Human Endometrial Explants Research Articles

Oxytocin antagonism reverses the effects of high oestrogen levels and oxytocin on decidualization and cyclooxygenase activity in endometrial tissues.

What is the in-vitro effect of oxytocin receptor (OTR) antagonism on parameters of receptivity in human endometrial explants and endometrial stromal cell lines cultured in oestradiol-rich conditions mimicking ovarian stimulation? Experimental in-vitro study on endometrial tissue explants collected by aspiration biopsy from 30 women undergoing fertility treatment and cultured endometrial tHESC cell line. The study examined the effects of high oestradiol, oxytocin and OTR antagonist on parameters of decidualization (cell viability and prolactin secretion) as well as cyclooxygenase-1/2 (COX-1/2) activity and prostaglandin F2α (PGF2α) secretion. Changes in expression of OXTR and COX-2 genes were examined using quantitative polymerase chain reaction (qPCR). In experiments on cultured endometrial cell line, high oestradiol and oxytocin similarly limited the viability of cells. In cultured endometrial explants both also decreased the secretion of prolactin (a marker of decidualization) and augmented endometrial COX-2 activity and formation of PGF2α. Oxytocin antagonist atosiban was confirmed to reverse the above effects, both in the endometrial line and endometrial explants. Addition of atosiban to cultures acted analogously in experiments employing both oxytocin and high oestradiol. Oxytocin antagonist reversed the effects of high oestradiol and oxytocin on parameters related to endometrial receptivity in conditions mimicking ovarian stimulation. This might point to a novel, endometrium-related mechanism to support embryo implantation achieved by the application of oxytocin antagonist prior to embryo transfer.

Effects of different progestins on prostaglandin biosynthesis in human endometrial explants

ObjectiveTo compare the effects of chlormadinone acetate (CMA), dienogest (DNG) and drospirenone (DRSP) on prostaglandin biosynthesis in a human endometrial explants model. Study designHuman endometrial explants obtained by aspiration curettage and human endometrial YHES cells were stimulated with interleukin-1β (IL-1β) and exposed to CMA, DNG, DRSP or dexamethasone (DEX; YHES cells). Cellular messenger RNA (mRNA) levels of cyclooxygenase-2 (COX-2) were analyzed by reverse-transcription quantitative real-time polymerase chain reaction. Concentrations of prostaglandin F2α (PGF2α) in culture supernatants were measured by enzyme-linked immunosorbent assay. ResultsCMA exerted after IL-1β stimulation a stronger down-regulation of COX-2 mRNA compared to DNG and DRSP in human explants (−55% vs. −40% and 46%, respectively). The effect of CMA on COX-2 mRNA was significantly stronger (p=.025) than that of DNG. Moreover, the effect of CMA was independent from cycle phase or presence of endometriosis. In order to evaluate the impact of the investigated progestins on effector molecules, PGF2α release was determined in supernatants. Again, CMA reduced the PGF2α release significantly by an average of −60% (p<.01). In contrast, no significant reduction was found for DNG and DRSP. In YHES cells, only DEX but not the progestins under study exerted a significant down-regulating effect (−79%, p<.01) on COX-2 mRNA after IL-1β stimulation. ConclusionAmong the tested progestins, CMA displayed the most consistent suppression of prostaglandin biosynthesis in human endometrial explants. ImplicationAmong three tested progestins, chlormadinone acetate had the most consistent suppressive effect on prostaglandins in endometrial explants. These findings support clinical observations about the efficacy of chlormadinone acetate in dysmenorrhea treatment.

Effects of an Antagonistic Analog of Growth Hormone-Releasing Hormone on Endometriosis in a Mouse Model and In Vitro.

