Effects of Psychological Status on The Oxidation Parameters of Semen and Blood in Azoospermic Men Kemal Gumus1*, Mehmet Gulum2, Ercan Yeni3, Halil Ciftci3, Yigit Akin4, Emre Huri2, Hakim Çelik5, Özcan Erel6 Purpose: In limited number of studies performed concerning the psychological moods of female, and male with the diagnosis of infertility, data related to increased incidence of depression, and anxiety have been reported. The objective of this study is to determine whether azoospermia has any psychological effects on men, and investigate the potential effects of psychological mood on seminal, and plasma oxidative parametres. Materials and Methods: Twenty-seven patients whose two consecutive semen analyses were reported as pellet –negative azoospermia constituted the azoospermic group, and 30 healthy individuals who applied to the infertility polyclinic with normal seminal parametres comprised the normozoospermic group. Results: BECK Anxiety scores were significantly higher in the azoospermic group (P = 0.009). When compared with the normozoospermic group, higher levels of oxidative parametres, but lower levels of the antioxidative parametre were detected in the azoospermic group (P < 0.05). In the azoospermic group, a positive correlation was detected between BECK Anxiety and total oxidant status. Anxiety may increase oxidative parametres in both plasma, and seminal fluid (r = 473, p = 0.026). Conclusion: Anxiety may increase oxidative parametres in both plasma, and seminal fluid. Oxidative milieu may impair sperm quality, and affect the success rates of assisted reproductive treatments. The determination of oxida- tive potential in infertile men, thiol, and prolidase may be used as biomarkers. Keywords: psychological status; oxidative parametres; azoospermia; infertility INTRODUCTION Infertility, defined as the inability to conceive after 1 year of regular unprotected sexual intercourse, affects around 15% of all couples of reproductive age, with about 50% being associated with abnormalities in the male, called male factor infertility(1,2). One of the causes of male factor infertility is azoospermia. Azoospermia refers to absence of sperm in the ejaculate after centrif- ugation and microscopic analysis of two semen samples obtained at different time points. This condition affects approximately 1 % of the general male population and 15 % of subfertile men(3). The term “oxidative stress” (OS) is defined as an imbalance between the production and the elimination of reactive oxygen species that re- sults in an accumulation of oxidative damage(1). Within the last decades, the knowledge concerning the link be- tween OS and psychiatric disorders has been surging(4). In limited number of studies performed concerning the psychological moods of female, and male with the diagnosis of infertility, data related to increased inci- dence of depression, and anxiety have been reported(5-7). It has been also reported that psychological conditions trigger the production of reactive free oxygen radicals 1Balikligol Hospital, Department of Urology, Sanliurfa, TURKEY, kemalag27@hotmail.com. 2Hacettepe University, Faculty of Medicine, Department of Urology, Ankara, TURKEY, mehmetgulum@hotmail.com. 3Harran University, Faculty of Medicine, Department of Urology, Sanliurfa,TURKEY,ercanyeni@hotmail.com. 4Izmir Katip Celebi University, Faculty of Medicine, Department of Urology, Izmir, TURKEY; yigitakin@yahoo.com. 5Harran University, Faculty of Medicine, Department of Physiology, Sanliurfa, TURKEY. Hakim Celik, hakimcell@yahoo.com. 6Yildirim Beyazit University, Faculty of Medicine, Department of Clinical Biochemistry, Ankara, TURKEY. *Correspondence: Balikligol hospital, Department of Urology, Sanliurfa, TURKEY, TR-63100, Ankara, Turkey. Tel: +90-5076159971.E mail: kemalag27@hotmail.com. Received April 2018 & Accepted August 2018 leading to disruption of balance between free radicals, and antioxidants with resultant induction of oxidative stress(8,9). Some biomarkers are used in the detection of oxidative stress including prolidase, and thiol/disulfide homeostasis(10-12). Prolidase (Pro) is an intracellular en- zyme necessary to release proline and hydroxyl-proline from the carboxyl terminus of imidodipeptides which take part in collagen degradation, recycle proline for protein synthesis, matrix remodeling and cell growth (8). Exposure of proline decreases antioxidant potential, suggesting its role in OS. Exposure of proline decreases antioxidant