The importance of pseudocapsule preservation during hysteroscopic myomectomy.

Minimally invasive techniques for myomectomy are based on the rationale of preserving the myometrial integrity, in order to spare muscular and fibro-neurovascular myometrial fibers and ensure complete and bloodless myoma removal. Post-operative myometrial vascularization is crucial in injured muscle regeneration. The post-surgical myometrial healing is needful for uterine reproductive function. Neurotransmitters and neurofibers were analyzed in the myoma pseudocapsule surrounding fibroid. They activate signaling molecule synthesis and release which, in turn, promote cell activation and induce muscle regeneration and growth. Pseudocapsule damage during myomectomy may lead to a reduction of neuropeptides and neurofibers at the hysterotomic site, to a poor physiological myometrial healing, with more fibrosis due to hypoxia, ischemia and necrosis. These pathophysiological events cause deficit in myometrial neurotransmission, muscular impulse and contractility, with ultimately impaired uterine muscle function during pregnancy, labor and delivery. Hence, during myomectomy, all manipulations should be performed as precisely and bloodlessly as possible, avoiding extensive, high wattage diathermocoagulation or excessive tissue manipulation or muscular trauma. Any iatrogenic pseudocapsule damage may alter neurotransmitter function during successive myometrial healing, impacting negatively on uterine repair and on eventual pregnancies. Hence the reasoned myomectomy on a biological basis, the "intracapsular myomectomy", satisfied these surgical and physiological requirements. It was described precisely and firstly by the hysteroscopy, with the image magnification of the preservation of the myoma pseudocapsule. The "intracapsular hysteroscopic myomectomy" demonstrated the safe and effective removal of submucous myomas with intramural development. It allowed to completely remove the myoma in one or two surgical steps, saving the pseudocapsule and the surrounding healthy myometrium. The respect of the myometrium and the reduced thermal injury, a part the excellent outcomes in terms of surgical complications prevention, post-surgical fibrosis and intrauterine synechiae reduction, highlighted the physiological development of a successive pregnancy, without any myometrial complications during pregnancy, labor and delivery.

Postablation neuroma of the myometrium-a report of 5 cases.

When hysterectomy is performed for chronic pelvic pain, routine pathology examination often provides no explanation. However, analysis of small uterine nerves using immunostains may help to address this deficiency. Small uterine nerves tend to be sparse or absent in wide areas of normal myometrium. Some studies of uterine nerves have suggested that endometriosis, adenomyosis, and fibroids are not inherently painful, with increased small nerves in the inner uterine wall associated with the history of pelvic pain. Although such areas may appear normal on hematoxylin and eosin (H&E), we have found a subtle inner wall lesion termed inner myometrial elastosis, best detected with trichrome or elastic stains, which may be a reaction to microscopic tears of inner myometrium. Such tears may induce increased inner wall innervation via the generation of nerve growth factor in granulation tissue. In the course of studying uterine nerves with immunostains, we found 5 cases with florid nerve proliferation, after deep endometrial ablation for abnormal uterine bleeding led to increased pelvic pain. We suggest that immunostains for postablation neuromas should be done in hysterectomies when pelvic pain increases after endometrial ablation. This may offer gynecologists and their patients an objective finding with a rational, scientific explanation for the pelvic pain.

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.

Estrogen-induced collagen reorientation correlates with sympathetic denervation of the rat myometrium.

Estrogen inhibits the growth and causes the degeneration (pruning) of sympathetic nerves supplying the rat myometrium. Previous cryoculture studies evidenced that substrate-bound signals contribute to diminish the ability of the estrogenized myometrium to support sympathetic nerve growth. Using electron microscopy, here we examined neurite-substrate interactions in myometrial cryocultures, observing that neurites grew associated to collagen fibrils present in the surface of the underlying cryosection. In addition, we assessed quantitatively the effects of estrogen on myometrial collagen organization in situ, using ovariectomized rats treated with estrogen and immature females undergoing puberty. Under low estrogen levels, most collagen fibrils were oriented in parallel to the muscle long axis (83% and 85%, respectively). Following estrogen treatment, 89% of fibrils was oriented perpendicularly to the muscle main axis; while after puberty, 57% of fibrils acquired this orientation. Immunohistochemistry combined with histology revealed that the vast majority of fine sympathetic nerve fibers supplying the myometrium courses within the areas where collagen realignment was observed. Finally, to assess whether depending on their orientation collagen fibrils can promote or inhibit neurite outgrowth, we employed cryocultures, now using as substrate tissue sections of rat-tail tendon. We observed that neurites grew extensively in the direction of the parallel-aligned collagen fibrils in the tendon main axis but were inhibited to grow perpendicularly to this axis. Collectively, these findings support the hypothesis that collagen reorientation may be one of the factors contributing to diminish the neuritogenic capacity of the estrogen-primed myometrial substrate.

