Association of Uterine Tissue Innervation and Peripheral Nerve Density with Adenomyosis Related Pain. A Systematic Review.

Adenomyosis is associated with dysmenorrhea and chronic pelvic pain; however, the triggering mechanisms of painful stimuli and the role of uterine nerve fibers in the manifestation of pain remain poorly understood. The objective of this study was to systematically review the role of uterine nerve fibers' presence and density in the occurrence of pain in patients with adenomyosis. An electronic search was performed using the Embase, PubMed/Medline, and Cochrane databases. We included all studies from inception to November 2023. A total of ten studies that compared uterine biopsies samples of women with and without adenomyosis were included. The biomarker antiprotein gene product 9.5 was decreased or absent in the endometrium of most included women with adenomyosis. None of the included studies observed a difference in neurofilament (NF) staining between the adenomyosis and non-adenomyosis groups. Studies that assessed nerve growth factor (NGF) staining were heterogeneous in design. One study reported no difference in immunohistochemistry staining in any endometrial layer between the adenomyosis and non-adenomyosis groups, while another reported increased staining in the adenomyosis functional endometrial layer, and a third study reported overexpression of NGF, synaptophysin (SYN), and microtubule-associated protein 2 mRNA in focal adenomyosis alone. Preliminary data from poor-quality studies suggest an increase in the uterine density of nerve fibers in patients with adenomyosis. Well-designed studies are essential to assess the cause-and-effect relationship between uterine nerve fibers and pain in patients with adenomyosis.

Reduced inflammatory state promotes reinnervation of endometriotic-like lesions in TNFRp55 deficient mice.

Endometriosis is a chronic gynecological disease, characterized by growth of endometrial tissue in ectopic sites due to alteration of peritoneal homeostasis and deregulation of apoptosis. Here we have examined whether TNFRp55 deficiency modulates the pro-inflammatory state and the reinnervation of endometriotic-like lesions in mice. Two-month-old female C57BL/6 mice, eight wild type (WT) and eight TNFRp55-/- (KO) were used in the study. Endometriotic-like lesions were induced experimentally. The right uterine horn was removed from the animal, divided longitudinally, cut in three square pieces and sutured to the intestine mesentery. After 4 weeks, the lesions and the peritoneal fluid were collected. The level of TNFα in the peritoneal fluid was evaluated by enzyme-linked immunosorbent assay (EIA). The expressions of COX2, GRα and GRß were evaluated in the lesions by western blot and immunohistochemistry. ß-III TUBULIN, BDNF and NGF protein concentrations were evaluated in the lesions by western blot. Gene expression of Pgp 9.5, SP and Th was analyzed by RT-PCR, whereas relative concentrations of TRKA, NTRp75, phosphorylated NFκB (pNFκB) and total NFκB in lesions were measured by EIA. Compared with the WT group, the KO mice showed lower TNFα levels in the peritoneal fluid and lower numbers of COX2 immunoreactive cells along with increased expression of GRα, ß-III TUBULIN, Pgp 9.5, SP, Th, BDNF, NGF, NTRp75 and pNFκB in the lesions. Future histological studies will be necessary to confirm the sensory/sympathetic imbalance in the endometriotic-like lesions of the KO mice. Our results suggest that a reduced inflammatory state promotes reinnervation of endometriotic-like lesions in TNFRp55-/- mice. Chronic deregulation of TNF receptors can have serious consequences for women with advanced endometriosis.

Invasion of human deep nodular endometriotic lesions is associated with collective cell migration and nerve development.

OBJECTIVE: To study the mechanisms of invasion and innervation of deep endometriosis in women. DESIGN: Morphologic and immunohistochemical analysis of human endometriotic lesions. SETTING: Academic research unit. PATIENT(S): Seventeen biopsy samples of deep endometriotic lesions were collected from patients undergoing surgery for deep endometriosis. INTERVENTION(S): The endometriotic samples were divided into two parts: the front (the most invasive area of lesions, approaching rectal infiltration) and center (the area close to the posterior part of the cervix). MAIN OUTCOME(S): To elucidate: gland morphology, proliferation, and expression of adhesion molecules (ß-catenin, E-cadherin, and N-cadherin) to determine the possible role of collective cell migration (CCM) in the invasion process; and nerve growth factor (NGF) and nerve fiber density (NFD) values to shed further light on the mechanism of innervation. RESULTS: Glands from the front showed significantly reduced thickness, but significantly higher proliferation. ß-Catenin expression was similar between the lesion center and front. E-cadherin levels were significantly lower and N-cadherin levels significantly higher in glands located at the front of the lesions. Expression of matrix metalloproteinase-9 was significantly higher in glands and stromal cells located at the invasion front. NFD and NGF expression were also significantly higher at the lesion front. CONCLUSION: Although some data in the literature point to features of epithelial to mesenchymal transition in human deep nodular endometriosis, our study suggests that gland invasion in these lesions is dominated by CCM. Innervation of deep nodular endometriotic lesions may be a consequence of nerve recruitment from surrounding organs.

