Vitrification as a tool to preserve women fertility in cancer patients

1
Vitrification as a tool to preserve women
fertility in cancer patients

• Safaa Al-Hasani
• Department of Gynecology and Obstetrics
• Reproductive Medicine
• University of Schleswig-Holstein, Campus Lübeck
• Germany

2
J. Liebermann, 2009
3

• Woldwide 4.0 millions children born through ART
• 20 of the children born through cryopreservation
  procedure

4
Introduction

• During the last decades there have been
  tremendous improvements in cancer treatment with
  the survival rates for most types of cancer
  increasing considerably.
• The dramatic increase in survival after cancer
  treatment is indeed true for the types of cancer
  that girls and young females may acquire.

5
Introduction

• The most common cancer type in children is
  leukemia followed by cancer of the central
  nervous system, neuroblastoma, Hodgkins,
  non-Hodgkins lymphoma, and Wilms tumour.

6
Introduction

• The most common type of malignancy among females
  during the reproductive age is breast cancer and
  64 of the patients are younger than 40 years
  old.
• It was also shown that the 5 year survival rates
  for breast cancer patients increased from 75 in
  the mid 1970s to 88 in the late 1990s.

7
Introduction

• Nowadays vitrification procedure showed better
  results in preservation of women fertility
  compared to the slow freezing method.

8

• In this presentation we will discuss the
  ultra-rapid method for the cryopreservation of
  human oocytes, embryos, ovarian tissue but not
  the whole ovary

9
Indications for fertility preservation

• Oncological
• Non-oncological
• Premature ovarian failure
• Autoimmune diseases
• Infections

10
Indications for fertility preservation

• Environmental factors
• Radiation
• Exposure to gonadotoxic agents
• Surgical menopause
• Women wishing to postpone motherhood

11

• Healthy delivery of a twin after transfer of
  embryos resulted from vitrified oocytes injected
  with sperm recovered from cryopreserved
  testicular tissue
• Safaa Al-Hasani
• Case report

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Cryopreservation of human Oocytes

• Slow Freezing Method
• Ultrarapid Freezing Method (Vitrification)

13
The principles of Slow Freezing procedure

• Low concentration of cryoprotectants
• Slow controlled cooling rates
• Slow process of dehaydration of the oocyte to
  reduce intracellular ice crystal formation and to
  reduce cell damage

14
Meiotic spindle

• Microtubule system chain of tubulin polymer hold
  the chromosomes in the metaphase plate
• The polymer formation is temperature dependant
• Lowering the temperature decrease polymerization
  shortening of the chain
• At 22C, the tubulin arm completely disappear
• This process is reversible

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Oocyte Freezing
16
Principles of cryopreservation

• Water in cell Around 90 of water is free
  (water) while the remaining 10 bounds to
  other molecular components of the cell (proteins,
  lipids, nucleic acids and other solutes). This
  water does not freeze and called hydrated water
• Removal of water is necessary during freezing to
  avoid ice crystal formation, dehydration is
  limited to the free water
• Removal of hydrated water could have adverse
  effect on the cell viability and the molecular
  function (freezing injuries)

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Factors causing cell injuries

• Cryopreservation involves chilling and even
  freezing or vitrifying cells in order to put
  their life on hold
• For either freezing or vitrification to maintain
  vital function of the cells, cooling warming and
  solute concentration must be managed in a way to
  favor survival and to minimize injury
• Living cells can be injured by reduction in
  temperature, by ice crystals, by osmotic forces,
  and by chemical toxicity
• All are factors related to slow freezing procedure

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Oocyte Freezing

• Experience
• A) From 1986 to 1996
• Chen, 1986
• Al-Hasani et al. 1987
• Van Uem et al. 1987
• Serafini et al. 1995

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Oocyte Freezing

• Experience
• B) from 1997
• Porcu et al., 1997
• Antinori et al., 1998
• Borini et al., 1998
• Polak de Fried et al., 1998
• Porcu et al., 1988
• Videli et al., 1998
• Yang et al., 1998
• Young et al., 1998

