Abstract
Background The bladder's regulatory function is influenced by central serotonergic modulation. T102C polymorphism of the serotonin 2A (5-HT2A) receptor gene is associated with urinary incontinence (UI) that has been reported by older community dwellers. We analyzed the association between 5-HT2A receptor gene polymorphism and urodynamic tests for UI in older women.
Methods A case-control study was performed with 68 older women submitted to urodynamic evaluation and 162 older women without urinary problems (self-reported), all community dwellers enrolled in the Gravataí-GENESIS Project, Brazil. Clinical interviews, complete urodynamic evaluation following the International Continence Society Report on Good Urodynamic Practice (case group), and molecular analyses were performed (case and control groups). Serotonin 2A receptor gene genotyping was carried out by polymerase chain reaction-restriction fragment length polymorphism using the restriction enzyme HpaII. We excluded patients with diabetes mellitus, neurologic damage, and diuretic intake.
Results The subjects' mean (SD) age was 68.1 (6.5) years (range, 60-92 years). We found an association between the TT genotype versus CC + CT genotypes and UI (P = 0.013; odds ratio, 2.69; 95% confidence interval, 1.37-5.29) and, in addition, an association with urgency UI, maximal cystometric capacity (TT, 349 mL; CC + CT, 429.5 mL [P = 0.047]), detrusor pressure at maximum cystometric capacity (TT, 11 cm H2O; CC + CT, 6.75 cm H2O [P = 0.032]), and detrusor compliance (TT, 34 mL/cm H2O; CC + CT, 61.25 mL/cm H2O [P = 0.006]).
Conclusions We confirmed our previous findings of a genetic influence of the TT genotype on UI involving the serotonergic pathway among older women. Further investigations including 5-HT2A expression in the bladder, pelvic floor, and striated sphincter muscle must be performed.
Urinary incontinence (UI) is a problem with a significant impact on quality of life, affecting social, psychological, physical, and financial aspects, and has a higher prevalence in women, which is age related. Overactive bladder, characterized as a syndrome consisting of urgency with or without urge UI, is usually accompanied by frequency and nocturia. Approximately 30% of overactive bladder carriers have urinary leak.1
Studies showed that serotonin (5-HT) metabolism is an important modulator of the procontinence voiding reflex. Serotonin is a monoamine neurotransmitter that is ubiquitously distributed throughout the peripheral and central nervous systems and modulates functions that are vital for survival.2Experimental studies in animals have revealed that spinal reflex circuits involved in voiding exhibit a dense serotonergic innervation because multiple 5-HT receptors are present. Activity in the serotonergic pathway generally enhances urine storage by facilitating the bladder sympathetic reflex pathway and inhibiting the parasympathetic voiding pathway.
Thus, 5-HT receptor antagonists and reuptake inhibitors are considered important targets for the development of new treatments of detrusor overactivity and UI, such as duloxetine.3Studies have shown that 5-HT receptors in the Onuf nucleus facilitate sphincter contraction, increasing urethral resistance. Agonists that stimulate these receptors facilitate the guarding reflex, whereas antagonists that block these receptors inhibit this reflex.4
The 5-HT2 receptor subfamily consists of 3 subtypes, A, B, and C, and the subtypes that are of interest in micturition are the 5-HT2A and 5-HT2C receptors. These receptors are believed to have an excitatory parasympathetic action, as they couple preferentially to Gq/11 to increase the hydrolysis of inositol phosphates and to elevate cytosolic (Ca2+), although little is known about its role in sphincter regulation.5
The regulation of bladder function via central serotonergic modulation of these sensory pathways has been a focus in the development of pharmacological treatments of bladder dysfunction.6de Groat7reviewed the influence of the central serotonergic mechanism on lower urinary tract function and concluded that in experimental animals, spinal reflex circuits involved in voiding exhibited (a) a dense serotonergic innervation, (b) multiple 5-HT receptors, and (c) sensitivity to 5-HT receptor agonists and antagonists and to 5-HT reuptake inhibitors. The author also postulated that although there is some evidence for serotonergic facilitation of voiding in the rat, most experiments in both the rat and the cat indicate that activation of the central serotonergic system can suppress voiding by enhancing the efferent control of the urethral outlet and by inhibiting parasympathetic excitatory input to the urinary bladder. In a review, Cohen8concluded that there is marked species variability in responsiveness to 5-HT and indicated that contraction in response to 5-HT in the canine bladder is mediated by activation of the 5-HT2 receptor.8
