Stesura Seveso Archivio Italiano di Urologia e Andrologia 2023; 95(3):11610 1 ORIGINAL PAPER Both genomic DNA (gDNA) and mitochondrial DNA (mtDNA) are released from dying cells, and circulating mtDNA fragments may trigger pro-inflammatory responses (4-12). Elevated levels of circulating cell-free mtDNA (cf-mtDNA) have been found in patients with various diseases, including urological malignancies, but the association with cancer is still debated (13-16). Different diseases have been associated with specific mtDNA fragmentation patterns related to cell death type (17-19). There may be a link between mtDNA fragments and prostate inflammation, but it remains unclear, and the studies have mainly focused on blood samples. Evaluating the fragmentation pattern of cf-mtDNA in urine might pro- vide insights into mtDNA-mediated inflammation and improve PCa biomarkers. Therefore, this study aimed to determine if the apoptosis- and necrosis-derived mtDNA fragments (79 bp and 230 bp, respectively) in post- prostate massage urine are related to prostate inflammation and can discriminate between PCa and inflammatory con- ditions, using serum PSA levels for comparison. MATERIALS AND METHODS Case selection This study was conducted with 93 male patients, who applied the Istanbul University-Cerrahpasa, Department of Urology. Patients aged 40 and older, PSA value > 2.5 ng/mL, and suspicious findings on rectal examination were included in the study (Table 1). Transrectal ultra- sound (TRUS) guided biopsies (12-28 cores) were per- formed for evaluation. The patients were divided into 3 groups according to the histopathological evaluations of TRUS biopsies: prostate cancer (PCa), prostatitis (Inflammation), and benign prostatic hyperplasia (BPH) groups. The results of biopsy showing adenocarcinoma and BPH were included in the PCa group. Similarly, the results showing prostatitis and BPH were included in the Inflammation group, whereas the results containing alone BPH were included in the BPH group. Additionally, according to the inflammation status in the results of TRUS biopsies, cases without inflammation (including cases of PCa and BPH groups) were defined as the Non- inflammation group. Patients with other malignant dis- ease, distant metastases, infectious or inflammatory dis- Background: We sought to determine whether two soluble forms with different size of mtDNA are linked to prostatic inflammation, and whether they discriminate prostate cancer (PCa) from inflammatory prostatic conditions. Methods: Histopathologically diagnosed prostatitis, PCa and benign prostatic hyperplasia patients (n = 93) were enrolled in this study and they were categorized as with and without prostate inflammation. Quantitative RT-PCR was used to ana- lyze the levels of 79-bp and 230-bp fragments in urine and blood samples collected following prostate massage. Results: The urine mtDNA-79 and mtDNA-230 were significant- ly increased in patients with prostate inflammation compared with those in without inflammation. Here, 79-bp fragment of apoptotic origin was significantly higher level than 230-bp frag- ment of necrotic origin. Although mtDNA-79 copy number in serum samples was also increased in patients with prostate inflammation, mtDNA-230 was similar in the two groups. Furthermore, mtDNA-79 and mtDNA-230 copy numbers in post- prostate massage urine were higher (about 16-fold and 22-fold, respectively) than those from serum samples. ROC analysis showed that, although post-prostate massage urine have relative- ly higher performance than blood, ability to discriminate cases of both fragments was not better than that of serum total PSA. Conclusions: Our results demonstrate that shorter cf-mtDNA fragment size in particular, increase in the presence of prostate inflammation in post-prostatic massage urine but both fragments could never improve serum total PSA performance. KEY WORDS: Prostate; Inflammation; Cancer; Mitochondrial DNA; Post-prostatic massage. Submitted 26 July 2023; Accepted 2 September 2023 