Late Proliferative Phase Research Articles

Analysis of aromatase and 17β-hydroxysteroid dehydrogenase type 2 messenger ribonucleic acid expression in deep endometriosis and eutopic endometrium using laser capture microdissection

To investigate mRNA expression of aromatase and 17beta-hydroxysteroid dehydrogenase type 2 (17betaHSD2) in epithelial and stromal cells from eutopic and ectopic endometrium of patients with deep endometriosis. Prospective study. University hospital. Patients with deep endometriosis and fertile women with macroscopically normal pelvic cavities. During surgery, 30 endometrial and 16 endometriotic samples were obtained from 30 patients with deep endometriosis. Control endometrial samples were obtained from 24 fertile women with macroscopically normal pelvic cavities who underwent laparoscopic tubal ligation or reversal of tubal sterilization. Epithelial cells and stromal cells from endometrial or endometriotic tissues were microdissected using laser capture microdissection. Expression levels of aromatase and 17betaHSD2 mRNA in microdissected epithelial and stromal cells were determined using quantitative real-time reverse transcriptase polymerase chain reaction. Aromatase mRNA expression was significantly higher in epithelial cells than in stromal cells in both eutopic and ectopic endometrium obtained from endometriosis patients. In the ectopic endometrium of 8 patients (8/16, 50%), 17betaHSD2 expression was not detected in either epithelial or stromal cells. In eutopic endometrium from endometriosis patients, 17betaHSD2 expression in epithelial cells was significantly increased during the early, middle, and late secretory phases compared with the late proliferative phase, whereas no significant cyclical difference was detected in control endometrium. Local estrogen concentration may be much higher in epithelial cells than in stromal cells in deep endometriotic tissue.

DNA microarray analysis of gene expression in eutopic endometrium from patients with deep endometriosis using laser capture microdissection

To investigate differentially expressed genes in epithelial and stromal cells of eutopic endometrium from patients with deep endometriosis and women with normal pelvic cavities using laser capture microdissection and complementary DNA microarrays. Prospective study. University hospital. Patients with deep endometriosis and fertile women who underwent laparoscopic tubal ligation or reversal of tubal sterilization. Endometrial tissue biopsies during the late proliferative phase and early, mid-, and late secretory phases. Genes that were regulated with a change greater than threefold were selected as differentially expressed genes. Validation was performed with real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Microarray analysis identified up-regulation during the late secretory phase (patients with endometriosis vs. controls) of several genes in two important signaling pathways: RAS/RAF/MAPK and PI3K. This included the genes RON, SOS, 14-3-3 protein eta, and uPAR in epithelial cells and KSR and PI3K p85 regulatory subunit alpha in stromal cells; real-time RT-PCR analysis validated up-regulation of all six genes. The RAS/RAF/MAPK and PI3K pathways may be involved in initial development of endometriosis.

Effect of the menstrual cycle and oral contraceptives on cyclooxygenase-2 expression in the endometrium

Objective. To compare the expression of cyclooxygenase-2 (COX-2) and proliferation markers (Ki-67) in the endometrium of patients with ovulatory cycles with those in the endometrium of patients using oral contraceptives.Patients and methods. Endometrial biopsies from 104 premenopausal patients with regular ovulatory cycles (n = 90) or using an oral contraceptive (n = 14) were selected for this study. Using immunohistochemical methods, both COX-2 (Novocastra clone 4H12) and Ki-67 (Dako clone MIB-1) expression were determined in the endometrium during the various phases of the menstrual cycle or following the use of oral contraceptives.Results. COX-2 expression in the glandular epithelium was maximal during menstruation, the late proliferative phase and the early luteal phase, and minimal during the late luteal phase. However, in the surface epithelium, COX-2 expression remained strongly positive throughout the luteal phase. Ki-67 positivity increased during the proliferative phase and diminished during the luteal phase in the glands. In contraceptive users, both Ki-67 and COX-2 expression in the endometrium was low.Conclusion. The increased expression of COX-2 during menstruation and at mid-cycle is eliminated by the continuous use of oral contraceptives. This may be the rationale for their therapeutic action in the treatment of dysmenorrhea and bleeding.

