Aseptic cryoprotectant-free vitrification of human ICSI spermatozoa

We would like to call your attention to the following One-on-One Training Course (hands-on workshop) on Aseptic cryoprotectant-free vitrification of human ICSI spermatozoa in Cologne and extend a cordial invitation to you. The course is an exclusive face-to-face individual training approach to facilitate learning that is anticipated to provide value-for-cost for participants because it is more effective than group training.

Course Design

This workshop is intended for postgraduate students and specialists in the field of mammalian reproductive cells cryopreservation (in medicine and veterinary medicine). 
At present, businesses that manufacture something new for clinical use find it much more difficult and time consuming than it was some years ago. We are announcing our workshop today because we are now able to present the plastic elements of the kits that had not previously been manufactured by firms and we had not the possibility of presenting it at our workshop. 
Our course contains short basic lectures and a film regarding the technology. The central part of our workshop is hands-on manipulations. 
During this workshop participants will obtain skills and kits (solutions, plastic and protocols) and will be ready when returning to their home laboratories to repeat the presented technology with respective results. 
All demonstrating manipulations with human spermatozoa will be performed on real objects: fresh ejaculates. 
The comparative cryopreservations will be offered for your review. For this comparison it will be possible during the workshop to use your home technology of sperm conventional (“slow”) freezing with permeable cryoprotectants. If you prefer to use your home freezing solutions for comparative cryopreservations, you can bring it (as well as instruments, plastic, containers etc.) to our workshop. 
During the workshop we will assist you to perform the comparative investigations, we will separately and blind note obtained results, and we will compare our notations after the end of experimental cryopreservations. During these experiments we will formulate the following parameters of technology: optimal volume of vitrifying spermatozoa suspension, optimal time elapsing from the expelling of capillaries from liquid nitrogen to the plunging into the warming medium and optimal volume of the warming (culture) medium. 

A few concise paragraphs on technologies and the workshop 

  1. Each element of the technology is a result of our research that we have already published [1–23, 50-52].
  2. Technologies of cryoprotectant-free vitrification of human, mouse, canine, stallion, donkey, ram, porcine, fish and cat ( spermatozoa are now realities [1–52].
  3. Technology we have developed is original. After warming, vitrified spermatozoa are immediately ready for further use without any additional treatment (centrifugation for removal of cryoprotectants, separation in the gradient and others). Technology allows obtaining the following: vitrifying 0.01 ml suspension of spermatozoa, warming this 0.01 ml and the same 0.01 ml use for ICSI. After warming, spermatozoa are free from sperm plasma.
  4. Vitrification kit includes only one osmotically neutral solution without permeable cryoprotectants.
  5. We recommend our technology not only for its simplicity but also because our technology better preserves spermatozoa from ejaculates of bed quality, like asthenozoospermia and cryptozoospermia compared to traditional “slow” freezing with permeable cryoprotectants. 

Course directors

  • Prof. Dr. Peter Mallmann, Director of University Maternal Hospital
  • Dr. (SU) Evgenia Isachenko, Biological Director of IVF-Laboratory, Department of Obstetrics and Gynaecology, Cologne University Hospital  
  • Prof. Dr. Gohar Rahimi, Medical Director of IVF-Laboratory, Department of Obstetrics and Gynaecology, Cologne University Hospital
  • Prof. Dr. Raul Sanchez, Director of Biomedical Center, La Frontera University, Temuco, Chile (during certain periods only)
  • Dr. (SU) Vladimir Isachenko, Leader of Research Group for Reproductive Medicine, Department of Obstetrics and Gynaecology, Cologne University Hospital

Further Information and Program

Date: Beginning October 2019 (any date at the participant’s convenience)
Duration: 2 days
Number of participants: One, a maximum of two per session
Location: University Hospital of Cologne, Department of Gynaecology and Obstetrics
Kerpener Str. 34, 50931 Cologne, Germany
Language: English and German
Course fee: 1000 EUR for a single participant, 1500 EUR for two participants

Information and registration:
Dr. (SU) Volodimir Isachenko
Phone +49 221 478-7349

About the place
Cologne is a famous tourist city. Staying in Cologne allows easy access to the Netherlands, Belgium, Luxemburg and France. For example, a tour to Amsterdam with a 5 to 6 hour stay in the city with transport Cologne-Amsterdam-Cologne occupies a little bit more than one light-day and costs up to 25 EUR. A 4-day tour to Paris (3 days in this city) including transport und hotel costs from 90 to 180 EUR.


