Ri-Cheng Chian, Ph.D.Ri-Cheng Chian, Ph.D.

McGill Reproductive CenterMcGill Reproductive CenterMcGill University Health CenterMcGill University Health Center

Department of Obstetrics and GynecologyDepartment of Obstetrics and GynecologyMcGill University, MontrealMcGill University, Montreal

CanadaCanada

Fertility cryopreservation with Fertility cryopreservation with oocyte vitrificationoocyte vitrification

Female fertility using cryopreservationFemale fertility using cryopreservation

1.1. Embryos - generated from IVF cycles;Embryos - generated from IVF cycles;

2.2. Ovarian tissues;Ovarian tissues;

3.3. Eggs;Eggs;

Egg freezingEgg freezing

• The development of an effective egg freezing system The development of an effective egg freezing system will have a significant impact on clinical practice of will have a significant impact on clinical practice of reproductive medicine;reproductive medicine;

• Fertility preservation for young women requiring Fertility preservation for young women requiring sterilizing medical and surgical treatments;sterilizing medical and surgical treatments;

• Cryobanking of eggs will benefit a large population Cryobanking of eggs will benefit a large population of single women who wish to delay motherhood of single women who wish to delay motherhood because of personal, professional and financial because of personal, professional and financial reason;reason;

• Women without partners;Women without partners;• Avoids ethical issues and legal Avoids ethical issues and legal

restrictions related to embryo banking;restrictions related to embryo banking;• Oocyte donation;Oocyte donation;• Option for delayed motherhood;Option for delayed motherhood;

Advantages of oocyte cryopreservationAdvantages of oocyte cryopreservation

• The first live birth was reported by Chen (1986);The first live birth was reported by Chen (1986);• Over two decades, very few live births were Over two decades, very few live births were

reported; reported; • Survival rate after thawing was approximately 50-Survival rate after thawing was approximately 50-

55%;55%;• Token together, less than 1,000 live births have Token together, less than 1,000 live births have

been reported by the conventional slow-freezing been reported by the conventional slow-freezing method;method;

• % of live births per thawed egg ranges from 1-10% % of live births per thawed egg ranges from 1-10% using this protocols;using this protocols;

Slow-freezing method for eggs:Slow-freezing method for eggs:

• Increased sucrose concentration in suspending Increased sucrose concentration in suspending solution (0.2M or 0.3M respectively) (Yang solution (0.2M or 0.3M respectively) (Yang et al.,et al., 1998; Fabbri 1998; Fabbri et al.,et al., 2001; Chen 2001; Chen et al.,et al., 2002; 2005); 2002; 2005);

• Choline-based freezing medium to replace sodium Choline-based freezing medium to replace sodium (Stachecki (Stachecki et al.,et al., 1998; Quintans 1998; Quintans et al.,et al., 2002; Boldt 2002; Boldt et al.,et al., 2003); 2003);

• The survival rate of the oocytes after thawing was The survival rate of the oocytes after thawing was increased to 65-70%;increased to 65-70%;

Modified slow-freezing methods for eggs:Modified slow-freezing methods for eggs:

Vitrification method for eggs:Vitrification method for eggs:

• Kuleshova Kuleshova et al. et al. (1999): Open pulled straw ((1999): Open pulled straw (Hum. Hum. Reprod.,Reprod., 14: 3077-3079); 14: 3077-3079);

• Yoon Yoon et al. et al. (2000; 2003): Electron microscope grid (2000; 2003): Electron microscope grid ((Fertil. Steril.,Fertil. Steril., 74:180-181; 74:180-181; Fertil. Steril.,Fertil. Steril., 79:1323- 79:1323-1326.);1326.);

• Katayama Katayama et al. et al. (2003): Cryotop ((2003): Cryotop (Fertil. Steril.,Fertil. Steril., 80: 80: 223-224.);223-224.);

• The survival rate of the oocytes after thawing was The survival rate of the oocytes after thawing was approximately 85-90%;approximately 85-90%;

What is vitrification?What is vitrification?

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

History of vitrificationHistory of vitrification

• Luyet B. (1937): Working hypotheses on the nature of life. Biodynamica, 1:1-7.

• Luyet B. (1937): The vitrification of organic colloids and protoplasm. Biodynamica, 1:7-14.

• Fahy GM. (1981): Prospect for vitrification whole organs. Cryobiology, 18:617-625.

