Consensus meeting on fertility preservation Barcelona, June 6 th -7 th 2011 Oocyte cryopreservation: slow freezing and vitrification Laura Rienzi, Rome, Italy Senior Clinical Embryologist
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
Oocyte cryopreservation
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
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.
Rienzi et al., Human Reproduction 2010
Patient population
Rienzi et al., Human Reproduction 2010
Laboratory outcomes
Rienzi et al., Human Reproduction 2010
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.
Ubaldi et al., Human Reproduction 2010
Laboratory results
Ubaldi et al., Human Reproduction 2010
44.6% of our patients,
39.9% of cycles
Clinical results
Ubaldi et al., Human Reproduction 2010
Clinical results
Ubaldi et al., Human Reproduction 2010
P=0,006
647 vitrified oocytes are still available
Oocyte vitrification: clinical application
in donor program
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