INTRODUCTION A possible link between inflammation and prostate cancer (PCa) has been suggested, but not yet confirmed. Several cross-sectional studies indicate that prostatic inflamma- tion is more common in patients without cancer (1, 2). The factors contributing to prostate inflammation are largely unknown, and inflammation could play a role in false positive prostate-specific antigen (PSA) results in can- cer screening (1, 3). Inflammatory lesions contain dead cells, including apop- tosis and necrosis, and evidence shows a bidirectional relationship between cell death forms and inflammation. Are elevated mitochondrial DNA fragments in prostatic inflammation a potential biomarker for prostate cancer? Ugur Aferin 1, Nurten Bahtiyar 2, Ilhan Onaran 3, Hamdi Ozkara 4 1 Department of Urology, Medical Faculty, Demiroglu Bilim University, Istanbul, Turkey; 2 Department of Biophysics, Cerrahpasa Medical Faculty, Istanbul University-Cerrahpasa, Istanbul, Turkey; 3 Department of Medical Biology, Cerrahpasa Medical Faculty, Istanbul University-Cerrahpasa, Istanbul, Turkey; 4 Department of Urology, Cerrahpasa Medical Faculty, Istanbul University-Cerrahpasa, Istanbul, Turkey. DOI: 10.4081/aiua.2023.11610 Summary Archivio Italiano di Urologia e Andrologia 2023; 95(3):11610 U. Aferin, N. Bahtiyar, I. Onaran, H. Ozkara 2 eases, histologically defined with both inflammation and cancer findings were excluded from the study. All patients provided written informed consent. This study was approved by the Istanbul University-Cerrahpasa Ethics Committee (approval no. 83045809; Istanbul, Turkey), and was performed according to the criteria set out by the Declaration of Helsinki. Sample collection Before TRUS biopsy, following a digital rectal examination of 3 strokes per prostate lobe, each subject provided 20- 30 ml urine in a first catch specimen. All urine specimens were centrifuged at 2500 x g for 10 min at 4°C, followed by storage of the urine and pellet at -80°C. Five milliliter venous blood samples were collected into anticoagulant- free tubes after prostatic massage and centrifugation was performed at 2500×g for 10 min at 4°C. Then obtained supernatants were stored at -80°C until analyses. DNA isolation, and Determination of mtDNA-79 and mtDNA-230 Copies The Plasma/Serum Cell-Free Circulating DNA Purification Kit (Norgen Biotek, Canada) and Urine Cell-Free Circulating DNA Purification Kit (Norgen Biotek, Canada) were used to isolate circulating DNA from serum and urine. Cell-free DNA was isolated according to the kit manufacturers' protocols. Quantitative analysis of mtDNA fragments in serum and urine was performed by quantitative real-time PCR (qPCR). In this study, two primer sets specific for the mitochondrial ribosomal 16S RNA were used. The first primer pair amplified a 79-bp fragment (mtDNA-79), and the second primer pair amplified a 230-bp fragment (mtDNA-230) The sequence of the forward primer spe- cific for both mtDNA fragments was 5’-CAGCCGCTAT- TAAAGGTTCG-3’. The sequence of the reverse primer specific for mtDNA-79 was 5’-CCTGGATTACTCCG- GTCTGA-3’, of the reverse primer specific for mtDNA- 230 was 5’-GGGCTCTGCCATCTTAACAA-3’. The qPCR was performed in duplicate on a Real Time PCR Detection system (Bio Rad Laboratories, Inc., Hercules, CA, USA). Each 20 µL reaction consisted of 2 mL DNA, 10 µl EvaGreen 2X qPCR MasterMix (abm, Canada) and 0.40 µL (10mM) forward/reverse primer. PCR conditions were 95 ºC for 10 min, followed by 40 cycles at 95 ºC for 15 s, 60 ºC for 60 s, and 72 ºC for 30 s. Each run included water blanks as a negative control. The specificity of the PCR products was confirmed by melting curve analysis. The quantity of each target gene in the samples was subse- quently calculated according to the corresponding stan- dard curve. The formula published by the Genomics and Sequencing Center of the University of Rhode Island (Kingston, RI, USA; cels.uri.edu/gsc/cndna.html) was used to calculate the mtDNA copy number (20). Statistical analysis Continuous variables