consensus meeting on fertility preservation barcelona, · pdf fileconsensus meeting on...

Consensus meeting on fertility preservation

Barcelona, June 6th-7th 2011

Oocyte cryopreservation: slow

freezing and vitrification

Laura Rienzi, Rome, Italy

Senior Clinical Embryologist

Cryopreservation of gametes, embryos and blastocysts is an essential

component of modern ART.

Successful cryopreservation program:

1. allows to reduce the number of embryos transferred, thereby reducing multiple pregnancies and maximizing cumulative pregnancy rates per oocyte retrieval.

2. allows delayed embryo transfer during a natural menstrual cycle.

3. allows to preserve male and female fertility.

Cryopreservation in ART

OUTLINES: oocyte cryopreservation

Need

Methods

Efficiency

Concerns

Need

Oocyte cryopreservation

Medical reason

Malignant diseases

Surgical ovary removal

Polycystic ovary

Hyperstimulation sydrome

Premature ovarian failure


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 donation

Oocyte banks may result in

- widespread availability

- shortened, eliminated waiting list

- safety (quarantine)

- choice

OUTLINES

Need

Methods

Efficiency

Concerns

Methods

Vitrification is not a new technique

1972 1973/1974 1985

Vitrification of

mouse embryos

Vitrification of

mouse oocytes

1989

Vitrification of

bovine

blastocysts

1993

Ultrarapid

Vitrification with

EM grids

1996

OPS Ultrarapid

Vitrification

1983 1999

Cryoloop

Cryotip

Ultrarapid

Vitrification

Slow freezing

of mouse

embryos

Slow freezing of

domestic animal

embryos

Slow freezing

of human

embryos

Slow

freezing of

human

oocytes

1935 1997

“it is more difficult to destroy a prejudice than an atom” Albert Einstein

1948

“Living matter can survive freezing, but only if the molecules are not ordered, but solidified where they are .. in disordered or uncrystallized form.” Luyet 1935

Vitrification: biophysical aspects

Types of cells

Volume of the sample

Cooling/warming rates * Viscosity Probability of vitrification =

Vitrification is a pseudo second order phase transition (IUPAC Compendium

of Chemical Terminology, 1997) converting a material into a glassy

amorphous solid that is free from crystalline structure

Biophysical aspects: binary phase diagram

T

0

C

~-40

C

-120

C

20

C

Concentration of solute

Heterogenous

Liquid phase

Glass

phase No cristal

Thermal

hysteresis

-100

C

Ice phase

CPs

1013 poise

10-4 poise

Cooling

rate

Minimum volume-direct contact approach

• Volume of 0.1 µl

• Cooling rate can be increased to -23.000ºC/min

• Required CPA concentration in VS 50% (v/v) to 30% (v/v)

• Osmolarity of VS ~8.000 to 4.000 mosm/l

Ana Cobo, personal comunication

OUTLINES

Need

Methods

Efficiency

Concerns

Efficiency

zona pellucida hardening

membrane permeability

Cytoplasmic and Cytoskeletron

damage

Meiotic spindle depolymerization

Impact on oocyte physiology

Polar body degeneration/fusion

Oocyte ageing

Ooocyte sensitivity

Slow freezing

Different protocols have been proposed: A) 1.5M PROH + 0.2/0.2 Sucrose: Gook et al., 1993; Porcu et al., 2000; Winslow et al., 2001; Yang et al.,2002 B) 1.5M PROH + 0.3/0.3 Sucrose: Fabbri et al.,2001; Fosas et al., 2003; Chen et al., 2005; Levi Setti et al., 2006; Borini et al.,2006; La sala et al., 2006; De Santis et al., 2007; Parmegiani et al., 2009 C) 1.5M PROH + 0.2/0.3 Sucrose: Bianchi et al., 2007; Borini et al., 2007 D) Sodium deplemented protocol: Quintas et al., 2002; Boldt et al., 2006; Petracco et al., 2006