Assessment of biomarkers in women with endometriosis-associated pain using the ENG contraceptive implant or the 52 mg LNG-IUS: a non-inferiority randomised clinical trial.

OBJECTIVE: The aim of the study was to assess the serum levels of the following biomarkers in women with endometriosis-associated pelvic pain before and after six months of using the etonogestrel (ENG) contraceptive implant or the 52 mg levonorgestrel-releasing intrauterine system (LNG-IUS): cancer antigen (CA)-125, cluster of differentiation (CD) 23 and endometrial nerve fibre density. METHODS: The study was conducted at the Department of Obstetrics and Gynaecology, University of Campinas Medical School, Brazil. A total of 103 women with endometriosis-associated pain diagnosed by surgery, transvaginal ultrasound and/or magnetic resonance imaging were included. Endometrial nerve fibre density and serum levels of CA-125 and soluble CD23 were assessed before and after six months of using the allocated method and were correlated to 10 cm visual analogue scale (VAS) scores for non-cyclical pelvic pain and dysmenorrhoea. RESULTS: Both contraceptive methods significantly reduced concentrations of serum soluble CD23 and endometrial nerve fibre density (p < .001); however, CA-125 was significantly reduced only among users of the ENG implant (p < .05). No correlation was observed between reduction of biomarkers and improvement of VAS pain and dysmenorrhoea scores. No differences were observed between the ENG implant and the LNG-IUS. CONCLUSION: Both progestin-only contraceptives significantly reduced two out of the three biomarkers evaluated. These two biomarkers could, therefore, be used as surrogate markers to follow up medical treatment of endometriosis-associated pain.

Important role of collective cell migration and nerve fiber density in the development of deep nodular endometriosis.

OBJECTIVE: To evaluate deep nodular endometriotic lesions induced in baboons over 12 months and analyze collective cell migration and nerve fiber density. DESIGN: Morphologic and immunohistochemical analysis of endometriotic lesions induced in baboons over the course of 1 year. SETTING: Academic research unit. ANIMAL(S): Three female baboons (Papio anubis). INTERVENTION(S): Recovery of induced deep nodular endometriotic nodules from baboons. MAIN OUTCOME MEASURE(S): Evaluation of the morphology of glands by analysis of the center of lesions and the invasion front; immunohistochemical staining with Ki67, E-cadherin, and ß-catenin for investigation of mitotic activity and cell-cell junctions, and with protein gene product 9.5 and nerve growth factor (NGF) for study of nerve fiber density (NFD). RESULT(S): All (100%) of the lesions were invasive 1 year after induction, compared with 42.29% after 6 months. Glands from the invasion front showed significantly reduced thickness but significantly higher mitotic activity. E-Cadherin and ß-catenin expression were similar between the center and front. NFD was significantly higher in lesions induced after 1 year than after 6 months, and NGF expression was significantly lower in 1-year lesions than in 6-month lesions. CONCLUSION(S): Nodular endometriotic lesions induced in the baboon model were found to be significantly more invasive and innervated after 12 months than after 6 months. The invasive phenotype was highly expressed in glands at the invasion front, and our study suggests that nerve fibers play a role in the development of lesions as observed in women.

Dienogest reduces proliferation, NGF expression and nerve fiber density in human adenomyosis.

