Urocortin and corticotrophin-releasing hormone receptor type 2 mRNA are highly expressed in deep infiltrating endometriotic lesions.

Ovarian endometrioma (OMA) and deep infiltrating endometriosis (DIE) are the most severe forms of endometriosis, but different pathogenetic mechanisms and clinical symptoms distinguish these two forms. Corticotrophin-releasing hormone (CRH) and urocortin (Ucn) are endometrial neuropeptides involved in tissue differentiation and inflammation. The expression of CRH, Ucn, Ucn2, CRH-receptors (type-1 and type-2) and inflammatory enzymes phospholipase-A2 group IIA (PLA2G2A) and cycloxygenase-2 (COX2) were evaluated in OMA (n = 22) and DIE (n = 26). The effect of CRH or Ucn on COX2 mRNA expression was evaluated in cultured human endometrial stromal cells. In DIE lesions, CRH, Ucn and CRH-R2 mRNA levels were significantly higher than in OMA (P < 0.01, P < 0.001 and P < 0.05, respectively); DIE lesions showed a higher expression of COX2 (P < 0.01) and PLA2G2A (P < 0.05) mRNA than OMA, which was positively correlated with CRH-R2 mRNA expression (P < 0.05). Intense immunostaining for CRH and Ucn was shown in DIE. Treatment of cultured endometrial stromal cells with Ucn significantly increased COX2 mRNA expression (P < 0.01); this effect was reversed by the CRH-R2 antagonist astressin-2B. In DIE, DIE lesions highly express neuropeptide and enzyme mRNAs, supporting a strong activation of inflammatory pathways.

Naproxen sodium decreases prostaglandins secretion from cultured human endometrial stromal cells modulating metabolizing enzymes mRNA expression.

Dysmenorrhea, defined as painful cramps occurring immediately before or during the menstrual period, is a common symptom of different gynecological diseases. An acute uterine inflammatory response driven by prostaglandins (PGs) is responsible for painful symptoms. Progesterone withdrawal is responsible for activation of cyclooxygenase (COX-2) enzyme and decrease of hydroxyprostaglandin dehydrogenase (HPDG) with consequent increased secretion of PGs secretion, inducing uterine contractility and pain. The most widely used drugs for the treatment of pelvic pain associated with menstrual cycle are non steroidal anti-inflammatory drugs (NSAIDs). The uterine site of action of these drugs is still not defined and the present study evaluated the effect of naproxen sodium in cultured human endometrial stromal cells (HESC) collected from healthy women. PGE2 release was measured by ELISA; COX-2 and HPDG mRNA expression were assessed by qRT-PCR. Naproxen sodium did not affect HESC vitality. Naproxen sodium significantly decreased PGE2 secretion (p < 0.01) and COX-2 mRNA expression (p < 0.01). TNF-α induced PGE2 release was reduced in presence of naproxen sodium (p < 0.05), in association with decreased COX-2 and increased HPDG mRNAs expression. Naproxen sodium decreases endometrial PGE2 release induced by inflammatory stimulus acting on endometrial COX-2 and HPDG expression, suggesting endometrial synthesis of prostaglandins as a possible target for reduction of uterine inflammatory mechanism in dysmenorrhea.

Expression of Inflammatory and Neurogenic Mediators in Adenomyosis.

OBJECTIVE: Adenomyosis is a uterine disorder characterized by dysmenorrhea, dyspareunia, abnormal uterine bleeding, and infertility. Pathogenesis indicates that endometrial cells invade and proliferate within myometrium, and inflammatory mediators participate to the intense painful symptoms. The aim of the present study was to investigate the messenger RNA (mRNA) and protein expression of inflammatory (interleukin 1ß [IL-1ß], corticotropin-releasing hormone [CRH], urocortin [Ucn]) and neurogenic (nerve growth factors [NGFs], synaptophysin [SYN], microtubule-associated protein 2 [MAP2]) factors in adenomyotic nodules. MATERIALS AND METHODS: This prospective study enrolled 16 women, 8 women with nodular adenomyosis and 8 control women undergoing to hysterectomy. Specimens from adenomyotic nodules and eutopic endometrium were collected after surgery. Endometrial tissue was also obtained from the control group and also used for preparing primary culture of human endometrial stromal cells (HESCs). Messenger RNA expression of inflammatory mediators (IL-1ß, CRH, and Ucn) and neurogenic factors (NGF, SYN, and MAP2) was analyzed by real-time polymerase chain reaction. The in vitro effects of CRH/Ucn on NGF or SYN mRNA expression were also investigated. RESULTS: Adenomyotic nodules highly expressed IL-1ß, CRH, and Ucn mRNAs, as well as NGF, SYN, and MAP2 mRNAs ( P < .001 vs eutopic endometrium and control). Endometrium of women with adenomyosis showed high expression of IL-1ß and CRH ( P < .001 vs control). Protein expression of CRH, NGF, and SYN in adenomyotic nodules was confirmed by immunohistochemical and immunofluorescence analyses. Urocortin increased NGF mRNA expression in cultured HESCs. CONCLUSION: The present study showed that adenomyotic nodules are novel site of expression of inflammatory and neurogenic factors, probably involved in the pathogenesis of adenomyosis.

