Characterization of epidermal growth factor-like domain 7 (EGFL7) expression in normal endometrium and in the endometrium of women with poor reproductive outcomes.

STUDY QUESTION: Could epidermal growth factor-like domain 7 (EGFL7) be a factor involved in the preparation of the endometrium for implantation and could its dysregulation be implicated in poor reproductive outcomes? SUMMARY ANSWER: EGFL7 is highly expressed in the endothelium and glandular epithelium throughout the menstrual cycle; it is upregulated by stromal cells in secretory phase and appears strongly reduced in endometrial biopsies and isolated stromal cells of women with unexplained recurrent pregnancy loss (uRPL) and recurrent implantation failure (RIF). WHAT IS KNOWN ALREADY: The secreted factor EGFL7, originally identified as a gene primarily expressed in endothelial cells, is also expressed by the mouse blastocyst and by mouse and human trophoblast cells. It regulates trophoblast migration and invasion by activating NOTCH1 signaling. NOTCH1 has been demonstrated to play a fundamental role in endometrial receptivity and its dysregulation may be involved in selected pregnancy complications characterized by altered endometrial receptivity, such as uRPL. STUDY DESIGN, SIZE, DURATION: This is an exploratory study for which 84 endometrial biopsies were collected from normally fertile women, as well as from women with uRPL and RIF. PARTICIPANTS/MATERIALS, SETTING, METHODS: Samples were collected from women in both the proliferative and secretory phases of the menstrual cycle and stratified into three sub-groups according to the patient clinical history: 20 fertile women (8 in proliferative and 12 in secretory phase), 41 women with uRPL (6 in proliferative and 35 in secretory phase), and 27 women with RIF (8 in proliferative and 19 in secretory phase). Immunohistochemistry, real-time PCR, and western blot analyses were performed to study the expression of EGFL7 and NOTCH1, as well as the NOTCH target genes. MAIN RESULTS AND THE ROLE OF CHANCE: Analysis of spatial and temporal distribution of EGFL7 in endometrial biopsies from fertile women revealed higher levels of EGFL7 in samples from the secretory phase compared to proliferative phase. The expected expression of EGFL7 in endothelial cells was shown as well as the novel, not previously reported, expression in endometrial glands and stromal cells. EGFL7 was significantly reduced in the endometrium of women with uRPL and RIF in the secretory phases and this was associated with a downregulation of the NOTCH1 signaling pathway. Human recombinant EGFL7 was able to activate the NOTCH1 signaling pathway in endometrial stromal cells (EndSCs) obtained from fertile women but not in cells from uRPL or RIF patients. EndSCs from fertile women and decidualized in vitro for three days showed an upregulation of EGFL7 expression, whereas cells obtained from women with uRPL and RIF and decidualized in vitro did not. LIMITATIONS, REASONS FOR CAUTION: This study was conducted with a relatively small number of patient samples. Although results are highly reproducible and consistent, additional observations from multicentric cohorts would strengthen the relevance of the data. Moreover, this is an in vitro study, which might only partially represent the in vivo conditions. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate for the first time that EGFL7 is new player involved in decidualization and provide new insights into the pathophysiology of selected implantation defects and early pregnancy complications. Our studies have revealed that alterations in EGFL7 expression and the consequent dysregulation of NOTCH signaling are potential underlying causes of RIF and uRPL. Our results might have therapeutic relevance, as the EGFL7/NOTCH pathway may represent a potential target for medical intervention. STUDY FUNDING/COMPETING INTEREST(S): This study has been supported by the Grant for Fertility Innovation 2017 (Merck KGaA). There are no competing interests to disclose. TRIAL REGISTRATION NUMBER: Not applicable.

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 QUESTION: Is 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 ANSWER: Women 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 ALREADY: Endometrial 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, DURATION: This 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, METHODS: The 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 CHANCE: Women 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 DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Small 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 FINDINGS: Since 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.

cAMP-Response Element-Binding 3-Like Protein 1 (CREB3L1) is Required for Decidualization and its Expression is Decreased in Women with Endometriosis.