potential, suggesting that proline induces OS. Statistically significantly higher oxidative status and prolidase activities have been demonstrated in gen- eralized anxiety disorder(8). Dynamic thiol/disulphide homeostasis was defined as a novel oxidative stress marker by Erel and Neselioglu (13). Dynamic thiol disulphide homeostasis status has critical roles in antioxidant protection, detoxification, signal transduction, apoptosis, regulation of enzymatic activity and transcription factors and cellular signalling mechanisms(14). Moreover, dynamic thiol disulphide homeostasis is being increasingly implicated in many disorders. There is also a growing body of evidence SEXUAL DYSFUNCTION AND ANDROLOGY Sexual Dysfunction and Andrology 295 Vol 16 No 03 May-June 2019 296 demonstrating that an abnormal thiol disulphide home- ostasis state is involved in the pathogenesis of a variety of diseases, including diabetes, cardiovascular diseases, cancer, rheumatoid arthritis, chronic kidney disease, Parkinson's disease, Alzheimer's disease, Friedreich's ataxia, multiple sclerosis(13). The objective of this study is to determine whether azoospermia which is a serious etiological factor of male infertility has any psycholog- ical effects on men, and investigate the potential effects of psychological mood on seminal, and plasma oxida- tive parametres. As far as we may know, this is the first study which investigated the effects of psychological mood on seminal, and plasma oxidative parametres of azoopermic men, and used both prolidase, and thiol/di- sulfide homeostasis as oxidative markers. MATERIALS AND METHODS Fifty seven patients who applied to infertility polyclinic of our hospital between January 2017 and June 2017 because of infertility and voluntarily participated in the trial were included in the study. Twenty-seven patients whose two consecutive semen analyses were reported as pellet –negative azoospermia constituted the azoo- spermic group, and 30 healthy individuals who applied to the infertility polyclinic with normal seminal para- metres comprised the normozoospermic group. Ap- proval of the local ethics commitee was obtained for the study (date of the approval: 05.01.2017 and registration #:01-18). The patients were informed about the study in detail, and then their undersigned consent forms were obtained. Exclusion criteria of the study were as follows: pres- ence of significant leucospermia, age > 45 years, BMI > 30 kg/m2, presence of varicocele, epididymo-orchitis, testicular torsion, testicular trauma, and tumour, smok- ers, and use of pentoxyfilline, vitamin preparations and antidepressants. In addition, participants were asked to fill in Turkish version of the Beck Depression Inventory (BDI) and Beck Anxiety Inventory (BAI). The BDI is composed of 21 questions that are scored on a four-point Likert scale (0-3 points). The BAI was used to assess the se- verity of clinical anxiety during the past week. It is a self-reported inventory containing 21 items on a four- point scale. Beck Anxiety Inventory, and Beck Depres- sion Inventory classify the severity of depression, and anxiety as follows: 0-13 pts, absence of depression or anxiety; 14-19 pts, mild; 20-28 pts, moderate, and 29- 63 pts, severe depression, and anxiety(15). For the investigation of infertility after 3 days of sexual abstinence, the patient ejaculated into a sterile a con- tained by masturbation without using lubricating gel. One drop from collected semen samples was dropped on Macler camera and examined under microscope at 20× magnification, and sperm counts, motility and leu- cocyte counts were determined. Besides, semen sample was spread on a slide, properly stained and dried be- fore examining sperm morphologies under microscope at 100 ×magnification. Based on the results of spermi- ogram according to WHO 2010 criteria, the patients were categorised in azoospermic and normozoospermic groups. The remaining semen samples were centrifuged at 3 000 × g for 15 min and stored at −80°C till oxida- tive parameters were analysed. Besides, venous blood samples were drawn from the participants after 12 hr of fasting and centrifuged at 4 000 × g for 10 min, and the separated serum portions were stored at −80°C ‘de till oxidative parameters (TAS, TOS ) were analysed. Measurement of total oxidant status Seminal plasma TOS levels were analysed using com- mercial Rel Assay kits developed by Erel. For the meas- urement of TOS levels, as described in the operating principles of the