Baboon Endometrium Research Articles

Retinoic acid action is altered within endometrium of baboons affected with endometriosis

Objective: Using a baboon model, we determined the changing expression of Retinoic Acid (RA) target genes during the menstrual cycle and during disease progression. This change could explain the cellular response and changes characteristic of endometriosis. In previous studies, we established that endometriosis affects the CRABP2:FABP5 ratio in an in vitro environment, shifting toward apoptosis and differentiation with higher CRABP2, and anti-apoptosis with higher levels of FABP5. Intervention(s): Endometriosis was induced in female baboons with intraperitoneal inoculation of menstrual endometrium ( n = 2–4). Tissue was harvested via endometrectomy during different stages of the menstrual cycle as well at 3, 6, and 12 month timepoints after inoculation with endometriosis. Main outcome measure(s): Real time PCR was used to quantify STRA6 (a gene responsible for retinol uptake), CRABP2 (a gene necessary for apoptotic and anti-apoptotic estrogenic RA effects), and FABP5 (a gene that mediates the anti-apoptotic actions of RA). Results: STRA6 and CRABP2 expression were highest in the proliferative phase and lowest in the late secretory phase. FABP5 expression remained stable throughout the 12 months following the induction of the disease, whereas STRA6 and CRABP2 continued to decrease during the same period. Conclusions: Our study confirms that a shift in the CRABP2:FABP5 ratio has similar in vivo effects as it does in vitro: changing RA expression with disease induction and progression. As CRABP2 may be important in determining cell fate in the endometrium, gene expression changes could contribute to the anti-apoptotic behavior of affected cells. As expression changes more during progression, earlier rather than later treatment becomes more critical in reducing the rate of disease progression.

Abnormally located SSEA1+/SOX9+ endometrial epithelial cells with a basalis-like phenotype in the eutopic functionalis layer may play a role in the pathogenesis of endometriosis.

STUDY QUESTIONIs endometriosis associated with abnormally located endometrial basalis-like (SSEA1+/SOX9+) cells in the secretory phase functionalis and could they contribute to ectopic endometriotic lesion formation?SUMMARY ANSWERWomen with endometriosis had an abnormally higher number of basalis-like SSEA1+/SOX9+ epithelial cells present in the stratum functionalis and, since these cells formed 3D structures in vitro with phenotypic similarities to ectopic endometriotic lesions, they may generate ectopic lesions following retrograde menstruation.WHAT IS KNOWN ALREADYEndometrial basalis cells with progenitor potential are postulated to play a role in the pathogenesis of endometriosis and SSEA1 and nuclear SOX9 (nSOX9) mark basalis epithelial cells that also have some adenogenic properties in vitro. Induction of ectopic endometriotic lesions in a baboon model of endometriosis produces characteristic changes in the eutopic endometrium. Retrograde menstruation of endometrial basalis cells is proposed to play a role in the pathogenesis of endometriosis.STUDY DESIGN, SIZE, DURATIONThis prospective study included endometrial samples from 102 women with and without endometriosis undergoing gynaecological surgery and from six baboons before and after induction of endometriosis, with in vitro assays examining the differentiation potential of human basalis-like cells.PARTICIPANTS/MATERIALS, SETTING, METHODSThe study was conducted at a University Research Institute. SSEA1 and SOX9 expression levels were examined in human endometrial samples from women aged 18–55 years (by immunohistochemistry (IHC) and qPCR) and from baboons (IHC). The differential gene expression and differentiation potential was assessed in freshly isolated SSEA1+ endometrial epithelial cells from women with and without endometriosis (n = 8/group) in vitro. In silico analysis of selected published microarray datasets identified differential regulation of genes of interest for the mid-secretory phase endometrium of women with endometriosis relative to that of healthy women without endometriosis.MAIN RESULTS AND THE ROLE OF CHANCEWomen with endometriosis demonstrated higher number of basalis-like cells (SSEA1+, nSOX9+) in the functionalis layer of the eutopic endometrium compared with the healthy women without endometriosis in the secretory phase of the cycle (P < 0.05). Induction of endometriosis resulted in a similar increase in basalis-like epithelial cells in the eutopic baboon endometrium. The isolated SSEA1+ epithelial cells from the eutopic endometrium of women with endometriosis had higher expression of OCT4, NANOG, FUT4 mRNA (P = 0.05, P = 0.007, P = 0.018, respectively) and they differentiated into ectopic endometriotic gland-like structures in 3D culture, but not into mesodermal lineages (adipose or bone cells).LARGE SCALE DATAN/ALIMITATIONS, REASONS FOR CAUTIONSmall sample size. Bioinformatics analysis and results depends on the quality of published microarray datasets and the stringency of patient selection criteria employed. Differentiation of SSEA-1+ cells was only examined for two mesodermal lineages (adipogenic and osteogenic).WIDER IMPLICATIONS OF THE FINDINGSSince endometrial epithelial cells with SSEA1+/nSOX9+ basalis-like phenotype generate endometriotic gland-like structures in vitro, they may potentially be a therapeutic target for endometriosis. An in depth analysis of the function of basalis-like eutopic endometrial epithelial cells might provide insights into their potential deregulation in other disorders of the endometrium including heavy menstrual bleeding and endometrial cancer where their function may be aberrant.STUDY FUNDING/COMPETING INTEREST(S)We acknowledge the support by Wellbeing of Women project grant RG1073 (D.K.H., C.E.G.) and R01 HD083273 from the National Institutes of Health (A.T.F.). We also acknowledge the support of Liverpool Women’s Hospital Foundation Trust (J.D.), Institute of Translational Medicine (L.D.S., H.A.L., A.J.V., D.K.H.), University of Liverpool, the National Health and Medical Research Council of Australia ID 1042298 (C.E.G.) and the Victorian Government Operational Infrastructure Support Fund. All authors declare no conflict of interest.