In vivo and in vitro estrogenic and progestagenic actions of Tibolone

Estrogen and progestin combination in hormone replacement therapy (HRT) increases the incidence of breast cancer, but decreases the endometrial cancer risk of unopposed estrogen. Therefore, a SERM such as Tibolone, that delivers the beneficial, but not the adverse side effects, of steroid hormones would be clinically advantageous. However, data from the Million Women Study suggests that Tibolone increases the risk of both breast and endometrial cancer. Herein, we assessed the estrogenic and progestagenic actions of Tibolone using transvaginal sonography studies and an in vitro model of breast (ZR-75, MCF7) and endometrial cancer (Ishikawa). The known cancer associated proteins (ER, EGFR, STATS, tissue factor and Bcl-xL) were selected for study. Transvaginal sonography demonstrated that postmenopausal women treated with Tibolone displayed a thinner endometrium than in the late proliferative phase, but had a phenotype characteristic of the secretory phase, thus demonstrating the estrogenic and progestagenic actions of this SERM. In vitro, Tibolone acted as an estrogen in downregulating ER and upregulating Bcl-xL, yet as progesterone, increasing STAT5 and tissue factor in breast cancer cells. The increase in tissue factor by Tibolone correlated with its coagulative potential. Interestingly, EGFR was up-regulated by progesterone in the breast and by estrogen in endometrial cells, while Tibolone increased protein levels in both cell types. In conclusion, this study further demonstrates the estrogenic and progestagenic nature of Tibolone. The pattern of regulation of known oncogenes in cells of breast and endometrial origin dictates caution and vigilance in the prescription of Tibolone and subsequent patient monitoring.

Cellular Expression and Hormonal Regulation of Neuropilin-1 and -2 Messenger Ribonucleic Acid in the Human and Rhesus Macaque Endometrium

Although much is known about the biology of vascular endothelial growth factor (VEGF) and its cognate receptors (VEGFRs), VEGFR1 and VEGFR2, little is known about the roles of the VEGFRs neuropilin (NP)-1 and NP-2 in the primate endometrium. In this study, we investigated the cellular localization and hormonal regulation of NP-1 and NP-2 mRNA by in situ hybridization in the endometrium of ovariectomized, hormonally cycled rhesus macaques and women during the natural menstrual cycle. NP-1 mRNA was highly expressed in vascular endothelium and in stromal cells, but in these cells, NP-1 expression did not change during the menstrual cycle. However, NP-1 mRNA was also expressed in the luminal epithelium (not the glands), and its expression in these cells was elevated during the mid- to late proliferative phase and completely suppressed during the secretory phase. The increase in NP-1 level in the luminal epithelium was estradiol dependent because such expression was not detectable in the absence of estradiol in ovariectomized, hormone-deprived animals. Moreover, NP-1 expression in the luminal epithelium was highly correlated with the degree of proliferation in these cells. A recent study showed that blockade of VEGF action can inhibit luminal epithelial cell proliferation, but there is no evidence of VEGFR1 and VEGFR2 expression in these cells. Therefore, NP-1 may be the relevant VEGFR that mediates proliferation in this epithelium. NP-2 mRNA, unlike NP-1, was expressed only by the endothelium of veins, and in these cells, its expression was hormonally regulated in the converse manner: it was very low during the proliferative phase and high during the secretory phase. The increased permeability and edema observed during the secretory phase in the primate endometrium may be mediated in part by VEGF-NP-2 interaction. In the human endometrium, the pattern of expression and cellular localization of both NP-1 and NP-2 during the menstrual cycle were essentially identical with that seen in the rhesus macaque endometrium. These are the first data to specify the hormonal regulation and cell-specific expression of NP-1 and NP-2 mRNA in the endometrium of both women and nonhuman primates. The findings extend our understanding of VEGF action in the primate endometrium.