Oktay et al., 2006

Slow Freezing literature 1996-2005

Vitrification

literature 2003-2005

Age, mean 33.7 32.3

Fertilization rate 64.9 (2,478/3,818) 74.2 (637/859)

Clinical pregnancies per thawed oocyte

2.3 x10-2 (153/6720) 4.5 x10-2 (61/1354)

Clinical Pregnancies per injected oocytes

4.0 x10-2 (153/3818) 7.2 x10-2 (61/859)

Clinical Pregnancies per transfer

20.6 (153/742) 45.5 (61/134)

Implantation rate 10.1 (185/1828) 17.2 (81/473)

Slow freezing vs vitrification

Oocyte vitrification: what is the evidence?

The clinical pregnancy rate has doubled with the introduction of

vitrification (Tulandi, 2008)

Efficiency in donation program not compromised with vitrification (RCT)

(Cobo et al., 2007; 2010; Nagy et al., 2007)

Prospective randomized study with own sibling oocytes demonstrates

the Lab efficiency of the technique (RCT) (Rienzi et al., 2010)

Cumulative ongoing pregnancy rate with oocyte vitrification without

embryo selection in a standard infertility program is comparable to what

obtained with embryo cryopreservation (Ubaldi et al., 2010)

Schoolcraft et al., 2009; Chian et al., 2008; Kim et al., 2010

Clinical application: infertile population

Study design

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

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


Laboratory outcomes


17.2% ongoing implantation rate per transferred embryo 12.9% ongoing implantation rate per warmed oocyte

Cumulative pregnancy rates

www.generaroma.it

Study design

o The study was design as a prospective longitudinal

cohort 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 fresh

and vitrified oocytes from the same stimulation cy-

cle was analyzed on a per patient basis.

Ubaldi et al., Human Reproduction 2010

Material & Methods

o All consecutives patients undergoing ICSI treatment

in the Centre for Reproductive Medicine GENERA

between September 2nd 2008 and May 15th 2009

were 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


44.6% of our patients,

39.9% of cycles

Clinical results


Clinical results


P=0,006

647 vitrified oocytes are still available

Oocyte vitrification: clinical application

in donor program

Oocyte vitrification: efficacy

o Embryo development in the laboratory is not affected by the

vitrification procedure

o High ongoing pregnancy rates are achieved in standard infertility

program and egg donation program with transfers of

embryos derived from vitrified oocytes

o Among various infertility factors, only female age

influenced significantly the outcome

o The overall efficiency justifies the application of this strategy in

routine infertility work

Multicentric longitudinal

cohort study All consecutive oocyte warming cycles performed in standard infertile population between september 2008 and May 2010 in 3 different centres: -GENERA Rome -IVI Valencia -Ospedale Mangiagalli Milan

Vitrification procedure was performed according to Cobo et al., 2007; 2010; Rienzi et al., 2010. AIM of the study is to evaluate the effect of patients and cycles characteristics (female age, infertily factor, stimulation protocol, sperm quality, number of oocytes retrieved, number of oocytes vitrified, oocyte incubation time, day of transfer) on outcomes. PRIMARY OUTCOME MEASURE: Delivery

Number %

Infertility factor

male 165/488 33.8

female

tubal 66/488 13.5

endometriosis 31/488 6.4

ovulatory 73/488 15.0

idiopathic 84/488 17.2

combined 26/488 5.3

other 43/488 8.8

Stimulation Protocol (fresh cycle)