As the expression of 5-HT2A receptors is controlled by nuclear genes, they can be influenced by genetic variations that could change their function. Epidemiological evidence suggests that some women have a genetic predisposition to the development of stress incontinence related to other structural and regulatory molecules other than 5-HT2A receptors.9
The genetic polymorphism related to 5-HT metabolism, the T102C polymorphism of the 5-HT2A receptor gene, has received considerable attention. Three genotypes, TT, CC, and TC, are possible. Although the T102C substitution does not alter amino acid sequence, there is evidence that total levels of 5-HT2A receptor messenger RNA and protein in healthy people with the CC genotype are lower than those in healthy people with the TT genotype.10Case-control studies have described an association between this polymorphism and several disturbances such as an association of the 102C allele with schizophrenia and other neuropsychiatric disorders11and with smoking12and an association between the T allele and nonfatal acute myocardial infarction.13Recent investigations also described an association between the heterozygous genotype (TC) and poorest performance of prefrontal executive cognition in healthy adults.14
A previous investigation by our team described an association between the TT genotype and UI among elderly community dwellers in Brazil.15In principle, we expected that TT carriers who have higher expression of membrane receptors should be more continent than TC and CC carriers because bladder relaxation is regulated indirectly by 5-HT and norepinephrine. Released at the medullary level, the role of both neurotransmitters is to facilitate bladder storage by increasing its capacity for accommodation (compliance). Serotonin acts by reducing acetylcholine activity (muscarinic receptors with parasympathetic activity), and norepinephrine causes relaxation of the detrusor by stimulation of its receptors, β3 subtype adrenergic receptors (in humans), localized in the bladder body.16
Because detrusor relaxation and the gain in compliance are not sufficient to maintain continence, stimuli of the pudendal nerve produce an adjuvant action in this function, through the release of acetylcholine (nicotinic receptors) that reinforces the contraction of the sphincter muscles and of the pelvic pathway (increased urethral resistance). In addition, the stimulation of the adrenoceptor (α1) of the proximal urethra, from the hypogastric nerves, reinforces the peripheral and central effector roles of the respective sympathetic and somatic controls or urethral function in the promotion of urinary continence.16
Because studies of urodynamic evaluation could be important in understanding the etiology of UI and in clinical application of this knowledge, we analyzed the association of T102C polymorphism of the 5-HT2A receptor gene and urodynamic parameters in older women.
PATIENTS AND METHODS
We performed a case-control observational study. (1) Case group included women who were defined by the International Continence Society as having "any involuntary leakage of urine."17First, we selected 84 incontinent older women who were seen at São Lucas Hospital during the period from July 2005 to December 2007, of whom 68 were without any associated diseases such as diabetes mellitus type 2 and use of diuretic drugs and neurodegenerative disturbances. It was possible not only to have the clinical and urodynamic analyses available but also to determine their 5-HT2A genotype. (2) Control group included continent older women from 1058 older participants of the GENESIS Project (Gravataí City) that studies gene-environmental interactions in successful aging and age-related disease. We selected 162 continent older women (self-reported) with previous analysis of the T102C polymorphism of the 5-HT2A receptor gene. (3) Population sample included 687 individuals (18-100 years old) who were previously investigated for the association of 5-HT2A and longevity. We excluded first- and second-degree relatives of subjects previously included to avoid genetic frequency bias.18We matched case and control subjects, and the sample age was 68.1 (6.5) years old (range, 60-99 years) showing no statistically significant difference between the 3 women groups evaluated.
