Archives of Gynecology and Obstetrics

, Volume 291, Issue 3, pp 689–694 | Cite as

A new method to process testicular sperm: combining enzymatic digestion, accumulation of spermatozoa, and stimulation of motility

  • Martina Wöber
  • Thomas Ebner
  • Sarah L. Steiner
  • Heinz Strohmer
  • Peter Oppelt
  • Eugen Plas
  • Andreas Obruca
Gynecologic Endocrinology and Reproductive Medicine



In azoospermia processing of the TESE material often results in a sample of reduced purity. This prospective study was set up to clarify whether a combination of enzymatic digestion, density gradient centrifugation and stimulation of motility (where indicated) is a feasible option in TESE patients.


A total of 63 samples (showing spermatozoa) were processed by the present tripartite processing method. The resulting sperm sample of high purity was directly used for ICSI and subsequent cryopreservation when quality of the accumulated sperm sample allowed for it (n = 39 cycles).


Compared to the control group blastocyst formation rate in the present tripartite processing technique was significantly (P < 0.01) higher (55.2 vs. 43.7 %). Fertilization rates differed significantly (P < 0.001) between cases in which motile sperm could be used (58.4 %) compared to ICSI with immotile sperm (45.0 %). Clinical pregnancy rate per transfer was 40.0 % (24/60) using fresh and 21.6 % (8/37) with cryopreserved TESE material. The calculated live birth rates were 31.7 and 21.6 %, respectively. Thirty-five healthy children were born.


A comparison with a control group suggests that the present approach using standardized ready-to-use products is efficient and reliable. Presumably healthy live births further indicate the safety of the procedure.


Collagenase Density gradient ICSI Processing of testicular sperm TESE Theophylline 