Endometriosis is a major cause of infertility and pelvic pain, affecting more than 10% of reproductive-aged women. Progesterone resistance has been observed in the endometrium of women with this disease, as evidenced by alterations in progesterone-responsive gene and protein expression. cAMPResponse Element-Binding 3-like protein 1 (Creb3l1) has previously been identified as a progesterone receptor (PR) target gene in mouse uterus via high density DNA microarray analysis. However, CREB3L1 function has not been studied in the context of endometriosis and uterine biology. In this study, we validated progesterone (P4) regulation of Creb3l1 in the uteri of wild-type and progesterone receptor knockout (PRKO) mice. Furthermore, we observed that CREB3L1 expression was significantly higher in secretory phase human endometrium compared to proliferative phase and that CREB3L1 expression was significantly decreased in the endometrium of women with endometriosis. Lastly, by transfecting CREB3L1 siRNA into cultured human endometrial stromal cells (hESCs) prior to hormonal induction of in vitro decidualization, we showed that CREB3L1 is required for the decidualization process. Interestingly, phosphorylation of ERK1/2, critical factor for decidualization, was also significantly reduced in CREB3L1-silenced hESCs. It is known that hESCs from patients with endometriosis show impaired decidualization and that dysregulation of the P4-PR signaling axis is linked to a variety of endometrial diseases including infertility and endometriosis. Therefore, these results suggest that CREB3L1 is required for decidualization in mice and humans and may be linked to the pathogenesis of endometriosis in a P4-dependent manner.

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

STUDY QUESTION: Are microRNAs (miRs) altered in the eutopic endometrium (EuE) of baboons following the induction of endometriosis? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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.

Lesion kinetics in a non-human primate model of endometriosis.

BACKGROUND: Endometriosis is a common cause of pelvic pain and infertility in women of reproductive age. It is characterized by the presence of endometrial tissue outside the normal location, predominantly in the pelvic peritoneum causing severe abdominal pain. However, the severity of the symptoms of endometriosis does not always correlate with the anatomic severity of the disease. This lack of correlation may be due to morphological lesion variation during disease progression. This study examined lesion kinetics in a non-human primate model of endometriosis to better understand lesion dynamics. METHODS: Endometriosis was experimentally induced in nine normal cycling female adult olive baboons (Papio anubis) by i.p. inoculation of autologous menstrual endometrium on Day 2 of menses for two consecutive menstrual cycles. Diagnostic laparoscopies were performed between Day 8-12 post-ovulation at 1, 3, 6, 9 and 12 months, followed by a necropsy at 15 months, after the second inoculation. In two animals, lesions were excised/ablated at 6 months and they were monitored for lesion recurrence and morphological changes by serial laparoscopy. Furthermore, five control animals underwent surgeries conducted at the same time points but without inoculation. RESULTS: A total of 542 endometriotic lesions were observed. The location, macroscopic (different colours) and microscopic appearance confirmed distinct endometriosis pathology in line with human disease. The majority of the lesions found 1 month after tissue inoculation were red lesions, which frequently changed colour during the disease progression. In contrast, blue lesions remained consistently blue while white lesions were evident at the later stages of the disease process and often regressed. There were significantly lower numbers of powder burn, blister and multicoloured lesions observed per animal in comparison to black and blue lesions (P-value≤0.05). New lesions were continually arising and persisted up to 15 months post-inoculation. Lesions reoccurred as early as 3 months after removal and 69% of lesions excised/ablated had reoccurred 9 months later. Interestingly, endometriotic lesions were also found in the non-inoculated animals, starting at the 6-month time point following multiple surgeries. CONCLUSIONS: Documentation of lesion turnover in baboons indicated that lesions changed their colour from red to white over time. Different lesion types underwent metamorphosis at different rates. A classification of lesions based on morphological appearance may help disease prognosis and examination of the effect of the lesion on disease symptoms, and provide new opportunities for targeted therapies in order to prevent or treat endometriosis. Surgical removal of endometriotic lesions resulted in a high incidence of recurrence. Spontaneous endometriosis developed in control baboons in the absence of inoculation suggesting that repetitive surgical procedures alone can induce the spontaneous evolution of the chronic disease. Although lesion excision/ablation may have short-term benefits (e.g. prior to an IVF cycle in subfertile women), for long-term relief of symptoms perhaps medical therapy is more effective than surgical therapy.