test, colorimetric method based on ox- idation of ferrous ion to ferric ion by oxidant molecules contained in the samples was used. The results were ex- pressed in μmol H 2 O 2 /L(16). Measurement of total antioxidant status Seminal plasma TAS was analysed using commercial Rel Assay kits developed by Erel. This method devel- oped by Erel is fully automated system and measures total antioxidant capacity (TAC) of the body against po- tent free radicals. Measurement of TOS levels is based on decolourisation of coloured ABTS cationic radical as a result of reduction by all antioxidant molecules contained in the sample in proportion with total con- centration of antioxidant molecules(17). As a calibrator, Trolox which is a water-soluble analogue of vitamin E is used. The results were expressed as mmol Trolox Equvalent/L. Measurement of oxidative stress index Oxidative stress index of the samples is calculated as the ratio of total oxidant levels of samples to total an- tioxidant levels of the samples and expressed as per- centages(17). Before calculation mmol unit of TAS is converted to the unit of micromole as performed for TOS test. Blood sampling and measurement of dynamic thi- ol/disulphide homeostasis Venous blood samples were drawn in tubes containing ethylenediaminetetraacetic acid and serum samples were immediately separated by centrifugation at 1500 g for 10 min. Samples were coded and stored at −80°C. Serum dynamic thiol/disulphide homeostasis was de- termined with a recently developed spectrophotomet- ric method described by Erel and Neselioglu using an automated clinical chemistry analyser (Roche, cobas 501, Mannheim, Germany)(13). Sodium borohydride is the essential agent in this method that is used to reduce dynamic bonds to functional thiol groups. To prevent extra reduction of 5,5′-dithiobis-2nitrobenzoic acid (DTNB), unused sodium borohydride is removed by formaldehyde addition. The total thiol content of the sample is measured using a modified Ellman reagent. Substracting native thiol contentf rom total thiol gives twice the disulphide amount. Having found the disul- phide amount, the disulphide/total thiol ratio, disul- phide/native thiol ratio and native thiol/total thiol ratio can be calculated. Determination of Prolidase Activity Serum was diluted 40-fold with 2.5 mmol/l Mn 2+,40 mmol/L trizma HCl buffer (pH 8.0) and pre incubat- ed at 37°C for 2 hr. The reaction mixture containing 30 mmol/L gly-pro, 40 mmol/L trizma HCl buffer (pH:8.0), and 100 μL of preincubation serum in 1 mL was incubated at 37°C for 30 min. Adding 0.5 mL of 20 % trichloroacetic acid solution then stopped the in- cubation reaction. The supernatant was used for meas- urement of proline by the method proposed by Myara et al.(18), which is a modification of Chinard’s method. (19) Intra-assay coefficient of variation of the assay was 3.8%. Psychological status in azoospermic men -Gumus et al. Statistical analysis Statistical analyses were performed using SPSS Ver- sion 15 (SPSS Inc. Chicago USA) software program. Distribution of numerous variables was evaluated us- ing Shapiro-Wilk test. Parametric tests were used for data with normal distribution. For intergroup compar- isons parametric Student ‘s t-test, and nonparametric Mann-Whitney U test were used. For correlation Spear- man’s correlation analysis was employed. The results were expressed as mean ± standard deviation, median (interquartile range); and p < .05 was accepted as the level of significance. RESULTS Demographic and BDI, and BAI data are shown in Ta- ble 1. The groups were similar in mean age, education, body mass index (BMI), duration of marriage and in- fertility, and volume of semen. A significant intergroup difference was not found concerning BECK D scores (P = 1.000). BECK A scores were statistically signif- icant higher in the azoospermic group (P = 0.009). In both azoospermic, and normozoospermic groups, any statistically significant correlation did not exist between BECK A (r = 366, p = 0.079; r = 366, p = 0.079 and D scores (r=103,p = 0.631;r = 180, p = 0.400), with dura- tion of marriage, and the infertility duration. Semen analyses When compared with the normozoospermic group, higher levels of oxidative parametres namely prol- idase, TOS, and OSI, but lower levels of the an- tioxidative parametre (ie. TAS) were detected in the azoospermic group (P < 0.05)(Table 2). In the normozoospermic group, the correlation between semen parameters (volume, sperm counts in one