Regulated expression of heparin-binding EGF-like growth factor (HB-EGF) in the human endometrium: a potential paracrine role during implantation

Heparin-binding epidermal growth factor (HB-EGF) is a recently identified member of the EGF growth factor family found to be expressed in the uterus of both mouse and human at the time of implantation. In the present study, we investigated the expression patterns of HB-EGF in normal cycling endometrium and compared its expression with the fertility-associated endometrial epithelial biomarkers alpha(v)beta(3) integrin, leukemia inhibitory factor (LIF) and homeobox gene, HOXA-10. RNase protection assay (RPA) using RNA made from endometrium collected from different phases of the menstrual cycle demonstrated increased HB-EGF expression during the mid-secretory phase, a pattern similar to, but slightly preceding the expression of alpha(v)beta(3) integrin and HOXA-10. In vitro studies demonstrated stimulation of HB-EGF expression by estradiol-17beta (E(2)) and progesterone (P(4)) alone or in combination in stromal cells. Combined treatment with E(2) + P(4) was, however, required to stimulate epithelial HB-EGF expression. In vitro experiments demonstrated the ability of HB-EGF to stimulate epithelial expression of the key endometrial proteins including LIF, HOXA-10, and the beta(3) integrin subunit. Each has previously been demonstrated to be an important epithelial biomarker expressed during the implantation window. In addition, conditioned media from endometrial stromal cells treated with E(2) + P(4) + relaxin mimicked the stimulatory effect of HB-EGF on epithelial expression of the beta(3) integrin subunit. The stimulatory effect of the stromal-conditioned medium was blocked by antibodies that neutralize a known receptor for HB-EGF. These data suggest that uterine receptivity may be regulated in part by the stromal-derived HB-EGF.     

Estrogen receptor-alpha (ER-alpha) and defects in uterine receptivity in women

Endometriosis is a disorder that affects 5% of the normal population but is present in up to 40% of women with pelvic pain and/or infertility. Recent evidence suggests that the endometrium of women with endometriosis exhibits progesterone insensitivity. One endometrial protein that fluctuates in response to progesterone is the estrogen receptor-alpha (ER alpha), being down-regulated at the time of peak progesterone secretion during the window of implantation. Here we demonstrate that the biomarker of uterine receptivity, beta 3 integrin subunit, is reduced or absent in some women with endometriosis and that such defects are accompanied by inappropriate over-expression of ER alpha during the mid-secretory phase. Using a well-differentiated endometrial cell line we showed that the beta 3 integrin protein is negatively regulated by estrogen and positively regulated by epidermal growth factor (EGF). By competing against estrogen with various selective estrogen receptor modulators (SERMs) and estrogen receptor agonists and antagonists, inhibition of expression of the beta 3 integrin by estrogen can be mitigated. In conclusion, we hypothesize that certain types of uterine receptivity defects may be caused by the loss of appropriate ER alpha down-regulation in the mid-secretory phase, leading to defects in uterine receptivity. Such changes might be effectively treated by timely administration of the appropriate anti-estrogens to artificially block ER alpha and restore normal patterns of gene expression. Such treatments will require further clinical studies.     

Characterization of androgen receptors in a well-differentiated endometrial adenocarcinoma cell line (Ishikawa)

Androgen receptors (AR) have been identified in the human endometrium, but their role in endometrial function and development towards endometrial receptivity remains poorly understood. In an effort to study the regulation and possible function in endometrial epithelium, we utilized the well-differentiated endometrial adenocarcinoma cell line, Ishikawa, as a model system. This cell line has proven to be stable, hormonally responsive, contains both estrogen and progesterone receptors, and has been shown to express endometrial proteins in a hormone responsive manner. In the present study, we demonstrate that Ishikawa cells also express AR, based on immunohistochemical staining, radioactive binding studies, RT-PCR and Northern blot analysis. The expression of AR is induced in Ishikawa cells by estrogens, similar to that reported for normal endometrium. Further, using an estrogen-responsive gene that has been characterized in this cell line, alkaline phosphatase, we show that androgens act as antiestrogens in diethylstilbestrol (DES) treated cells, inhibiting enzymatic activity in a dose-dependent manner. These data support a physiologic role for AR in the endometrium. Elevations in endometrial AR in certain clinical situations such as polycystic ovarian syndrome (PCOS) may amplify the effects of androgens on the endometrium leading to suspected defects in uterine receptivity, higher than expected infertility and high miscarriage rates observed in patients with this disorder.     