Altered expression of microRNA-451 in eutopic endometrium of baboons (Papio anubis) with endometriosis.

Are microRNAs (miRs) altered in the eutopic endometrium (EuE) of baboons following the induction of endometriosis? Induction of endometriosis causes significant changes in the expression of eight miRs, including miR-451, in the baboon endometrium as early as 3 months following induction of the disease. Endometriosis is one of the most common gynecological disorders and causes chronic pelvic pain and infertility in women of reproductive age. Altered expression of miRs has been reported in women and has been suggested to play an important role in the pathophysiology of several gynecological disorders including endometriosis. EuE was obtained from the same group of baboons before and 3 months after the induction of endometriosis. The altered expression of miR-451 was validated in the eutopic and ectopic endometrium of additional baboons between 3 and 15 months following disease induction. Timed endometrial biopsies from women with and without endometriosis were also used to validate the expression of miR-451. Total RNA was extracted from EuE samples before and after the induction of endometriosis, and miRNA expression was analyzed using a 8 × 15 K miR microarray. Microarray signal data were preprocessed by AgiMiRna software, and an empirical Bayes model was used to estimate the changes. The present study focused on quantitative RT-PCR validation of the microarray data, specifically on miR-451 and its target genes in both baboons (n = 3) and women [control (n = 7) and endometriosis (n = 19)]. Descriptive and correlative analysis of miR-451 and target gene expression was conducted using in situ hybridization and immunohistochemistry, while functional analysis utilized an in vitro 3' untranslated region (UTR) luciferase assay and overexpression of miR-451 in human endometrial and endometriotic cell lines. Induction of endometriosis results in the altered expression of miR-451, -141, -29c, -21, -424, -19b, -200a and -181a in the baboon endometrium. In the baboon, induction of endometriosis significantly decreased the expression of miR-451 at 3 months (P < 0.001), which was also associated with increased expression of its target gene YWHAZ (14.3.3ζ). A similar significant (P < 0.0001) decrease in miR-451 expression was observed in women with endometriosis. The 3' UTR luciferase assay confirmed the regulation of YWHAZ expression by miR-451. Furthermore, overexpression of miR-451 in 12Z cells (immortalized human endometriotic epithelial cell line) led to the decreased expression of its target YWHAZ and this was correlated with decreased cell proliferation. The study focused only on miR-451 and one of its targets, namely YWHAZ. A single miR could target number of genes and a single gene could also be regulated by number of miRs; hence, it is possible that other miRs and their regulated genes may contribute to the pathophysiology of endometriosis. Our data suggest that the presence of ectopic lesions in baboon causes changes in EuE miR expression as early as 3 months postinduction of the disease, and some of these changes may persist throughout the course of the disease. We propose that the marked down-regulation of miR-451 in both baboons and women with endometriosis increases the expression of multiple target genes. Increased expression of one of the target genes, YWHAZ, increases proliferation, likely contributing to the pathophysiology of the disease.