  1. 1.
    Silber SJ, Nagy ZP, Liu J, Godoy H, Devroey P, Van Steirteghem AC (1994) Conventional in vitro fertilization versus intracytoplasmic sperm injection for patients requiring microsurgical sperm aspiration. Hum Reprod 9:1705–1709PubMedGoogle Scholar
  2. 2.
    Devroey P, Liu J, Nagy ZP, Goossens A, Tournaye H, Camus M, Van Steirteghem A, Silber S (1995) Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum Reprod 10:1457–1460PubMedCrossRefGoogle Scholar
  3. 3.
    Patrizio P, Silber S, Ord T, Balmaceda JP, Asch RH (1988) Two births after microsurgical sperm aspiration in congenital absence of vas deferens. Lancet 2(8624):1364PubMedCrossRefGoogle Scholar
  4. 4.
    Maiburg MC, Hoppenbrouwers AC, van Stel HF, Giltay JC (2011) Attitudes of Klinefelter men and their relatives towards TESE-ICSI. J Assist Reprod Genet 28:809–814PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Akarsu C, Caglar G, Vicdan K, Isik AZ, Tuncay G (2009) Pregnancies achieved by testicular sperm recovery in male hypogonadotrophic hypogonadism with persistent azoospermia. Reprod Biomed Online 18:455–459PubMedCrossRefGoogle Scholar
  6. 6.
    Fahmy I, Kamal A, Aboulghar M, Mansour R, Serour GI, Shamloul R (2004) Percutaneous aspiration biopsy using an intravenous catheter for testicular sperm retrieval in patients with obstructive azoospermia. Reprod Biomed Online 9:102–105PubMedCrossRefGoogle Scholar
  7. 7.
    Dafopoulos K, Griesinger G, Schultze-Mosgau A, Orief Y, Schöpper B, Nikolettos N, Diedrich K, Al-Hasani S (2005) Factors affecting outcome after ICSI with spermatozoa retrieved from cryopreserved testicular tissue in non-obstructive azoospermia. Reprod Biomed Online 10:455–460PubMedCrossRefGoogle Scholar
  8. 8.
    Dafopoulos K, Griesinger G, Schultze-Mosgau A, Orief Y, Schöpper B, Nikolettos N, Diedrich K, Al-Hasani S (2005) Cumulative pregnancy rate after ICSI with cryopreserved testicular tissue in non-obstructive azoospermia. Reprod Biomed Online 10:461–466PubMedCrossRefGoogle Scholar
  9. 9.
    Silber SJ (2000) Microsurgical TESE and the distribution of spermatogenesis in non-obstructive azoospermia. Hum Reprod 15:2278–2284PubMedCrossRefGoogle Scholar
  10. 10.
    Colpi GM, Colpi EM, Piediferro G, Giacchetta D, Gazzano G, Castiglioni FM, Magli MC, Gianaroli L (2009) Microsurgical TESE versus conventional TESE for ICSI in non-obstructive azoospermia: a randomized controlled study. Reprod Biomed Online 18:315–319PubMedCrossRefGoogle Scholar
  11. 11.
    Turunc T, Gul U, Haydardedeoglu B, Bal N, Kuzgunbay B, Peskircioglu L, Ozkarde H (2010) Conventional testicular sperm extraction combined with the microdissection technique in nonobstructive azoospermic patients: a prospective comparative study. Fertil Steril 94:2157–2160PubMedCrossRefGoogle Scholar
  12. 12.
    Turek PJ, Givens CR, Schriock ED, Meng MV, Pedersen RA, Conaghan J (1999) Testis sperm extraction and intracytoplasmic sperm injection guided by prior fine-needle aspiration mapping in patients with nonobstructive azoospermia. Fertil Steril 71:552–557PubMedCrossRefGoogle Scholar
  13. 13.
    Herwig R, Tosun K, Pinggera GM, Soelder E, Moeller KT, Pallwein L, Frauscher E, Bartsch G, Wildt L, Illmensee K (2004) Tissue perfusion essential for spermatogenesis and outcome of testicular sperm extraction (TESE) for assisted reproduction. J Assist Reprod Genet 21:175–180PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Friedler S, Raziel A, Soffer Y, Strassburger D, Komarovsky D, Ron-El R (1997) Intracytoplasmic injection of fresh and cryopreserved testicular spermatozoa in patients with nonobstructive azoospermia: a comparative study. Feril Steril 68:892–897CrossRefGoogle Scholar
  15. 15.
    Habermann H, Seo R, Cieslak J, Niederberger C, Prins GS, Ross L (2000) In vitro fertilization outcomes after intracytoplasmic sperm injection with fresh or frozen-thawed testicular spermatozoa. Fertil Steril 73:955–960PubMedCrossRefGoogle Scholar
  16. 16.
    Baukloh V (2002) German Society for Human Reproductive Biology. Retrospective multicentre study on mechanical and enzymatic preparation of fresh and cryopreserved testicular biopsies. Hum Reprod 17:1788–1794PubMedCrossRefGoogle Scholar
  17. 17.
    Hammitt DG, Sattler CA, Ferrigni RG, Singh AP (2002) Development of a new and efficient laboratory method for processing testicular sperm. J Assist Reprod Genetics 19:335–342CrossRefGoogle Scholar
  18. 18.
    Crabbé E, Verheyen G, Tournaye H, Van Steirteghem A (1997) The use of enzymatic procedures to recover testicular germ cells. Hum Reprod 12:1682–1687PubMedCrossRefGoogle Scholar