OBJECTIVES: To evaluate the in vivo effect of dienogest on proliferation, apoptosis, aromatase expression, vascular density, nerve growth factor (NGF) expression and nerve fiber density in human adenomyosis tissue. STUDY DESIGN: Twelve women who underwent hysterectomy for adenomyosis were enrolled. Six patients received dienogest treatment prior to hysterectomy (dienogest group), and age-matched six patients who had not received any hormonal treatment for ≥3 months before surgery (control group). Cell proliferation, vascular and nerve fiber density in adenomyosis tissue were evaluated by staining for Ki67, von Willebrand factor and PGP9.5, respectively. Apoptosis was detected using the TUNEL assay. The expression aromatase and NGF were evaluated by staining for corresponding antibodies. RESULTS: The proportion of Ki67 positive epithelial cells was significantly lower in samples from dienogest-treated patients in comparison with controls (p<0.05). The density of blood vessels in adenomyosis was marginally lower in the dienogest group in comparison with controls but statistical significance was not reached (p=0.07). The intensity of NGF expression and the density of nerve fibers were significantly lower in the dienogest group compared with controls (p<0.05 for both). CONCLUSION: This study demonstrates that adenomyosis, taken from patients treated with dienogest, shows remarkable histological features, such as reductions in proliferation, NGF expression and nerve fiber density. These findings indicate the impact of dienogest on local histological events, and explains its therapeutic effect on adenomyosis.

Reinnervation of rat endometrium in the anterior eye chamber model of experimental endometriosis: Old methods for new questions.

Endometriosis is a benign estrogen-dependent chronic gynecological disease characterized by the presence of endometrial-like tissue outside the uterine cavity. In both women and experimental endometriotic rats, endometriosis lesions endow autonomic and sensory nerves, which are thought to contribute to the disease-associated pain. Some evidence indicates that the reinnervation of lesions is regulated by factors produced by the endometrial tissue as well as by environmental factors from the peritoneum. In this study, we examined the reinnervation of the rat endometrial tissue in an ectopic environment different from the peritoneum employing the anterior eye chamber model of experimental endometriosis. At 3 and 6weeks following transplantation, endometrial grafts retained many histological features of the eutopic tissue. Both sympathetic and sensory nerves reinnervated endometrial grafts and distributed in the stroma-like tissue, around blood vessels and in close proximity to the glands and lining epithelium. Sympathetic innervation was more robust than sensory innervation. No significant topographical relationship between sympathetic nerves and macrophages was observed. These results suggest that the rat endometrium possesses intrinsic neuritogenic capacities and can be reinnervated by sympathetic and sensory nerves in ectopic sites different from the peritoneum.

Effects of catecholaminergic nerve lesion on endometrial development during early pregnancy in Mice.

Maternal stress is common during pregnancy and the postnatal period. This stress typically activates the sympathetic nervous system which releases catecholamines. This study explored the influence of sympathectomy by using neurotoxin 6-hydroxydopamine (6-OHDA) on embryo implantation, and investigated the influence mechanism of sympathectomy on reconstruction of endometrial structure during early pregnancy. In the 6-OHDA-treated mice, uterine glands in the endometrium developed poorly, and the gland epithelia were arranged irregularly during early pregnancy. Furthermore, vacuoles, karyopykosis and plasmarrhexis appeared in some gland epithelia. The percentage of uterine glands and the density of proliferating cell nuclear antigen (PCNA) positivity were dramatically decreased, and Fas ligand (FasL) expression was decreased in cells from pregnancy days 5-9 (E5-9) in the treated group. Antioxidant enzyme activity levels in uteri were lower but the malondialdehyde (MDA) levels were higher in the 6-OHDA mice than those in the control mice at E5-9. Similarly, the number of inducible nitric oxide synthase (iNOS) positive cells was significantly increased during early pregnancy following treatment with 6-OHDA. Our results have indicated that peripheral catecholaminergic nerve lesions induced by 6-OHDA cause adverse pregnancy outcomes through disruption of endometrial gland development, which increases oxidative stress and iNOS expression in the endometrium. Thus, catecholaminergic nerves might favourably influence blastocyst implantation, foetal survival and development during early pregnancy by oxidative state regulation and endometrial gland reconstruction.

Hysteroscopic local anesthetic intrauterine cornual block in office endometrial ablation: a randomized controlled trial.