First day
TimeProgramCourse director
08:00 - 08:45Lecture 
Cryobiology as a field of knowledge and application in reproductive medicine and oncology.
Dr. (SU) Vladimir Isachenko
09:00 - 09:45Lecture 
Cryoprotectant-free vitrification of human spermatozoa: theory and practical application.
Dr. (SU) Evgenia Isachenko
10:00 - 10:45Film with comments 
Cryoprotectant-free vitrification of mammalian spermatozoa. 
Dr. (SU) Vladimir Isachenko
11:00 - 12:45 and
13:40 - 16:00
  1. Preparation of vitrification medium
  2. Dilution of spermatozoa by vitrification medium.
Dr. (SU) Vladimir Isachenko; Dr. (SU) Evgenia Isachenko; Prof. Dr. Gohar Rahimi; Prof. Dr. Raul Sanchez
Second day
TimeProgramCourse director
08:00 - 08:45Lecture 
Integration of spermatozoa in oocytes: assisted technology for artificial activation and correction of position of the pronuclei.
Prof. Dr. Peter Mallmann
09:00 - 12:50 and 13:40 - 16:00Hands-on
  1. Vitrification of 10 μl and 5 μl spermatozoa suspension for ICSI and IVF in micro volumes. 

    1.1. Aspiration of spermatozoa into capillary, relocation of capillary into isolating straw, sealing of isolating straw and plunging of capillary into liquid nitrogen (cooling).

    1.2. Warming of spermatozoa by plunging of capillary into pre-warmed medium. 
  2. Quality control of warmed spermatozoa, in accordance with a principle “it is better to do and see it once than read about it ten times”

    2.1. Cryoprotectant-free vitrification of spermatozoa vs. traditional slow freezing with glycerol or DMSO: comparison of effectiveness. For this type of comparative cryopreservations, spermatozoa will be distributed for two parts: cryoprotectants-free vitrification and conventional freezing with permeable cryorotectants. 
Dr. (SU) Vladimir Isachenko; Dr. (SU) Evgenia Isachenko; Prof. Dr. Gohar Rahimi; Prof. Dr. Raul Sanchez
16:00 - 17:00Extensive discussions and repeated hands-on manipulations
Prof. Dr. Peter Mallmann; Dr. (SU) Evgenia Isachenko; Dr. (SU) Vladimir Isachenko; Prof. Dr. Gohar Rahimi; Prof. Dr. Raul Sanchez
17:00 - 17:15Presentation of certificates and departure 