Endometriosis is a benign gynecologic disorder causing dysmenorrhea, pelvic pain, and subfertility. Receptors for the growth hormone-releasing hormone (GHRH) were found in endometriotic tissues. Antagonists of GHRH have been used to inhibit the growth of endometriotic endometrial stromal cells. In this study, the GHRH receptor splice variant (SV) 1 was detected in human endometrial tissue samples by Western blots and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The highest messenger RNA (mRNA) and protein levels of SV1 were found in eutopic endometrium from patients with endometriosis compared to ectopic endometriotic tissues and endometrium from normal patients. The highest expression for GHRH mRNA was found by qRT-PCR in ectopic endometriosis lesions. In an in vivo mouse model with human endometrial explants from patients with endometriosis, 10 μg MIA-602 per day resulted in significantly smaller human endometrial xenotransplants after 4 weeks compared to mice treated with vehicle. The endometrial tissues expressed SV1 before and after xenotransplantation. The proliferation of endometrial stromal cells as well as the endometriosis cell lines 12-Z and 49-Z was decreased by exposure to 1 μM MIA-602 after 72 hours. The protein levels of epithelial growth factor receptors in 12-Z and 49-Z cell lines were reduced 48 and 72 hours after the administration of 1 μM MIA-602. MIA-602 decreased the activation of the MAP-kinases ERK-1/2. Our study demonstrates the presence of SV1 receptor as a target for treatment with GHRH antagonist in endometriosis. Endometrial tissues respond to MIA-602 with inhibition of proliferation in vitro and in vivo. The use of MIA-602 could be an effective supplement to the treatment strategies in endometriosis.

Androgens Upregulate Endometrial Epithelial Progesterone Receptor Expression: Potential Implications for Endometriosis

Androgenic compounds have been implicated in induction of endometrial atrophy yet the mechanisms of androgen effects on human endometrium have not been well studied. We hypothesized that androgens may promote their endometrial effects via modulation of progesterone receptor (PR) expression. Proliferative phase endometrial samples were collected at the time of hysterectomy. We evaluated the effect of the potent androgen 5α-dihydrotestosterone (DHT) on endometrial PR expression by treating human endometrial explants, endometrial stromal cells, and Ishikawa cells with DHT. Ishikawa cells were also treated with DHT ± the androgen receptor (AR) blocker flutamide. The PR-B, total PR messenger RNA (mRNA), and PR protein expression were assessed. Expression of cyclin D1 and D2 was checked as markers of cell proliferation. As expected, estradiol induced PR expression in isolated stromal cells, endometrial epithelial cells, and tissue explants. The DHT treatment also resulted in increased PR expression in endometrial explants and Ishikawa cells but not in stromal cells. Further, protein levels of both nuclear PR isoforms (PR-A and PR-B) were induced with the DHT treatment. Although flutamide treatment alone did not affect PR expression, flutamide diminished androgen-induced upregulation of PR in both endometrial explants and Ishikawa cells. Although estradiol induced both cyclin D1 and cyclin D2 mRNA, DHT did not induce these markers of cell proliferation. Androgens may mediate endometrial effects through upregulation of PR gene and protein expression. Endometrial PR upregulation by androgens is mediated, at least in part, through AR.

Seminal Plasma Promotes Lesion Development in a Xenograft Model of Endometriosis

The factors that predispose one-tenth of reproductive-aged women to endometriosis are poorly understood. We determined that genetic deficiency in transforming growth factor β1 impairs endometriosis-like lesion growth in mice. Given that seminal plasma is an abundant source of transforming growth factor β, we evaluated the effect of exposure to seminal plasma on the growth of endometrial lesions. Human endometrial explants were exposed to seminal plasma or to control medium before transfer to Prkdc(scid)-mutant (severe combined immunodeficient) mice. Xenografts exposed to seminal plasma showed an eightfold increase in volume and a 4.3-fold increase in weight after 14 days. These increases were associated with increased proliferation of endometrial epithelial cells and enhanced survival and proliferation of human stromal cells compared with those in control lesions, in which human stromal cell persistence was negligible. Although the distribution of macrophages was altered, their number and activation status did not change in response to seminal plasma. Seminal plasma stimulated the production of a variety of cytokines in endometrial tissue, including growth-regulated oncogene, granulocyte macrophage colony-stimulating factor, and IL-1β. These data suggest that seminal plasma enhances the formation of endometriosis-like lesion via a direct effect on endometrial cell survival and proliferation, rather than via macrophage-mediated mechanisms. These findings raise the possibility that endometrial exposure to seminal plasma could contribute to endometriotic disease progression in women.