agonist 278/488 58.0

antagonist 210/488 42.0

Sperm origin

>1000,00/ml (ejaculated) 469/488 96.1

<1000,00/ml (ejaculated) 4/488 0.8

Surgically extracted 15/488 3.1

Patients and cycles characteristics

Fresh and warming

cycles characteristics N (mean) SD

Fresh cycles

Female age 35.99 3.93

CCOCS retrieved 4949 (10.9) 6.34

MII 3899 (8.63) 4.71

Vitrified MII 2965 (6.56) 4.01

Warming cycles

Warmed MII 2740 (5.61) 3.37

Survived MII 2321 (4.76) 0.85

Inseminated oocytes 2183 (4.47) 0.94

Fertilized oocytes 1655 (3.39) 2.41

Excellent and good quality embryos 915 (1.88) 1.64

Embryo transferred 929 (1.90) 0.92

Embryos crypreserved 187 (0.38) 1.05

Warming cycles clinical

outcomes

N %

Embryo transfers 436/488 89.3

Clinical Pregnancy rate per cycle 166/488 34.0

Clinical Pregnancy rate per transfer 166/436 38.1

Implantation rate 186/929 20.0

Abortion rate 37/166 22.3

Delivery rate per cycle 129/488 26.4

Delivery rate per transfer 129/436 29.6

Babies born 147/929 15.8

Factors influencing the

outcome: delivery rate

B P OR 95% CI

Female age -0.05 0.03 0.94 0.88-0.98

Number of vitrified oocytes 0.06 0.01 1.07 1.01-1.13

Day of transfer 0.33 0.02 1.39 1.04-1.88

Multivariate Logistic Regression Analysis

DELIVERY RATE PER CYCLE 0 = not obtaind 1 = obtained

Number of vitrified MII

Improvement = 0.015

≤ 8 MII > 8 MII

% n

0.000 73.6 359

1.000 26.4 129

total 100.0 488

Day of transfer

Improvement = 0.015

≤ 3 days

% n

0.000 59.7 80

1.000 40.3 54

total 100.0 134

> 3 days

% n

0.000 69.0 69

1.000 31.0 31

total 20.5 100

% n

0.000 32.4 11

1.000 67.6 23

total 7.0 34

Female age

Improvement = 0.006

≤ 38 years

% n

0.000 78.8 279

1.000 21.2 75

total 72.5 354

> 38 years

% n

0.000 74.4 183

1.000 25.6 63

total 50.4 246

% n

0.000 88.9 96

1.000 11.1 12

total 22.1 108

OUTLINES

Need

Methods

Efficiency

Concerns Concerns

Obstetric outcomes

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

Toxicity and Sterility

Concerns “The most widely emphasized concerns… are toxicity and danger of contamination. Unfortunately, available vitrification methods still struggle with these problems to date” Son and Tan, 2009

Toxicity and Sterility

OPEN systems are required for success (lower cryoprotectants

concentration higher cooling warming rate)

CLOSED systems are required for safety?? (same for slow freezing)

Sterile LN2 for vitrification would be the solution. Unfortunately, sterilization

in large volume is very difficult (impossible?) ... but...

Sterilization in small volume is possible:

FILTRATION (Vajta et al., 1998)

UV (Arav et al., 1998, Parmegiani et al., 2009)

The problem of storage can be solved: in Vapor phase LN2 tanks or closed

containers

Sterile Nitrogen: UV irradiation

EGA

Survival 85 –90% 90 – 95% 85 – 95% 70 – 95% Implantation 15%- 18% 15 – 20% 20- 30% 25- 40%

Excellent survival and development

ability;

Consistent and reproducible results;

Optimal timing of cryopreservation;

Vitrification in ART

“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

Conclusions

- Vitrification procedure maintain oocyte competence to develop in vitro

(also in the population of infertile patients).

- Oocyte vitrification is effective to improve clinical results in ART

(~60 ongoing cumulative clinical pregnancy rate in young

patients) and can be applied for fertility preservation.

- In a more general sense, oocytes cryopreservation may be regarded

as a step towards decreasing differences between genders

regarding choice in reproduction.

CLINICA VALLE GIULIA, Roma

www.generaroma.it

Gynecology:

Filippo Ubaldi

Elena Baroni

Silvia Colamaria

Maddalena Giuliani

Fabio Sapienza

Embryology:

Laura Rienzi

Stefania Romano

Laura Albricci

Antonio Capalbo

Roberta Maggiulli

Benedetta Iussig

Nicoletta Barnocchi

SALUS – ASI MEDICAL, Marostica GENERA UMBERTIDE, Perugia

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