• Porcu et al., 1999
• Porcu et al., 1999a
• Porcu et al., 1999b
• Yang et al., 1999
• Porcu et al., 2000
• Fabbri et al., 2001
• Porcu et al., 2002
• Yang et al., 2002

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Oocyte Freezing

• Factors that have improved results
• The use of mature eggs
• Cryoprotective solutions
• Freezing speed slow
• Thawing speed rapid
• ICSI
• Vitrification

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Pregnancies and births from frozen human
oocytes (slow cooling)

• Authors
  Year Cryoprotectant Oocyte
  stage Pregnancies/Births

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Oocyte Freezing

• Pregnancies per cycle
• With frozen eggs 17.2
• With frozen embryos 18.7
• Porcu et al., 2002

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Vitrification of human oocytes and embryos
24

• In 1937, Luyet wrote that crystallization is
  incompatible with living systems and should be
  avoided whenever possible

Luyet. Biodynamica 1937 1 114
25
Historical review

• It was described at the end of the 18th Century
• Tammann, 1898
• Vitrification of mouse embryos at 196C
• Rall Fahy, 1985 Ali Shelton, 1993
• Blastocyst development from bovine oocytes
• Martino et al., 1996
• Blastocyst development, pregnancies, deliveries
  from human vitrified oocytes, zygotes, cleaved
  eggs and blastocyst

Tammann. Z Phys Chem 1898 25 441-479 Rall
Fahy. Nature 1985 313 (6003) 573575 Ali
Shelton. J Reprod Fertil 1993 98 (2)
459465 Martino et al. Biol Reprod 1996 54 (5)
10591069
26
Vitrification Two droplets of different
solutions plunged directly into liquid
nitrogen left droplet is pure Dulbeccos
phosphate-buffered saline (DPBS) with ice
crystallization, in contrast to the right droplet
containing an equimolar combination of 20
ethylene glycol and dimethyl sulphoxide with 0.4
M sucrose in DPBS without ice crystallization
(glassy, vitrified state).
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Vitrification

• Successful vitrification requires
• Minimum volume of holding media (lt1ml)
• Increased viscosity
• High CPAs concentration
• High cooling rate (-50 000 C and warming rate
  (36 000 C)
• Special vehicle device (carrier) (Cryotops)
• Direct plunging in LN2

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Risk of Contamination of Germplasm during
Cryopreservation and Cryobanking in IVF
UnitsBielanski and Vajta 2009
-It has to be stated that none of the reported infections after insemination or embryo transfer in humans and demostic animals can be clearly attributed to the applied cryopreservation and storage procedure. -To ensure rapid cooling in some vitrification techniques requiring direct contact, sterile LN2 should be used, then samples should be safely sealed into pre-cooled secondary containers. Human Reprod. 24, 2457-2467

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Shortly before loading on Cryotop
30
Cryotop (Kuwayama)
31
Risk of Contamination of Germplasm during
Cryopreservation and Cryobanking in IVF
UnitsBielanski and Vajta 2009
-Unfortunately, the open system and direct contact might be indispensable to achieve the required cooling and warming rates for every sensitive samples so far, commercially available closed analogues (Cryotips, CBS and SSV analogues) have failed to demonstrate the same efficiency for human oocytes when compared with their counterparts using the direct contact approach. Human Reprod. 24, 2457-2467

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Slow cooling (langsames
Einfrieren)
Vitrifikation
(ultra-rapides Einfrieren)
- 0.3C/min
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Example of cooling rates

• -2500C/min by using 0.25 mL straws
• thick straws and large volumes of medium do not
  allow a high cooling rate and thawing rate
• -25.000 -50.000C/min by using a carrier that
  allows very small volumes
• direct contact with LN2

34

• The physical definition of vitrification is the
  solidification of a solution (water is rapidly
  cooled and formed into a glassy, vitrified state
  from the liquid phase) at low temperature, not by
  ice crystallization but by extreme elevation in
  viscosity during cooling
• Fahy, 1984

Fahy et al. Cryobiology 1984 21 407426
35

• In contrast to slow-rate freezing protocols,
  during vitrification the entire solution remains
  unchanged and water does not precipitate, so no
  ice crystals are formed