Induction of endometriosis alters the peripheral and endometrial regulatory T cell population in the non-human primate.

BACKGROUND: Endometriosis is a gynecological condition that is characterized by extreme abdominal pain and also decreased fertility. Regulatory T cells (Tregs) have immunosuppressive activity critical for embryonic implantation and likewise the acceptance of tissue engraftment. Utilizing the induced non-human primate (Papio anubis) model of endometriosis, we hypothesize that endometriosis decreases the peripheral and endomet rial Treg profile, whereas ectopic lesions have increased Treg localization. METHODS: Peripheral blood and endometrium were obtained throughout the menstrual cycle prior to and after induction of disease. Animals were randomly assigned to control (n = 7) or diseased (n = 16) treatment groups. Endometriosis was induced by i.p. injection of autologous menstrual tissue for 2 consecutive months during menses. Peripheral blood and endometrial tissue were collected at d9-11PO at 1, 3, 6, 9, 12 and 15 months post-induction of disease for fluorescence-activated cell sorting, quantitative RT-PCR and immunohistochemistry. Ectopic lesions were excised at 1 and 6 months post-inoculation and also harvested at necropsy (15 months) and processed for RNA of IHC. Identification of Tregs through analysis of FOXP3 expression was conducted utlilizing several methodologies. Differences were determined by non-parametric statistical analysis between all treatment groups and time points. RESULTS: In control animals, the proportion of peripheral natural Tregs (nTregs) was reduced (P < 0.05) during the mid- and late secretory stages of the menstrual cycle compared with menses. The induction of disease decreased peripheral Treg expression at early time points (P < 0.05) and this remained low throughout the time course, compared with the pre-inoculatory level of an individual. FOXP3 gene expression and Treg populations were also decreased in the eutopic endometrium (P < 0.05) compared with control animals, whereas these parameters were increased in ectopic lesions (P < 0.05), compared with the eutopic endometrium. CONCLUSIONS: Our data suggest that a reduction in peripheral Tregs may be a causative factor for endometriosis-associated infertility, while the increase in ectopic Treg expression may aid lesion development. Furthermore, endometriosis appears to disrupt Treg recruitment in both eutopic and ectopic endometrium.

Endometrial stromal cells and immune cell populations within lymph nodes in a nonhuman primate model of endometriosis.

Mounting evidence suggests that immunological responses may be altered in endometriosis. The baboon (Papio anubis) is generally considered the best model of endometriosis pathogenesis. The objective of the current study was to investigate for the first time immunological changes within uterine and peritoneal draining lymph nodes in a nonhuman primate baboon model of endometriosis. Paraffin-embedded femoral lymph nodes were obtained from 22 normally cycling female baboons (induced endometriosis n = 11; control n = 11). Immunohistochemical staining was performed with antibodies for endometrial stromal cells, T cells, immature and mature dendritic cells, and B cells. Lymph nodes were evaluated using an automated cellular imaging system. Endometrial stromal cells were significantly increased in lymph nodes from animals with induced endometriosis, compared to control animals (P = .033). In animals with induced endometriosis, some lymph node immune cell populations including T cells, dendritic cells and B cells were increased, suggesting an efficient early response or peritoneal drainage.

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.

Aberrant expression of regulators of cell-fate found in eutopic endometrium is found in matched ectopic endometrium among women and in a baboon model of endometriosis.