Chemokine receptor expression in the human ectocervix: implications for infection by the human immunodeficiency virus-type I.

Human immunodeficiency virus-type 1 (HIV-1) is a sexually transmitted pathogen that can infect cells in the female reproductive tract (FRT). The mechanism of viral transmission within the FRT and the mode of viral spread to the periphery are not well understood. To characterize the frequency of potential targets of HIV infection within the FRT, we performed a systematic study of the expression of HIV receptors (CD4, galactosyl ceramide (GalCer)) and coreceptors (CXCR4 and CCR5) on epithelial cells and leucocytes from the ectocervix. The ectocervix is a likely first site of contact with HIV-1 following heterosexual transmission, and expression of these receptors is likely to correlate with susceptibility to viral infection. We obtained ectocervical tissue specimens from women undergoing hysterectomy, and compared expression of these receptors among patients who were classified as being in the proliferative or secretory phases of their menstrual cycle at the time of hysterectomy, as well as from postmenopausal tissues. Epithelial cells from tissues at early and mid-proliferative stages of the menstrual cycle express CD4, although by late proliferative and secretory phases, CD4 expression was absent or weak. In contrast, GalCer expression was uniform in all stages of the menstrual cycle. CXCR4 expression was not detected on ectocervical epithelial cells and positive staining was only evident on individual leucocytes. In contrast, CCR5 expression was detected on ectocervical epithelial cells from tissues at all stages of the menstrual cycle. Overall, our results suggest that HIV infection of cells in the ectocervix could most likely occur through GalCer and CCR5. These findings are important to define potential targets of HIV-1 infection within the FRT, and for the future design of approaches to reduce the susceptibility of women to infection by HIV-1.

The Immune Environment in Human Endometrium during the Window of Implantation

Changes in the immune environment in the endometrium are believed to be important for successful implantation and maintenance of pregnancy. We have previously investigated global gene profiling in human endometrium during the window of implantation by oligonucleotide microarray technology, and analysis of these data underscore the regulation of a group of immune-related genes. The present study was therefore conducted to examine the pattern of expression and regulation of these genes including decay accelerating factor (DAF), indoleamine 2,3 dioxygenase (IDO), interleukin-15 (IL-15), IL-15 receptor alpha subunit (IL-15Ralpha), interferon regulatory factor-1 (IRF-1), lymphotactin (Lpn), natural killer-associated transcript 2 (NKAT2) and NKG5 in secretory and proliferative human endometrium. Endometrial biopsies were obtained from normally cycling women in the late proliferative and mid-secretory phase of the menstrual cycle. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis were used to determine the expression and regulation of these genes in secretory and proliferative human endometrium. Cellular localization of NKG5, Lpn and IDO by in situ hybridization in secretory-phase endometrium was also examined. Semi-quantitative RT-PCR and Northern blot results demonstrate that there is a coordinated upregulation of this group of genes during the window of implantation. We demonstrate the upregulation of immune-related genes IL-15Ralpha, Lpn and NKG5 in secretory versus proliferative human endometrium. We also demonstrate a similar upregulation in secretory endometrium of other immune-related genes, viz, DAF, IDO, IL-15, IRF-1 and NKAT2. The functions of these genes include stimulation of proliferation of uterine natural killer (uNK) cells, inhibition of cytolytic activity of uNK cells, inhibition of cell growth of T cells and other pathogens and inhibition of the classical complement pathway. Upregulation of these immune-related genes in the window of implantation suggests their role during the process of implantation and in immune tolerance of the implanting conceptus.

