Natsuko Tokushige

Rich innervation of deep infiltrating endometriosis

BACKGROUND Deep infiltrating endometriosis (DIE) is a specific type of endometriosis, which can be associated with more severe pelvic pain than other forms of endometriotic lesions. However, the mechanisms by which pain is generated are not well understood. METHODS DIE (n = 31) and peritoneal endometriotic (n = 40) lesions were sectioned and stained immunohistochemically with antibodies against protein gene product 9.5, neurofilament, nerve growth factor (NGF), NGF receptors tyrosine kinase receptor-A (Trk-A) and p75, substance P, calcitonin gene-related peptide, vesicular acetylcholine transporter, neuropeptide Y, vasoactive intestinal peptide and tyrosine hydroxylase to demonstrate myelinated, unmyelinated, sensory and autonomic nerve fibres. RESULTS There were significantly more nerve fibres in DIE (67.6 +/- 65.1/mm(2)) than in peritoneal endometriotic lesions (16.3 +/- 10.0/mm(2)) (P < 0.01). DIE was innervated abundantly by sensory Adelta, sensory C, cholinergic and adrenergic nerve fibres; NGF, Trk-A and p75 were strongly expressed in endometriotic glands and stroma of DIE. CONCLUSIONS The rich innervation of DIE may help to explain why patients with this type of lesion have severe pelvic pain.

Diagnosis of endometriosis by detection of nerve fibres in an endometrial biopsy: a double blind study

BACKGROUND Diagnosis of endometriosis currently requires a laparoscopy and this need probably contributes to the considerable average delay in diagnosis. We have reported the presence of nerve fibres in the functional layer of endometrium in women with endometriosis, which could be used as a diagnostic test. Our aim was to assess efficacy of nerve fibre detection in endometrial biopsy for making a diagnosis of endometriosis in a double-blind comparison with expert diagnostic laparoscopy. METHODS Endometrial biopsies, with immunohistochemical nerve fibre detection using protein gene product 9.5 as marker, taken from 99 consecutive women presenting with pelvic pain and/or infertility undergoing diagnostic laparoscopy by experienced gynaecologic laparoscopists, were compared with surgical diagnosis. RESULTS In women with laparoscopic diagnosis of endometriosis (n = 64) the mean nerve fibre density in the functional layer of the endometrial biopsy was 2.7 nerve fibres per mm(2) (+/-3.5 SD). Only one woman with endometriosis had no detectable nerve fibres. Six women had endometrial nerve fibres but no active endometriosis seen at laparoscopy. The specificity and sensitivity were 83 and 98%, respectively, positive predictive value was 91% and negative predictive value was 96%. Nerve fibre density did not differ between different menstrual cycle phases. Women with endometriosis and pain symptoms had significantly higher nerve fibre density in comparison with women with infertility but no pain (2.3 and 0.8 nerve fibre per mm(2), respectively, P = 0.005). CONCLUSIONS Endometrial biopsy, with detection of nerve fibres, provided a reliability of diagnosis of endometriosis which is close to the accuracy of laparoscopic assessment by experienced gynaecological laparoscopists. This study was registered with the Australian Clinical Trials Registry (ACTR) 00082242 (registered: 12/12/2007). The study was approved by the Ethics Review Committee (RPAH Zone) of the Sydney South West Area Health Service (Protocol number X05-0345) and The University of Sydney Human Research Ethics Committee (Ref. No. 10761) and all women gave their informed consent for participation.

The role of Foxp3+ regulatory T-cells in endometriosis: a potential controlling mechanism for a complex, chronic immunological condition

BACKGROUND Endometriosis is an inflammatory condition, associated with highly dysregulated immune response at both uterine and peritoneal levels. Surprisingly, Foxp3+ regulatory T-cells, which control and suppress a range of immune responses, have not previously been investigated in endometriosis. METHODS AND RESULTS Immunohistochemical analysis of Foxp3+ cells in 127 eutopic endometrial samples and 59 ectopic peritoneal lesions revealed that these immune cell populations are highly disturbed in women suffering from endometriosis. We showed that Foxp3+ cells remained highly up-regulated during the secretory phase of the menstrual cycle, while at this time their expression is significantly down-regulated in women without endometriosis (P < 0.001). Foxp3+ cells were detected in the stroma of 18 of the 59 peritoneal endometriotic lesions, but not in the surrounding or control peritoneal tissue. CONCLUSIONS We propose that in eutopic endometrium in women with endometriosis Foxp3+ cells decrease the ability of newly recruited immune cell populations to effectively recognize and target endometrial antigens shed during menstruation, allowing their survival and ability to implant in ectopic sites. At these ectopic sites, variable expression of Foxp3+ cells within some peritoneal endometriotic lesions is likely to be linked to the characteristics and stage of individual lesion development and be playing key roles in pathogenesis and progression of this unique condition.

