Differences in Poly(ADP-ribose) Polymerase1- (PARP1-) and Proliferative Cell Nuclear Antigen (PCNA) Immunoreactivity in Patients Who Experienced Successful and Unsuccessful Microdissection Testicular Sperm Extraction Procedures Purpose: The aim of this study is to evaluate expression of deoxyribonucleic acid (DNA) synthesis and repair markers in testicular tissues of azoospermic men in whom sperm retrieval could and could not be achieved as a result of microdissection testicular sperm extraction (micro-TESE) procedure. Materials and Methods: In this prospective cohort study, testicular tissues were retrieved from 60 Non-obstruc- tive Azoospermia (NOA) patients who underwent micro-TESE procedure. These patients were divided into two groups: micro-TESE positive group, which included 30 NOA patients from whom sperm could be extracted via micro-TESE procedure; and micro-TESE negative group, which included 30 NOA patients from whom sperm retrieval could not be achieved via micro-TESE procedure. Expression and distribution patterns of poly(ADP-ri- bose) polymerase-1 (PARP-1) and proliferative cell nuclear antigen (PCNA) in extracted tissues were assessed by immunohistochemical staining to reveal any differences in DNA synthesis and repair between the two groups. Results: Micro-TESE positive group exhibited significantly stronger immunoreactivity for both PCNA and PARP- 1 (P = .001 and P = .001 respectively). The results of this study reveal that both DNA synthesis and repair markers were expressed strongly in patients who experienced successful micro-TESE procedure. Conclusion: Although further studies are needed to support these findings, PARP-1 and PCNA expression in tes- ticular tissues of NOA patients could be promising predictive factors for micro-TESE procedure success. Key words: Azoospermia, Micro-Tese, PCNA, PARP-1 INTRODUCTION Male factor is an important cause of infertility, contributing to 40-50% of all cases. Varicocele, cryptorchidism, infections, obstructive lesions, cystic fibrosis, erectile dysfunction, trauma, and tumors are very well known etiologies of male infertility as well as defective spermatozoa.(1) Non-obstructive azoospermia (NOA), which is de- fined as the absence of spermatozoa in semen due to impaired spermatogenesis, is the contributing factor for approximately 10% of cases of male factor infertility. (2) Sexual chromosomal abnormalities, Y chromosome translocations and microdeletions, trauma, oxidative stress, cryptorchidism, and radiation are possible etiol- ogies for NOA.(3) Retrieval of spermatozoa is achieved by testicular sperm extraction (TESE) in 50% of NOA cases, which was described for the first time in 1994.(4,5) Over time, micro-TESE, which allows direct visualiza- tion of tubules containing more germ cells with active spermatogenesis via an operating microscope, became the method of choice for spermatozoa retrieval (SR).(6) Poly ADP-ribosylation is a post-translational modifica- tion of proteins that plays a key role in the maintenance 1 Izmir Medical Park Hospital IVF Unit, Izmir, Turkey. 2 Irenbe IVF Center, Izmir, Turkey. 3 Manisa Celal Bayar University, School of Vocational Health Service, Manisa, Turkey. 4 Izmir Medical Park Hospital Urology Department, Izmir,Turkey. 5 Izmir Medical Park Hospital Obstetric and Gynecology Department, Izmir,Turkey 6 Izmir Medical Park Hospital, Biostatistics Department, Izmir,Turkey. *Correspondence: IRENBE IVF center, Talatpasa Avenue, No:6, 35220 Konak/Izmir/TURKEY. E mail: barisbuke@hotmail.com Received March 2017 & Accepted June 2017 of genomic integrity and DNA repair.(7) Poly (ADP-ri- bose) polymerase-1 (PARP-1) is involved in many molecular and cellular processes such as DNA damage detection and repair, cell differentiation, apoptosis, and chromatin structure modulation.(8) Sufficient spermatogenesis depends on the proliferative activity of spermatogonia and the loss of germ cells during meiosis and spermiogenesis.(9) Proliferative cell nuclear antigen (PCNA) is a nuclear matrix protein in- volved in DNA synthesis and repair. PCNA is also a useful tool in the diagnosis of germinal arrest, which is a result of DNA synthesis deterioration.