Characterization of cells isolated from suburethral mucosa of women with stress urinary incontinence (SUI) and pelvic organ prolapse (POP). A comparative study

Flores A I1, Perez-Lorenzo M J1, de la Torre P1, Muñoz-Gálligo E2, Masero-Casasola A R2, García-García-Porrero A2, Gutiérrez-Vélez M C2, Medina-Polo J2, García-Muñoz H2, Grande-García J2, Alcazar-Garrido A1, Vielsa-Gordillo I2

Research Type

Basic Science / Translational

Abstract Category

Female Stress Urinary Incontinence (SUI)

Abstract 286
Female Incontinence
Scientific Podium Short Oral Session 16
Thursday 30th August 2018
10:20 - 10:27
Hall C
Basic Science Cell Culture Stress Urinary Incontinence Pelvic Organ Prolapse Molecular Biology
1. Instituto de Investigacion Hospital 12 de Octubre (imas12), 2. Instituto de Investigacion Hospital 12 de Octubre (imas12), Hospital 12 de Octubre

Ana I Flores



Hypothesis / aims of study
Urinary incontinence (UI) in women is a common and costly problem. Stress urinary incontinence (SUI) happens when a physical movement or activity places pressure or stress over the bladder, and the anatomic structures which maintain continence –urethral sphincter, urethra, pelvic floor muscles and so on- are unable to counteract it. In this way, SUI occurs when the pelvic floor muscles and the muscles of the urinary sphincter weaken. Pregnancy, vaginal delivery, being overweight and age are some of the related risk factors to develop SUI. 
Some authors have proposed that pelvic floor tissues of women with SUI are altered, not only at an anatomical way, but also, at a cellular and a molecular level. Due to the alterations observed in SUI patients, there should be some mechanisms of reparation. When a tissue is injured, a sequence of overlapping phases takes place to repair the injured tissue. Tissue repair occurs in three phases, the inflammatory, proliferative, and remodeling phases. Myofibroblasts play a crucial role in tissue repair. Myofibroblasts are found, in both, the proliferative and the remodeling phases playing an important role in producing extracellular matrix, including collagen. Myofibroblast are defined as fibroblast-like cells that express α-smooth muscle actin (α-SMA). However, except for the contractility of myofibroblasts, functional similarities and differences between fibroblasts and myofibroblasts have not been fully elucidated. Moreover, myofibroblasts have morphological characteristics similar to those of both fibroblasts and smooth muscle cells. 
Our aim was to isolate and characterized individual populations of myofibroblast cells in culture from the suburethral mucosa of patients with stress urinary incontinence (SUI) and pelvic organ prolapse (POP) without SUI and to analyze the possible differences between them in both populations.
Study design, materials and methods
Patients were recruited at the Department of Obstetrics and Gynecology from the Hospital. Informed consent was approved by the Ethics Committee and after informed consent was obtained, biopsies were collected from both groups of patients.
Myofibroblast cell cultures were established from suburothelial layer (1 cm x 0.5 cm) by mincing the tissue with 0.25% trypsin-EDTA with 0.15% collagenase type II for 30 min at 37ºC. Cells were pelleted at 1500g for 10 min and resuspended in DMEM with 10% fetal bovine serum. Culture medium was changed every 3 days. Cells were counted at different passages and the number of accumulated cell number was calculated. The expression of several genes was analyzed by quantitative real-time PCR in alternate passages during the expansion culture, from passage 3 to passage 11. Immuflorescence technique was analyzed the presence of α-SMA protein in early (p3), intermediate (p7) and late (p11) passages. Total fluorescence per cell was calculated using the Image J software. An average of 12-15 photos were analyzed for each passage. To calculate the corrected total cell fluorescence (CTCF) we used the formula CTCF = Integrated Density – (Area of selected cell X Mean fluorescence of background readings). 
Data are expressed as means ± SEM or SD. Statistical differences between experimental groups were determined by Student’s t test (unpaired, two-tailed). A p value of less than 0.05 was considered significant. Prism 6.0 (Graphpad) was used for statistical analyses.
The results showed that the suburethral myofibroblasts isolated from the mucosa of patients with SUI or POP have different growth capacity (Figure 1). Besides, the expression of genes and proteins characteristic of the myofibroblasts was also different in the two types of patients (Figure 2). α-SMA positive cells were observed by immunofluorescence in myofibroblast cell culture of SUI patients (Figure 3A). The total cell fluorescence (CTCF) showed that SUI patients have higher number of α-SMA positive cells and the fluorescence was significantly higher than the CTCF from POP patients (Figure 3B).
Interpretation of results
POP myofibroblasts have significantly higher growth capacity than SUI myofibroblats suggesting differences between the two populations of cells. 
Desmin, CD90 and α-SMA are characteristic genes of myofibroblasts. SUI patients present an elevated expresión in these genes compared to POP patients. CD90 is a marker for a variety of stem cells suggesting that SUI patients have a population of de-differentited cells, the myofibroblast. These myofibroblasts are probably involved in the pathology of the disease and are trying to repair the injured tissue.
Isolated cells from SUI patients have a bigger number of positive α-SMA cells as observed by immunofluorescence. In addition, the fluorescence of the positive cells was more intense as calculated by Image J, indicating a higher presence of the protein inside of the cells. These results suggest that these cells are myofibroblasts compared to cells isolated from POP patients where cells seem to have a morphology more similar to fibroblasts.
Concluding message
These findings could be important in order to achieve a better understanding of the mechanism involved in the pathogenesis of SUI in women, and in the physiological ways of restoration of continence. Perhaps these ways could be effective in some patients, but not in others –spontaneous regression of SUI has been described- but this needs to be elucidated.
Figure 1
Funding This work was funded by project PI 15/01803, from the Instituto de Salud Carlos III (Ministry of Economy, Industry and Competitiveness) and cofunded by the European Regional Development Fund. Clinical Trial No Subjects Human Ethics Committee Ethics Committee Hospital 12 Octubre (16/275). Helsinki Yes Informed Consent Yes