Top

Published in:

01-01-2014 | Reproductive Medicine

Evaluation of conventional semen parameters, intracellular reactive oxygen species, DNA fragmentation and dysfunction of mitochondrial membrane potential after semen preparation techniques: a flow cytometric study

Authors: Leila Rashki Ghaleno, Mojtaba Rezazadeh Valojerdi, Ehsan Janzamin, Mohammad Chehrazi, Mina Sharbatoghli, Reza Salman Yazdi

Published in: Archives of Gynecology and Obstetrics | Issue 1/2014

Abstract

Purpose

The aim of this study was to evaluate the conventional sperm parameters, level of intracellular reactive oxygen species (ROS), DNA fragmentation (DF) and dysfunction of mitochondrial membrane potential (MMP) after semen preparation techniques with flow cytometry.

Methods

Semen samples were obtained from 28 men with normal semen analysis according to WHO (world health organization). Each was divided into three equal parts for processing with routine techniques: conventional swim up (CSW), direct swim up (DSW) and density gradient centrifugation (DGC). The conventional sperm parameters were evaluated with computer-assisted sperm analyzer (CASA) and the level of intracellular ROS, dysfunction of MMP and DF were determined with flow cytometry procedure.

Results

Conventional sperm parameters such as motility, progressive motility and normal morphology increased after sperm processing by CSW and DGC compared to DSW. A significant increase in intracellular H₂O₂ (p < 0.05) was demonstrated in the CSW versus DSW technique, while processed sperm by the DSW procedure showed a significant increase in the percentage of dysfunction of MMP and intracellular O ₂ ^•− (p < 0.05) when compared with CSW and DGC techniques. Additionally, a high mean of DF (p < 0.05) was observed in the DGC technique as compared to CSW.

Conclusion

Data from flow cytometry study demonstrated that intracellular H₂O₂ and DF increased after CSW and DGC processing techniques, respectively, whereas the level of intracellular O ₂ ^•− and dysfunction of MMP only increased after the DSW processing technique.

de Lamirande E, OFlaherty C (2008) Sperm activation: role of reactive oxygen species and kinases. Biochimica et Biophysica Acta (BBA). Proteins Proteomics 1784(1):106–115CrossRef

Agarwal A, Said TM, Bedaiwy MA, Banerjee J, Alvarez JG (2006) Oxidative stress in an assisted reproductive techniques setting. Fertil Steril 86(3):503–512. doi:10.1016/j.fertnstert.2006.02.088 PubMedCrossRef

Henkel R, Schill W-B (2003) Sperm preparation for ART. Reprod Biol Endocrinol 1(1):108PubMedCentralPubMedCrossRef

Saleh RA, Agarwal A (2002) Oxidative stress and male infertility: from research bench to clinical practice. J Androl 23(6):737–752PubMed

Khajavi N, Rs MM, Marzieh Tavalaei, Deymeh MR, Hossein Nasresfahani Mohammad (2009) Can Zeta sperm selection method recover sperm with higher DNA integrity compare to density gradient centrifugation? Iran J Reprod Med 7(2):73–77

Li Z, Zhou Y, Liu R, Lin H, Liu W, Xiao W, Lin Q (2011) Effects of semen processing on the generation of reactive oxygen species and mitochondrial membrane potential of human spermatozoa. Andrologia. doi:10.1111/j.1439-0272.2010.01123.x

O’Connell M, McClure N, Powell LA, Steele EK, Lewis SE (2003) Differences in mitochondrial and nuclear DNA status of high-density and low-density sperm fractions after density centrifugation preparation. Fertil Steril 79(Suppl 1):754–762 (pii: S0015028202048276)PubMedCrossRef

Aitken RJ, Clarkson JS (1988) Significance of reactive oxygen species and antioxidants in defining the efficacy of sperm preparation techniques. J Androl 9(6):367–376PubMed

Wang X, Sharma RK, Gupta A, George V, Thomas AJ, Falcone T, Agarwal A (2003) Alterations in mitochondria membrane potential and oxidative stress in infertile men: a prospective observational study. Fertil Steril 80:844–850PubMedCrossRef

10.

Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK, Meyer A, Nelson DR, Thomas AJ (2003) Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril 79:1597–1605PubMedCrossRef

11.

Sakkas D, Manicardi GC, Tomlinson M, Mandrioli M, Bizzaro D, Bianchi PG, Bianchi U (2000) The use of two density gradient centrifugation techniques and the swim-up method to separate spermatozoa with chromatin and nuclear DNA anomalies. Hum Reprod 15(5):1112–1116. doi:10.1093/humrep/15.5.1112 PubMedCrossRef

12.

Lachaud C, Tesarik J, CaÃ ± adas ML, Mendoza C (2004) Apoptosis and necrosis in human ejaculated spermatozoa. Hum Reprod 19(3):607–610. doi:10.1093/humrep/deh130 PubMedCrossRef

13.

Guthrie HD, Welch GR (2006) Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry. J Anim Sci 84(8):2089–2100. doi:10.2527/jas.2005-766 PubMedCrossRef

14.