Altered expression of microRNA-451 in eutopic endometrium of baboons ( Papio anubis ) with endometriosis

Altered expression of microRNA-451 in eutopic endometrium of baboons ( Papio anubis ) with endometriosis

Decreased Notch pathway signaling in the endometrium of women with endometriosis impairs decidualization.

Endometriosis is a common gynecological disease affecting one in 10 women of reproductive age and is a major cause of pelvic pain and impaired fertility. Endometrial stromal cells of women with endometriosis exhibit a reduced response to in vitro decidualization. NOTCH1 is critical for decidualization of both mouse and human uterine stromal cells. This study aimed to determine whether decidualization failure in women with endometriosis is a consequence of impaired Notch signaling. We investigated expression levels of Notch signaling components in the endometrium of women and baboons with or without endometriosis. We identified NOTCH1-regulated genes during decidualization of human uterine fibroblast (HuF) cells by microarray and quantified their expression levels in in vitro-decidualized endometrial stromal cells isolated from women with or without endometriosis. Notch signaling receptors NOTCH1 and NOTCH4, ligands JAGGED2 and DLL4, as well as direct target genes HES5 and HEY1 were decreased in the eutopic endometrium of women and baboons with endometriosis. Notch signaling was decreased in stromal cells isolated from women with endometriosis, which was associated with impaired in vitro decidualization. Genes that were down-regulated by NOTCH1 silencing in decidualized HuF cells were also decreased in decidualized endometrial stromal cells of women with endometriosis. FOXO1 acts as a downstream target of Notch signaling and endometriosis is associated with decreased expression of NOTCH1-regulated, FOXO1-responsive genes during decidualization. Decreased Notch signaling is associated with endometriosis and contributes to impaired decidualization through the down-regulation of FOXO1.

Nerve fiber density in deep nodular endometriotic lesions induced in a baboon experimental model

To study the occurrence of nerve fibers in deep nodular endometriotic lesions after nodules were induced in baboons and nerve fiber densities measured 6 months after the grafting procedure. Experimental animal study. Academic gynecology research unit. Ten baboons (Papio anubis). Recovery of induced endometriotic nodules and eutopic endometrium. Protein gene product (PGP) 9.5 and nerve growth factor (NGF) immunohistochemistries were performed to evaluate nerve fiber density and NGF expression in induced endometriotic lesions and eutopic endometrium. Eutopic (basalis) endometrium, myometrium, and invasive and noninvasive nodular lesions were analyzed separately. The highest nerve fiber densities were observed in normal myometrium and in the basal layer of eutopic endometrium. No significant differences were observed between the two lesion types. However, the NGF staining intensity score was found to be higher in glands of deep invasive lesions than in glands of eutopic baboon endometrium. This is the first study to show the presence of nerve fibers in eutopic baboon endometrium and induced deep endometriotic nodules. Long-term studies are now warranted to determine if nerves still grow in invasive and noninvasive lesions >6 months after grafting, and to evaluate the role of the lesion environment.

Induced Endometriosis in Nonhuman Primates1

Induced Endometriosis in Nonhuman Primates1

Changes in Mesenchymal Stem Cell-Associated Gene Expression in Secretory and Menstrual Endometrium of Baboons after Experimental Induction of Endometriosis.

Changes in Mesenchymal Stem Cell-Associated Gene Expression in Secretory and Menstrual Endometrium of Baboons after Experimental Induction of Endometriosis.

Decreased Expression of MicroRNA 451 (MIR) Alters Cell Cycle Genes and Ptgs2 in the Eutopic Endometrium (EUE) in a Baboon Model of Induced Endometriosis.

