ANDROLOGY Stem Cell Factor Receptor Immunoexpression in Adolescent Varicocele Arena Salvatore1*, Impellizzeri Pietro1, Fazzari Carmine2, Peri Flora Maria1, Antonelli Enrica1, Calabrese Ugo1, Centorrino Antonio1, Alibrandi Angela3, Romeo Carmelo1 Purpose: Stem cell factor receptor (c-kit) plays a crucial role in regulating proliferation and survival of germ cells. The aim of this study was to find the correlation between the number of c-kit positive germ cells, testicular asym- metry and histological grade in varicocele affected testis samples of adolescents. Materials and Methods: Twenty testicular biopsy samples of adolescents affected by varicocele and eight normal control testes were included. The relationship between percentage of testicular asymmetry, number of tubular c-kit positive germ cells and severity of spermatogenic failure was assessed. Results: The mean (SD; median) histological grade for spermatogenic failure in controls was 1.37(0.52; 1), while in the varicocele group, it was 2.70(1.08; 3) (P = .0052). Mean(SD; median) number c-kit positive germ cells in the control group were 20.1(2.52; 20), while in the varicocele group it was 12.35(7.16; 12.5) (P = .0059). Spearman test documented a significant positive correlation between percentage of hypotrophy and histological grade of spermatogenic failure (r = 0.5544 , 95% CI: 0.1345 to 0.8055, P = .0112) but a negative correlation with the num- ber of c-kit positive cells (r = - 0.5871, 95% CI: - 0.8219 to -0.1817, P = .0065). Moreover, a significant negative correlation was found between grade of histological changes and number of c-kit positive germ cells (P < .0001). Conclusion: A significant correlation between hypotrophy, histological lesions and c-kit positive germ cells exists in varicocele testes. This finding suggests a possible role for c-kit in the pathogenesis of germ cell impairment in varicocele. Histological changes and lack of c-kit germ cells were also noted in testes not displaying hypotrophy. We believe that reliable markers should be found as better predictors of testicular function in adolescent with varicocele. Keywords: c-kit; histology; male infertility; testicular hypotrophy; varicocele INTRODUCTION Varicocele affects testicular function and is consid-ered a main but corrigible cause of male infertil- ity(1-2). Even if medical management promises a poten- tial solution (3-9), surgery is considered the mainstay of varicocele treatment(10). The pathogenesis of testicular damage remains controversial and no mechanism has exclusively explained the exact way in which varico- cele impairs spermatogenesis. Multifactorial pathogen- esis of testicular damage includes hypoxia by small ves- sel occlusion and venous stasis and(11), enhancement of scrotal and testicular temperature(12), retrograde blood flow of renal and adrenal metabolites in the spermatic vein(13), reduction of gonadotrophins and androgens(14), testicular aquaporin 9 down-regulation(15), and oxida- tive stress (16). It has been reported that the expression of c-kit receptor plays a pivotal role in the survival and proliferation of type A spermatogonia(17), mature germ cells, and regulation of gametogenesis(18-19). Moreover, its expression in testicular tubular cells has also been 1Unit of Pediatric Surgery, Department of Human Pathology of Adult and Childhood “Gaetano Barresi”, University of Messina, Messi- na, Italy. 2Department of Human Pathology, Ospedale Umberto I, Siracusa, Italy. 3Unit of Statistical and Mathematical Sciences, Department of Economics, University of Messina, Messina, Italy. *Correspondence: Unit of Pediatric Surgery, Department of Human Pathology of Adult and Childhood “Gaetano Barresi”, University of Messina, Messina, Italy, Tel: +390902213014. Email: salarena@unime.it. Received May 2019 & Accepted February 2020 reported to be inversely related to germ cell apoptosis and hypoplasia(20-22). Of note, alteration of transcription levels or deregulated actions of the c-kit signaling sys- tem have been associated with reproductive disorders and male infertility(21-23). In this regard, W (dominant White spotting) and Sl (Steel) loci mutations in mice which encode the c-kit receptor, cause sterility, as they are necessary for the migration and proliferation of pri- mordial germ cells(24). To the best of our knowledge, the immunoexpression of c-kit in adolescent human testicular samples affected by varicocele has not yet been investigated. The aim of the present study was to immunolocalize c-kit and to correlate it with the number of c-kit positive germ cells per tubule. Testicular volumetric asymmetry and grade of histological changes were also assessed. MATERIALS AND METHODS Ethical approval for this study was received from the Local