Three UI subtypes were considered: incontinence of urgency, mixed, and stress incontinence. Stress UI is the complaint of involuntary leakage of urine on effort or exertion, or on sneezing or coughing. Urge UI is the complaint of involuntary leakage accompanied by, or immediately preceded by, a strong desire to void. Mixed incontinence is the complaint of involuntary leakage associated with urgency and also with exertion, effort, sneezing, or coughing; nocturnal enuresis is the complaint of loss of urine during sleep (the original definition of enuresis has been reasserted as that of any involuntary loss of urine).19
The study was submitted to the research ethics committee of Pontifícia Universidade Católica do Rio Grande do Sul affiliated with Conselho Nacional de Saúde, and the study protocol was approved (document numbers 226/01, 797/01, and 481/06). Written informed consent was obtained from all participants.
Urinary Evaluation
The urinary evaluation consisted of a clinical history, targeted physical examination, and urodynamic evaluation, which were all performed by the principal investigator. The apparatus used was the multichannel Urosystem Viotti (São Paulo, Brazil) using the criteria recommended by the Subcommittee on Standardization and Terminology of the International Continence Society (uroflowmetry, measure of post-voiding or bladder residual, filling cystometry, and pressure flow studies).20Control subjects were first evaluated by geriatric investigators, and the UI state was then confirmed by the same investigator who performed additional analysis in UI subjects.
Polymorphism Analysis
Serotonin 2A receptor gene polymorphism was determined from DNA isolated from lymphocytes using an extraction kit. Genotyping of the T102C polymorphism was performed by the polymerase chain reaction (PCR) method proposed by Warren et al.,21with minor modifications performed by do Prado-Lima et al.12Briefly, standard PCR was carried out in a 25-μL volume containing 100-ng genomic DNA, 200 μmol/L of each deoxyribonucleotide triphosphate, 1.5-mmol/L MgCl2, 250 nmol/L of the sense (5'-TGTGCTACAAG TTCTGGCTT-3') and anti-sense (5'-GTGCA GTTTTTCT CTAGGG-3') primers, and 0.6-U DNA Taq polymerase. The PCR products were digested with HpaII (BM), and the digestion products were separated by electrophoresis and visualized under UV light. The 102T allele PCR products remained uncut, with a single-DNA band of 342 base pairs, whereas the 102C allele showed 2 bands of 216 and 126 base pairs.
The genotyping data were subjected to the following quality measures. (a) Before the sample analysis was performed, a pilot genotyping analysis (N = 50) was conducted to verify the reliability of the 2 investigators in charge of these analyses in the study, which showed 100% concordance. (b) All genotyping data were recorded in a notebook with numbered pages. (c) When the image conditions were unsatisfactory, the genotyping sample was prepared anew. Furthermore, to improve the accuracy of the interpretation of our genotyping analyses, we used the Image Master VDS and Fuji Film Photography System FTI-500 (Pharmacia Biotech, Uppsala, Sweden).
Statistical Analysis
The allele and genotype frequencies were tested for Hardy-Weinberg equilibrium among cases, controls, and population groups. The significance of allele frequency or genotype distribution among case-control volunteers was examined by the nonparametric χ2 or Fisher exact test (2 tailed). Statistical analyses were performed using the SPSS/PC Statistical Package Version 11.0 (SPSS Inc, Chicago, IL). All P values were 2 tailed, and P < 0.05 was considered as statistically significant.
RESULTS
Baseline characteristics and T102C genotype and allele frequencies of control and case volunteers included in the study are described in Table 1.
The genotype frequencies for the 3 groups investigated were in Hardy-Weinberg equilibrium. Women with UI had a frequency of the TT genotype significantly greater in relation to continent women and in relation to the general sample of the population.
Additional statistical analysis was carried out to test dose-effect for the association of incontinence and T allele. The results showed that women with TT genotype had a significantly greater risk of UI than TC + CC women (P = 0.013; odds ratio, 2.69; 95% confidence interval, 1.37-5.29). On the other hand, women with CC genotype did not show this significant difference compared with TT women (P = 0.385; odds ratio, 0.68; 95% confidence interval, 0.33-1.4).
The urodynamic data compared in the 3 genotypes are presented in Table 2 as variations in medians and interquartiles (analyzed by a nonparametric test because most of the quantitative variables did not show a normal distribution).
The physical examination and urodynamic data are presented in Table 3.
As can be seen, genotype TT carriers showed significantly greater detrusor pressure at first desire to void than did CC carriers. The TT group also displayed a lower maximum cystometric capacity (than did TC and CC carriers) and reduced bladder compliance.
