Association of Longitudinal Changes in Symptoms and Urinary Biomarkers in Patients with Urological Chronic Pelvic Pain Syndrome: A MAPP Research Network Study.

PURPOSE: We analyzed a series of novel noninvasive urinary biomarkers for their ability to objectively monitor the longitudinal clinical status of patients with urological chronic pelvic pain syndrome. MATERIALS AND METHODS: Baseline, 6 and 12-month urine samples were collected (216) and used to quantify vascular endothelial growth factor, vascular endothelial growth factor (VEGF) receptor 1 (R1), neutrophil gelatinase associated lipocalin (NGAL), matrix metalloproteinase-2, matrix metalloproteinase (MMP)-9, and MMP-9/NGAL complex by enzyme-linked immunosorbent assays. Patient symptom changes were classified as improved, stable or worse using a functional clustering algorithm. Proportional odds models were used to evaluate the association between symptom change and urinary biomarkers. RESULTS: Across all sampled participants, longitudinal decreases in normalized VEGF concentration (pg/µg) were associated with pain severity improvement, and decreases in MMP-9, NGAL and VEGF-R1 concentration (pg/ml) as well as NGAL normalized concentration were associated with improved urinary symptoms. Longitudinal decreases in normalized VEGF-R1 were associated with pain improvement in patients with moderate widespreadness, no bladder symptoms and no painful filling. Lower baseline normalized VEGF-R1 concentration was associated with pain improvement in patients with pelvic pain only. Higher baseline MMP-9/NGAL levels were associated with pain and urinary improvement across all participants. Moreover, longitudinal increases in MMP-2 concentration was associated with improved pain in men and patients with painful filling. CONCLUSIONS: Our results suggest these urinary biomarkers may be useful in monitoring urological chronic pelvic pain syndrome symptom changes with respect to both urinary severity and pain severity. With further testing, they may represent objective biological measures of urological chronic pelvic pain syndrome progression and/or resolution while also providing insight into the pathophysiology of urological chronic pelvic pain syndrome.

Culture-independent Next Generation Sequencing of Urine and Expressed Prostatic Secretions in Men With Chronic Pelvic Pain Syndrome.

OBJECTIVE: To compare standard cultures and next-generation sequencing (NGS) in men with chronic prostatitis/chronic pelvic pain syndrome (CPPS). CPPS shares clinical features with urinary tract infections, but bacteria are seldom found. NGS is more sensitive than standard cultures. MATERIALS AND METHODS: Men diagnosed with CPPS (National Institute of Health Category III) underwent traditional cultures and NGS of their urine and expressed prostatic secretions (EPS). Characteristics between groups were compared statistically. RESULTS: Thirty-one men with CPPS were included (mean age 44.5). All standard urine cultures were negative, and 3 EPS cultures were positive. Seventy-eight unique microbes were detected with NGS, including uropathogens in 10 of the men. There were no bacteria identified by NGS in EPS that were not also found in the urine. Men with positive NGS did not differ from those without in age, symptom severity or phenotype. Men with typical urinary tract infection symptoms (eg, dysuria, chills) were more likely to have uropathogens detected on NGS relative to men without such symptoms. Nine patients were prescribed antibiotics based on their NGS findings, but only 1 exhibited symptom improvement (11%). CONCLUSION: NGS commonly identified bacteria in CPPS patients, but these did not localize to the prostate. NGS positivity did not correlate with symptom severity and antibiotic therapy was seldom effective. NGS detected uropathogens more frequently in those with clinical symptoms suggestive of urinary tract infection. Clinical trials are needed to examine the utility of NGS-guided antibiotics in this subpopulation.

Pelvic floor myofascial pain in patients with symptoms of urinary tract infection.

