Sylvia Mechsner

A pilot study to evaluate the clinical relevance of endometriosis-associated nerve fibers in peritoneal endometriotic lesions.

OBJECTIVE To investigate the clinical relevance of endometriosis-associated nerve fibers in the development of endometriosis-associated symptoms. DESIGN Prospective nonrandomized study. SETTING University hospital endometriosis center. PATIENT(S) Fifty-one premenopausal patients underwent surgical laparoscopy because of chronic pelvic pain, dysmenorrhea, or for ovarian cysts. Endometriosis was diagnosed in 44 patients. INTERVENTION(S) The preoperative and postoperative pain scores were determined using a standardized questionnaire with a visual analogue scale from 1-10. Patients with peritoneal endometriosis were divided into two groups depending on their preoperative pain score: group A with a pain score of at least 3 or more and group B with a pain score of 2 or less. Patients without peritoneal endometriosis were classified as group C and patients without endometriosis were classified as group D. Immunohistochemical analysis of neurofilament and protein gene product 9.5 were used for nerve fiber detection. Occurrence of endometriosis-associated nerve fibers was correlated with the severity of pelvic pain and/or dysmenorrhea. RESULT(S) Peritoneal endometriosis-associated nerve fibers were found significantly more frequently in group A than in group B (82.6% vs. 33.3%). CONCLUSION(S) The present study suggests that the presence of endometriosis-associated nerve fibers in the peritoneum is important for the development of endometriosis-associated pelvic pain and dysmenorrhea.

Overexpression of nerve growth factor in peritoneal fluid from women with endometriosis may promote neurite outgrowth in endometriotic lesions

To investigate the role of the nerve growth factor (NGF) in the endometriosis-associated innervation in the development of endometriosis-associated symptoms, 41 peritoneal fluid samples (PF) from patients with surgically and histologically proven endometriosis and 20 PF from patients with other gynecologic conditions were analyzed with Western blot and a novel in vitro model using dorsal root ganglia (DRG) to show neuronal outgrowth; endometrial cells also were analyzed. The results suggest that the PF of endometriosis patients and endometriotic lesions have neurotropic properties, because the Western blot analysis and the cell culture stainings showed NGF expression, and the neurite outgrowth of DRG treated with PF of patients with endometriosis was significantly higher than when treated with PF of patients without endometriosis. Furthermore, blocking NGF with both anti-NGF and K252a leads to a significant decrease in neurite outgrowth.

Imbalance between sympathetic and sensory innervation in peritoneal endometriosis

To investigate possible mechanisms of pain pathophysiology in patients with peritoneal endometriosis, a clinical study on sensory and sympathetic nerve fibre sprouting in endometriosis was performed. Peritoneal lesions (n=40) and healthy peritoneum (n=12) were immunostained and analysed with anti-protein gene product 9.5 (PGP 9.5), anti-substance P (SP) and anti-tyrosine hydroxylase (TH), specific markers for intact nerve fibres, sensory nerve fibres and sympathetic nerve fibres, respectively, to identify the ratio of sympathetic and sensory nerve fibres. In addition, immune cell infiltrates in peritoneal endometriotic lesions were analysed and the nerve growth factor (NGF) and interleukin (IL)-1β expression was correlate with the nerve fibre density. Peritoneal fluids from patients with endometriosis (n=40) and without endometriosis (n=20) were used for the in vitro neuronal growth assay. Cultured chicken dorsal root ganglia (DRG) and sympathetic ganglia were stained with anti-growth associated protein 43 (anti-GAP 43), anti-SP and anti-TH. We could detect an increased sensory and decreased sympathetic nerve fibres density in peritoneal lesions compared to healthy peritoneum. Peritoneal fluids of patients with endometriosis compared to patients without endometriosis induced an increased sprouting of sensory neurites from DRG and decreased neurite outgrowth from sympathetic ganglia. In conclusion, this study demonstrates an imbalance between sympathetic and sensory nerve fibres in peritoneal endometriosis, as well as an altered modulation of peritoneal fluids from patients with endometriosis on sympathetic and sensory innervation which might directly be involved in the maintenance of inflammation and pain.

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.

