vitrification as a tool to preserve women fertility in cancer patients safaa al-hasani department of...

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

J. Liebermann, 2009

• Woldwide 4.0 millions children born through ART

• 20% of the children born through cryopreservation procedure

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.

Introduction

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

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.

Introduction

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

In this presentation we will discuss the ultra-rapid method for the

cryopreservation of human oocytes, embryos, ovarian tissue but not the

whole ovary

Indications for fertility preservation

a) Oncological

b) Non-oncological1. Premature ovarian failure

2. Autoimmune diseases

3. Infections

Indications for fertility preservation

c) Environmental factors1. Radiation

d) Exposure to gonadotoxic agents

e) Surgical menopause

f) Women wishing to postpone motherhood

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

Cryopreservation of human Oocytes

Slow Freezing Method Ultrarapid Freezing Method (Vitrification)

1. Low concentration of cryoprotectants

2. Slow controlled cooling rates

3. Slow process of dehaydration of the oocyte to reduce intracellular ice crystal formation and to reduce cell damage

The principles of Slow Freezing procedure:

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 22˚C, the tubulin arm completely disappear

– This process is reversible

Oocyte Freezing

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)

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

Oocyte Freezing

• Experience

A) From 1986 to 1996

• Chen, 1986

• Al-Hasani et al. 1987

• Van Uem et al. 1987

• Serafini et al. 1995

• 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

Oocyte Freezing

Factors that have improved results

• The use of mature eggs

• Cryoprotective solutions

• Freezing speed: slow

• Thawing speed: rapid

• ICSI

• Vitrification

Oocyte Freezing

Pregnancies and births from frozen human oocytes (slow cooling)

Authors Year Cryoprotectant Oocyte stage Pregnancies/Births

1986, 1987

1987

1989

1995

1996

1998

1998

1998

1998

1998

1999

2000

2002

2002

2003

2003

Chen-Al-Hasani

Diedrich -Van Uem

Siebzehnrubl

Serafini

Tucker

Tucker - Polak de Fried

Young- Yang

Naworth and Kissing

Antinori-Borini

Tucker

Lanzerndof

Porcu - Chia

Chen - Quintans

Albani

Boldt - Fosas

DM-PR/DMSO

DMSO

PR/DMSO

PR

PR

PR

PR

PR

PR

PR

PR

PR

PR

PR

PR

PR

MII

MII

MII

MII

MII

MII

MII

MII

MII

GV

MII

MII

MII

MII

MII

MII

2/2 - 2/?

1/? - 1/1

1/?

2/0

3/0

5/2 - 1/1

1/? - 1/?

1/0

1/1 - 3/3

1/1

1/2

19/19 - 1/0

1/? - 6/2

1/?

4/4 - 4/4

1/? - 1/?

Pregnancies per cycle

• With frozen eggs: 17.2%• With frozen embryos: 18.7%

Porcu et al., 2002

Oocyte Freezing

Vitrification of human oocytes and embryos

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

Luyet. Biodynamica 1937; 1: 1–14

Historical review

• It was described at the end of the 18th Century Tammann, 1898

• Vitrification of mouse embryos at –196°C 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: 441-479Rall & Fahy. Nature 1985; 313 (6003): 573–575Ali & Shelton. J Reprod Fertil 1993; 98 (2): 459–465Martino et al. Biol Reprod 1996; 54 (5): 1059–1069

Vitrification: Two droplets of different solutions plunged directly into liquid nitrogen:left droplet is pure Dulbecco’s 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).

DPBSEG+DMSO+0.4M sucrose

Ice crystallization

Vitreous, glassy state

Vitrification

Successful vitrification requires:

– Minimum volume of holding media (<1l)– 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

Risk of Contamination of Germplasm during Cryopreservation and Cryobanking in IVF Units

Bielanski 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

Shortly before loading on Cryotop

Cryotop (Kuwayama)

Risk of Contamination of Germplasm during Cryopreservation and Cryobanking in IVF Units

Bielanski 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

Slow cooling (langsames Einfrieren)

- 0.3°C/min

Vitrifikation(verlusrieren)

- 50.000°C/min

1 sec.