OBJECTIVE: To correlate lower urinary tract symptoms typically associated with a urinary tract infection (UTI) with physical examination findings of pelvic floor myofascial pain (PFMP). METHODS: This retrospective review included all new patients presenting to a urogynecology clinic between August 2 and December 19, 2016. Patients completed validated questionnaires, had a catheterized urine specimen, and underwent pelvic examination. Associations between demographics, symptoms, urine culture, and PFMP were analyzed. RESULTS: We included 250 patients with urinary frequency (n=160, 64.0%), urgency (n=155, 62.0%), urgency incontinence (n=140, 56.0%), pelvic pain (n=43, 17.2%), and dysuria (n=25, 10.0%). PFMP was detected in 125 (50.0%) patients and culture-proven UTI in 15 (6.0%) patients. Demographics associated with PFMP were lower prolapse stage (P<0.001), age younger than 50 years (P<0.001), lower parity (P=0.028), and non-white ethnicity (P=0.003). Symptoms associated with PFMP were dysuria (adjusted odds ratio 4.13, 95% confidence interval 1.08-15.78), urgency/frequency (2.72, 1.47-5.04), and patient-reported pelvic pain (2.57, 1.08-6.12). These symptoms were independent predictors in multivariable logistic regression analysis. CONCLUSIONS: Most patients had symptoms associated with UTI; however, culture-confirmed diagnosis was infrequent and PFMT was diagnosed in half of participants. Clinicians treating women with these symptoms are advised to examine the pelvic floor muscles.

Changes in brain white matter structure are associated with urine proteins in urologic chronic pelvic pain syndrome (UCPPS): A MAPP Network study.

The Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network has yielded neuroimaging and urinary biomarker findings that highlight unique alterations in brain structure and in urinary proteins related to tissue remodeling and vascular structure in patients with Urological Chronic Pelvic Pain Syndrome (UCPPS). We hypothesized that localized changes in diffusion tensor imaging (DTI) measurements might be associated with corresponding changes in urinary protein levels in UCPPS. To test this hypothesis, we created statistical parameter maps depicting the linear correlation between DTI measurements (fractional anisotropy (FA) and apparent diffusion coefficient (ADC)) and urinary protein quantification (MMP2, MMP9, NGAL, MMP9/NGAL complex, and VEGF) in 30 UCPPS patients from the MAPP Research Network, after accounting for clinical covariates. Results identified a brainstem region that showed a strong correlation between both ADC (R2 = 0.49, P<0.0001) and FA (R2 = 0.39, P = 0.0002) with urinary MMP9 levels as well as a correlation between both ADC (R2 = 0.42, P = 0.0001) and FA (R2 = 0.29, P = 0.0020) and urinary MMP9/NGAL complex. Results also identified significant correlations between FA and urinary MMP9 in white matter adjacent to sensorimotor regions (R2 = 0.30, P = 0.002; R2 = 0.36, P = 0.0005, respectively), as well as a correlation in similar sensorimotor regions when examining ADC and urinary MMP2 levels (R2 = 0.42, P<0.0001) as well as FA and urinary MMP9/NGAL complex (R2 = 0.33, P = 0.0008). A large, diffuse cluster of white matter was identified as having a strong correlation between both ADC (R2 = 0.35, P = 0.0006) and FA (R2 = 0.43, P<0.0001) with urinary NGAL levels. In contrast, no significant association between DTI measurements and VEGF was observed. Results suggest that elevated MMP9 or MMP9/NGAL in UCPPS may be related to degenerative neuronal changes in brainstem nuclei through excitotoxicity, while also facilitating synaptic plasticity in sensorimotor regions.

Predictors of pain, urinary symptoms and quality of life in patients with chronic pelvic pain syndrome (CPPS): A prospective 12-month follow-up study.

OBJECTIVE: Chronic pelvic pain syndrome (CPPS) can affect both men and women and often causes substantial impairment to quality of life. Although cross-sectional studies have suggested that psychosocial aspects may constitute important factors in the etiology and maintenance of CPPS, longitudinal studies are rare. Therefore, the present study examines psychosocial factors as prospective predictors of pain intensity, urinary symptoms and impediments to quality of life in men and women with CPPS. METHODS: Data were collected from patients during visits to a specialized, interdisciplinary outpatient clinic and after 12 months. Outcomes included pain intensity, urinary symptoms and impediments to quality of life, all of which were measured with the NIH-CPSI. Age, sex, depressive-anxious symptomatology (PHQ-ADS), pain catastrophizing (PCS), health anxiety (WI-7) and social support (FSozU) were examined as predictors in multivariate linear regressions. RESULTS: Data from 109 patients (59.6% female; age M = 49.3, SD = 16.7) were analyzed. Pain severity (ß = .30, p = .004), age (ß = .22, p = .02), urinary symptoms (ß = .24, p = .01) and depressive-anxious symptomatology (ß = .29, p = .009) at baseline emerged as predictors of pain at follow-up. Urinary symptoms were predicted by urinary symptoms (ß = .53, p < .001) and depressive-anxious symptomatology (ß = .25, p = .01) at baseline; impediments to quality of life were predicted by depressive-anxious symptomatology (ß = .27, p = .01). CONCLUSION: Psychological factors, especially depressive-anxious symptomatology, predict CPPS-specific symptom severity and impediments to quality of life after 12 months and thus substantially contribute to the chronification of CPPS. It is recommended to address anxiety and depression in patients with CPPS as early as possible in biopsychosocially oriented treatment approaches.