were presented as means ± SD or medians, and categorical variables were presented as fre- quencies with percentages. Differences between groups were compared with Mann Whitney-U, and Kruskal- Wallis tests. Receiver operating characteristic (ROC) analy- sis, and area under the ROC curve (AUC) were used for determination of diagnostic performance of mtDNA lev- els. Point-biserial correlation was used for correlation between categorical variables and mtDNA levels. All sta- tistical analyses were performed using Prism 5.0 (GraphPad Software, San Diego, CA). A value of p < 0.05 was considered statistically significant. RESULTS Assessment of mtDNA levels in urine and serum according to inflammation status Urine mtDNA-79 (median: 1.52 x 109 vs. 0.66 x 109 copies/ml) and mtDNA-230 (1.59 x 108 vs. 1.71 x 107 copies/ml) levels were increased in the Inflammation group compared to the Non-inflammation group (p < 0.001 and p < 0.01, respectively) (Figure 1 A-B). Serum mtDNA-79 (median: 2.45 x 107 vs. 1.05 x 107 copies/ml) levels were higher in the Inflammation group compared with the Non-inflammation group (p < 0.05), but the differences were not significant in serum mtDNA- 230 levels (median: 0.87 x 106 vs. 0.51 x 106 copies/ml) between these groups (p > 0.05) (Figure 1 C-D). Assessment of urine and serum mtDNA levels in Inflammation, PCa, and BPH groups Urine mtDNA-79 (median: 1.52 x 109 vs. 0.58 x 109 copies/ml) and mtDNA-230 (1.59 x 108 vs. 0.81 x 107 copies/ml) levels were increased in the Inflammation group compared to the BPH group (p < 0.01, for both) (Figure 2 A-B) whereas the differences were not signifi- cant in serum mtDNA-79 (median: 2.69 x 107 vs. 1.01 x 107 copies/ml) and mtDNA-230 levels (median: 0.51 x 106 vs. 0.88 x 106 copies/ml) between these groups (p > 0.05) (Figure 2 C-D). There were statistically significant differences in urine Table 1. Demographic data of all groups. Non-inflammation Inflammation Variable BPH PCa Prostatitis (n: 29) (n: 34) (n: 30) Age (years) 65.51 ± 6.85 65.09 ± 9.18 63.32 ± 6.71 PSA (ng/ml) 5.72 ± 2.80 18.24 ± 21.04 a*** 11.36 ± 9.81 b *** Histology Adenocarcinoma, n (%) NA 34 (100%) NA Pathological tumour stage pT1, n (%) NA 15 (44.12%) NA pT2, n (%) NA 19 (55.88%) NA Gleason Grading Gleason Score < 7, n (%) NA 12 (35.29%) NA Gleason Score = 7, n (%) NA 15 (44.12%) NA Gleason Score > 7, n (%) NA 7 (20.59%) NA Histology Biopsy core with cancer Median, n NA 4.5 NA Range, n NA 1-12 NA Histology Biopsy core with inflammation Median, n NA NA 6 Range, n NA NA 3-15 Values were presented as Mean ± standard deviation; BPH: benign prostatic hyperplasia; PCa: prostate cancer; NA: not applicable; a PCa vs BPH; b Inflammation vs. BPH; *** p < 0.001. Archivio Italiano di Urologia e Andrologia 2023; 95(3):11610 3 DNA fragments in prostatic inflammation mtDNA-79 (median: 1.52 x 109 vs. 0.67 x 109 copies/ml) (p < 0.05), but not in urine mtDNA-230 (median: 1.59 x 108 vs. 2.23 x 107 copies/ml), serum mtDNA-79 (median: 2.69 x 107 vs. 1.18 x 107 copies/ml), and mtDNA-230 (median: 0.51 x 106 vs. 0.41 x 106 copies/ml) levels between the Inflammation and the PCa groups (p > 0.05, for all). Additionally, there were no significant changes in urine mtDNA-79 (median: 0.67 x 109 vs. 0.57 x 109 copies/ml), urine mtDNA-230 (median: 2.23 x 107 vs. 0.81 x 107 copies/ml), serum mtDNA-79 (median: 1.18 x 107 vs. 1.01 x 107 copies/ml) and serum mtDNA-230 (median: 0.41 x 106 vs. 0.88 x 106 copies/ml) levels between the compari- son of the PCa and BPH groups (Figure 2 A-D). Figure 1. Comparison of the A. urine mtDNA-79, B. urine mtDNA-230, C. serum mtDNA-79, and D. serum mtDNA- 230 between the Inflammation and Non- inflammation groups, *p < 0.05, **p < 0.01, ***p < 0.001. The bold line in the box plots represents the median value. Figure 2. Pairwise comparisons of the A. urine mtDNA-79, B. urine mtDNA-230, C. serum mtDNA-79, and D. serum mtDNA- 230 among the Inflammation, PCa, and BPH groups; *p < 0.05, **p < 0.01, The bold line in the box plots represents the median