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.

Comparative study of nerve fiber density between adenomyosis patients with moderate to severe pain and mild pain.

BACKGROUND: Since some retrospective studies have given inconsistent findings about innervation in adenomyosis, its role in the pain mechanism is still inconclusive. OBJECTIVE: Define the nerve fiber density in adenomyotic tissue as it correlated to pain symptoms. MATERIAL AND METHOD: A cross-sectional study was performed in twenty-five uterine samples from reproductive age women with adenomyosis who underwent either laparotomy or laparoscopic surgery. The nerve fiber density from hysterectomized specimens as measured by immunohistochemistry staining for Protein gene product (PGP) 9.5 and Neurofilament (NF) were compared with the level of pain in the patients as defined by a visual analogue scale and a verbal rating scale. RESULTS: Nerve fibers as detected by PGP9.5 and NF staining in the myometrium were significantly increased in the group of women with adenomyosis experiencing moderate and severe pain as compared to the group experiencing less pain (4 (0, 7) vs. 1.55 (0, 7)/mm2, p-value <0.001, and 6 (3, 10) vs. 0 (0, 4)/mm2, p-value <0.001 respectively). At both phases of the menstrual cycle, the densities of nerve fibers stained with PGP9.5 and NF showed no significant difference. CONCLUSION: These results suggested that the increased of nerve fibers shown in the more severe pain group might play a role in the pathogenesis or symptoms of adenomyosis.

Immunohistochemical localization of nerve fibers in the pseudocapsule of fibroids.

The pseudocapsule surrounding fibroids consists of compressed myometrium containing nerves and blood vessels that continue into adjacent myometrium. Oxytocin (OXT) is thought to affect wound healing after myomectomy. We determined the presence of OXT and protein gene product 9.5 (PGP9.5) immunoreactive nerve fibers in pseudocapsule compared to adjacent myometrium. Samples (N=106) of pseudocapsule and adjacent myometrium were collected from 57 women with uterine fibroids undergoing myomectomy, and stained with anti-OXT and PGP 9.5 antibodies to demonstrate the presence of nerve fibers. Nerve fibers in the pseudocapsule stained positively with OXT (89/106, 84.0%) and PGP 9.5 (94/106, 88.7%). The densities of nerve fibers staining with PGP 9.5 and OXT in the pseudocapsule were highest in the isthmus (23.68±22.45/mm2 and 43.35±40.74/mm2, respectively). There were no significant differences in the density of nerve fibers, stained with either OXT or PGP 9.5, between the pseudocapsule, and adjacent normal myometrium regardless of the fibroid location in the uterus (P>0.05). These results suggest that the pseudocapsule should avoid to be damaged during the myomectomy procedure.

[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.

NT, NPY and PGP 9.5 presence in myomeytrium and in fibroid pseudocapsule and their possible impact on muscular physiology.

The uterine myoma pseudocapsule is a neurovascular bundle surrounding fibroid, containing neuropeptides, probably involved in uterine scar healing. We studied neurotensin (NT), neuropeptide tyrosine (NPY), and protein gene product 9.5 (PGP 9.5) nerve fibres in the pseudocapsule neurovascular bundle of intramural uterine fibroids on 67 no pregnant women by intracapsular myomectomy sparing the neurovascular bundle, sampling full thickness specimens of the pseudocapsule of uterine fibroids (PUF) and normal myometrium (NM) obtained from the fundus uteri (FU) and the uterine body (UB). The samples were sent for histological and immunofluorescent analyses and compared by morphometrical quantification. The Conventional Unit (C.U.) difference of NT, NPY, and PGP 9.5 nerve fibres was statistically analyzed. Our results showed that NT, NPY, and PGP 9.5 neurofibers are almost equally present in PUF as in NM of a no pregnant uterus. As all of these neuropeptides are present in the uterine muscle and can affect muscle contractility, uterine peristalsis and muscular healing. A myomectomy respecting the pseudocapsule neurofibers should facilitate smooth muscle scarring and promote restoration of normal uterine peristalsis with a possible positive influence on fertility.

