Oocyte cryopreservation has been at the heart of this debate
within applied cryobiology. Originally, the early reports of oocyte
cryopreservation in both human and animal oocytes were made
using CRSC [13–20]. Since then pregnancies and live births resulting from cryopreserved oocytes have been reported in a number of
species [21–24] and human [25]. Clinical programmes were developed in the early 2000s using CRSC with optimized CPA mixtures
[26, 27]. At the same time VS techniques were becoming optimized [4, 28]. It became apparent that experienced practitioners
could recover higher percentages of oocyte from cryogenic storage
using VS methods [29–31].
Historically, oocytes have been considered to be difficult cells
to cryopreserve for a number of reasons. The oocyte is a large single
cell with relatively low permeability to water and cryoprotective
agents (CPA). This means that oocytes have a tendency to retain
water as ice starts to form in the suspending medium during cooling and, if this leads to formation of intracellular ice as cooling
proceeds, damage to the cell results. Permeability of oocytes varies
between species, strains and maturational status of the oocyte. The
mature oocyte is also a short-lived cell that must undergo fertilization in order for it to continue to survive and develop. For fertilization to occur naturally, the oocyte must retain integrity of a
number of its’ unique structural features. These include the surrounding zona pellucida, the cortical granules, and the microtubular spindle on which the chromosomes are organized. The zona
pellucida is a glycoprotein coat surrounding the oocyte. Changes to
this layer, triggered by the action of a single sperm binding to its’
receptor, induce the cortical granules to release their contents. The
enzymes released from the cortical granules act to crosslink the
glycoproteins of the zona pellucida, thus rendering it impenetrable
to further sperm [32]. Cryopreservation has been shown to result
in the premature release of the contents of cortical granules, thus
creating a block to sperm penetration [33]. CPAs themselves can
cause a transient increase in intracellular calcium similar to the
initial increase caused by sperm entry and subsequent induction
of zona hardening [34]. Conversely cryopreservation can lead to
physical damage to the zona pellucida resulting in multiple sperm
entry. Both of these problems are overcome by application of the
intracytoplasmic sperm injection (ICSI) technique, whereby a single sperm is injected into the oocyte to achieve fertilization. The
microtubular spindle is the structure upon which the condensed
chromosomes are aligned in mature oocytes and is responsible for
the movement of chromosomes during cell division. Damage to
this structure can lead to aneuploidy after fertilization. During
cooling the microtubular spindle has been shown to disassemble,
although there is growing evidence that the spindle is capable of
repair on warming and post-thaw culture [35–37]. One way of
avoiding the potential for damage to the microtubular spindle is
Cryopreservation of Oocytes
439
within applied cryobiology. Originally, the early reports of oocyte
cryopreservation in both human and animal oocytes were made
using CRSC [13–20]. Since then pregnancies and live births resulting from cryopreserved oocytes have been reported in a number of
species [21–24] and human [25]. Clinical programmes were developed in the early 2000s using CRSC with optimized CPA mixtures
[26, 27]. At the same time VS techniques were becoming optimized [4, 28]. It became apparent that experienced practitioners
could recover higher percentages of oocyte from cryogenic storage
using VS methods [29–31].
Historically, oocytes have been considered to be difficult cells
to cryopreserve for a number of reasons. The oocyte is a large single
cell with relatively low permeability to water and cryoprotective
agents (CPA). This means that oocytes have a tendency to retain
water as ice starts to form in the suspending medium during cooling and, if this leads to formation of intracellular ice as cooling
proceeds, damage to the cell results. Permeability of oocytes varies
between species, strains and maturational status of the oocyte. The
mature oocyte is also a short-lived cell that must undergo fertilization in order for it to continue to survive and develop. For fertilization to occur naturally, the oocyte must retain integrity of a
number of its’ unique structural features. These include the surrounding zona pellucida, the cortical granules, and the microtubular spindle on which the chromosomes are organized. The zona
pellucida is a glycoprotein coat surrounding the oocyte. Changes to
this layer, triggered by the action of a single sperm binding to its’
receptor, induce the cortical granules to release their contents. The
enzymes released from the cortical granules act to crosslink the
glycoproteins of the zona pellucida, thus rendering it impenetrable
to further sperm [32]. Cryopreservation has been shown to result
in the premature release of the contents of cortical granules, thus
creating a block to sperm penetration [33]. CPAs themselves can
cause a transient increase in intracellular calcium similar to the
initial increase caused by sperm entry and subsequent induction
of zona hardening [34]. Conversely cryopreservation can lead to
physical damage to the zona pellucida resulting in multiple sperm
entry. Both of these problems are overcome by application of the
intracytoplasmic sperm injection (ICSI) technique, whereby a single sperm is injected into the oocyte to achieve fertilization. The
microtubular spindle is the structure upon which the condensed
chromosomes are aligned in mature oocytes and is responsible for
the movement of chromosomes during cell division. Damage to
this structure can lead to aneuploidy after fertilization. During
cooling the microtubular spindle has been shown to disassemble,
although there is growing evidence that the spindle is capable of
repair on warming and post-thaw culture [35–37]. One way of
avoiding the potential for damage to the microtubular spindle is
Cryopreservation of Oocytes
439