{"paper_id":"24b3af48-3d50-4b81-9235-dfacec4c0161","body_text":"Chronic pelvic pain (CPP) is a highly prevalent (2 to 25%) clinical problem among women\n( 1 ). It is described as continuous or\nintermittent pain in the anatomic pelvis (anterior abdominal wall at or below the\numbilicus) that lasts at least 6 months, is not exclusively related to menstruation or\nsexual intercourse, and is sufficiently severe to cause functional disability or to lead\nto medical care ( 2 ). Its etiology is often\nunknown but may result from a complex interaction between the gastrointestinal, urinary,\ngynecologic, musculoskeletal, neurologic, and endocrine systems. It is also influenced\nby psychological and sociocultural factors. Among women seen at gynecology outpatient\nclinics, one of the diseases most often associated with CPP is endometriosis. Abdominal\nmyofascial pain syndrome, characterized by hyperirritable trigger points is another\nfrequent underdiagnosed condition causing CPP, especially in countries where the\nprevalence of cesarean section and other abdominal surgeries is common ( 3 ).\nResearchers, physicians, gynecologists and patients believe that CPP can be caused by\nthe endometriosis lesions ( 4 ), but determining\nhow the lesions cause CPP has proved difficult. Various pathophysiological mechanisms\nhave been proposed to explain the development of myofascial pain syndromes, but little\nis known about the cause of this condition. The chronic inflammatory response in\nendometriosis has been identified as an important factor in the pathophysiology of the\ndisease ( 5 ). Similarly, elevated levels of\ninflammatory mediators have been reported in sites close to or far from active\nmyofascial trigger points ( 6 ). Some studies\nindicate a possible role for nitric oxide (NO) in the pathogenesis of endometriosis\n( 7 ). It is now well appreciated that NO can\nact as a mediator and regulator of the inflammatory response ( 8 ) and of the modulation of nociception at both the peripheral and\nthe central level ( 9 ). Studies on animals have\ndemonstrated that NO enhances the sensitivity of peripheral nociceptors ( 10 ) and can contribute to hyperalgesia secondary to\ncentral sensitization ( 11 ). Most studies have\nshown pronociceptive properties of NO in the spinal cord. Nevertheless, NO has a dual\nrole in pain pathways. There is also evidence of its antinociceptive and analgesic\neffect, particularly when present at low concentrations within the spinal cord ( 12 ). In addition to this direct analgesic effect, it\nmay also mediate the peripheral and central antinociceptive effect of other analgesic\ncompounds (e.g., opioids and NSAIDs) ( 9 ). In\nhumans, intracutaneous NO injection can evoke pain ( 13 ), and increased NO levels have been detected in the plasma of patients\nwith chronic pain ( 14 ), temporomandibular joint\ndisorders ( 15 ), chronic orofacial pain ( 16 ), and bladder pain syndrome/interstitial cystitis\n( 17 ). Although it has been observed that\ngreater amounts of NO are present in the endometrial tissues of women with endometriosis\n( 7 ), no studies have investigated plasma NO\nlevels in women with CPP secondary to endometriosis, nor has it been determined whether\nthere are differences between women with CPP and those with abdominal myofascial pain\nsyndrome. Thus, the objective of the present study was to determine plasma NO levels in\nwomen with CPP secondary to those conditions and to explore their association with pain\nintensity and pain threshold.\n\nThis prospective study was conducted in women of reproductive age without infertility\nand with regular menstrual cycles. The Ethics Committee of the Hospital das Clínicas,\nFaculdade de Medicina de Ribeirão Preto, Universidade de São Paulo approved the study\n(protocol #3502/2007, national registry 0112.0.004.000-07). The study samples were\nobtained when all inclusion criteria were satisfied and written informed consent was\nobtained. The research subjects were women with CPP and treated at the Gynecologic\nEndoscopy and Chronic Pelvic Pain Center of the same institution. Data from healthy\ncontrol women during routine gynecological follow-up were matched with the study\nsubjects by date of blood collection, age and parity, and were obtained from a\ndatabase of primary health care unit records maintained by the university.\nWe selected 25 healthy subjects and 40 women with CPP without previous treatment.