Proteomic Pattern in Female Stress Urinary Incontinence: a Pilot Study

Methods:

Sample preparation and analysis will include:

• Sample collection and immediate addition of protease inhibitor cocktail (Roche, Complete Protease Inhibitor Cocktail).
• Urine centrifugation and filtration for removal of cell debris.
• Protein precipitation by applying methanol/chloroform separation for removal of all possible non-proteinic substances.
• Protein digestion applying in-solution trypsin, pepsin, and Glu-C.
• Peptide separation using nano HPLC and different chromatographic approaches.

• Reversed phase (RP) separation of peptides and MS detection
• Separation of peptides using HILIC (hydrophilic Interaction Liquid Chromatography) for discrimination of polar peptides.
• In addition to RP and HILIC directly coupled to MS, digested peptides will be separated using multidimensional approaches. Weak anion exchange columns operated under HILIC conditions will be used to separate peptides carrying posttranslational modifications such as phosphorylation or acetylation, thus, increasing the dynamic range of detection. During this separation, fractions will be collected and re-injected onto the RP and HILIC with MS detection.
• All nano HPLC separations will be performed using biocompatible separation system.

Mass spectrometric analysis of digested peptides will be performed using two different detection methods: ion-trap and the time-of-flight (qToF) MS. qToF mass spectrometric detection and analysis will also be used for the label-free quantitation of peptides and proteins detected in samples. All measurements will be performed in triplicate to provide corrections for technical variability of separation and ionization.

General database search will be performed using the Human SwissProt Database in its actual version at the time of analysis. Data search will be performed using Mascot (http://www.matrixscience.com/) and X!Tandem (http://www.thegpm.org/tandem/) search machines, and the final data allocation and filtering by using Scaffold (www.proteomsoftware.com).

Statistical analysis:

Data analysis will be conducted with the bioconductor package limma. Before data analysis, data will be filtered by excluding proteins with measurements with a low interquartile range. Groups will be compared by moderated paired t-statistics adjusting for age. Adjustment for multiple testing will be done by Benjamini-Hochberg correction controlling the FDR at 0.05.

Inclusion criteria

• patients with stress urinary incontinence: a history of symptoms of stress urinary incontinence for at least 3 months (including anamnestic complaints of involuntary leakage on effort or exertion or on sneezing or coughing) and a positive provocative stress test (defined as an observed transurethral loss of urine that was simultaneous with a cough or Valsalva maneuver at a bladder volume of 300ml or less)
• patients capable of independent toiling
• patients who are able to fully understand all study procedures and to provide written informed consent to study participation
• age ≥ 18
• patients after vaginal delivery

Exclusion criteria

• patients who have previously been treated for SUI (both surgical or pharmacological treatment)
• patients who have a medical history of uncontrolled overactive bladder (OAB) or urinary incontinence other than SUI (including anamnestic complaints on involuntary urine leakage accompanied by or immediately preceded by urgency, not stress induced)
• patients with well established neurological disorder (e.g. Multiple Sclerosis, Parkinson's disease, Alzheimer's disease)
• patients with pelvic organ prolapse stage ≥ II (ICS classification)
• patients with a clinically significant bladder outlet obstruction and/or patients with a post void residual volume (PVR) > 100ml
• patients with a history of acute urinary retention or history of repeated catheterizations due to acute urinary retention within the last 3 months prior to the day of informed consent
• patients with an indwelling catheter and patients practicing intermittent self- catheterization
• patients who have undergone a bladder biopsy or any other minor pelvic surgical intervention less than 30 days prior to the day of informed consent
• patients with a history of bladder cancer
• patients with acute or recurrent urinary tract infection and/or unexplained haematuria
• patients with stone(s) in the bladder or urethra and upper tract stone disease causing symptoms
• patients with evidence of renal insufficiency (creatinine > 1.5x upper limit of normal)
• patients with evidence of hepatic disease (total bilirubin >1.5x upper limit of normal, or AST or ALT or alkaline phosphatase >2x upper limit of normal)
• patients with a history of alcohol and/or other drug abuse
• patients who are unable and/or unlikely to comprehend and follow the study procedures and instructions
• patients who are pregnant or lactating
• patients with serious medical conditions who, in the opinion of the investigators, should not participate in this study

The control group is represented by patients with the same inclusion and exclusion criteria as the case group, except stress urinary incontinence.