Surgical management of neurovascular bundle in uterine fibroid pseudocapsule.

The uterine fibroid pseudocapsule is a fibro-neurovascular structure surrounding a leiomyoma, separating it from normal peripheral myometrium. The fibroid pseudocapsule is composed of a neurovascular network rich in neurofibers similar to the neurovascular bundle surrounding a prostate. The nerve-sparing radical prostatectomy has several intriguing parallels to myomectomy. It may serve either as a useful model in modern fibroid surgical removal, or it may accelerate our understanding of the role of the fibrovascular bundle and neurotransmitters in the healing and restoration of reproductive potential after intracapsular myomectomy. Surgical innovations, such as laparoscopic or robotic myomectomy applied to the intracapsular technique with magnification of the fibroid pseudocapsule surrounding a leiomyoma, originated from the radical prostatectomy method that highlighted a careful dissection of the neurovascular bundle to preserve sexual functioning after prostatectomy. Gentle uterine leiomyoma detachment from the pseudocapsule neurovascular bundle has allowed a reduction in uterine bleeding and uterine musculature trauma with sparing of the pseudocapsule neuropeptide fibers. This technique has had a favorable impact on functionality in reproduction and has improved fertility outcomes. Further research should determine the role of the myoma pseudocapsule neurovascular bundle in the formation, growth, and pathophysiological consequences of fibroids, including pain, infertility, and reproductive outcomes.

Uterine distension differentially affects remodelling and distensibility of the uterine vasculature in non-pregnant rats.

During pregnancy the mammalian uterine circulation undergoes significant expansive remodelling necessary for normal pregnancy outcome. The underlying mechanisms are poorly defined. The goal of this study was to test the hypothesis that myometrial stretch actively stimulates uterine vascular remodelling by developing a new surgical approach to induce unilateral uterine distension in non-pregnant rats. Three weeks after surgery, which consisted of an infusion of medical-grade silicone into the uterine lumen, main and mesometrial uterine artery and vein length, diameter and distensibility were recorded. Radial artery diameter, distensibility and vascular smooth muscle mitotic rate (Ki67 staining) were also measured. Unilateral uterine distension resulted in significant increases in the length of main uterine artery and vein and mesometrial segments but had no effect on vessel diameter or distensibility. In contrast, there were significant increases in the diameter of the radial arteries associated with the distended uterus. These changes were accompanied by reduced arterial distensibility and increased vascular muscle hyperplasia. In summary, this is the first report to show that myometrial stretch is a sufficient stimulus to induce significant remodelling of uterine vessels in non-pregnant rats. Moreover, the results indicate differential regulation of these growth processes as a function of vessel size and type.

Estrogen up-regulation of semaphorin 3F correlates with sympathetic denervation of the rat uterus.

Current evidence indicates that rises in systemic levels of estrogen create in the uterus an inhibitory environment for sympathetic nerves. However, molecular insights of these changes are far from complete. We evaluated if semaphorin 3F mRNA, a sympathetic nerve repellent, was produced by the rat uterus and if its expression was modulated by estrogen. We also analyzed whether uterine nerves express the semaphorin 3F binding receptor, neuropilin-2. Uterine levels of semaphorin 3F mRNA were measured using real time reverse transcriptase-polymerase chain reaction in prepubertal rat controls and following chronic estrogen treatment. Localization of semaphorin 3F transcripts was determined by in situ hybridization and the expression of neuropilin-2 was assessed by immunohistochemistry. These studies showed that: (1) chronic estrogen treatment led to a 5-fold induction of semaphorin 3F mRNA in the immature uterus; (2) estrogen provoked a tissue-specific induction of semaphorin 3F which was particularly localized in the connective tissue that borders muscle bundles and surrounds intrauterine blood vessels; (3) two major cell-types were recognized in the areas where transcripts were concentrated, fibroblast-like cells and infiltrating eosinophil leukocytes; and (4) some delicate nerve terminal profiles present in the estrogenized uterus were immunoreactive for neuropilin-2. Temporal and spatial expression patterns of semaphorin 3F/neuropilin-2 are consistent with a possible role of this guidance cue in the remodeling of uterine sympathetic innervation by estrogen. Though correlative in its nature, these data support a model whereby semaphorin 3F, in combination with other inhibitory molecules, converts the estrogenized myometrium to an inhospitable environment for sympathetic nerves.