Macrophage expression in endometrium of women with and without endometriosis

BACKGROUND Endometriosis is an inflammatory condition, characterized by the presence of endometrial-like tissue outside the uterus. The immune system provides a defence mechanism in response to foreign pathogens, and macrophages play important roles in this response. Activation of macrophages has been reported in peritoneal fluid and ectopic endometriotic lesions; however, controversy exists regarding the composition and function of macrophage populations in eutopic endometrium of women with and without endometriosis. This study aimed to quantify macrophages in eutopic endometrium of women with and without endometriosis, during the early, mid and late proliferative and menstrual phases of the cycle. METHODS Paraffin-embedded endometrial curettage blocks were selected from pathology archives. Seventy-six specimens from women with and without endometriosis were analysed using standard immunohistochemical techniques with CD68-PGM1 (phosphoglucomutase 1) clone antibody. Macrophages were counted according to their morphology over several fields of view. RESULTS A significant increase in macrophage cell numbers was shown in eutopic endometrium in women with endometriosis (mean +/- SD, 182.7 +/- 72.9/mm(2)) during all stages of the proliferative phase compared with normal controls (101.6 +/- 53.4/mm(2); P < 0.001). Significant increase in macrophage density occurred in the control group during the mid-menstrual phase, Days 3-4 (P < 0.01), which was not observed in women with endometriosis. CONCLUSIONS This study further supports an association between immune changes in eutopic endometrium and presence of endometriosis. However, it remains uncertain if eutopic immune changes are primary or secondary occurrences.

Dendritic cell populations in the eutopic and ectopic endometrium of women with endometriosis

BACKGROUND Immune alterations may be involved in the pathogenesis and progression of endometriosis. Dendritic cells (DCs) are potent antigen presenting cells that are highly involved in the initiation of the immune response. The aim of this study was to investigate DC populations in the eutopic and ectopic endometrium of women with endometriosis compared with controls. METHODS Hysterectomy samples were obtained from premenopausal women with (n = 33) and without (n = 28) endometriosis. In addition, paired peritoneal endometriotic lesions and uterine curettings were collected from 32 women with endometriosis. Specimen sections were stained immunohistochemically using antibodies for monoclonal mouse antibodies directed against human CD1a and CD83, which are specific for immature and mature DCs, respectively. RESULTS The mean density of endometrial CD1a+ DCs in the basal layer was significantly increased in women with endometriosis compared with controls during the proliferative phase only (P = 0.001). There was a highly significant decrease in the density of endometrial CD83+ DCs in women with endometriosis compared with controls in both layers of the endometrium across all phases of the menstrual cycle (P = 0.001). The density of CD1a+ DCs was significantly increased in peritoneal endometriotic lesions (P = 0.003) and in the surrounding peritoneum (P = 0.001) compared with paired uterine curettings and peritoneum distant from the lesion. CONCLUSIONS Both CD1a+ and CD83+ DC populations were altered in the eutopic and ectopic endometrium of women with endometriosis compared with controls. Alterations in these cells, which play a crucial role in the coordination of the immune response, may be involved in pain generation and the pathogenesis of endometriosis.

Macrophages and nerve fibres in peritoneal endometriosis

BACKGROUND Endometriosis is considered to be an inflammatory disease, and macrophages are the most numerous immune cells in endometriotic lesions. However, the mechanisms underlying the elevation of macrophages and their role in the pathogenesis and manifestations of endometriosis still remain unclear. METHODS The number of macrophages stained for CD68 in endometriotic lesions (n = 24) and in peritoneum distant from the lesions (n = 14) from women with endometriosis was compared with the number of macrophages in normal peritoneum from women without endometriosis (n = 18). Peritoneal lesions were also double-stained for CD68 and protein gene product 9.5 to study the relationship between macrophages and nerve fibres. RESULTS The densities of macrophages in peritoneal endometriotic lesions and unaffected peritoneum from women with endometriosis were both significantly higher than that in normal peritoneum from women without endometriosis (P < 0.001). More nerve fibres were also found in the areas where increased numbers of macrophages were identified. CONCLUSIONS There was a significant elevation of macrophages in both normal peritoneum and peritoneal lesions from women with endometriosis compared with normal peritoneum from women without endometriosis. These cells may well play roles in the growth and development of endometriotic lesions and in the generation of pain through interaction with nerve fibres.

