When and how to cryopreserve oocytes analysis of oocyte physiology and molecular markers to improve cryopreservation methods-alpha rienzi-laura_2010

When and how to cryopreserve oocytes?

Analysis of oocyte physiology and molecular markers to improve

cryopreservation methods

ALPHA 20108TH BIENNIAL CONFERENCE BUDAPEST 2010

Laura Rienzi, Rome, Italy

CLINICA VALLE GIULIA, Rome

Oocyte cryopreservation:when and how?

www.generaroma.it

Medical reason

Malignant diseases

Surgical ovary removal

Polycystic ovary

Hyperstimulation sydrome

Premature menopause etc.

Oocyte cryopreservation : when and how?

www.generaroma.it

Logistic reasons

Sperm collection problem

Legal reasons

Restrictions in embryo cryopreservation

Fate of embryos of separated couples

Social reasons

Wish to delay motherhood

Moral reasons

Oocyte cryopreservation: when and how?

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Oocyte donation program

Oocyte banks may result in

- widespread availability

- shortened, eliminated waiting list

- safety (quarantine)

- choice

Traditional freezing and/or vitrification?

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Efficiency in donation program not compromised with vitrification (Cobo et al., 2007; Nagy et al., 2007)

Prospective randomized study with own oocytesno difference (Rienzi et al., 2010)

The clinical pregnancy rate has doubled with theintroduction of vitrification (Tulandi, 2008)

Cumulative ongoing pregnancy rate with oocyte vitrification without embryo selection in a standard infertility program (Ubaldi, 2010)

Laboratory outcomes: Laboratory outcomes: Slow freezing infertile populationSlow freezing infertile population

Clinical application: infertile population

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Study design

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In order to validate the effectiveness of a vitrification approach for oocyte cryopreservation a prospective comparison was designed in our population of infertile patients (september 08 - march 09).

This study was set-up as a non-inferiority trial with a prospective target of 240 sibling metaphase II oocytes obtained from an estimated 40 ICSI patients

Oocyte fertilization rates after ICSI (per warmed oocyte and per injected oocyte) were evaluated as primary outcomes. Secondary outcomes were pronuclear morphology and embryo development

Rienzi et al., Human Reproduction 2010

Material & Methods

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The general idea of the study was to minimize extra stress on oocytes often related with cryopreservation procedures, namely:

1. Long exposure to Hepes buffered media, with uncertain temperature control, for oocyte denudation and selection under the inverted microscope

2. Prolonged oocyte in vitro culture without the protection of cumulus and corona cells

3. Oocyte ageing

In this way, by using randomized sibling oocytes the only difference between the fresh and the vitrified group was the vitrification procedure itself followed by 2 hours of in vitro culture.


Patient population

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Laboratory outcomes

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Survival rate


Laboratory outcomes: Laboratory outcomes: Vitrification egg donation programVitrification egg donation program

Clinical outcomes:Slow freezing infertile population

èè+Clinical outcomes:Slow freezing infertile population

Cumulative ongoing pregnancy rates: vitrification

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Study design

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o The study was design as a prospective longitudinalcohort study.

o The baseline characteristics, embryological data,clinical and ongoing pregnancy rate were analyzed on a per cycle basis.

o The cumulative pregnancy rate obtained with freshand vitrified oocytes from the same stimulation cy-cle was analyzed on a per patient basis.

Ubaldi et al., Human Reproduction 2010

Material & Methods

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o All consecutives patients undergoing ICSI treatment in the Centre for Reproductive Medicine GENERAbetween September 2nd 2008 and May 15th 2009were considered for this study

o Only patients with supernumerary oocytes available for cryopreservation were included. A single fresh attempt was included for each patient.


Laboratory results

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44.6% of our patients, 39.9% of cycles

Clinical results

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Results

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P=0,006

647 vitrified oocytes are still available

Oocyte vitrification: clinical application infertile population

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o Embryo development is not affected by the vitrification procedure up to day 2

o High cumulative ongoing pregnancy rates were achievedin a standard infertility program with transfers of embryos derived from fresh and subsequently vitrified eggs

o Among various infertility factors, only female age influenced significantly the outcome

o The overall efficiency justifies the application of this stra-tegy in routine infertility work

Obstetric outcomes

www.generaroma.it

Chian RC, Huang JY, Tan SL, Lucena E, Saa A, Rojas A, Castellón LA, García Amador MI, Montoya Sarmiento JE.