OBJECTIVE: To evaluate the efficacy of a hysteroscopic local anesthetic intrauterine cornual block (ICOB) on pain experienced during office endometrial ablation (EA) in addition to a traditional direct local anesthetic cervical block (DCB). DESIGN: Prospective, randomized, double-blind, placebo-controlled trial. SETTING: University teaching hospital. PATIENT(S): Women with heavy menstrual bleeding scheduled for an office endometrial ablation. INTERVENTION(S): Before office EA, DCB plus hysteroscopic ICOB just medial to each tubal ostium using local anesthetic mixture made up of 1 mL 3% mepivacaine plus 1 mL 0.5% bupivacaine versus control group receiving DBC plus ICOB with 2 mL of placebo (saline). PRIMARY OUTCOME: pain reported during procedure via visual analogue scale (VAS) from 0 to 10; secondary outcomes: postoperative pain, rescue analgesic requirement, and duration of hospital stay. RESULT(S): Most characteristics were similar across groups. The mean VAS score during the procedure was statistically significantly lower by 1.44 (95% confidence interval, -2.65 to -0.21) in the active group compared with the placebo group. There were no statistically significant differences between the two groups in the postprocedural mean VAS scores, rescue analgesic requirement, or duration of hospital stay. CONCLUSION(S): Used in addition to DCB, ICOB reduces the pain experienced during office EA compared with DCB alone. CLINICAL TRIAL REGISTRATION NUMBER: NCT01808898.

Are endometrial nerve fibres unique to endometriosis? A prospective case-control study of endometrial biopsy as a diagnostic test for endometriosis in women with pelvic pain.

STUDY QUESTION: Can the presence of endometrial nerve fibres be used as a diagnostic test for endometriosis in women with pelvic pain? SUMMARY ANSWER: Endometrial fine nerve fibres were seen in the endometrium of women both with and without endometriosis, making their detection a poor diagnostic tool for endometriosis. WHAT IS KNOWN ALREADY: Laparoscopy and biopsy are currently the gold standard for making a diagnosis of endometriosis. It has been reported that small density nerve fibres in the functional layer of the endometrium are unique to women with endometriosis and hence nerve fibre detection could function as a less invasive diagnostic test of endometriosis. However, it may be that other painful conditions of the pelvis are also associated with these nerve fibres. We therefore focused this prospective study on women with pelvic pain to examine the efficacy of endometrial nerve fibre detection as a diagnostic test for endometriosis. STUDY DESIGN, SIZE, DURATION: This prospective case-control study conducted between July 2009 and July 2013 included 44 women with pelvic pain undergoing laparoscopic examination for the diagnosis of endometriosis. Immunohistochemical nerve fibre detection in endometrial curettings and biopsies using anti-protein gene product 9.5 was compared with surgical diagnosis. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Paired endometrial biopsies and curettings were taken from patients with (n = 22, study group) and without (n = 22, control group) endometriosis. Tissue was analysed by immunohistochemistry and nerve fibres were counted whenever they were present in the functional layer of the endometrium. MAIN RESULTS AND THE ROLE OF CHANCE: Fine nerve fibres were present in the eutopic endometrium of patients both with and without endometriosis. The presence of nerve fibres in curettings was not effective for either diagnosing or excluding endometriosis; sensitivity and specificity were 31.8 and 45.5% respectively, positive predictive value was 36.8% and negative predictive value was 40.0%. Few endometrial biopsy specimens were found to have nerve fibres present; sensitivity and specificity for endometrial biopsy were 13.6 and 68.2% respectively, positive predictive value was 30.0% and negative predictive value was 44.1%. LIMITATIONS, REASONS FOR CAUTION: This was a relatively small sample size and studies like this are subject to the heterogeneous nature of the patient population and tissue samples, despite our best efforts to regulate these parameters. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate that fine nerve fibres are present in women with and without endometriosis. Future work should focus on the function of endometrial nerves and whether these nerves are involved with the subfertility or pain that endometriosis sufferers experience. Our study does not support the detection of endometrial nerve fibres as a non-invasive diagnostic test of endometriosis in women with pelvic pain.

The importance of pelvic nerve fibers in endometriosis.

Several lines of recent evidence suggest that pelvic innervation is altered in endometriosis-affected women, and there is a strong presumption that nerve fibers demonstrated in eutopic endometrium (of women with endometriosis) and in endometriotic lesions play roles in the generation of chronic pelvic pain. The recent observation of sensory C, sensory A-delta, sympathetic and parasympathetic nerve fibers in the functional layer of endometrium of most women affected by endometriosis, but not demonstrated in most women who do not have endometriosis, was a surprise. Nerve fiber densities were also greatly increased in myometrium of women with endometriosis and in endometriotic lesions compared with normal peritoneum. Chronic pelvic pain is complex, and endometriosis is only one condition which contributes to this pain. The relationship between the presence of certain nerve fibers and the potential for local pain generation requires much future research. This paper reviews current knowledge concerning nerve fibers in endometrium, myometrium and endometriotic lesions, and discusses avenues of research that may improve our knowledge and lead to enriched understanding and management of endometriotic pain symptoms.