Ethics Committee (ethics code: 90/19). We ret- rospectively evaluated testicular incisional biopsies of Urology Journal/Vol 17 No. 4/ July-August 2020/ pp. 391-396. [DOI: 10.22037/uj.v0i0.5351 ] 20 post-pubertal adolescents (mean age (SD):14.3 (1.4) years old, range 13–18) with grade II or III idiopath- ic left varicocele which were obtained during inguinal varicocelectomy before the ligation of spermatic veins. Post-mortem tissue samples from eight age-matched subjects with normal lesion-free adolescent testes were also processed by employing archival blocks. Diagno- sis of varicocele was made after performing physical examination and was confirmed by echo color Doppler ultrasonography. The percentage of volume difference between the right (unaffected) and left testis was cal- culated according to medical records and decreased volume of the left testis compared with the right was considered as testicular hypotrophy. Histological grading Histological spermatogenesis was graded according to the Johnsen score. As previously described, samples were assigned to the following groups: (26) • Normal spermatogenesis: A majority (>50%) of the spermatogenic tubules have a Johnsen’s score of 10 (normal) or 9 (disorganized architecture, but with ≥ 10 sperm cells). • Spermatogenic impairment: <50% of the spermato- genic tubules exhibit a Johnsen’s score of 10 or 9. To investigate the severity of the spermatogenic impair- ment, this group was further divided into the following histological subgroups: (i) Moderate hypospermatogenesis: < 50% of the sem- iniferous tubules have a Johnsen’s score of 9 or 10 but >10% of the seminiferous tubules have a Johnsen’s score of 8 (<10 sperm cells); (ii) Severe hypospermatogenesis with few sperm cells: no seminiferous tubules have a Johnsen’s score of 9 or 10, and ≤ 10% of the seminiferous tubules have a Johnsen’s score of 8 (<10 sperm cells). (iii) Severe hypospermatogenesis without sperm cells: no mature sperm cells found but at least one seminifer- ous tubule has a Johnsen’s score between 7 and 3 (pres- ence of immature germ cells). (iv) Sertoli cell-only syndrome: the seminiferous tu- bules have a Johnsen’s score ≤ to 2 (no germ cells). Immunohistochemical evaluation Immunostaining was carried out using a 1:50 dilution of the rabbit polyclonal anti-human c-kit (CD117) anti- body (Dako- Cytomation A/S,Glostrup, Denmark) after microwave treatment. The streptavidin-biotin meth- od (LSAB, Dako), with diaminobenzidine develop- ment (Vector VIP substrate, Vector Lab, Burlingame, CA), and methylgreen or toluidine blue nuclear coun- ter-staining was performed. Positive controls were car- ried out using specimens from lung small cell carcino- ma for c-kit labelling. Negative controls were obtained by omitting incubation with the primary antibody. Statistical analysis In our study, we focused on the primary variable which was the immunoexpression of c-kit in human testicular samples. Secondary variables were the histology of the testis, the grade of histological spermatogenesis and the volume of testis. For a quantitative analysis, the percentage of intratubu- lar positive cells in each tubule was counted in five tu- bules of each specimen. As the number of observations in each group was not enough to assess the normali- ty of the distributions, the results of each group were compared by the non-parametric Mann–Whitney U test. The relationship between the quantitative param- eters (percentage of left testicular hypotrophy, number of c-kit positive germ cells per tubule) and the histolog- ical grade of spermatogenesis according to modified Johnsen’s score(26) was studied by the non-parametric Spearman’s Rank correlation test between the para- metric values and values 1 (normal spermatogenesis), 2 (moderate hypospermatogenesis), 3 (severe hypo- spermatogenesis with few sperm cells), 4 (severe hy- Variables Left control testes Left testes with varicocele P value Mean ± SD Median Q1-Q3 Mean ± SD Median Q1-Q3 Histological grade 1.37 ± 0.52 1 1-2 2.70 ± 1.08 3 2-4 .0052 c-kit+ germ cells 20.1 ± 2.52 20 17.5-22 12.35 ± 7.16 12.5 6.5-18 .0059 Table 1. Mean (SD) of histological grade and number of c-kit positive cells in each group and the relative p-value. Figure 1. A-C: Light microscopy: A) Normal control testis (Johnsen's score 9-10, H&H, original magnification 200x); B) Varicocele affected testis showing detachment and sloughing of basal germ cells and a depletion of sperm cells (Jonhsen's score 5, H&H, original magnification 200x); C) Severe grade of histological lesion of a varicocele affected testis, with intra and extratubular edema, venular ectasia, peritubular ectasia and marked depletion of germ cells (Johnsen's score 3, H&H, original magnification 200x). C-kit expression