FEMALE UROLOGY Neuronal Nitric Oxide Synthase Expression in the Anterior Vaginal Wall of Patients with Stress Urinary Incontinence Levent Ozcan1*, Emre Can Polat2, Efe Onen1, Alper Otunctemur2, Emin Ozbek2, Adnan Somay3, Nurver Ozbay3 Purpose: Stress urinary incontinence (SUI) and pelvic organ prolapse (POP) are common medical problems, par- ticularly among older women. In this study, we aim to explore the relationship between the neurotransmitter nNOS in the vaginal epithelium, and the occurrence of SUI and changes of nNOS levels according to menopausal status. Matherials and Methods: Fourty women were enrolled. The patients were divided into four groups according to menstruaiton status and SUI. The vagina specimens were taken during transobturator tape application. The speci- mens were examined pathologically in terms of n-NOS expression. nNOS expression was compared between SUI and control groups. The results were evaluated statistically. Result: Epithelial total nNOS score in group 1 and group 3 were 2.4 ± 0.5 and 1.4 ± 0.5 respectively (P = .003). Stromal total nNOS score was found 2.2 ± 0.4 in group 1 and 1.3 ± 0.5 in group 3 (P = .001). Epithelial total nNOS score in group 2 and group 4 were 4.4 ± 0.5 and 3.5 ± 0.5 respectively (P = .003). Stromal total nNOS score was found 4.4 ± 0.5 in group 2 and 3.6 ± 0.5 in group 4 ( P = .006). Conclusion: Our results show that expression of nNOs in the anterior vaginal epithelium decreased significantly in the SUI group. Altough our findings indicate important results, well designed further studies are needed to com- prehend the role of NOS pathways better in SUI pathophysiology. Key words: human vaginal tissue; nitric oxide; pathophysiology; pelvic floor disorders; stress urinary incontinence INTRODUCTION Stress urinary incontinence (SUI) and pelvic organ prolapse (POP) are common medical problems, par- ticularly among older women. They can have a signifi- cantly negative impact on the quality of life, and yet less than half of women with urinary incontinence seek med- ical attention. These two diseases have an etiologically close relationship.(1)Pelvic floor tissues, including vagi- nal wall tissues, are rich in nerve fibers and functionally protect the normal position of pelvic organs.(2) The ef- fects of pelvic support weakness may induce the devel- opment of SUI.(3) Changes in some neuropeptides in the pelvic floor tissue have been observed in SUI and POP patients.(4) Vasoactive intestinal peptide (VIP) is one of the neuropeptides widely distributed in the vaginal wall that serves as a local neurotransmitter or neuromodu- lator. However, the underlying neuropathophysiology of SUI is unclear. Nitric oxide (NO) is an anorganic free-radical gas that stimulates soluble guanylyl cyclase activity and creates smooth muscle relaxation.(5) There are three NOS isoforms:, neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). Neuronal NOS (nNOS) is present in neuronal tissues; neuronal excitation in- creases Ca+2 concentration within nerves, which leads to synthesis of NO from L-arginine. Elucidation of the physiologic role of NO and nitric oxide synthase (NOS) is not only of research interest but may also provide a basis for therapeutic interventions in patients with low- er urinary tract dysfunction. It is also important as a me- 1Derince Training and Research Hospital, Department of Urology, Kocaeli, Turkey. 2Okmeydani Training and Research Hospital, Department of Urology, Istanbul, Turkey. 3Fatih Sultan Mehmet Training and Research Hospital, Department of Pathology, Istanbul, Turkey. *Correspondence: Derince Training and Research Hospital, Department of Urology, 41900 Derince, Kocaeli, Turkey. Tel: (+90) -262-317 8000. Fax: (+90)- 262- 233 4641. E-mail: drleventozcan@yahoo.com. Received May 2016 & Accepted December 2017 diator in the female and male reproductive tracts.