Publications are mentioned in this announcement
  1. F. Nawroth et al. Vitrification of human spermatozoa without cryoprotectants. CryoLetters, 23:93-102 (2002)
  2. E. Isachenko et al. Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from practical difficulties to present success. Reprod. Biomed. Online, 6:191-200 (2003) 
  3. V. Isachenko et al. Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biol. Reprod. 71:1167-1173 (2004) 
  4. E. Isachenko et al. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Hum. Reprod. 19:932-939 (2004) 
  5. V. Isachenko et al. Clean technology for cryoprotectant-free vitrification of human spermatozoa. Reprod. Biomed. Online 10:350-354 (2005)
  6. E. Isachenko et al. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction 136:167-73 (2008)
  7. V. Isachenko et al. Cryoprotectant-free vitrification of human spermatozoa in large (up to 0.5 mL) volume: a novel technology. Clin. Lab. 57:643-650 (2011)
  8. V. Isachenko et al. Human spermatozoa vitrified in the absence of permeable cryoprotectants: birth of two healthy babies. Reprod. Fertil. Dev. 24:323-326 (2011)
  9. O. Merino et al. Fish (Oncorhynchus mykiss) spermatozoa cryoprotectant-free vitrification: stability of mitochondrion as criterion of effectiveness. Anim. Reprod. Sci. 124:125-131 (2011) 
  10. R. Sanchez et al. Live birth after intrauterine insemination with spermatozoa from an oligo-astheno-zoospermic patient vitrified without permeable cryoprotectants. J. Androl. 33:559-562 (2011) 
  11. R. Sánchez et al. Canine sperm vitrification with sucrose: effect on sperm function. Andrologia 43:233-241 (2011) 
  12. V. Isachenko et al. Vitrification of human ICSI/IVF spermatozoa without cryoprotectants: new capillary technology. J. Androl. 33:462-468 (2012) 
  13. O. Merino et al. Cryoprotectant-free vitrification of fish (Oncorhynchus mykiss) spermatozoa: first report. Andrologia 44 Suppl. 1:390-395 (2012) 
  14. R. Sánchez et al. Vitrified sperm banks: the new aseptic technique for human spermatozoa allows cryopreservation at -86 °C. Andrologia 44:433-435 (2012) 
  15. R. Sánchez et al. Vitrificación de espermatozoides: una alternativa a la inyección intracitoplasmatica de espermatozoides en paciente con oligoastenozoospérmia severa. Rev. Int. Andrologia 11: 36-39 (2013). 
  16. E. Figueroa et al. Effect of seminal plasma on Atlantic salmon (Salmo salar) sperm vitrification. Theriogenology 83:238-245 (2015) 
  17. O. Merino et al. Protective effect of butylated hydroxytoluene on sperm function in human spermatozoa cryopreserved by vitrification technique. Andrologia 47:186-193 (2015) 
  18. M. Mansilla et al. High temperature is essential for preserved human sperm function during the devitrification process. Andrologia 48:111-113 (2016) 
  19. V. Isachenko et al. Technologies of cryoprotectant-free vitrification of human spermatozoa: asepticity as criterion of effectiveness. Andrology 5:1055-1063 (2017) 
  20. V. Isachenko et al. Chapter 6. Technology of Aseptic Cryoprotectant-Free Vitrification of Human ICSI Spermatozoa. Methods Mol. Biol. 1568:79-84 (2017)
  21. P. Uribe et al. Effect of incubation temperature after devitrification on quality parameters in human sperm cells. Cryobiology, 79:78-81(2017) 
  22. M. Schulz et al. Trehalose sustains a higher post-thaw sperm motility than sucrose in vitrified human sperm. Andrologia, 49:e12757 (2017) 
  23. V. Isachenko et al. Cryoprotectant-free vitrification of spermatozoa: fish as a model of human. Andrologia, in press (2018)
  24. Y. Chen et al. Small-volume vitrification for human spermatozoa in the absence of cryoprotectants by using Cryotop. Andrologia 47:694-699 (2014) 
  25. M. A. Khalili et al. Vitrification of neat semen alters sperm parameters and DNA integrity. Urol. J. 11:1465-1470 (2014)
  26. A. Agha-Rahimi et al. Vitrification is not superior to rapid freezing of normozoospermic spermatozoa: effects on sperm parameters, DNA fragmentation and hyaluronan binding. Reprod. Biomed. Online 28:352-358 (2014) 
  27. M. Mansilla et al. High temperature is essential for preserved human sperm function during the devitrification process. Andrologia doi: 10.1111/and.12406 (2015)