Increased nerve fibers in placental bed myometrium in women with preeclampsia.

Narrowing of the uterine spiral arterioles below the deciduomyometrial junction is 1 of the key pathophysiological changes in women with preeclampsia. The contribution of pelvic autonomic nerves to decidualization and impaired placentation in preeclampsia is not clear. Placental bed biopsies were obtained from 10 women with preeclampsia and 23 nornotensive women at caesarean section. We stained them with anti-S100 and CD34 antibodies to detect the presence of nerve fibers and blood vessels, respectively. We detected S100-immunoactive nerve fibers in the myometrium but not in the decidua in both groups of women. S100-immunoactive nerve fiber density in the placental bed myometrium was significantly increased in women with preeclampsia compared to normotensive women. There was no clear relationship between the densities of nerve fibers and CD34-positive blood vessels in these biopsies. These results suggest increased nerve fibers in the placental bed myometrium may play a role in the pathogenesis of the preeclampsia.

Mysteries of endometriosis pain: Chien-Tien Hsu Memorial Lecture 2009.

The more that one looks at the condition endometriosis, the more one realises that it is a unique and complex condition exhibiting a bizarre range of deviations from normal endometrial and myometrial physiology, and presenting with a challenging range of pain-related symptoms. The changing nature of the pain is not well defined, and the molecular mechanisms leading to pain generation are far from clear. Recent research has begun to reveal some of these links between expression of unusual molecules in the eutopic endometrium and ectopic lesions, microanatomical changes in the pelvic nervous system, neuronal dysfunction and the later development of neuropathic pain. A better understanding of these mechanisms will undoubtedly lead to improved use of current medical and surgical treatments, and to the development of novel treatments in the future.

Innervation of endometrium and myometrium in women with painful adenomyosis and uterine fibroids.

OBJECTIVE: To determine whether nerve fibers can be detected in the endometrium and myometrium in women with painful uterine fibroids and adenomyosis. DESIGN: A retrospective immunohistochemical study. SETTING: An academic training hospital. PATIENT(S): Thirty-seven women with uterine fibroids and 29 women with adenomyosis. INTERVENTION(S): Histologic sections of contiguous endometrial and myometrial tissues were stained immunohistochemically using the highly specific polyclonal rabbit antiprotein gene product 9.5 (PGP9.5) and monoclonal mouse antineurofilament protein (NF). MAIN OUTCOME MEASURE(S): Results were determined through immunohistochemical staining using PGP9.5 and NF. RESULT(S): We detected PGP9.5-immunoactive nerve fibers in the functional layer of the endometrium in women with pain but not in women without pain. PGP9.5-immunoactive nerve fiber density in the basal layer of the endometrium or myometrium significantly increased in women with pain, however, PGP9.5-immunoactive nerve fiber density had no statistical differences between women with adenomyosis and uterine fibroids. We identified NF-immunoactive nerve fibers in the basal layer of the endometrium and myometrium in women with adenomyosis and uterine fibroids, but found no significant differences. CONCLUSION(S): These results suggest that PGP9.5-immunoactive nerve fibers appearing in the endometrium and myometrium of women with painful adenomyosis and uterine fibroids may play a role in pain generation in these two disorders.

Endometriosis-associated nerve fibers and pain.