(6-8) In this study, we aim to explore the relationship between the neurotransmitter, nNOS in the vaginal epithelium, and the occurrence of SUI and changes of nNOS levels according to menopausal status. PATIENTS AND METHODS Study population Institutional review board approval was obtained and all participants signed an informed consent before being enrolled in the study. Patients who attended our clinic with the complaints of incontinence and were detect- ed to have SUI were included in the study. To evaluate incontinence, detailed incontinence history, age, body mass index (BMI), number of deliveries and previous surgical history were recorded. On physical examina- tion, we accessed the women for SUI at gynecologic position, and we performed cough stress test. Fourty women were enrolled. The patients were cat- egorized into four groups according to menstruaiton status and SUI. Premenopausal SUI formed group 1, premenopausal controls formed group 2 and postmen- opausal SUI formed group 3, postmenopausal controls formed group 4. Each group had 10 cases. The vagina specimens were taken during transobturator tape appli- cation. The specimens were examined pathologically in terms of n-NOS expression. The results were evaluated statistically. Control groups were choosen among heal- ty women who had surgery for benign reasons such as Female Urology 280 Vol 15 No 05 September-October 2018 281 ureterorenoscopy or urethral caruncle. Excluded from the study were women who had previous endometriosis, adenomyosis, uterine fibroids, connec- tive tissue disorders, pelvic inflammatory conditions, or pelvic surgery. None of the patients took hormonal drugs during the 3 months before surgery. And none of the patients had POP. Immunohistochemistry The tissue sections were fixed in 10% buffered formalin for about 24 h, embedded in paraffin, and 5 µm sec- tions were then deparaffinized. Then the sections were immersed in antigen retrival solution (Biogenex) and treated in microwave oven for 10 minutes. After cool- ing, the sections were washed with Phosphate Buffered Solution (PBS). The incubation with the primary anti- body was done in a solution of 0.8% BSA and 20 mM NaN3 in PBS containing the nNOS–specific mouse antibody (1:100 dilution) for 1 h at room temperature. After rinsing with PBS the sections were incubated with the secondary biotinylated goat antimouse antibody for 30 min at room temperature. Then, an alkalene phos- phatase complex was utilized as a detection system (1:200 dilution) for another 1 hr. Finaly, the staining was developed for 15 min with fast red solution and counterstained with Mayer’s hematoxylin. Negative controls sections were incubated in the absence of the primary antibody. The nNOS immunoreactivity of the specimens were rated according to a score that was calculated using in- tensity and area scores. The intensity of staining was on the following scale: 0, no staining of nNOS in the vagina; 1+, mild staining; 2+, moderate staining and 3+, marked staining. The area of staining was evaluated as follows: 0, no staining of in the vagina in any micro- scopic field; 1+, 0-25% of the vagina stains positive; 2+, 25-50% staining positive; 3+, 50-75% staining pos- itive; 4+, 75-100% staining positive. Statictical Analysis All statistical analysis was performed by using SPSS ver. 15.0 (SPSS Inc., Chicago, IL, USA). Epithelial and stromal scores of nNOS were compared in four groups using Kruskal-Wallis H test and Mann-Whitney U test. In all comparisons of values, p-values of less than 0.05 were considered to be statistically significant. RESULTS There were no significant differences in age, BMI (cal- culated as weight in kilograms divided by height in me- ters squared), or parity among the four groups. Charac- teristics features of the patients are shown in Table 1. nNOS immunostainings were shown in the anterior vaginal wall in all groups. nNOS immunostainings at different density were observed , as demonstrated by a mild, moderate, or marked staining accordingly. In women with SUI, only scattered nNOS with weak stain- ing was observed. (Figures 1, 2 and 3). However, in