  19. 19.
    Ebner T, Tews G, Mayer RB, Ziehr S, Arzt W, Costamoling W, Shebl O (2011) Pharmacological stimulation of sperm motility in frozen and thawed testicular sperm using the dimethylxanthine theophylline. Fertil Steril 96:1331–1336PubMedCrossRefGoogle Scholar
  20. 20.
    Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB (2000) Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 73:1155–1158PubMedCrossRefGoogle Scholar
  21. 21.
    Oktay K, Nugent D, Newton H, Salha O, Chatterjee P, Gosden RG (1997) Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue. Fertil Steril 67:481–486PubMedCrossRefGoogle Scholar
  22. 22.
    Verheyen G, De Croo I, Tournaye H, Pletincx I, Devroey P, van Steirteghem AC (1995) Comparison of four mechanical methods to retrieve spermatozoa from testicular tissue. Hum Reprod 10:2956–2959PubMedGoogle Scholar
  23. 23.
    Salzbrunn A, Benson DM, Holstein AF, Schulze W (1996) A new concept for the extraction of testicular spermatozoa as a tool for assisted fertilization (ICSI). Hum Reprod 11:752–755PubMedCrossRefGoogle Scholar
  24. 24.
    Crabbé E, Verheyen G, Silber S, Tournaye H, Van de Velde H, Goossens A, Van Steirtheghem A (1998) Enzymatic digestion of testicular tissue may rescue the intracytoplasmic sperm injection cycle in some patients with non-obstructive azoospermia. Hum Reprod 13:2791–2796PubMedCrossRefGoogle Scholar
  25. 25.
    Aydos K, Demirel LC, Baltaci V, Unlü C (2005) Enzymatic digestion plus mechanical searching improves testicular sperm retrieval in non-obstructive azoospermia cases. Eur J Obstet Gynecol Reprod Biol 120:80–86PubMedCrossRefGoogle Scholar
  26. 26.
    Ramasamy R, Reifsnyder JE, Bryson C, Zaninovic N, Liotta D, Cook CA, Hariprashad J, Weiss D, Neri Q, Palermo GD, Schlegel PN (2011) Role of tissue digestion and extensive sperm search after microdissection testicular sperm extraction. Fertil Steril 96:299–302PubMedCrossRefGoogle Scholar
  27. 27.
    Scholtes MCW, van Hoogstraten DG, Schmoutziguer A, Zeilmaker GH (1999) Extraction of testicular sperm from previously cryopreserved tissue in couples with or without transport of oocytes and testicular tissue. Fertil Steril 72:785–791PubMedCrossRefGoogle Scholar
  28. 28.
    Thompson-Cree ME, McClure N, Donnelly ET, Steele KE, Lewis SE (2003) Effects of cryopreservation on testicular sperm nuclear DNA fragmentation and its relationship with assisted conception outcome following ICSI with testicular spermatozoa. Reprod Biomed Online 7:449–455PubMedCrossRefGoogle Scholar
  29. 29.
    Balaban B, Urman B, Sertac A, Alatas C, Aksoy S, Mercan R, Nuhoglu A (1999) In-vitro culture of spermatozoa induces motility and increases implantation and pregnancy rates after testicular sperm extraction and intracytoplasmic sperm injection. Hum Reprod 14:2808–2811PubMedCrossRefGoogle Scholar
  30. 30.
    Terriou P, Hans E, Giorgetti C, Spach JL, Salzmann J, Urrutia V, Roulier R (2000) Pentoxifylline initiates motility in spontaneously immotile epididymal and testicular spermatozoa and allows normal fertilization, pregnancy, and birth after intracytoplasmic sperm injection. J Assist Reprod Genetics 7:194–199CrossRefGoogle Scholar
  31. 31.
    Nordhoff V, Schüring AN, Krallmann C, Zitzmann M, Schlatt S, Kiesel L, Kliesch S (2013) Optimizing TESE-ICSI by laser-assisted selection of immotile spermatozoa and polarization microscopy for selection of oocytes. Andrologia 1:67–74CrossRefGoogle Scholar
  32. 32.
    Griveau JF, Lobel B, Laurent MC, Michardière L, Le Lannou D (2006) Interest of pentoxifylline in ICSI with frozen-thawed testicular spermatozoa from patients with non-obstructive azoospermia. Reprod Biomed Online 12:14–18PubMedCrossRefGoogle Scholar
  33. 33.
    Calogero AE, Fishel S, Hall J, Ferrara E, Vicari E, Green S, Hunter A, Burrello N, Thornton S, DÁgata R (1998) Correlation between intracellular cAMP content, kinematic parameters and hyperactivation of human spermatozoa after incubation with pentoxifylline. Hum Reprod 13:911–915PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Martina Wöber
    • 1
  • Thomas Ebner
    • 2
    • 3
  • Sarah L. Steiner
    • 1
  • Heinz Strohmer
    • 1
  • Peter Oppelt
    • 2
    • 3
  • Eugen Plas
    • 4
  • Andreas Obruca
    • 1
  1. 1.Kinderwunschzentrum Goldenes KreuzViennaAustria
  2. 2.Department of Gynecological Endocrinology and Kinderwunsch ZentrumLandes- Frauen- und KinderklinikLinzAustria
  3. 3.Faculty of MedicineJohannes Kepler UniversityLinzAustria
  4. 4.Department of UrologyHospital HietzingViennaAustria

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