The assessment and diagnosis of endometriosis remain elusive targets. Patient and medical-related factors add to delays in the detection and treatment. Recently, investigators have revealed specific nerve fibers present in endometriotic tissue, with existing parallels between density and pain severity. The aim of this review is to compile a comprehensive review of existing literature on endometriosis-related nerve fiber detection, and the effects of medical therapy on these neural fibers. We performed a systematic literature-based review using Medline and PubMed of nerve fibers detected in eutopic endometrium, endometriotic lesions, and the peritoneum. Various arrangements of significant medical terms and phrases consisting of endometriosis, pelvic pain, nerve fiber detection/density in endometriosis, and diagnoses methodology, including treatment and detection were applied in the search. Subsequent references used were cross-matched with existing sources to compile all additional similar reports. Similar nerve fibers were detected within lesions, endometrium, and myometrium, though at varying degrees of density. Hormonal therapy is widely used to treat endometriosis and was shown to be related to a reduction in fiber density. A direct result of specific nerve fiber detection within eutopic endometrial layers points to the use of a minimally invasive endometrial biopsy technique in reducing delay in diagnosis and subsequent possible preservation of fertility.

[Distribution of nerve fibers in endometrium and its clinical significance in adenomyosis].

OBJECTIVE: To investigate nerve fibers distribution in endometrium of adenomyosis and their relationship with dysmenorrhea. METHODS: Endometrial tissue was sampled from 74 hysterectomy specimens including 32 cases with adenomyosis and 42 cases with uterine fibroids. Two-step Envision immunohistochemical staining was used to detect distribution of nerve fibers in endometrium. Highly specific polyclonal rabbit anti-protein gene product 9.5 (PGP9.5) and monoclonal mouse anti-neurofilament protein (NF) were used to demonstrate both myelinated and unmyelinated nerve fibers in endometrium in women with adenomyosis and uterine fibroids. RESULTS: The positive rate of PGP9.5 immunoreactive nerve fibers in the functional layer of endometrium of pain patients were with 64% (14/22) in adenomyosis and 67% (10/15) in uterine fibroids. And their density were 0.6 (0 - 9.4)/mm(2) and 0.6 (0 - 6.0)/mm(2) without reaching statistical difference (P > 0.05). No expression of NF could be detected in the functional layer of endometrium of adenomyosis and uterine fibroids. There were no PGP9.5 immunoreactive nerve fibers in the functional layer of endometrium in non-pain women with adenomyosis and uterine fibroids. Moreover, No NF immunoreactive nerve fibers in the functional layer of endometrium were shown in non-pain patients with adenomyosis and uterine fibroids. PGP9.5 immunoreactive nerve fibers and the nerve density in the basal layer of endometrium were 64% (14/22), 1.1 (0 - 12.0)/mm(2) in pain adenomyosis and 50% (5/10), 0.6 (0 - 3.0)/mm(2) in non-pain adenomyosis. NF immunoreactive nerve fibers and the density in the basal layer of endometrium were 23% (5/22), (0 - 0.6)/mm(2) in pain adenomyosis and 20% (2/10), (0 - 1.0)/mm(2) in non-pain adenomyosis. PGP9.5 immunoreactive nerve fibers and the nerve density in the basal layer of endometrium were 80% (12/15) and 1.6 (0 - 10.0)/mm(2) in pain fibroids and 44% (12/27), 0 (0 - 5.0)/mm(2) in non-pain fibroids. NF immunoreactive nerve fibers and the nerve density in the basal layer of endometrium were 40% (6/15), 0 (0 - 0.4)/mm(2) in pain fibroids and 15% (4/27), 0 (0 - 1.0)/mm(2) in non-pain fibroids. There was no statistical different PGP9.5 and NF immunoreactive nerve fibers distribution in basal layer of endometrium between pain adenomyosis and pain fibroids or between non-pain adenomyosis and non-pain fibroids (all P > 0.05). However, PGP9.5 immunoreactive nerve fibers density in basal layer of endometrium was higher in pain adenomyosis and fibroids when compared with non-pain adenomyosis and fibroids (P < 0.05). CONCLUSIONS: PGP9.5 immunoreactive nerve fibers might confer the occurrence of pelvic pain, however, NF immunoreactive nerve fibers may not involved in the pathogenesis of pain.

Neurotrimin is an estrogen-regulated determinant of peripheral sympathetic innervation.