Vitrifikation (ultra-rapides

Einfrieren)

Example of cooling rates

• -2500°C/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 – -50.000°C/min by using a carrier that allows very small volumes

– direct contact with LN2

“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: 407–426

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

Slow freezing versus ultra-rapid freezing

Traditional Vitrification

CPA concentration 1.5 M 3.0–5.0 M

Volume 0.3–1.0 mL <1 µL

Contact between N2 and cell

No Yes

Cooling rate ~0.5°C/min ~25.000–50.000°C/min

Freezing Slow Ultra-rapid

Thawing / warming Slow Rapid

Time consuming ≥180 min 1 sec

Dehydration Not controlled Controlled

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

> 4 Hrs

Less

Much less

10 min

Slow freezing versus ultra-rapid freezing

AuthorWarmedOocytes

N°survived

N°2 PN

cleavedembryos

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 233165

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)

Vitrification

Kuleshova (1999)

Hong (1999)

Katayama (2003)

Yoon (2003)

11/17 (65)

30/33 (90)

42/46 (94)

325/474 (69)

45

69

91

72

1(33)

1

2/6 (33)

6/28 (21)

Fertilized % Pregnancies/transfer N (%)

Oocytes

AUTHORS Survived N (%)

Vitrification of Human Oocytes

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

Figs. 5 Human oocytes before and after vitrification, ICSI and IVC.

Before Vitrification 2hrs after CultureJust after Thawing

4-cell stage (Day2)

Blastocyst (Day5)PN stage (Day 1)

Vitrified / warmedgroup

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%)

Antinori et al. Reprod Biomed Online 2007; 14 (1): 72-79

Vitrified / warmedgroup

Update up to July 2007

No. of transfers 120 270No. 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

Antinori et al. Reprod Biomed Online 2007; 14 (1): 72-79

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

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

Oocyte Donation & Vitrification

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

Comparison between fresh and frozen-thawed embryo transfer

Vitrification of Zygotes (Luebeck)Fresh ET Frozen-Thawed

ETNo 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)

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

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

Can fresh embryo transfers be replaced by cryo-preserved-thawed embryo transfers in assisted

reproductive cycles? A prospective controlled trial.

Fresh ET (n=191)

FET (n=184) 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)

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): 434–437

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

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

Outcome of Blastocyst Vitrification by using the “Cryoloop” Method


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

Outcome of Blastocyst Vitrification by using the “Cryotop” Method


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

Post-thaw survival rates of human zygotes, embryos and blastocysts after

vitrification

0

10

20

30

40

50

60

70

80

90

100

Sur

viva

l rat

e (%

)

PN zygote 4-cell Blastocyst

n=5881 n=897 n=1175

Publicis User

Vitrification demonstrates significant improvement versus slow freezing of human

blastocystsStehlik et al. (2005)

Day 5 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

Stehlik et al. Reprod Biomed Online 2005; 11 (1): 53–57

• 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

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): 20–26

“…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:

1.Cryosurvival seems more consistent, allowing greater case of patient management, with transfers being almost certain to occur2.Vitrification is able to be undertaken on a more flexible basis by laboratory staff




...and:

3.Vitrification allows for the potential reduction in personnel time needed during the entire vitrification process

4.It may enable more optimal timing of embryo cryopreservation, e.g., individual blastocysts may be cryopreserved at their optimal stage of development and expansion…”




• ovarian biopsy and

cryopreservation

• in vitro Growth („IVG“)

followed by In vitro

Maturation („IVM“)

In the future Cryopreservation of „Ovarian Cortex“

Oktay et al. 2004

• Woldwide 10-12 children born through retransplantation of ovarian cortex

The number of unsuccessful traials is unknown !!

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.

Successful vitrification of bovine and human ovarian tissue

Kagawa et al., RBMOnline 2009

Gross morphology of vitrifed human ovarian tissue using the Cryotissue method. Vitrifed human ovarian tissue was translucent in liquid nitrogen (–196°C). Scale bar represents 10 mm.



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.



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. Haematoxylin–eosin 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.



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.



Surviving oocytes (arrows) of pre-antral follicles of vitrifed–thawed ovarian tissue in human (Hoechst/propidium iodide stain). Nuclei of living oocytes were blue. Scale bar represents 50 µm.



No. of samplesNumber of Oocytes (%)

Collected Surviving

Vitrified 7 954 (100) 855 (89.6)

Survival of oocytes in vitrified-warmed human ovarian tissue





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. […]

Successful vitrification

– High cooling rate (> –50.000°C)– Fast cooling period (<1 sec.)– Low volume (<1 µL)– High concentration of cryoprotectants

=> This will avoid crystal formation

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

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

• Quality is evolution• Vitrification is a revolution

Professor Josiane Van der Elst

19 January 2007

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

Thank you for your attention!

vitrification as a tool to preserve women fertility in cancer patients safaa al-hasani department of...

Documents

cancer treatment

types of cancer

common cancer type

breast cancer patients

slow freezing method

cell viability

cell proteins

cell injuriescryopreservation