Vitrification

Report
Page 1
Effect of cumulus cells and vitrification protocol
on survival and subsequent development
Golestan jahromi
PhD student
Introduction
Several lines of evidence indicate that
surrounding cumulus cells play a fundamental
role in the maturation process and full
development competence
Introduction
cumulus cells are beneficial to oocyte survival
after cryopreservation
may minimize the release of cortical granules
prevent premature zona hardening
maintaining fertilization capacity of
cryopreserved oocytes
Introduction
Chian et al. reported that bovine oocytes
matured without cumulus cells had a higher
survival rate after vitrification.
Moreover, the rate of embryo development to
the 8-cell stage in cumulus-cells free oocytes
was significantly higher than that of cumulus
cell-intact oocytes.
Introduction
Ice growth and recrystallization are considered
to be important factors in determining
vitrification outcomes.
Synthetic ice blockers, which specifically inhibit
the formation/emergence of ice nuclei and ice
crystal growth, have recently been used to
supplement vitrification solutions
Introduction
Unlike conventional cryoprotectants that inhibit
freezing by interacting with water, ice blockers are
believed to bind to the surface of growing ice
crystals and inhibit the addition of any further
water molecules in specific planes of growth
This selective attraction to surfaces of ice growth
permits ice blockers to exert significant effects even
while present at very low concentrations.
Introduction
Small quantities of ice blocker can therefore
modify the number and size of ice crystals and
thereby change the vitrification tendency of a
solution without adding additional toxicity
Introduction
The commercially available ice blockers are
SuperCool X-1000 and SuperCool Z-1000.
To evaluate the effect of the presence of
cumulus cells on the outcome of vitrification of
GV or MII oocytes
The effect of adding ice blockers SuperCool X1000 and SuperCool Z-1000 to vitrification
media on oocyte survival and subsequent
embryonic development
IVF
Collection of
oocytes
IVM
warming
Vitrification
According to the manufacturer, the cooling and
warming rates of the Cryotop are 23,000 and
42,000 °C/min, respectively
Motile spermatozoa were obtained by
centrifugation of frozen–thawed semen
Day 2
after insemination
Cleavage rates
Day 8 after
insemination
proportion of oocytes
developing to the blastocyst stage
Experiment 1
Cumulusenclosed
GV
COCs
partiallydenuded
oocytes
Cumulusenclosed
MII
partiallydenuded
oocytes
Experiment 2: Effect of ice blocker X-1000
and Z-1000
COCs
Control
Basic media
Basic media +
X-1000
VS +1% (v/v) X1000
Basic media +
Z-1000
VS +1% (v/v)
Z-1000
Basic media +
X-1000 + Z1000
VS +1% (v/v)
Z-1000 and 1%
(v/v) X-1000
Statistical analysis
The data for survival, cleavage and blastocyst
rates were expressed as mean ± SD and
analyzed using one-way ANOVA.
Differences were considered significant at a
level of P < 0.05.
development of bovine GV stage
Oocytes
treated
N
Survived, n
(%)
Cleaved, n
(%)
Blastocyst, n Blastocyst/cl
(%)
eavage (%)
Cumulusenclosed
control
141
141
(100 ± 0.0)
121
(86.3 ± 1.9)
47
(33.8 ± 1.8)
(39.5 ± 2.2)
Partiallydenuded
control
118
117
(99.3 ±0.8)
89
(75.8 ± 3.9)
14
(11.5 ± 4.2)
(14.8 ± 5.2)
Cumulusenclosed
vitrified
177
166
(93.8 ± 2.5)
108
(65.8 ± 5.6)
19
(11.3 ± 1.7)
(18.0 ± 3.5)b
Partiallydenuded
vitrified
143
117
(81.3 ± 3.6)c
56
(47.3 ±
4.0)c
4
(4.0 ± 2.3)c
(7.8 ± 4.5)b
embryo development of bovine MII stage
Oocytes
treated
N
Survived, n
(%)
Cleaved, n
(%)
Blastocyst, n (%)
115
45 (35.6 ± 2.8)a
(91.6 ± 2.5)a
Blastocyst/cl
eavage (%)
Cumulusenclosed
control
130
126
(96.8 ± 1.5)a
(39.0 ± 4.1)a
Partiallydenuded
control
122
119
92
32 (27.2 ± 2.3)b
(97.4 ± 1.1)a (78.4 ± 4.7)b
(34.6 ± 2.7)
a,b
Cumulusenclosed
vitrified
158
147
(93.0 ± 2.3)a
51
7 (5.0 ± 4.3)c
(35.2 ± 4.6)c
(12.6 ± 9.7)
b,c
Partiallydenuded
vitrified
167
153
(91.8 ± 2.4)a
57
7 (4.4 ± 1.4)c
(36.8 ± 3.2)c
(10.8 ± 3.5)c
development of bovine GV stage cumulus-enclosed oocytes
vitrified with different ice blocker media
Oocytes treated
N
Survived, n
(%)
Cleaved, n (%) Blastocyst, n
(%)
Blastocyst/cle
avage (%)
Control
110
104
(94.4 ± 1.9)a
77
(74.6 ± 3.2)
24
(23.0 ± 10.4)
(31.2 ± 1.9)
Basic media
114
98
(86.0 ± 2.7)b
38
(38.0 ± 3.2)
2 (2.0 ± 1.3)
(6.2 ± 4.1)
Basic media +
X-1000
111
97
(88.4 ±2.9)
36
(37.2 ± 2.4)
3 (2.8 ± 1.2)
(7.8 ± 3.2)
Basic media
+ Z-1000
110
93
(83.6 ± 4.8)
37
(40.0 ± 3.0)
2(2.2 ± 1.4)
(4.8 ± 3.0)
Basic media
+ X-1000
+ Z-1000
114
101
(88.0 ± 3.6)
43
(41.4 ± 6.7)
0 (0)
(0)
Cumulus-enclosed oocytes vitrified at the GV
stage exhibited a significantly higher cleavage
rate and blastocyst rate than those vitrified at
MII stage (P < 0.05).