value. Archivio Italiano di Urologia e Andrologia 2023; 95(3):11610 U. Aferin, N. Bahtiyar, I. Onaran, H. Ozkara 4 ROC curve analysis of serum and urine mtDNA, and serum PSA according to inflammation status Generally, AUC values demonstrated moderate discrimi- natory power, and urine AUC results (mtDNA-79, and mtDNA-230, 0.712, and 0.707, respectively) were higher than serum (mtDNA-79, and mtDNA-230, 0.653, and 0.585, respectively) in the Inflammation group compared to the Non-inflammation group. Similarly, urine mtDNA- 79 and mtDNA-230 AUC values were higher than serum PSA AUC values (0.712, 0.707, and 0.585, respectively). Urine mtDNA-79 and mtDNA-230 levels showed similar diagnostic performance with urine combined mtDNA-79 and mtDNA-230 results (Figure 3). Sensitivity and speci- ficity values for all testing were given in Tables 2, 3. ROC curve analysis of serum and urine mtDNA and serum PSA in Inflammation, PCa, and BPH groups AUC values of ROC curve analysis were deter- mined for urine and serum mtDNA, and serum PSA in the Inflammation, PCa, and BPH groups. Mostly the results in Inflammation vs. BPH, and Inflammation vs. PCa groups had moderate diagnostic performance (Figure 4). Urine mtDNA-79 and mtDNA-230 had better diagnostic performance (AUC range 0.725- 0.691) than serum mtDNA-79, and mtDNA- 230 (AUC range 0.682-0.572). AUC values of serum PSA were higher than urine mtDNA-79, and mtDNA-230. The combination of urine mtDNA-79 and mtDNA-230 results had mod- erate AUC values similarly as urine mtDNA-79 and mtDNA-230. ROC curve analysis between the PCa and the BPH groups indicated that except for serum PSA (AUC:0.793) , all other parameters had mild AUC values (range 0.534-0.594). Urine mtDNA-79 and mtDNA-230 values were high- er than serum, but all of them were in mild AUC levels (Tables 2, 3). Correlation analyzes of urine and serum mtDNA The correlation analysis showed that the pres- ence of prostatic inflammation was positively correlated with urine mtDNA-79, urine mtDNA-230, and serum mtDNA-79 levels. Also, urine mtDNA-79 levels were positive correlated with urine mtDNA-230 results. Similarly, serum mtDNA-79 levels were posi- tively associated with serum mtDNA-230 (Table 4). DISCUSSION To date, a few studies have quantified cf- mtDNA levels and mtDNA fragmentation pat- tern in urologic cancer patients. Some studies found elevated cf-mtDNA content in PCa (15, 21, 22), but another study on cf-mtDNA could- Table 2. Diagnostic performance of serum PSA, serum and post-prostatic massage urine specimens mtDNA. Sensitivity Specificity AUC (95%Cl) P Urine mtDNA-79 Inflammation vs. Non-Inflammation Inflammation vs. BPH Inflammation vs. PCa PCa vs. BPH Urine mtDNA-230 Inflammation vs. Non-Inflammation Inflammation vs. BPH Inflammation vs. PCa PCa vs. BPH Serum mtDNA-79 Inflammation vs. Non-Inflammation Inflammation vs. BPH Inflammation vs. PCa PCa vs. BPH Serum mtDNA-230 Inflammation vs. Non-Inflammation Inflammation vs. BPH Inflammation vs. PCa PCa vs. BPH Serum PSA Inflammation vs. Non-Inflammation Inflammation vs. BPH Inflammation vs. PCa PCa vs. BPH AUC: area under an ROC curve; BPH: benign prostatic hyperplasia; PCa: prostate cancer; NS: not significant. %73.3 %73.4 %73.2 %41.2 %70.1 %63.3 %66.7 %58.8 %54.2 %56.1 %48.1 %42.3 %47.8 %52.2 %47.8 %56.1 70.0% 70.1% 56.7% 64.7% 0.001 0.006 0.003 NS 0.001 0.002 0.008 NS 0.04 0.03 NS NS NS NS NS NS NS < 0.001 NS < 0.001 %71.4 %72.4 %70.6 %68.9 %66.7 %72.4 %73.5 %58.6 %60.4 %63.6 %65.4 %63.6 %75.6 %75.0 %68.1 %75.0 60.3% 89.7% 61.8% 89.6% 0.712 (0.588-0.836) 0.709 (0.570-0.847) 0.714 (0.580-0.849) 0.534 (0.388-0.679) 0.707 (0.591-0.823) 0.725 (0.594-0.857) 0.691 (0,557-0.825) 0.595 (0.449-0.741) 0.653 (0.511-0.794) 0.682 (0.528-0.836) 0.651 (0.498-0.803) 0.542 (0.377-0.707) 0.585 (0.427-0.744) 0.602 (0.429-0.776) 0.572 (0.403-0.741) 0.580 (0.408-0.752) 0.609 (0.495-0.725) 0.823 (0.711-0.935) 0.572 (0.429-0.716) 0.793 (0.679-0.907) Table 3. Diagnostic performances of mtDNA combinations in serum and post-prostatic massage urine specimens. Sensitivity Specificity AUC (95%Cl) P Combination of urine mtDNA-79 and mtDNA-230 Inflammation vs. Non-Inflammation Inflammation vs. BPH Inflammation vs. PCa PCa vs. BPH Combination of serum mtDNA-79 and mtDNA-230 Inflammation vs. Non-Inflammation Inflammation vs. BPH Inflammation vs. PCa PCa vs. BPH AUC: area under an ROC curve; BPH: benign prostatic hyperplasia; PCa: prostate cancer; NS: not significant. 