BACKGROUND: We have recently shown that women with endometriosis express an increased amount of telomerase and nucleolin, with concomitant loss of γ-H2AX in eutopic endometrium. To further examine these selected factors that regulate cell fate, in the pathogenesis of endometriosis, we studied the expression of telomerase, nucleolin, proliferating cell nuclear antigen and γ-H2AX in ectopic endometriotic deposits from women, and in matched eutopic and ectopic endometrial tissue from a baboon model of endometriosis. METHODS: Ectopic active peritoneal endometriotic lesions were collected from seven symptomatic women. Endometriosis was induced in six baboons by intra-peritoneal autologous inoculation of menstrual endometrium. Eutopic and matched ectopic endometrial tissues were collected prior to and 6, 12 and 15 months after the induction of endometriosis as previously described. Eutopic endometrium was also obtained from eight healthy fertile control baboons. Immunohistochemistry was performed as previously described, and telomerase activity was confirmed using the telomeric repeat amplification protocol assay. RESULTS: All active human endometriotic lesions expressed the proliferative markers but showed weak or absent staining for γ-H2AX. A similar expression pattern of these markers was seen in the ectopic lesions of the baboons with induced disease. In these baboons, the eutopic endometrium also showed intense immunoreactivity for all proliferative markers 6-12 months after induction with a parallel loss of γ-H2AX. The opposite staining pattern was seen in eutopic endometrium of healthy animals and in pre-induction endometrium of animals with induced disease. CONCLUSIONS: Endometriotic lesions have excess proliferative potential; in baboons, these were present within 12 months of the initiation of the disease. In eutopic tissue, these changes appear to be induced by the development of endometriosis.

The endometrial response to chorionic gonadotropin is blunted in a baboon model of endometriosis.

Endometriosis-associated infertility has a multifactorial etiology. We tested the hypothesis that the endometrial response to the early embryonic signal, human chorionic gonadotropin (hCG), alters over time in a nonhuman primate model of endometriosis. Animals with experimental or spontaneous endometriosis were treated with hCG (30 IU/d), from d 6 after ovulation for 5 d, via an oviductal cannula. Microarray analysis of endometrial transcripts from baboons treated with hCG at 3 and 6 months of disease (n=6) identified 22 and 165 genes, respectively, whose levels differed more than 2-fold compared with disease-free (DF) animals treated with hCG (P<0.01). Quantitative RT-PCR confirmed abnormal responses of known hCG-regulated genes. APOA1, SFRP4, and PAPPA, which are normally down-regulated by hCG were up-regulated by hCG in animals with endometriosis. In contrast, the ability of hCG to induce SERPINA3 was lost. Immunohistochemistry demonstrated dysregulation of C3 and superoxide dismutase 2 proteins. We demonstrate that this abnormal response to hCG persists for up to 15 months after disease induction and that the nature of the abnormal response changes as the disease progresses. Immunohistochemistry showed that this aberrant gene expression was not a consequence of altered LH/choriogonadotropin receptor distribution in the endometrium of animals with endometriosis. We have shown that endometriosis induces complex changes in the response of eutopic endometrium to hCG, which may prevent the acquisition of the full endometrial molecular repertoire necessary for decidualization and tolerance of the fetal allograft. This may in part explain endometriosis-associated implantation failure.

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.

The non-human primate model of endometriosis: research and implications for fecundity.

The development of an animal model of endometriosis is crucial for the investigation of disease pathogenesis and therapeutic intervention. These models will enhance our ability to evaluate the causes for the subfertility associated with disease and provide a first-line validation of treatment modulators. Currently rodents and non-human primate models have been developed, but each model has their limitations. The aim of this manuscript is to summarize the current findings and theories on the development of endometriosis and disease progression and the effectiveness of therapeutic targets using the experimental induced model of endometriosis in the baboon (Papio anubis).

IFPA meeting 2008 workshops report.

