Wilder A. Palomino

ECC-1 Cells: A Well-Differentiated Steroid-Responsive Endometrial Cell Line with Characteristics of Luminal Epithelium

Abstract Endometrial cancer cell lines have provided a valuable model to study endometrial epithelial cells in vitro. Since the first development of HEC1B over 35 yr ago, many different cell lines have been isolated and described. One valuable cell line that maintains hormone responsiveness and unique stability over time is the ECC-1 cell line, developed originally by the late P.G. Satyaswaroop. In this study, we investigated some of the properties of these cells and present their salient characteristics. Like Ishikawa cells, ECC-1 cells maintain both estrogen receptors (ESR1 [ER alpha] and ESR2 [ER beta]), progesterone receptors (PR A and B; PGRs), and androgen receptors (ARs), along with the p160 steroid receptor coactivators NCOA1 (formerly SRC1), NCOA2 (formerly TIF2), and NCOA3 (formerly AIB1). The karyotype of these cells is abnormal, with multiple structural rearrangements in all cells analyzed. Unlike Ishikawa cells that express glandular epithelial antigens, ECC-1 cells maintain a luminal phenotype, with expression of KRT13 (cytokeratin 13) and KRT18 (cytokeratin 18). Apparent differences in the regulation of ESR2 also were evident in ECC-1 cells compared to Ishikawa cells. Like other endometrial cell lines, ECC-1 cells express the steroid receptor coactivators and exhibit epidermal growth factor-stimulated expression of known luminal proteins thought to be involved in implantation, including the hyaluronate receptor CD44 and SPP1 (formerly osteopontin) and CD55 (decay-accelerating factor). These characteristics appear to be stable and persistent over multiple cell passages, making this well-differentiated cell line an excellent choice to study endocrine and paracrine regulation of endometrial epithelium in vitro.

A single midcycle dose of levonorgestrel similar to emergency contraceptive does not alter the expression of the L-selectin ligand or molecular markers of endometrial receptivity.

OBJECTIVE To examine the effects of a single-dose of 1.5 mg of levonorgestrel (commonly used as emergency contraceptive) on endometrial receptivity biomarkers through the oral or vaginal route. DESIGN Prospective randomized single-blinded trial. SETTING Affiliated Hospital and University Research Center. PATIENT(S) Fertile normal women previously sterilized by tubal ligation. INTERVENTION(S) Levonorgestrel (1.5 mg) was administered on the day of LH surge either orally (n = 14) or vaginally (n = 13). MAIN OUTCOME MEASURE(S) Molecular assessment of endometrial progesterone receptors, L-selectin ligand, glicodelin-A and αvβ3 integrin by Immunohistochemistry and reverse transcriptase-polymerase chain reaction. RESULT(S) Plasma progesterone concentration and endometrial dating were not different. The pattern of progesterone receptors and glycodelin-A expression was not affected during the early and midsecretory phase. Some endometrial biopsies from the group in which levonorgetrel was orally administered showed areas of glandular atrophy and stromal decidualization. However, the expression of the progesterone receptor, L-selectin ligand, αvβ3 integrin, and glycodelin-A were not different between the groups. CONCLUSION(S) Levonorgestrel, given as emergency contraceptive on the day of LH surge, does not disrupt either ovulation or progesterone production by the corpus luteum. The contraceptive mechanism of levonorgestrel at the time of LH surge does not include changes in the progesterone receptors or the endometrial receptivity biomarkers.

