Following approval by our institutional ethical committee, we conducted a retrospective database review that identified 43 boys who had undergone surgery for non-palpable testis. Inclusion criteria consisted of a preoperative diagnosis of a nonpalpable undescended testis and either the presence of a testicular nubbin or absent testis with blind-ending gonadal vessels and vas deferens at surgical exploration. During surgery, all testicular remnants (nubbins) detected were removed. Vanishing testes were confirmed during inguinal surgeries. In addition, the scrotal descended testis was biopsied in each case and fixed by subdartos pouch or suture.
Boys with monorchidism with or without a nubbin that completely lacked testicular tissue were included in the study. Investigated patients neither had chromosomal anomalies nor “re-do” surgeries performed earlier in life. Four boys with normal and 3 boys with abnormal testicular histology were hormonally treated prior to the surgery. All tissue samples were immediately fixed in 2% glutaraldehyde in phosphate buffer, embedded in epoxy resin, sectioned at a thickness of 1 μm, and stained with toluidine blue. Histological analysis was performed using light microscopy at 60× total magnification. For each biopsy, at least 100 seminiferous tubules were examined. Histological data included the total number of germ cells, the number of A dark (Ad) spermatogonia, and the presence of primary spermatocytes. Exclusion criteria were age > 18 years and missing data for any of the histological variables. Germ cell counts from the contralateral descended testis group and from a historical control group were compared. For the latter, data from 26 control testes, most of them obtained post-mortem, were selected from data published and age-matched [8, 9].
Immunohistochemistry
16 testicular biopsies were analyzed (8 in each group). Fresh performed slides were blindly evaluated by two examiners experienced in immunohistology.
Immunohistochemistry was performed for steroidogenic acute regulatory (STAR) protein which is important in steroid hormone synthesis. It enhances the metabolism of cholesterol into pregnenolone by mediating the transfer of cholesterol to the inner mitochondrial membrane [10]. RNA-binding protein Sam68 belongs to the evolutionary conserved signal transduction and activation of RNA (STAR) family. It is required for the correct progression of spermatogenesis and for male fertility in the mouse and probably human [11]. Moreover, the decreased expression of Sam68 has been shown in human testes with spermatogenic defects demonstrating its role in the regulation of germ cells [12].
For immunohistochemical analysis, epon was removed from the tissue sections. The sections were treated with 2% bovine serum albumin to reduce non-specific binding and then incubated with the primary antibody overnight at 4 °C. All samples were washed with phosphate-buffered saline between incubations. We used an antibody targeting the steroidogenic acute regulatory (STAR) protein (Santa Cruz sc-166,821), labeled with horseradish peroxidase-polymer (HRP; goat polyclonal anti-rabbit IgG, mouse IgG and IgM, prediluted ab2891, Abcam, Cambridge, UK) to detect the binding of the primary antibody. The chromogenic reaction was developed by adding a freshly prepared solution of 3,3-diaminobenzidine solution (DAB + chromogen; DAKO). The DAB reaction was terminated by washing in Tris-buffered saline (0.05 M Tris-buffered saline and 0.85 M NaCl, pH 7.6). To allow visualization of testicular cells, the samples were counterstained with toluidine blue. Antibody binding was indicated by a brown precipitate. Different cell types were identified based on their nuclear morphology and position within the gonad. Immunohistochemistry experiments were performed at least three times on samples from at least eight patients from each group, and only those with identical results between experiments for each sample were included in the study. Controls for non-specific binding of the secondary antibody were performed in all experiments by omitting the primary antibody; these experiments consistently yielded no signal within the seminiferous epithelium or the interstitial space. Experimental design, biomaterials and treatments, reporters, staining, imaging data, and image characterization were performed in compliance with the minimum information specification for immunohistochemistry experiments [13].
Statistical analysis
The software package StatXact 6.30 (2004, CYTEL Corporation) was used to apply the Mann–Whitney U test of unpaired data. In addition, Fisher’s exact test was applied, and p < 0.01 was considered to indicate significance.