In the comparison of the additional urodynamic findings, a significant difference was observed in the type of UI in relation to genotype (Table 3). In the case group, TT carriers showed greater prevalence of urgency UI, whereas carriers of the genotypes CC and TC showed a greater prevalence of stress UI. The prevalence of mixed UI was similar among these genotypes.
DISCUSSION
In the present study, we investigated the association of the T102C polymorphism of the 5-HT2A receptor gene with UI in older women who live in a senior community and submitted to urodynamic analysis.
We found a significant association between TT and general UI when we compared the genotype and allele frequencies between case and control older women and when we compared the case group and a general population sample. However, when we analyzed the incontinence subtypes just in incontinent older women who underwent a urodynamic evaluation, we also found differences between 5-HT2A genotypes carriers. Older women with TT showed a higher frequency of urge incontinence than did CC and TC carriers. This association is explained when we observed in urodynamic evaluations that TT carriers had a detrusor pressure of first desire to void, higher detrusor pressure, lower volume at maximum cystometric capacity, and reduction of bladder compliance. Therefore, we suggest that the TT genotype is associated with risk of UI and that among the different UI subtypes, TT carriers have higher risk of urge incontinence than stress and mixed incontinences when compared with CC and TC carriers.
The results obtained confirmed the association between voiding dysfunction and the polymorphism investigated that was previously described by Schwanke et al.15The importance of these results is based on the following aspects. (1) The study conducted by Schwanke et al. had a different experimental design in which older community dwellers were selected and genotyped for the T102C polymorphism of the 5-HT2A receptor gene, and tests were performed as indicators of their health including UI. (2) The sample size was relatively small, and UI was evaluated only based on self-reporting by the older individuals. Thus, the presence of biases could have influenced the results obtained producing a false-positive association; (3) because it was a preliminary investigation, no complementary urologic evaluation with a urologic and urodynamic clinical anamnesis had been done. (4) Despite the limitations of the study by Schwanke et al., it is of extreme importance because investigations of the role of 5-HT receptors in urinary incontinency are a current theme in urologic research, being very actively pursued in animal models and in humans in a more limited manner. The importance of these studies is related, for example, to the use of 5-HT reuptake inhibitors that are used in the treatment of UI. Despite the wide use of these medications, as in the case of duloxetine, studies are lacking in serotonergic physiologic dynamics. (5) In addition, there is a need for more studies on the influence of the gene-environment interaction in UI associated with biological aging because this disturbance is highly prevalent in older women independent of other risks.
The importance of investigating urodynamic aspects in the association of UI with genetics is based on the following considerations. An adequate investigation of UI should be based primarily on clinical history. Urodynamics as a science applicable in the clinic is relatively recent. Technological advances have broadened their use in the investigation of physiopathologic states of bladder function. The indication for complementary diagnostic urodynamic investigation has been discussed for female patients with a classic history of stress UI and for older individuals with good response to initial conservative treatment of UI. Despite being an invasive method, it is considered the method of choice in the investigation of dysfunction of the lower urinary tract. In the case group, the clinical and urodynamic evaluation of UI of older persons should be indicated for patients in situations of obtaining some type of benefit from this assessment, that is, if it influences the choice and success of treatment.22
When the urodynamic data were compared among the 3 genotypes, it was seen that TT carriers showed a significantly higher pressure at first desire to void, higher detrusor pressure, and smaller volume at maximum cystometric capacity, besides reduced bladder compliance. This picture is compatible with the physiologic aspects directly related to the symptoms of UI. In addition, these results were reinforced by the occurrence of a greater prevalence of urge UI in TT carriers, whereas women with the CC and TC genotypes had a higher prevalence of stress UI.
What is the possible functional role of the 5-HT2A in increasing the risk or worsening of UI in older individuals? If we consider the role of 5-HT/receptors in the control of micturition and T102C polymorphism, we can make the following statements:
(1)There is evidence that the TT genotype results in approximately 20% more 5-HT2A than CC genotype and thereby would show greater response to 5-HT in its target tissues.