\nWomen with CPP were divided into 2 groups. Group 1 included 24 women diagnosed with\nendometriosis confirmed by histology and classified during laparoscopy according to\nthe American Society for Reproductive Medicine ( 18 ) as moderate (n=14) or severe (n=10). Women with intestinal, bladder,\nor rectovaginal septum invasion by endometriosis, as well as women with minimal or\nmild endometriosis because of extensive surgery or lack of histologic confirmation,\nrespectively, were excluded from the study. Group 2 included 16 women diagnosed\nexclusively with abdominal myofascial pain syndrome ( 19 ). The study thus included a healthy control group, an endometriosis\ngroup, and a myofascial group. Subjects with a history of inflammation including\nfever, a history of acute diseases such as flu; chronic diseases such as rheumatoid\narthritis, lupus, asthma, and allergic reactions; hypertension, smoking habit,\nalcoholism, using hormonal contraceptives, or without an imputable cause for CPP,\nwere also excluded ( Figure 1 ).\nWomen with endometriosis underwent laparoscopy under general anesthesia and with\npneumoperitoneum obtained using carbon dioxide. Judicious inspection of the\nabdominopelvic cavity was performed before any other procedures were carried out.\nEndometriotic lesions were classified according to the American Society of\nReproductive Medicine classification system ( 18 ). Electrosurgical ablation and/or excision of superficial lesions that\nwere identified, removal of deep lesions, and/or cystectomy for endometriomas were\nperformed. All women underwent conservative surgery, preserving the ovaries.\nThe diagnosis of abdominal myofascial pain syndrome was clinically confirmed by\nSimons' criteria ( 19 ). All women with this\nsyndrome were examined by abdominal wall ultrasound in order to exclude any other\nlocal conditions such as hernias or abdominal wall cystic endometriosis. Women with\nsigns of ilioinguinal/iliohypogastric/genitofemoral neuropathies were excluded. No\nwomen in this group presented with significant dysmenorrhea. As specified in our\nservice protocol, all women in the myofascial group received injections of 2 mL 0.5%\nlidocaine ( 20 ), without a vasoconstrictor,\ndirectly in and perpendicular to the trigger point at 5 weekly sessions.\nPain was quantified using a visual analogue scale (VAS) before and after treatment;\nthe McGill pain questionnaire was applied only before treatment ( 21 ). We considered the reduction of pain to be\nclinically relevant when subjects showed a 50% reduction in the VAS, or at least a 20\nmm change with significant improvement reported in daily activities ( 22 ).\nThe pressure pain threshold (i.e., the minimal pressure that induced pain) was\nassessed with an Instrutherm DD-500 pressure algometer with digital traction and\ncompression and a measuring capacity of 5 kg (Digital Dynamometer, Model: DD200,\nSerial No. 08030300338819, Instrutherm Measuring Instruments Ltd., Brazil). The\napparatus consisted of a 1-cm diameter rubber disk attached to the plunger of a\npressure (force) gauge. The dial of the gauge was calibrated in kg/cm 2 . We\nroutinely measured the threshold at a single point on the thenar region of the\nnondominant hand. Three consecutive measurements were taken with an interval of 20\nmin and the means were used in the analysis. The Hospital Anxiety and Depression\nScale ( 23 , 24 ) was also used as a psychometric instrument.\nBlood samples were obtained from women in the follicular phase of the menstrual cycle\n(fifth to ninth day) between 7:00 and 9:00 am ( 25 ). The first sample was collected immediately before treatment and the\nsecond at 4 weeks after treatment. Subjects whose data were included in the database\nwere routinely contacted by phone 3 to 4 days before the collection and advised about\ndiet (to avoid foods such as meats in excessive amounts, coffee, teas, chocolates,\nspices, pepper, salt, among others) and drug intake (to avoid analgesics and\nnonsteroidal anti-inflammatory drugs). Plasma samples were stored at -70°C until\nnitrate was assayed by the Griess colorimetric reaction ( 26 ).\nNitrite (NO 2 ) plus nitrate (NO 3 ) levels were determined in\nplasma samples by enzymatically reducing nitrate with bacterial nitrate reductase.\nBriefly, duplicate plasma samples (40 µL) were incubated overnight at 37°C with an\nequal volume of reductase buffer (0.5 M potassium phosphate, pH 7.5, containing NADPH\nand nitrate reductase). The total amount of nitrate was then determined by the\ncolorimetric Griess reaction. Briefly, samples were incubated with 80 µL freshly\nprepared Griess reagent (2% sulphanylamide in 5% phosphoric acid and 0.2%\nnaphthylethylenediamine dihydrochloride). After 15 min of color development at room\ntemperature, the sample absorbance at 540 nm was compared with values on a standard\nnitrate curve. The results are reported as µM NO 3 +NO 2 .