Obstetric and perinatal outcome in 200 infants conceived from vitrified oocytes. Reprod Biomed online 2008

Noyes N, Porcu E, Borini A.

Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. Reprod Biomed online 2009

Stress tollerance

“To him who devotes his life to science, nothing can give more happiness than increasing the number of discoveries, but his cup of joy is full when the results of

his studies immediately find practical applications.”Louis Pasteur

Analysis of oocyte physiology and molecular markers

membrane membrane permeabilitypermeability


60-65% 35-40% 75-80%

Survival rates of human oocytes frozen with the

same slow freezing protocol

(Lassalle et al., 1985)

Osmotic response to glycerol of mouse oocytes injected with

Aquaporin-3 cRNA

Edashige et al., 2003

Aqua-3

Aquaporin-9 expression

Aquaporin-9, a protein channel that can transport water and other solutes through the plasmalemma is expressed in rat GV-stage but notmature oocytes (Ford et al., 2000)

-+

++ Permeability

Membrane permeabilityMembrane permeability

CG release and CG release and ZP hardeningZP hardening



Ghetler et al., 2006

Fresh

Frozen

No evidence of cortical granule discharge in cryopreserved oocytes

Failed Fertilized

Frozen Non-frozen

Gook et al., 1993

“The immunostaining examination for CG of the frozen–thawed oocytes did not reveal evidence of the premature release of CG.”

Li et al., 2005

Cortical granules release

Control(non-vitrified)

Vitrified DMSO/EG0

100

200

300

400

500

600

700

800

900Ti

me

for

zona

disso

lution

(s)

n = greater than 60 oocytes per treatment with 3 replicatesLarman et al., 2006

Zona Pellucida HardeningZona Pellucida Hardening


Polar body Polar body degeneration/fusiondegeneration/fusion



Early reports on failure of PBII extrusion and increase of aneuploidy in thawed mouse oocytes

Glenister et al, 1987; Carroll et al., 1989

Frozen

+-

No. of Oocytes (%)

Scored % Aneuploidy % Retention PB

352 6.4 2.6218 8.0 4.4

No increase in the rates of aneuploidy/digyny in parthenogenetically activated mouse oocytes after cryopreservation with DMSO/slow freezing

Bos-Mikich and Whittingham, 1995

Aneuploidy and PB retention

http://av.rds.yahoo.com/_ylt=A9ibyKuk3hxFpsMAhn1nNaMX;_ylu=X3oDMTBvMmFkM29rBHBndANhdl9pbWdfcmVzdWx0BHNlYwNzcg--/SIG=125auae83/EXP=1159606308/**http%3a//www.petwebsite.com/mice/selecting_mice.htm


Meiotic spindle Meiotic spindle depolymerizationdepolymerization




3h 373h 37°°CCTS2TS2TS1TS1 PBSPBSTS3TS3

THAWING

PBSPBS FS1FS1 FS2FS2

FREEZING

Rienzi et al., 2004

Meiotic spindle analysis during slow freezing

Coticchio Coticchio et alet al., 2006., 2006

50.8% vs

73.1%

1.5 PrOH sucrose 0.1mol/l


Bianchi Bianchi et alet al., Human Reproduction, 2005., Human Reproduction, 2005

Det

ecta

ble

mei

otic

spi

ndle

(%)

1.5 PrOH sucrose 0.3 mol/l

69.7% vs

73.1%

Coticchio Coticchio et alet al., 2006., 2006


PrePre--vitrificationvitrification PostPost--vitrificationvitrification

PostPost--culture 2hculture 2h

Meiotic spindle analysis during vitrification

0

0.5

1

1.5

2

2.5

pre-vit 0 h 2 h

Meiotic Spindle view and vitrificationHUMAN OOCYTES

Mei

otic

spi

ndle

inte

nsity

Larman, Larman, RBM on line 2007RBM on line 2007

Meiotic spindle analysis during vitrification




CG release and CG release and ZP hardeningZP hardeningCytoplasmic and Cytoplasmic and

