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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

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Vitrification as a tool to preserve women fertility in cancer patients

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


Vitrification as a tool to preserve women fertility in cancer patients

J. Liebermann, 2009 cancer patients


Vitrification as a tool to preserve women fertility in cancer patients


Introduction
Introduction cancer patients

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.


Introduction1
Introduction cancer patients

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.


Introduction2
Introduction cancer patients

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.


Introduction3
Introduction cancer patients

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


Vitrification as a tool to preserve women fertility in cancer patients

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
Indications for for the cryopreservation of human oocytes, embryos, ovarian tissue but not the whole ovaryfertility preservation

  • Oncological

  • Non-oncological

    • Premature ovarian failure

    • Autoimmune diseases

    • Infections


Indications for fertility preservation1
Indications for for the cryopreservation of human oocytes, embryos, ovarian tissue but not the whole ovaryfertility preservation

  • Environmental factors

    • Radiation

  • Exposure to gonadotoxic agents

  • Surgical menopause

  • Women wishing to postpone motherhood


Vitrification as a tool to preserve women fertility in cancer patients

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
Cryopreservation of human Oocytes resulted from vitrified oocytes injected with sperm recovered from cryopreserved testicular tissue

  • Slow Freezing Method

  • Ultrarapid Freezing Method (Vitrification)


The principles of slow freezing procedure
The principles of Slow Freezing procedure resulted from vitrified oocytes injected with sperm recovered from cryopreserved testicular tissue:

  • 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


Meiotic spindle
Meiotic spindle resulted from vitrified oocytes injected with sperm recovered from cryopreserved testicular tissue

  • 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


Vitrification as a tool to preserve women fertility in cancer patients

Oocyte Freezing resulted from vitrified oocytes injected with sperm recovered from cryopreserved testicular tissue


Principles of cryopreservation
Principles of cryopreservation resulted from vitrified oocytes injected with sperm recovered from cryopreserved testicular tissue

  • 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
Factors causing cell injuries resulted from vitrified oocytes injected with sperm recovered from cryopreserved testicular tissue

  • 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


Vitrification as a tool to preserve women fertility in cancer patients

Oocyte Freezing resulted from vitrified oocytes injected with sperm recovered from cryopreserved testicular tissue

  • Experience

    • A) From 1986 to 1996

      • Chen, 1986

      • Al-Hasani et al. 1987

      • Van Uem et al. 1987

      • Serafini et al. 1995


Vitrification as a tool to preserve women fertility in cancer patients

Oocyte Freezing resulted from vitrified oocytes injected with sperm recovered from cryopreserved testicular tissue

  • 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


Vitrification as a tool to preserve women fertility in cancer patients

Oocyte Freezing resulted from vitrified oocytes injected with sperm recovered from cryopreserved testicular tissue

  • Factors that have improved results

    • The use of mature eggs

    • Cryoprotective solutions

    • Freezing speed: slow

    • Thawing speed: rapid

    • ICSI

    • Vitrification


Pregnancies and births from frozen human oocytes slow cooling

Chen-Al-Hasani resulted from vitrified oocytes injected with sperm recovered from cryopreserved testicular tissue

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/?

1986, 1987

1987

1989

1995

1996

1998

1998

1998

1998

1998

1999

2000

2002

2002

2003

2003

Pregnancies and births from frozen human oocytes (slow cooling)

Authors Year Cryoprotectant Oocyte stage Pregnancies/Births


Vitrification as a tool to preserve women fertility in cancer patients

Oocyte Freezing resulted from vitrified oocytes injected with sperm recovered from cryopreserved testicular tissue

  • Pregnancies per cycle

    • With frozen eggs: 17.2%

    • With frozen embryos: 18.7%

    • Porcu et al., 2002


Vitrification of human oocytes and embryos
Vitrification of human oocytes and embryos resulted from vitrified oocytes injected with sperm recovered from cryopreserved testicular tissue


Vitrification as a tool to preserve women fertility in cancer patients

Luyet. Biodynamica 1937; 1: 1–14


Historical review
Historical review with living systems and should be avoided whenever possible”

  • 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: 441-479

Rall & Fahy. Nature 1985; 313 (6003): 573–575

Ali & Shelton. J Reprod Fertil 1993; 98 (2): 459–465

Martino et al. Biol Reprod 1996; 54 (5): 1059–1069


Vitrification as a tool to preserve women fertility in cancer patients

Vitrification: with living systems and should be avoided whenever possible”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).

