Fertility cryopreservation with oocyte vitrification

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Fertility cryopreservation with oocyte
vitrification
Ri-Cheng Chian, Ph.D. McGill Reproductive
Center McGill University Health Center Department
of Obstetrics and Gynecology McGill University,
Montreal Canada
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Female fertility using cryopreservation

1. Embryos - generated from IVF cycles
2. Ovarian tissues
3. Eggs

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Egg freezing

• The development of an effective egg freezing
  system will have a significant impact on clinical
  practice of reproductive medicine
• Fertility preservation for young women requiring
  sterilizing medical and surgical treatments
• Cryobanking of eggs will benefit a large
  population of single women who wish to delay
  motherhood because of personal, professional and
  financial reason

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Advantages of oocyte cryopreservation

• Women without partners
• Avoids ethical issues and legal restrictions
  related to embryo banking
• Oocyte donation
• Option for delayed motherhood

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Slow-freezing method for eggs

• The first live birth was reported by Chen (1986)
• Over two decades, very few live births were
  reported
• Survival rate after thawing was approximately
  50-55
• Token together, less than 1,000 live births have
  been reported by the conventional slow-freezing
  method
• of live births per thawed egg ranges from 1-10
  using this protocols

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Modified slow-freezing methods for eggs

• Increased sucrose concentration in suspending
  solution (0.2M or 0.3M respectively) (Yang et
  al., 1998 Fabbri et al., 2001 Chen et al.,
  2002 2005)
• Choline-based freezing medium to replace sodium
  (Stachecki et al., 1998 Quintans et al., 2002
  Boldt et al., 2003)
• The survival rate of the oocytes after thawing
  was increased to 65-70

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Vitrification method for eggs

• Kuleshova et al. (1999) Open pulled straw (Hum.
  Reprod., 14 3077-3079)
• Yoon et al. (2000 2003) Electron microscope
  grid (Fertil. Steril., 74180-181 Fertil.
  Steril., 791323-1326.)
• Katayama et al. (2003) Cryotop (Fertil. Steril.,
  80 223-224.)
• The survival rate of the oocytes after thawing
  was approximately 85-90

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What is vitrification?

• Vitrification is a process which allows glasslike
  solidication of water without ice-crystal
  formation in the living cells.

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History of vitrification

• Luyet B. (1937) Working hypotheses on the nature
  of life. Biodynamica, 11-7.
• Luyet B. (1937) The vitrification of organic
  colloids and protoplasm. Biodynamica, 17-14.
• Fahy GM. (1981) Prospect for vitrification whole
  organs. Cryobiology, 18617-625.
• Rall WF and Fahy GM. (1985) Ice-free
  cryopreservation of mouse embryos at -196C by
  vitrification. Nature, 313573-575.

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Nature of water

• The temperature below 0ºC will introduce
  formation of water ice-crystal
• Below -130ºC is the glass transition temperature
  of water

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Cryobiology

• Theoretically, if the formation of intracellular
  and extracellular ice-crystal prevented and the
  glass transition occurred, the cells will be
  survival after freezing-thawing
• However, the cells may have other injuries during
  freezing-thawing procedures

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Cryobiology (cont.)

• Chilling injury
• The temperature between 30ºC and 0ºC may
  compromise cell membrane integrity, metabolism
  and cytoskeleton
• Cryoprotectant may be required to add into
  freezing solution

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Cryoprotectants

Glycerol
Dimethylsulphoxide (DMSO)
Propylene glycerol (PROH)
Ethylene glycol (EG)
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Cryoprotectant (cont.)

• The mechanism of the protective action of
  cryoprotectants is the same, but their toxicities
  are different
• Permeation ability is different with different
  cryoprotectants and temperatures
• Therefore, the toxicity of cryoprotectants must
  be considered for freezing

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Cryoprotectant (cont.)

• There are osmotic change before and after
  freezing in cryopreservation solution
• These osmotic changes may cause the death of
  cells, normally it is referred to osmotic
  injury

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Cryoprotectant (cont.)

• Hypertonic solution is required, i.e. sucrose is
  added to prevent swelling and shrinkage of the
  cells

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Factors affect successful frozen-thawing

• Chilling injury
• Cryoprotectant (toxicity and temperature)
• Osmotic injury
• Speed of freezing and thawing

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Is it difficult to freeze oocytes?