Larsen L, Scheike T, Jensen TK, Bonde JP, Ernst E, Hjollund NH, Zhou Y, SkakkebÃ¦k NE, Giwercman A, The Danish First Pregnancy Planner Study Team (2000) Computer-assisted semen analysis parameters as predictors for fertility of men from the general population. Hum Reprod 15(7):1562–1567. doi:10.1093/humrep/15.7.1562 PubMedCrossRef

15.

Gil-Guzman E, Ollero M, Lopez MC, Sharma RK, Alvarez JG, Thomas AJ, Agarwal A (2001) Differential production of reactive oxygen species by subsets of human spermatozoa at different stages of maturation. Hum Reprod 16(9):1922–1930. doi:10.1093/humrep/16.9.1922 PubMedCrossRef

16.

Mahfouz RZ, du Plessis SS, Aziz N, Sharma R, Sabanegh E, Agarwal A (2010) Sperm viability, apoptosis, and intracellular reactive oxygen species levels in human spermatozoa before and after induction of oxidative stress. Fertil Steril 93(3):814–821. doi:10.1016/j.fertnstert.2008.10.068 PubMedCrossRef

17.

Mahfouz R, Sharma R, Lackner J, Aziz N, Agarwal A (2009) Evaluation of chemiluminescence and flow cytometry as tools in assessing production of hydrogen peroxide and superoxide anion in human spermatozoa. Fertil Steril 92(2):819–827PubMedCrossRef

18.

Espinoza JA, Schulz MA, Sánchez R, Villegas JV (2009) Integrity of mitochondrial membrane potential reflects human sperm quality. Andrologia 41(1):51–54. doi:10.1111/j.1439-0272.2008.00878.x PubMedCrossRef

19.

Yang Y, Monserud RA, Huang S (2004) An evaluation of diagnostic tests and their roles in validating forest biometric models. Can J For Res 34(3):619–629CrossRef

20.

Saleh RA, Agarwal A (2002) Oxidative stress and male infertility: from research bench to clinical practice. J Androl 23(6):737–752PubMed

21.

Jameel T (2008) Sperm swim-up: a simple and effective technique of semen processing for intrauterine insemination. J Pak Med Assoc 58(2):71–74PubMed

22.

Mortimer D (2000) Sperm preparation methods. J Androl 21(3):357–366PubMed

23.

Henkel R, Franken D, Lombard C, Schill W-B (1994) Selective capacity of glass-wool filtration for the separation of human spermatozoa with condensed chromatin: a possible therapeutic modality for male-factor cases? J Assist Reprod Genet 11(8):395–400. doi:10.1007/bf02211725 PubMedCrossRef

24.

Guthrie HD, Welch GR (2012) Effects of reactive oxygen species on sperm function. Theriogenology. doi:10.1016/j.theriogenology.2012.05.002 PubMed

25.

Le Lannou D, Blanchard Y (1988) Nuclear maturity and morphology of human spermatozoa selected by Percoll density gradient centrifugation or swim-up procedure. J Reprod Fertil 84(2):551–556. doi:10.1530/jrf.0.0840551 PubMedCrossRef

26.

Moohan JM, Lindsay KS (1995) Spermatozoa selected by a discontinuous Percoll density gradient exhibit better motion characteristics, more hyperactivation, and longer survival than direct swim-up. Fertil Steril 64(1):160–165PubMed

27.

Yamamoto Y, Maenosono S, Okada H, Miyagawa I, Sofikitis N (1997) Comparisons of sperm quality, morphometry and function among human sperm populations recovered via SpermPrep II filtration, swim-up and Percoll density gradient methods. Andrologia 29(6):303–310PubMedCrossRef

28.

Mortimer D (1990) Semen analysis and sperm washing techniques. In: Gagnon C (ed) Control of sperm motility: biological clinical aspects. CRC Press, Boca Raton, F1; 1990:263–284

29.

Mortimer S (1997) A critical review of the physiological importance and analysis of sperm movement in mammals. Hum Reprod Update 3(5):403–439. doi:10.1093/humupd/3.5.403 PubMedCrossRef

30.

Mortimer D, Leslie EE, Kelly RW, Templeton AA (1982) Morphological selection of human spermatozoa in vivo and in vitro. J Reprod Fertil 64(2):391–399. doi:10.1530/jrf.0.0640391 PubMedCrossRef

31.

McGovern P, Quagliarello J, Arny M (1989) Relationship of within-patient semen variability to outcome of intrauterine insemination. Fertil Steril 51(6):1019–1023PubMed

32.

Marchetti C, Obert G, Deffosez A, Formstecher P, Marchetti P (2002) Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm. Hum Reprod 17(5):1257–1265PubMedCrossRef

33.

Soderlund B, Lundin K (2000) The use of silane-coated silica particles for density gradient centrifugation in in vitro fertilization. Hum Reprod 15(4):857–860. doi:10.1093/humrep/15.4.857 PubMedCrossRef

34.

Ren SS, Sun GH, Ku CH, Chen DC, Wu GJ (2004) Comparison of four methods for sperm preparation for IUI. Arch Androl 50(3):139–143. doi:10.1080/01485010490425566 PubMedCrossRef

35.