Effect of a dienogest for an experimental three-dimensional endometrial culture model for endometriosis

The pathogenesis of endometriosis remains poorly understood at least in part because early stages of the disease process are difficult to investigate. Previous studies have proposed a three-dimensional fibrin matrix culture model to study human endometriosis. We examined the ultrastructural features of the endometriosis in this model and assessed the effect of a progestin on endometrial outgrowth and apoptosis in this culture system. Endometrial explants were placed in three-dimensional fibrin matrix culture and treated with and without various concentrations of the progestin dienogest. By the second week, endometrial gland-like formation was established in outgrowths both attached to and at a distance from the explants. These cells formed a combination of clumps and tubular monolayers surrounding a central cavity. Electron microscopy demonstrated that these cells are polarized with microvilli on the apical surface, desmosome-like structures, and basement membrane; features consistent with glandular epithelial cells. Outgrowth of endometrial stromal cells and glandular formation was impaired in response to dienogest in a dose-dependent manner. Our study shows that the human endometrial explants cultured in three-dimensional fibrin matrix establish outgrowths that ultrastructurally resemble ectopic endometrial implants. This model may provide insight into the cellular processes leading to endometriosis formation and enables screening of therapeutic compounds.

Low-dose mifepristone increases uterine expression of aquaporin 1/aquaporin 2 at the time of implantation

BackgroundThe aim of this study was to investigate the mechanism by which low-dose mifepristone serves as an antiimplantation contraceptive drug. A human endometrial explant system was used to study the effects of low-dose mifepristone (65 nmol/L and 200 nmol/L) on expression of the water channel family aquaporins, aquaporin-1 and aquaporin-2 (AQP1/AQP2), at the time of implantation. Study DesignEndometrial samples from 17 normally cycling patients at the “window of implantation” were treated with different concentrations of mifepristone. The protein and mRNA expression of AQP1/AQP2 in the endometrium was examined using immunohistochemistry (IHC) and reverse transcriptase–polymerase chain reaction (RT-PCR), respectively. ResultsThe IHC and RT-PCR analyses demonstrated that expression of AQP1/AQP2 was increased by mifepristone in a dose-dependent manner, with the highest AQP1/AQP2 expression levels detected in subjects treated with 200-nmol/L mifepristone. ConclusionLow-dose mifepristone may negatively regulate implantation by increasing AQP1/AQP2 protein and mRNA expression. The findings from this study provide further evidence to support the potential contraceptive activity of low-dose mifepristone.

Chlormadinone acetate suppresses prostaglandin biosynthesis in human endometrial explants

To elucidate the mode of action of chlormadinone acetate (CMA) in reducing dysmenorrheic pain by studying the effects of CMA and dexamethasone (DEX) on messenger RNA (mRNA) abundance of cyclo-oxygenase-2 (COX-2), annexin-1 (ANXA1), glucocorticoid receptor (GR), progesterone receptor (PR), and concentrations of prostaglandin F(2α) (PGF(2α)) and leukotrienes B(4) (LTB(4)) and C(4) (LTC(4)) in human endometrial explants. Ex vivo study. University hospital. Fifteen premenopausal patients undergoing surgery for benign gynecologic disorders. Endometrial explants were obtained by aspiration curettage and stimulated ex vivo with interleukin-1β before exposure to CMA or DEX; mRNA levels were determined via reverse transcription-quantitative real-time polymerase chain reaction, and concentrations of arachidonic acid metabolites by enzyme immunoassays. Messenger RNA levels of COX-2, ANXA1, PR, and GR; concentrations of PGF(2α), LTB(4), and LTC(4) in endometrial explants treated with CMA or DEX. In IL-1β-treated explants COX-2 mRNA and PGF(2α), concentrations were significantly down-regulated by CMA but not by DEX. Chlormadinone acetate did not affect mRNA abundance of ANXA1, PR, and GR. Our data suggest that CMA is a suppressor of COX-2 expression. Comparison with DEX revealed that progestin-specific activity of CMA may mainly be responsible for suppression of prostaglandin biosynthesis in human endometrium.

