From pathophysiology to practice: addressing oxidative stress and sperm DNA fragmentation in Varicocele-affected subfertile men

Lira Neto, Filipe Tenório; Campos, Lucas Ribeiro; Roque, Matheus; Esteves, Sandro C.

doi:10.1590/S1677-5538.IBJU.2024.9917

Filipe Tenório Lira Neto

Andros Recife, Recife, PE, Brasil

Departamento de Urologia, Instituto de Medicina Integral Prof. Fernando Figueira, Recife, PE, Brasil

Hospital Santa Joana Recife, Recife, PE, Brasil

https://orcid.org/0000-0002-6783-307X

Lucas Ribeiro Campos

Departamento de Urologia, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brasil

Matheus Roque

Departamento de Medicina Reprodutiva, Mater Prime, São Paulo, SP, Brasil

Sandro C. Esteves Correspondence address:Sandro C. Esteves, MD, PhD ANDROFERT - Clínica de Andrologia e Reprodução Humana, Centro de Ref. em Reprodução Masculina Av. Dr. Heitor Penteado, 1464 Campinas, Brasil E-mail: s.esteves@androfert.com.br

ANDROFERT - Clínica de Andrologia e Reprodução Humana, Centro de Referência. em Reprodução Masculina, Campinas, SP, Brasil

Departamento de Cirurgia, Disciplina de Urologia da Universidade Estadual de Campinas - UNICAMP, Campinas, SP, Brasil

Department of Clinical Medicine, Faculty of Health, Aarhus University, Aarhus, Denmark

Correspondence address:
Sandro C. Esteves, MD, PhD ANDROFERT - Clínica de Andrologia e Reprodução Humana, Centro de Ref. em Reprodução Masculina Av. Dr. Heitor Penteado, 1464 Campinas, Brasil E-mail: s.esteves@androfert.com.br

CONFLICT OF INTEREST

None declared.

