which comprise gap junctionchannels, is regulated by ovarian hormones in the female reproductivetract of rodents. In order to determine if these hormones alsoaffect connexin expression in the human uterus, the distributionpatterns of different connexins (cx26, cx32, cx43) were investigatedby immunohistochemistry in human endometrial tissue collectedthroughout the menstrual cycle. During the early proliferativephase of the cycle extremely low staining for connexin 43 wasobserved and connexin 26 antigens could not detected. An increasein the amount of connexin 43 stromal cells and of connexin 26in glandular and luminal epithelial cells was seen from days11–15 of the cycle. Following ovulation, the expressionof both connexinswas suppressed and was completely abolishedin the late secretory phase. Weak staining for connexin 32 wasfoundmainly in the late proliferative and the early secretoryphase and was restricted to the basal membrane region the glandularcells. These results suggest that the different onnexins couldrepresent cell biological markers for the proliferation anddifferentiation of the human endometrium throughout the menstrualcycle.     

Endothelin synthesis and receptors in human endometrium throughout the normal menstrual cycle

This study was undertaken to investigate the presence of messengerRNA (mRNA) for prepro-endothelin-I (ET-1) and the known receptorsubtypes (ETA and ETB) in human endometrium at different stagesof the menstrual cycle obtained at hysterectomy. Northern blotanalysis revealed expression of ET-1 mRNA in human endometriumduring the normal menstrual cycle. The concentration of ET-1mRNA in endometrial tissue was greater during the menstrualand proliferative phases than during the ovulatory and secretoryphases. Immunoreactive ET-1 was secreted into the medium ofisolated endometrial stromal cells. Oestradiol and progesteronesignificantly attenuated ET-1 release in endometrial stromalcells cultured for 6 days. ETA and ETB mRNA were also presentin endometrial tissue of the normal cycle. The concentrationof ETA receptor mRNA was greater in the proliferative phasethan in the secretory phase, whereas expression of ETB mRNAincreased in menstrual phase. ET-1 significantly increased extracellularaccumulation of cyclic AMP (cAMP), intracellular generationof inositol phosphates and significantly enhanced DNA synthesisin cultured endometrial stromal cells from the proliferativephase. Our results showed that human endometrial cells synthesizedand released ET-1, and contained ETA and ETB receptors whichwere functionally coupled to phosphoinositide breakdown andto adenylate cyclase with the increase of cAMP by ET-1 stimulation.Our findings suggest that ET-1 may have a potential autocrineand/or paracrine function in human endometrial stromal cells.     

Gene expression pattern and immunoreactive protein localization of LGR7 receptor in human endometrium throughout the menstrual cycle

Relaxin (RLX) is a pregnancy-associated polypeptide hormone. In non-pregnant women, the peak of circulating relaxin coincides with the window of endometrial receptivity and both in vivo and in vitro experiments showed that it plays a role in the decidualization process. Recently, two receptors, LGR7 and LGR8, have been identified as high affinity receptors for relaxin. Here we describe LGR7 mRNA and protein expression in human endometrium using semi-quantitative and quantitative fluorescent PCR (Q-PCR) and immunohistochemical analyses. Three different experimental designs were used. First, endometrial biopsies from five different phases of the menstrual cycle were analysed. Secondly, we assessed the early luteal phase in more detail. Finally we analysed the expression at LH+2 (2 days after the natural LH surge, pre-receptive endometrium) versus LH+7 (receptive endometrium) within the same menstrual cycle from the same patient to avoid inter-cycle or inter-person variations in gene expression. Our results indicate that there is no consistent regulation of LGR7 mRNA expression, neither during the menstrual cycle nor during the early-mid-luteal phase. In general, we observed a large degree of variation in LGR7 mRNA expression levels between patients. LGR7 immunoreactive protein was identified in all stages of the menstrual cycle. LGR7 protein was localized in both the epithelial and the stromal compartments, except for the mid-luteal phase when the expression was restricted to the endometrial epithelium. We conclude that no consistent regulation of LGR7 mRNA expression can be detected in human endometrium during the menstrual cycle.     

白细胞介素-8在妊娠早期小鼠子宫内膜的表达及对胚泡着床的影响

目的：研究白细胞介素-8（IL-8）在妊娠早期小鼠子宫内膜的表达及其对小鼠胚泡着床的影响。方法：用免疫组化法及图像分析技术对IL-8在妊娠1～6d小鼠子宫内膜的表达进行定位和测定;子宫角注入IL-8抗体,探讨IL-8对胚泡着床的影响。结果：在子宫内膜腔上皮,IL-8主要定位于细胞的游离面,妊娠1d阳性反应最弱,4d表达最强,5d有所下降,6d又开始增强;在子宫内膜腺上皮,妊娠4d表达最弱,5d和6d最强;在子宫内膜的基质细胞,随妊娠天数增加,IL-8的表达逐渐增强。IL-8Ab明显抑制小鼠胚泡着床。结论：IL-8在妊娠早期小鼠子宫内膜持续表达,并参与胚泡的着床调控过程。     

Perivascular location of a chemokine interleukin-8 in human endometrium: a preliminary report

The cytokine interleukin-8 (IL-8) is capable of inducing selectiveneutrophil chemotaxis and activation and has been postulatedas the signal for neutrophil recruitment and activation in reproductivetissues. The aim of this study was to examine the localizationof IL-8 in non-pregnant human endometrium in view of its potentialas a local modulator of endometrial function. Endometrial biopsies(proliferative, n=8; secretory, n= 7) from 15 subjects wereavailable for immunolocalization of IL-8. Primary antibody (rabbitpolyclonal against IL-8) application was followed by eithera horseradish peroxidase streptavidin detection system (proliferative,n= 5; secretory, n= 4) or an avidin–biotin alkaline phosphatasedetection method (proliferative, n=3; secretory, n= 3). Allendometrial biopsies showed heterogeneous positive stainingfor IL-8 in association with blood vessels in both proliferativeand secretory phase biopsies. The immunostaining was apparentlynot associated with endothelial cells but rather appeared tobe associated with the smooth muscle layer of arterioles. Secretoryphase biopsies exhibited immunoreactivity in association withsmall blood vessels and spiral arterioles. The perivascularlocation of IL-8 throughout the stages of the human menstrualcycle is consistent with its proposed biological role as a modulatorof endometrial function, especially synergism with prostaglandinE and the transmigration of leukocytes.