Low-dose mifepristone increases uterine natural killer cell cytotoxicity and perforin expression during the receptive phase

To investigate the immunologic mechanism by which low-dose mifepristone serves as an anti-implantation contraceptive drug. In vitro study. University hospital and research laboratory. Fifteen normally cycling patients at the "window of implantation." A human endometrial explant system was used to study the effects of low-dose mifepristone (65 and 200 nmol/L) on uterine natural killer (uNK) cells. Endometrial samples were treated with different concentrations of mifepristone. The cytotoxicity of uNK cells to K562 target cells and the expression of perforin (PFN) by uNK cells were examined using a methyl thiazolyl tetrazolium (MTT) assay and double immunohistochemistry, respectively. Both uNK cell cytotoxicity and expression of PFN were increased after treatment with 65 or 200 nmol/L mifepristone, and these effects were dose-dependent. Mifepristone may negatively regulate implantation by increasing the cytotoxicity of uNK cells, and this increased cytotoxicity may result from increased PFN expression. These findings provide further evidence to support the potential contraceptive activity of low-dose mifepristone.

Inhibition of steroid sulfatase decreases endometriosis in an in vivo murine model

Steroid sulfatase (STS) is involved in estrogen biosynthesis and expressed in eutopic and ectopic endometrium of disease-free and endometriosis patients. The present study was designed to investigate its role in endometriosis development. Human endometrial explants were cultured on inserts for 24 h to assess the effectiveness of an STS inhibitor (STS-I), estradiol-3-O-sulfamate (E2MATE), on STS activity in endometrial tissue. Endometriosis was induced in mice, and E2MATE (or vehicle alone) was given orally for 21 days. Plasma estradiol levels were measured, and STS activity was assessed in murine organs (uterus, liver and leukocytes) and in lesions. Lesion number, weight and size (morphometry) were quantified. Lesion STS and progesterone receptor (PR) expression, proliferation and apoptosis rates were determined by immunohistochemistry. In vitro, addition of 1 µM E2MATE to the culture medium resulted in decreased STS activity in endometrial explants (P < 0.001). Treatment of mice with E2MATE (1.0 and 0.5 mg/kg) did not modify plasma estradiol levels, but inhibited STS activity in murine uterus (P < 0.05), liver (P < 0.001) and leukocytes (P < 0.001), as well as in induced lesions (P < 0.05). E2MATE reduced lesion weight (P < 0.01) and size (P < 0.05), but had no impact on proliferation or apoptosis rates, nor STS protein expression. Stromal edema was observed in the uterus of animals treated with E2MATE, but not in the stroma of lesions. Increased PR expression was detected in endometriotic lesions (P < 0.001). E2MATE was shown to effectively inhibit STS activity in endometrial tissue in vitro. In vivo, E2MATE decreased endometriosis development without affecting systemic estradiol levels. Use of STS-I could therefore be of potential interest in endometriosis treatment.

Critical evaluation of human endometrial explants as an ex vivo model system: a molecular approach

The human endometrium is unique among adult tissues. Its functions are modulated by numerous hormones and mediators. The aim of this study was to evaluate the suitability of human endometrial explants for studying functional effects of chemicals and drugs on gene expression biomarkers. Endometrial tissues were obtained by aspiration curettage and cultivated for up to 24 h. Relative mRNA concentrations were determined by reverse transcription quantitative real-time PCR. Viability was assessed by light microscopy, lactate dehydrogenase assay and scanning electron microscopy. It was acceptable after 6 h of culture but reduced after 24 h. Culture-induced alterations of mRNA levels were found for progesterone receptor, estrogen receptor(α), leukemia inhibitory factor and cyclooxygenase-2 in tissues from all cycle stages. The suitability of the model to detect chemical effects was demonstrated by the down-regulation of cyclooxygenase-2 mRNA by chlormadinone acetate in proliferative and secretory endometrium. The model is mainly restricted by interindividual variations and varying tissue quality. An advantage is the preservation of tissue composition. We conclude that human endometrial explants are a complex model due to limited viability, difficult standardization and intrinsic alterations during culture. Experiments with this model should be performed over a limited time period under strictly controlled conditions.