A genome-scale DNA methylation study in women with interstitial cystitis/bladder pain syndrome.

AIMS: To assess the feasibility of using voided urine samples to perform a DNA methylation study in females with interstitial cystitis/bladder pain syndrome (IC/BPS) as compared to age- and race-matched controls. A unique methylation profile could lead to a non-invasive, reproducible, and objective biomarker that would aid clinicians in the diagnosis of IC/BPS. METHODS: Nineteen IC/BPS patients and 17 controls were included. IC/BPS patients had an Interstitial Cystitis Symptom Index score of >8; controls had no bladder symptoms. DNA was extracted from pelleted urine sediment. Samples with >500 ng of genomic DNA underwent quantitative DNA methylation assessment using the Illumina Infinium MethylationEPIC BeadChip. Age- and race-matching was applied prior to analysis. Linear regression models were used to compare average methylation between IC/BPS cases and controls at each cytosine guanine dinucleotide site (loci where methylation can occur). RESULTS: Sixteen participants (eight IC/BPS age- and race-matched to eight controls) had adequate DNA for methylation analysis. The median age was 43.5 years (interquartile range 33.8, 65.0), the median BMI was 27.1 (IQR 22.7, 31.4), and 14 were Caucasian (87.5%). A total of 688 417 CpG sites were analyzed. In exploratory pathway analysis utilizing the top 1000 differentially methylated CpG sites, the mitogen-activated protein kinase (MAPK) pathway was overrepresented by member genes. CONCLUSIONS: The results demonstrate the feasibility of using voided urine specimens from women with IC/BPS to perform DNA methylation assessments. Additionally, the data suggest genes within or downstream of the MAPK pathway exhibit altered methylation in IC/BPS.

Crowdsourcing Disease Biomarker Discovery Research: The IP4IC Study.

PURPOSE: Biomarker discovery is limited by readily assessable, cost efficient human samples available in large numbers that represent the entire heterogeneity of the disease. We developed a novel, active participation crowdsourcing method to determine BP-RS (Bladder Permeability Defect Risk Score). It is based on noninvasive urinary cytokines to discriminate patients with interstitial cystitis/bladder pain syndrome who had Hunner lesions from controls and patients with interstitial cystitis/bladder pain syndrome but without Hunner lesions. MATERIALS AND METHODS: We performed a national crowdsourcing study in cooperation with the Interstitial Cystitis Association. Patients answered demographic, symptom severity and urinary frequency questionnaires on a HIPAA (Health Insurance Portability and Accountability Act) compliant website. Urine samples were collected at home, stabilized with a preservative and sent to Beaumont Hospital for analysis. The expression of 3 urinary cytokines was used in a machine learning algorithm to develop BP-RS. RESULTS: The IP4IC study collected a total of 448 urine samples, representing 153 patients (147 females and 6 males) with interstitial cystitis/bladder pain syndrome, of whom 54 (50 females and 4 males) had Hunner lesions. A total of 159 female and 136 male controls also participated, who were age matched. A defined BP-RS was calculated to predict interstitial cystitis/bladder pain syndrome with Hunner lesions or a bladder permeability defect etiology with 89% validity. CONCLUSIONS: In this novel participation crowdsourcing study we obtained a large number of urine samples from 46 states, which were collected at home, shipped and stored at room temperature. Using a machine learning algorithm we developed BP-RS to quantify the risk of interstitial cystitis/bladder pain syndrome with Hunner lesions, which is indicative of a bladder permeability defect etiology. To our knowledge BP-RS is the first validated urine biomarker assay for interstitial cystitis/bladder pain syndrome and one of the first biomarker assays to be developed using crowdsourcing.