in adolescent varicocele- Salvatore et al. Andrology 392 Vol 17 No 04 July-August 2020 393 pospermatogenesis without sperm cells) and 5 (Sertoli cell only syndrome) regarding the histologic subgroups (26). P < .05 was considered as statistically significant. RESULTS Changes in tubular and extra-tubular compartments were seen in the studied varicocele testes, displaying more or less disarranged tubular compartments. Slough- ing and detachment of basal germ cells occurred with a varying degree of germ and Sertoli cell impairment, up to spermiogenesis failure. Unlike normal testes (Figure 1A), varicocele testes showed dilated microvessels, more or less expanded extra-tubule fluid and extracel- lular matrix, and many extracellular vesicles inside the seminiferous tubules. Leydig cell clusters were con- stantly observed. A varying degree of interstitial ede- ma prevailed, with notable peritubular and extratubu- lar fibrosis (Figure 1B & C). Mean (SD) histological grade of spermatogenesis in the control group was 1.37 (0.52), while in the varicocele group, this figure was 2.70 (1.08), indicating a statistically significant differ- ence (P = .0052). Specifically, in the control group, 5 testes had value 1 and 3 testes demonstrated value 2. On the other hand, in the varicocele group, 3 testes had value 1, 6 had value 2, 5 demonstrated value 3, and 6 samples were value 4. Mean (SD) of c-kit positive germ cells in the control group was 20.1 (2.52) and 12.35 (7.16) in the varicocele group (P = .0059) (Figure 2A- C) (Table 1). The mean percentage (SD) of testicular volume discrepancy between right (unaffected) and Figure 2. A-C: C-kit immunohistochimestry: A) Control testis: about 20% of c-kit positive germ cells are shown in a control testis (original magnification 200x); B) Varicocele testis with moderate hypospermatogenesis: about 10% of c-kit positive cells are present in sloughed germ cells (original magnification 200x); C) Varicocele testis with severe hypospermatogenesis without sperm cells: no positive cells are present (original magnification 200x) Figure 3. Distribution of percentage of left testicular asymmetry and histological grade of spermatogenesis in varicocele group Figure 4. Distribution of percentage of left testicular asymmetry and number of c-kit positive germ cells in the varicocele group. C-kit expression in adolescent varicocele- Salvatore et al. left (varicocele affected) testes was %14.1 (%22.5), median %12.5, Q1 = 0, and Q3 = 29.7. Spearman test documented a significant positive correlation between percentage of testicular volumetric discrepancy and histological grade of spermatogenesis (r = .5544, 95% confidence interval (CI): 0.1345 to 0.8055, P = .0112) (Figure 3) but a negative correlation with number of c-kit positive cells (r = - 0.5871, 95% CI: - 0.8219 to -0.1817, P = .0065) (Figure 4). Moreover, a significant negative correlation was found between histological grade of spermatogenesis and number of c-kit positive germ cells (p <.0001, r = - 0.7621, 95% CI: 0.9035 to -0.4714) (Figure 5). DISCUSSION Although surgical correction of varicocele in adoles- cents before onset of infertility may be advantageous, it has been reported that fertility problems will arise lat- er in about 20% of adolescents with varicocele(26). As sperm analysis is not obtained worldwide from pediatric patients, both for legal and ethical reasons and because patients' hormonal profiles are not fully developed, es- tablishing the potential existence of prognostic markers that would make it possible to decide for surgery and to determine, when necessary, the appropriate time of surgery is still a controversial issue. While waiting for a reliable marker to be identified for early detection of testicular impairment in adolescents affected by vari- cocele, current international guidelines recommended treatment of clinical varicocele in adolescents only in selected cases such as objective evidence of reduced ip- silateral testicular size(9). However, recently, it has been reported that 100% of adolescent patients varicocele af- fected with a testicular volumetric discrepancy > 20% calculated by ultrasonography fail to fulfill the World Health Organisation adult criteria for normal spermat- ogenesis in comparison with 50% of those with sym- metrical testis(27). Not only is it suggested that a positive correlation between testicular hypotrophy and spermat- ogenic impairment exists but also that a symmetrical testis affected by varicocele does not always reflect normal semen parameters(28). In our study, we found that there is a significant positive correlation between the percentage of testicular volumetric discrepancy and the histological grade of spermatogenesis and that tes- ticular symmetry does