Rapamycin inhibits cell proliferation in type I and type II endometrial carcinomas: A search for biomarkers of sensitivity to treatment

Objectives Our goal was to evaluate the effect of rapamycin, an mTOR inhibitor, in type I and II human endometrial cancer tumor explants. Methods Short-term tissue culture with fresh endometrial cancer tumor explants was performed. Cell proliferation was assessed by MTS assay after treatment with rapamycin. Akt and PTEN status were documented by Western blotting. The effect of rapamycin on phosphorylated-S6 and 4E-BP-1 was also assessed by Western blotting. Real-time RT-PCR was used to quantify hTERT mRNA expression. Telomere length was determined by terminal restriction fragment Southern blotting. Results Thirteen fresh endometrial cancer tumor explants (nine Type I, four Type II) were placed in short-term culture and treated with rapamycin. Nine of the endometrial cancer tumors responded to rapamycin, with a median IC 50 of 11.4 nM. Sensitivity to rapamycin was independent of PTEN and Akt status. Tumors (13/13) had a reduction in phosphorylated-S6 and 10/13 had a reduction in phosphorylated 4E-BP-1. Rapamycin decreased hTERT mRNA expression in all of the endometrial cancer tumors. Telomere length did not correspond with responsiveness to this drug. Conclusions Rapamycin demonstrated activity in fresh endometrial tumor explants independent of PTEN and Akt status. Some tumors demonstrated a reduction in phosphorylated-S6 and 4E-BP-1 without a significant change in cellular proliferation, suggesting that additional pathways may modulate cellular proliferation. Thus, mTOR inhibitors may be a useful targeted therapy for both type I and type II endometrial cancers, but the search remains for a predictive biomarker of sensitivity to this therapy.

“The Curse”: A 21st Century Perspective of Models of Its Molecular Basis

“The Curse,” the “Period,” “Riding the Crimson Wave,” “Aunt Flo,” and “the Red Coats are Coming” (euphemisms for menstrual bleeding) capture the long history of taboo and mystery of menstruation, a critical component of reproductive capability that has had major effects on social structures and equality movements (1). With the advent of steroid hormone contraceptives, withdrawal bleeding seemingly mimicked the natural menstrual cycle with its predictable bleeding pattern, although unpredictable bleeding has limited their usefulness for long-term use for many women, depending on the preparation, dose, dosing schedule, and mode of delivery. Indeed, some have questioned the value of having menstruation at all, and in appropriate clinical situations, this can be of value. There is also a silent epidemic that prevails in women not on contraceptive steroids and who experience abnormal (“dysfunctional”) uterine bleeding, a major cause of hysterectomy (2). Despite millions of women over millennia experiencing the normal physiologic process of menstruation and abnormalities in uterine bleeding, models of menstruation and mechanisms underlying endometrial tissue desquamation and bleeding, repair and regeneration, and abnormal bleeding have had mixed results and applicability to human biology and pathophysiology. In this issue of Endocrinology (3), a unique approach using cultured human endometrial explants and advanced techniques of laser capture microscopy and whole-genome transcriptomics has contributed to our basic knowledge of initiation of menstrual bleeding. However, much remains for this and future generations to investigate to prevent and treat disorders of endometrial/uterine bleeding that affect millions of women worldwide. Menstruation is the shedding of most of the functionalis layer of the endometrium, accompanied by bleeding, that occurs at the end of each menstrual cycle during a woman’s reproductive life. The shedding is not uniform, it occurs at focal points, progressing over 4–5 d until most of the tissue is lost, and it repairs very rapidly. Scanning electron microscopy (4) reveals that small lesions are apparent in the luminal epithelium as early as d 28 of the normalized cycle. The very rapid degeneration of the functionalis layer exposes open blood vessels and glands. It can therefore be surmised that the primary event initiating menstruation is tissue destruction and that loss of blood vessel integrity is one consequence of this. Shedding and repair occur simultaneously at adjacent sites, making these processes exceedingly difficult to study. Menstruation differs from wounding of other tissues in that menstrual blood does not easily clot and that the repair occurs without scarring. Menstrual bleeding is initiated by the fall in circulating levels of estradiol (E2) and progesterone (P) that result from the demise of the corpus luteum. The key evidence that menstruation is triggered by a loss of steroid support of the tissue is that 1) if P levels are artificially maintained, menstruation does not occur; 2) administration of mifepristone, a P receptor (R) antagonist, during the secretory phase results in uterine bleeding; and 3) withdrawal of E2 and P, as in the case of cyclic contraceptive use, results in uterine bleeding. Importantly, menstruation occurs only in women, a few old world monkeys, the elephant shrew, and certain bats. In these species, differentiation (“decidualization”) of the endometrial stroma is initiated during each cycle,