Author, year, (country)	Assays	Study groups	Main results
Hendin et al., 1999 (USA) (⁴⁹49 Hendin BN, Kolettis PN, Sharma RK, Thomas AJ, Jr., Agarwal A. Varicocele is associated with elevated spermatozoal reactive oxygen species production and diminished seminal plasma antioxidant capacity. J Urol. 1999;161:1831-4.)	Seminal ROS by chemiluminescence with luminol; seminal TAC by enhanced chemiluminescence	17 normozoospermic men without varicocele; 15 men with incidental varicocele; 21 infertile men with palpable varicocele	ROS levels: Controls: 1.3 ± 0.33 log[ROS+1] Men with incidental varicocele: 1.99 ± 0.26 log[ROS+1] (P<0.05 versus controls); Infertile men with varicocele:2.18 ± 0.25 log[ROS+1] (P<0.05 versus controls); TAC: Controls: 1443.0 ± 105.0 molar Trolox; Men with incidental varicocele: 939.0 ± 107.0 molar Trolox (P<0.05 versus controls); Infertile men with varicocele: 1186.0 ± 96.9 molar Trolox (P<0.05 versus controls)
Sharma et al., 1999 (USA) (⁴³43 Sharma RK, Pasqualotto FF, Nelson DR, Thomas AJ, Jr., Agarwal A. The reactive oxygen species-total antioxidant capacity score is a new measure of oxidative stress to predict male infertility. Hum Reprod. 1999;14:2801-7.)	Seminal ROS by chemiluminescence with luminol; seminal TAC by enhanced chemiluminescence	25 normozoospermic fertile, healthy men; 55 infertile men with palpable varicocele	ROS levels: Controls: 1.39 ± 0.73 log[ROS+1]; Infertile men with varicocele: 2.10 ± 1.21 log[ROS+1] (P<0.05 versus controls) TAC: Controls: 1650.93 ± 532.22 molar Trolox; Infertile men with varicocele: 1100.11 ± 410.13 molar Trolox (P<0.05 versus controls)
Köksal et al., 2000 (Turkey) (⁵⁶56 Köksal IT, Tefekli A, Usta M, Erol H, Abbasoglu S, Kadioglu A. The role of reactive oxygen species in testicular dysfunction associated with varicocele. BJU international. 2000;86:549-52.)	Intratesticular MDA by thiobarbituric acid reaction	10 infertile men without varicocele; 15 infertile men with palpable varicocele	MDA levels: Infertile men without varicocele: 33.5 ± 18.93 pmol/mg; Infertile men with varicocele: 38.3 ± 22.92 pmol/mg (P NS versus controls); MDA levels in men with grade III varicocele were higher than in men with lower grade varicocele (P<0.05)
Pasqualotto et al., 2000 (USA) (⁴⁰40 Pasqualotto FF, Sharma RK, Nelson DR, Thomas AJ, Agarwal A. Relationship between oxidative stress, semen characteristics, and clinical diagnosis in men undergoing infertility investigation. Fertility and sterility. 2000;73:459-64.)	Seminal ROS by chemiluminescence with luminol; Seminal TAC by enhanced chemiluminescence	19 normozoospermic men without varicocele; 77 infertile men with palpable varicocele	ROS levels: Controls: 1.3 ± 0.3 log[ROS+1]; Infertile men with varicocele: 2.2 ± 0.13 log[ROS+1] (P<0.05 versus controls) TAC: Controls: 1653.98 ± 115.29 molar Trolox; Infertile men with varicocele: 1173.05 ± 58.07 molar Trolox (P<0.05 versus controls)
Saleh et al., 2003 (USA) (⁴⁴44 Saleh RA, Agarwal A, Sharma RK, Said TM, Sikka SC, Thomas AJ, Jr. Evaluation of nuclear DNA damage in spermatozoa from infertile men with varicocele. Fertility and sterility. 2003;80:1431-6.)	Seminal ROS by chemiluminescence with luminol; Seminal TAC by enhanced chemiluminescence	16 fertile men without varicocele; 15 infertile men without varicocele; 16 infertile men with palpable varicocele	ROS levels: Controls: 0.36 (IQR: 0.1, 2) (cpm)/20×106 sperm/mL; Infertile men without varicocele: 1.7 (IQR: 0.1, 5.4) (cpm)/20×106 sperm/mL (P NS versus controls); Infertile men with varicocele: 12 (IQR: 1.3, 53.4) (cpm)/20×106 sperm/mL (P<0.05 versus controls) TAC: Controls: 871 (IQR: 699, 1288) molar Trolox; Infertile men without varicocele: 904 (IQR: 693, 978) molar Trolox (P NS versus controls); Infertile men with varicocele: 693 (IQR: 499, 822) molar Trolox (P<0.05 versus controls)
Allamaneni et al., 2004 (USA) (⁵⁷57 Allamaneni SS, Naughton CK, Sharma RK, Thomas AJ, Jr., Agarwal A. Increased seminal reactive oxygen species levels in patients with varicoceles correlate with varicocele grade but not with testis size. Fertility and sterility. 2004;82:1684-6.)	Seminal ROS by chemiluminescence with luminol; Seminal TAC by enhanced chemiluminescence	46 infertile men with palpable left varicocele	Median ROS level 119 (13, 2475) x10⁴cpm ROS levels positively correlated with varicocele grade
Mehraban et al., 2005 (Iran) (³⁷37 Mehraban D, Ansari M, Keyhan H, Sedighi Gilani M, Naderi G, Esfehani F. Comparison of nitric oxide concentration in seminal fluid between infertile patients with and without varicocele and normal fertile men. Urology journal. 2005;2:106-10.)	Seminal total nitrite and nitrate levels	40 fertile men without varicocele; 40 infertile men without varicocele; 40 infertile men with palpable varicocele	Seminal total nitrite and nitrate levels: Controls: 37.06 ± 20.39 μmol/L; Infertile men without varicocele: 33.7 ± 18.99 μmol/L (P NS versus controls); Infertile men with varicocele: 52.34 ± 26.62 μmol/L (P<0.05 versus controls and infertile men without varicocele)
Smith et al., 2006 (Chile) (⁹⁸98 Smith R, Kaune H, Parodi D, Madariaga M, Rios R, Morales I, et al. Increased sperm DNA damage in patients with varicocele: relationship with seminal oxidative stress. Hum Reprod. 2006;21:986-93.)	Seminal ROS by chemiluminescence with luminol; Seminal TAC by enhanced chemiluminescence	25 normozoospermic healthy donors 37 men with varicocele and normal SA; 18 men with varicocele and abnormal SA	ROS levels: Controls: 2.8 ± 0.9 log[ROS+1]; Men with varicocele and normal SA: 3.3 ± 1.2 log[ROS+1] (P<0.05 versus controls); Men with varicocele and abnormal SA: 4.3 ± 1.1 log[ROS+1] (P<0.05 versus controls) TAC: Controls: 1.2 ± 0.1 mM Trolox; Men with varicocele and normal SA: 1.1 ± 0.4 mM Trolox (P NS versus controls); Men with varicocele and abnormal SA: 1.1 ± 0.5 mM Trolox (P NS versus controls)