milli- litre, sperm motility, and morphology), and TAS, TOS, OSI, prolidase, BECK A and D values was examined. Statistically significantly negative correlations were de- tected between prolidase, and sperm counts (r = -566, P = 0.009), motility (r= -526, P = 0.017) , and mor- phology (r= -545, P = 0.013) , and also between OSI, and sperm counts (r = -650, P = 0.002). A significant correlation was not detected between other parametres. Serum analyses Levels of serum prolidase, TOS, OSI were higher in the azoospermic group relative to normozoospermic group (P < 0.05), but antioxidant parametres (TAS, native thi- ol, total thiol and disulphide) were lower than those of the normozoospermic group (P < 0.05) (Table 3). In the azoospermic group, a positive correlation was de- tected between BECK A and TOS (r = 473, P = 0.026). A negative correlation was detected between BECK A and plasma prolidase levels (r = 493, P = 0.017). DISCUSSION As a generally accepted corollary, major sources of seminal oxidative stress are leucocytes, and defective sperm cells. In our previous study(1), contrary to our pre- diction during planning of the study, we found higher oxidative stress, but lower antioxidative parametres in seminal plasma of azoospermic men devoid of leuko- cytes, and spermazoa in comparison with normozoo- spermic men. As a possible cause of this condition, we reported that azoospermia which is a serious etiological factor of infertility exerts psychological effects on men, and subsequently higher levels of plasma oxidative parametres increasing with stress pass through semi- nal vesicles, and prostate into seminal fluid leading to enhanced oxidative stress, but lower antioxidative par- ametres in seminal plasma of azoospermic men. How- ever we did not analyze the psychological mood of azo- ospermic men in our previous study. Therefore in the Table 1. Study subgroup participants’ demographic, anxiety - depression scores and semen parameters Variables Azoospermic group, n = 27 Normozoospermic group, n = 30 p Value (Mean ± SD) (Mean ± SD) Mean age (years) 29,4 ± 5,7 30.4 ± 6.4 0.535 Education (years) 9.1 ± 4.45 10.5 ± 5.10 0.546 Body Mass Index (BMI) 25.4 ± 5.8 26.5 ± 4.6 0.469 Duration of marriage (years)* 4 (8) 3.5 (3.7) 0.887 Duration of infertility (years)* 4.2 (1,9) --- NA Volume*, ml 1,4 (1) 1,6 (1) 0.605 Concentration**(x106/mL) 0 34.6 ± 37,9 NA Motility** (%motile sperm) 0 42.9 ± 30,0 NA Morfology** (%normal sperm) 0 8.3 ± 6,7 NA BAI** score 20,4 ± 10,1 7.9 ± 5,3 0.003 BDI score * 11 (22) 10.5 (14,2) 1.000 Abbreviations: BDI, Beck Depression Inventory; BAI, Beck Anxiety Inventory *Median (IQR), **Mean ± standard deviation (SD) Statistically significant p values were written as bold Azoospermic group, n = 27 Normozoospermic group, n = 30 p Value Prolidase* (U/I) 36.4 (25,2) 21.7 (12.2) 0.019 TAS**, mmol Trolox Equivalent/L 0.94 ± 0.33 1.30 ± 0.32 0.001 TOS*, μmol H 2 O 2 /L 18.7 (3.5) 15.3 (4.0) 0.037 OSI** 1.99 ± 0.65 1.23 ± 0.52 0.000 Abbreviations: TAS, total antioxidant status; TOS, total oxidant status; OSI, oxidative stress index Table 2. Seminal plasma oxidative/antioxidative parameters in azoospermic and normozoospermic groups. Psychological status in azoospermic men -Gumus et al. Sexual Dysfunction and Andrology 297 Vol 16 No 03 May-June 2019 298 present study we aimed to evaluate psychological mood of the azoospermic men, and investigate the correlation between psychological mood, and oxidative parametres of blood, and seminal fluid. Multiple number of studies which investigated the cor- relation between infertility, and psychological state of both men, and women have been published. In a meta-analysis, Greil et al. analyzed the correlation between infertility, and psychological distress, and indicated that infertility is generally related to psycho- logical distress which may be both the cause, and out- come of infertility(7). The authors also emphasized that the duration of infertility, and treatment should be taken into consideration in studies which would be performed concerning infertility, and psychological distress. Very limited number of studies have investigated psycholog- ical mood in azoospermia which is a serious etiologi- cal cause of infertility. Akbal et al. reported increase in symptoms of erectile dysfunction, anxiety, and depres- sion in azoospermic men in whom TESE could not find any viable spermatozoa relative to normozoospermic men(20). In recent studies, Castro et al. demonstrated presence of a correlation