Tamoxifen-Induced Adduct Formation and Cell Stress in Human Endometrial Glands

The beneficial effects of tamoxifen in the prevention and treatment of breast cancer are compromised by an increased risk of endometrial polyps, hyperplasia, and cancer. Tamoxifen is metabolized to an array of metabolites with estrogenic effects but also to reactive intermediates that may form protein and DNA adducts. The aim of this study was to investigate cellular [(3)H]tamoxifen adduct formation by light microscopic autoradiography and cell stress by immunohistochemical analysis of glucose-regulating protein 78 (GRP78), nuclear factor kappaB (NF-kappaB), and caspase 3 in human endometrial explants after short-term incubation with tamoxifen. The cellular expression of tamoxifen-metabolizing enzymes in human endometrial biopsy samples was also determined by immunohistochemistry. The results showed selective [(3)H]tamoxifen adduct formation in glandular and surface epithelia after incubation with a nontoxic concentration of [(3)H]tamoxifen (6 nM). There was also a selective expression of the endoplasmic reticulum stress chaperone GRP78 and activated caspase 3 at these sites after incubation with cytotoxic concentrations of tamoxifen (10-100 microM). The cell stress was preferentially observed in samples from women in the proliferative menstrual phase. No treatment-related expression of NF-kappaB was observed. Constitutive expression of the tamoxifen-metabolizing enzymes CYP1B1, CYP2A6, CYP2B6, CYP2C8/9/19, CYP2D6, and SULT2A1 in glandular and surface epithelia was shown, but there was a large interindividual variation. The colocalization of [(3)H]tamoxifen adducts, expression of GRP78, caspase 3, and tamoxifen-metabolizing enzymes in human glandular and surface epithelia suggest a local bioactivation of tamoxifen at these sites and that epithelial cells are early target sites for tamoxifen-induced cell stress.

Morphology of human endometrial explants and secretion of stromal marker proteins in short- and long-term cultures

Human endometrial tissue is frequently biopsied under surgical and laparoscopic procedures for the investigation of infertility, abdominal, or menstrual pain. These symptoms often but not always are the consequence of endometriosis, which is characterised by the growth of endometrial tissue outside the uterine cavity and affecting 8–10% of women during the fertile age. First-line treatment is often by surgery. Biopsied endometrial tissue is not only used for immunohistochemical examination but has also been cultured in vitro. Explant culture systems maintain the three-dimensional structure of the tissue, but so far no morphological validation studies are available for the stromal cells which are responsible for the production of hormones and inflammatory cytokines in the endometrium. We have documented, by transmission electron microscopy, the morphological alterations of stromal cells in short- (12 h) and long-term (7 days) cultures of endometrial explants biopsied in the postovulatory phase. The production of prolactin, a stromal cell marker, was determined. We found that the morphological integrity of these cells was starting to be disrupted from as early as 12 h in culture. Some stromal cells, however, developed into predecidual cells. After 96 h, a large fraction of the cell population was necrotic, and after 7 days, the cytoplasm had disappeared. In presence of progesterone, the decay of stromal cell integrity was slowed down. The release of prolactin and IGF-binding protein-1 during culture followed the morphological pattern. We conclude that the explant culture model is viable for not more than 48 h in vitro for stromal cells, but that this interval can be prolonged by the addition of progesterone which initiates decidualisation.