• Rall WF and Fahy GM. (1985): Ice-free cryopreservation of mouse embryos at -196°C by vitrification. Nature, 313:573-575.

Nature of waterNature of water

• The temperature below 0ºC will introduce formation of water ice-crystal;

• Below -130ºC is the glass transition temperature of water;

• 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;

CryobiologyCryobiology

Cryobiology (cont.)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;

CryoprotectantsCryoprotectants

CH3–S–CH3

||O

Glycerol Dimethylsulphoxide(DMSO)

Propylene glycerol(PROH)

Ethylene glycol(EG)

Cryoprotectant (cont.)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;

• 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’;

Cryoprotectant (cont.)Cryoprotectant (cont.)

Cryoprotectant (cont.)Cryoprotectant (cont.)

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

• Chilling injury;• Cryoprotectant (toxicity and

temperature);• Osmotic injury;• Speed of freezing and thawing;

Factors affect successful Factors affect successful frozen-thawing frozen-thawing

Is it difficult to freeze oocytes?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;

Vitrification procedureVitrification procedure

EM (5 min) VM (1 min) Loading ontoMcGill Cryoleaf

Plunge intoLN2(-196ºC)

7.5% EG+PROH 15.0%EG+PROH0.5 sucrose

Morphological change in EM and VM

Before in EM 1 min in EM 2 min 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

McGill CryoleafMcGill Cryoleaf

Thawing procedureThawing procedure

TM (37ºC) DM-I (3 min) DM-II (3 min) WM (3 min)

1.0M sucrose 0.5M sucrose 0.25M sucrose Culture medium

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

Initial data for survival rates of human oocytesInitial 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)

Immuno-fluorescent staining of Immuno-fluorescent staining of meiotic spindles and chromosomesmeiotic spindles and chromosomes

Aneuploidy screening of mouse oocytes Aneuploidy screening of mouse oocytes following vitrification and slow-freezingfollowing vitrification and slow-freezing

• Slow-freezing of oocytes results in more spindle and chromosome abnormalities than vitrification;

• However, incidence of aneuploidy is similar between vitrification and slow freezing.

InterpretationInterpretation

Viability and pregnancy outcome of vitrified in-Viability and pregnancy outcome of vitrified in-vivo oocytes following thawing and ICSI vivo oocytes following thawing and ICSI

at McGill Reproductive Centerat McGill Reproductive Center

Patients (cycles) 38 (38)Age 31.5 ± 0.5No. of oocytes thawed 463No. of oocytes survived (%) 383 (82.7)No. of oocytes fertilized (%) 287 (74.9)No. of embryos transferred 133 (3.5±1.1)No. of clinical pregnancies (%) 17 (44.7)No. of implantation (%) 25 (18.8)

Chian et al (Fertil & Steril., In press)

Clinical pregnancy outcome of vitrified in-vivo Clinical pregnancy outcome of vitrified in-vivo oocytes following thawing and ICSI oocytes following thawing and ICSI

at McGill Reproductive Centerat McGill Reproductive Center

Patients (cycles) 38 (38)No. of clinical pregnancies (%) 17 (44.7)No. of live birth (%) 15 (39.5)No. of miscarriages 2No. of singleton 9No. of Twins 5No. of triplets 1No. of newborn 22

Chian et al (Fertil & Steril., In press)

Patients 20

Mean age (years) 30.8 ±0.9

Mature oocytes retrieved 6

Immature oocytes retrieved 290

Mean oocyte maturation rate (%) 67.3±4.9

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 (9.6±5.4)

Pregnancies per cycle (%) 4 (20.0)

Clinical pregnancies per cycle (%) 4 (20.0)

Ongoing pregnancies (%) 0

Live births 4

Mean birth weight (grams) 4,049±413.7

IVM-Vitrification trial at MRCIVM-Vitrification trial at MRC

Chian et al (Fertil & Steril., In press)

Pregnancies conceived following oocyte vitrification are not associated with adverse obstetric and perinatal outcomes.

InterpretationInterpretation

ConclusionsConclusions• 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.

• Staff at McGill Reproductive Center; • Dr. Ruvalcaba Castellon, L.A. at Instituto

Mexicano de Infertilidad, Jalisco, Mexico;• Dr. Lucena, E. at CECOLFES, Bogota, Colombia.

AcknowledgementsAcknowledgements

Thank you!

ri-cheng.chian@muhc.mcgill.ca