Endometriosis is one of the most common gynecological disorders which are a cause of chronic pelvic pain and infertility. The molecular mechanisms associated with the pathophysiology of the endometriosis related infertility are poorly understood. Several mechanisms have been postulated and recently the involvement of miR's which regulate gene expression have been postulated to play a role in endometriosis. To identify miR's in the EUE of baboons that are altered following the induction of endometriosis and to validate their potential target genes at different stages of the disease. Total RNA from the EUE extracted from the same group of baboons (n=3) before and 3 months after the induction of endometriosis were analyzed on a 8x15K microRNA microarray (Agilent). The signal data were preprocessed by AgiMiRna and an empirical Bayes model was used to estimate the p-values. The potential mRNA targets of differentially expressed miR's were predicated using TargetScan 5.1. The present study was restricted to the analysis of the significantly down regulated miR-451 and its 3 targets CDKN2D (inhibits CDK4-cell cycle regulation), GATAD2B (encodes transcription repressor protein p66 beta-component of the MeCP1 complex) and PTGS2 (involved in prostaglandins synthesis-Inflammation) by qRT-PCR analysis at different time points of the disease. Induction of endometriosis leads to rapid and significant changes in the expression of several miRs (miR 451, 181a, 200a, 19b, 424, 21, 29c and 141). miR-451 was highly down regulated (p< 0.000036) at 3 months and qRT-PCR analysis for miR-451 confirmed the microarray data. Further, detailed analysis of miR-451 in additional baboons with endometriosis revealed a continuous decrease in expression at 1, 6, 9, and 15 m in both the EUE and in endometriotic lesions. The temporal decrease in miR-451 expression leads to significant increases in the expression of its targets CDKN2D, GATAD2B and PTGS2. Induction of endometriosis causes rapid changes in the expression of miRNAs 181a, 200a, 19b, 424, 21, 29c and 141in the baboon endometrium. These miRs, previously reported to be altered in women with endometriosis, further validates baboon model. This study also identified a previously unreported miR-451 whose significant down regulation is associated with increased expression of its predicted targets CDKN2D, GATAD2B and PTGS2. Present study demonstrated that altered cell cycle and inflammatory response might be due to altered expression of CDKN2D and GATAD2B mediated by miR-451 under endometriotic condition. Protein p66 beta, product of GATAD2B, is a component of MeCP1 (Methyl-CpG-binding protein) complex, this complex represses transcription by binding and remodeling the methylated nucleosomes. This study reveals crucial role of miR-451 in regulating the expression of genes involved in cell cycle, nucleosome remodeling and inflammation. These physiological responses are associated with endometriotic pathology. (U54-HD40093) (poster)

Extracellular matrix metalloproteinase inducer expression in the baboon endometrium: menstrual cycle and endometriosis

Extracellular matrix metalloproteinase inducer (EMMPRIN; BSG) regulates tissue remodeling through matrix metalloproteinases (MMPs). In human and non-human primates, endometrial remodeling is important for menstruation and the pathogenesis of endometriosis. We hypothesized that as in humans, BSG and MMPs are expressed in the endometrium of cycling baboons, and their expression is hormonally regulated by ovarian hormones, but endometriosis disrupts this regulation. BSG expression was evaluated in the baboon endometrium by q-PCR and immunohistochemistry. In the endometrium of control cycling animals, BSG mRNA levels were highest in late secretory stage tissue. BSG protein localized to glandular epithelial cells during the proliferative phase; whereas, secretory stage tissues expressed BSG in glandular and luminal epithelia with weak stromal staining. Several MMPs were differentially expressed throughout the menstrual cycle with the highest levels found during menstruation. In ovariectomized animals, BSG endometrial mRNA levels were highest with treatment of both estrogen and progesterone than that with only estrogen. Estrogen alone resulted in BSG protein localization primarily in the endometrial glandular epithelia, while estrogen and progesterone treatment displayed BSG protein localization in both the glandular and stromal cells. Exogenous hormone treatment resulted in differential expression patterns of all MMPs compared with the control cycling animals. In the eutopic endometrium of endometriotic animals, BSG mRNA levels and protein were elevated early but decreased later in disease progression. Endometriosis elevated the expression of all MMPs except MMP7 compared with the control animals. In baboons, BSG and MMP endometrial expression is regulated by both ovarian hormones, and their expression patterns are dysregulated in endometriotic animals.