between azoospermia, and neuropsychiatric diseases(21). Bak et al. reported that pa- tients with the established diagnosis of nonobstructive azoospermia which signifies absence of sperm produc- tion had experienced intense panicky feelings with in- creased tendency to insomnia, anxiety and depression. (22) In our present study, we found statistically signifi- cantly higher anxiety scores in azoospermic men when compared with the normozoospermic group. While de- pression scores were not so high in azoospermic men. Levels of anxiety did not correlate with the duration of marriage, and disease in our study, relatively shorter average duration of marriage, and disease in the azoo- spermic group may be the reason why we couldn’t find any correlation between these parametres. In groups with longer duration of infertility, more realistic out- comes may be obtained. It is already known that oxidative parameters increase, while antioxidative parameters decrease in both plas- ma, and seminal fluid of infertile men. In our study, we found increases in oxidative parameters (TOS, OSI, prolidase), but decreases in antioxidative param- eters (TAS, thiol) in plasma, and seminal fluid of azo- ospermic men. Despite contradictory arguments made in many studies, as is the case with multiple number of diseases including anxiety, and depression, prolidase has been associated with increased oxidative stress, and decreased antioxidant levels.(23) It has been estimated that this oxidative effect is caused by release of proline mediated by the action of prolidase. In support with the study which detected higher levels of prolidase in anx- iety, we found a positive correlation between BECK-A scores and levels of TOS, and prolidase in the plasma of azoospermic men. Besides, prolidase and increased ox- idative stress negatively affected sperm counts, motili- ty, and morphology. Ozcan et al. reported the presence of a negative correlation between prolidase activity on the wall of the varicocele, and sperm counts in azoo- spermic men with varicocele.(24) They indicated that this condition might be the result of an oxidative milieu sec- ondary to increased levels of prolidase in the spermatic vein, and duct. In the same study, they reported lack of any correlation between sperm motility, and enzymatic activity of prolidase. Thiol is a novel biomarker which can be measured only in blood, and thiols are antioxidant buffers which bal- ance both intracellular, and extracellular oxidative pro- cesses.(25) In accordance with decreased antioxidative potential in infertile men, we found lower thiol levels in azoospermic infertile men when compared with normo- zoospermic individuals. Though any relevant study has not been performed in infertile men, some studies have demonstrated antioxidative contribution of thiol levels to oxidative processes. Tokgöz et al. detected marked decreases in native, and total thiol levels in the sera of the patients who had undergone prostate biopsies(12). They also indicated that these decreases might be re- lated to acute oxidative stress due to the procedure of prostate biopsies. Kozanhan et al. performed a study with health care professionals who had been exposed to anesthetic gases, and reported decreased thiol, but in- creased disulfide levels(11). They stated that these chang- es stemmed from oxidative effects of anesthetic gases, and thiol/disulfide balance might be used as a biomark- er in the diagnosis of oxidative stress. CONCLUSIONS Infertility is an important health problem which may induce neuropsychiatric disorders as anxiety. Anxiety may increase oxidative parametres in both plasma, and seminal fluid. Oxidative milieu may impair sperm qual- ity, and affect the success rates of assisted reproductive treatments. We think that in the determination of oxida- tive potential in infertile men, thiol, and prolidase may be used as biomarkers in infertility. Table 3. Serum oxidative/antioxidative parameters in azoospermic and normozoospermic groups. Azoospermic group, n = 27 Normozoospermic group, n = 30 p Value Prolidase* 8.8 (9.1) 6,5 (1.9) 0.032 TAS, mmol Trolox Equivalent/L 0.87 ± 0.28 1,13 ± 0.35 0.010 TOS*, μmol H2O2/L 16.7 (3.3) 14.0 (2.7) 0.003 OSI* 1,9 (1.6) 1.25 (0.6) 0.001 Native thiol (μmol/L) 403.9 ± 79.4 475.4 ± 86.8 0.007 Total thiol (μmol/L) 460.5 ± 79.5 549.2 ± 93.9 0.002 Disulphide (μmol/L) 28.3 ± 9.4 36.3 ± 13.5 0.029 Disulphide/native thiol ratio 7.3 ± 2.7 7.9 ± 3.2 0.476 Disulphide/total thiol ratio 6.3 ± 2.1 6.7 ± 2.3 0.496 Native thiol/total thiol ratio 87.4 ± 4.2 86.5 ± 4.6 0.495 Abbreviations: TAS, total antioxidant status; TOS, total oxidant status; OSI, oxidative stress index *Median (IQR), Mean ± SD Psychological status in azoospermic men -Gumus et al. ACKNOWLEDGEMENT This study was approved in Balikligöl Hospital Depart- ment of Urology and Yildirim Beyazit University, Fac- ulty of Medicine, Department of Clinical Biochemistry as a research project. CONFLICT OF INTEREST The authors report no conflict of interest. REFERENCES 1. Gulum M, Gumus K, Yeni E, et al. Blood and semen paraoxonase—arylesterase activities in normozoospermic and azoospermic men. Andrologia. 