(10) This study aimed to explore the differences in DNA synthesis and repair in testicular tissues of NOA pa- tients from whom sperm retrieval could and could not be achieved, by way of evaluating the expression and distribution patterns of PARP-1 and PCNA immunohis- tochemically. PATIENTS AND METHODS Study Population The study population included 60 primary infertile azo- ospermic men with no endocrinopathy, known chromo- SEXUAL DYSFUNCTION AND ANDROLOGY Süleyman Akarsu1, Baris Büke2*, Seren Gülsen Gürgen3, Serkan Akdemir4, Funda Gode1, Merve Biçer5, Mustafa Agah Tekindal6 , Ahmet Zeki Isik1 Sexual Dysfunction and Andrology 5018 some disorder, retrograde ejaculation, history of uro- genital surgery or erectile dysfunction, aged between 31-39 years, who applied to Izmir University IVF Clin- ic, between September 2015 and April 2016. Patients with numerical, structural chromosome abnormalities and with partial translocation were not included in the study. The participants were subjected to micro-TESE procedure by the same urologist and divided into two matched groups, those with sperm in tissue specimens, and those without In 30 men, SR could not be achieved (micro-TESE negative group), while in the remaining 30 men, SR could be achieved (micro-TESE positive group). The local ethics committee approved the study protocol. After obtaining informed written consent from each of the participants, general health data were collected. Procedure For each individual, chromosome number and structures were studied by examining 20 metaphase numbers us- ing lymphocyte cell culture from peripheral blood and GTG banding. All of the participants had normal karyo- types and hormonal status including FSH. Azoospermia was confirmed in at least two different semen analyses of 60 primary infertile men, according to World Health Organization guidelines.(11) To evaluate testicular size and texture, all participants underwent testicular ultra- sound and Doppler sonographic examination. Testicular tissue samples were extracted via microdis- section-TESE method as defined by Schlegel.(12) The extracted tissues were fixed in neutral formalin for 24 hours. After washing the tissues in tap water for a night long, they were dehydrated through ascending grades of alcohol and cleared in xylene. 5 micron thick sections were taken to poly-lysine coated glass slides and parted for immunohistochemistry. Evaluation Immunohistochemistry The sections were incubated at 60 °C for overnight, dep- araffinized in xylene, and dehydrated through descend- ing grades of alcohol. For antigen retrieval, the sections were boiled in citrate buffer (10 mM, pH 6.0) for 15 minutes in a microwave processor (Thermo, CA, USA). To prevent endogenous peroxidase activity, the sections were treated with 3% hydrogen peroxide (Thermo, CA, USA) for 15 minutes. The sections were then blocked with blocking serum (Ultra V Block, Thermo, CA, USA) for 10 minutes. Subsequently, the sections were incubated with primary antibodies PARP-1 (Scbt, CA, USA) and PCNA (Thermo, CA, USA) for 60 minutes at room temperature and humid air. Then, antigen-an- tibody complex was fixed with biotinylated secondary antibody and streptavidin-peroxidase complex (20 min- utes). AEC (Thermo, CA, USA) was used for labeling. The sections were then counterstained with Mayer’s hematoxylin and mounted for microscopic evaluation. The images were recorded via an Olympus microscope with an attached camera (CX31 Germany). Ten fields on each slide were chosen randomly under X400 magnification, and the H score was established according to density and percentage of involvement. The density of involvement was scored as 0 (no involve- ment), 1(+, weak immunoreactivity), 2(++, moderate immunoreactivity), or 3(+++, strong immunoreactivi- ty). The percentage of involvement/immunoreactivity was calculated by division of the number of immunore- active cells to total cells and scored as 1 (0-10%, focal), 2 (11-50%, regional), and 3 (51-100%, diffuse). For each field, density and amount scores were calculated with the formula: “ HScore= ΣPi.(i+1)”. The sum of 10 fields’ scores in each slide made up the individual slide score.