Use of hormonal therapy is associated with reduced nerve fiber density in deep infiltrating, rectovaginal endometriosis.

OBJECTIVE: To study the density of nerve fibers in cases of deep infiltrating endometriosis (DIE) of the rectovaginal septum in relation to various clinical factors. DESIGN: A research laboratory-based study. SETTING: A tertiary center together with a research laboratory. METHODS: Archived DIE tissue samples from 45 women operated upon for rectovaginal septum DIE were re-examined histologically, and by immunohistochemistry. MAIN OUTCOME MEASURES: The effect of progestogens or combined oral contraceptives on the density of nerve fibers, and the expression of nerve growth factor (NGF) and its high-affinity receptor (tyrosine kinase receptor A, Trk-A). RESULTS: The use of hormonal therapy was associated with reduced densities of sympathetic, parasympathetic and sensory nerve fibers in DIE lesions. Density of total nerve fibers (with pan-neuronal marker PGP9.5) was significantly lower (p < 0.05) in lesions collected from hormone-treated women (8.6/mm², 4.2-20.8/mm²; median density, from 25th to 75th quartiles) compared with that in lesions from untreated women (24.9/mm², 11.2-34.9/mm²). DIE lesions stained strongly for NGF and its receptor Trk-A. Expression of NGF, but not of Trk-A, was significantly reduced during use of hormonal therapy. CONCLUSIONS: Use of hormonal therapy was associated with significantly reduced nerve fiber density in DIE lesions. This may be an important mechanism of action of hormonal therapy for controlling DIE pain symptoms. The expression of estrogen-regulated NGF and its receptor was only partially suppressed during the use of hormonal therapy, suggesting that local estrogen action is often maintained during conventional hormonal therapy in cases of DIE.

Potential Role of Semaphorin 3A and Its Receptors in Regulating Aberrant Sympathetic Innervation in Peritoneal and Deep Infiltrating Endometriosis.

Previous studies have demonstrated the involvement of nerve repellent factors in regulation of the imbalanced innervation of endometriosis. This prospective study aims to explore the role of Sema 3A in regulating aberrant sympathetic innervation in peritoneal and deep infiltrating endometriosis. Ectopic endometriotic lesion were collected from patients with peritoneal endometriosis (n = 24) and deep infiltrating endometriosis of uterosacral ligament (n = 20) undergoing surgery for endometriosis. Eutopic endometrial samples were collected from patients with endometriosis (n = 22) or without endometriosis (n = 26). Healthy peritoneum (n = 13) from the lateral pelvic wall and healthy uterosacral ligament (n = 13) were obtained from patients who had no surgical and histological proof of endometriosis during hysterectomy for uterine fibroids. Firstly, we studied the immunostaining of Sema 3A, Plexin A1 and NRP-1 in all the tissues described above. Then we studied the nerve fiber density (NFD) of endometriosis-associated (sympathetic) nerve and para-endometriotic (sympathetic) nerve by double immunofluorescence staining. Finally we analyzed the relationship between expression of Sema 3A in stromal cells of endometriotic lesion and the aberrant innervation of endometriosis. Semi-quantitative immunostaining demonstrated that (1) Higher immunostaining of Sema 3A were found in the eutopic endometrial glandular epithelial cells from patients with endometriosis (p = 0.041) than those without endometriosis; (2) Sema 3A immunostaining was higher in glandular epithelial cells of peritoneal endometriosis (P<0.001) and deep infiltrating endometriotic lesions of uterosacral ligament (P = 0.028)compared with glandular epithelial cells of the endometrium from women with endometriosis, while its expression in ectopic stormal cells in both groups were significantly lower than that from eutopic endometrium of women without endometirosis (P<0.001, P<0.001, respectively). NFDs of Anti-TH (+) endometriosis-associated sympathetic nerve of peritoneal endometriosis (p<0.001) and deep endometriosis of uterosacral ligament (p<0.001) were significantly lower than NFDs of para-endometriotic sympathetic nerve. Our results suggest that Sema 3A may contribute to the regulation of aberrant sympathetic innervation in peritoneal and deep infiltrating endometriosis.

