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Reproductive Biology and Endocrinology
Published in: Reproductive Biology and Endocrinology 1/2009

Open Access 01-12-2009 | Research

Leukocytospermia and sperm preparation - a flow cytometric study

Authors: Giuseppe Ricci, Sandra Perticarari, Rita Boscolo, Roberto Simeone, Monica Martinelli, Leo Fischer-Tamaro, Secondo Guaschino, Gianni Presani

Published in: Reproductive Biology and Endocrinology | Issue 1/2009

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Abstract

Background

Leukocytes represent the predominant source of reactive oxygen species both in seminal plasma and in sperm suspensions and have been demonstrated to negatively influence sperm function and fertilization rate in assisted reproduction procedures. Peroxidase test is the standard method recommended by WHO to detect semen leukocytes but it may be inaccurate. The aims of this study were (i) to compare the efficiency of swim-up and density-gradient centrifugation techniques in removing seminal leukocytes, (ii) to examine the effect of leukocytes on sperm preparation, and (iii) to compare flow cytometry and peroxidase test in determining leukocyte concentration in semen using a multiparameter flow cytometric method.

Methods

Semen samples from 126 male partners of couples undergoing infertility investigations were analyzed for leukocytospermia using standard optical microscopy and flow cytometry. Sixty-nine out of 126 samples were also processed using simultaneously the swim-up and density-gradient centrifugation techniques. A multiparameter flow cytometric analysis to assess simultaneously sperm concentration, sperm viability, sperm apoptosis, and leukocyte concentration was carried out on neat and prepared sperm.

Results

Both sperm preparation methods removed most seminal leukocytes. However, the concentration of leukocytes was significantly lower after swim-up compared to that after density-gradient centrifugation preparation. Leukocytes concentration, either initial or in prepared fractions, was not correlated with sperm parameters (optical microscopy and flow cytometry parameters) after semen processing. There was no correlation between leukocyte concentration in the ejaculate and sperm recovery rate, whereas a significant correlation was found between the concentration of the residual leukocytes in prepared fractions and viable sperm recovery rate. Although the overall concordance between the flow cytometry and the optical microscopy was satisfactory, the sensitivity of peroxidase test for the detection of leukocytospermia resulted low.