Early changes in micro RNA (miRNA) expression in the eutopic endometrium (EUE) in a baboon model of induced endometriosis

OBJECTIVE: To identify miRNAs in the EUE of baboons (Papio anubis) at 3 months following the induction of endometriosis.DESIGN: In the cycle prior to the induction of endometriosis, control EUE samples were obtained at laparotomy on day 10 post ovulation (PO) (n=4). Endometriosis was then induced in the same four animals by intraperitoneal inoculation of autologous menstrual tissue. Following laparoscopic confirmation of endometriosis, EUE samples were obtained at 3 months of disease.MATERIALS AND METHODS: Total RNA from the EUE was extracted and purified. A 8x15K miRNA microarray (Agilent) was applied. The signal data were preprocessed by AgiMiRna, a Limma package for R environment. Empirical Bayes model was used to estimate the P-values. The potential mRNA targets of differentially expressed miRNAs were predicted and compared against Affymetrix Human Genome Plus 2.0 expression data.Tabled 1Differentially Expressed miRNAs with Fold Change (FC) and P-values in baboons with endometriosis compared to controlsmiRNADisease/Control FCP-Valuehsa-miR-451-14.940.000036hsa-miR-181a-1.460.068hsa-miR-200a1.880.037hsa-miR-19b2.190.073hsa-miR-4242.430.027hsa-miR-212.490.014hsa-miR-29c4.460.009hsa-miR-1415.190.032 Open table in a new tab CONCLUSION: Induction of endometriosis causes rapid changes in the expression of several miRNAs in the baboon endometrium. miRNAs 200a, 424, 181a, 21, 29c, and 141, previously reported to be altered in women with endometriosis, were also altered in the endometrium of baboons with endometriosis, further validating this animal model. In addition, miR451, a previously unreported miRNA associated with the apoptotic pathway and cell survival, was significantly down regulated. OBJECTIVE: To identify miRNAs in the EUE of baboons (Papio anubis) at 3 months following the induction of endometriosis. DESIGN: In the cycle prior to the induction of endometriosis, control EUE samples were obtained at laparotomy on day 10 post ovulation (PO) (n=4). Endometriosis was then induced in the same four animals by intraperitoneal inoculation of autologous menstrual tissue. Following laparoscopic confirmation of endometriosis, EUE samples were obtained at 3 months of disease. MATERIALS AND METHODS: Total RNA from the EUE was extracted and purified. A 8x15K miRNA microarray (Agilent) was applied. The signal data were preprocessed by AgiMiRna, a Limma package for R environment. Empirical Bayes model was used to estimate the P-values. The potential mRNA targets of differentially expressed miRNAs were predicted and compared against Affymetrix Human Genome Plus 2.0 expression data. CONCLUSION: Induction of endometriosis causes rapid changes in the expression of several miRNAs in the baboon endometrium. miRNAs 200a, 424, 181a, 21, 29c, and 141, previously reported to be altered in women with endometriosis, were also altered in the endometrium of baboons with endometriosis, further validating this animal model. In addition, miR451, a previously unreported miRNA associated with the apoptotic pathway and cell survival, was significantly down regulated.

The Transcription Factor C/EBPβ is a Marker of Uterine Receptivity and Expressed at the Implantation Site in the Primate

During early pregnancy, the endometrium undergoes pronounced hormone-dependent functional changes in preparation for embryo implantation. Local autocrine-paracrine signaling at the fetal-maternal interface is crucial for the establishment of pregnancy. We previously reported that the transcription factor C/ EBPbeta, which is expressed at the implantation sites (ISs) in pregnant mice, acts as a key mediator of steroid hormone responsiveness in the endometrium. Mice lacking C/EBPbeta fail to support implantation due to defects in epithelial proliferation and stromal cell differentiation. In the current study, C/EBPbeta expression was dramatically stimulated in the endometrium of baboons (Papio anubis) during the window of uterine receptivity in response to a local infusion of chorionic gonadotropin, an embryonic signal. A robust induction of C/EBPbeta expression was also seen at the IS in the baboon and the human. Collectively, our results indicate that C/EBPbeta is a biomarker of endometrial receptivity and plays a conserved functional role during implantation in the primate.