Circulating angiogenic factors during periovulation and the luteal phase of normal menstrual cycles

Objective To measure serial serum concentrations of the angiogenic factors vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and angiogenin (ANG) in the periovulatory and secretory phase of normal menstrual cycles in healthy women and to determine their peaks, which might reflect the stage of their critical angiogenic action. Design Prospective study. Setting University departments of obstetrics and gynecology. Participant(s) Thirty-three healthy Swedish women with regular menstrual cycles. Intervention(s) Serial blood samples were collected from each woman. Luteinizing hormone surge was identified by testing morning urine. Main outcome measure(s) Circulating levels of VEGF, bFGF, and ANG. Result(s) Circulating peak concentrations were determined for VEGF on day 0 and 9 after ovulation, for bFGF on day 1 before ovulation and day 9 after ovulation, and for ANG on day 3 after ovulation. Conclusion(s) Circulating VEGF increased in a stage-dependent cyclic fashion. Basic FGF peaked during the late proliferative and mid secretory phase. Circulating ANG showed increased expression around the early secretory phase of the cycle.

Expression of keratinocyte growth factor receptor (KGFR/FGFR2 IIIb) in human uterine cervical cancer

Keratinocyte growth factor receptor (KGFR), also known as FGFR2 IIIb, is a splice variant of FGFR-2. KGFR is expressed in many types of epithelial cell and is activated with four known ligands [FGF-1, FGF-3, FGF-7 (also known as KGF) and FGF-10] that are predominantly synthesized by mesenchymal cells. KGFR is highly expressed in the late-proliferative phase of a normal endometrium and in endometrial adenocarcinoma. In the present study, we attempted to determine the expression and localization of KGFR in human cervical cancer cell lines and cervical cancer tissues. The KGFR protein was detected in CaSki and HeLa cells, but not in ME-180 cells of cervical cancer cell lines. In non-cancer cervical tissues, KGFR immunoreactivity was weakly expressed in the surface of squamous epithelial cells and vascular smooth muscle cells. Immunohistochemically, the KGFR protein was detected in squamous cell carcinoma in 36 of 42 (86%) cervical cancer patients. In cervical cancer tissues, KGFR was detected in 17 of 18 (94%) of patients with the keratinizing type and 19 of 24 (79%) of patients with the non-keratinizing type of cervical cancer. Furthermore, KGFR was prominently localized in proliferating reserve cells and squamous metaplastic reserve cells adjacent to cancer cells. In contrast, KGFR was not detected in cervical ductal cells in cancer or non-cancer cervical tissues. These findings may indicate that KGFR mediates the growth and differentiation of reserve cells and squamous cell carcinoma in the cervix.

Ubiquitin is associated with the survival of ectopic stromal cells in endometriosis

BackgroundEndometriosis is a condition that affects women of reproductive age, where the glandular and/or stromal tissues from the eutopic endometrium implant in ectopic locations. It is well established that the survival of ectopic implants is due to lower levels of apoptosis, but no consensus exists as to which pathway/s this is mediated by. The ubiquitin protein shares a similar sequence homology to an anti-apoptotic protein called BAG-1 and is expressed in the normal endometrium. Currently, no studies have been conducted to determine ubiquitin expression and its possible anti-apoptotic effects in endometriosis.MethodsArchived endometrial tissues from endometriosis patients and women undergoing laparoscopic diagnosis (controls) from January 2000 to July 2003 at Westmead Hospital were examined, where 14 cases of endometriosis and 55 controls were included in the study.ResultsBoth the ubiquitin protein and apoptosis were expressed in both glandular and stromal cells throughout the menstrual cycle of the eutopic endometrium, in which ubiquitin exhibited a cyclic expression, reaching a peak in late proliferative phase. In contrast, ubiquitin was predominantly expressed in cells of stromal origin in endometriosis, was no longer regulated by a cyclic pattern and was associated with an aberrant level of cell survival.ConclusionsFor the first time, this study shows that ubiquitin is expressed in endometriotic cells and may contribute to a reduced sensitivity of ectopic endometrial tissue to apoptosis. These findings also suggest that stromal cells contribute differentially to the development of ectopic endometrial tissue.