Possible roles of oxytocin receptor and vasopressin-1α receptor in the pathomechanism of dysperistalsis and dysmenorrhea in patients with adenomyosis uteri

OBJECTIVE To investigate the expression of oxytocin (OTR) and/or vasopressin (VP1αR) receptor in patients with and without adenomyosis uteri. DESIGN Retrospective nonrandomized study. SETTING University hospital endometriosis research center. PATIENT(S) Forty patients with histologically proven adenomyosis and 40 patients without adenomyosis who had undergone hysterectomy for dysmenorrhea, bleeding disorders, and fibroids. INTERVENTION(S) Immunohistochemical examination of both OTR and VP1αR expression in endometrium, myometrium, and adenomyotic lesions, and identification of smooth muscle cells using antibodies against OTR, VP1αR, and smooth muscle actin (sm-actin). MAIN OUTCOME MEASURE(S) The immunoreactive score (IRS) was used for expression of OTR, VP1αR, and sm-actin. RESULT(S) Expression of OTR in epithelial cells of adenomyotic lesions and surrounding myometrial cells was detectable. VP1αR was expressed only in myometrial cells and blood vessels. Using a specific anti-sm-actin antibody, another spindle cell population was characterized to represent smooth muscle cells which are in direct contact with the adenomyotic stroma. Compared with the unaffected myometrium, the surrounding adenomyosis-associated myometrium overexpressed OTR and showed changes in morphology. In the uteri of patients with adenomyosis, the junctional zone was often seen to be quite fissured. CONCLUSION(S) In addition to the specific expression of VP1αR, OTR expression and morphologic changes in the myometrial architecture of uteri having adenomyosis support the hypothesis that dysperistalsis plays an essential role in the development of endometriosis and dysmenorrhea. In the near future, specific inhibition of this receptor might yield a promising treatment for therapy.

Estrogen and progestogen receptor positive endometriotic lesions and disseminated cells in pelvic sentinel lymph nodes of patients with deep infiltrating rectovaginal endometriosis: a pilot study

BACKGROUND Deep infiltrating endometriosis (DIE) shows similarities to malignant diseases. A recent study involving DIE patients found endometriosis in mesorectal lymph nodes (LNs) after segmental bowel resection. However, it is unclear whether this observation is a local phenomenon or a sign of systemic disease. Therefore, we conducted a prospective study to investigate the occurrence of endometriosis in pelvic sentinel lymph nodes (SLNs) in patients with DIE. METHODS Fourteen patients underwent primary surgery for symptomatic DIE. Combined vaginal laparoscopic-assisted resection of the rectovaginal septum was performed. Dye was injected into the visible/palpable nodule. SLNs were removed from the iliac region. In order to identify endometriotic cells, immunohistochemical analysis of estrogen and progestogen receptors, CD10 and cytokeratin was performed. RESULTS In 12 out of 14 patients with DIE, SLNs were detected. The localization of the SLN followed the typical LN spread of the upper vagina. In three patients, we could detect typical endometriotic lesions in the LNs. Ten out of 12 (83.3%) SLNs showed disseminated estrogen and/or progestogen positive cells. CONCLUSIONS By using immunohistochemistry, we could demonstrate endometriotic lesions and endometriotic-like cells in pelvic SLNs of patients with DIE suggesting the potential for lymphatic spread of the disease.

Influence of nerve growth factor in endometriosis-associated symptoms.

To investigate the role of the nerve growth factor (NGF) in the development of dysmenorrhea/pelvic pain in patients with endometriosis, we performed a prospective, clinical, blind study. Peritoneal fluids (PFs) were obtained from patients with histologically proven endometriosis. Patients with endometriosis were divided into 7 different groups depending on their preoperative pain score and symptomatology: patients with no pain, patients with minimal pain (dysmenorrhea, pelvic pain, or both), and patients with severe pain (dysmenorrhea, pelvic pain, or both) and were used for the neuronal growth assay with cultured chicken dorsal root ganglia (DRG) and for Western blot analyses. Dorsal root ganglia were stained with anti-calcitonin gene-related peptide (CGRP) and anti-growth-associated protein 43 (GAP 43). Peritoneal fluids from patients with endometriosis induce neurite outgrowth. There was no significant difference in the outgrowth between the 7 pain groups. Western blot analyses showed a moderate NGF expression in the PFs from patients with endometriosis, without significant differences in the 7 pain groups. The present study suggests that the neurotrophic properties of endometriotic tissues are endometriosis- and not pain-associated.