Divergent regulation of angiopoietin‐1 and ‐2, Tie‐2, and thrombospondin‐1 expression by estrogen in the baboon endometrium

Estrogen has an important role in the reconstruction of a new vascular network in the endometrium during each menstrual cycle; however, the underlying mechanisms are incompletely understood. Angiopoietin-1 (Ang-1) promotes vessel assembly, whereas Ang-2 and thrombospondin-1 (TSP-1) cause vessel breakdown. To determine the potential effect of estrogen on the expression of these angioregulatory factors in the endometrium, Ang-1, Ang-2, TSP-1, and Tie-2 receptor mRNA levels were assessed by real-time reverse transcriptase polymerase chain reaction in glandular epithelial and stromal cells isolated from the endometrium of ovariectomized baboons treated acutely with estradiol. Corresponding protein expression was assessed by immunocytochemistry and the proximity ligation assay (PLA) during advancing stages of the baboon menstrual cycle. Serum estradiol levels in ovariectomized baboons were 400 pg/ml within 4-6 hr of estradiol treatment. Ang-1 mRNA levels in glandular epithelial cells increased threefold (P < 0.01) within 4 hr of estradiol administration. In contrast, TSP-1 mRNA levels decreased four- to fivefold (P < 0.01) in endometrial glandular epithelial and stromal cells 4-6 hr after estradiol, whereas Ang-2 and Tie-2 expression was unaltered. Immunostaining for Ang-1 increased, TSP-1 decreased, and Ang-2 and Tie-2 were unaltered in the endometrium during the secretory compared with the proliferative phase of the cycle. Endometrial Ang-1 protein expression, quantified by PLA, increased 10-fold (P < 0.05) between the early proliferative and late proliferative/mid-secretory phases of the menstrual cycle in association with the rise in estrogen. In summary, estrogen induced a rapid, divergent, and cell-specific change in expression of angiostimulatory and angioinhibitory growth factors in the endometrium of the nonhuman primate.

Connexin expression pattern in the endometrium of baboons is influenced by hormonal changes and the presence of endometriotic lesions

Experimentally induced endometriosis in baboons serves as an elegant model to discriminate between endometrial genes which are primarily associated with normal endometrial function and those that are changed by the presence of endometriotic lesions. Since connexin genes are characteristic of the hormonally regulated differentiation of the endometrium, we have examined connexin expression in baboon endometrium to delineate if they are altered in response to the presence of endometriotic lesions. Connexin expression in the endometrium of cycling baboons is similar to that of the human endometrium with Connexin(Cx)43 being primarily seen in the stromal compartment and Cx26 and Cx32 being present predominantly in the epithelium. Although Cx32 is up-regulated during the secretory phase, Cx26 and Cx43 are down-regulated. In the baboon model of induced endometriosis a change in connexin pattern was evident in the presence of endometriotic lesions. In the secretory phase, Cx26 and Cx32 are no longer present in the epithelium but Cx26 is now observed primarily in the stromal cells. Infusion of chorionic gonadotrophin in a manner that mimics blastocyst transit in utero failed to rescue the aberrant stromal expression of Cx26 that is associated with the presence of endometriotic lesions suggesting an impairment of the implantation process. The altered connexin pattern coupled with a loss of the channel protein in the epithelium and a gain of Cx26 in the stromal compartment suggests that the presence of lesions changes the uterine environment and thereby the differentiation programme. This aberrant expression of connexins may be an additional factor that contributes to endometriosis-associated infertility.

Divergent Regulation of Vascular Endothelial Growth Factor, Angiopoietin-1 and -2, and Thrombospondin-1 Expression by Estrogen in the Baboon Endometrium.

Divergent Regulation of Vascular Endothelial Growth Factor, Angiopoietin-1 and -2, and Thrombospondin-1 Expression by Estrogen in the Baboon Endometrium.