COUP-TFII Regulates Human Endometrial Stromal Genes Involved in Inflammation

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII; NR2F2) is an orphan nuclear receptor involved in cell-fate specification, organogenesis, angiogenesis, and metabolism. Ablation of COUP-TFII in the mouse uterus causes infertility due to defects in embryo attachment and impaired uterine stromal cell decidualization. Although the function of COUP-TFII in uterine decidualization has been described in mice, its role in the human uterus remains unknown. We observed that, as in mice, COUP-TFII is robustly expressed in the endometrial stroma of healthy women, and its expression is reduced in the ectopic lesions of women with endometriosis. To interrogate the role of COUP-TFII in human endometrial function, we used a small interfering RNA-mediated loss of function approach in primary human endometrial stromal cells. Attenuation of COUP-TFII expression did not completely block decidualization; rather it had a selective effect on gene expression. To better elucidate the role of COUP-TFII in endometrial stroma cell biology, the COUP-TFII transcriptome was defined by pairing microarray comparison with chromatin immunoprecipitation followed by deep sequencing. Gene ontology analysis demonstrates that COUP-TFII regulates a subset of genes in endometrial stroma cell decidualization such as those involved in cell adhesion, angiogenesis, and inflammation. Importantly this analysis shows that COUP-TFII plays a role in controlling the expression of inflammatory cytokines. The determination that COUP-TFII plays a role in inflammation may add insight into the role of COUP-TFII in embryo implantation and in endometrial diseases such as endometriosis.

Human chorionic gonadotropin (hCG) modulation of TIMP1 secretion by human endometrial stromal cells facilitates extravillous trophoblast invasion in vitro

STUDY QUESTION Are secreted extracellular matrix (ECM) remodelling elements, relevant to embryo implantation and placentation, modified by hCG in endometrial stromal cells (ESCs)? SUMMARY ANSWER hCG decreases tissue inhibitor of metalloproteinase 1 (TIMP-1) secretion in ESCs, thereby facilitating extravillous trophoblast invasion in vitro. WHAT IS KNOWN ALREADY Successful embryo implantation and placentation depend on the appropriate invasion of the trophoblast into the maternal endometrial stroma. hCG is one of the earliest embryo-derived secreted signals in the endometrium which abundantly expresses hCG receptors. However, there is little data concerning the effects of hCG on endometrial ECM remodelling with respect to embryo implantation. PARTICIPANTS/MATERIALS, SETTING, METHODS This study was conducted in an academic research laboratory within a tertiary-care hospital. Samples were collected from 36 women undergoing benign gynaecological surgery during the mid-secretory phase. ESCs were isolated and stimulated with hCG (10 UI/ml) or vehicle. Conditioned media (CM) were analysed to determine changes in the secreted profile of nine matrix metalloproteinases (MMPs) and three tissue-specific inhibitors of MMPs (TIMPs) using an ELISA array. Data were confirmed by gelatine zymography, western blot and ELISA. The HTR8/SVneo cell line served as a model for trophoblast cells. The invasive potential of trophoblast cells was assessed using Transwell invasion assays under CM or co-culture conditions with ECS and the role of regulated molecules was examined by using immunoprecipitation in CM prior to the assessment of invasive potential. MAIN RESULTS AND THE ROLE OF CHANCE MMP-2 levels increased 30%, whereas TIMP-1 levels decreased 20% in CM from ESCs stimulated with hCG (P < 0.05). Gelatine zymography confirmed an increase in MMP-2 activity (P < 0.05). ELISA and western blotting also confirmed the reduction in TIMP-1 upon hCG treatment (P < 0.05). Invasion assays revealed a ∼50% increase in invading HTR8/SVneo cells in chambers with hCG-stimulated ESCs compared with the control (P < 0.05). Immunodepletion of TIMP-1 from control ESC-CM partially resembled the effect of CM from hCG-stimulated ESCs in the trophoblast invasion assays. LIMITATIONS, REASONS FOR CAUTION The assays were performed in vitro and ESCs were not decidualized, therefore they reflected the very early stages of embryo implantation or the advanced stages when decidualization fails. WIDER IMPLICATIONS OF THE FINDINGS Our data suggest that hCG induces endometrial stromal extracellular remodelling by modulating secreted MMP-2 and TIMP-1. This regulation may be physiologically relevant because it increases the invasive potential of trophoblast-derived cells. At present, few data exist concerning the implications of hCG and endometrial ECM remodelling in embryo implantation. Hence, our results should be confirmed by further in vivo studies. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by FONDECYT 11100443, PBCT-PSD51 (IDIMI) and FONDAP 15010006. None of the authors have any conflicts of interest to declare.