• Mammalian oocytes have proven to be more
  difficult to cryopreserve than cleavage-stage
  embryos because it is relatively large cell and
  contains more water in the cell
• Mature oocytes with its special structures, like
  metaphase spindle is fragile for freezing

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Vitrification procedure
7.5 EGPROH
15.0EGPROH 0.5 sucrose
EM (5 min)
Loading onto McGill Cryoleaf
Plunge into LN2(-196ºC)
VM (1 min)
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Morphological change in EM and VM
1 min in EM
2 min in EM
Before in EM
3 min in EM
4 min in EM
5 min in EM
10 sec in VM
30 sec in VM
1 min in VM
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McGill Cryoleaf
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Thawing procedure
1.0M sucrose
0.5M sucrose
0.25M sucrose
Culture medium
TM (37ºC)
DM-I (3 min)
DM-II (3 min)
WM (3 min)
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Morphological changes in thawing media
0.5 min in TM
3.0 min in DM-1
3.0 min in DM-2
3.0 min in WM-1
3.0 min in WM-2
Transfer to culture
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Initial data for survival rates of human oocytes
Stage No. of oocytes Survived ()
GV 32 32 (100)
M-I 30 30 (100)
M-II 19 19 (100)
Total 81 81 (100)
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Immuno-fluorescent staining of meiotic spindles
and chromosomes
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Aneuploidy screening of mouse oocytes following
vitrification and slow-freezing
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Interpretation

• Slow-freezing of oocytes results in more spindle
  and chromosome abnormalities than vitrification
• However, incidence of aneuploidy is similar
  between vitrification and slow freezing.

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Viability and pregnancy outcome of vitrified
in-vivo oocytes following thawing and ICSI at
McGill Reproductive Center
Patients (cycles) 38
(38) Age 31.5 0.5 No. of oocytes
thawed 463 No. of oocytes survived ()
383 (82.7) No. of oocytes fertilized () 287
(74.9) No. of embryos transferred 133
(3.51.1) No. of clinical pregnancies () 17
(44.7) No. of implantation () 25 (18.8)
Chian et al (Fertil Steril., In press)
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Clinical pregnancy outcome of vitrified in-vivo
oocytes following thawing and ICSI at McGill
Reproductive Center
Patients (cycles) 38
(38) No. of clinical pregnancies () 17
(44.7) No. of live birth () 15 (39.5) No.
of miscarriages 2 No. of singleton
9 No. of Twins 5 No. of triplets
1 No. of newborn 22
Chian et al (Fertil Steril., In press)
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IVM-Vitrification trial at MRC
Patients 20
Mean age (years) 30.8 0.9
Mature oocytes retrieved 6
Immature oocytes retrieved 290
Mean oocyte maturation rate () 67.34.9
Oocytes vitrified and thawed 215
Oocytes survived (mean SEM) 148 (67.5 5.8)
Oocytes fertilized (mean SEM) 96 (64.2 4.5)
Embryos transferred (median range) 64 (4 range 1-6)
Implantation (mean SEM) 4 (9.65.4)
Pregnancies per cycle () 4 (20.0)
Clinical pregnancies per cycle () 4 (20.0)
Ongoing pregnancies () 0
Live births 4
Mean birth weight (grams) 4,049413.7
Chian et al (Fertil Steril., In press)
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Interpretation
Pregnancies conceived following oocyte
vitrification are not associated with adverse
obstetric and perinatal outcomes.
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Conclusions

• Vitrification of human oocytes is associated with
  acceptable pregnancy rate and normal obstetrical
  and neonatal outcomes
• The offspring derived from vitrified oocytes are
  healthy
• Vitrification of oocytes can be used safely for
  human reproductive medicine
• Oocyte vitrification may offer cancer patients
  for fertility preservation.

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Acknowledgements

• Staff at McGill Reproductive Center
• Dr. Ruvalcaba Castellon, L.A. at Instituto
  Mexicano de Infertilidad, Jalisco, Mexico
• Dr. Lucena, E. at CECOLFES, Bogota, Colombia.

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Thank you!
ri-cheng.chian_at_muhc.mcgill.ca