The eutopic endometrial response to chorionic gonadotrophin is abnormal in a baboon model of endometriosis: a possible explanation for endometriosis associated implantation failure

OBJECTIVE: To test the hypothesis that the endometrial response to Chorionic Gonadotrophin (CG) the major early, embryo derived signal, is altered by the presence of endometriosis. DESIGN: We assessed endometrial morphology, protein and gene expression changes occurring after in vivo intra-oviductal CG treatment of baboons with endometriosis. MATERIALS AND METHODS: Endometriosis was experimentally induced in female baboons (n=5) by intraperitoneal inoculation with menstrual endometrium. Three and six months following disease induction, human CG (hCG; 30IU/day) was infused into the oviduct via a subcutaneous osmotic mini-pump and polyurethane cannula. HCG infusion began on day 6 post ovulation (PO). Endometrial tissue was subsequently harvested by endometriectomy between days 9 to 11 PO, corresponding to the approximate time of implantation in the baboon. Control endometria were similarly harvested from disease free animals (n=6). Total RNA from each animal was hybridized to HumanRef-6 v3.0 Expression BeadChips (Illumina) containing 23,000 unique genes. Microarray data analyses were performed using BRB Array Tools. Verification of microarray data was performed using real time (RT) PCR; protein abundance changes were assayed by immunohistochemistry. All experimental procedures were approved by the Animal Care Committee of the University of Illinois, Chicago. RESULTS: Assessment of an early morphological response of baboon endometrium to hCG, the development of an epithelial plaque, revealed that the endometrium of all control animals was stimulated by hCG. However, an epithelial plaque was only elicited by hCG in two of five animals with endometriosis. Microarray analysis of endometria from baboons three and six months following induction of disease identified 22 and 116 genes, respectively, whose transcript levels differed more than 2-fold from those of control endometria (p<0.05). Several of these genes were previously shown to be regulated by hCG in disease free baboons. RT PCR analysis confirmed aberrant levels of LIF, complement factor C3, sFRP4 and SERPA3 in endometria of baboons with endometriosis. Immunohistochemical analysis revealed that this aberrant gene expression profile was not a result of altered CG receptor distribution in the endometrium of animals with endometriosis. CONCLUSIONS: An altered response to CG may prevent the acquisition of the full endometrial molecular repertoire necessary for implantation and may in part explain the implantation failure associated with endometriosis.

Vascular Endothelial Growth Factor Mediates the Estrogen-Induced Breakdown of Tight Junctions between and Increase in Proliferation of Microvessel Endothelial Cells in the Baboon Endometrium

To assess whether there is a link between estrogen, vascular endothelial growth factor (VEGF), and early aspects of uterine angiogenesis, an acute temporal study was conducted in which ovariectomized baboons were pretreated with VEGF Trap, which sequesters endogenous VEGF, and administered estradiol at time 0 h. Serum estradiol levels approximated 500 pg/ml 4-6 h after estradiol administration. VEGF mRNA levels in endometrial glandular epithelial and stromal cells were increased to values 6 h after estradiol that were 3.74 +/- 0.99-fold (mean +/- se) and 5.70 +/- 1.60-fold greater (P < 0.05), respectively, than at 0 h. Microvessel interendothelial cell tight junctions, which control paracellular permeability, were present in the endometrium at time 0 h, but not evident 6 h after estradiol administration. Thus, microvessel paracellular cleft width increased (P < 0.01, ANOVA) from 5.03 +/- 0.22 nm at 0 h to 7.27 +/- 0.48 nm 6 h after estrogen. In contrast, tight junctions remained intact, and paracellular cleft widths were unaltered in estradiol/VEGF Trap and vehicle-treated animals. Endometrial microvessel endothelial cell mitosis, i.e. percent Ki67+/Ki67- immunolabeled endothelial cells, increased (P < 0.05) from 2.9 +/- 0.3% at 0 h to 21.4 +/- 7.0% 6 h after estrogen treatment but was unchanged in estradiol/VEGF Trap and vehicle-treated animals. In summary, the estrogen-induced disruption of endometrial microvessel endothelial tight junctions and increase in endothelial cell proliferation were prevented by VEGF Trap. Therefore, we propose that VEGF mediates the estrogen-induced increase in microvessel permeability and endothelial cell proliferation as early steps in angiogenesis in the primate endometrium.