Mechanisms underlying axon degeneration in peripheral neuropathies and during normal remodeling are poorly understood. Because estrogen induces widespread sympathetic axon degeneration in female reproductive tract smooth muscle, we surveyed estrogen-regulated genes in rat myometrium. Microarray analysis revealed that the neural cell adhesion protein neurotrimin (Ntm) was markedly up-regulated 6 hr and down-regulated 24 hr after injection of 17beta-estradiol, and real time RT-PCR confirmed this pattern of expression. Protein analysis by Western blotting showed that uterine Ntm protein is also up-regulated in vivo 6-24 hr following estrogen injection and that Ntm protein is increased selectively in the myometrium during the high-estrogen phase of the estrous cycle. Cultured myometrial smooth muscle cells display perinuclear accumulations of Ntm protein, and 17beta-estradiol also increases intracellular levels of Ntm and its secretion into the culture medium. To determine if neurotrimin is required for estrogen-induced sympathetic pruning, sympathetic neurons were cocultured with uterine smooth muscle cells transfected with siRNA directed against Ntm. Although estrogen inhibited neurite outgrowth in nontransfected cocultures, estrogen's ability to reduce sympathetic outgrowth was impaired substantially following Ntm down-regulation. This supports a role for neurotrimin in mediating estrogen-induced sympathetic pruning in some peripheral targets. Together with earlier studies, these findings support the idea that physiological sympathetic axon degeneration is a multifactorial process requiring dynamic regulation of multiple repellant proteins.

Effects of hormonal treatment on nerve fibers in endometrium and myometrium in women with endometriosis.

OBJECTIVE: To investigate how hormonal treatment can change nerve fiber density and to identify types of nerve fibers in endometrium and myometrium in women with endometriosis. DESIGN: Laboratory study using human tissue. SETTING: University-based laboratory. PATIENT(S): Hormonally treated and untreated women with endometriosis undergoing hysterectomy or curettage. INTERVENTION(S): Endometrial and myometrial tissues were prepared from women with hormonally treated endometriosis and women with untreated endometriosis. MAIN OUTCOME MEASURE(S): Types and density of nerve fibers in endometrium and myometrium in women with hormonally treated and untreated endometriosis were determined immunohistochemically. RESULT(S): The nerve fiber density (mean density +/- SD per square millimeter) in the functional and the basal layers of endometrium (0.2 +/- 0.7/mm(2) and 0.9 +/- 1.3/mm(2), respectively) and myometrium (1.5 +/- 0.8/mm(2)) from women with hormonally treated endometriosis was much lower than that of endometrium (functional layer: 11 +/- 5/mm(2), basal layer: 18 +/- 8/mm(2), respectively) and myometrium (3 +/- 1/mm(2)) from women with untreated endometriosis. Nerve growth factor and nerve growth factor receptor p75 expression was also significantly reduced in women with hormonally treated endometriosis compared with women with untreated endometriosis. CONCLUSION(S): Hormonal treatment significantly reduced nerve fiber density in endometrium and myometrium in women with endometriosis.

Different types of small nerve fibers in eutopic endometrium and myometrium in women with endometriosis.

OBJECTIVE: To investigate types of nerve fibers in endometrium and myometrium in women with endometriosis. DESIGN: Laboratory study using human tissue. SETTING: University-based laboratory. PATIENT(S): Women with and without endometriosis undergoing hysterectomy. INTERVENTION(S): Histologic sections of contiguous endometrial and myometrial tissues were prepared from hysterectomies performed on women with and without endometriosis. MAIN OUTCOME MEASURE(S): Types and density of nerve fibers in endometrium and myometrium in women with and without endometriosis were determined using a series of specific markers for neuronal structure and function: PGP9.5, NF, SP, CGRP, TH, VAChT, VIP, and NPY. RESULT(S): Nerve fibers stained with PGP9.5 and NF in endometrium and myometrium were significantly increased in women with endometriosis compared with women without endometriosis. Nerve fibers in the functional layer of endometrium in women with endometriosis were likely to be sensory C, a mixture of sensory A delta, sensory C, and adrenergic fibers in the basal layer of the endometrium, a mixture of sensory A delta, sensory C, adrenergic and cholinergic fibers in the myometrium. CONCLUSION(S): Increased nerve fiber density in endometrium and myometrium, and sensory C fibers and adrenergic nerve fibers in the endometrium in women with endometriosis may play an important role in the mechanisms of pain generation in this condition.