Balkan Journal of Medical Genetics

COMPARISON OF FGF -8, FGF -10, FGF- RECEPTOR 2, ANDROGEN RECEPTOR, ESTROGEN RECEPTOR-Α AND SS IN HEALTHY AND HYPOSPADIAC CHILDREN
Emaratpardaz N, Turkyilmaz Z, Karabulut R, Dayanir D, Kaya C, Sert AAE, Arkan G, Ucaner FA, Kapisiz A, Eryilmaz S, Atan A, Sonmez K
*Corresponding Author: Prof. Ramazan Karabulut, MD, Gazi University Faculty of Medicine, Department of Pediatric Surgery, Besevler, 06550, Ankara-Turkey. Tel: +90 312 2026210; Fax: +90 312 2230528. E-mail: karabulutr@yahoo.com, ramazank@gazi.edu.tr
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MATERIAL AND METHODS

Approval from the Gazi University Ethical Commit- tee was obtained before the study began (07/03/2022-191). The study includes the children with hypospadias and the ones without who came to our clinic for circumcision. The children who underwent circumcision were the control group. After giving detailed information, written informed consent was obtained from their legal guardians (parents). Children who had previous hypospadias surgery, received hormone therapy, had accompanying urological anomalies such as undescended testis, had endocrine disorders and syndromes were excluded from the study. Between June 2022 and September 2022, 20 children with hypospadias and 20 children who applied just for circumcision were included. All children met the criteria. During circumcision and hypospadias correction surgery, the foreskin tissues about 1x1 cm from healthy and children with hypospa- dias were taken for histomorphological and immunohistochemical (IHC) studies. Levels of AR, ER-α and ER-ß, FGF-8, FGF-10, FGFR-2 were evaluated in these tissues. Using Microsoft Office Excel Professional Plus 2016 (Microsoft Corporation, Redmond, WA, USA), statistical analysis was carried out. To compare the quantitative data between two groups that did not exhibit a normal distribu- tion, the Mann-Whitney U test was employed. Histomorphological Evaluation Tissue samples were fixed in 10% neutral formalde- hyde for 48 h. After washing the fixed tissues in running water for 24 hours, they were kept in 70% ethyl alcohol, 80% ethyl alcohol, 96% ethyl alcohol, Acetone I, Acetone II, Acetone III and Acetone IV, for 20 minutes each, respec- tively. Tissues extracted from acetone were kept in Xylene I and Xylene II for 30 minutes each. Before the tissues were embedded, they were kept in molten Paraffin I and Paraffin II for 1 hour in an oven at 60 o C and embedded in paraffin blocks. Sections of 6μm thickness obtained from the blocks were kept in xylol for 2 times for 15 minutes. Samples extracted from xylol were kept in ethyl alcohol solutions at 100%, 96%, 90%, 80%, 70% and 50% con- centrations, respectively, for 10 minutes, then rehydrated by soaking them in distilled water for 5 minutes, twice. Sections obtained from the blocks were then stained with Hematoxylin Eosin (H&E) for histological evaluation. The samples, which were dehydrated by passing through the alcohol series, were kept in xylol and covered with a lamella using entellan. All specimens were evaluated histopathologically by taking photographs with the Leica Q Vin 3 program with the help of Leica DM4000 (Ger- many) computer aided imaging system. These evaluations were examined separately by 2 instructors working in the Histology and Embryology Department. Immunohistochemical evaluation Tissue samples were processed similar to the histo- morphological stage. Afterwards, the tissues were lined up on the immunohistochemistry bar in a humid environment and were scratched with PAP-Pen and washed 3 times with PBS (Phosphate Buffer Saline, pH: 7.4) for 3 minutes. Samples were treated with serum blocking solution for 10 minutes to prevent non-specific binding, AR Ab-1(Cat. No. MS-443-PO, Thermo Fisher Scientific, Cheshire, UK), ER-α (Cat. No. RM-9101-SO), Thermo Fisher Scientif- ic, Cheshire, UK), ER-ß (Cat. No. orb448242, Biorbyt Ltd., Cambridge, UK), FGFR-2 (Cat. No. STJ91850, St Johns Laboratory Ltd., London, UK UK), FGF-8 (Cat. No. PA1216, Boster Biological Technology, Pleasanton, CA, USA), FGF-10 (Cat. No. E-AB-65862, Elabscience Biotechnology Inc., Houston, TX, USA) primary antibodies were incubated at +4°C for 1 night. After incuba- tion, a 3% hydrogen peroxide solution was applied to the samples washed with PBS for 15 minutes to inhibit endogenous peroxidase activity. After washing the samples with PBS, a secondary antibody with biotin was applied. Again, the samples were washed 3 times with PBS for 3 minutes, and chromogen containing diaminobenzidine (DAB) substrate was applied and left until a visible im- mune reaction occurred. Mayer’s Hematoxylin was used as background dye for the samples washed with PBS. The samples, which were dehydrated by passing through the alcohol series, were kept in xylol and covered with a lamella using entellan. All samples were evaluated with the help of the Leica DM4000 (Germany) computer aided imaging system. Photographs were taken using the Leica Q Vin 3 program. Uptake of AR, ER-α and β in cell counts provided in 10 independent fields selected for each slide and were evaluated with the scoring system specified by Qiao et al. [9] (Table 1). The evaluation of FGF-8, FGF-10 and FGFR-2 was done as follows, taking the study of Haid et al. as an ex- ample [7]; Epidermis assessment l Pattern 1: Limited to basal involvement l Pattern 2: Less than 50% of keratinocytes are in- volved l Pattern 3: Involvement of more than 50% of ke- ratinocytes Dermis evaluation: l Pattern A: No positive cells l Pattern B: diffuse involvement in less than 50% of all visible dermal cells l Pattern C: Clustered positive cells more than 50% of all visible dermal cells.

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