Berberine improves ovulation and endometrial receptivity in polycystic ovary syndrome

BackgroundPolycystic ovary syndrome (PCOS) is a clinical syndrome with reproductive and endocrine disorders. Berberine is a monomer from Chinese herbs such as Coptis chinensis, whose effect on improving ovulation and endometrial receptivity of PCOS is uncertain.PurposeTo evaluate the effect of berberine on improving PCOS and explore the mechanism.MethodsThe rat model of PCOS was induced by intraperitoneal injection of testosterone propionate. Then they was divided into model (Mod) group, low-dose of berberine (BL) group, high-dose of berberine (BH) group and metformin (Met) group as well as a control (Con) group was established. Ovary morphology, hormone level, glucolipid metabolism were measured. UID-mRNA-seq of ovary tissue was conducted to seek the mechanism of berberine on improving ovulation. Three biomarkers of endometrial receptivity were also examined in endometrium by immunohistochemistry.ResultsThe number of cystic follicles was increased while the number of corpus luteum was decreased in the rats of Mod group. These changes could be reversed by high-dose of berberine intervention. Berberine could also decrease the levels of serum luteinizing hormone (LH) and total cholesterol (TC) in PCOS rats. Meanwhile, berberine improved the impairment of abnormal oral glucose tolerance without affecting fasting insulin level and Homeostasis model assessment-insulin resistance (HOMA-IR). Luteinizing hormone/ choriogonadotropin receptor (LHCGR) and cytochrome P450 Family 19 Subfamily A Member 1 (CYP19A1) were focused via RNA-seq of ovary. Protein expression in ovary and mRNA expression in granulosa cell of LHCGR and CYP19A1 were decreased in Mod group and rescued by the intervention of berberine. A decrease of endometrial thickness and an increase of integrin αvβ3 and lysophosphatidic acid receptor 3 (LPAR3) protein expression were observed in Mod group, which could be also reversed by berberbine.ConclusionsBerberine could improve ovulation in PCOS and the mechanism might be associated with up-regulating LHCGR and CYP19A1. Berberine could also improve endometrial receptivity through down-regualting αvβ3 and LPAR3.     

Immunolocalisation of phosphorylated STAT3, interleukin 11 and leukaemia inhibitory factor in endometrium of women with unexplained infertility during the implantation window

Background  

Uterine receptivity and embryo implantation are critical in the establishment of pregnancy. The diagnosis of endometrial fertility requires more precise measurements of endometrial receptivity. Interleukin (IL-11) and leukemia inhibitory factor (LIF) are essential for murine implantation and signal via intracellular phosphorylation (p) of STAT3 in the endometrium. Both cytokines are present in the endometrium of women duiring the receptive window. Endometrial IL-11, IL-11 receptor alpha (IL-11Ralpha), LIF and pSTAT3 in women with primary unexplained infertility was compared to normal fertile women during the implantation window.     

Proteomic Insights into Endometrial Receptivity and Embryo‐Endometrial Epithelium Interaction for Implantation Reveal Critical Determinants of Fertility

In vitro fertilization has overcome infertility issues for many couples. However, achieving implantation of a viable embryo into the maternal endometrium remains a limiting step in optimizing pregnancy success. The molecular mechanisms which characterize the transient state of endometrial receptivity, critical in enabling embryo‐endometrial interactions, and proteins which underpin adhesion at the implantation interface, are limited in humans despite these temporally regulated processes fundamental to life. Hence, failure of implantation remains the “final frontier” in infertility. A human coculture model is utilized utilizing spheroids of a trophectoderm (trophoblast stem) cell line, derived from pre‐implantation human embryos, and primary human endometrial epithelial cells, to functionally identify “fertile” versus “infertile” endometrial epithelium based on adhesion between these cell types. Quantitative proteomics identified proteins associated with human endometrial epithelial receptivity (“epithelial receptome”) and trophectoderm adhesion (“adhesome”). As validation, key “epithelial receptome” proteins (MAGT‐1/CDA/LGMN/KYNU/PC4) localized to the epithelium of receptive phase (mid‐secretory) endometrium obtained from fertile, normally cycling women but is largely absent from non‐receptive (proliferative) phase tissues. Factors involved in embryo‐epithelium interaction in successive temporal stages of endometrial receptivity and implantation are demonstrated and potential targets for improving fertility are provided, enhancing potential to become pregnant either naturally or in a clinical setting.     