Workshops are an important part of the IFPA annual meeting. At the IFPA meeting 2008 diverse topics were discussed in 12 themed workshops. Topics covered included: immunology of placentation; galectins and trophoblast invasion; signaling in implantation and invasion; markers to identify trophoblast subpopulations; placental pathology; placental toxicology; stereology; placental transport of fatty acids; placental mesenchymal stem cells; comparative placentation; trophoblast and neoplasia; trophoblast differentiation. This report is a summary of the various topics covered.

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.

A commentary on gestational programming and functions of HLA-G in pregnancy.

HLA-G is an HLA class Ib gene that is highly expressed in human trophoblast cells. The single HLA-G mRNA is alternatively spliced to generate at least seven transcripts, three of which encode soluble isoforms. Many studies have shown that high levels of soluble antigens are associated with successful implantation and graft acceptance. To study expression, regulation and functions of two of the soluble isoforms, HLA-G5 and HLA-G6, we generated recombinant proteins in eukaryotic cells and developed monoclonal antibodies specific for each of the two proteins. In addition, we investigated the olive baboon Paan-AG gene as a potential functional correlate of HLA-G. Here, we present summaries of the studies that have been conducted in our laboratory using these tools and discuss the results within the context of the research on this topic that is ongoing in ours and other laboratories worldwide. Collectively, the data indicate that soluble HLA-G is a critical contributor to immune privilege in pregnancy and imply that this placenta-derived substance may impact other pathways leading to successful reproduction.

Decidual stromal cell response to paracrine signals from the trophoblast: amplification of immune and angiogenic modulators.

During the invasive phase of implantation, trophoblasts and maternal decidual stromal cells secrete products that regulate trophoblast differentiation and migration into the maternal endometrium. Paracrine interactions between the extravillous trophoblast and the maternal decidua are important for successful embryonic implantation, including establishing the placental vasculature, anchoring the placenta to the uterine wall, and promoting the immunoacceptance of the fetal allograph. To our knowledge, global crosstalk between the trophoblast and the decidua has not been elucidated to date, and the present study used a functional genomics approach to investigate these paracrine interactions. Human endometrial stromal cells were decidualized with progesterone and further treated with conditioned media from human trophoblasts (TCM) or, as a control, with control conditioned media (CCM) from nondecidualized stromal cells for 0, 3, and 12 h. Total RNA was isolated and processed for analysis on whole-genome, high-density oligonucleotide arrays containing 54,600 genes. We found that 1374 genes were significantly upregulated and that 3443 genes were significantly downregulated after 12 h of coincubation of stromal cells with TCM, compared to CCM. Among the most upregulated genes were the chemokines CXCL1 (GRO1) and IL8,CXCR4, and other genes involved in the immune response (CCL8 [SCYA8], pentraxin 3 (PTX3), IL6, and interferon-regulated and -related genes) as well as TNFAIP6 (tumor necrosis factor alpha-induced protein 6) and metalloproteinases (MMP1, MMP10, and MMP14). Among the downregulated genes were growth factors, e.g., IGF1, FGF1, TGFB1, and angiopoietin-1, and genes involved in Wnt signaling (WNT4 and FZD). Real-time RT-PCR and ELISAs, as well as immunohistochemical analysis of human placental bed specimens, confirmed these data for representative genes of both up- and downregulated groups. The data demonstrate a significant induction of proinflammatory cytokines and chemokines, as well as angiogenic/static factors in decidualized endometrial stromal cells in response to trophoblast-secreted products. The data suggest that the trophoblast acts to alter the local immune environment of the decidua to facilitate the process of implantation and ensure an enriched cytokine/chemokine environment while limiting the mitotic activity of the stromal cells during the invasive phase of implantation.

Identification of novel genes regulated by chorionic gonadotropin in baboon endometrium during the window of implantation.