Involvement of prostaglandins in inflammation induced by latex of Calotropis procera.

INTRODUCTION:The aerial parts of the plant Calotropis procera produce milky white latex that causes inflammation of the skin and mucous membranes. Prostaglandins are one of the mediators released in an inflammatory response following induction of cyclooxygenase (COX). In the present study, we have evaluated the role of prostaglandins in inflammatory response elicited by the latex of C. procera. METHODS: Aqueous extract of dried latex of C. procera was injected into the 6-day air-pouch in the rat. The inflammatory response was evaluated by studying the air-pouch fluid for its volume, protein and prostaglandin (PG) E2 concentrations, and leucocyte counts. The granulation tissue from the pouch was quantified and studied for COX-2 expression by reverse transcriptase-polymerase chain reaction. The inhibitory effect of celecoxib and dexamethasone was evaluated on the aforementioned parameters. RESULTS: Dried latex produced an inflammatory response that was maximum at 6 h. It was associated with the accumulation of protein-rich fluid, leucocytes and PGE2 production. It also resulted in granulation of the pouch cavity that was a maximum on day 3. COX-2 expression could be detected in the granulation tissue on day 1 and it increased progressively up to day 5. The anti-inflammatory drugs celecoxib and dexamethasone significantly attenuated the inflammatory response and inhibited COX-2 expression in granulation tissue. CONCLUSIONS: Latex of C. procera induces an inflammatory response characterized by an early exudative phase accompanied by PGE2 production and a late proliferative phase associated with COX-2 induction. Both the phases were effectively inhibited by COX-2 inhibitors.

Use of oxytocin receptor expression in distinguishing between uterine smooth muscle tumors and endometrial stromal sarcoma.

The present study aimed to investigate oxytocin receptor (OTR) expression in the normal uterus, and particularly in uterine smooth muscle tumors and endometrial stromal sarcomas (ESSs) because these tumors can be difficult to distinguish. The expressions of OTR, CD10, h-caldesmon, calponin, smooth muscle actin, and desmin were analyzed in 10 conventional leiomyomas (LMs), 10 highly cellular leiomyomas (HCLs), eight leiomyosarcomas (LMSs), and nine ESSs. In five normal uteri and five cases of adenomyosis, OTR was strongly expressed in the myometrium and showed expression pronounced in the surface epithelium during the late proliferative phase and at the time of ovulation, whereas the endometrial stromal cells were negative. All LMs and HCLs were strongly positive for OTR. Five cases of LMS showed moderate to strong OTR expression in 100% of the tumor cells, whereas three cases were weakly positive in 10-20% of the tumor cells. Every ESS was negative for OTR, except in regions of smooth muscle differentiation. All ESSs were positive for CD10, as were one LM, six HCLs, and five LMSs. The ESSs were negative for h-caldesmon and showed desmin positivity mainly in regions of smooth muscle metaplasia. h-Caldesmon, calponin, smooth muscle actin, and desmin were expressed in all LMs, HCLs, and LMSs except for one leiomyosarcoma with epithelioid features, which was negative for h-caldesmon and calponin. Our study indicates that the evaluation of OTR expression is useful in the distinction of uterine smooth muscle tumors from ESSs, and that the OTR is expressed in normal and neoplastic uterine smooth muscle cells.