Steroids modulate the expression of alpha4 integrin in mouse blastocysts and uterus during implantation

Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and the maternal uterine milieu, which are controlled at the embryo-maternal interface by the coordinated interplay of a variety of growth factors, cytokines, hormones, and cell adhesion molecules expressed by both the decidualized endometrium and the trophoblast cells. Proper implantation of the embryo is solely dependent on the initial endometrial receptivity and the preparation of the blastocyst to glue itself to the uterine wall. Both these events are considered to be mediated by cell adhesion molecules and integrins expressed by the blastocyst as well by as the maternal endometrium. Integrin expression by the blastocyst and the uterus is a dynamic process. However, reports on the expression and the hormonal modulation of integrins and their role in blastocyst activation and uterine receptivity during implantation are meager. The present study investigates the expression and hormonal regulation of alpha4beta1 integrin by steroid hormones in the blastocyst and the receptive uterus using an in vivo, delayed-implantation mouse model system. The dormant and activated blastocysts as well as the uteri were recovered from ovariectomized mice after progesterone-alone and progesterone-plus-estrogen therapy, respectively. Immunolocalization of protein expression of alpha4 and beta1 integrin subunits indicate that steroids modulate the expression of alpha4beta1 integrin receptor in the mouse blastocyst as well as the uterus and that a differential expression is observed with exposure to progesterone and estrogen. Intrauterine blocking of alpha4 integrin by specific antibody resulted in implantation failure in normal as well as in delayed-implantation mice. Based on our data, we propose here, to our knowledge for the first time, that alpha4beta1 integrin, which is responsible for binding to fibronectin and vascular cell adhesion molecule-1, is induced by estradiol and is down-regulated by progesterone in mice during implantation. Furthermore, the results also indicate the direct role of alpha4 integrin in the process of implantation.     

Endometrial contraception: modulation of molecular determinants of uterine receptivity

Modulation of endometrial receptivity is a promising approach for fertility regulation since it allows a contraceptive to act specifically at the endometrium. This was corroborated by our previous observations that treatment with low doses of a pure progesterone antagonist (PA, antiprogestin), onapristone (ZK 98299), in bonnet monkeys inhibited fertility by selectively retarding endometrial development, without affecting the hypophyseal-hypothalamic function. In the present study, further investigations, undertaken to analyze the molecular repertoire of a nonreceptive primate endometrium, determined expression of: steroid hormone receptors, i.e. progesterone receptor (PR) and estrogen receptor (ER); cytokines, i.e. leukemia inhibitory factor (LIF): transforming growth factor beta (TGFbeta) and its receptor (TGFbetaR); and cell adhesion molecules, i.e. integrins (alpha(v)beta(3), alpha(1)beta(1)). These studies were conducted during the different phases of the normal menstrual cycle and following treatment with different doses of onapristone (2.5 mg, 5 mg, or 10 mg every third day for one cycle) in bonnet monkeys. The molecules were analysed collectively to explore the possibility of a correlation between expression of these markers and endometrial receptivity and to investigate whether there exists a regulatory link between expression of these molecules under in vivo conditions. Three types of expression patterns of endometrial factors were observed during the peri-implantation period following onapristone treatment: 1) LIF, alpha(v)beta(3), and alpha(1)beta(1) showed significant (P < 0.02) down regulation in glandular epithelium of endometria in animals treated with all three doses of onapristone as compared to the control group. This was indicative of their critical role in the progesterone-driven cascade leading to implantation. 2) PR, TGFbeta, and TGFbetaR remained unaffected in the endometria from 2.5 mg treated animals and showed down regulation in animals treated with 5 and 10 mg onapristone as compared to the control group, thereby suggesting that the expression of these markers may not truely reflect endometrial receptivity per se. However, their facilitatory role in preparing the endometrium for implantation can not be ruled out since continued perturbation in the expression of these molecules may affect endometrial growth, remodelling, and differentiation, which in turn may render the endometrium nonreceptive; 3) ER remained unaltered in endometria of animals rendered infertile with 2.5, 5, and 10 mg onapristone. This observation indirectly suggests that onapristone-induced endometrial changes are mediated via some specific mechanisms. The present study clearly demonstrates that endometrial non-receptivity induced at low doses of onapristone is associated with changes in the expression pattern of specific molecular markers. However, no direct correlation was observed between in vivo expression of TGFbeta, LIF, and integrins, thereby lending support to the concept that there exists redundancy or multiple pathways which regulate implantation events.     