66.67% 80.02% 72.72% 51.51% 47.6% 50.02% 54.17% 50.20% 0.001 < 0.001 < 0.001 NS NS NS NS NS 85.36% 76.67% 73.33% 53.57% 60.97% 63.16% 68.18% 55.56% 0.715 (0.592-0.837) 0.810 (0.694-0.926) 0.756 (0.634-0.877) 0.488 (0.342-0.634) 0.647 (0.494-0.800) 0.651 (0.481-0.821) 0.671 (0.489-0.811) 0.542 (0.364-0.719 Table 4. Correlation analyzes of urine and serum mtDNA according to inflammation status. Parameter Correlation Coefficient Sig. (2-tailed) Inflammation status vs. urine mtDNA-79 r = 0.343 p = 0.001 Inflammation status vs. urine mtDNA-230 r = 0.335 p = 0.001 Inflammation status vs. serum mtDNA-79 r = 0.250 p = 0.035 Urine mtDNA-79 vs. urine mtDNA-230 r = 0.601 p < 0.001 Serum mtDNA-79 vs. serum mtDNA-230 r = 0.262 p = 0.035 Archivio Italiano di Urologia e Andrologia 2023; 95(3):11610 5 DNA fragments in prostatic inflammation n't distinguish between PCa and BPH (15). In addition to previous publications on blood samples (23), this study found higher levels of mtDNA fragments in post-prostate massage urine in cases with prostate inflammation than those without inflammation. Different studies revealed a positive link between elevat- Figure 3. Receiver operating characteristic (ROC) curves of the A. urine mtDNA-79, B. urine mtDNA-230, C. serum mtDNA-79, and D. serum mtDNA- 230 between the Inflammation and Non-inflammation groups. Figure 4. Pairwise comparisons of the receiver operating characteristic (ROC) curves of the A. urine mtDNA-79, B. urine mtDNA-230, C. serum mtDNA-79, and D. serum mtDNA- 230 among the Inflammation, PCa, and BPH groups. Archivio Italiano di Urologia e Andrologia 2023; 95(3):11610 U. Aferin, N. Bahtiyar, I. Onaran, H. Ozkara 6 ed systemic mtDNA levels and inflammation-associated diseases (9, 24, 25) and inflammatory cell death is often necrotic (7). We expected that a possible increase in the long mtDNA fragments (> 200 bp) derived from non- apoptotic types of cell death (i.e. necrosis) may be associ- ated with prostate inflammation according to the histopathological examination. Surprisingly, we found that 230 bp fragment was significantly lower than 79 bp fragment (an implication of an active apoptotic phenom- enon) in prostate inflammation. Additionally, our results show no correlation between urine and serum mtDNA- 230 fragment in patients with inflammation, implying different factors contribute to elevated cf-mtDNA levels in each compartment. Prostate massage can stimulate the release of tissue’s viable cells as well dying/dead cells and increase the distribution concentration of circulating cf-mtDNA in urine. The influ- ence of exfoliated inflammatory or endothelial cells on mtDNA fragments cannot be excluded, complicating our results. The cause of changes in cf-mtDNA fragment levels due to prostate inflammation remains unclear with our experimental design and existing literature. Although the origin of cf-mtDNA and cf-DNA lacks consensus (26), excessive release by apoptotic or necrotic cells and reduced clearance by inflammatory cells may alter mtDNA content. The mtDNA-79 fragment might be more resistant to DNAse activity than long fragments. Furthermore, whether the higher levels of cf-mtDNA fragments is a cause or a consequence for prostate inflammation in the context of mitochondrial damage, is the chicken or the egg causality dilemma. Although cf-mtDNA is found in blood, urine, or saliva samples, data for prostate diseases associated with malig- nancy were mostly based on blood samples. Urine sam- ples collected after prostate massage may provide more evidence for prostate-associated conditions due to fewer confounding