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Slow freezing versus ultra-rapid freezing
Traditional Vitrification
CPA concentration 1.5 M 3.05.0 M
Volume 0.31.0 mL lt1 µL
Contact between N2 and cell No Yes
Cooling rate 0.5C/min 25.00050.000C/min
Freezing Slow Ultra-rapid
Thawing / warming Slow Rapid
Time consuming 180 min 1 sec
Dehydration Not controlled Controlled
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Slow freezing versus ultra-rapid freezing
Traditional Vitrification
Reduced osmotic injury No Yes
Zona pellucida fracture Possible No
Ice crystal formation Yes No
Seeding Yes No need
Procedure Complicated Simple
Device Yes No need
Costs Liquid nitrogen amount Duration out Incubator High High gt 4 Hrs Less Much less 10 min
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Author Warmed Oocytes N survived N 2 PN cleaved embryos N ET N Preg
Cha, 1999 7 7 7 - - 1
Kuleshova, 1999 17 11 5 3 3 1
Kuwayama, 2000 - - 2 2 1 1
Yoon, 2000 90 57 39 32 7 3
Yoon, 2003 474 325 142 125 28 6
Katayama, 2003 46 42 38 34 6 2
Kim, 2005 186 139 58 39 10 4
Kim, 2005 233 165 77 49 12 7
Chian, 2005 180 169 126 54 15 7
Ruvalcaba, 2005 60 46 38 34 10 8
Kyono, 2005 5 5 5 1 1 1
Kuwayama 2005 107 86 77 64 29 12
Lucena, 2006 159 120 105 97 23 13
Selman, 2006 24 18 14 14 6 2
Total 1588 1190 (75) 733 (61.6) 548 (74.7) 151 68 (45)
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Vitrification
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Vitrification of Human Oocytes
Kuwayama 2005 Kuwayama 2005
Vitrified oocytes 64
Survived oocytes after warming 58(91)
Fertilized oocytes 52(89.6)
Embryo Transfer 29
N of clinical pregnancy 12(41)
Deliveries 7
Ongoing pregnancies 3
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Before Vitrification
2hrs after Culture
Just after Thawing
4-cell stage (Day2)
Blastocyst (Day5)
PN stage (Day 1)
Figs. 5 Human oocytes before and after
vitrification, ICSI and IVC.
42
Antinori et al. Reprod Biomed Online 2007 14
(1) 72-79
Vitrified / warmed group Update up to July 2007
No. of cycles 120 270
No. of warmed oocytes 330 707
No. of oocytes survived () 328 (99.3) 699 (98.8)
No. of injected oocytes 328 699
No. of fertilised oocytes (2PN) 305 (92.9) 639 (91.4)
No. of cleaved oocytes 295 (96.7) 624 (97.6)
43
Antinori et al. Reprod Biomed Online 2007 14
(1) 72-79
Vitrified / warmed group Update up to July 2007
No. of transfers 120 270
No. of transferred embryo 295 624
No. of embryos per transfer 2.45 2.31
No. of clinical pregnancies 39 (32.5) 76 (28.1), 5 twins
No. of ongoing pregnancies 24
No. of abortions 8 (20.5) 16 (21)
No. of deliveries 31 36, all singleton
IR per transferred embryo 13.2 12.9
IR per thawed oocyte 11.8 11.6
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Embryo Development of Fresh Versus Vitrified
Metaphase II Oocytes after ICSI A Prospective
Randomised Sibling-Oocyte Study

• Conclussion Our results indicate that oocyte
  vitrification procedure followed by ICSI is not
  inferior to fresh insemination procedure, with
  regard to fertilization and embryo developmental
  rates. Moreover, ongoing clinical pregnancy is
  comparable with this procedure, even with a
  restricted number of oocytes available for
  inseminat-ion. We believe that these results will
  help the spread of vitrification for human
  oocytes cryopreservation.The promising clinical
  results obtained, in a population of infertile
  patients, need to be confirmed on a larger scale.
• 
  
• 
  
  Rienzi et al., 2010
• 
  Human
  Reprod., 25, 66-73

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Oocyte Donation Vitrification
Vitrified
M II oocytes 231
Survival 96.9
Fertilization 76.3
No. of transfers 23
Mean number of embryos 2.1
Ongoing pregnancy rate 48
Cobo et al., 2008
46
Obsteric and perinatal outcome in 200 infants
conceived from vitrified oocytes