postmenopausal women without SUI, the intensity and distribution of nNOS decreased and showed dispersed and moderate staining (Figures 4 and 5). Marked ep- ithelial and stromal staining with nNOS was observed in a premenopausal patient without SUI (Figures 6,7). Comparison of nNOS expression is shown in Table 1. As shown in Table 1, the group with the most decreased Table 1. Characteristics of patients and comparison of nNOS expression among the four groups. Group1 Group2 Group3 Group4 p Age 47.1 ± 5.1 46.5 ± 4.1 50.2 ± 2.7 49.6 ± 2.9 .126 Paritiy 3.2 ± 1.1 2.8 ± 1.4 3.2 ± 1.3 2.9 ± 1 .781 BMI (kg/m2) 30.5 ± 3.3 27.8 ± 2.9 29.6 ± 1.3 28.3 ± 2.5 .126 nNOS epithelial 2.4 ± 0.5 4.4 ± 0.5 1.4 ± 0.5 3.5 ± 0.5 .001 nNOS stromal 2.2 ± 0.4 4.4 ± 0.5 1.3 ± 0.5 3.6 ± 0.5 .001 nNOS epithelial 2.4 ± 0.5 1.4 ± 0.5 .003 nNOS stromal 2.2 ± 0.4 1.3 ± 0.5 .001 nNOS epithelial 4.4 ± 0.5 3.5 ± 0.5 .003 nNOS stromal 4.4 ± 0.5 3.6 ± 0.5 .006 Figure 1. Mild epithelial staining (1+) with nNOS in a patient pre- menopausal with sui Immunostaining x100 Figure 2. Mild epithelial staining (1+) with nNOS in a patient postmenopausal with sui Immunostaining x40 Stress urinary incontinence and nNOS-Ozcan et al. expression of nNOS is Group 3. Epithelial total nNOS scores in group 1 and group 3 were 2.4 ± 0.5and 1.4 ± 0.5, respectively with statisti- cally significant differences between the two groups (P = .003). Stromal total nNOS score was found to be 2.2 ± 0.4 in group 1 and 1.3 ± 0.5 in group 3. There were sta- tistically significant differences between the two groups ( P = .001) (Table 1) (Figures 1,5) Epithelial total nNOS score in group 2 and group 4 were 4.4 ± 0.5and 3.5 ± 0.5, respectively with statistically significant differences between the two groups (P = .003). Stromal total nNOS score was found to be 4.4 ± 0.5 in group 2 and 3.6 ± 0.5 in group 4. There were sta- tistically significant differences between the two groups ( P = .006) (Table 1) (Figures 3,4). DISCUSSION Pelvic floor disorders (PFD),including POP and SUI, are major health problems in women. The causes of PFD are multifactorial, including defect in the pelvic floor musculature and connective tissue weaknesses.(9) Another study has suggested that damage to the inner- vation of the pelvic floor can be an important factor in the etiology of SUI.(10) Neuropeptides, which are con- sidered a marker of nerve damage, are used in the study of pelvic floor dysfunction. A neuropeptide is a pep- tide released by different tissues, that acts as a neural messenger. Neuropeptides act as neurohormones, neu- rotransmitters, or neural modulators. Neuropeptides are widely distributed in the central and peripheral nervous system and in many areas of the human body, including the gut, pancreas, heart, lung, and genital tract.(11) Neu- ropeptides are well-represented in the innervation of the human female reproductive tract. Neurons that contain vasoactive intestinal polypeptide (VIP) are abundant in the vagina, where they innervate blood vessels and smooth muscle in the vaginal wall, and form a plexus beneath the epithelial layer.(12) Neuropeptide Y (NPY) is also abundant in neurons in the human female gen- ital tract, and is contained in nerves innervating blood vessels, as well as in nerves that form a subepithelial plexus.(13) Previous studies have shown that VIP levels were sig- nificantly decreased in the anterior vaginal wall in pre- menopausal and postmenopausal SUI or POP patients. Hu et al. found that the expression of VIP in the vaginal epithelium significantly decreased in POP patients.(14) In Figure 5. Moderate stromal staining (2+) with nNOS in a patient postmenopausal without sui Immunostaining x100 Figure 6. Marked epithelial staining (3+) with nNOS in a patient premenopausal without sui Immunostaining x100 Figure 3. Mild stromal staining (1+) with nNOS in a patient post- menopausal with sui Immunostaining x100 Figure 4. Moderate epithelial staining (2+) with nNOS in a patient postmenopausal without sui Immunostaining x100 Stress urinary incontinence and nNOS-Ozcan et al. Female Urology 282 Vol 15 No 05 September-October 2018 283 another study, Falconer et al. showed that women with SUI have a significantly lower total innervation of the paraurethral vaginal epithelium than controls without incontinence.