The potential role of endometrial nerve fibers in the pathogenesis of pain during endometrial biopsy at office hysteroscopy.

We aimed to evaluate whether nerve fibers are present in the endometrial layer of patients submitted to office hysteroscopy and their potential contribution to the pathogenesis of pain during that procedure. Through a prospective case-control study performed in tertiary centers for women's health, endometrium samples were collected during operative office hysteroscopy from 198 cycling women who previously underwent laparoscopy and/or magnetic resonance imaging investigation for infertility assessment. Samples were classified according to the degree of the pain patients experienced and scored from values ranging from 0 (absence of discomfort/pain) to 10 (intolerable pain) on a 10-cm visual analog scale (VAS). The presence of nerve fiber markers (S100, NSE, SP, VIP, NPY, NKA, NKB, NKR1, NKR2, and NKR3) in the endometrium was also evaluated by morphologic and immunohistochemical analyses. We found that S-100, NSE, NKR1, NK-A, NK-B, VIP, and NPY, were immunolocalized in samples of endometrium, in significantly (P < .01, for all) higher levels in samples collected from patients with VAS score > 5 (group A) than ≤ 5 (group B) and significantly (P < .0001 for all) positively correlated with VAS levels. A statistically significant (P = .018) higher prevalence of endometriosis and/or adenomyosis was depicted in patients of group A than group B. Data from the present study led us to conclude that nerve fibers are expressed at the level of the functional layer of the endometrium and may contribute to pain generation during office hysteroscopy, mainly in women affected by endometriosis and adenomyosis.

Peripheral changes in endometriosis-associated pain.

BACKGROUND: Pain remains the cardinal symptom of endometriosis. However, to date, the underlying mechanisms are still only poorly understood. Increasing evidence points towards a close interaction between peripheral nerves, the peritoneal environment and the central nervous system in pain generation and processing. Recently, studies demonstrating nerve fibres and neurotrophic and angiogenic factors in endometriotic lesions and their vicinity have led to increased interest in peripheral changes in endometriosis-associated pain. This review focuses on the origin and function of these nerves and factors as well as possible peripheral mechanisms that may contribute to the generation and modulation of pain in women with endometriosis. METHODS: We conducted a systematic search using several databases (PubMed, MEDLINE, EMBASE and CINAHL) of publications from January 1977 to October 2013 to evaluate the possible roles of the peripheral nervous system in endometriosis pathophysiology and how it can contribute to endometriosis-associated pain. RESULTS: Endometriotic lesions and peritoneal fluid from women with endometriosis had pronounced neuroangiogenic properties with increased expression of new nerve fibres, a shift in the distribution of sensory and autonomic fibres in some locations, and up-regulation of several neurotrophins. In women suffering from deep infiltrating endometriosis and bowel endometriosis, in which the anatomical distribution of lesions is generally more closely related to pelvic pain symptoms, endometriotic lesions and surrounding tissues present higher nerve fibre densities compared with peritoneal lesions and endometriomas. More data are needed to fully confirm a direct correlation between fibre density in these locations and the amount of perceived pain. A better correlation between the presence of nerve fibres and pain symptoms seems to exist for eutopic endometrium. However, this appears not to be exclusive to endometriosis. No correlation between elevated neurotrophin levels and pain severity appears to exist, suggesting the involvement of other mediators in the modulation of pain. CONCLUSIONS: The increased expression of neurotrophic factors and nerve fibres in endometriotic lesions, eutopic endometrium and the peritoneum imply a role of such peripheral changes in the pathogenesis of endometriosis-associated pain. However, a clear link between these findings and pain in patients with endometriosis has so far not been demonstrated.

[Expression of nerve growth factor produced by ectopic endometrium from patients with adenomyosis and its relationship with pain scales and innervation].

