Computational protocol: Immunohistochemical expression of hypoxia-inducible factor-1α in stromal cells of vaginal tissue in post-menopausal women with pelvic organ prolapse

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Protocol publication

[…] Patient characteristics and tissue collection: Vaginal wall tissue samples were obtained during surgeries from 120 post-menopausal women. There were 60 women each in POP and control groups. Control group included women with other benign gynaecological diseases such as ovarian cysts and uterine myomas. Biopsy samples of 1 cm × 1 cm of the anterior vaginal wall tissue were taken from the same part of the wall in each case i.e. from the part right next to the connection between the anterior vaginal wall and the cervix. POP was assessed using POP-quantification (POP-Q) score. In this study, women with the POP-Q 4 (complete eversion) were included in the POP group, while women with the POP-Q 0 were included in the control group. All women included in the study were multiparous. Women with any additional diseases, such as diabetes, malignant diseases, pelvic inflammatory disease and endometriosis were excluded from the study. None of the women has ever used hormone replacement therapy or has been smoking cigarettes. Biopsy samples were obtained during vaginal hysterectomy for POP group and abdominal hysterectomy for control group at the department of Gynecology and Obstetrics, University Hospital in Split, Croatia. Each patient has signed written informed consent to participate in the study. Convenience sample criterion of selection was used when choosing our POP and the control groups. The RECORD (REporting of studies Conducted using Observational Routinely- collected health Data) statement guidelines were followed during the study. Power of the study and effect size were calculated using G*Power statistical power analysis program (version, Heinrich Heine University Düsseldorf, Germany). Power of the study was calculated for all variables and effect size for the variables where null hypothesis was rejected. The study was approved by the Research Ethics Committee of University Hospital Center, Split. This study was a part of a larger project which included a whole series of researches pertaining to pelvic floor damage. The study was conducted between 2009 and 2014.Immunohistochemistry: Tissue samples were fixed in 10 per cent buffered formalin and processed through standard processes in the automatic tissue processor (Shandon Excelsior, Thermo Fisher Scientific, United Kingdom), embedded in paraffin, cut at 4 μm and placed on positive charged slides (Superfrost Plus Adhesion Slides, Thermo Scientific). Immunohistochemistry was performed using the BenchMark ULTRA Automated IHC/ISH slide staining system (Ventana Medical Systems, Inc., USA), using horseradish peroxidase detection system. After tissue deparaffinization for 10 min at 72°C, slides were pretreated with Tris-based buffer for 52 min at 95°C and incubated with 3 per cent H2O2 for four minutes at 36°C to inactivate endogenous peroxidase. Slides were incubated with primary antibody for HIF-1α (SC-10790, Santa Cruz Biotechnology, Dallas, USA) for 92 min at 37°C. Reaction was visualized with a Olympus BX41 Microscope using diaminobenzidine (DAB) chromogen and counterstained with haematoxylin (ULTRAVIEW Universal DAB Detection Kit, Ventana Medical Systems, Inc.). All washes between the various steps were done with phosphate-buffered saline solution. The same immunohistochemical protocol was followed for the negative controls, with the omission of the primary antibodies. Human colon cancer tissues were used as positive controls.HIF-1α expression was scored by counting 100 stromal cells nuclei in the representative slides of the vaginal wall using Olympus Image Analyzer (magnification ×200). Counting was performed at the hot spots in the fields with the most prominent immunohistochemical reaction. Data were expressed in the form of the number of positive stromal cells nuclei/total number of stromal cell nuclei in the area scored. All immunohistochemical slides were evaluated by two investigators who were blinded to the study group.Statistical analysis: Data were tested for normality of distribution using the Kolmogorov-Smirnov test. As data were not normally distributed (data not shown), non-parametric Mann–Whitney test was used. All analyses were conducted using SPSS (version 18; SPSS Inc., Chicago, IL, USA), with the significance level set at P<0.05. […]

Pipeline specifications

Software tools G*Power, SPSS
Application Miscellaneous
Organisms Homo sapiens