Hann-Chorng Kuo, Department of Urology, Buddhist Tzu Chi General Hospital, 707, Section 3, Chung Yang Road, Hualien, Taiwan. e-mail: firstname.lastname@example.org
Study Type – Aetiology (case series)
Level of Evidence 4
What’s known on the subject? and What does the study add?
Urinary nerve growth factor levels were higher in women with OAB-dry and OAB-wet compared to the controls. The link between female OAB and risk factors such as obesity and menopause has not been determined yet.
This study found ageing, menopause, or higher BMI did not influence the urinary NGF levels in OAB women. Higher urinary NGF levels in OAB women could be an inflammatory disorder unrelated to ageing or obesity.
• To measure urinary nerve growth factor (NGF) in women with overactive bladder (OAB)-dry and OAB-wet and investigate the association of urinary NGF expression with these factors.
PATIENTS AND METHODS
• Differentiation between OAB-wet and OAB-dry was based on symptoms and a 3-day voiding diary.
• Urinary NGF levels were measured by enzyme-linked immunosorbent assay (ELISA).
• The urinary NGF levels were compared among controls, OAB-dry and OAB-wet subgroups, and also between OAB patients ≥55 years and <55 years, as well as between patients with a body mass index (BMI, kg/m2) <20, 20–30 and >30.
• A total of 113 women with OAB-dry, 106 with OAB-wet and 84 controls were enrolled. The urinary NGF/creatinine (Cr) levels were significantly highest in OAB-wet (2.13 ± 3.87) and second highest in OAB-dry (0.265 ± 0.59) compared to controls (0.07 ± 0.21).
• Analysis of urinary NGF or NGF/Cr levels among controls, OAB-dry and OAB-wet groups by age and BMI showed no significant differences, except for the OAB-dry group.
• Urinary NGF/Cr was not significantly correlated with age (P= 0.088) or BMI (P= 0.886) in women with OAB-dry and OAB-wet.
• Urinary NGF levels were significantly higher in women with OAB-dry and even higher in women with OAB-wet.
• The urinary NGF level was not associated with ageing, menopause or higher BMI either in controls or OAB patients.
Overactive bladder (OAB) syndrome is a condition of urinary urgency, usually accompanied by frequency and nocturia, with or without urgency urinary incontinence . Urgency is the core symptom for the presence of OAB. OAB is highly prevalent in Asian women. The prevalence ranges from 8% in China to 28.4% in Korea [2,3]. OAB not only is a substantial economic burden, but also has great impact on the health-related quality of life  and negatively affects women’s sexual health, reducing sexual desire and the ability to achieve orgasm . In addition, OAB is significantly associated with obesity (body mass index, BMI, kg/m2, >30) , especially among obese pre-menopausal women (<55 years) . The risk of OAB incontinence is greater in women with higher BMIs . However, other studies do not confirm that menopause or BMI are risk factors for female OAB or nocturia [9,10].
Clinically, an urgency perceptive scale or urgency severity score is used to grade the severity of OAB, although these instruments are based on subjective reporting of symptoms and patients might not able to identify urgency from strongly urge to void when the bladder is extremely full. Patients with OAB may have detrusor overactivity (DO) or increased bladder sensation, which have been postulated to be of the same spectrum of bladder dysfunction .
Clinical and experimental data indicate a direct link between higher levels of nerve growth factor (NGF) in bladder tissue with lower urinary tract conditions such as BOO, OAB and interstitial cystitis/painful bladder syndrome [12,13]. Bladder inflammation results in altered NGF levels and morphological changes in the sensory and motor neurones innervating the bladder . Higher urinary NGF levels have been reported in patients with OAB and DO [15–17]. The higher urinary NGF levels in patients with DO have also been reported to decrease after antimuscainic therapy or intravesical botulinum toxin injection .
Chronic inflammation, whether triggered by a urinary tract infection or otherwise, is considered to be a possible underlying mechanism for OAB or interstitial cystitis/painful bladder syndrome . Bladder biopsy findings in patients with neurogenic and idiopathic DO showed signs of chronic inflammation in 59.1% of biopsies before botulinum toxin A administration, indicating that chronic inflammation is present in some DO bladders . A recent pilot study with 17 OAB patients and eight controls also showed that higher levels of urinary inflammatory biomarkers are identified in patients with OAB . Urinary NGF plays a potential role as a biomarker of OAB . The present study investigated urinary NGF expression in women with OAB symptoms with respect to OAB subtype, age and BMI.
PATIENTS AND METHODS
Women with urgency and/or urgency incontinence who visited our outpatient clinic from 2007 to 2009 were enrolled prospectively for analysis. A group of women without LUTS served as controls. The present study was approved by the Institutional Review Board of the hospital. Informed consent was obtained from all subjects before collecting urine. Urine samples were collected for measurement of NGF before any treatment was given.