Cytoskeletron Cytoskeletron damagedamage


0,4

0,5

0,6

0,7

0,8

0,9

1

0 60 120 180 240 300 360 420 480 540

Time (secs)

Nor

mal

ised

vol

ume

ExperimentalPredicted

Coticchio et al., 2004 VITRIFICATION

SLOW FREEZING

Osmotic toxicityOsmotic toxicity

Osmotic toxicityOsmotic toxicity

Van den Abbeel et al., 2007

OOCYTE OSMOTIC TOLERENCE AND OOLEMMA PERMEABILITY

Temperature of exposure influence shrinking (swelling) patterns

- Oocyte shrinkage tolerance is about 30% of their initial volume

- At 22°C, EG has a lower permeability coefficient relative to DMSO and PG

- The membrane is more selective for EG and DMSO than for PG (mean reflection coefficient Sigma lower for PG)

- Permeability coefficients of individual oocytes varied substantially (inherent biological variability)

zona pellucidazona pellucidahardeninghardening


Cytoplasmic and Cytoplasmic and Cytoskeletron Cytoskeletron

damagedamage


Impact on oocyte Impact on oocyte physiologyphysiology



METABOLISM MONITORING THROUGH PYRUVATE UPTAKE (mouse oocytes):

Mouse oocytes and developing embryos following slow freezing were metabolically impaired compared with those that were vitrified

Lane and Gardner, 2001; Lane et al., 2002

…although vitrification was also associated with a decrease in nutrient utilization by the oocyte compared to controls the decrease was significantly smaller than that induced by slow freezing.

Oocyte metabolism post-cryopreservation

Lane and Gardner., 2001

Pyru

vate

Upt

ake

(pmol/o

ocyt

e/h)

0

1

2

3

4

5

a

b

c

Control Vitrification Slow-freezing

Oocyte metabolism post-cryopreservation

PROTEOMIC ANALYSIS OF OOCYTE PROTEIN PROFILES (mouse oocytes) by SELDI-TOF MS:

Mouse oocytes following slow freezing revealed major alterations compared with those that were vitrified.

Vitrified oocyctes appeared to be similar to the non-cryopreserved control oocytes...

Larman et al., 2006

Oocyte protein profile post-cryopreservation

Slow Frozen Oocytes

In Vivo & VitrifiedOocytes

= Upregulated= Downregulated

Heat Map

Hierarchical Clustering of Anionic Protein Profile

Larman et al., 2006Larman et al., 2006

zona pellucidazona pellucidahardeninghardening


Cytoplasmic and Cytoplasmic and Cytoskeletron Cytoskeletron

damagedamage


Impact on oocyte Impact on oocyte physiologyphysiology


Oocyte Oocyte ageingageing


Possible injuriesPossible injuriesOocyte agingOocyte aging

Oocyte cryopreservation poses certainly specific problems:

- The oolemma and not the size of MII oocyte is the key to explain the low survival rates obtained with slow freezing.

- Release of cortical granules (controversial)

- Chemical toxicity from cryoprotectants (type specific)

- Osmotic toxicity

- Meiotic spindle depolymerization (slow freezing)

- Oocyte physiology alteration (metabolism and protein profile) especially true for slow freezing

Oocyte safety

Safety of the procedures

Concerns

“The most widely emphasized concerns… aretoxicity and danger of contamination.

Unfortunately, available vitrification methods still struggle with these problems to date”

Son and Tan, 2009

CLINICA VALLE GIULIA, Roma

www.generaroma.it

Ginecologia:

Filippo Ubaldi

Elena Baroni

Silvia Colamaria

Maddalena Giuliani

Fabio Sapienza

Matteo Buccheri

Embriologia:

Laura Rienzi

Stefania Romano

Laura Albricci

Antonio Capalbo

Roberta Maggiulli

Benedetta Iussig

Nicoletta Barnocchi

SALUS – ASI MEDICAL, Marostica

When and how to cryopreserve oocytes analysis of oocyte physiology and molecular markers to improve cryopreservation methods-alpha rienzi-laura_2010

Health & Medicine

oocyte vitrification

human reproduction

oocyte cryopreservation

vitrified oocytes

warmed oocyte

injected oocyte

oocyte denudation

vitrification procedure