Urine peptide patterns for non-invasive diagnosis of endometriosis: a preliminary prospective study.

OBJECTIVE: To detect endometriosis by urine peptide biomarkers using magnetic beads-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and to identify interesting peptides using liquid chromatography tandem mass spectrometry. STUDY DESIGN: Prospective case-control study in a university-based gynecological department and central laboratory. A total of 122 patients suffering from dysmenorrhea, pelvic pain and infertility were enrolled in the study. Urine samples were collected before laparoscopy. Urine samples were analyzed by the MALDI-TOF technique to generate peptide profiling and ClinProTools software was used to set up a diagnostic model for endometriosis. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to identify interesting peptides. RESULTS: At laparoscopy 60 patients were diagnosed with endometriosis and 62 patients were disease-free. There were 36 different peptides expressed in endometriosis patients detected by MALDI-TOF compared with controls. We established a genetic algorithm as a diagnostic model with the combination of five peptides (m/z=1433.9, 1599.4, 2085.6, 6798.0 and 3217.2). The model showed a sensitivity of 90.9% and specificity of 92.9%. Urine from another 26 symptomatic patients before laparoscopy were randomly selected and analyzed accordingly. A genetic algorithm showed a sensitivity of 90.9% and specificity of 92.9% in predicting endometriosis before laparoscopy. We also identified two peptides not belonging to the diagnostic model as collagen precursors. CONCLUSIONS: Patients with endometriosis have a unique cluster of peptides in urine. Peptide proteomic profiling provides a novel method for non-invasive diagnosis of endometriosis.

Treatment of chronic prostatitis/chronic pelvic pain syndrome category IIIA with Serenoa repens plus selenium and lycopene (Profluss) versus S. repens alone: an Italian randomized multicenter-controlled study.

OBJECTIVES: To evaluate the efficacy and safety of Serenoa repens + selenium and lycopene (Profluss) versus S. repens alone for the treatment of category IIIa chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). PATIENTS AND METHODS: 102 patients with IIIa CP/CPPS were enrolled and randomized into two groups each to receive Profluss or S. repens alone for 8 weeks. Evaluation was based on results of the National Institutes of Health-Chronic Prostatitis Symptom Index (NIH-CPSI), IPSS, maximum peak flow rate (MPFR), and PSA measurements at baseline and at weeks 4, 8 and 8 after the end of treatment. The primary endpoint was a >50% reduction in NIH-CPSI score. Secondary endpoints evaluated were MPFR, IPSS, PSA and white blood cell count. RESULTS: No patients withdrew from the study. The mean NIH-CPSI score decreased significantly (p < 0.001) in both groups; we observed a decrease in the total score from 27.45 to 13.27 in group 1 (-51.64%) and from 27.76 to 20.62 in group 2 (-26.06%). IPSS improved significantly (p < 0.001) in both arms, but more in group 1. PSA and white blood cell count decreased significantly (p < 0.007) only in group 1. The MPFR improved more in group 1 (p < 0.005). CONCLUSION: Profluss is a triple therapy that is safe and well tolerated. It ameliorates symptoms associated with IIIa CP/CPPS.

Effect of antibiotic therapy on interleukin-6 in fresh semen and postmasturbation urine samples of patients with chronic prostatitis/chronic pelvic pain syndrome.