Expression of Endometrial Protein Kinase A During Early Pregnancy in Bonnet Monkeys (Macaca radiata)1

Embryo-induced signaling pathways are considered to be important for initiation and sustenance of pregnancy. However many of these pathways remain to be deciphered in primates. In the present study, differential display RT-PCR was used to identify genes or gene fragments that are differentially expressed in endometrium of bonnet monkeys (Macaca radiata) on Day 6 of pregnancy. Of several fragments found to be differentially expressed, a fragment of 567 base pair (named GG1) was characterized in detail. GG1 was highly represented in endometrium of pregnant animals compared with that of nonpregnant animals. Sequencing analysis revealed homology of this fragment to exons 7, 8, 9, and 10 and surprisingly to intron 6 of cAMP-dependent protein kinase A (PKA) regulatory type I alpha (tissue-specific extinguisher 1) (PRKAR1A). The increased expression of this fragment in gestational endometrium was confirmed by quantitative PCR studies. Two transcripts of 3.0 kilobase (kb) and 1.5 kb were detected in Northern blot probed with labeled GG1. Protein expressions of alpha regulatory (PRKAR1A) and alpha catalytic (PRKCA) subunits of PKA were also higher in gestational endometrium compared with that in nongestational endometrium. Further in vitro studies using human endometrial explants demonstrated regulation of PRKAR1A (or GG1) and prostaglandin-endoperoxide synthase 2 or cyclooxygenase 2 (PTGS2) by estradiol. This is the first study to date on the differential expression of PKA in primate endometrium during early pregnancy and its in vitro regulation by estradiol.

Matrix metalloproteinase messenger RNA expression in human endometriosis grafts cultured on a chicken chorioallantoic membrane

To investigate the role of matrix metalloproteinase 1 and 2 (MMP-1/MMP-2) in human endometrial explants in a chicken chorioallantoic membrane model (CAM) of endometriosis. Experimental prospective study. University hospital. Endometrium samples obtained from ovulating women, both healthy and patients with endometriosis, who were undergoing curettage and diagnostic laparoscopy for benign gynecologic conditions. Endometrial grafts were transplanted to the CAM and cultured for 0, 24, 48, 72, and 96 hours. Expression of MMP-1 and MMP-2 messenger RNA (mRNA) was quantified by competitive reverse-transcriptase polymerase chain reaction (RT-PCR) and normalized to expression of the housekeeping gene human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. All grafts expressed MMP-1 and MMP-2 mRNA. The endometrium of healthy women and patients with endometriosis showed a statistically significant increase of MMP-1 mRNA expression 24, 48, 72, and 96 hours after transfer to the CAM. An increase of MMP-2 mRNA expression was only detected after 96 hours of CAM culture in patients with and without endometriosis. No statistically significant difference regarding the MMP-1 and MMP-2 mRNA expression could be shown in healthy women or endometriosis patients. Our data suggest that MMP-1 and MMP-2 are major factors involved in the invasion of endometrium into the peritoneum and in vascularization of endometriosis. Whether MMPs are suitable targets for treatment of endometriosis has to be examined in further studies.