(2)The physiologic process of urination, which involves the relaxation of the bladder's body and the contraction of the urethral complex, is influenced by 5-HT, through an indirect action, in combination with other neurotransmitters;
(3)The pharmacologic treatment of this condition uses 5-HT reuptake inhibitors in the synapse to enhance continence.
It would be expected that TT individuals make up procontinents instead of proincontinents. This apparent paradox seems to have its origin in a complex network of relationships that involve the interaction of various 5-HT receptors with respect to their expression and localization and the differential interaction of 5-HT with other regulatory substances with or without neural action.
Animal model studies are currently exploring the role of serotonergic receptors in different tissues of the lower urinary tract via medullary or supramedullary routes that are involved in micturition control. Most investigations analyze the action of agonists and antagonists of these receptors in the voiding response. Mbaki and Ramage23published a study on the role of 5-HT2A receptors in the regulation of micturition. These authors pointed out that these receptors are especially implicated in the control of the urethra, which is the target in the treatment of UI, such as duloxetine. The study was conducted in anesthetized female rats for the purpose of determining the role of each of 3 subtypes of 5-HT2 receptors (A, B, and C) in sphincter regulation. The effects of agonists and antagonists were determined in each of the receptor subtypes about the pressure of the urethra, the bladder, and the external urethral sphincter, to voiding reflex induced by bladder distention and also to systemic arterial pressure and heart rate. The results showed that the 5-HT2A and 5-HT2C receptors located in the sacral medulla activated the striated external sphincter, whereas the 2C receptors inhibited voiding reflex and the 2B receptors probably acted at the level of the urethra, augmenting the tone of the sphincter smooth muscles. Besides this, the study showed that the 5-HT2B and 5-HT2C receptors did not play an important role in the physiologic regulation of micturition. However, the 2A receptors seem to be involved in the voiding reflex through the activation of the striated urethral sphincter. The reason is that in the presence of antagonists of the 2A receptor, as in the case of ketanserin and MDL100907, there was an increase in the volumetric capacity of the bladder and inhibition of its isovolumetric contractions. Although this was a study in an experimental model with limitations with respect to translation to humans, this information suggests that the increase in the expression in the 2A receptor in the detrusor muscle of the bladder could diminish the induction of its relaxation and cause difficulty in urine storage and, consequently, setting off the voiding reflex.
Another study examined the changes in response of the detrusor muscle via 5-HT receptors in diabetic rats by analyzing segments of detrusor muscle treated with saline to induce isotonic contraction and with 5-HT receptor antagonists to induce relaxation. Regarding these results, it is important to point out that the 5-HT2A receptor antagonists inhibited the contractile response of 5-HT in a dose-dependent manner.24
Xu et al.25found a preferential localization of 5-HT2A expression in striated muscles in pelvic floor with a paracrine mode of action (showing inhibitory or excitatory responses or even variable intensity through the regulation of other serotonergic or nonserotonergic receptors such as noradrenergic and cholinergic receptors) and very weak expression in Onuf nucleus in female rats.
Although the previously cited studies were carried out in experimental models, the results of these 2 studies are in agreement with our urodynamic findings described here. The reason is that carriers of the TT genotype showed only higher pressure and smaller volume of maximal cystometric capacity and reduced compliance, which indicates an action of the 5-HT2A receptor in the modulation of bladder contraction. Unfortunately, a review of the literature did not show similar studies in humans that could support this hypothesis.
Tissue expression of the receptors in various anatomic segments could confirm and elucidate the role of the 2A receptor in the modulation of bladder and sphincter activities. Such strategy is part of the future aims of our research.
CONCLUSIONS
We confirmed the previous findings of a genetic influence of 5-HT2A on UI in older women related to the TT genotype. Further investigations including 5-HT2A expression in the bladder, pelvic floor, and striated sphincter muscle must be performed in humans.
ACKNOWLEDGMENTS
We are indebted to Dr Albert Leyva (Research Publications, Kansas City, MO) for English editing, Dr Ceres Oliveira for statistical analysis, Rosária Geremia for review of references, and Pontifícia Universidade Católica do Rio Grande do Sul for the doctoral fellowship awarded to Jorge Noronha.