Deep Infiltrating Endometriosis and Endometrial Adenocarcinoma Express High Levels of Myostatin and Its Receptors Messenger RNAs.

Myostatin is a growth factor member of the transforming growth factor ß superfamily, which is known to play major roles in cell proliferation and differentiation. The present study investigated the messenger RNA (mRNA) expression of myostatin and myostatin receptors (activin receptor-like kinase 4 [ALK4], transforming growth factor (TGF)-ß type I receptor kinase [ALK5] and activin receptor type IIB [ActRIIB]) in endometrium of healthy women during menstrual cycle as well as in benign (endometriosis, polyps) and malignant (endometrial adenocarcinoma) conditions. Endometrial specimens were collected by hysteroscopy, whereas endometriotic lesions were collected by laparoscopy, and adenocarcinomas were sampled after hysterectomy. Total RNA was extracted from tissue homogenates, and gene expression was assessed by quantitative real-time polymerase chain reaction. Myostatin and myostatin receptors mRNAs were expressed by healthy endometrium throughout the menstrual cycle, with no differences between the proliferative and secretory phase. The highest myostatin mRNA expression was found in patients with deep infiltrating endometriosis (DIE) and in endometrial carcinoma; expression was also found in ovarian endometrioma (OMA ) and endometrial polyps. Myostatin receptors mRNA expression was higher in DIE and adenocarcinomas compared to control endometrium. The expression of ALK5 and ActRIIB in OMA was higher than in controls, whereas polyps had an increased expression of ALK5 mRNA. In conclusion, the present data showed for the first time the expression of myostatin in healthy endometrium and a higher expression in endometriosis and endometrial cancer, suggesting myostatin involvement in human endometrial physiology and related pathologies.

Expression and regulation of 11ß-hydroxysteroid dehydrogenase type 1 in first trimester human decidua cells: Implication in preeclampsia.

Glucocorticoids are implicated in successful blastocyst implantation, whereas alterations in glucocorticoid levels are associated with various pregnancy disorders including preeclampsia. Tissue concentration of active glucocorticoids depends on the expression of 11ß-hydroxysteroid dehydrogenase (11ß-HSD). This study investigated the contribution of first trimester decidua to glucocorticoid availability at the fetal-maternal interface by assessing the expression and regulation of 11ß-HSD in human first trimester decidual tissues and cells and by evaluating 11ß-HSD levels in preeclamptic vs. gestational age-matched decidua. 11ß-HSD1 was the predominant isoform in first trimester decidua. In vitro, decidual cell 11ß-HSD1 levels and enzymatic activity were up-regulated by ovarian steroids and inflammatory cytokines. Higher levels of 11ß-HSD1 were found in preeclamptic decidua compared to controls. The present study indicates the predominance of 11ß-HSD oxoreductase isoform in early decidua. Observations that ovarian hormones and inflammatory cytokines up-regulate 11ß-HSD1, together with increased 11ß-HSD1 expression in preeclampsia, highlight a role for decidual cells in controlling biologically active glucocorticoids in early pregnancy.

Expression of microtubule associated protein 2 and synaptophysin in endometrium: high levels in deep infiltrating endometriosis lesions.