Ishikawa et al. 2007 (Japan) (⁵⁸58 Ishikawa T, Fujioka H, Ishimura T, Takenaka A, Fujisawa M. Increased testicular 8-hydroxy-2'-deoxyguanosine in patients with varicocele. BJU international. 2007;100:863-6.)	Intratesticular 8-OHdG positive cell by immunostaining	5 healthy fertile men; 36 infertile men with palpable varicocele and abnormal SA	Incidence of 8-OHdG immunostained germ cells: Controls: 29 ± 5.4%; Varicocele grade I:38 ±10%, (P<0.05 versus controls); Varicocele grade II: 41 ± 9.1% (P<0.05 versus controls); Varicocele grade III: 57 ± 9.3% (P<0.05 versus controls and grade I+II)
Sakamoto et al., 2008 (Japan) (³⁸38 Sakamoto Y, Ishikawa T, Kondo Y, Yamaguchi K, Fujisawa M. The assessment of oxidative stress in infertile patients with varicocele. BJU international. 2008;101:1547-52.)	Seminal NO levels; Seminal 8-OHdG levels; Seminal SOD activity	15 normozoospermic men without varicocele; 15 infertile men with varicocele and normal SA; 15 infertile men with palpable varicocele and oligozoospermia	NO levels: Controls: 8.2 ± 4.3μmol/L; Men with varicocele and normal SA:15.4 ± 0.3μmol/L (P<0.05 versus controls); Men with varicocele and oligozoospermia: 7.8 ± 4.0 μmol/L (P NS versus controls); 8-OHdG levels: Controls: 14.7 ± 8.3 μmol/L; Men with varicocele and normal SA:10.0 ± 5.4 μmol/L (P NS versus controls); Men with varicocele and oligozoospermia: 10.8 ± 7.5 μmol/L (P NS versus controls); SOD activity: Controls: 75.6 ± 13.1%; Men with varicocele and normal SA: 84 ± 6.7% (P<0.05 versus controls); Men with varicocele and oligozoospermia: 89.4 ± 4.4 % (P<0.05 versus controls)
Mostafa et al., 2009 (Egypt) (⁴⁷47 Mostafa T, Anis T, Imam H, El-Nashar AR, Osman IA. Seminal reactive oxygen species-antioxidant relationship in fertile males with and without varicocele. Andrologia. 2009;41:125-9.)	Seminal MDA by thiobarbituric acid reaction; Seminal H2O2 by spectrophotometric Method; Seminal SOD; Seminal GPx; Seminal Cat	45 fertile men without varicocele; 45 fertile men with varicocele; 42 infertile men with unilateral palpable varicocele and abnormal SA; 44 infertile men with abnormal SA and without varicocele	MDA and H2O2 were significantly higher, and antioxidants were significantly lower in fertile men with varicocele, OA men with and without varicocele compared with controls; All ROS parameters were increased, and all antioxidants were decreased in infertile men with varicocele compared to infertile men without varicocele
Abd-Elmoaty et al., 2010 (Egypt) (⁶²62 Abd-Elmoaty MA, Saleh R, Sharma R, Agarwal A. Increased levels of oxidants and reduced antioxidants in semen of infertile men with varicocele. Fertility and sterility. 2010;94:1531-4.)	Seminal NO levels by colorimetric method; Seminal MDA by colorimetric method; Seminal SOD; Seminal GPx; Seminal Cat	18 fertile men without varicocele; 42 infertile men with varicocele	MDA levels: Controls: 8.4 ± 1.3 pmol/mL; Infertile men with varicocele: 13.5 ± 2.8 pmol/mL (P<0.05 versus controls) NO levels: Controls: 11.3 ± 1.0 nmol/L; Infertile men with varicocele: 17.9 ± 4.1 nmol/L (P<0.05 versus controls); CAT, SOD, GPX, and ascorbic acid were significantly lower in infertile men with varicocele compared with fertile men (P values <.05, .01, .01, and .05, respectively)
Blumer et al., 2011 (Brazil) (¹⁶⁵165 Blumer CG, Restelli AE, Giudice PT, Soler TB, Fraietta R, Nichi M, et al. Effect of varicocele on sperm function and semen oxidative stress. BJU international. 2012;109:259-65.)	Seminal MDA by thiobarbituric acid reaction	19 men without varicocele; 12 men with varicocele (fertility status not informed);	MDA levels: Controls: 301.4 ± 95.9 ng/mL Men with varicoceles: 287.1 ± 127.7 ng/mL (P NS versus controls)
Mostafa et al., 2012 (Egypt) (⁵⁹59 Mostafa T, Anis T, El Nashar A, Imam H, Osman I. Seminal plasma reactive oxygen species-antioxidants relationship with varicocele grade. Andrologia. 2012;44:66-9.)	Seminal MDA by thiobarbituric acid reaction; Seminal H2O2 by spectrophotometric Method; Seminal SOD; Seminal GPx; Seminal Cat	20 fertile men without varicocele; 22 infertile men with grade 1varicocele; 43 infertile men with grade II varicocele; 23 infertile men with grade III varicocele	Levels of MDA and H2O2 were increased and antioxidants; SOD, Cat, GPx, vit. C levels were decreased in men with varicocele of all grades (I, II, III) compared with the controls; Men with grade II and III varicocele demonstrated higher MDA and H2O2 levels as well as decreased activities of SOD, Cat, GPx, and levels of vit. C compared with men with grade I varicocele.
Mostafa et al., 2016 (Egypt) (⁵⁰50 Mostafa T, Rashed L, Taymour M. Seminal cyclooxygenase relationship with oxidative stress in infertile oligoasthenoteratozoospermic men with varicocele. Andrologia. 2016;48(2):137-42.)	Seminal MDA by thiobarbituric acid reaction; Seminal GPx	24 fertile men without varicocele; 22 fertile men with varicocele; 34 infertile men with palpable varicocele and abnormal SA; 24 infertile men with abnormal SA and without varicocele	MDA levels: Controls: 1.2 ± 0.17 nmol/mL; Fertile men with varicocele: 1.9 ± 0.69 nmol/mL (P<0.05 versus controls); Infertile men without varicocele and abnormal SA: 2.4 ± 0.47 nmol/mL (P<0.05 versus controls and other groups); Infertile men with varicocele and abnormal SA: 3.02 ± 0.47 nmol/mL (P<0.05 versus controls and other groups) GPx activity: Controls: 0.47 ± 0.6 U/mL; Fertile men with varicocele: 0.36 ± 0.8 U/mL (P<0.05 versus controls); Infertile men without varicocele and abnormal SA: 0.3 ± 0.03 U/mL (P<0.05 versus controls and other groups); Infertile men with varicocele and abnormal SA: 0.21 ± 0.04 U/mL (P<0.05 versus controls and other groups)