(13) Image analysis software (Leica Q Win V3 Plus Image, Leica, Germany) was used for each field’s score calculation. Statistical Analysis Statistical analyses were performed using Statistical Package for Social Sciences version 20.0 (SPSS Sta- tistics for Windows, Version 20.0. Armonk, NY: IBM Corp.). For discrete and continuous variables, descrip- tive statistics (mean, standard deviation, median, mini- mum value, maximum value, and percentile) are given. In addition, the homogeneity of the variances, which is one of the prerequisites of parametric tests, was checked through Levene’s test. The assumption of normality was tested via the Shapiro–Wilk test. A total of 60 in- dividuals participated in the study. All the individuals were independent of each other. For this reason, two in- dependent group comparisons were used for the study. To compare the differences between the two groups, the Student’s t-test was used when the parametric test pre- requisites were fulfilled, and the Mann–Whitney U test was used when such prerequisites were not fulfilled. RESULTS Table 1 summarizes the statistical differences between groups. In the micro-TESE negative group, fewer spermato- genic cells showed PARP-1 expression, and immuno- reactivity was weaker as well. In the micro-TESE pos- itive group, spermatogenic cells that showed PARP-1 expression were more frequent, and immunoreactivity, especially in primary spermatocytes, was stronger (Fig- ure 1). Regarding PCNA staining, the micro-TESE negative group included few immunoreactive cells, while in the micro-TESE positive group, the number of immunore- active cells was greater (Figure 2). DISCUSSION The main purpose of this study was to determine wheth- er there is a difference in PARP-1 and PCNA immuno- reactivity in two groups of infertile men suffering from Sexual Dysfunction and Infertility 4039 Table 1. Comparisons of PARP-1 and PCNA immunoreactivity in TESE negative and positive groups. Groupa n Mean ±SD Min-Max p Parp_1 TESE-negative 30 73,30 ± 20,31 45-117 ,001 TESE-positive 30 203,97 ± 18,23 176-240 PCNA TESE-negative 30 28,23 ± 4,71 19-37 ,001 TESE-positive 30 61,90 ± 7,71 50-75 aData is presented as mean ± SD or number (percent) PARP-1 and PCNA Immunoreactivity in TESE Tissues-Akarsu et al. Vol 14 No 05 September-October 2017 5019 non-obstructive azoospermia. The first group of pa- tients consisted of men in whom SR could be achieved as a result of micro-TESE procedure, while the other group included azoospermic men in whom SR could not be achieved. Our data revealed that the micro-TESE positive group showed stronger PARP-1 and PCNA im- munoreactivity, which indicates better DNA synthesis and repair. Apart from genetic disorders, the etiologic factors for NOA, such as alkylating agents, radiation, and toxins, are suggested to contribute NOA by accumulating re- active oxygen species (ROS). Accumulation of ROS causes DNA damage and impairs DNA integrity.(14) As a result of DNA damage and loss of integrity, spermat- ogenesis and sperm-oocyte interaction is destructed.(15) Furthermore, DNA damage in sperm cells is related to fertilization failure, lower IVF success, poor placenta- tion, and pregnancy complications.(16) ROS-related DNA damage causes a substantial amount of single and double strand DNA breaks that lead to activation of PARP enzymes.(17) PARP enzymes act in many cellular processes including DNA repair, cellular proliferation, differentiation, apoptosis, and necrosis. PARP-1 is the enzyme responsible for more than 90% of PARP activity in human testes.(18) PARP-1 achieves DNA repair by DNA base excision, homologous recom- bination, and non-homologous end-joining processes. There is a large amount of evidence in the literature that poly ADP-ribosylation plays an important role in safe- guarding DNA integrity in spermatogenesis.(19) Howev- er, there is evidence regarding a possible role of PARP overactivation due to excessive amount of ROS that can lead to male reproductive disorders via necroptotic cell death. It is considered to be related with the type and amplitude of the factor that brings out ROS.