(15) Alm et al.(16) reported the existence of vasoactive intestinal peptide (VIP) containing nerves in the vaginal wall. VIP-containing nerves have been de- scribed throughout the human female genital tract, and are most abundant in the vagina, cervix, and clitoris.(2) From studies on laboratory animals it has been suggest- ed that NO may also function as a neurotransmitter in male and female genital organs, including the uterus and vagina.(17-20) and can mediate neurogenic vasodila- tation. Hoyle et al. reported that nerves that utilise nitric oxide, NPY, VIP, or CGRP as a neurotransmitter may play a role in controlling blood flow and capillary per- meability in the human vagina.(21) Nerve and supportive tissue injury caused by prior pregnancy and delivery increasingly deteriorates as the patients ages.(22) As a component of the “hammock” according to the theory presented by Delancey(23), the anterior vaginal wall with decreased neurotransmitter content might alter the tension of the hammock, which contributes to the pathogenesis of SUI. In light of previous studies we hypothesized that NOS secreted by the neuropeptidergic fiber terminals take part in the pathogenesis of SUI. In the present study we investigated nNOS expression in the anterior vaginal wall because the anterior vaginal wall plays a more im- portant role than the posterior vaginal wall in the etiol- ogy of SUI(11). Our results confirmed that nNOS levels decreased significantly in the anterior vaginal wall in SUI patients, supporting the possible role of nNOS in the pathophysiologic process of SUI. Another result of this study is that, nNOS expressions are related to menopausal status. Compared to preman- opausal control patients with postmenopausal control patients, nNOS levels decreased in postmenopausal period. This result shows clearly nNOS expressions is decrease in postmenopausal period. The precise mechanism for the decrease of nNOS re- mains unclear. Lower nNOS in patients with SUI may either be related to the lower neuronal production of NOS or to age-induced nerve degeneration. Another possible explanation is that nNOS alterations affect the pathophysiological process via the local blood supply and nutrition state. Figure 7. Marked stromal staining (3+) with nNOS in a patient premenopausal without sui Immunostaining x100 In summary, the present study constituted a specific as- sessment of the relationship between nNOS expression and the development of SUI. The present data could not determine whether differences in nNOS innervation are the cause of SUI or whether they result from the patho- logical changes caused by SUI. CONCLUSIONS In SUI patients, pelvic support tissue becomes weak- ened with age, and has a stronger correlation with nNOS containing nerves. However, the underlying neu- ropathophysiology of SUI is still unclear. The lack of western blotting analysis is a limitation of our study. Well designed further studies are needed to comprehend the role of nNOS pathways in SUI pathophysiology. CONFLICT OF INTEREST None declared. REFERENCES 1. Molander U, Milsom I, Ekelund P, Mellström D. An epidemiological study of urinary incontinence and related urogenital symptoms in elderly women. Maturitas. 1990; 12:51–60. 2. Hong X, Huang L, Song Y. Role of vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide in the vaginal wall of women with stress urinary incontinence and pelvic organ prolapse. Int Urogynecol J Pelvic Floor Dysfunct. 2008; 19:1151–7. 3. Smith AR, Hosker GL,Warrell DW. The role of partial denervation of the pelvic floor in the aetiology of genitourinary prolapse and stress incontinence of urine. A neurophysiological study. Br J Obstet Gynecol. 1989; 96:24–8. 