Ni et al., 2016 (China) (⁶¹61 Ni K, Steger K, Yang H, Wang H, Hu K, Zhang T, et al. A comprehensive investigation of sperm DNA damage and oxidative stress injury in infertile patients with subclinical, normozoospermic, and astheno/oligozoospermic clinical varicocoele. Andrology. 2016;4:816-24.)	Seminal MDA by thiobarbituric acid reaction	25 healthy normozoospermic men without varicocele; 15 infertile men with subclinical varicocele; 19 infertile men with grade I varicocele; 18 infertile men with grade II varicocele; 14 infertile men with grade III varicocele	MDA levels: Control group: 7.45 ± 3.58 nmol/mL; Varicocele subclinical group: 7.22 ± 3.33 nmol/mL; Varicocele grade I group: 12.18 ± 4.86 nmol/mL (P<0.05 versus controls); Varicocele grade II group: 14.12 ± 5.42 nmol/mL (P<0.05 versus controls); Varicocele grade III group: 15.86 ± 6.78 nmol/mL (P<0.05 versus controls)
Abdelbaki et al., 2017 (Egypt) (⁴²42 Abdelbaki SA, Sabry JH, Al-Adl AM, Sabry HH. The impact of coexisting sperm DNA fragmentation and seminal oxidative stress on the outcome of varicocelectomy in infertile patients: A prospective controlled study. Arab journal of urology. 2017;15:131-9.)	Seminal ROS by chemiluminescence with luminol; Seminal TAC by Colorimetric assay kit	20 normozoospermic fertile men without varicocele; 60 infertile men with palpable varicocele	ROS levels: Controls: 2.62 ± 0.8 log[ROS+1]; Infertile men with varicocele: 4.49 ± 0.9 log[ROS+1] (P<0.05 versus controls); TAC: Controls: 1.5 ± 0.5 mM Trolox; Infertile men with varicocele: 0.97 ± 0.4 mM Trolox (P<0.05 versus controls)
Alkan et al., 2018 (Turkey) (⁶⁰60 Alkan İ, Yüksel M, Canat HL, Atalay HA, Can O, Özveri H, et al. Superoxide Anion Production by the Spermatozoa of Men with Varicocele: Relationship with Varicocele Grade and Semen Parameters. World J Mens Health. 2018;36:255-62.)	Seminal ROS by chemiluminescence with luminol; Seminal superoxide anion by chemiluminescence with lucigenin	13 normozoospermic men without varicocele; 17 men with grade II varicocele; 17 men with grade III varicocele	ROS levels: Controls: 2.4 ± 0.1 log[ROS+1]; Men with grade II varicocele: 2.7 ± 0.4 log[ROS+1] (P<0.05 versus controls); Men with grade III varicocele: 3.2 ± 0.5 log[ROS+1] (P<0.05 versus controls and grade II group); Superoxide anion levels: Controls: 2.3 ± 0.2 log[ROS+1]; Men with grade II varicocele: 2.5 ± 0.3 log[ROS+1] (P<0.05 versus controls); Men with grade III varicocele: 3.0 ± 0.5 log[ROS+1] (P<0.05 versus controls and grade II group)
Tanaka et al., 2020 (Japan) (⁴⁵45 Tanaka T, Kobori Y, Terai K, Inoue Y, Osaka A, Yoshikawa N, et al. Seminal oxidation-reduction potential and sperm DNA fragmentation index increase among infertile men with varicocele. Human fertility (Cambridge, England). 2022;25:142-6.)	Seminal ORP measured by MiOXSYS	102 normozoospermic men without varicocele; 138 infertile men with palpable varicocele	ORP: Men without varicocele: 9.82 ± 10.31 mV/10⁶ sperm/mL; Infertile men with varicocele: 16.73 ± 12.13 mV/10⁶ sperm/mL (P<0.05 versus controls)
Ammar et al., 2021 (Tunisia) (⁵¹51 Ammar O, Tekeya O, Hannachi I, Sallem A, Haouas Z, Mehdi M. Increased Sperm DNA Fragmentation in Infertile Men with Varicocele: Relationship with Apoptosis, Seminal Oxidative Stress, and Spermatic Parameters. Reprod Sci. 2021;28:909-19.)	Seminal MDA by thiobarbituric acid reaction; Seminal SOD; Seminal Cat; Seminal GPx	29 fertile men without varicocele; 11 infertile men with palpable varicocele and normal SA; 40 infertile men with palpable varicocele and abnormal SA	MDA levels: Controls: 0.56 ± 0.25 nmol/mL; Infertile men with varicocele and normal SA: 1.43 ± 1.2 nmol/mL (P<0.05 versus controls); Infertile men with varicocele and abnormal SA: 1.63 ± 1.38 nmol/mL (P<0.05 versus controls); GPx and CAT activities were decreased in both groups with varicocele, and SOD activity was decreased only in infertile men with varicocele and abnormal SA when compared to controls (P<0.05)
Camargo et al., 2021 (Brazil) (¹⁶⁶166 Camargo M, Ibrahim E, Intasqui P, Belardin LB, Antoniassi MP, Lynne CM, et al. Seminal inflammasome activity in the adult varicocele. Human fertility (Cambridge, England). 2021:1-15.)	Seminal MDA by thiobarbituric acid reaction	15 normozoospermic men without varicocele; 15 infertile men with grade 2 or 3 varicocele	MDA levels: Controls: 20.1 ± 4.59 nmol/mL Infertile men with varicocele: 21.6 ± 8.97 nmol/mL (P NS versus controls)
Gill et al., 2021 (Poland) (⁴⁶46 Gill K, Kups M, Harasny P, Machalowski T, Grabowska M, Lukaszuk M, et al. The Negative Impact of Varicocele on Basic Semen Parameters, Sperm Nuclear DNA Dispersion and Oxidation-Reduction Potential in Semen. Int J Environ Res Public Health. 2021;18(11).)	Seminal ORP measured by MiOXSYS	105 normozoospermic men without varicocele; 64 men with proven fertility; 71 infertile men with clinical varicocele; 95 infertile men without varicocele	ORP: Normozoospermic group: 1.68 ± 0.91 mV/106 sperm/mL; Proven fertility group: 1.00 ± 0.8 mV/106 sperm/mL; Varicocele group: 36.10 ± 60.97 mV/106 sperm/mL (P < 0.05 versus normozoospermic and proven fertility);