\nComparisons of NO levels between groups were performed using the nonparametric\nWilcoxon test for each pair. The Wilcoxon signed rank test was used to compare NO\nbetween pre- and post-treatment samples. Correlations between NO, VAS, and pain\nthreshold were evaluated by the multivariate restricted maximum likelihood method.\nFor that analysis, we defined pain intensity variation (ΔVAS) as (post-treatment\nVAS-pretreatment VAS)/pretreatment VAS) NO level variation (ΔNO) was (post-treatment\nNO-pretreatment NO)/pretreatment NO. Pain threshold variation (Δthreshold) was\n(post-treatment threshold-pretreatment threshold)/pretreatment threshold). A negative\nsign was used to indicate a reduction of measured value. The statistical tests and\nplots were performed using JMP 10 statistical software for Mac (SAS Institute, USA)\nwith the level of significance set at P<0.05.\n\nThe characteristics of the study participants are presented in  Table 1 . There was a clinically relevant reduction of pain in 91.7%\n(n=22/24) of women with endometriosis and 75.0% (n=12/16) of women with abdominal\nmyofascial pain syndrome. Pretreatment NO levels (μM) were lower in healthy volunteers\n(47.0±12.7) than in the myofascial (64.2±5.0, P=0.01) or endometriosis (99.5±12.9,\nP<0.0001) groups. The latter group had higher pretreatment NO levels than the\nmyofascial group (P =0.005).\nThe pain thresholds (kg/cm 2 ) of healthy women (2.6±0.2) were significantly\nhigher than those of women with endometriosis (1.0±0.1, P<0.0001) or myofascial\nsyndrome (1.9±0.2, P=0.007). This last group had higher pretreatment pain thresholds\nthan the group of women with endometriosis (P=0.0004).\nThere was a reduction of plasma NO levels after treatment in the endometriosis group\n(99.5±12.9  vs  61.6±5.9, P=0.002), but not in the myofascial group\n(64.2±5.0  vs  61.1±8.2; P=0.70). No differences were observed in\npost-treatment NO levels between the study groups and controls even though the analysis\nhad sufficient power to demonstrate it (P=0.09).  Table\n2  shows post-treatment data regarding the VAS pain intensity, pain threshold\nand NO levels in the endometriosis and myofascial groups.\nWe observed a correlation between ΔVAS and ΔNO level in the endometriosis group\n[correlation = 0.67 (95%CI: 0.35 to 0.85), P<0.0001], but not in the myofascial group\n[correlation = -0.64 (-0.89 to 0.10), P=0.20]. We also observed a correlation between\nΔthreshold and ΔNO level in the endometriosis group [correlation = -0.53 (-0.78 to\n-0.14), P<0.0001] but not in the myofascial group [correlation = -0.12 (-0.65 to\n0.49), P=0.88].\n\nThe present study showed that women with CPP secondary to endometriosis had\nsignificantly elevated plasma NO levels compared with healthy controls and women with\nCPP secondary to abdominal myofascial pain syndrome. Clinical improvement of CCP after\nsurgical treatment of endometriosis was associated with a reduction of plasma NO levels.\nHowever, these findings were not observed in women with myofascial syndrome. This\nsuggests that ablation or excision of endometriosis may be responsible for reducing\nperipheral levels of NO. Indeed, injection of anesthetic into myofascial syndrome\ntrigger points does not eliminate the injury and probably only modulates the afferents.\nAdditionally, we observed that NO reduction in women with endometriosis was directly\nassociated with reduction of pain threshold, suggesting that NO may be involved in the\nprocess of central sensitization. Interestingly, NO levels did not reach those seen in\nhealthy women, even after appropriate treatment of their condition. It is possible that\nother, particularly central, pathophysiological events may be associated with the origin\nof NO, but our study does not allow further evaluation.\nPublished studies have shown that NO may be involved in the pathophysiology of\nendometriosis. It has been observed that NO synthesis is increased in endometrial tissue\nfrom women with endometriosis ( 7 ), and changes\nhave been observed in endothelial nitric oxide synthase (eNOS) expression in the\nendometrium of women with endometriosis ( 27 , 28 ) that can be associated with specific gene\npolymorphisms of eNOS ( 29 ). However, eNOS has not\nbeen involved in pain processing ( 30 ).