factors. Therefore, we expected urine mtDNA fragments to reflect prostate inflammation mag- nitude better than blood ones. As anticipated, post- prostate massage urine from patients with prostate inflammation showed higher mean mtDNA-79 and mtDNA-230 copy numbers (approximately 16-fold and 22-fold, respectively) compared to serum samples. Serum mtDNA-79 was significantly increased in patients with inflammation (p < 0.05), while serum mtDNA-230 remained similar between inflamed and non-inflamed patients (p > 0.05). Post-prostate massage urine mtDNA- 79 and mtDNA-230 copy numbers showed slightly high- er AUC values for discriminating prostate inflammation than serum. The differences in fragment levels between urine and serum could be due to urine's enrichment in prostate-derived cf-mtDNA and the rapid clearance of nucleic acids from blood. Furthermore, urinary cf- mtDNA concentrations have lower coefficient of variations (CV) than serum, but further investigation is needed as their CVs still exceed 100%. In this work, we also performed ROC curve analysis for urine cf-mtDNA fragments to differentiate PCa from inflammatory prostatic conditions, and compared it with the diagnostic performance of serum PSA levels. Considering the AUC, sensitivity and specificity, the lev- els of post-prostate massage urine mtDNA-79 and mtDNA-230 had slightly higher performance in distin- guishing prostate inflammation than their serum levels. Here, the discrimination performance of single parameter for prostate inflammation is moderate with an AUC of about 0.7. Nevertheless, each fragment yielded perform- ance similar to than serum PSA levels. Furthermore, when we tested the ability of these fragments in urine to discriminate PCa from BPH, PCa from prostate inflam- mation, and prostate inflammation from BPH, the AUC values of both fragment were within the range of 0.53- 0.73, which were close to those (0.58-0.82) for the serum PSA. Moreover, combining of two fragment copy num- bers did not significantly improve performance over using a single fragment alone. Therefore, mtDNA frag- ments alone does not seem to be a reliable predictor of differentiating PCa or BPH from inflammatory prostatic conditions. This study had several limitations, including focusing only on histologically measurable inflammation, being a single- center trial, and lacking adjustment for prostate volume- related variables and other potential confounding factors due to small sample sizes. Additionally, although this study was conducted on the samples from PCa patients without prostatitis, it is essential to keep in mind that presence of prostatitis in PCa is not an infrequent event. In conclusion, the present data demonstrate that cf- mtDNA fragments in post-prostatic massage urine increase in the presence of prostate inflammation; short- er cf-mtDNA fragment size in particular, may be a result of mostly an active apoptotic phenomenon associated with prostate inflammation. Even though both mtDNA fragments in the urine samples show relatively higher performance than blood, they do not seem to help iden- tify patients with PCa from non-PCa patients more than the conventional PSA test. 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Correspondence Ugur Aferin, MD ugur.aferin@demiroglu.bilim.edu.tr Department of Urology, Medical Faculty, Demiroglu Bilim University, Istanbul, Turkey Nurten Bahtiyar, PhD (Corresponding Author) nurten.bahtiyar@iuc.edu.tr Department of Biophysics, Cerrahpasa Medical Faculty, Istanbul University-Cerrahpasa, Istanbul, Turkey Ilhan Onaran, PhD ilonaran@iuc.edu.tr Department of Medical Biology, Cerrahpasa Medical Faculty, Istanbul University-Cerrahpasa, Istanbul, Turkey Hamdi Ozkara, MD hozkara@iuc.edu.tr Department of Urology, Cerrahpasa Medical Faculty, Istanbul University- Cerrahpasa, Istanbul, Turkey Conflict of interest: The authors declare no potential conflict of interest. The present study was supported by the Research Fund of the Istanbul University-Cerrahpasa (Grant no. 30381).