Complement C3 and decay-accelerating factor expression levels are modulated by human chorionic gonadotropin in endometrial compartments during the implantation window.

The control of complement activation in the embryo–maternal environment has been demonstrated to be critical for embryo survival. Complement proteins are expressed in the human endometrium; however, the modulation of this expression by embryo signals has not been explored. To assess the expression of complement proteins in response to human chorionic gonadotropin (hCG), we designed an experimental study using in vivo and in vitro models. Twelve fertile women were treated with hCG or left untreated during the mid-luteal phase, and an endometrial biopsy was performed 24 hours later. The localizations of C3, membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF; CD55), and protectin (CD59) were assessed by immunohistochemistry, and the messenger RNA (mRNA) levels of these proteins were quantified by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) in cells harvested from endometrial compartments using laser capture microdissection. Endometrial explants were cultured with or without hCG for 24 hours, and the C3 and DAF protein levels were measured by Western blotting. Elevated C3 mRNA levels in stromal cells and elevated DAF levels in epithelial luminal cells were detected after hCG treatment. In the endometrial explant model, the progesterone receptor antagonist RU486 inhibited the increases in the levels of C3 and DAF in response to hCG. The findings of this study indicate that hCG plays a role in embryo–endometrium communication and affects the expression of complement proteins in endometrial compartments during the implantation window.

Endometrial BCL6 Overexpression in Eutopic Endometrium of Women With Endometriosis.

The objective of this study was to examine B-cell CLL/lymphoma 6 (BCL6) expression in human eutopic endometrium across the menstrual cycle in women with and without endometriosis and to establish a cutoff for future studies. This design was a series of case–control studies in tertiary University teaching hospitals. We examined BCL6 expression by messenger RNA and immunohistochemically in prospectively collected samples in both the proliferative (P) and the secretory phases. BCL6 is minimally increased in the mid-secretory phase of the menstrual cycle compared to the P phase in normal patients. BCL6 protein expression was significantly higher in the secretory phase of patients with endometriosis (n = 29) versus fertile controls without endometriosis at laparoscopy (n = 20; P < .0001). Normal fertile controls (n = 28) recruited for endometrial biopsy also had low levels of secretory phase BCL6 expression compared to women with unexplained infertility (UI; n = 119). A receiving–operator characteristic analysis of these data revealed an area under the curve of 94% (95% confidence interval 85%-100%; P < .0001) with an HSCORE cutoff of 1.4 to differentiate cases with and without endometriosis. Using this cutoff value, BCL6 was positive in 88% of cases with UI. Laparoscopic examination of a subset of 65 patients confirmed abnormalities in 98% of cases; 61 (93.8%) were found to have endometriosis, 3 (4.6%) with hydrosalpinx, and 1 (1.5%) with a normal pelvis. These data suggest that BCL6 is a promising candidate as a single diagnostic biomarker for detection of endometriosis in women with otherwise UI and may be associated with endometrial dysfunction, including progesterone resistance.

2-Methoxyestradiol in the human corpus luteum throughout the luteal phase and its influence on lutein cell steroidogenesis and angiogenic activity

OBJECTIVE To quantitate 2-methoxyestradiol (2-ME) in human corpus luteum (CL) of different ages and to determine the expression of cytochrome-P450-1A1 (CYP1A1) and catechol-O-methyl transferase (COMT) in CL and the action of 2-ME on P, vascular endothelial growth factor (VEGF) secretion, and luteal angiogenesis. DESIGN Experimental study. SETTING University division of reproductive endocrinology. PATIENT(S) Twenty-four women of reproductive age. INTERVENTION(S) CL was collected from 15 women during the minilaparotomy for tubal sterilization. Granulosa lutein cells were harvested 36 hours after hCG administration in patients undergoing IVF. MAIN OUTCOMES MEASURE(S) Levels of 2-ME were determined by high-performance liquid chromatography in CL. CYP1A1 and COMT were assessed by immunohistochemistry and Western blot. P and VEGF were measured by radioimmunoassay and ELISA. The angiogenic potential was analyzed using EA.hy926 cells. RESULT(S) Plasma levels of E₂ decreased in the late luteal phase in association with an increase in luteal tissue of 2-ME concentrations. Concomitantly, there was a significant reduction of angiogenic activity in late CL. There was no significant variation in CYP1A1 and COMT expression in all CL. In physiological doses, 2-ME inhibited basal VEGF by granulosa lutein cells and diminished the angiogenic activity in conditioned media but did not prevent P and VEGF production stimulated by hCG. CONCLUSION(S) These data suggest the participation of 2-ME in physiological luteolysis by reducing angiogenesis. However, 2-ME did not prevent in vitro hCG stimulation of P biosynthesis, providing a mechanism for CL rescue in the cycle of conception.