Effect of progestogens and combined oral contraceptives on nerve fibers in peritoneal endometriosis

OBJECTIVE To investigate how progestogens and combined oral contraceptives change nerve fiber density in peritoneal endometriotic lesions and to identify the types of nerve fibers still present during hormone treatment. DESIGN Laboratory study using human tissue. SETTING University-based laboratory. PATIENT(S) Hormonally treated and untreated women with endometriosis undergoing laparoscopy, hysteroscopy, and curettage. INTERVENTION(S) Biopsy samples from peritoneal endometriotic lesions in hormonally treated and untreated women with endometriosis. MAIN OUTCOME MEASURE(S) Types and density of nerve fibers were immunohistochemically determined in peritoneal endometriotic lesions from hormonally treated and untreated women with endometriosis. RESULT(S) The nerve fiber density (mean +/- standard deviation/mm(2)) in peritoneal endometriotic lesions from hormone-treated women with endometriosis (10.6 +/- 2.2/mm(2)) was statistically significantly lower than in peritoneal endometriotic lesions from untreated women with endometriosis (16.3 +/-10.0/mm(2)). Nerve growth factor and nerve growth factor receptor p75 expression in peritoneal endometriotic lesions were slightly reduced in hormone-treated women with endometriosis compared with untreated women with endometriosis. CONCLUSION(S) Progestogens and combined oral contraceptives reduced nerve fiber density and nerve growth factor and nerve growth factor receptor p75 expression in peritoneal endometriotic lesions.

Hyperinnervation in Intestinal Deep Infiltrating Endometriosis

STUDY OBJECTIVE To investigate the extent and types of innervation of endometriotic lesions in various regions of the bowel. DESIGN Retrospective nonrandomized immunohistochemical study (Canadian Task Force classification II-3. SETTING University-based laboratory. PATIENTS Thirty-six women undergoing laparoscopy or laparotomy because of deep infiltrating endometriosis in various regions of the bowel, including the sigmoid colon, appendix, and rectum. INTERVENTIONS Immunohistochemical staining of endometriotic specimens with antibodies against protein gene product 9.5, neurofilament, nerve growth factor, nerve growth factor receptors tyrosine kinase receptor A and p75, growth-associated protein 43, substance P, neuropeptide Y, and vasoactive intestinal peptide to demonstrate myelinated, unmyelinated, sensory, and autonomic nerve fibers. MEASUREMENTS AND MAIN RESULTS There were significantly more nerve fibers in intestinal deep infiltrating endometriosis (mean [SD] 172.6 [94.2]/mm(2)) than in other deep infiltrating endometriotic lesions (e.g., cul-de-sac and uterosacral ligament) (67.6 [65.1]/mm(2); p<.01). Intestinal deep infiltrating endometriosis was innervated abundantly by sensory Adelta,sensory C, cholinergic, and adrenergic nerve fibers. Nerve growth factor, tyrosine kinase receptor A, and p75 were strongly expressed in endometriotic lesions, and growth-associated protein-43 was also strongly expressed in the endometriosis-associated nerve fibers. CONCLUSION The hyperinnervation in intestinal deep infiltrating endometriosis may help to explain why patients with this type of lesion have more severe pain.

Discovery of a novel biomarker in the urine in women with endometriosis

OBJECTIVE To investigate whether proteins secreted in urine differ between women with and without endometriosis. DESIGN Laboratory study using human urine. SETTING University-based laboratory. PATIENT(S) Women with and without endometriosis undergoing laparoscopy, hysteroscopy and curettage. INTERVENTION(S) Urine collection from women with and without endometriosis. MAIN OUTCOME MEASURE(S) Proteomic techniques and mass spectrometry to identify proteins secreted in the urine of women with and without endometriosis. RESULT(S) On average, 133 proteins were significantly different between women with and without endometriosis. Cytokeratin-19 was highly up-regulated in the urine of women with endometriosis. CONCLUSION(S) Cytokeratin-19 may be a valuable urinary biomarker for endometriosis.

Nerve fibers in ovarian endometriomas

Ovarian endometriomas (n = 29) were innervated by mainly sympathetic and sensory fibers. These fibers may be involved in the generation of pain symptoms.