to cryopreserve the immature oocyte before the spindle is formed,
when the chromosomes are contained within the germinal vesicle
and the oocyte is termed immature [38]. However, success following cryopreservation of immature oocytes is less than that following
cryopreservation of mature oocytes [39]. If protocols for hormonal
stimulation of ovaries to induce release of numerous mature
oocytes are not available or not easily applicable, for example, in
patients with polycystic ovary syndrome or in cancer patients, when
harvesting of ovarian tissue is the only option for fertility preservation, then only immature oocytes, contained within the ovaries,
may be available for storage. The major problem with cryopreserving immature oocytes is that they must be matured in vitro in
order to become fertilizable. This maturation involves communication with the cumulus cells that surround the oocyte [40]. The
cumulus cells are much smaller than the oocyte and are connected
with it by numerous gap junctions. Development of cryopreservation protocols that allow survival of the oocyte, the cumulus cells,
and connections between the two is difficult [41]. However, it was
shown that preservation of this cell-to-cell connection can be
achieved even within such a complex ovarian tissue when the
method of VS was applied [42]. Also, techniques for maturation
in vitro require further refinement in most species. In vitro maturation of oocytes prior to cryopreservation may produce poor quality
oocytes that are more prone to damage during cryopreservation
[43]. As well as these general problems, the oocytes of some species
have further characteristics that make them still more difficult to
freeze; for example, porcine oocytes have a high lipid content and
are sensitive to chilling [44].
The protocols used for the cryopreservation of unfertilized
oocytes have largely been adopted from embryo cryopreservation
techniques. As with embryo cryopreservation, both CRSC and VS
techniques have been successfully applied. Murine oocytes have
been successfully cryopreserved using a range of CPA such as
glycerol or dimethyl sulfoxide, whilst the technique most commonly applied to the controlled rate cryopreservation of human
oocytes is that of slow cooling in the presence of propanediol and
sucrose. This technique, combined with ICSI, first yielded a live
birth from cryopreserved human oocytes in 1997 [25]. Since then,
the number of human live births has been increasing steadily, with
live births escalating into hundreds [36, 45]. VS has also produced
accumulating numbers of human live births [46, 47], with more
than 250 recorded by 2009 [48] and coming to more than
thousands babies born till 2016–2017 [49–52], following significant improvement of oocyte VS by the Cryotop method
[53, 54]. Combination of (i) stepwise equilibration at the room
temperature in increasing concentrations of permeating CPA with
addition of non-permeating agents to the VS solution (minimising
the toxic effect by using a mixture of CPAs and equilibration at
440
Victoria Keros and Barry J. Fuller
when the chromosomes are contained within the germinal vesicle
and the oocyte is termed immature [38]. However, success following cryopreservation of immature oocytes is less than that following
cryopreservation of mature oocytes [39]. If protocols for hormonal
stimulation of ovaries to induce release of numerous mature
oocytes are not available or not easily applicable, for example, in
patients with polycystic ovary syndrome or in cancer patients, when
harvesting of ovarian tissue is the only option for fertility preservation, then only immature oocytes, contained within the ovaries,
may be available for storage. The major problem with cryopreserving immature oocytes is that they must be matured in vitro in
order to become fertilizable. This maturation involves communication with the cumulus cells that surround the oocyte [40]. The
cumulus cells are much smaller than the oocyte and are connected
with it by numerous gap junctions. Development of cryopreservation protocols that allow survival of the oocyte, the cumulus cells,
and connections between the two is difficult [41]. However, it was
shown that preservation of this cell-to-cell connection can be
achieved even within such a complex ovarian tissue when the
method of VS was applied [42]. Also, techniques for maturation
in vitro require further refinement in most species. In vitro maturation of oocytes prior to cryopreservation may produce poor quality
oocytes that are more prone to damage during cryopreservation
[43]. As well as these general problems, the oocytes of some species
have further characteristics that make them still more difficult to
freeze; for example, porcine oocytes have a high lipid content and
are sensitive to chilling [44].
The protocols used for the cryopreservation of unfertilized
oocytes have largely been adopted from embryo cryopreservation
techniques. As with embryo cryopreservation, both CRSC and VS
techniques have been successfully applied. Murine oocytes have
been successfully cryopreserved using a range of CPA such as
glycerol or dimethyl sulfoxide, whilst the technique most commonly applied to the controlled rate cryopreservation of human
oocytes is that of slow cooling in the presence of propanediol and
sucrose. This technique, combined with ICSI, first yielded a live
birth from cryopreserved human oocytes in 1997 [25]. Since then,
the number of human live births has been increasing steadily, with
live births escalating into hundreds [36, 45]. VS has also produced
accumulating numbers of human live births [46, 47], with more
than 250 recorded by 2009 [48] and coming to more than
thousands babies born till 2016–2017 [49–52], following significant improvement of oocyte VS by the Cryotop method
[53, 54]. Combination of (i) stepwise equilibration at the room
temperature in increasing concentrations of permeating CPA with
addition of non-permeating agents to the VS solution (minimising
the toxic effect by using a mixture of CPAs and equilibration at
440
Victoria Keros and Barry J. Fuller