Conclusion

Seminal leukocytes do not seem to influence sperm preparation results. However, for assisted conception, semen samples containing leukocytes should be processed using swim-up method. Although peroxidase-test is recommended by WHO as the standard method for determining semen leukocytes, it should not be used in clinical research study.
Appendix
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Literature
1.
Henkel RR, Schill WB: Sperm preparation for ART. Reprod Biol Endocrinol. 2003, 14: 108-129. 10.1186/1477-7827-1-108.CrossRef
2.
Aitken RJ, West KM: Analysis of the relationship between reactive oxygen species production and leukocyte infiltration in fractions of human semen separated on percoll gradients. Int J Androl. 1990, 13: 433-451. 10.1111/j.1365-2605.1990.tb01051.x.CrossRefPubMed
3.
Plante M, de Lamirande E, Gagnon C: Reactive oxygen species released by activated neutrophils, but not by deficient spermatozoa, are sufficient to affect normal sperm motility. Fertil Steril. 1994, 62: 387-393.PubMed
4.
Agarwal A, Saleh RA, Bedaiwy MA: Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril. 2003, 79: 829-843. 10.1016/S0015-0282(02)04948-8.CrossRefPubMed
5.
Quayle AJ, Xu C, Mayer KH, Anderson DJ: T lymphocytes and macrophages, but not motile spermatozoa, are a significant source of human immunodeficiency virus in semen. J Infect Dis. 1997, 176: 960-968. 10.1086/516541.CrossRefPubMed
6.
Tremellen K: Oxidative stress and male infertility - a clinical perspective. Hum Reprod Update. 2008, 14: 243-258. 10.1093/humupd/dmn004.CrossRefPubMed
7.
Tomlinson MJ, White A, Barratt CL, Bolton AE, Cooke ID: The removal of morphologically abnormal sperm forms by phagocytes: a positive role for seminal leukocytes?. Hum Reprod. 1992, 7: 517-522.PubMed
8.
Aitken RJ, West K, Buckingham D: Leukocytic infiltration into the human ejaculate and its association with semen quality, oxidative stress and sperm function. J Androl. 1994, 15: 343-352.PubMed
9.
Yanushpolsky EH, Politch JA, Hill JA, Anderson DJ: Is leukocytospermia clinically relevant?. Fertil Steril. 1996, 66: 822-825.PubMed
10.
Sharma R, Pasqualotto F, Nelson D, Thomas A, Agarwal A: Relationship between seminal white blood cells counts and oxidative stress in men treated at an infertility clinic. J Androl. 2001, 22: 575-583.PubMed
11.
Ricci G, Perticarari S, Fragonas E, Giolo E, Canova S, Pozzobon C, Guaschino S, Presani G: Apoptosis in human sperm: its correlation with semen quality and the presence of leukocytes. Hum Reprod. 2002, 17: 2665-2672. 10.1093/humrep/17.10.2665.CrossRefPubMed
12.
Aziz N, Agarwal A, Lewis-Jones I, Sharma RK, Thomas AJ: Novel associations between specific sperm morphological defects and leukocytospermia. Fertil Steril. 2004, 82: 621-627. 10.1016/j.fertnstert.2004.02.112.CrossRefPubMed
13.
Henkel R, Kierspel E, Stalf T, Mehnert C, Menkveld R, Tinneberg HR, Schill WB, Kruger TF: Effect of reactive oxygen species produced by spermatozoa and leukocytes on sperm functions in non-leukocytospermic patients. Fertil Steril. 2005, 83: 635-642. 10.1016/j.fertnstert.2004.11.022.CrossRefPubMed
14.
Yilmaz S, Koyuturk M, Kilic G, Alpak O, Aytoz A: Effects of leucocytospermia on semen parameters and outcomes of intracytoplasmic sperm injection. Int J Androl. 2005, 28: 337-342. 10.1111/j.1365-2605.2005.00562.x.CrossRefPubMed
15.
Moskovtsev SI, Willis J, White J, Mullen JB: Leukocytospermia: relationship to sperm deoxyribonucleic acid integrity in patients evaluated for male factor infertility. Fertil Steril. 2007, 88: 737-740. 10.1016/j.fertnstert.2006.11.132.CrossRefPubMed
16.
Fariello RM, Del Giudice PT, Spaine DM, Fraietta R, Bertolla RP, Cedenho AP: Effect of leukocytospermia and processing by discontinuous density gradient on sperm nuclear DNA fragmentation and mitochondrial activity. J Assist Reprod Genet. 2009, 26: 151-157. 10.1007/s10815-008-9288-0.PubMedCentralCrossRefPubMed
17.
Krausz C, West K, Buckingham D, Aitken RJ: Development of a technique for monitoring the contamination of human semen samples with leukocytes. Fertil Steril. 1992, 57: 1317-1325.PubMed
18.
Aitken RJ, Buckingham DW, Brindle J, Gomez E, Baker HWG, Irvine DS: Analysis of sperm movement in relation to the oxidative stress created by leukocytes in washed sperm preparations and seminal plasma. Hum Reprod. 1995, 10: 2061-2071.PubMed
19.
Aitken RJ, Buckingham D, West K, Wu FC, Zikopoulos K, Richardson DW: Differential contribution of leukocytes and spermatozoa to the generation of reactive oxygen species in the ejaculates of oligozoospermic patients and fertile donors. J Reprod Fertil. 1992, 94: 451-462.CrossRefPubMed
20.
Kessopoulou E, Tomlinson MJ, Barratt CL, Bolton AE, Cooke ID: Origin of reactive oxygen species in human semen: spermatozoa or leucocytes?. J Reprod Fertil. 1992, 94: 463-470.CrossRefPubMed
21.
Whittington K, Ford WCL: Relative contribution of leukocytes and of spermatozoa to reactive oxygen species production in human sperm suspensions. Int J Androl. 1999, 22: 229-235. 10.1046/j.1365-2605.1999.00173.x.CrossRefPubMed