Differential regulation of insulin-like growth factor binding protein-1 and -2 by insulin in the baboon (Papio anubis) endometrium

BackgroundThe purpose of this study was to examine the effect of insulin on expression and synthesis of IGFBP-1 and IGFBP-2 in the baboon endometrium in vitro.MethodsBaboon endometrial explants collected from cycling, ovariectomized, steroid-treated, simulated-pregnant and pregnant animals were cultured for 48 h in the presence or absence of insulin, with or without estradiol, progesterone and hCG.ResultsInsulin clearly inhibited IGFBP-1 production and mRNA expression in a time- and dose-dependent manner, whereas IGFBP-2 synthesis was not significantly affected. The inhibitory effects of insulin on IGFBP-1 were more evident in explants of non-pregnant tissue or tissue away from the implantation site. In the absence of insulin, synthesis of IGFBP-1 was induced in explants with low levels of de novo synthesis whereas IGFBP-2 synthesis was inhibited. This effect was potentiated by steroids and hCG in the explant cultures.ConclusionInsulin differentially regulates endometrial IGFBP-1 and IGFBP-2 secretion in the baboon.

Altered expression of HOXA10 in endometriosis: potential role in decidualization

Endometriosis is a poorly understood gynaecologic disorder that is associated with infertility. In this study, we examined the expression of HOXA10 in the eutopic endometrium of baboons with induced endometriosis. A decrease in HOXA10 mRNA was observed after 3, 6, 12 and 16 months of disease, which reached statistical significance at 12 and 16 months. HOXA10 protein levels were decreased in both the epithelial and stromal cells of the endometrium. Furthermore, expression of beta3 integrin (ITGB3), which is upregulated by HOXA10, was decreased, whereas EMX2, a gene that is inhibited by HOXA10, was increased. Next, methylation patterns of the HOXA10 gene were analysed in the diseased and control animals. The F1 region on the promoter was found to be the most significantly methylated in the endometriosis animals and this may account for the decrease in HOXA10 expression. Finally, we demonstrate that stromal cells from the eutopic endometrium of baboons with endometriosis expressed significantly higher levels of insulin-like growth factor binding protein-1 (IGFBP1) mRNA than disease-free animals in response to estradiol, medroxyprogesterone acetate and dibutyryl cAMP (H + dbcAMP). The functional role of HOXA10 in IGFBP1 expression was further explored using human endometrial stromal cells (HSC). Overexpression of HOXA10 in HSC resulted in a decrease of IGFBP1 mRNA, whereas silencing HOXA10 caused an increase of IGFBP1 mRNA, even in the presence of H + dbcAMP. These data demonstrate that HOXA10 negatively influences IGFBP1 expression in decidualizing cells. Thus, the decrease in HOXA10 levels may in part be involved with the altered uterine environment associated with endometriosis.

The Altered Distribution of the Steroid Hormone Receptors and the Chaperone Immunophilin FKBP52 in a Baboon Model of Endometriosis Is Associated With Progesterone Resistance During the Window of Uterine Receptivity

This study examines the distribution of estrogen receptors (ESR), progesterone receptors (Pgr), and the chaperone immunophilin FKBP52 in the eutopic endometrium in a baboon model of endometriosis during the window of receptivity to determine if their aberrant distribution contributes to reduced fecundity. Endometriosis was induced by inoculation of menstrual endometrium into the peritoneal cavity. Eutopic endometrium was collected at 3, 6, 9, 12, and 15 months postinoculation. Western blot (WB) and immunohistochemical analyses were performed. Isolated endometrial stromal cells were cultured in the presence or absence of steroid hormones. In animals with endometriosis, ESR-1 (ER-alpha) decreased in endometrial stromal cells, while ESR-2 (ER-beta) was reduced in both glandular epithelial (GE) and stromal cells. Immunoreactive total Pgr was markedly diminished in the GE, which was confirmed by WB analysis. Furthermore, treatment of isolated stromal cells from baboons with endometriosis with hormones did not increase levels of PRA or PRB as in control baboons. FKBP52 was also reduced in the eutopic endometrium of baboons with endometriosis. Endometriosis results in an aberrant distribution of ESR-1, ESR-2, Pgr, and FKBP52 in the eutopic endometrium. The authors propose that a dysregulation in the paracrine signaling between the endometrial stromal and GE cells reduces the responsiveness of Pgr, creating an endometrial environment that is unsuitable for implantation.