• Statement These preliminary findings may provide
  reassuring evidence that pregnancies and infants
  conceived following oocyte vitrification are not
  associated with increased risk of adverse
  obstetric and perinatal outcomes.
• Chian et al,
  RBM online 16, May 2008

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Comparison between fresh and frozen-thawed embryo
transferVitrification of Zygotes (Luebeck)
Fresh ET Frozen-Thawed ET
No of patients 52 59
No of cycles 53 61
No of vitrified Zygotes / 259
No of survived zygotes / 250 (96.5)
No of transferred embryos 114 (2.5) 240 (2.6)
No of embryo transfers 53 83
No of pregancies 13 (24.5) 29 (34.1)
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Our Results in Avoiding Hyperstimulation
Patients Triggered with GnRH-Agonist
No. of Patients No. of Zygotes vitrif. No. of Zygotes re-warmed No. () Zygotes survived No. () Preg. No. Of Children born () of live birth
59 433 163 158 (97) 25 (42) 13 (25)
No. of Patients received warmed Zygotes 45
Two Twins
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The blastocyst is characterized by early
cavitation resulting in the formation of an
eccentric and then expanded cavity lined by a
distinct inner cell mass region and trophectoderm
layer. The blastocele is less than half the
volume of the embryo
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Can fresh embryo transfers be replaced by
cryo-preserved-thawed embryo transfers in
assisted reproductive cycles? A prospective
controlled trial.
Fresh ET (n191) FET (n184) p value
No. of oocytes retrievd 14.2 14 NS
No. Of M II oocytes retrieved 11 10.8 NS
E2 day of hCG (pg/ml) 2861.2 2793.4 NS
Fertilization rate 72.7 73 NS
No. of embryos transferred 2.2 0.4 2.1 0.3 NS
Implantation rate () 14.1 23.0 0.004
Clinical pregnancy rate () 24.6 36.4 0.013
Ongoing pregnancy rate () 22.5 34.2 0.012
Multiple pregnancy rate () 14.9 26.4 NS
Aflatoonian et al. 2009, Human Reprod. (submitted)
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Vitrification of human 8-cell embryos, a modified
protocol for better pregnancy rates Rama Raju et
al. (2005)
Vitrification Slow freezing
Embryos, n 436 420
Embryos thawed, n 127 120
Embryos survival, n () 121 (95.3) 72 (60)
Pregnancy, n () 14 (35) 4 (17.4)
40 ethylene glycol 0.6 mol sucrose, nylon
loop Ethylene glycol is a good croyprotectant
to preserve 8-cell embryos because of its low
toxicity as shown by the high survival rate, and
vitrification is a promising alternate to the
conventional slow-freezing method.
Rama Raju et al. Reprod Biomed Online 2005 11
(4) 434437
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Outcome of Blastocyst Cryopreservation by using
the Slow Freezing Method
Reference No. of thawed blastocysts Survival rate Implantation rate Pregnancy rate
Troup et al. 1990 34 38 (13/34) NA no pregnancy
Menezo et al. 1992 106 NA NA 21.0
Kaufmann et al. 1995 1239 83 (1033/1239) 13.4 21.7
Nakayama et al. 1995 69 78 (54/69) 18.8 1.7
Martin et al. 2003 624 86 23.3 30.6
Andersen et al. 2004 202 81 (164/202) 43.0 69.0
Veeck et al. 2004 628 76 (479/628) NA 59.2
Kuwayama et al. 2005 156 84 (131/156) NA 51.0
Stehlik et al. 2005 71 83 (59/71) 6.8 16.7
Liebermann Tucker 2006 254 91 29.6 42.8
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Outcome of Human Blastocyst Vitrification by
using the Hemi-straw Method
Reference No. of vitrif. blastocysts Survival rate Implantation rate Pregnancy rate
Vanderzwalmen et al. 2002 167 58.5 NA 20.5
Vanderzwalmen et al. 2003 281 60 NA 27
Zech et al. 2005 177 82 NA 35
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Outcome of Blastocyst Vitrification by using the
Cryoloop Method
Reference No. of vitrif. blastocysts Survival rate Implantation rate Pregnancy rate
Mukaida et al. 2001 60 63 NA 31.5
Reed et al. 2002 15 100 15.4 25
Mukaida et al. 2003b 725 87 NA 37
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Outcome of Blastocyst Vitrification by using the
Cryotop Method
Reference No. of vitrif. blastocysts Survival rate Implantation rate Pregnancy rate
Hiraoka et al. 2003 49 98 33 50
Stehlik et al. 2005 41 100 NA 50
Kuwayama et al. 2005 6484 90 NA 53
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Post-thaw survival rates of human zygotes,
embryos and blastocysts after vitrification
n5881
n897
n1175
57
Vitrification demonstrates significant
improvement versus slow freezing of human
blastocystsStehlik et al. (2005)
Day 5 Day 5 Day 6 Day 6
Slow freezing Vitrification Slow freezing Vitrification
Embryos transferred, No. 24 20 27 15
Embryos survival, 83 100 89.5 100
Pregnancy, 16.7 50 18.5 33.3

