13. There is evidence to suggest that the microtubular spindle is
capable of repair during a period of post-thaw culture.
14. The presence of the sucrose solution on the internal sides of the
straw would dilute the concentration of CPA in the VSDP
column and, more importantly, may allow the propagation of
ice crystals from the sucrose solution into the area within which
the oocytes are contained.
15. Care must be taken not to dilute the CPA concentration in
these small droplets, the use of small droplets aids location of
the oocytes in these viscous solutions.
16. The straw is held at À140
C (below the glass transition
temperature range for aqueous CPS mixtures), rather than
plunging directly into liquid nitrogen in order to reduce the
occurrence of cracks in the vitrified glass which, on warming,
may form sites of ice nucleation. It is important to measure the
temperature as extreme fluctuations in temperature are present
across a few centimeters in liquid nitrogen vapor. Higher cooling/warming rates are recommended when lower CPA concentrations are used and oocyte is vitrified in the low—less than
1 μL volume—of the VS solution. Immediate warming in a
large volume of warming solution at 37
C gives better survival
if oocytes are vitrified in a closed system [74].
17. Oocytes are particularly difficult to locate in this solution, and
operator practice is important. The oocytes must spend no
longer than 5 min in 1 M sucrose solution at this point, and
this time can pass quickly. Other groups have developed postvitrification dilution steps using 0.5 M sucrose solutions [73],
which could be applied here, although we have not direct
evidence about their use with VSDP protocols.
Acknowledgments
We would like to gratefully acknowledge the significant role played
by Dr. Sharon Paynter, who as a collaborator over many years
developed the practical aspects of the two methods described in
this chapter.
References
1. Fuller B, Paynter S (2004) Fundamentals of
cryobiology in reproductive medicine. Reprod
Biomed Online 9:680–691
2. Keros V, Fuller BJ (2015) Cryopreservation of
mammalian oocytes. In: Wolkers WF, Oldenhof H (eds) Methods in cryopreservation and
freeze-drying, Methods in molecular biology.
Springer, pp 289–304
3. Yurchuk T, Petrushko M, Fuller B (2018) Science of cryopreservation in reproductive medicine - embryos and oocytes as exemplars. Early
Hum Dev 126:6–9
4. Iussig B, Maggiulli R, Fabozzi G et al (2019) A
brief history of oocyte cryopreservation: arguments and facts. Acta Obstet Gynecol Scand
98:550–558
Cryopreservation of Oocytes
451
capable of repair during a period of post-thaw culture.
14. The presence of the sucrose solution on the internal sides of the
straw would dilute the concentration of CPA in the VSDP
column and, more importantly, may allow the propagation of
ice crystals from the sucrose solution into the area within which
the oocytes are contained.
15. Care must be taken not to dilute the CPA concentration in
these small droplets, the use of small droplets aids location of
the oocytes in these viscous solutions.
16. The straw is held at À140
C (below the glass transition
temperature range for aqueous CPS mixtures), rather than
plunging directly into liquid nitrogen in order to reduce the
occurrence of cracks in the vitrified glass which, on warming,
may form sites of ice nucleation. It is important to measure the
temperature as extreme fluctuations in temperature are present
across a few centimeters in liquid nitrogen vapor. Higher cooling/warming rates are recommended when lower CPA concentrations are used and oocyte is vitrified in the low—less than
1 μL volume—of the VS solution. Immediate warming in a
large volume of warming solution at 37
C gives better survival
if oocytes are vitrified in a closed system [74].
17. Oocytes are particularly difficult to locate in this solution, and
operator practice is important. The oocytes must spend no
longer than 5 min in 1 M sucrose solution at this point, and
this time can pass quickly. Other groups have developed postvitrification dilution steps using 0.5 M sucrose solutions [73],
which could be applied here, although we have not direct
evidence about their use with VSDP protocols.
Acknowledgments
We would like to gratefully acknowledge the significant role played
by Dr. Sharon Paynter, who as a collaborator over many years
developed the practical aspects of the two methods described in
this chapter.
References
1. Fuller B, Paynter S (2004) Fundamentals of
cryobiology in reproductive medicine. Reprod
Biomed Online 9:680–691
2. Keros V, Fuller BJ (2015) Cryopreservation of
mammalian oocytes. In: Wolkers WF, Oldenhof H (eds) Methods in cryopreservation and
freeze-drying, Methods in molecular biology.
Springer, pp 289–304
3. Yurchuk T, Petrushko M, Fuller B (2018) Science of cryopreservation in reproductive medicine - embryos and oocytes as exemplars. Early
Hum Dev 126:6–9
4. Iussig B, Maggiulli R, Fabozzi G et al (2019) A
brief history of oocyte cryopreservation: arguments and facts. Acta Obstet Gynecol Scand
98:550–558
Cryopreservation of Oocytes
451