4. Busacchi P, Perri T, Paradisi R et al. Abnormalities of somatic peptide-containing nerves supplying thepelvic floor of women with genitourinary prolapse and stress urinary incontinence. Urology. 2004;63:591–595. 5. Dokita S, Smith SD, Nishimoto T, Wheeler MA, Weiss RM. Involvement of nitric oxide and cyclic GMP in rabbit urethral relaxation. Eur J Pharmacol. 1994; 15;266:269-75. 6. Yallampalli, C, Izumi, H., Byam-Smith, M. and Garfield, R.E: An Larginine- nitric-oxide- cyclic guanosine monophosphate system exists in the uterus and inhibits contractility during pregnancy. Am. J. Obstet. Gynecol. 1993; 170, 175-85. 7. Rosselli, M., Imthurn, B., Macas, E. et al. Endogenous nitric oxide modulate endothelin-1 induced contraction of bovine oviduct. Biochem. Biophys. Res. Comm. 1994; 201, 143-6. 8. Adams, M.L., Nock, B., Troung, R. and Cicero, T.J. Nitric oxide control of steroidogenesis; endocrine effects of Nl-nitro-L-arginine and comparisons to alcohol. Life Sci. 1992; 50, 35-40. 9. Ozbek E, Polat EC, Ozcan L, Otunctemur A, Emrence Z, Ustek D.TT polymorphism Stress urinary incontinence and nNOS-Ozcan et al. in rs2165241 and rs1048661 region in lysyl oxidase like-1 gene may have a role in stress urinary incontinence physiopathology. J Obstet Gynaecol Res. 2013;39:237-42. 10. Gilpin SA, Gosling JA, Smith AR, Warrell DW. The pathogenesis of genitourinary prolapse and stress incontinence of urine. A histological and histochemical study. Br J Obstet Gynaecol. 1989; 96:15–23. 11. Zhu L, Lang J, Jiang F, Jiang X, Chen J. Vasoactive intestinal peptide in vaginal epithelium of patients with pelvic organ prolapse and stress urinary incontinence. Int J Gynaecol Obstet.2009; 105:223-5. 12. Palle C, Ottesen B, Jorgensen J, Fahrenkrug J. Peptide histidine methionine and vasoactive intestinal polypeptide: occurrence and relaxant effect in the human female reproductive tract. Biology of Reproduction 41, 1103-1111, 1989. 13. Jorgensen JC, Sheikh SP, Forman A, Norgard M, Schwartz TW, Ottesen B. Neuropeptide Y in the human female genital tract: localization and biological action. American Journal of Physiology. 1989; 257, E220-E227. 14. Hu JM, Cheng X, Wang L, Zhu JN, Zhou LH. Vasoactive intestinal peptide expression in the vaginal anterior wall of patients with pelvic organ prolapse.Taiwan J Obstet Gynecol. 2013;52:233-40. 15. Falconer C, Ekman-Ordeberg G, Hilliges M, Johansson O: Decreased innervation of the paraurethral epithelium in stress urinary incontinence women. Eur J Obstet Gynecol Reprod Biol.1997; 72:195–198. 16. Alm P, Alumets J, Hakanson R et al. Vasoactive intestinal polypeptide nerves in the human female genital tract. Am J Obstet Gynecol. 1980; 136:349–51. 17. Kummer W, Fischer A, Mundel P et al. Nitric oxide synthase in VIP-containing vasodilator nerve fibres in the guinea-pig. Neuroreport. 1992; 3, 653-655. 18. Ding Y-Q, Wang Y-Q, Qin B-Z, Li J-S. The major pelvic ganglion is the main source of nitric oxide synthase-containing nerve fibers in penile erectile tissue of the rat. Neuroscience Letters. 1993; 164, 187-189. 19. Grozdanovic Z, Mayer B, Baumgarten HG, Brutning G. Nitric oxide synthase-containing nerve fibers and neurons in thegenital tract of the female mouse. Cell and Tissue Research. 1994; 275, 355-360. 20. Lincoln J, Hoyle CHV, Burnstock G: Transmission: nitric oxide. In Autonomic Neuroeffector Mechanisms (ed. Burnstock G, Hoyle CHV), pp. 509-539. Chur: Harwood Academic, 1995. 21. Hoyle CH, Stones RW, Robson T, Whitley K, Burnstock G.Innervation of vasculature and microvasculature of the human vagina by NOS and neuropeptide-containing nerves.J Anat Jun;188 ( Pt 3):633-44, 1996. 22. Kerns JM, DamaserMS, Kane JM et al. Effects of pudendal nerve injury in the female rat. Neurourol Urodyn. 2000; 19:53–69. 23. DeLancey JOL: Structural support of the urethra as it relates to stress urinary incontinence: the hammock hypothesis. Am J Obstet Gynecol. 1994; 170:1713–23. Stress urinary incontinence and nNOS-Ozcan et al. Female Urology 284