Differential expression of angiopoietins 1 and 2 and their receptor Tie-2 in human endometrium

Angiogenesis, the growth of new capillaries from pre-existing blood vessels, is a physiological process involved in both normal menstrual cycling and implantation of the embryo. So far, very little is known about the expression of angiopoietins, growth factors involved in angiogenesis, in human endometrium. Both angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are ligands for the endothelial cell-specific receptor tyrosine kinase Tie-2. In this study we determined the mRNA expression of Ang-1, Ang-2 and Tie-2 by quantitative competitive RT/(QC)-PCR (including specifically designed competitor cDNA) in biopsied human endometrium throughout the menstrual cycle. We detected the mRNA for the angiopoietins in 30 out of 32 endometrial biopsies (94%), covering early proliferative (n = 4), mid proliferative (n = 12), late proliferative (n = 3), early secretory (n = 3), mid secretory (n = 5) and late secretory (n = 3) phases. Analysis of the target/competitor ratios (QC-PCR) revealed that Ang-1 mRNA expression was significantly up-regulated (P = 0.027) during the secretory phase of the menstrual cycle. In contrast, the expression levels of both Ang-2 mRNA and Tie-2 mRNA showed only minor variations at different cycle stages. These findings were confirmed by the relative expression ratio of Ang-1 versus Ang-2 in a multiplex PCR. The expression of Ang-1, Ang-2 and Tie-2 mRNA was detected in both isolated endometrial epithelial and stromal cell fractions. Immunohistochemical localization of the proteins revealed qualitative differences in both cell type and cycle stage expression. In conclusion, the enhanced Ang-1 expression during the secretory phase might serve to stabilize the newly developed blood vessels.

Estradiol inhibits IL-1a-induced IκBa phosphorylation in endometrial cells

Estradiol inhibits IL-1a-induced IκBa phosphorylation in endometrial cells

Effects of leptin on the production of cytokines by cultured human endometrial stromal and epithelial cells

Objective To evaluate the effects of leptin on the production of interleukin (IL)-6 family cytokines and chemokines by human endometrial stromal cells (ESC) and epithelial cells. Design The effects of leptin on the production of IL-6, IL-11, leukemia inhibitory factor (LIF), IL-8, growth-regulated oncogene (GRO)-α, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-3α by ESC and the endometrial epithelial cell line HHUA were investigated. Setting Research laboratory at a medical university. Patient(s) Eight endometrial specimens in the late proliferative phase were used for the isolation of ESC. Intervention(s) ESC and HHUA were incubated for 24 hours with recombinant human leptin. Main outcome measure(s) The concentration of IL-6, IL-11, LIF, IL-8, GRO-α, MCP-1, and MIP-3α were measured using ELISAs. Result(s) Unstimulated ESC and HHUA constitutively secreted IL-6, IL-11, LIF, IL-8, GRO-α, MCP-1, and MIP-3α. The increase in levels of IL-6, IL-8, GRO-α, MCP-1, and MIP-3α in the culture media of ESC and HHUA paralleled the addition of increasing amounts of leptin. In contrast, the levels of IL-11 and LIF were not affected by leptin administration. Conclusion(s) The present findings suggest that leptin may be an additional modulator of IL-6 and chemokine expression in the endometrium. Leptin may contribute to the normal and pathological processes of human reproduction by the regulation of these cytokines in the local environment.

Cell type-specific expression and regulation of neuropilin-1 and -2 in the rhesus macaque endometrium suggests roles for VEGF-Neuropilin in endometrial remodeling during the menstrual cycle in primates