OBJECTIVE: To investigate the expression of nerve growth factor (NGF) in the ectopic endometrium in adenomyosis patients, and explore the relationship between NGF expression and innervation or pain scales. METHODS: From Mar. 2009 to Oct. 2009, 45 adenomyosis patients undergoing hysterectomy in Obstetrics and Gynecology Hospital of Fudan University were enrolled in this study, which were classified into 33 cases in pain group and 12 cases in non-pain group based on symptom. The degree of dysmenoreal, chronic pelvic pain and dyspareunia was evaluated by visual analogue scale, including no pain, mild to moderate pain and severe pain group. In the mean time, 26 patients with leiomyoma or cervical intraepithelial neoplasia III (CIN III) undergoing hysterectomy were defined as control group. Ectopic endometrium from experimental group and eutopic endometrium from control group were collected in the surgery. The expression of NGF was examined by immunohistochemistry. The density of protein gene product (PGP) 9.5 positive nerve fibers was detected by immuno-fluorescence. RESULTS: The NGF level and the density of PGP 9.5 positive nerve fibers in adenomyosis pain group (0.25 ± 0.08, 16 ± 8 )were higher than adenomyosis painless (0.19 ± 0.05, P = 0.007;11 ± 5, P = 0.018) and control group (0.18 ± 0.05, P = 0.000; 9 ± 4, P = 0.000) . The NGF level and the density of PGP9.5 positive nerve fibers in severe dysmenorrheal group (0.29 ± 0.07, 19 ± 10) were higher than mild to moderate dysmenorrheal (0.22 ± 0.07, P = 0.018;13 ± 4, P = 0.035) and painless group (0.18 ± 0.05, P = 0.000;11 ± 5, P = 0.006) of adenomyosis patients. There was no difference of NGF level and the density of PGP 9.5 positive nerve fibers in chronic pelvic pain group and no chronic pelvic pain group of adenomyosis patients, so was dyspareunia group and no dyspareunia group. CONCLUSION: The increased NGF level of adenomyosis nodules and improving innervation might be involved in the mechanism of adenomyosis related pain.

Nerve fiber density in deep nodular endometriotic lesions induced in a baboon experimental model.

OBJECTIVE: To study the occurrence of nerve fibers in deep nodular endometriotic lesions after nodules were induced in baboons and nerve fiber densities measured 6 months after the grafting procedure. DESIGN: Experimental animal study. SETTING: Academic gynecology research unit. ANIMALS: Ten baboons (Papio anubis). INTERVENTION(S): Recovery of induced endometriotic nodules and eutopic endometrium. MAIN OUTCOME MEASURE(S): Protein gene product (PGP) 9.5 and nerve growth factor (NGF) immunohistochemistries were performed to evaluate nerve fiber density and NGF expression in induced endometriotic lesions and eutopic endometrium. RESULT(S): Eutopic (basalis) endometrium, myometrium, and invasive and noninvasive nodular lesions were analyzed separately. The highest nerve fiber densities were observed in normal myometrium and in the basal layer of eutopic endometrium. No significant differences were observed between the two lesion types. However, the NGF staining intensity score was found to be higher in glands of deep invasive lesions than in glands of eutopic baboon endometrium. CONCLUSION(S): This is the first study to show the presence of nerve fibers in eutopic baboon endometrium and induced deep endometriotic nodules. Long-term studies are now warranted to determine if nerves still grow in invasive and noninvasive lesions >6 months after grafting, and to evaluate the role of the lesion environment.

Expression of neuronal markers in the endometrium of women with and those without endometriosis.