Author, year, (country)	SDF assay	Study groups	Main SDF results
Saleh et al., 2003 (USA) (⁴⁴44 Saleh RA, Agarwal A, Sharma RK, Said TM, Sikka SC, Thomas AJ, Jr. Evaluation of nuclear DNA damage in spermatozoa from infertile men with varicocele. Fertility and sterility. 2003;80:1431-6.)	SCSA	16 fertile men without varicocele, 16 infertile men with palpable varicocele and 15 infertile men without varicocele	Control group: 15.0% (IQR: 10.0%, 22.0%); Infertile with varicocele: 25.0% (IQR: 20.0%, 35.0%) (P < 0.05 versus control); Infertile without varicocele: 20.0% (IQR: 13.0%, 28.0%)
Smith et al., 2005 (Chile) (⁹⁸98 Smith R, Kaune H, Parodi D, Madariaga M, Rios R, Morales I, et al. Increased sperm DNA damage in patients with varicocele: relationship with seminal oxidative stress. Hum Reprod. 2006;21:986-93.)	TUNEL and SCSA	25 healthy men without varicocele, 37 men with grade 2 and 3 varicocele and normal SA, 18 men with grade 2 or 3 varicocele and abnormal SA (fertility status not informed)	Control group: TUNEL 14.2% ± 1.2%; SCSA 7.1% ± 0.9%; Varicocele and normal SA group: TUNEL 26.1% ± 3.2%; SCSA 20.7% ± 4.0% (P < 0.05 versus control); Varicocele and abnormal SA group: TUNEL 32.2 ± 4.1%; SCSA 35.5% ± 9.0 % (P < 0.05 versus control)
Talebi et al., 2008 (Iran) (¹⁶⁷167 Talebi AR, Moein MR, Tabibnejad N, Ghasemzadeh J. Effect of varicocele on chromatin condensation and DNA integrity of ejaculated spermatozoa using cytochemical tests. Andrologia. 2008;40:245-51.)	SCSA	20 fertile men without varicocele and 20 infertile men with grade 2 or 3 varicocele	Control group: 17.3% ± 7.4%; Varicocele group: 60.5% ± 15.5% (P < 0.05 versus control)
Wu et al., 2009 (Taiwan) (¹⁶⁸168 Wu GJ, Chang FW, Lee SS, Cheng YY, Chen CH, Chen IC. Apoptosis-related phenotype of ejaculated spermatozoa in patients with varicocele. Fertility and sterility. 2009;91:831-7.)	Comet	5 healthy men without varicocele and 15 men with grade 2 or 3 varicocele (fertility status not informed)	Control group: 4.5% ± 0.9%; Varicocele group: 8.4% ± 3.1% (P < 0.05 versus control)
Blumer et al., 2011 (Brazil) (¹⁶⁵165 Blumer CG, Restelli AE, Giudice PT, Soler TB, Fraietta R, Nichi M, et al. Effect of varicocele on sperm function and semen oxidative stress. BJU international. 2012;109:259-65.)	Comet	19 men without varicocele; 12 men with varicocele (fertility status not informed);	Class II sperm DNA fragmentation: Control: 51.3% ± 14.7 %; Men with varicocele: 59.4% ± 14.8% (P<0.05 versus control); There were no differences regarding the other three classes of sperm DNA fragmentation.
La Vignera et al., 2012 (Italy) (¹⁵⁴154 La Vignera S, Condorelli R, Vicari E, D'Agata R, Calogero AE. Effects of varicocelectomy on sperm DNA fragmentation, mitochondrial function, chromatin condensation, and apoptosis. J Androl. 2012;33:389-96.)	TUNEL	30 fertile men without varicocele, 30 infertile men with grade 3 left varicocele and abnormal SA	Control group: 2.0% ± 1.0%; Varicocele group: 5.0% ± 3.0% (P < 0.05 versus control)
Li et al., 2012 (China) (¹⁶⁹169 Li F, Yamaguchi K, Okada K, Matsushita K, Ando M, Chiba K, et al. Significant improvement of sperm DNA quality after microsurgical repair of varicocele. Systems biology in reproductive medicine. 2012;58:274-7.)	SCSA	19 healthy normozoospermic men and 19 infertile men with palpable varicocele and abnormal semen parameters	Control group: 17.4% ± 5.3%; Varicocele group: 28.4% ± 15.6% (P < 0.05 versus control)
Esteves et al., 2015 (Brazil) (¹⁰¹101 Esteves SC, Gosalvez J, Lopez-Fernandez C, Nunez-Calonge R, Caballero P, Agarwal A, et al. Diagnostic accuracy of sperm DNA degradation index (DDSi) as a potential noninvasive biomarker to identify men with varicocele-associated infertility. International urology and nephrology. 2015;47:1471-7.)	SCD	80 fertile donors and 98 infertile men with varicocele	Control group: 11.3% ± 5.5%; Varicocele group: 33.5% ± 18,3% (P < 0.05 versus control)
Alhathal et al., 2016 (Canada) (¹⁷⁰170 Alhathal N, San Gabriel M, Zini A. Beneficial effects of microsurgical varicocoelectomy on sperm maturation, DNA fragmentation, and nuclear sulfhydryl groups: a prospective trial. Andrology. 2016;4:1204-8.)	SCSA	6 healthy normozoospermic men without varicocele, and 29 infertile men with palpable varicocele and abnormal semen parameters	Control group: 7.4% ± 5.0%; Varicocele group: 20.0% ± 10.6% (P < 0.05 versus control)
Ni et al., 2016 (China) (⁶¹61 Ni K, Steger K, Yang H, Wang H, Hu K, Zhang T, et al. A comprehensive investigation of sperm DNA damage and oxidative stress injury in infertile patients with subclinical, normozoospermic, and astheno/oligozoospermic clinical varicocoele. Andrology. 2016;4:816-24.)	SCSA	25 healthy normozoospermic men without varicocele, 19 infertile men with grade 1varicocele, 18 infertile men with grade 2 varicocele, and 14 infertile men with grade 3varicocele	Control group: 12.0% ± 7.9%; Varicocele grade 1 group: 23.6% ± 7.5% (P < 0.05 versus control); Varicocele grade 2 group: 27.7% ± 9.0% (P < 0.05 versus control); Varicocele grade 3 group: 30.0 % ± 8.3% (P < 0.05 versus control)
Abdelbaki et al., 2017 (Egypt) (⁴²42 Abdelbaki SA, Sabry JH, Al-Adl AM, Sabry HH. The impact of coexisting sperm DNA fragmentation and seminal oxidative stress on the outcome of varicocelectomy in infertile patients: A prospective controlled study. Arab journal of urology. 2017;15:131-9.)	SCSA	20 fertile normozoospermic men without varicocele, and 60 infertile men with palpable varicocele and abnormal semen parameters	Control group: 7.6% ± 2.8%; Varicocele group: 29.9% ± 8.3% (P< 0.05 versus control)
Dieamant et al., 2017 (Brazil) (¹⁷¹171 Dieamant F, Petersen CG, Mauri AL, Conmar V, Mattila M, Vagnini LD, et al. Semen parameters in men with varicocele: DNA fragmentation, chromatin packaging, mitochondrial membrane potential, and apoptosis. JBRA assisted reproduction. 2017;21:295-301.)	TUNEL	2008 men without varicocele and 391 men with palpable varicocele (fertility status not informed)	Control group: 15.3% ± 8.5%; Varicocele group: 16.3% ± 8.8% (P < 0.05 versus control)
Santana et al., 2019 (Brazil) (¹⁰⁷107 Santana VP, Miranda-Furtado CL, Pedroso DCC, Eiras MC, Vasconcelos MAC, Ramos ES, et al. The relationship among sperm global DNA methylation, telomere length, and DNA fragmentation in varicocele: a cross-sectional study of 20 cases. Systems biology in reproductive medicine. 2019;65:95-104.)	SCD	20 men without varicocele, and 19 men with varicocele (fertility status not informed)	Control group: 26.0% ± 10.0%; Varicocele group: 37.0% ± 20.0% (P = 0.09 versus control)
Lara-Cerrillo et al., 2020 (Spain) (¹⁷²172 Lara-Cerrillo S, Gual-Frau J, Benet J, Abad C, Prats J, Amengual MJ, et al. Microsurgical varicocelectomy effect on sperm telomere length, DNA fragmentation and seminal parameters. Human fertility (Cambridge, England). 2020:1-7.)	Comet	12 fertile men without varicocele and 20 infertile men with grades 2 or 3 varicocele	Control group: 45.0% ± 56.0%; Varicocele group: 53.0% ± 45.0% (P value not informed)
Tanaka et al., 2020 (Japan) (⁴⁵45 Tanaka T, Kobori Y, Terai K, Inoue Y, Osaka A, Yoshikawa N, et al. Seminal oxidation-reduction potential and sperm DNA fragmentation index increase among infertile men with varicocele. Human fertility (Cambridge, England). 2022;25:142-6.)	SCSA	102 normozoospermic men without varicocele and 138 infertile men with palpable varicocele	Control group: 9.8% ± 10.3%; Varicocele group: 16.7% ± 12.1% (P < 0.05 versus control)
Ammar et al., 2021 (Tunisia) (⁵¹51 Ammar O, Tekeya O, Hannachi I, Sallem A, Haouas Z, Mehdi M. Increased Sperm DNA Fragmentation in Infertile Men with Varicocele: Relationship with Apoptosis, Seminal Oxidative Stress, and Spermatic Parameters. Reprod Sci. 2021;28:909-19.)	TUNEL	29 fertile men without varicocele; 11 infertile men with palpable varicocele and normal SA; 40 infertile men with varicocele and abnormal SA	Control group: 8.14% ± 6.86%; Varicocele and normal SA group: 60.87% ± 8.61% (P < 0.05 versus control); Varicocele and abnormal SA group: 69.88% ± 5.87% (P < 0.05 versus control)
Camargo et al., 2021 (Brazil) (¹⁶⁶166 Camargo M, Ibrahim E, Intasqui P, Belardin LB, Antoniassi MP, Lynne CM, et al. Seminal inflammasome activity in the adult varicocele. Human fertility (Cambridge, England). 2021:1-15.)	Comet	15 normozoospermic men without varicocele; 15 infertile men with grade 2 or 3 varicocele	Control group: 39.3% ± 11.69%; Varicocele group: 43.6% ± 11.9% (P = NS versus control)
Gil et al., 2021 (Poland) (⁴⁶46 Gill K, Kups M, Harasny P, Machalowski T, Grabowska M, Lukaszuk M, et al. The Negative Impact of Varicocele on Basic Semen Parameters, Sperm Nuclear DNA Dispersion and Oxidation-Reduction Potential in Semen. Int J Environ Res Public Health. 2021;18(11).)	SCD	105 normozoospermic men without varicocele; 64 men with proven fertility; 71 infertile men with clinical varicocele; 95 infertile men without varicocele	Normozoospermic group: 13.3% ± 5.9%; Proven fertility group: 13.9% ± 7.1%; Varicocele group: 23.3% ± 11.9% (P < 0.05 versus normozoospermic and proven fertility); Infertile without varicocele: 19.4% ± 5.9% (P < 0.05 versus normozoospermic and proven fertility)
Jellad et al., 2021 (Tunisia) (¹⁰⁶106 Jellad S, Hammami F, Khalbous A, Messousi M, Khiari R, Ghozzi S, et al. Sperm DNA status in infertile patients with clinical varicocele. Prog Urol. 2021;31:105-11.)	TUNEL	15 normozoospermic men without varicocele; 30 infertile men with palpable varicocele	Control group: 64.5% ± 17.7%; Varicocele group: 72.0% ± 15.3% (P < 0.05 versus control)
Jeremias et al., 2021 (Brazil) (⁹⁹99 Jeremias JT, Belardin LB, Okada FK, Antoniassi MP, Fraietta R, Bertolla RP, et al. Oxidative origin of sperm DNA fragmentation in the adult varicocele. Int Braz J Urol. 2021;47:275-83. doi: 10.1590/S1677-5538.IBJU.2019.0827. https://doi.org/10.1590/S1677-5538.IBJU.... )	Comet	39 normozoospermic men without varicocele; 55 normozoospermic men with palpable varicocele	Control group: 39.3% ± 11.69%; Varicocele group: 43.6% ± 11.9% (P = NS versus control)