Objective: Neuropilin-1 and -2 (NP-1/NP-2) are transmembrane receptors that play a role in axonal guidance by binding class III semaphorins, and in angiogenesis by binding vascular endothelial growth factor (VEGF). Although much is known about the biology of VEGF and its cognate receptors, VEGFR1 and VEGFR2, the roles of NP-1 and NP-2 in the primate endometrium are unknown. The goal of this study was to investigate the temporal and spatial expression of NP-1 and NP-2 in the rhesus macaque endometrium during the menstrual cycle. Design: We used in situ hybridization to investigate the cellular localization of NP-1 and NP-2 mRNA in the endometrium of ovarectomized, artificially cycled rhesus macaques. Materials and Methods: Twenty-four ovariectomized rhesus macaques were sequentially treated with estradiol (E) for 14 days and E plus progesterone (P) for 14 days. Then P was withdrawn from all animals to induce menstruation, and E was maintained. Uteri were obtained from one set of animals on days 5–6 (early-proliferative, n = 4), 8-10 (mid-proliferative, n = 3) and on day 14 (late-proliferative, n = 4) after P withdrawal. In the rest of the animals, P was added back after 14 days E treatment and tissue was sampled 3 (early-secretory, n = 4), 7/8 (mid-secretory, n = 4), and 14 (late-secretory, n = 3) days later. In situ hybridization was performed using self-designed probes by RT-PCR and cloning into GEM-T /Teasy vector systems to evaluate the expression and localization of NP-1 and -2 mRNA. Results: NP-1 mRNA was highly expressed in the endothelium of both arteries and veins, but there were no marked changes in its expression during the cycle. However, the luminal epithelium, which also expressed NP-1 mRNA, showed increased expression during the mid- to late-proliferative phase followed by a substantial decrease in expression of this receptor during the luteal phase. NP-1 mRNA was also abundantly expressed in the endometrial stromal cells (but not the glands) with a marked gradient of expression from the surface to the lower-functionalis. Stromal cell NP-1 mRNA changed little during the cycle except for a slight down-regulation during the mid- to late-secretory phase. NP-2 mRNA expression was strictly localized to the endothelium of the veins, was very low during the proliferative phase, and became substantially elevated during the secretory phase. Conclusion: This is the first report in the primate endometrium showing dynamic cell-specific expression and regulation of NP-1 and -2 mRNA during the menstrual cycle. The cyclic expression of NP-1 in the luminal epithelium suggests that VEGF-NP-1 interactions may influence the cyclic changes in the surface properties of this epithelium. In the vascular endothelium, VEGF-neuropilin interactions may result in proliferative effects when only NP-1 is present, and other effects (perhaps related to permeability) when both NP-1 and NP-2 are present during the secretory phase. Stromal NP-1 may mediate other, nonendothelial effects of VEGF through paracrine or autocrine routes. Supported by:German Research Foundation GE 1173/1-1 (AG) and Mellon Foundation (NRN).

Submucous and outer myometrium leiomyomas are two distinct clinical entities

Submucous and outer myometrium leiomyomas are two distinct clinical entities

Different basic fibroblast growth factor and fibroblast growth factor-antisense expression in eutopic endometrial stromal cells derived from women with and without endometriosis.

In all species studied, the basic fibroblast growth factor (bFGF) gene is transcribed into multiple mRNAs, one of which is an antisense RNA (1B FGF-AS) probably involved in regulating the stability of the sense transcript. In this study we investigated whether the regulatory mechanisms of bFGF expression might be altered in endometrial stromal cells derived from women with endometriosis. bFGF and 1B FGF-AS mRNA levels were quantified in primary cultures of eutopic endometrial stromal cells derived from 29 women without endometriosis and 24 patients affected by the disease. When the data were analyzed according to the phase of the menstrual cycle, endometrial stromal cells derived from patients in the late proliferative phase showed significantly higher bFGF mRNA values and significantly lower 1B FGF-AS mRNA levels compared with control samples. Furthermore, the mean bFGF/1B FGF-AS mRNA ratio was significantly higher in endometrial stromal cells derived from patients compared with that in controls (mean +/- SEM, 2.31 +/- 0,55 and 0.77 +/- 0.14, respectively; P = 0.009). Moreover, for bFGF expression the differences existing at the mRNA level were maintained at the protein level. These findings support the hypothesis that 1B FGF-AS mRNA could regulate the expression of the sense transcript and suggest that in endometrial cells derived from patients, the presence of higher bFGF levels could improve their ability to proliferate at the ectopic site.