Estrogen stimulates the human endometrium to express a factor(s) that promotes vascular smooth muscle cell migration as an early step in microvessel remodeling

Vascular smooth muscle cell (VSMC) migration is a pivotal early step in blood vessel remodeling; however, very little is known about the regulation of this process in the human endometrium during the menstrual cycle. In this study, explants of human endometrium were incubated with estradiol and/or progesterone and the conditioned medium (CM) applied to cultures of VSMC to test the hypothesis that estrogen and progesterone stimulate endometrial cells to secrete a factor(s) that promotes VSMC migration. Endometrial explants were composed of highly organized glands and stroma. VSMC migration (cells migrated in 21 h/mm(2) fibronectin-coated semipermeable membrane) in the presence of CM from human endometrial explants obtained in the proliferative phase of the menstrual cycle and incubated for 24 h with estradiol was approximately threefold greater (P < 0.001) than with medium alone and greater (P < 0.05) than with CM from explants treated with estradiol plus progesterone or progesterone. It is concluded, therefore, that estrogen stimulates endometrial secretion of a factor(s) that promotes VSMC migration as an early step in vessel remodeling within the endometrium.

Androstenedione Up-Regulation of Endometrial Aromatase Expression via Local Conversion to Estrogen: Potential Relevance to the Pathogenesis of Endometriosis

Up-regulation of aromatase expression in endometrial cells disseminated into the peritoneal cavity may enhance their survival via local estrogen synthesis, which may lead to endometriosis. The factors that mediate induction of aromatase in the endometrium are not well defined, but increased expression of steroidogenic factor (SF)-1 may play a role. The objective of the study was to determine whether androstenedione (A4), the predominant sex steroid in peritoneal fluid, regulates endometrial aromatase expression. This was a cell/tissue culture study. The study was conducted at an academic center. Quantitative real-time PCR, HPLC, and chromatin immunoprecipitation were used in this study. Treatment of cultured human endometrial explants and stromal cells with A4 (10 nm) significantly up-regulated expression of aromatase mRNA transcripts containing exon IIa at their 5'-ends. In endometrial stromal cells and the human endometrial surface epithelial (HES) cell line, induction of aromatase mRNA by A4 was associated with increased expression of SF-1. In HES cells, tritiated A4 was metabolized to estradiol, testosterone (T), dihydrotestosterone, and androstanediol. Both estradiol and T, but not nonaromatizable androgens, up-regulated aromatase and SF-1 mRNA in HES cells. Chromatin immunoprecipitation revealed that A4 enhanced recruitment of SF-1 to its response element (-136 bp) upstream of CYP19 exon IIa. This, together with the findings that both estrogen receptor antagonist, ICI 182,780, and aromatase inhibitor, fadrozole, suppressed A4 and T induction of aromatase and SF-1 mRNA, indicates that the inductive effects of A4 and T are mediated by their conversion to estrogens. Exposure of endometrial cells to A4 may enhance CYP19 gene expression through its aromatization to estrogens.

Letrozole stimulates the growth of human endometrial explants cultured in three-dimensional fibrin matrix

To investigate the effects of an aromatase inhibitor, letrozole, on the growth of human endometrium in a three-dimensional fibrin matrix model of endometriosis. Experimental study of human endometrial biopsies in a three-dimensional fibrin matrix culture system. Academic research center. Eight normal women with benign gynecologic problems. Endometrial biopsy samples were washed, cut into small pieces, and placed between two layers of fibrin gel in the presence or absence of letrozole in the culture medium. Tissue changes were assessed by histological and immunohistochemical staining using an inverted microscope, image analysis, and a semiquantitative scoring system. Stromal and epithelial cell outgrowth into the fibrin matrix and angiogenesis comprising endothelial cell invasion of the matrix. Letrozole (0.1 micromol/L, 1 micromol/L, and 10 micromol/L) exerted a significant growth stimulation effect on endometrial tissue in this model. In contrast to our expectations, letrozole stimulated growth of normal human endometrium in an in vitro model of endometriosis. Normal endometrium may respond differently than endometriotic lesions to therapeutic agents. Our findings should be kept in mind when considering future research to explore new clinical treatments for endometriosis.