22.
Ochsendorf FR: Infections in the male genital tract and reactive oxygen species. Int J Androl. 1999, 5: 399-420.
23.
Ford WC: Regulation of sperm function by reactive oxygen species. Hum Reprod Update. 2004, 10: 387-399. 10.1093/humupd/dmh034. Epub 2004 Jun 24.CrossRefPubMed
24.
World Health Organization: WHO Laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 1999, Cambridge: Cambridge University Press, 4
25.
Villegas J, Schulz M, Vallejos V, Henkel R, Miska W, Sánchez R: Indirect immunofluorescence using monoclonal antibodies for the detection of leukocytospermia: comparison with peroxidase staining. Andrologia. 2002, 34: 69-73. 10.1046/j.0303-4569.2001.00476.x.CrossRefPubMed
26.
Henkel R, Maass G, Hajimohammad M, Menkveld R, Stalf T, Villegas J, Sánchez R, Kruger TF, Schill WB: Urogenital inflammation: changes of leucocytes and ROS. Andrologia. 2003, 35: 309-313.CrossRefPubMed
27.
Perticarari S, Ricci G, Granzotto M, Boscolo R, Pozzobon C, Guarnieri S, Sartore A, Presani G: A new multiparameter flow cytometric method for human semen analysis. Hum Reprod. 2007, 22: 485-494. 10.1093/humrep/del415.CrossRefPubMed
28.
Cohen J: A coefficient of agreement for nominal scales. Educational and Psychological Measurement. 1960, 20: 37-46. 10.1177/001316446002000104.CrossRef
29.
Landis JR, Koch GG: The measurement of observer agreement for categorical data. Biometrics. 1977, 33: 159-174. 10.2307/2529310.CrossRefPubMed
30.
De Geyter C, De Geyter M, Behre HM, Schneider HP, Nieschlag E: Peroxidase-positive round cells and microorganisms in human semen together with antibiotic treatment adversely influence the outcome of in-vitro fertilization and embryo transfer. Int J Androl. 1994, 17: 127-34. 10.1111/j.1365-2605.1994.tb01231.x.CrossRefPubMed
31.
Sukcharoen N, Keith J, Irvine DS: Predicting the fertilizing potential of human sperm suspensions in vitro: importance of sperm morphology and leukocyte contamination. Fertil Steril. 1995, 63: 1293-1300.PubMed
32.
Lackner JE, Märk I, Sator K, Huber J, Sator M: Effect of leukocytospermia on fertilization and pregnancy rates of artificial reproductive technologies. Fertil Steril. 2008, 90: 869-871. 10.1016/j.fertnstert.2007.07.1310. Epub 2007 Nov 14.CrossRefPubMed
33.
Erel CT, Sentürk LM, Demir F, Irez T, Ertüngealp E: Antibiotic therapy in men with leukocytospermia. Int J Fertil Womens Med. 1997, 42: 206-210.PubMed
34.
Lackner JE, Herwig R, Schmidbauer J, Schatzl G, Kratzik C, Marberger M: Correlation of leukocytospermia with clinical infection and the positive effect of anti-inflammatory treatment on semen quality. Fertil Steril. 2000, 20: 1-6.
35.
Krisp A, Hörster S, Skrzypek J, Krause W: Treatment with levofloxacin does not resolve asymptomatic leucocytospermia -a randomized controlled study. Andrologia. 2003, 35: 244-247. 10.1046/j.1439-0272.2003.00580.x.CrossRefPubMed
36.
Boomsma CM, Heineman MJ, Cohlen BJ, Farquhar C: Semen preparation techniques for intrauterine insemination. Cochrane Database Syst Rev. 2004, 3: CD004507-PubMed
37.
Moustafa MH, Sharma RK, Thornton J, Mascha E, Abdel-Hafez MA, Thomas AJ, Agarwal A: Relationship between ROS production, apoptosis and DNA denaturation in spermatozoa from patients examined for infertility. Hum Reprod. 2004, 19: 129-138. 10.1093/humrep/deh024.CrossRefPubMed
38.
Weng SL, Taylor SL, Morshedi M, Schuffner A, Duran EH, Beebe S, Oehninger S: Caspase activity and apoptotic markers in ejaculated human sperm. Mol Hum Reprod. 2002, 8: 984-891. 10.1093/molehr/8.11.984.CrossRefPubMed
39.
Mahfouz RZ, du Plessis SS, Aziz N, Sharma R, Sabanegh E, Agarwal A: Sperm viability, apoptosis, and intracellular reactive oxygen species levels in human spermatozoa before and after induction of oxidative stress. Fertil Steril. 2008,
40.
Twigg J, Irvine DS, Houston P, Fulton N, Michael L: Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: protective significance of seminal plasma. Mol Hum Reprod. 1998, 4: 439-445. 10.1093/molehr/4.5.439.CrossRefPubMed
41.
Barroso G, Morshedi M, Oehninger S: Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod. 2000, 15: 1338-1344. 10.1093/humrep/15.6.1338.CrossRefPubMed
42.
Attaran M, Pasqualotto E, Falcone T, Goldberg JM, Miller KF, Agarwal A, Sharma RK: The effect of follicular fluid reactive oxygen species on the outcome of in vitro fertilization. Int J Fertil Womens Med. 2000, 45: 314-320.PubMed
43.
Ricci G, Presani G, Guaschino S, Simeone R, Perticarari S: Leukocyte detection in human semen using flow cytometry. Hum Reprod. 2000, 15: 1329-1337. 10.1093/humrep/15.6.1329.CrossRefPubMed
Metadata
Title
Leukocytospermia and sperm preparation - a flow cytometric study
Authors
Giuseppe Ricci
Sandra Perticarari
Rita Boscolo
Roberto Simeone
Monica Martinelli
Leo Fischer-Tamaro
Secondo Guaschino
Gianni Presani
Publication date
01-12-2009
Publisher
BioMed Central
Published in
Reproductive Biology and Endocrinology / Issue 1/2009
Electronic ISSN: 1477-7827
DOI
https://doi.org/10.1186/1477-7827-7-128

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