Author, year, (country)	ROS assay	Study groups	Main results
Mancini et al., 2004 (Italy) (¹⁴⁰140 Mancini A, Meucci E, Milardi D, Giacchi E, Bianchi A, Pantano AL, et al. Seminal antioxidant capacity in pre- and postoperative varicocele. J Androl. 2004;25:44-9.)	Seminal TAC by Colorimetric assay kit	25 infertile men with varicocele 14 infertile men who underwent varicocelectomy 10-24 months before	TAC: Preoperative: 106.6 ± 8.9 seconds; Postoperative: 105.8 ± 8.7 seconds (P NS versus preoperative)
Sakamoto et al., 2008 (Japan) (³⁸38 Sakamoto Y, Ishikawa T, Kondo Y, Yamaguchi K, Fujisawa M. The assessment of oxidative stress in infertile patients with varicocele. BJU international. 2008;101:1547-52.)	Seminal NO levels; Seminal 8-OHdG levels; Seminal SOD activity	Not reported	NO levels: Preoperative: 17.1 ± 9.1μmol/L; 6 months postoperative: 7.5 ± 4.5μmol/L (P<0.05 versus preoperative) 8-OHdG levels: Preoperative: 10.3 ± 4.7 μmol/L; 6 months postoperative: 6.2 ± 2.5 μmol/L (P<0.05 versus preoperative) SOD activity: Preoperative: 85.8 ± 5.8%; 6 months postoperative: 78.1 ± 8.1% (P<0.05 versus preoperative)
Dada et al., 2010 (India) (¹³⁵135 Dada R, Shamsi MB, Venkatesh S, Gupta NP, Kumar R. Attenuation of oxidative stress & DNA damage in varicocelectomy: implications in infertility management. The Indian journal of medical research. 2010;132:728-30.)	Seminal ROS by chemiluminescence with luminol	11 infertile men with palpable varicocele	ROS levels: Preoperative: 142,897.704 RLU per 20 million sperm/min 1 month postoperative: 10,776.736 RLU per 20 million sperm/min (P < 0.05 versus preoperative); 3 months postoperative: 6,456.249 RLU per 20 million sperm/min (P < 0.05 versus preoperative)
Baker et al., 2013 (USA)	Seminal ROS by chemiluminescence with luminol; Seminal TAC by Colorimetric assay kit	24 infertile men with palpable varicocele	ROS levels: Preoperative: 1185.1 RLU/sec/10⁶; 3 months postoperative: 2710.911851 RLU/sec/10⁶ (P not reported) TAC: Preoperative: 2292 µM Trolox; 3 months postoperative: 1885 mM Trolox (P < 0.05 versus preoperative); % patients with TAC above normal: Preoperative: 86%; 3 months postoperative: 71% (P value not reported)
Tavalaee et al., 2015 (Iran) (¹³⁶136 Tavalaee M, Bahreinian M, Barekat F, Abbasi H, Nasr-Esfahani MH. Effect of varicocelectomy on sperm functional characteristics and DNA methylation. Andrologia. 2015;47:904-9.)	Seminal OS by DCFH-DA staining	23 infertile men with varicocele grade II and III	DCFH-DA negative spermatozoa: Preoperative: 37.2% ± 3.6 %; 3 months postoperative: 61.3% ± 5.3 % (P < 0.05 versus preoperative)
Barekat et al., 2016 (Iran) (¹³⁷137 Barekat F, Tavalaee M, Deemeh MR, Bahreinian M, Azadi L, Abbasi H, et al. A Preliminary Study: N-acetyl-L-cysteine Improves Semen Quality following Varicocelectomy. International journal of fertility & sterility. 2016;10:120-6.)	Seminal OS by DCFH-DA staining	20 infertile men with varicocele grade II and III	DCFH-DA positive spermatozoa: Preoperative: 47.6% ± 6.6 %; 3 months postoperative: 36.6% ± 3.8 % (P < 0.05 versus preoperative)
Abdelbaki et al., 2017 (Egypt) (⁴²42 Abdelbaki SA, Sabry JH, Al-Adl AM, Sabry HH. The impact of coexisting sperm DNA fragmentation and seminal oxidative stress on the outcome of varicocelectomy in infertile patients: A prospective controlled study. Arab journal of urology. 2017;15:131-9.)	Seminal ROS by chemiluminescence with luminol; Seminal TAC by Colorimetric assay kit	55 infertile men with palpable varicocele	ROS levels: Preoperative: 4.49 ± 0.9 log[ROS+1]; 3 months postoperative: 3.27 ± 1.3 log[ROS+1] (P < 0.05 versus preoperative) TAC: Preoperative:1.01 ± 0.4 mM Trolox; 3 months postoperative: 2.05 ± 0.5 mM Trolox (P < 0.05 versus preoperative)
Ni et al., 2016 (China) (⁶¹61 Ni K, Steger K, Yang H, Wang H, Hu K, Zhang T, et al. A comprehensive investigation of sperm DNA damage and oxidative stress injury in infertile patients with subclinical, normozoospermic, and astheno/oligozoospermic clinical varicocoele. Andrology. 2016;4:816-24.)	Seminal MDA by thiobarbituric acid reaction	19 infertile men with grade I varicocele; 18 infertile men with grade II varicocele; 14 infertile men with grade III varicocele	MDA levels: Preoperative varicocele grade I group: 12.18 ± 4.86 nmol/mL; 3 months postoperative varicocele grade I group: 9.88 ± 3.98 nmol/mL (P NS versus preoperative); 6 months postoperative varicocele grade I group: 8.76 ± 2.73 nmol/mL (P < 0.05 versus preoperative); Varicocele grade II group: 14.12 ± 5.42 nmol/mL; 3 months postoperative varicocele grade II group: 9.22 ±3.75 nmol/mL (P < 0.05 versus preoperative) 6 months postoperative varicocele grade II group: 9.71 ± 2.83 nmol/mL (P < 0.05 versus preoperative); Varicocele grade III group: 15.86 ± 6.78 nmol/mL; 3 months postoperative varicocele grade II group: 11.38 ± 3.94 nmol/mL (P < 0.05 versus preoperative); 6 months postoperative varicocele grade III group: 9.50 ± 3.28 nmol/mL (P < 0.05 versus preoperative)
Abbasi et al., 2020 (Iran) (¹³⁸138 Abbasi B, Molavi N, Tavalaee M, Abbasi H, Nasr-Esfahani MH. Alpha-lipoic acid improves sperm motility in infertile men after varicocelectomy: a triple-blind randomized controlled trial. Reproductive biomedicine online. 2020;41:1084-91.)	Lipid peroxidation by the BODIPY probe	22 infertile men with varicocele grade II and III	BODIPY-positive spermatozoa: Preoperative: 36.22% ± 3.38 %; 80 days postoperative: 24.04% ± 1.80 % (P < 0.05 versus preoperative)
Kavoussi et al., 2022 (USA) (¹³⁹139 Kavoussi PK, Gilkey MS, Machen GL, Kavoussi SK, Dorsey C. Varicocele Repair Improves Static Oxidation Reduction Potential as a Measure of Seminal Oxidative Stress Levels in Infertile Men: A Prospective Clinical Trial Using the MiOXSYS System. Urology. 2022;165:193-7.)	ORP by MiOXSYS System	49 infertile men with palpable varicocele	ORP: Preoperative:4.73 mV/10⁶ sperm/mL; 3 months postoperative: 2.03 mV/106 sperm/mL (P < 0.05 versus preoperative)