(20,21) Most of the studies regarding PARP expression in tes- ticular tissue have been implemented in rats. Moreover, in the context of male fertility disorders, only a few studies exist. Two different studies investigated PARP expression in testicular tissue of varicocele patients and reported stronger immunoreactivity in the varicocele group, indicating increased DNA repair.(22,23) Two other studies investigated the effect of aging on PARP ex- pression in testicular tissues, and both studies reported stronger immunoreactivity in elder patients, similar to varicocele patients, indicating more DNA repair due to increased amount of DNA strand breaks.(24,25) The pres- ent study is the first to investigate PARP expression in testicular tissues of NOA patients undergoing mi- cro-TESE procedure. The results of this study revealed stronger immunoreactivity in micro-TESE positive group regarding PARP-1 staining, possibly indicating a better response to stress stimulus via increased DNA repair. Proliferative cell nuclear antigen (PCNA) is a well- known marker of DNA synthesis that has a key role in DNA replication. It also has a role in cell cycle regula- tion and DNA repair.(10) The PCNA index, which was developed to assess aggressiveness of tumors, has also been shown to be a useful marker to assess germ cell kinetics.(26,27) Our data revealed stronger immunore- activity for PCNA in the micro-TESE positive group, indicating increased DNA synthesis and proliferation activity in this group. The testicular cell number in the seminiferous tubules is maintained by a dynamic balance between cell pro- liferation and apoptotic cell death(28) Fragmentation of DNA in the nucleus is one of the morphological chang- es in the apoptotic process and can be detected in his- tological sections using the TUNEL (In situ terminal deoxynucleotidlytransferase-mediated deoxy-UTP nick end labeling) method.(29) This assay identifies single and double-stranded DNA breaks, labeling free 3αOH termini with modified nucleotides in a template-inde- pendent manner(30) In this experimental study, we did not perform the apoptosis pathway. However, we are planning to investigate apoptotic signal relations in testicular tissue using the micro-TESE procedure as a continuation of the present study. CONCLUSIONS In summary, the results of the present study revealed in- PARP-1 and PCNA Immunoreactivity in TESE Tissues-Akarsu et al. Figure 1. Immunohistochemical expression of PARP-1 in testicu- lar tissues of NOA patients. A1 and A2 belong to TESE negative group; B1 and B2 belong to TESE positive group. α: Immun pos- itive spermotogenic cells. A1 and B1 X100 = 50 µm, X400 Bar = 10 µm. Mayer’s hematoxylin background staining. Figure 2. Immunohistochemical expression of PCNA in testicu- lar tissues of NOA patients. A1 and A2 belong to TESE negative group; B1 and B2 belong to TESE positive group. : Immun pos- itive spermotogenic cells. A1 and B1 X100 = 50 µm, X400 Bar = 10 µm. Mayer’s hematoxylin background staining. Sexual Dysfunction and Andrology 5020 creased PARP-1 and PCNA immunoreactivity reflecting greater DNA repair and synthesis activity in testicular tissues of NOA patients in whom SR could be achieved by micro-TESE procedure. In other words, increased PARP-1 and PCNA immunoreactivity was evident in successful TESE procedures. Based on these findings, it is possible to suggest that stronger PARP-1 and PCNA immunoreactivity in testicular tissue of NOA patients can be a promising predictor of successful SR following TESE procedures. However, we should emphasize that it would be inappropriate to make such a decision solely on the basis of the immunohistochemical findings of a single study. Further studies with additional DNA re- pair, synthesis, and apoptosis markers will be useful to enlighten the issue. ACKNOWLEDGEMENT This study was approved in IVF Center, Izmir Medical Park Hospital, as a research project. The authors would like to thank Dr. Mustafa Agah Tekindal and appreciate her support for the preparing of this manuscript. CONFLICT OF INTEREST The authors report no conflict of interest. REFERENCES 1. Kumar N, Sıngh A K. Trends Of Male Factor Infertility, An mportant Cause Of Infertility: A Review Of Literature. J Hum Reprod Sci. 2015; 8: 191. 2. Palermo GD, Schlegel PN, Harıprashad JJ, et al. 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