\nFurthermore, a significant increase in NO level was also detected in the peritoneal\nfluid of women with endometriosis ( 19 ), probably\ndue to increased expression of inducible nitric oxide synthase (iNOS) in peritoneal\nmacrophages ( 31 ).\nMost previous studies have focused on women with infertility, and we did not find any\nthat were specific to NO in women with CPP. The association between NO and pain is\ncomplex, but our results are similar to those reported by some other authors ( 14 , 17 ). Under\nchronic pain conditions, we know that the activation of N-methyl-D-aspartate (NMDA)\nreceptors in the spinal cord can produce persistent enhancement of pain via production\nof NO and/or prostaglandins ( 32 , 33 ). There is also evidence that NO may be directly\ninvolved in the process of central sensitization following a painful stimulus ( 11 , 34 ). Our\nresults ( 9 , 12 ) suggest that this may be happening in women with CPP secondary to\nendometriosis, since there was a positive correlation between the reduction in plasma NO\nlevels and pain thresholds measured at a distance from the primary site of pain.\nMoreover, it has been suggested that such a mechanism might not be important in other\nconditions such as abdominal myofascial pain syndrome.\nThe number of patients studied here was small, but the inclusion criteria were very\nstrict; thus we believe that the data can be generalized with confidence. We\nrecognize, however, that an issue that might be questioned is the interference of\nanalgesic drugs, most commonly NSAIDs ( 35 ).\nAlthough all women claimed that they had not taken painkillers within the previous 3\nor 4 days, this could not be verified because they tended to deny this\nself-medication behavior. In any case, the chronic anti-inflammatory action of NSAIDs\nmay be due not only to inhibition of prostaglandin synthesis but also through\nactivation of arginine-NO-cGMP pathways ( 36 ),\ninhibition of inducible nitric oxide synthase gene expression, and NO synthesis\n( 37 ). Thus, we would have expected to\ndetect lower NO levels in women with CPP before treatment, which was not the case.\nFurther, it is known that neuronal nitric oxide synthase (nNOS) is the most important\nenzyme in the spinal cord during both development and maintenance of neuropathic and\ninflammatory pain ( 38 , 39 ). However, our study does not allow for determining or making\ninferences about the origin of NO identified peripherally.\nNO may represent an objective systemic marker of reduced disease activity of\nendometriosis after treatment, and perhaps of reduced central sensitization.\nAdditionally, the knowledge that inhibitors of nitric oxide synthase may act as\nanalgesic agents ( 40 ) opens an opportunity for\ntheir use as a therapeutic target for this condition.\nThe present study does not permit us to make any conclusions about the source of NO.\nIt may be locally produced in large amounts, and may reach the bloodstream after\nwashout from the interstitial space in the affected region. The reasons are not\nentirely clear but it is known that persistent activation of nociceptors results in\nactivation of the three known NO synthases, particularly inducible and neuronal\nnitric oxide synthase, hence increasing NO synthesis ( 14 ). On the other hand, we cannot explain why this would not occur in\nwomen with abdominal myofascial pain syndrome, and it is possible that, in women with\nCPP, more NO is produced by the vascular system in response to the prolonged and\nnoncontinuous use of analgesic and/or anti-inflammatory drugs (about 80% of our\npatients self-medicate with these substances more than twice a week) ( 3 ). Under these conditions, more NO would be\nproduced in endothelial cells to compensate for the inhibitory effect of these\nanalgesics. We consider this last hypothesis to be less probable because, assuming\nthe same rates of self-medication, no difference would be expected between\ngroups.\nIn summary, NO levels were elevated in women with CPP secondary to endometriosis, but\nnot in women with CPP secondary to abdominal myofascial pain syndrome. Surgical\ntreatment of endometriosis was associated with reduction in NO levels, which was\ncorrelated with a reduction in pain intensity and in distant pain thresholds. It is\npossible that NO is involved in the pathophysiology of pain secondary to\nendometriosis, possibly associated with central sensitization. In our opinion, this\ncorrelation supports NO as a potential marker in patients with CPP and endometriosis,\na characteristic that has not been reported previously.","source_license":"CC0","license_restricted":false}