OBJECTIVES: To investigate the effect of antibiotic therapy on interleukin (IL)-6 in fresh semen and postmasturbation urine samples of patients with chronic prostatitis or chronic pelvic pain syndrome (CP/CPPS). METHODS: A total of 128 patients with CP/CPPS prospectively underwent the evaluation of IL-6 in fresh semen and postmasturbation urine samples. At 6 weeks after 4 weeks antibiotic therapy, the IL-6 levels were re-evaluated. RESULTS: Of the 128 patients, 109 (85.2%) were available for our analysis. Of the 109 patients, 72 (66.1%) met the criteria for National Institutes of Health (NIH) classification for inflammatory CP/CPPS (type IIIa) and 37 (33.9%) met the NIH criteria for noninflammatory CP/CPPS (type IIIb). Before antibiotic therapy, 86 patients (78.9%), irrespective of NIH classification, had an increased IL-6 level in fresh semen; 64 (88.9%) patients with CP/CPPS type IIIa and 22 (59.5%) with type IIIb CP/CPPS had increased IL-6 levels. After 4 weeks of therapy, a significant reduction was found in the IL-6 level, with only 44 (40.4%, P = .009) patients showing an increased IL-6 level: 34 patients with type IIIa (47.2%, P = .0000) and 10 with type IIIb (27.0%, P = .0033). An increased IL-6 level was found in the postmasturbation urine sample in 37 patients (33.9%), irrespective of NIH classification: 28 (38.9%) with type IIIa and 9 (24.3%) with type IIIb. At 6 weeks after therapy, only 3 patients (2.8%, P = .000) had an increased IL-6 level: 2 with type IIIa (2.8%, P = .0000) and 1 with type IIIb (2.7%, P = .02). CONCLUSIONS: The IL-6 levels had decreased significantly after antibiotic therapy in patients with CP/CPPS, suggesting a bacterial inflammatory character. The determination of IL-6 in seminal plasma and postmasturbation urine samples is useful as an addition to the diagnostic test for the patient with CP/CPPS and as an efficacy marker for therapy.

Antiproliferative factor, heparin-binding epidermal growth factor-like growth factor, and epidermal growth factor in men with interstitial cystitis versus chronic pelvic pain syndrome.

OBJECTIVES: To determine whether men with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) have urine markers previously described for patients with interstitial cystitis (IC; presence of antiproliferative factor [APF] activity, decreased levels of heparin-binding epidermal growth factor-like growth factor [HB-EGF], and increased levels of epidermal growth factor). METHODS: Clean catch urine specimens were collected from 41 symptomatic patients with CP/CPPS, 36 asymptomatic men without bladder disease who served as the control group, and 24 men with IC. APF activity was determined by (3)H-thymidine incorporation into primary normal adult human bladder epithelial cells. HB-EGF and epidermal growth factor levels were determined by enzyme-linked immunosorbent assay. RESULTS: Men with CP/CPPS did not differ significantly from asymptomatic controls for any of the three markers tested (P >0.49). In contrast, APF activity was present significantly more often and HB-EGF levels were significantly lower in the urine specimens from men with IC than in the specimens from controls or patients with CP/CPPS (P <0.00001 for all four comparisons). Although the epidermal growth factor levels also tended to be higher in the urine from patients with IC than in the urine from controls, the difference did not reach statistical significance (P = 0.06). CONCLUSIONS: These findings indicate that at least two of the urine biomarkers previously identified in women with IC (presence of APF activity and decreased levels of HB-EGF) are also found in men with IC, but not in men with CP/CPPS. This finding suggests that IC and CP/CPPS may be two different disorders with distinct pathophysiologies. It also confirms the utility of the presence of APF activity and HB-EGF levels as markers for IC in men, as well as in women, with this disorder.

Seminal fluid analysis in chronic prostatitis/chronic pelvic pain syndrome.

Prostatitis is a common cause of morbidity among adult men. There are more than 2,000,000 doctor visits per year in the United States, approximately half to urologists (Collins et al., 1998, J Urol 159:1224; Roberts et al., 1998, Urology 51:578; Krieger et al., 2003, Urology). The problem is that very few patients have obvious infections, or functional or structural abnormalities. The aim of this study is to examine our experience with seminal fluid analysis in this patient population, and to outline the potential utility of this examination in patient evaluation.

Symptoms and inflammation in chronic prostatitis/chronic pelvic pain syndrome.