Discussion
The role of the cumulus cells during vitrification of
MII oocytes remains controversial.
Some investigators reported that cumulus presence
would protect MII oocytes against vitrificationinduced damage.
Discussion
Zhang et al. found no difference in the
development of vitrified ovine MII oocytes with
or without cumulus cells.
Gasparrini et al. reported that the presence of
cumulus cells severely reduced the cleavage rate
of MII buffalo oocytes following vitrification
Discussion
It is generally accepted that cumulus-oocyte
communication via an intact corona radiata is
necessary for oocytes to attain full
cytoplasmic maturation during IVM and
improve fertilization rates during IVF
Discussion
we established that the cleavage rate of
denuded (GV and MII) bovine oocytes was
significantly reduced compared to cumulusenclosed oocytes, and almost no denuded
bovine oocytes developed up to blastocyst
stage after in vitro fertilization.
In this study:
The survival, cleavage and blastocyst rate of
cumulus-enclosed vitrified oocytes are
significantly higher than that of partially-denuded
vitrified oocytes.
Discussion
In the present study:
No significant differences were detected
between vitrified cumulus-enclosed and
partially-denuded oocytes in the survival,
cleavage and blastocyst rate.
Discussion
The possible explanation is the cumulus was
detrimental to vitrification, which comprises the
benefits of cumulus in IVF procedure.
From another point of view, the
intracytoplasmic sperm injection technique
rather than conventional IVF has been used to
achieve fertilization, which can circumvent the
detrimental effects of removing the cumulus on
subsequent zona penetrability.
Discussion
The cell cycle stage during meiosis appears to
affect the results of bovine oocyte vitrification
due to varying sensitivity to cooling procedures.
Chilling injury is reported to be higher in vitrified
immature oocytes, owing to low membrane
stability and susceptibility of the cytoskeleton
Discussion
However, an increase in chromosomal
abnormality has been observed in vitrified
mature oocytes, owing to alterations in the
meiotic spindle.
Discussion
The results indicate that cumulus-enclosed
oocytes vitrified at the GV stage exhibited a
significantly higher cleavage and blastocyst rate
than those vitrified at MII.
Discussion
This may be due to the increase in volume
associated with cumulus expansion during
maturation.
It may also be due to the higher water permeability
(Lp) and solute permeability (Ps) of MII than GV
bovine oocytes.
That means the changes of cell volume and
intracellular CPA concentrations are more severe in
MII than GV bovine oocytes during CPA addition and
dilution process, which make it more sensitive.
Discussion
In the present study, we report for the first time
the effect of ice blockers on the bovine oocytes,
however, the results indicate that the survival
rate and development competence of bovine
oocytes vitrified in solutions supplemented with
or without X-1000 and/or Z-1000 by Cryotop
method are not significantly different.
Discussion
Ice blockers did not affect the survival rate and
developmental competence of vitrified bovine
oocytes.
Discussion
When the vitrification systems are large volume,
such as organs, in which a large quantity of
nucleators exit, ice blockers can suppress
nucleation and recrystallization by binding to
nucleators in solutions during vitrification and
warming. Therefore, the ice growth was
inhibited and damage to the systems was
reduced.
Discussion
In this study, the combination of the two ice
blocker agents inhibit blastocyst development
maybe because 1% X-1000 and 1% Z-1000 is not
the ideal balance, which shows less effective
than either agent alone.
Conclusion
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