STUDY QUESTION: How do the expression patterns of neuronal markers differ in the endometrium of women with and without endometriosis? SUMMARY ANSWER: The neuronal markers, PGP9.5, NGFp75 and VR1, are expressed in the endometrium at levels that do not differ between women with and without endometriosis. WHAT IS KNOWN ALREADY: Aberrant neuronal growth within the uterus may contribute to abnormal fertility and uterine dysfunction. However, controversy still exists as to whether aberrant innervation in the endometrium is associated with gynaecological pathology such as endometriosis. This may reflect the use of subjective methods such as histology to assess the innervation of the endometrium. We, therefore, employed a quantitative method, western blotting, to study markers of endometrial innervation in the presence and absence of endometriosis. STUDY DESIGN, SIZE, DURATION: This study included 45 women undergoing laparoscopic examination for the diagnosis of endometriosis. Endometrial samples were analysed by western blot for the expression of neuronal and neurotrophic markers, PGP9.5, VR1 and NGFp75. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Endometrial pipelle biopsies were obtained from patients with (n = 20, study group) and without (n = 25, control group) endometriosis. Tissue was analysed by immunohistochemistry and western blot analysis for the expression of pan-neuronal marker, PGP9.5, sensory nociceptive marker, TPVR1, and low-affinity neurotrophic growth factor receptor, NGFRp75. MAIN RESULTS AND THE ROLE OF CHANCE: PGP9.5, NGFp75 and VR1 were expressed in the endometrium of women, independent of the presence of endometriosis. Furthermore, the expression level of PGP9.5, VR1 and NGFp75 did not alter between the two cohorts of women. LIMITATIONS, REASONS FOR CAUTION: Studies of this nature are subject to the heterogeneous nature of patient population and tissue samples despite attempts to standardize these parameters. Hence, further studies using similar methodology will be required to confirm our results. WIDER IMPLICATIONS OF THE FINDINGS: Our results highlight that sensory neuronal markers are present in women with and without endometriosis. Future work will assess what the targets of the endometrial nerves are and investigate their function, their impact on endometrial biology and, in particular, whether aberrant neuronal function, rather than the mere presence of neuronal function, could be the root cause of subfertility and/or pain affecting many endometriosis sufferers. Our results do not, however, confirm the previous paradigm of increased innervation in the endometrium of women with endometriosis, nor the use of nerve cell detection from pipelle biopsies to diagnose endometriosis.

Combination of non-invasive and semi-invasive tests for diagnosis of minimal to mild endometriosis.

OBJECTIVES: Non-surgical diagnostic approach for endometriosis would be of great gain to both physicians and patients. The aim of this study was to evaluate the diagnostic value of serum measurement of IL-6 combined with the presence of nerve fibres in the functional layer of endometrium for diagnosis of minimal-mild endometriosis. METHODS: In this prospective study 114 women who underwent laparoscopy for infertility and/or pelvic pain were divided into two groups: control cases (40 cases) with no pathologic findings; and endometriosis patients (74 cases) [subdivided into stages 1-2 or minimal-mild (MM) and stages 3-4 or moderate-severe cases]. Blood was drawn one day before laparoscopy and stored for subsequent analysis of IL-6. Endometrial biopsy was obtained prior to laparoscopy and Immunohistochemistry was performed using the pan-neuronal marker protein gene product 9.5(PGP9.5). Then laparoscopic diagnosis of endometriosis confirmed by histopathology was done. RESULTS: Serum IL-6 with a threshold of 15.4 pg/ml was found to be able to diagnose MM endometriosis with 89.5 % sensitivity and 82.5 % specificity, but sensitivity and specificity of presence of nerve fibres in the functional layer of endometrium were 92 % and 80 % respectively. When two diagnostic modalities were combined the sensitivity and specificity were raised to 100 and 92.5 % respectively. CONCLUSIONS: Combination of both serum IL-6 and presence of nerve fibres in the endometrium is more reliable method for diagnosis of MM endometriosis than in single test.

Is the detection of endometrial nerve fibers useful in the diagnosis of endometriosis?

Laparoscopy is currently considered to be the gold standard investigation in patients suspected to have endometriosis, but this is an invasive and relatively costly procedure and there may be significant delays in diagnosis. As the eutopic endometrium is recognized to be abnormal in patients with endometriosis, it has been suggested that endometrial sampling could provide an indirect diagnostic approach. In particular, recent reports have suggested that the presence of nerve fibers within the endometrial functional layer could represent a specific and sensitive marker of concurrent peritoneal endometriosis. However, such studies have been performed in select patient groups and using novel sampling and analytic techniques that are not used routinely in clinical pathology laboratories. The present study was performed upon conventional endometrial biopsies from 68 patients who underwent laparoscopy for suspected endometriosis. The biopsies were stained immunohistochemically for the neural marker PGP 9.5 and examined in a blinded manner. Endometrial functional layer nerve fibers were identified in 15 (22%) biopsies overall including 9/47 (19%) cases with histologically confirmed peritoneal endometriosis and 6/21 (29% cases) without endometriosis. There was no correlation between the presence of functional layer nerve fibers and the presenting symptoms, endometrial histology, or current hormonal therapy. In our experience, endometrial functional layer nerve fibers assessment performed using standard immunohistochemical techniques on routine biopsy specimens proved neither sensitive nor specific for the diagnosis of endometriosis. Pathologists and gynecologists considering this diagnostic approach should carefully consider the methodological factors that may influence its reliability.