OBJECTIVES: To evaluate the possibility that patients with inflammatory and noninflammatory chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) might present with different symptoms. Patients with CP/CPPS present with characteristic symptoms without bacteriuria. The new National Institutes of Health consensus suggests that CP/CPPS can be divided into inflammatory and noninflammatory categories. METHODS: Standardized symptom surveys were completed by 130 subjects who met the criteria for CP/CPPS after clinical examination and urethral, urine, expressed prostatic secretion (EPS), and seminal fluid analysis evaluations. RESULTS: When classified by either EPS or postprostatic massage urine (VB3) findings, subjects with and without inflammation had similar symptoms. However, when classified using the combination of EPS, VB3, and seminal fluid analysis, subjects with inflammatory CP/CPPS had more severe (P <0.02) and more frequent symptoms, in particular, difficulty reaching erection (P <0.01), weak urinary stream (P <0.01), urinary frequency (P = 0.03), and penile pain (P = 0.04). CONCLUSIONS: The increased severity and frequency of symptoms among patients with inflammatory CP/CPPS provide empirical support for the new consensus classification on the basis of the combination of EPS, VB3, and seminal fluid analysis findings.

Seminal secretory capacity of the male accessory sex glands in chronic pelvic pain syndrome (CPPS)/chronic prostatitis with special focus on the new prostatitis classification.

OBJECTIVE: The aim of the study was to evaluate the secretory dysfunction of the male accessory glands in men with inflammatory versus non-inflammatory chronic pelvic pain syndrome (CPPS). METHODS: One hundred and twelve consecutive patients symptomatic for chronic pelvic pain were included into the study. All underwent a combined granulocyte analysis in expressed prostatic secretions (EPS) and a four-glass-test followed by ejaculate analysis. Patients were subgrouped according to elevated granulocyte counts in prostatic secretions, leukocytes in semen, or any of both. The content/total enzyme activity of the secretory seminal plasma parameters gamma-glutamyl-transferase (gamma-GT), fructose, and alpha-glucosidase representing the secretory capacity of the prostate gland, the seminal vesicles, and the epididymes, respectively, were investigated. RESULTS: The only significant findings were a reduced total enzyme activity of gamma-GT in men stratified according to elevated granulocyte counts in prostatic secretions (p=0.022; cutpoint 9.85U per ejaculate; sensitivity 61.1%, specificity 58.8%, AUC 0.6347) and in men with any inflammatory sign (p=0.033; cutpoint 9.9U per ejaculate, sensitivity 63%, specificity 58.33%, AUC 0.6404). CONCLUSIONS: Secretory damage of the prostate gland in men with inflammatory CPPS is demonstrable provided that increased granulocytes in prostatic secretions are part of the diagnostic criteria. However, because of the low sensitivity and specificity of gamma-GT it cannot be recommended as diagnostic tool to detect inflammatory disease on the basis of reduced secretory capacity.

Comparison of expressed prostatic secretions with urine after prostatic massage--a means to diagnose chronic prostatitis/inflammatory chronic pelvic pain syndrome.

OBJECTIVES: To compare the analysis of urine after prostatic massage (VB3) with expressed prostatic secretions (EPS) to assess the significance of leukocyte analysis in VB3 and to give a first hint of the diagnosis of inflammatory chronic pelvic pain syndrome (CPPS) when EPS cannot be obtained. METHODS: Three hundred twenty-eight men (mean age 38 years, range 18 to 70) with expressible prostatic secretions were investigated. EPS were stained using the Papanicolaou stain and analyzed for leukocytes per high power field (HPF) (x1000). Additionally, identical aliquots of first voided urine (VB1), midstream urine (VB2), and VB3 were centrifuged, stained (Papanicolaou), and analyzed for leukocytes (x400). Patients with increased numbers of leukocytes in VB1 and VB2 (2 or more per x400) were excluded. For statistical analysis, Spearman's correlation coefficient for nonparametric tests was used. RESULTS: Of 180 men with less than 10 leukocytes per HPF in EPS, 178 (98.9%) had less than 10 leukocytes per view field in VB3. In 148 men with 10 or more leukocytes per HPF in EPS, 136 (91.9%) also had elevated leukocyte counts in VB3. The presence of elevated leukocytes in VB3 predicted the presence of increased leukocytes in EPS with a high certainty: 91.9% sensitivity, 98.9% specificity, and 95.7% accuracy, with a positive and negative predictive value of 98.6% and 93.7%, respectively. CONCLUSIONS: We conclude that the determination of leukocytes in VB3 is a feasible and reliable method compared with the analysis of EPS. However, although this association does not directly prove the significance of VB3 in those patients from whom no EPS can be obtained, we suggest this method be taken into account as an indirect indicator in the diagnosis of inflammation.