Author, year, (country)	Population	Type of Included Studies	SDF assay	Varicocelectomy technique	Number of studies and participants	Decrease %SDF after varicocelectomy	Limitations
Wang et al., 2012 (China) (¹⁰³103 Wang YJ, Zhang RQ, Lin YJ, Zhang RG, Zhang WL. Relationship between varicocele and sperm DNA damage and the effect of varicocele repair: a meta-analysis. Reproductive biomedicine online. 2012;25:307-14.)	Infertile men with palpable varicocele and abnormal SA	Retrospective and prospective cohort	SCSA, TUNEL and Comet	Open non-microsurgical and open microsurgical	6 studies; 177 participants	WMD -3.37%; 95% CI: -4.09 to -2.65, P<0.05	One study included men using antioxidants, and another study included adolescents; Data were pooled irrespective of SDF assay type; Pregnancy and live birth rates not evaluated
Qiu et al., 2020 (China) (¹⁴⁴144 Qiu D, Shi Q, Pan L. Efficacy of varicocelectomy for sperm DNA integrity improvement: A meta-analysis. Andrologia. 2021;53(1):e13885.)	Men with varicocele	Prospective cohort and case-control	SCSA, SCD, TUNEL, Comet, and AOT	Open non-microsurgical and open microsurgical	11 studies; 394 participants	WMD -5.79%; 95% CI -7.39 to -4.19, P<0.05	One study included fertile men, another included men with subclinical varicocele; one study included adolescents, and another trial assessed SDF by a sperm chromatin protamination test; data was pooled irrespective of SDF assay type; pregnancy and live birth rates not evaluated
Birowo et al., 2020 (Indonesia) (¹⁴⁷147 Birowo P, Rahendra Wijaya J, Atmoko W, Rasyid N. The effects of varicocelectomy on the DNA fragmentation index and other sperm parameters: a meta-analysis. Basic Clin Androl. 2020;30:15.)	Infertile men with palpable varicocele	Prospective cohort	SCSA and TUNEL	Open non-microsurgical and open microsurgical	7 studies; 289 participants	WMD -6.86%; 95% CI -10.04 to -3.69, P<0.05	Low number of studies and participants; data was pooled irrespective of SDF assay type; pregnancy and live birth rates not evaluated
Lira Neto et al., 2020 (Brazil) (²³23 Lira Neto FT, Roque M, Esteves SC. Effect of varicocelectomy on sperm deoxyribonucleic acid fragmentation rates in infertile men with clinical varicocele: a systematic review and meta-analysis. Fertility and sterility. 2021;116:696-712.)	Infertile men with palpable varicocele	Retrospective and prospective cohort	SCSA, SCD, TUNEL and Comet	Open non-microsurgical, open microsurgical, and laparoscopic	19 studies; 1070 participants	WMD -7.23%; 95% CI -8.86; -5.59; P<0.05	Pregnancy and live birth rates not assessed