Prostaglandin (PG) F2αReceptor Expression and Signaling in Human Endometrium: Role of PGF2αin Epithelial Cell Proliferation

Prostaglandin (PG) F(2alpha), a member of the prostanoid bioactive lipid family, is secreted by human endometrium throughout the menstrual cycle and is present in both menstrual fluid and medium of endometrial explants in culture. PGF(2alpha) mediates its effects through a seven-transmembrane G-protein-coupled receptor (FP). The aim of this study was to examine the temporal expression, signaling, and role of FP receptor in the human endometrium. Quantitative RT-PCR analysis demonstrated highest expression of FP receptor in the mid- to late-proliferative phase, compared with early-proliferative and secretory phase endometrium. In situ hybridization studies localized FP receptor mRNA expression to the epithelial cell compartment during the mid- to late-proliferative phase. Moreover, treatment of endometrial tissue with 1-100 nM PGF(2alpha) induced a concentration-dependent increase in inositol phosphate mobilization, indicating functional FP receptor expression. The Ishikawa human endometrial epithelial cell line was used to investigate further the signaling and role of PGF(2alpha) in endometrial epithelial cells. Ishikawa cells endogenously express the FP receptor, and treatment with 1-100 nM PGF(2alpha) elicits a concentration-dependent increase in inositol phosphate release. Moreover, treatment of Ishikawa cells with 100 nM PGF(2alpha) induced phosphorylation of ERK1/2 that was abolished when cells were cotreated with 50 micro M PD98059 (MAPK kinase inhibitor) or 10 micro M U73122 [phospholipase C (PLC) inhibitor]. Treatment of Ishikawa cells with PGF(2alpha) for 24 h induced a significant concentration-dependent increase in Ishikawa cell proliferation. Coincubation of the cells with 50 micro M PD98059 or 2 micro M U73122 demonstrated that PLC inhibition significantly reduced PGF(2alpha)-induced proliferation, whereas MAPK kinase inhibition had no effect. In summary, these studies demonstrate increased FP receptor expression in endometrial epithelial cells during the proliferative phase of the menstrual cycle and identify a role for PGF(2alpha) in epithelial cell proliferation via a PLC-dependent pathway.

Interleukin-13 and tumor necrosis factor-β differentially regulate the production of cytokines by cultured human endometrial stromal cells

Objective To evaluate the effects of interleukin (IL)-13, a T-helper (Th)2 cytokine, and tumor necrosis factor (TNF)-β, a Th1 cytokine, on the production of IL-6 family cytokines and chemokines by endometrial stromal cells (ESC). Design The effects of IL-13 and TNF-β, on the production of IL-6, IL-11, leukemia inhibitory factor (LIF), IL-8, growth-regulated oncogene α (GROα), monocyte chemoattractant protein-1 (MCP-1), regulated on activation, T-cell expressed and secreted (RANTES), and eotaxin were investigated. Setting Research laboratory at a medical university. Patient(s) Thirteen endometrial specimens in the late proliferative phase were used. Intervention(s) The ESC were incubated for 24 hours with recombinant human IL-13 and recombinant human TNF-β. Main outcome measure(s) The concentration of IL-6, IL-11, LIF, IL-8, GROα, MCP-1, RANTES, and eotaxin in the culture media was measured using ELISA. Result(s) The increase in levels of IL-6, IL-8, MCP-1, and eotaxin in the culture media of ESC paralleled the addition of increasing amounts of IL-13 and TNF-β, whereas the levels of IL-11 and LIF were decreased with increasing amounts of IL-13, but were increased with increasing amounts of TNF-β. Tumor necrosis factor-β enhanced the production of GROα and RANTES in dose-dependent manner; however, IL-13 did not affect the expression of GROα or RANTES. Conclusion(s) These results suggest that IL-13 and TNF-β secreted in the cyclic endometrial tissue and in the decidua may differentially regulate the production of IL-6 family cytokines and chemokines by ESC. The controlled expression of these cytokines in the endometrium may contribute to the modulation of the immune reaction during the menstrual cycle and in early pregnancy by the regulation of leukocyte trafficking and functions.