Vitreous, glassy state

Ice crystallization

EG+DMSO+0.4M sucrose

DPBS


Vitrification
Vitrification with living systems and should be avoided whenever possible”

Successful vitrification requires:

  • Minimum volume of holding media (<1ml)

  • 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


Vitrification as a tool to preserve women fertility in cancer patients
Risk of Contamination of Germplasm during Cryopreservation and Cryobanking in IVF UnitsBielanski and Vajta: 2009


Shortly before loading on cryotop
Shortly before loading on Cryotop and Cryobanking in IVF Units


Vitrification as a tool to preserve women fertility in cancer patients

Cryotop (Kuwayama) and Cryobanking in IVF Units


Vitrification as a tool to preserve women fertility in cancer patients
Risk of Contamination of Germplasm during Cryopreservation and Cryobanking in IVF UnitsBielanski and Vajta: 2009


Vitrification as a tool to preserve women fertility in cancer patients

Slow cooling and Cryobanking in IVF Units(langsames Einfrieren)

Vitrifikation(verlusrieren) 

Vitrifikation(ultra-rapides Einfrieren)

1 sec.

- 0.3°C/min

- 50.000°C/min


Example of cooling rates
Example of cooling rates and Cryobanking in IVF Units

  • -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


Vitrification as a tool to preserve women fertility in cancer patients

“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


Vitrification as a tool to preserve women fertility in cancer patients

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
Slow freezing versus ultra-rapid freezing vitrification the entire solution remains unchanged and water does not precipitate, so no ice crystals are formed


Vitrification as a tool to preserve women fertility in cancer patients

Slow freezing versus ultra-rapid freezing vitrification the entire solution remains unchanged and water does not precipitate, so no ice crystals are formed


Vitrification as a tool to preserve women fertility in cancer patients

Vitrification vitrification the entire solution remains unchanged and water does not precipitate, so no ice crystals are formed

Oocytes

Pregnancies/

transfer N (%)

AUTHORS

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)

Survived N (%)

Fertilized %


Vitrification of human oocytes
Vitrification of Human Oocytes vitrification the entire solution remains unchanged and water does not precipitate, so no ice crystals are formed


Vitrification as a tool to preserve women fertility in cancer patients

Before Vitrification vitrification the entire solution remains unchanged and water does not precipitate, so no ice crystals are formed

Just after Thawing

2hrs after Culture

PN stage (Day 1)

4-cell stage (Day2)

Blastocyst (Day5)

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


Vitrification as a tool to preserve women fertility in cancer patients

Antinori et al. Reprod Biomed Online 2007; 14 (1): 72-79 vitrification the entire solution remains unchanged and water does not precipitate, so no ice crystals are formed


Vitrification as a tool to preserve women fertility in cancer patients

Antinori et al. Reprod Biomed Online 2007; 14 (1): 72-79 vitrification the entire solution remains unchanged and water does not precipitate, so no ice crystals are formed


Vitrification as a tool to preserve women fertility in cancer patients
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


Vitrification as a tool to preserve women fertility in cancer patients

Oocyte II Oocytes after ICSI: A Prospective Randomised Sibling-Oocyte Study Donation & Vitrification

Cobo et al., 2008


Obsteric and perinatal outcome in 200 infants conceived from vitrified oocytes
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
Comparison between fresh and frozen-thawed embryo transfer vitrified oocytesVitrification of Zygotes (Luebeck)


Our results in avoiding hyperstimulation patients triggered with gnrh agonist
Our vitrified oocytesResults in Avoiding Hyperstimulation PatientsTriggeredwithGnRH-Agonist

* No. of Patients received warmed Zygotes 45

** Two Twins


Vitrification as a tool to preserve women fertility in cancer patients

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


Vitrification as a tool to preserve women fertility in cancer patients

Can fresh embryo transfers be replaced by 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 embryocryo-preserved-thawed embryo transfers in assisted reproductive cycles?

