cryopreservation with open carrier systems. Liquid air has the
same temperature as liquid nitrogen (À195.7
C) [37].
3. Spermatozoa analyses are performed according to published
guidelines of the World Health Organization (WHO) [40].
4. Specimens used for the cryopreservation protocol described
here have the following quality criteria: >15 Â 10
6 spermatozoa/mL, >32% progressively motile sperm, and >4.0% morphologically normal spermatozoa.
5. Cooling rates achieved with liquefied air and liquid nitrogen
both are greater than 20,000
C/min for volumes of 50, 25,
and 10 μL [37].
6. After about 8–10 s in liquid nitrogen and 10–12 s in liquid air,
the spheres solidify and fall to the bottom of the strainer. Once
this process has taken place, the procedure is repeated to obtain
more spheres.
7. The solid spheres were packaged into 1.8 mL cryotubes and
stored for at least 24 h. A metal strainer was immersed in the
liquid nitrogen for easy collection of the spheres. Specimens
need to be stored for at least 24 h in a tank with liquid nitrogen
before warming.
8. Warming was performed by quickly submerging spheres one by
one (not more than five spheres) into 5 mL warming media
accompanied by gentle vortexing for 5–10 s. It only takes
2~3 s for the spheres to melt. Roughly estimated, the warming
rate ranges from ~6990 to 4660
C/min.
References
1. Sanger WG, Olson JH, Sherman JK (1992)
Semen cryobanking for men with cancercriteria change. Fert Steril 58:1024–1027
2. Isachenko V, Montag M, Isachenko E,
Nawroth F, Dessole S, Van der Ven H (2004)
Developmental rate and ultrastructure of vitrified human pronuclear oocytes after step wise
versus direct rehydration. Hum Reprod
19:660–665
3. Isachenko V, Isachenko E, Katkov II,
Montag M, Dessole S, Nawroth F, van der
Ven H (2004) Cryoprotectant-free cryopreservation of human spermatozoa by vitrification
and freezing in vapour: effect on motility, DNA
integrity, and fertilization ability. Biol Reprod
71:1167–1173
4. Jensen JR, Morbeck DE, Coddington CC III
(2011) Fertility preservation. Clin Endocrinol
86:45–49
5. Polge EJC (1957) Low-temperature storage of
mammalian spermatozoa. Proc R Soc Lond B
Biol Sci 147:498–508
6. Nawroth F, Isachenko V, Dessole S, Rahimi G,
Farina M, Vargiu N, Orth I (2002) Vitrification of human spermatozoa without cryoprotectants. CryoLetters 23:93–102
7. Isachenko E, Isachenko V, Weiss JM,
Kreienberg R, Katkov II, Schulz M, Sanchez
R (2008) Acrosomal status and mitochondrial
activity of human spermatozoa vitrified with
sucrose. Reproduction 136:167–174
8. Di Santo M, Tarozzi N, Nadalini M, Borini A
(2012) Human sperm cryopreservation:
update on techniques, effect on DNA integrity,
and implications for art. Adv Urol
2012:854837
9. Pabo ´n D, Meseguer M, Sevillano G, Cobo A,
Romero JL, Remohı ´ J, de los Santos MJ
(2019) A new system of sperm cryopreservation: evaluation of survival, motility, DNA oxidation, and mitochondrial activity. Andrology
7:293–301
10. Said TM, Gaglani A, Agarwal A (2010) Implication of apoptosis in sperm cryoinjury. Reprod
Biomed Online 21:456–462
434
Mengying Wang et al.
same temperature as liquid nitrogen (À195.7
C) [37].
3. Spermatozoa analyses are performed according to published
guidelines of the World Health Organization (WHO) [40].
4. Specimens used for the cryopreservation protocol described
here have the following quality criteria: >15 Â 10
6 spermatozoa/mL, >32% progressively motile sperm, and >4.0% morphologically normal spermatozoa.
5. Cooling rates achieved with liquefied air and liquid nitrogen
both are greater than 20,000
C/min for volumes of 50, 25,
and 10 μL [37].
6. After about 8–10 s in liquid nitrogen and 10–12 s in liquid air,
the spheres solidify and fall to the bottom of the strainer. Once
this process has taken place, the procedure is repeated to obtain
more spheres.
7. The solid spheres were packaged into 1.8 mL cryotubes and
stored for at least 24 h. A metal strainer was immersed in the
liquid nitrogen for easy collection of the spheres. Specimens
need to be stored for at least 24 h in a tank with liquid nitrogen
before warming.
8. Warming was performed by quickly submerging spheres one by
one (not more than five spheres) into 5 mL warming media
accompanied by gentle vortexing for 5–10 s. It only takes
2~3 s for the spheres to melt. Roughly estimated, the warming
rate ranges from ~6990 to 4660
C/min.
References
1. Sanger WG, Olson JH, Sherman JK (1992)
Semen cryobanking for men with cancercriteria change. Fert Steril 58:1024–1027
2. Isachenko V, Montag M, Isachenko E,
Nawroth F, Dessole S, Van der Ven H (2004)
Developmental rate and ultrastructure of vitrified human pronuclear oocytes after step wise
versus direct rehydration. Hum Reprod
19:660–665
3. Isachenko V, Isachenko E, Katkov II,
Montag M, Dessole S, Nawroth F, van der
Ven H (2004) Cryoprotectant-free cryopreservation of human spermatozoa by vitrification
and freezing in vapour: effect on motility, DNA
integrity, and fertilization ability. Biol Reprod
71:1167–1173
4. Jensen JR, Morbeck DE, Coddington CC III
(2011) Fertility preservation. Clin Endocrinol
86:45–49
5. Polge EJC (1957) Low-temperature storage of
mammalian spermatozoa. Proc R Soc Lond B
Biol Sci 147:498–508
6. Nawroth F, Isachenko V, Dessole S, Rahimi G,
Farina M, Vargiu N, Orth I (2002) Vitrification of human spermatozoa without cryoprotectants. CryoLetters 23:93–102
7. Isachenko E, Isachenko V, Weiss JM,
Kreienberg R, Katkov II, Schulz M, Sanchez
R (2008) Acrosomal status and mitochondrial
activity of human spermatozoa vitrified with
sucrose. Reproduction 136:167–174
8. Di Santo M, Tarozzi N, Nadalini M, Borini A
(2012) Human sperm cryopreservation:
update on techniques, effect on DNA integrity,
and implications for art. Adv Urol
2012:854837
9. Pabo ´n D, Meseguer M, Sevillano G, Cobo A,
Romero JL, Remohı ´ J, de los Santos MJ
(2019) A new system of sperm cryopreservation: evaluation of survival, motility, DNA oxidation, and mitochondrial activity. Andrology
7:293–301
10. Said TM, Gaglani A, Agarwal A (2010) Implication of apoptosis in sperm cryoinjury. Reprod
Biomed Online 21:456–462
434
Mengying Wang et al.