Differentiation between OAB-wet and OAB-dry was based on symptoms and a 3-day voiding diary. All subjects were requested to maintain a 3-day voiding diary to verify the occurrence of OAB-dry (i.e. urgency at least once per day without urgency incontinence) or OAB-wet (i.e. at least one episode of urgency incontinence per three days). The control groups consisted of women who were hospital employees and patients with disorders other than lower urinary tract disorders and who were free of LUTS and had a normal voiding condition. Patients with previous bladder or urethral surgery, active urinary tract infections within 3 months, neurogenic lesions or post-void residual volumes of more than 50 mL were excluded.
All subjects were requested to drink 1000 mL of water to create a strong desire to void. They were then allowed to void into uroflowmeter. The voided volume was measured and urine samples were collected. The urine samples were put on ice immediately and transferred to the laboratory for preparation. The urine samples were centrifuged at 3000 g for 10 min at 4 °C. The supernatant was separated into aliquots in 1.5-mL tubes and preserved in a freezer at −80 °C. Concurrently, 3 mL of urine was taken to measure the urinary creatinine (Cr) level.
Urinary NGF levels were measured by enzyme-linked immunosorbent assay (ELISA) according to previously reported methods . The amount of NGF in each urine sample is determined from an NGF standard curve. All samples were tested in triplicate and the values were averaged. The total urinary NGF levels were further normalized to the concentration of urinary Cr (NGF/Cr level).
The total urinary NGF and NGF/Cr levels were compared among control, OAB-dry and OAB-wet groups using one-way ANOVA. The NGF levels were also compared between OAB patients ≥55 years (post-menopause) and <55 years (pre-menopause), as well as between patients with a BMI < 20, 20–30 and greater than 30. The Mann-Whitney U-test was used for statistical analysis between subgroups without making the assumption of normality and univariate nature. Pearson’s correlation was used for analyzing the association between urinary NGF levels and age as well as BMI. Receiver-operator characteristics curves were used for calculation of areas below curves and to analyze the sensitivity and specificity of threshold values of urinary NGF or NGF/Cr for differentiation of OAB-wet and OAB-dry subgroups. P < 0.05 was considered statistically significant.
A total of 113 women with OAB-dry, 106 with OAB-wet and 84 controls were enrolled in the present study. The mean age was 61.5 ± 14.3 years for the OAB group and 42.6 ± 16.9 years for the control group (P < 0.001). There was no significant difference in mean age between women with OAB-dry and OAB-wet.
The urinary NGF and NGF/Cr levels were significantly highest in OAB-wet and second highest in OAB-dry compared to controls (Table 1). Using receiver-operator characteristics analysis, the areas below the curves were highest in urinary NGF/Cr (0.901) and NGF (0.897) between OAB-wet and controls. A threshold urinary NGF value of 1.265 pg/mL provided a sensitivity of 87.7% and specificity of 80%, whereas a threshold value of urinary NGF/Cr level of 0.085 provided a sensitivity of 84.9% and specificity of 84.5% for differentiation of OAB-wet and controls (Fig. 1A). The areas below the curves were 0.766 and 0.765 for urinary NGF/Cr and NGF, respectively, for differentiation of overall OAB patients and controls (Fig. 1B).
Table 1. The urinary nerve growth factor (NGF) and NGF/creatinine (Cr) levels in the controls and women with overactive bladder (OAB) of different age and body mass index (BMI) subgroups
Control (n= 84)
OAB-dry (n= 113)
OAB-wet (n= 106)
The number of subjects is given in parenthesis. *Significant difference between OAB-dry and OAB-wet women.
Age significantly correlated with BMI among the controls (r= 0.593, P < 0.001) and OAB women (r= 0.146, P= 0.037). Analysis of urinary NGF and NGF/Cr levels among the controls, OAB-dry and OAB-wet women by age (≥55 vs <55 years) and BMI (<20, 20–30 and >30) showed no significant differences between the subgroups within the OAB group, except for OAB-dry. A significantly higher urinary NGF/Cr level was noted in women with OAB-dry and age <55 years (P= 0.038). However, this result was not observed in women with OAB-wet.
Using Pearson’s correlation, urinary NGF/Cr levels significantly correlated with age (P= 0.003) but not with BMI (P= 0.288) in the overall subjects, including controls and OAB women. Urinary NGF/Cr did not significantly correlate with age (P= 0.234) or BMI (P= 0.767) in women with OAB-dry and OAB-wet (Fig. 2).
Increasing evidence shows that OAB could be an inflammatory disorder. Tyagi and Chancellor  proposed the hypothesis that local inflammation is a cause and plays a central role in the aetiology of OAB. Pre-clinical studies show that increased urinary levels of monocyte chemoattractant protein 1 and CXC chemokine CXCL1 are evidence of bladder inflammation . Along with the presence of chronic bladder inflammation in some bladder biopsies from idiopathic DO , urinary NGF, PGE2 and cytokines are also higher in OAB patients [15,16,20,21]. Additionally, urinary NGF decreases in response to effective antimuscarinic treatment or intravesical botulinum toxin A injection . This evidence suggests that a local inflammation might be present in some OAB bladders.