Knowledge gaps	Suggested studies
Mechanisms by which varicocele causes OS.	Proteomics and Metabolomics studies in men with and without varicocele.
Impact of varicocele laterality on OS.	Cross-sectional studies in infertile men with unilateral versus bilateral varicocele.
Impact of subclinical varicocele on OS.	Cross-sectional studies in infertile men with subclinical versus palpable varicocele.
Definition of cut-off levels for the different OS markers for men with varicocele	Cross-sectional studies including healthy fertile normozoospermic men (controls), fertile men with varicocele, and infertile men with varicocele, using several markers of OS (Total ROS, MDA, 8-OHdG, and TAC) simultaneously, providing ROC curve analysis for each marker.
Impact of time on varicocele-induced OS.	Prospective cohort studies with fertile and infertile men with varicocele measuring OS markers in serial time points.
Impact of varicocelectomy on varicocele-induced OS	Prospective cohort studies including infertile men measuring various OS markers before and after varicocele repair, including subanalyses by varicocele grade, laterality, baseline OS marker levels, and surgical technique. Measuring various OS markers in serial time points is also recommended.
Impact of time on varicocele-induced sperm DNA fragmentation.	Prospective cohort studies with fertile and infertile men measuring sperm DNA fragmentation in serial time points.
Proportion of men with varicocele and increased sperm DNA fragmentation levels	Cross-sectional studies including healthy, fertile normozoospermic men (controls), fertile men with varicocele, and infertile men with varicocele. Cut-off levels should be defined for each assay based on the literature.
Impact of varicocele grade on sperm DNA fragmentation.	Cross-sectional studies in infertile men grouped by varicocele grade (including subclinical).
Impact of varicocele laterality on sperm DNA fragmentation.	Case-control studies in infertile men with unilateral versus bilateral varicocele.
Impact of varicocelectomy on varicocele-induced sperm DNA fragmentation	Prospective cohort studies including infertile men with increased sperm DNA fragmentation levels undergoing varicocele repair. Subanalyses by varicocele grade, laterality, and surgical technique should be performed. Measuring sperm DNA fragmentation in serial time points is also recommended.
Association between OS and sperm DNA fragmentation levels in men with varicocele	Cross-sectional studies including healthy fertile normozoospermic men (controls), fertile men with varicocele, and infertile men with varicocele, and measuring simultaneously several OS markers as well as sperm DNA fragmentation
Association between the improvement of OS and sperm DNA fragmentation levels after varicocele repair.	Prospective cohort studies including infertile men with increased OS markers and sperm DNA fragmentation levels undergoing varicocele repair. Several OS markers and sperm DNA fragmentation should be measured simultaneously and in serial time points.
Association between the improvement of OS and sperm DNA fragmentation levels after varicocele repair with natural pregnancy outcomes.	Prospective cohort studies including infertile men with increased OS markers and sperm DNA fragmentation levels undergoing varicocele repair. Participants should be followed up for at least 12 months after surgery in couples actively trying to conceive.
Association between the improvement of OS and sperm DNA fragmentation levels after varicocele repair with ART outcomes.	Prospective cohort studies including infertile men with increased OS markers and sperm DNA fragmentation levels undergoing varicocele repair. Participants should wait at least 3 months after surgery for ART treatments and should be followed up until the end of each treatment.

[1] Correspondence address:
Sandro C. Esteves, MD, PhD ANDROFERT - Clínica de Andrologia e Reprodução Humana, Centro de Ref. em Reprodução Masculina Av. Dr. Heitor Penteado, 1464 Campinas, Brasil E-mail: s.esteves@androfert.com.br

Brasil

Brasil

From pathophysiology to practice: addressing oxidative stress and sperm DNA fragmentation in Varicocele-affected subfertile men

ABSTRACT