• Day 5 vitrified blastocysts showed significantly
  increased survival and pregnancy rates compared
  with Day 5 slow-frozen blastocysts
• A similar trend was observed with Day 6
  blastocysts

Stehlik et al. Reprod Biomed Online 2005 11 (1)
5357
58
Comparison of vitrification and conventional
cryopreservation of Day 5 and Day 6 blastocysts
during clinical application Liebermann Tucker
(2006)
Vitrification Conventional
Blastocysts warmed, n 547 570
Blastocysts survival, n () 528 (96.5) 525 (92)
Blastocysts transferred, n 523 518
Implantation, n () 160 (30.6) 152 (28)
Ongoing pregnancies, n () 117 (88.6) 109 (79.6)

• Vitrification technique yields the same
  implantation and pregnancy rate as slow-frozen
  blastocyst transfer

Liebermann Tucker. Fertil Steril 2006 86 (1)
2026
59
Comparison of vitrification and conventional
cryopreservation of Day 5 and Day 6 blastocysts
during clinical application Liebermann Tucker
(2006)

• We believe that vitrification shows much
  promise as a successful alternative to
  conventional freezing technology.
• Even without significant clinical improvement,
  the evident advantages of vitrification are that
• Cryosurvival seems more consistent, allowing
  greater case of patient management, with
  transfers being almost certain to occur
• Vitrification is able to be undertaken on a more
  flexible basis by laboratory staff

Liebermann Tucker. Fertil Steril 2006 86 (1)
2026
60
Comparison of vitrification and conventional
cryopreservation of Day 5 and Day 6 blastocysts
during clinical application Liebermann Tucker
(2006)

• ...and
• Vitrification allows for the potential reduction
  in personnel time needed during the entire
  vitrification process
• It may enable more optimal timing of embryo
  cryopreservation, e.g., individual blastocysts
  may be cryopreserved at their optimal stage of
  development and expansion

Liebermann Tucker. Fertil Steril 2006 86 (1)
2026
61
In the future Cryopreservation of Ovarian
Cortex

• ovarian biopsy and cryopreservation
• in vitro Growth (IVG) followed by In vitro
  Maturation (IVM)

Oktay et al. 2004
62

• Woldwide 10-12 children born through
  retransplantation of ovarian cortex
• The number of unsuccessful traials is unknown !!