Evidence of neurotrophic events due to peritoneal endometriotic lesions

To investigate the neurotrophic properties of endometriosis, as well as the involvement of neurotrophic factors in the development of chronic pelvic pain in patients with endometriosis, we performed a prospective clinical study. The presence of neurotrophins was investigated in the peritoneal fluid (PF) of patients with peritoneal endometriotic lesions or adenomyosis, as well as from women with non-endometriotic adhesions and from women without endometriosis/adenomyosis/adhesions. The PF from patients with peritoneal endometriotic lesions was divided in three groups: asymptomatic endometriosis, minimal pain and severe pain. PF from patients with adenomyosis or with non-endometriotic adhesions and the control group were divided in patients without pain and with pain. Neurotrophin expression in PF was analyzed using Elisa and the neuronal growth assay with cultured chicken sensory ganglia (dorsal-root-ganglia, DRG) and sympathetic ganglia. PF from women with peritoneal endometriotic lesions overexpress nerve growth factor (NGF) and neurotrophin-3 (NT-3), but not brain derived neurotrophic factor (BDNF), whereas the PF of women with adenomyosis or adhesions seems to express normal amounts of these factors. Neurotrophin expression did not differ among the pain groups. Furthermore, the PF from patients with peritoneal endometriotic lesions induced a strong sensory and a marginal sympathetic neurite outgrowth, while the PF from women with adenomyosis and non-endometriotic adhesions induced an outgrowth similar to the control group. The induced neurite outgrowth could only be inhibited in DRG incubated with peritoneal endometriotic lesions. Interestingly, the outgrowth of sympathetic ganglia was inhibited in all studied groups. The present study suggests that only peritoneal endometriotic lesions lead to an increased release of NGF and NT-3 into the PF and that NGF modulates the nerve fiber growth in endometriosis.

Immunohistochemical characterization of endometriosis-associated smooth muscle cells in human peritoneal endometriotic lesions

BACKGROUND Smooth muscle cells (SMC) are common components of endometriotic lesions. SMC have been characterized previously in peritoneal, ovarian and deep infiltrating endometriotic lesions and adenomyosis. The aim of this retrospective study was to investigate the extent of differentiation in endometriosis-associated SMC (EMaSMC) in peritoneal endometriotic lesions. METHODS We obtained biopsies from peritoneal endometriotic lesions (n = 60) and peritoneal sites distant from the endometriotic lesion (n = 60), as well as healthy peritoneum from patients without endometriosis (control tissue, n = 10). These controls were hysterectomy specimens from patients without endometriosis or adenomyosis. Histopathological examination of peritoneal specimens using antibodies against oxytocin receptor (OTR), vasopressin receptor (VPR), smooth muscle myosin heavy chain (SM-MHC), estrogen receptor (ER) or progesterone receptor (PR) was performed. To identify SMC and their level of differentiation, antibodies for smooth muscle actin desmin and caldesmon were used. RESULTS SMC were detected in all endometriotic lesions. SMC were more abundant in unaffected peritoneum of women with endometriosis (38%) compared with women without endometriosis (6%; P < 0.0001). Depending on the level of differentiation, SMC stained for SM-MHC, OTR, VPR, ER and PR. OTR was only detected in fully differentiated SMC. CONCLUSIONS Identification of OTR, VPR, ER and PR leads to the hypothesis that the EMaSMC might be functionally active and possibly involved in the generation of pain associated with endometriosis.

Lymphangiogenesis in deep infiltrating endometriosis

BACKGROUND In patients diagnosed with deep infiltrating endometriosis (DIE), foci of endometriosis are detected in mesorectal lymph nodes (LNs) after segmental bowel resection and in pelvic sentinel LNs. Lymph vessels (LVs) seem to be the possible routes for the dissemination of endometriotic cells from DIE-lesions to LN. Therefore, we conducted a study to investigate the occurrence and density of LV and lymphangiogenic growth factors in DIE. METHODS Included in this study were 38 premenopausal women who underwent surgery due to symptomatic rectovaginal DIE. In order to identify LV, immunohistochemical analysis with anti-Podoplanin (D2-40), LYVE-1 and Prox-1 was performed. Furthermore, the expression of VEGF-C and VEGF-D in endometriotic tissue was investigated. RESULTS LV density (LVD) of DIE lesions was significantly higher compared with healthy corresponding tissue. All LV makers could be detected, and the density of LYVE-1- or Prox-1-positive LV was significantly higher than that of D2-40-positive LV. Endometriotic epithelial cells and stromal cells showed a moderate to strong VEGF-C and VEDF-D expression. CONCLUSIONS DIE lesions have lymphangiogenic properties, probably leading to endometriosis-like cells in lymphatic vessels and LNs featuring a loco-regional disease.