Pranav P, Lucy L, Zuying C, Thomas T, Raja S, Isaac S, Keith I (1998) Preparation by differential gradient centrifugation is better than swim-up in selecting sperm with normal morphology (strict criteria). Fertil Steril 69(4):722–726CrossRef

36.

Nisarg D, Rakesh S, Kartikeya M, Edmund S, Ashok A (2009) Physiologic and pathologic levels of reactive oxygen species in neat semen of infertile men. Fertil Steril 92(5):1626–1631CrossRef

37.

Andolz P, Bielsa MA, Genesca A, Benet J, Egozcue J (1987) Improvement of sperm quality in abnormal semen samples using a modified swim-up procedure. Hum Reprod 2(2):99–101PubMed

38.

Barroso G, Taylor S, Morshedi M, Fl Manzur, Gavino F, Oehninger S (2006) Mitochondrial membrane potential integrity and plasma membrane translocation of phosphatidylserine as early apoptotic markers: a comparison of two different sperm subpopulations. Fertil Steril 85(1):149–154PubMedCrossRef

39.

Espinoza JA, Paasch U, Villegas JV (2009) Mitochondrial membrane potential disruption pattern in human sperm. Hum Reprod 24(9):2079–2085. doi:10.1093/humrep/dep120 PubMedCrossRef

40.

Zini A, Finelli A, Phang D, Jarvi K (2000) Influence of semen processing technique on human sperm DNA integrity. Urology 56(6):1081–1084 (pii: S0090-4295(00)00770-6)PubMedCrossRef

41.

Santiso R, Tamayo M, Gosalvez J, Meseguer M, Garrido N, Fernandez JL (2010) Swim-up procedure selects spermatozoa with longer telomere length. Mutat Res 688(1–2):88–90. doi:10.1016/j.mrfmmm.2010.03.003 PubMedCrossRef

42.

Irvine DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ (2000) DNA integrity in human spermatozoa: relationships with semen quality. J Androl 21(1):33–44PubMed

43.

Sakkas D, Mariethoz E, Manicardi G, Bizzaro D, Bianchi P, Bianchi U (1999) Origin of DNA damage in ejaculated human spermatozoa. Rev Reprod 4(1):31–37. doi:10.1530/ror.0.0040031 PubMedCrossRef

44.

Zhang XD, Chen MY, Gao Y, Han W, Liu DY, Huang GN (2011) The effects of different sperm preparation methods and incubation time on the sperm DNA fragmentation. Hum Fertil (Camb) 14(3):187–191. doi:10.3109/14647273.2011.604817 CrossRef

45.

Sun JG, Jurisicova A, Casper RF (1997) Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 56(3):602–607. doi:10.1095/biolreprod56.3.602 PubMedCrossRef

46.

Oosterhuis GJE, Andre BM, Ellen K-B, Cornelis BL, Joop S, Istvan V (2000) Measuring apoptosis in human spermatozoa: a biological assay for semen quality? Fertil Steril 74(2):245–250PubMedCrossRef

47.

Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, Irvine DS (1998) Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod 59(5):1037–1046. doi:10.1095/biolreprod59.5.1037 PubMedCrossRef

48.

Younglai EV, Holt D, Brown P, Jurisicova A, Casper RF (2001) Sperm swim-up techniques and DNA fragmentation. Hum Reprod 16(9):1950–1953. doi:10.1093/humrep/16.9.1950 PubMedCrossRef

Title: Evaluation of conventional semen parameters, intracellular reactive oxygen species, DNA fragmentation and dysfunction of mitochondrial membrane potential after semen preparation techniques: a flow cytometric study
Authors: Leila Rashki Ghaleno
Mojtaba Rezazadeh Valojerdi
Ehsan Janzamin
Mohammad Chehrazi
Mina Sharbatoghli
Reza Salman Yazdi
Publication date: 01-01-2014
Publisher: Springer Berlin Heidelberg
Published in: Archives of Gynecology and Obstetrics / Issue 1/2014
Print ISSN: 0932-0067
Electronic ISSN: 1432-0711
DOI: https://doi.org/10.1007/s00404-013-2946-1

Keynote webinar | Spotlight on sleep in brain health

Springer Medicine

Evaluation of conventional semen parameters, intracellular reactive oxygen species, DNA fragmentation and dysfunction of mitochondrial membrane potential after semen preparation techniques: a flow cytometric study

Abstract

Purpose

Methods

Results

Conclusion

Keynote webinar | Spotlight on sleep in brain health

Springer Medicine

Abstract

Purpose

Methods

Results

Conclusion

Please log in to get access to this content

Other articles of this Issue 1/2014

Role of hysteroscopy and endometrial biopsy in women with unexplained infertility

Work–life balance of German gynecologists: a web-based survey on satisfaction with work and private life

Fetal mortality at and beyond term in singleton pregnancies in Baden-Wuerttemberg/Germany 2004–2009

Blastocyst formation, pregnancy and birth (more than 10 years after the birth of the first child from the same IVF cycle), derived from human PN-stage cryopreserved for 10 years

Measure DRAM with a purpose: diagnose or evaluate

Bethlem myopathy: pregnancy and delivery