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Successful vitrification of bovine and human
ovarian tissueKagawa et al., RBMOnline 2009
Vitrifcation procedure the Cryotissue method.
The ovarian tissue slicer was developed, with a
plate to produce 1 10 10 mm slices. (1) The
tissue slicer was put on the surface of ovary.
(2) Then another plate was put on the tissue
slicer, the ovary was cut between the slicer and
the surface of ovary by using a sharp edge. (3)
The ovarian tissue was cut into 1 10 10 mm
slices.
65
Successful vitrification of bovine and human
ovarian tissueKagawa et al., RBMOnline 2009
Gross morphology of vitrifed human ovarian
tissue using the Cryotissue method. Vitrifed
human ovarian tissue was translucent in liquid
nitrogen (196C). Scale bar represents 10 mm.
66
Successful vitrification of bovine and human
ovarian tissueKagawa et al., RBMOnline 2009
All oocytes (arrows) were located in the cortical
area of the human ovarian tissue. Note that they
were all located within 0.75 mm of the surface,
allowing much thinner slices to be made than can
be obtained by hand, or than have been used in
previous studies. Scale bar represents 1 µm.
67
Successful vitrification of bovine and human
ovarian tissueKagawa et al., RBMOnline 2009
Morphologically normal oocyte in a pre-antral
follicle from vitrifed-warmed human ovarian
tissue. (A) Normal oocyte was surrounded by one
or two layers of somatic cells in normal
interstitial tissue of vitrifed ovarian tissue.
Haematoxylineosin staining. (B) Normal oocyte
was surrounded by three or four layers of somatic
cells in normal interstitial tissue of vitrifed
ovarian tissue. Scale bar represents 50 µm.
68
Successful vitrification of bovine and human
ovarian tissueKagawa et al., RBMOnline 2009
Histological section of vitrifed human ovarian
tissue. Immunohistochemical staining for
proliferating cell nuclear antigen (PCNA), a
marker protein for proliferating cells. Abundant
PCNA-positive proliferating cells were
demonstrated in the interstitial tissue cells and
a few proliferating cells were noted in granulose
cells (arrows) of vitrifed human ovarian tissue.
Scale bar represents 50 µm.
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Successful vitrification of bovine and human
ovarian tissueKagawa et al., RBMOnline 2009
Surviving oocytes (arrows) of pre-antral
follicles of vitrifedthawed ovarian tissue in
human (Hoechst/propidium iodide stain). Nuclei
of living oocytes were blue. Scale bar
represents 50 µm.
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Successful vitrification of bovine and human
ovarian tissueKagawa et al., RBMOnline 2009
Survival of oocytes in vitrified-warmed human
ovarian tissue
No. of samples Number of Oocytes () Number of Oocytes ()
No. of samples Collected Surviving
Vitrified 7 954 (100) 855 (89.6)
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Successful vitrification of bovine and human
ovarian tissueKagawa et al., RBMOnline 2009

• Abstract
• In addition, human ovarian tissue from
  cancer patients, and from ovary transplant
  donors was
• also vitrifed by the Cryotissue method. After
  warming, high oocyte survival in human tissue
  (similar to bovine tissue) was
• obtained. These results indicate that an
  ultra-rapid cooling vitrifcation method has the
  potential for clinical use in human
• ovarian tissue cryopreservation.

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Successful vitrification

• High cooling rate (gt 50.000C)
• Fast cooling period (lt1 sec.)
• Low volume (lt1 µL)
• High concentration of cryoprotectants
• gt This will avoid crystal formation

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Why do we prefer the vitrification procedure now?

• No mechanical injury (extracellular crystal
  formation)
• Less osmotic stress to cells
• No intracellular crystal formation
• Less labour in laboratory daily work
• Simple protocol
• Useful for oocytes and blastocysts, which have
  less success with slow freezing
• No need for expensive devices

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Future Aspects

• Avoiding hyperstimulation syndrome in patients
  with PCOS by vitrification of all 2PN and
  replaced in a programmed cycle
• Cancelling of fresh ET in case of more than 10
  Follicles
• Vitrification of all zygotes resulted from IVM
  programme
• An option for cancer patients to vitrify the
  oocytes instead of ovarian tissue
• In oocytes donation programme
• Vitrification of the oocytes to postpone
  fertility
• Mantains viability of specimens during long term
  storage

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• Quality is evolution
• Vitrification is a revolution
• Professor Josiane Van der Elst
• 19 January 2007

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Conclusion

• Easy to perform
• Low cost
• Future first choice procedure
• It was shown to be superior to slow freezing
  procedure
• Very high survival rates of oocytes and embryos
  at all stages of development
• It seems that the cryotop method is the most
  efficient procedure
• Revitrification is possible
• Ovarian Cortex is now also possible

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Thank you for your attention!