2017;49. 2. Agarwal A, Durairajanayagam D, Halabi J, Peng J, Vazquez-Levin M. Proteomics, oxidative stress and male infertility. Reprod Biomed Online. 2014;29:32-58. 3. Macaluso M, Wright-Schnapp TJ, Chandra A, et al. A public health focus on infertility prevention, detection, and management. Fertil Steril. 2010;93:16 e1-0. 4. Ng F, Berk M, Dean O, Bush AI. Oxidative stress in psychiatric disorders: evidence base and therapeutic implications. Int J Neuropsychopharmacol. 2008;11:851-76. 5. Drosdzol A, Skrzypulec V. Depression and anxiety among Polish infertile couples--an evaluative prevalence study. J Psychosom Obstet Gynaecol. 2009;30:11-20. 6. Eisenberg ML, Li S, Cullen MR, Baker LC. Increased risk of incident chronic medical conditions in infertile men: analysis of United States claims data. Fertil Steril. 2016;105:629- 36. 7. Greil AL. Infertility and psychological distress: a critical review of the literature. Soc Sci Med. 1997;45:1679-704. 8. Ercan AC, Bahceci B, Polat S, et al. Oxidative status and prolidase activities in generalized anxiety disorder. Asian J Psychiatr. 2017;25:118-22. 9. Rodrigues R, Petersen RB, Perry G. Parallels between major depressive disorder and Alzheimer's disease: role of oxidative stress and genetic vulnerability. Cell Mol Neurobiol. 2014;34:925-49. 10. Gecit I, Eryilmaz R, Kavak S, et al. The Prolidase Activity, Oxidative Stress, and Nitric Oxide Levels of Bladder Tissues with or Without Tumor in Patients with Bladder Cancer. J Membr Biol. 2017;250:455-9. 11. Kozanhan B, Inanli I, Deniz CD, et al. Dynamic thiol disulphide homeostasis in operating theater personnel exposed to anesthetic gases. Am J Ind Med. 2017;60:1003-9. 12. Tokgoz H, Tas S, Giray O, et al. The change in serum Thiol/Disulphide homeostasis after transrectal ultrasound guided prostate biopsy. Int Braz J Urol. 2017;43:455-61. 13. Erel O, Neselioglu S. A novel and automated assay for thiol/disulphide homeostasis. Clin Biochem. 2014;47:326-32. 14. Circu ML, Aw TY. Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic Biol Med. 2010;48:749-62. 15. Beck AT, Ward CH, Mendelson M, Mock J, Erbaugh J. An inventory for measuring depression. Arch Gen Psychiatry. 1961;4:561- 71. 16. Erel O. A new automated colorimetric method for measuring total oxidant status. Clin Biochem. 2005;38:1103-11. 17. Erel O. A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin Biochem. 2004;37:277-85. 18. Myara I, Charpentier C, Lemonnier A. Optimal conditions for prolidase assay by proline colorimetric determination: application to iminodipeptiduria. Clin Chim Acta. 1982;125:193-205. 19. Chinard FP. Photometric estimation of proline and ornithine. J Biol Chem. 1952;199:91-5. 20. Akbal C, Mangir N, Tavukcu HH, Ozgur O, Simsek F. Effect of testicular sperm extraction outcome on sexual function in patients with male factor infertility. Urology. 2010;75:598- 601. 21. Castro A, Rodriguez F, Florez M, et al. Pseudoautosomal abnormalities in terminal AZFb+c deletions are associated with isochromosomes Yp and may lead to abnormal growth and neuropsychiatric function. Hum Reprod. 2017;32:465-75. 22. Bak CW, Seok HH, Song SH, Kim ES, Her YS, Yoon TK. Hormonal imbalances and psychological scars left behind in infertile men. J Androl. 2012;33:181-9. 23. Pirincci N, Kaba M, Gecit I, et al. Serum prolidase activity, oxidative stress, and antioxidant enzyme levels in patients with renal cell carcinoma. Toxicol Ind Health. 2016;32:193-9. 24. Ozcan O, Malkoc E, Cosar A, et al. Prolidase enzyme activity in varicose venous walls related to sperm count in patients with varicocele. Scand J Clin Lab Invest. 2013;73:97-101. 25. Cremers CM, Jakob U. Oxidant sensing by reversible disulfide bond formation. J Biol Chem. 2013;288:26489-96. Psychological status in azoospermic men -Gumus et al. Sexual Dysfunction and Andrology 299