Endometrial function in vervet monkeys (Cercopithecus aethiops): morphology,beta3 integrin and insulin-like growth factor binding protein-1 expression during the menstrual cycle and pregnancy in the normal and disrupted endometrium

The expression of endometrial beta3 integrin and insulin-like growth factor binding protein-1 (IGFBP-1) was studied in cycling and pregnant vervet monkeys. There were clear changes of beta3 integrin expression during the menstrual cycle, with the strongest immunostaining observed on day 26. Moderate to strong expression was observed during pregnancy. The expression of IGFBP-1 during the menstrual cycle was weak but upregulated during pregnancy with moderate to strong staining. The administration of a single dose of onapristone at 10 mg/kg on days 17, 21 and 22 of the menstrual cycle, followed by a biopsy on days 22, 22 and 26, respectively, and during pregnancy (34-44 days menstrual age) 24 h before the biopsy, disrupted and desynchronized the endometrium. However, no effect on beta3 integrin expression could be observed and staining reflected the untreated patterns. The same applied to IGFBP-1 except that during pregnancy the expression of this protein was reduced or abolished. The results suggest that beta3 integrin is associated with endometrial receptivity in vervet monkeys and that IGFBP-1 plays an important role during pregnancy in this species. The administration of onapristone appeared to only influence IGFBP-1 expression. To our knowledge, this is the first time that these endometrial proteins have been investigated in vervet monkeys. This study should therefore contribute to improving our understanding of the reproductive function of this species.     

Expression of apoptosis-related genes in the endometrium of polycystic ovary syndrome patients during the window of implantation

The present study investigated the expression of the apoptosis-related genes fas-associated via death domain (FADD) and Bcl-2 in the endometrium during the window of implantation in polycystic ovary syndrome (PCOS) patients. The aim was to explore the role of cell apoptosis in endometrial receptivity during this period. The subjects were divided into experimental and control group. The experimental group comprised 12 infertile women with PCOS, and the control group comprised 12 women who were infertile because of tubal pathological factors but had normal menstrual cycles. Endometria were collected by biopsy 7d after ovulation. Six samples from each group were randomly selected and subjected to gene chip analyses. The expression of endometrial FADD and Bcl-2 was determined by immunohistochemistry, and cell apoptosis was detected by the TUNEL method. Compared with the control group, 194 differentially expressed genes were found in the PCOS group, 102 of which were upregulated and 92 were downregulated. The differentially expressed genes were divided into 15 types according to function. Among the nine genes related to cell apoptosis, five (including Bcl-2) were upregulated and four were downregulated (including FADD). Bcl-2 expression during the window of implantation in the PCOS group increased compared with the control group, showing a significant difference (P<0.05). FADD expression in the PCOS group notably decreased compared with that in the control group, which also showed a significant difference (P<0.05). Cell apoptosis analysis showed a significant difference between the average apoptotic indices in the PCOS and control groups (P<0.05). Significant differences were observed between the endometrial gene expression in the PCOS and control groups. The decrease in cell apoptosis during the window of implantation in PCOS patients may be one of the causes of the reduced endometrial receptivity.     

The expression pattern of MUC1 glycoforms and other biomarkers of endometrial receptivity in fertile and infertile women

Changes in the surface epithelium of the endometrium, characterized in part by alterations in cell-surface molecules, sex steroid receptors and the appearance of pinopodes, coincide with the window of endometrial receptivity in the menstrual cycle. This study was performed to evaluate the usefulness of hematoxylin and eosin staining, scanning and transmission microscopy, and MUC1 glycoform, sex steroid receptor, and interleukin receptor (type 1) expression as biomarkers of endometrial receptivity using carefully characterized clinical fertile and infertile groups of women. Using a combination of immunohistochemistry and scanning electron microscopy (SEM) called scanning immunoelectron microscopy (SIM), we confirmed that MUC1 mucin was not associated with the endometrial pinopodes, which have been linked with embryo adhesion. We also showed that failure of embryo implantation was associated with an abnormal endometrial expression of MUC1 mucin, and retention of nuclear progesterone receptor (PR) particularly in epithelial cells. Hematoxylin and eosin staining, transmission electron microscopy (TEM), SEM in isolation and immunohistochemistry for interleukin receptor were not shown to be useful markers. Progesterone-dependent regulation of MUC1 appears to be an important factor in determining endometrial receptivity.     