Women with increased bladder sensation without symptoms of urgency had urinary NGF levels similar to those of controls, although women with OAB-dry or OAB-wet had significantly higher urinary NGF levels , suggesting that a higher urinary NGF level plays an important role in mediating the sensation of urgency in OAB. Increased bladder sensation could be caused by an increased alertness of bladder fullness, whereas polyuria may contribute to urinary frequency. Clinically, it is sometimes difficult to differentiate these women from women with OAB-dry. The results of the present study provide evidence that women with OAB-wet had eightfold higher urinary NGF/Cr levels than women with OAB-dry, suggesting that OAB-wet is a chronic inflammatory disorder with highly expressed urinary NGF. By contrast, OAB-dry could be a mixture of increased bladder sensation and DO. Therefore, urinary NGF is a possible biomarker for differentiation between OAB subtypes.
The results obtained in the present study show that women with OAB-wet had significantly higher urinary NGF levels than those with OAB-dry. A possible reason for the difference in NGF levels between OAB-dry and OAB-wet could be the result of different degrees of inflammation and a higher percentage of DO in patients with OAB-wet. Hashim and Abrams  found that 44% of women with urgency (OAB-dry) had DO, whereas 58% of women with urgency and urgency incontinence (OAB-wet) had DO. Hyman et al.  also found a higher incidence of DO associated with urgency incontinence (OAB-wet) than in those with symptoms of urgency and frequency, nocturia or difficult urination (75% vs 36%) among men with LUTS . Taken together, these clinical observations suggest that urinary NGF is strongly associated with severe urgency symptoms, and a higher urinary NGF level could impact the occurrence of DO.
Previous epidemiological studies showed that the prevalence of OAB increases with age in women [2–4,24]. Menopause has been considered a risk factor for the higher prevalence of OAB and incontinence. However, recent investigations and the results obtained in the present study do not support this concept [9,10]. The present study showed that urinary NGF levels were significantly corerelated with age in overall women, although the mean age of the controls was significantly younger than OAB women. Therefore, this age-related increased NGF level in overall women could be a significant confounding element as a result of sampling bias. No significant difference of urinary NGF levels was fond between pre-menopausal and post-menopausal women with OAB-wet. Furthermore, a higher NGF level was noted in pre-menopausal than post-menopausal women with OAB-dry. These results indicate that increased age or menopause do not result in a higher urinary NGF level in OAB women.
Obesity is well recognized as a risk factor for female urinary incontinence [6–8]. Obesity measured by BMI is associated with higher prevalence of OAB and LUTS in women , and is associated with more severe OAB disease . Sympathetic hyperactivity is hypothetically considered to be a cause of obesity and the metabolic syndrome, which is associated with LUTS . In the present study, there was no significant association between urinary NGF levels and BMI in either controls or women with OAB. Urinary NGF levels in OAB women with low BMI were as high as those in women with high BMI. If OAB is considered as a local inflammatory bladder disorder, the results obtained in the present study imply that bladder inflammation (as shown by urinary NGF levels) in OAB-dry or OAB-wet is not significantly affected by an obesity factor. The association betwen obesity and OAB prevalence in women might be associated with contributing factors other than NGF.
The pathophysiology of OAB is not well elucidated. Whether OAB-dry and OAB-wet are of the same disease spectrum is still a matter of debate. A longitudinal study of women with OAB showed a dynamic exchange of OAB-dry and OAB-wet in a sample of the women, suggesting that OAB is a neuroendocrine disorder and chronic inflammation could be one of the underlying heterogenous pathophysiologies of OAB . Urinary NGF measurements in women with OAB provide insight into the underlying pathophysiology of these conditions. Although ageing and obesity could be contributing factors for the prevalence of OAB in women, risk factors of OAB, such as menopause and BMI, did not correlate well with urinary NGF expression in the present study. This observation could be a result of the multiple aetiologies of OAB, such as neurogenic inflammation, urothelial dysfunction or myogenic instability.
One major limitation of the present study is that the mean age of the control group was significantly younger than that of women with OAB-dry and OAB-wet. The cause of this limitation is the difficulty encountered in enrolling ageing women without any LUTS and voiding dysfunction. This is the likely explanation for the significant correlation between age and urinary NGF in overall women. When only the women with OAB were included, the correlation between age and urinary NGF became insignificant.
In conclusion, urinary NGF levels were significantly higher in women with OAB-dry and OAB-wet. The urinary NGF level was not associated with ageing, menopause or higher BMI in the controls or OAB patients. Higher urinary NGF levels in OAB women could be a local inflammatory disorder that is unrelated to ageing or obesity.