KRAS Activation and over-expression of SIRT1/BCL6 Contributes to the Pathogenesis of Endometriosis and Progesterone Resistance

Endometriosis is an inflammatory condition that is associated with progesterone resistance and cell proliferation, resulting in pain, infertility and pregnancy loss. We previously demonstrated phosphorylation of STAT3 in eutopic endometrium of infertile women with this disorder leading to over-expression of the oncogene BCL6 and stabilization of hypoxia-induced factor 1 alpha (HIF-1α). Here we report coordinated activation of KRAS and over-expression of Sirtuin 1 (SIRT1), a histone deacetylase and gene silencer, in the eutopic endometrium from women with endometriosis throughout the menstrual cycle. The mice with conditional activation of KRAS in the PGR positive cells reveal an increase of SIRT1 expression in the endometrium compared to control mice. The expression of progesterone receptor target genes including the Indian Hedgehog pathway genes are significantly down-regulated in the mutant mice. SIRT1 co-localizes with BCL6 in the nuclei of affected individuals and both proteins bind to and suppress the promoter of GLI1, a critical mediator of progesterone action in the Indian Hedgehog pathway, by ChIP analysis. In eutopic endometrium, GLI1 expression is reduced in women with endometriosis. Together, these data suggest that KRAS, SIRT1 and BCL6 are coordinately over-expressed in eutopic endometrium of women with endometriosis and likely participate in the pathogenesis of endometriosis.

The Endometria of Women With Endometriosis Exhibit Dysfunctional Expression of Complement Regulatory Proteins During the Mid Secretory Phase

The control of complement activation within embryo-endometrium environment is critical for embryo survival. Cell evasion from complement attack requires interaction of complement regulatory proteins (CRPs) with cell adhesion αvβ3 integrin. We aim to compare the expression of CRPs in endometria of women with and without endometriosis and to examine the molecular interaction of decay accelerating factor (DAF) with αvβ3 integrin. Endometrial expression of Membrane cofactor protein (CD46), Decay accelerating factor (DAF), Membrane attack complex inhibitory factor (CD59) and β3 integrin subunit were determined through menstrual cycle by immunohistochemistry. DAF protein quantity was determined by Western blot and mRNA levels measured in epithelial cells isolated by laser capture microdissection (LCM). Using in vitro assay, we examined DAF and β3 integrin expression through paracrine regulation between endometrial compartments. To determine whether β3 integrin and DAF interacts in vivo, endometrial samples were subjected to immunoprecipitation and colocalization using dual immunofluorescence technique. DAF and β3 integrin expression were significantly low in samples from women with endometriosis during mid secretory phase. This observation was supported by decreased DAF protein quantity; faint DAF and β3 integrin interaction and reduced mRNA levels in cells dissected by LCM. Moreover epithelial DAF and β3 integrin expression through paracrine regulation by progesterone from stromal compartment was disrupted in endometriosis. Endometria from women with endometriosis exhibits aberrant expression of complement proteins. The abnormal DAF expression potentially compromises embryo survival, contributing to understand the implantation failure in women with endometriosis.

Endometrial progesterone receptors and levonorgestrel as emergency contraceptive

This editorial focuses on levonorgestrel as emergency contraceptive (LNG-EC) and its mechanism of action as well as its effectiveness.