not exclude the probability of an even severe testicular impairment, triggering a potential subsequent infertility. This data suggests that testicular volume should not be considered as an early, reliable marker of testicular impairment in adolescent varico- cele. Moreover, in this study, a lack of c-kit positive germ cells was found in varicocele affected testes, with a sig- nificant positive correlation with histological changes. C-kit is closely associated with developing germ cells contributing to germ-cell homoeostasis(29). A dimin- ished expression of c-kit has been found in the testis of infertile men(30), particularly in Sertoli-cell-only syn- drome(31, 32). These observations highlight the importance of c-kit signaling for germ-cell survival and successful spermatogenesis, implying crucial participation of c-kit in the onset and maintenance of spermatogenesis(29,33,34). A reduced expression of c-kit in the tubular compart- ment of damaged varicocele-affected testes substanti- ates the role of this proto-oncogene in the pathogen- esis of infertility. Moreover, unexpected data which emerged from our study was that there is a significant difference in histological grade of spermatogenesis be- tween the subgroup of varicocele affected testes with a hypotrophy ≤ 10% and control testes. In this regard, 9 out of 20 varicocele-affected testes showed a volumet- ric testicular discrepancy ≤ 10% and in this subgroup, the histological grade of spermatogenesis was 2.33 ± 1.00 which was significantly higher compared to the control group (P = .0495). Furthermore, a lower but not significant reduction of c-kit cells was recorded in varicocele group compared with control group (14.89 ± 6.918 versus 20.13 ± 2.532, P = .1193). It suggests that testes affected by varicocele during adolescence can display a significant impairment of the tubular testic- ular function irrespective of testicular volume, and that left testicular volumetric loss is strictly correlated with histological changes, which based on our experience, is the rule. Even if current international guidelines, which provide a framework of standardized care, restrict the surgical management of idiopathic monolateral asymp- tomatic varicocele to conditions such as the presence of ipsilateral testicular hypotrophy, at times when sperm analysis is not available, our study suggests that a reli- able and more sensitive indicator of testicular damage should be identified to avoid progressive impairment of future fertility in adolescents with varicocele. On this basis, we believe that the effort of both basic and clin- ical research should be focused on the identification of a reliable predictor of future infertility of adolescent affected by varicocele even when volumetric testicular discrepancy is not present. On the other hand, until a highly sensitive, specific and non-invasive indicator is found, it is at least questionable to correct all varicoce- les in adolescents, since only few adolescents with var- icocele will show infertility impairment in adulthood. The main limitations of this study were its retrospective nature, small sample size and the unavailability of ac- cess to testicular volume in the control group Figure 5. Distribution of percentage of histological grade of sper- matogenesis and number of c-kit positive germ cells in the varico- cele group. C-kit expression in adolescent varicocele- Salvatore et al. Andrology 394 Vol 17 No 04 July-August 2020 395 CONCLUSIONS Our study showed a significant correlation between the percentage of left testicular asymmetry, histological grade of spermatogenesis and c-kit positive germ cells in varicocele affected testes. It suggests a role of c-kit in the pathogenesis of germ cell impairment in testes of adolescents affected by varicocele. However, histo- logical changes and a lack of c-kit germ cells have also been noted in testes not displaying a significant left hy- potrophy. This suggests that testicular hypotrophy is a specific but not sensitive-enough indicator of testicular damage. We believe that other markers should be inves- tigated as better and more reliable predictors of testicu- lar function in adolescents with varicocele. ACKNOWLEDGEMENT The authors would like to thank Miss Piscilla Haass for the linguistic revision. CONFLICT OF INTEREST None declared by the authors. REFERENCES 1. Haddad NG, Houk CP, Lee PA. Varicocele: a dilemma in adolescent males. Pediatr Endocrinol Rev 2014; 11:274-83 2. Pasqualotto FF, Lucon AM, de Góes PM et al. Testicular growth, sperm concentration, percent motility, and pregnancy outcome after varicocelectomy based on testicular histology. Fertil Steril 2005; 83:362-6 3. Oliva A, Dotta A, Multigner L. 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