Injury and EGF mediate the expression of alpha6beta4 integrin subunits in corneal epithelium

Our goal was to evaluate the role of epidermal growth factor and injury on the expression of integrin subunits alpha6(alpha6) and beta4(beta4). An in vitro wound model was used to evaluate corneal wound repair and cellular migration. Primary rabbit corneal epithelial cell cultures were serum-starved and injured in the presence or absence of EGF or tyrphostin AG1478, an inhibitor of EGF receptor kinase activity. Repair was monitored morphologically and expression was analyzed using in situ hybridization and immunohistochemistry accompanied by confocal microscopy. The addition of EGF to cell cultures induced a dose-dependent increase in beta4 mRNA expression but the constitutive expression of alpha6 was several fold greater. In the wounded cultures there was a rapid change in expression at the edge of the wound that was enhanced with EGF. In our model there was an increase in beta4 and alpha6 protein in migrating cells. Changes in integrin expression were accompanied by a transient increase in activation of the EGF receptor. The addition of tyrphostin inhibited migration of cells and wound repair, the activation of the EGF receptor and phosphorylation of beta4 in the cytoplasm. These data indicate that the activation of the EGF receptor plays a critical role in the regulation of integrin receptors and the mediation of cellular migration.     

MUC16对子宫内膜容受性的影响及其意义

目的：多囊卵巢综合征（Polycystic Ovary Syndrome,PCOS）系由于体内复杂的内分泌和代谢环境影响了子宫内膜稳态,导致子宫内膜容受性下降,造成患者生育力减弱和不良妊娠结局。通过测定多囊卵巢综合征患者子宫内膜与正常生育力妇女增生期、分泌早、中、晚期子宫内膜中MUC16的相对表达量,本文探讨MUC16与PCOS患者子宫内膜容受性下降的关系,为临床上改善PCOS患者子宫内膜容受性,提高PCOS患者的妊娠率,降低流产率提供一条新的可能途径。方法：选择PCOS患者子宫内膜、正常生育力妇女增生期、分泌早、中、晚期子宫内膜各20例,用免疫组化SP法检测MUC16在各组的表达情况。结果：（1）MUC16在月经周期各期均有表达,在分泌中期表达最强。（2）PCOS组MUC16的表达较分泌中期组弱,差异有显著性（P〈0.05）。结论：（1）在子宫内膜中的表达呈周期性变化。（2）PCOS患者子宫内膜中MUC16表达异常可能使子宫内膜容受性下降,推测其与胚胎不能正常着床或着床后发育不良、导致流产有关。     

Cyclic modulation of integrin expression in bovine endometrium.

Integrins are heterodimeric glycoproteins involved in cell-cell and cell-extracellular matrix adhesion. In this study, the spatial and temporal distribution of selected integrins and extracellular matrix proteins was determined in bovine endometrium from cycling and ovariectomized animals using indirect immunohistochemistry. The expression of integrins alpha(6) and alpha(v)beta(3) was estrous cycle-dependent. Strong immunostaining for alpha(v)beta(3) occurred in the basement membrane region of intercaruncular luminal epithelium except on Day 16 (P<0.05). Staining of subepithelial stromal cells declined in diestrous samples (P<0.05). In all samples, there was reduced alpha(v)beta(3) reactivity in the caruncles. Staining for alpha(6) decreased in the epithelial basement membrane at proestrus through estrus (Days 18-0). Expression of integrin subunits alpha(3) and alpha(4) was cycle-independent. Moderate staining for alpha(3) was detected on epithelium and alpha(4) was present on stromal cells. The distribution of beta(1) suggested dimerization with alpha(3), alpha(4), and alpha(6). Laminin was detected in the epithelial and vasculature basement membranes. Collagen IV was present in the glandular epithelium basement membrane and subepithelial stromal cells, whereas fibronectin was found only in the stroma. Estrous cycle-dependent distribution and expression of alpha(v)beta(3) and alpha(6) suggest their regulation by ovarian steroids, growth factors, and prostaglandins.