Chorionic gonadotropin (CG) is an early embryo-derived signal that is known to support the corpus luteum. An in vivo baboon model was used to study the direct actions of human CG (hCG) on the endometrium, during the periimplantation period. Endometrial gene expression was analyzed using microarrays. The endometrial biopsies were taken from hCG-treated (n = 5) and control (n = 6) animals on d 10 after ovulation. Class comparison identified 61 genes whose transcript levels differed between control and hCG-treated samples (48 increased, 13 decreased in mean expression level more than 2.5-fold; P < 0.01). Real-time PCR of transcript abundance confirmed up-regulation of several of these, including SerpinA3, matrix metalloproteinase 7, leukemia inhibitory factor (LIF), IL-6, and Complement 3 (P

Morphological and glycosylation changes associated with the endometrium and ectopic lesions in a baboon model of endometriosis.

BACKGROUND: Endometriosis is one of the most common causes of infertility and pelvic pain. A baboon model has recently been developed whereby the intrapelvic injection of menstrual endometrium results in the induction of endometriotic lesions. We have used this model to investigate changes in ultrastructure and glycosylation of endometria from normal and diseased baboons. METHODS: Endometriosis was induced in eight female baboons; endometrial tissue and endometriotic lesions were removed on days 9-11 post ovulation between 3 and 16 months of disease and compared with endometrium from 17 control animals, using electron microscopy and lectin histochemistry. RESULTS: Ultrastructurally, diseased endometrial glands showed abnormalities in secretory vacuoles and an intracellular accumulation of glycogen; in later stages of the disease, glands resembled those of the late secretory phase endometrium. The abnormalities were mirrored by changes in glycan expression. In early disease, there was an increased binding of lectin from Dolichos biflorus agglutinin (DBA) to fucosylated N-acetylglucosamine residues, whereas in later stages, this binding generally decreased in association with the appearance of a late secretory phenotype. CONCLUSIONS: Endometriosis is accompanied by progressive changes in the gland architecture and biochemistry resulting in dyssynchrony within the window of uterine receptivity, which may result in the reduced fertility associated with this disease.

Extracellular matrix metalloproteinase inducer regulates metalloproteinases in human uterine endometrium.

CONTEXT: Endometrial remodeling occurs during each menstrual cycle in women and also during the establishment of endometriosis. Both processes involve the production of metalloproteinases (MMPs) by uterine endometrial cells. OBJECTIVE: The objective of this study was to determine whether tissue remodeling and endometrial invasion involve activation of MMPs by extracellular matrix metalloproteinase inducer (EMMPRIN). MAIN OUTCOME MEASURES: EMMPRIN expression was examined by immunohistochemistry and real-time PCR in ectopic and eutopic endometria. For functional assays, human uterine fibroblasts were treated in the absence or presence of IL-1beta (10 ng/ml) or purified native EMMPRIN (0.5 or 1 microg/ml) for 24 h. Cellular RNA and conditioned medium were assayed by real-time PCR or immunoblotting. RESULTS: EMMPRIN protein localized to epithelial and fibroblast cells of eutopic and ectopic endometria. The pattern of localization was regulated by ovarian hormones. EMMPRIN mRNA levels varied throughout the menstrual cycle in parallel with the cyclic changes in estradiol. EMMPRIN treatment (0.5 microg/ml) of human uterine fibroblast cells stimulated MMP-1 (5.23-fold) and MMP-2 (8.55-fold), but not MMP-3, mRNA levels over levels in control cells (P < 0.05). EMMPRIN treatment (1 microg/ml) stimulated endogenous EMMPRIN (1.6-fold) mRNA levels (P > 0.05). IL-1beta stimulated MMP-1 (5.6-fold), MMP-2 (2.8-fold), and MMP-3 (75-fold) gene expression, but not EMMPRIN, over levels in control cells (P < 0.05). Both EMMPRIN and IL-1beta treatments stimulated MMP-1, -2, and -3, but not EMMPRIN protein secretion, with 0.5 microg/ml producing the greatest response. CONCLUSIONS: The ability of EMMPRIN to stimulate MMP secretion by endometrial fibroblasts indicates its potential role in uterine remodeling and the pathogenesis of endometriosis.