An effective closure of the female urethra in stress situations is dependent on an integrated action of various anatomical intra- and extraurethral structures. The most important extraurethral structures - from a functional aspect - are the suburethral vaginal wall, the pubourethral ligaments, the pubococcygeus muscles and the paraurethral connective tissue. An important ingredient in the supportive structures of the genitourinary region is fibrous connective tissue, consisting mainly of collagen and structural glycoproteins. Hence, defects in the actual connective tissue – in particular the paraurethral connective tissue that connects the aforementioned structures to each other and to the urethra – will bring about an ineffective urethral closure. Female genuine stress incontinence (GSI) may be caused by defective connective tissue per se and / or by a disconnection to extraurethral structures, whereby the urethra cannot be closed at stress situations. Genital prolapse may or may not be associated with GSI, or may even be masked due to the extent of pelvic floor relaxation. However, little knowledge exists why some patients with prolapse develop GSI and some do not.

 

Aims of study:

The status of paraurethral connective tissue in postmenopausal female patients was investigated for relevant changes of paraurethral connective tissue between continent and incontinent women with genital prolapse.

 

Methods:

Before pelvic reconstructive surgery patients underwent a complete urogynecologic assessment including history, urinalysis, evaluation of residual bladder volume, medium fill cystometry, urethral pressure profile measurements (UPP) at rest and during stress, and a clinical stress test in the supine and standing position. A pronounced prolapse was repositioned during UPP measurements and during a clinical stress test to assess or exclude GSI. All patients underwent pelvic floor reconstructive surgery (anterior colporrhaphy: n=19, sacrospinous fixation: n=8). Patients with proven GSI received additional tension-free vaginal tape procedure.

 

During pelvic floor reconstructive surgery biopsies from both paraurethral regions were obtained. Biopsies were investigated for localization and distribution of both collagen (types I, III, IV, V, VI) and glycoproteins (fibronectin, laminin, vitronectin) using immunofluorescent microscopic techniques. Frozen biopsies were incubated with primary polyclonal antibodies (1:25 dilution in PBS) at room temperature for 60 minutes. Three washings with a 0.2% PBS-BSA solution and exposure to Trimethylrhodamin-isothiocyanat (TRITC)-conjugated second antibodies (swine anti-rabbit immunglobulins [Dakopatts, Denmark] diluted 1:20 dilution in PBS) were performed to visualize the protein. After another washing with PBS-BSA solution, photographs of the samples were taken (Kodak Ektachrome 400) for documentation.

 

Results:

GSI was present in 10/19 women (Group A, mean age: 56,2 years), whereas 9/19 patients (Group B, mean age: 59,6 years, p > 0.05) were continent.

Irrespective of the presence or absence of GSI all types of collagen (I,III, IV, V, VI) were found in the biopsies of the whole study group. The tissues of Group A patients showed a marked weaker immunohistochemical reaction of type I, III and VI collagen compared with the specimen of group B patients.

No difference of Type IV and V collagen was observed between the biopsies of group A and B patients. Type V collagen was located in the subepithelial connective tissue zone of the stroma, touching the basement membrane and forming a fibrillar meshwork. Type IV collagen was selectively found in the zone of basal membrane and vessel walls.

 

Among the structural glycoproteins fibronectin and laminin were found in the specimen of all patients. However staining of fibronectin was less pronounced than that of collagen. Nevertheless, fibronectin was distinctly found in the extracellular matrix. The stroma revealed a fine fibrillar matrix reaching to the basement membrane. Laminin showed a similar distribution in the basement membrane as type IV collagen.

Vitronectin was be observed in the paraurethral connective tissue of all group B patients, whereas this glycoprotein was lacking in the biopsies of group A women. In vitronectin positive tissues the stained structures were granular-like or fibrous and sometimes amorphous. A striking association of vitronectin with elastic fibres was seen.

 

Conclusions:

There is a complex architecture of the extracellular matrix in the female paraurethral region with marked differences between postmenopausal, continent women and patients with GSI, irrespective of the presence of pelvic floor relaxation. Our findings suggest a selective and altered metabolism of connective tissue in the paraurethral region responsible for the onset of GSI in patients with pelvic floor relaxation.