OBJECTIVE: To assess whether healthy endometrium, eutopic endometrium, and endometriotic lesions express nerve growth factor (NGF), microtubule-associated protein 2 (MAP-2), and synaptophysin (SYP). DESIGN: Molecular study in tissue extracts. SETTING: University hospital. PATIENT(S): A group of women (n = 70), divided as [1] healthy controls (n = 30) and [2] with endometriosis (n = 40), was included. INTERVENTION(S): From the healthy control group an endometrial specimen was collected by hysteroscopy (proliferative phase, n = 16; secretive phase, n = 14). Endometriotic and endometrial specimens were collected from women undergoing laparoscopic surgery for endometriosis, endometrioma (OMA) (n = 20), or deep infiltrating endometriosis (DIE) (n = 20). MAIN OUTCOME MEASURE(S): To assess expression of NGF, MAP-2, and SYP messenger RNA (mRNA) levels in endometrium and in endometriosis by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and protein localization by immunofluorescence. Cultures of human endometrial stromal cells were used to evaluate the effect of tumor necrosis factor (TNF)-α on NGF and SYP. RESULT(S): Endometrial tissue from control expressed mRNA for NGF, MAP-2, and SYP, without any difference between proliferative and secretive phase. The DIE and OMA lesions showed the highest NGF mRNA expression, significantly higher than in eutopic endometrium and control. In DIE lesions SYP mRNA expression was higher than in OMA or in eutopic endometrium or controls. Immunofluorescence analysis of NGF, MAP-2, and SYP showed a slightly more intense positive signal in endometriotic lesions. Exposure to TNF-α increased NGF and SYP mRNA expression in endometrial culture cells. CONCLUSION(S): The present study revealed the presence of two selected neuronal markers, MAP-2 and SYP mRNAs and protein expression, in eutopic endometrium and in endometriotic lesions.

Corticotropin releasing hormone and Urocortin 2 activate inflammatory pathways in cultured trophoblast cell lines.

OBJECTIVE: Embryo implantation and parturition are recognized as inflammatory events involving endocrine and immune system. NF-kB and MAPK are two transcription factor families involved in inflammation. A possible role of neuroendocrine mechanism in early pregnancy and delivery was proposed for the neuropeptides related to corticotropin releasing hormones (CRH), named Urocortins (Ucns). Experimental and clinical studies support a role for CRH, Ucn, Ucn2 and Ucn3 in the endocrine/immune modulation of inflammation in human trophoblast; however the intracellular mechanisms are not yet recognized. The aim of the present study was to evaluate which of these neuropeptides modulate NF-kB or MAPKs pathways. STUDY DESIGN: In Jeg-3 placental cell line the effect of CRH, Ucn, Ucn2 or Ucn3 on NF-kB and MAPKs pathways were evaluated using Western blot analysis. RESULTS: CRH induced the phosphorylation of MAPK subunits; Ucn2 was able to induce the phosphorylation of both NF-kB and MAPK subunits. Ucn and Ucn3 had no effects on these pathways. CONCLUSIONS: These data provide novel information on inflammatory process in trophoblast cells: Ucn2 is a potent pro-inflammatory neuropeptide via NF-kB and MAPK pathways and CRH via MAPK, and CRH and Ucn2 network participates in the inflammatory mechanisms of pregnancy and parturition.

Myostatin, follistatin and activin type II receptors are highly expressed in adenomyosis.

OBJECTIVE: To evaluate the expression pattern of activins and related growth factor messenger RNA (mRNA) levels in adenomyotic nodules and in their endometrium. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Symptomatic premenopausal women scheduled to undergo hysterectomy for adenomyosis. INTERVENTION(S): Samples from adenomyotic nodules and homologous endometria were collected. Endometrial tissue was also obtained from a control group. MAIN OUTCOME MEASURE(S): Quantitative real-time polymerase chain reaction (PCR) analysis and immunohistochemical localization of activin-related growth factors (activin A, activin B, and myostatin), binding protein (follistatin), antagonists (inhibin-α, cripto), and receptors (ActRIIa, ActRIIb) were performed. RESULT(S): Myostatin mRNA levels in adenomyotic nodule were higher than in eutopic endometrium and myostatin, activin A, and follistatin concentrations were higher than in control endometrium. No difference was observed for inhibin-α, activin B, and cripto mRNA levels. Increased mRNA levels of ActRIIa and ActRIIb were observed in adenomyotic nodules compared with eutopic endometrium and control endometrium. Immunofluorescent staining for myostatin and follistatin confirmed higher protein expression in both glands and stroma of patients with adenomyosis than in controls. CONCLUSION(S): The present study showed for the first time that adenomyotic tissues express high levels of myostatin, follistatin, and activin A (growth factors involved in proliferation, apoptosis, and angiogenesis). Increased expression of their receptors supports the hypothesis of a possible local effect of these growth factors in adenomyosis. The augmented expression of ActRIIa, ActRIIb, and follistatin in the endometrium of these patients may play a role in adenomyosis-related infertility.