  28. V. Kuznyetsov et al.  Vitrification of a small number of spermatozoa in normozoospermic and severely oligozoospermic samples. Syst. Biol. Reprod. Med. 61:13-17 (2015)
  29. M. Slabbert et al. Large volume cryoprotectant-free vitrification: an alternative to conventional cryopreservation for human spermatozoa. Andrologia 47:594-599 (2015)  
  30. M. Ali Mohamed. Slow cryopreservation is not superior to vitrification in human spermatozoa; an experimental controlled study. Iran. J. Reprod. Med. 13: 633–44 (2015). 
  31. A. Agha-Rahimi et al. Cryoprotectant-free vitrification of human spermatozoa in new artificial seminal fluid. Andrology 4: 1037–44 (2016)
  32. M. Diaz-Jimenez et al. Effect of different extenders for donkey sperm vitrification in straws. Reprod Domest Anim. 52 Suppl 4:55-57 (2017)
  33. J. Aizpurua et al. New permeable cryoprotectant-free vitrification method for native human sperm. Hum. Reprod. 32:2007-2015 (2017)
  34. A. Nabi et al. In-vitro application of pentoxifylline preserved ultrastructure of spermatozoa after vitrification in asthenozoospermic patients. Urol. J. 14:4038-4043 (2017)
  35. A. Arando et al. Storage temperature and sucrose concentrations affect ram sperm quality after vitrification. Anim. Reprod. Sci. 181:175-185 (2017)
  36. C. C. Arraztoa et al. Porcine sperm vitrification II: Spheres method. Andrologia 49 doi: 10.1111/and.12738 (2017)
  37. C. C. Arraztoa et al. Porcine sperm vitrification I: cryoloops method. Andrologia  49 doi: 10.1111/and.12706 (2017)
  38. W. F. Swanson et al. Urethral catheterization and sperm vitrification for simplified semen banking in felids. Reprod. Domest. Anim. 52 Suppl 2:255-260 (2017)
  39. F. Horta et al. Minimal volume vitrification of epididymal spermatozoa results in successful in vitro fertilization and embryo development in mice. Asian J. Androl. 19:107-112 (2017)
  40. J. Sun et al.  Successful delivery derived from cryopreserved rare human spermatozoa with novel cryopiece. Andrology 5:832-837 (2017)
  41. N. Riva et al.  Comparative analysis between slow freezing and ultra-rapid freezing for human sperm cryopreservation. JBRA Assist. Reprod. doi: 10.5935/1518-0557.20180060 (2018)
  42. M. Hidalgo et al.  Concentrations of non-permeable cryoprotectants and equilibration temperatures are key factors for stallion sperm vitrification success. Anim. Reprod. Sci. 196:91-98 (2018)
  43. I.I. Katkov et al.  KrioBlastTM as a new technology of ultrafast cryopreservation of cells and tissues. 2. Kinetic vitrification of human pluripotent stem cells and spermatozoa. Bull Exp Biol Med. 165:171-175 (2018)
  44. C. C. Pérez-Marín et al. Effect of trehalose- and sucrose-based extenders on equine sperm quality after vitrification: Preliminary results. Cryobiology 80:62-69 (2018) 
  45. E. Caturla-Sánchez et al. Vitrification of dog spermatozoa: Effects of two cryoprotectants (sucrose or trehalose) and two warming procedures. Cryobiology 80:126-129 (2018)
  46. M.  Karthikeyan et al. Comparison of conventional slow freeze versus permeable cryoprotectant-free vitrification of abnormal semen sample: a randomized controlled trial. J. Hum. Reprod. Sci. 12:150-155 (2019)
  47. C. Consuegra et al. Vitrification of stallion sperm using 0.25 ml straws: Effect of volume, concentration and carbohydrates (sucrose/trehalose/raffinose). Anim Reprod Sci. 206:69-77 (2019)
  48. M.  Diaz-Jimenez et al. Optimization of donkey sperm vitrification: Effect of sucrose, sperm concentration, volume and package (0.25 and 0.5 mL straws). Anim Reprod Sci. 204:31-38 (2019)
  49. L. Medrano et al. First birth of a healthy infant following intra cytoplasmic sperm injection using a new permeable cryoprotectant- free sperm vitrification protocol. Cryobiology 87:117-119 (2019)
  50. P. Uribe et al. Nitrosative stress in human spermatozoa causes cell death characterized by induction of mitochondrial permeability transition-driven necrosis. Asian J Androl. 20:600-607 (2018)
  51. Wang M. et al. Non-sterile liquid nitrogen vs. sterile liquid air: Aseptic technology for cryoprotectant-free vitrification of human spermatozoa by direct dropping into a cooling agent. Andrologia, In press.
  52. E. Spis et al. Conventional freezing vs. cryoprotectant-free vitrification of epididymal (MESA) and testicular (TESE) spermatozoa: Three live births. Cryobiology. In press.