A prospective controlled trial.

Aflatoonian et al. 2009, Human Reprod. (submitted)


Vitrification as a tool to preserve women fertility in cancer patients
Vitrification of human 8-cell embryos, a modified protocol for better pregnancy rates Rama Raju et al. (2005)

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






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

n=5881

n=897

n=1175


Vitrification as a tool to preserve women fertility in cancer patients
Vitrification demonstrates significant improvement versus slow freezing of human blastocystsStehlik et al. (2005)

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): 53–57


Vitrification as a tool to preserve women fertility in cancer patients

Comparison of vitrification and conventional cryopreservation of Day 5 and Day 6 blastocysts during clinical application Liebermann & Tucker (2006)

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

Liebermann & Tucker. Fertil Steril 2006; 86 (1): 20–26


Vitrification as a tool to preserve women fertility in cancer patients

“…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

Comparison of vitrification and conventional cryopreservation of Day 5 and Day 6 blastocysts during clinical application Liebermann & Tucker (2006)

Liebermann & Tucker. Fertil Steril 2006; 86 (1): 20–26


Vitrification as a tool to preserve women fertility in cancer patients

...and: successful alternative to conventional freezing technology.

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…”

Comparison of vitrification and conventional cryopreservation of Day 5 and Day 6 blastocysts during clinical application Liebermann & Tucker (2006)

Liebermann & Tucker. Fertil Steril 2006; 86 (1): 20–26


Vitrification as a tool to preserve women fertility in cancer patients

In the future Cryopreservation of successful alternative to conventional freezing technology. „Ovarian Cortex“

  • ovarian biopsy and cryopreservation

  • in vitro Growth („IVG“) followed by In vitro Maturation („IVM“)

Oktay et al. 2004


Vitrification as a tool to preserve women fertility in cancer patients


Successful vitrification of bovine and human ovarian tissue kagawa et al rbmonline 2009
Successful vitrification of bovine and human ovarian tissue ovarian cortexKagawa 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.


Successful vitrification of bovine and human ovarian tissue kagawa et al rbmonline 20091
Successful vitrification of bovine and human ovarian tissue ovarian cortexKagawa 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.


Successful vitrification of bovine and human ovarian tissue kagawa et al rbmonline 20092
Successful vitrification of bovine and human ovarian tissue ovarian cortexKagawa 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.


Successful vitrification of bovine and human ovarian tissue kagawa et al rbmonline 20093
Successful vitrification of bovine and human ovarian tissue ovarian cortexKagawa 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. 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.


Successful vitrification of bovine and human ovarian tissue kagawa et al rbmonline 20094
Successful vitrification of bovine and human ovarian tissue ovarian cortexKagawa 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.


Successful vitrification of bovine and human ovarian tissue kagawa et al rbmonline 20095
Successful vitrification of bovine and human ovarian tissue ovarian cortexKagawa et al., RBMOnline 2009

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.


Successful vitrification of bovine and human ovarian tissue kagawa et al rbmonline 20096
Successful vitrification of bovine and human ovarian tissue ovarian cortexKagawa et al., RBMOnline 2009

Survival of oocytes in vitrified-warmed human ovarian tissue


Successful vitrification of bovine and human ovarian tissue kagawa et al rbmonline 20097
Successful vitrification of bovine and human ovarian tissue ovarian cortexKagawa 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. […]


Successful vitrification
Successful vitrification ovarian cortex

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
Why do we prefer the vitrification procedure now? ovarian cortex

  • 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
Future Aspects ovarian cortex

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


Vitrification as a tool to preserve women fertility in cancer patients

  • Quality is evolution ovarian cortex

  • Vitrification is a revolution

    Professor Josiane Van der Elst

    19 January 2007


Conclusion
Conclusion ovarian cortex

  • 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