Neca% FINDIKLI, Ph.D.
Director of IVF Laboratories
Bahceci Women’s Health Group, Turkey
Oocyte Cryopreserva%on: Efficiency and Safety
A Brief introduc%on
• 1978 -‐ First IVF birth, in 1978 (UK)
• 1982 -‐ First birth from frozen oocyte (Australia)
• Late 1980s : successful pregnancies by slow freeze – rapid thaw protocols
• There was a lack of progress due to
technical concerns & low success rates
• Due to:
• Extraordinary volume and poor membrane conduc6vity
• instability of microtubules & microfilaments
• Zona hardening
Recent Progress
• Early 2000s:
• Enforcing effect of legisla%ve restric%ons
• Introduc%on of vitrifica%on
• Use of ICSI a`er warming of oocytes
has improved the outcome and oocyte cryopreserva%on has found wider applica%ons.
• In 2013: ASRM has li`ed the experimental label on oocyte cryopreserva%on following four RCTs:
• Cobo et al., 2008
• Cobo et al., 2010
• Rienzi et al., 2010
• Permegiani et al., 2011
(Studies stated that vitrified/warmed oocytes can result in similar fer%liza%on and pregnancy rates compared to fresh oocytes)
Programlı Dondurma
0 20 40 60 80 100
Oocyte Day 1 Day 2 Day 3 Day 5
Survival (%)
Slow Freezing
Survival rates in the early 2000s
• 7.5% Ethylene Glycol
• 7.5% DMSO
15% Ethylene Glycol 15% DMSO
0.5 M Sucrose
Equilibria%on solu%on(ES)
Vitrifica%on solu%on(VS)
(Dura%on of incuba%on in VS is cri%cal)
Oocyte cryopreserva%on
Vitrifica%on
Oocyte cryopreserva%on
Oocyte cryopreserva%on
• 1.0 M Sucrose
• 0.5 M Sucrose
•
• No Sucrose
Thawing Solu%on
Dilu%on Solu%on
Washing Solu%on
Oocyte Warming
Oocyte cryopreserva%on/warming
Slow Freezing Vitrifica%on
Permeable Low MW
Non-‐permeable Low MW
High MW
Vitrifica%on
Ehylene glycol DMSO
1,2-‐Propanediol
Intracellular Cryoprotectants
Sucrose Trehalose
Dehydra%on
Ficoll, PEG
Extracellular Cryoprotectants 10-‐20 M
0.5-‐0.75 M
10 mg/ml
Slow Freezing
1,2 -‐propanediol DMSO
Glycerol
Sucrose Trehalose 0.2-‐1 M 1-‐ 1.5 M
Cryoptotectants
What is really happening during cryopreserva%on?
Kopeika et al., 2015
What is really happening during cryopreserva%on?
Kopeika et al., 2015
Is a frozen oocyte equal to a fresh one?
Tachibana et al. 2013-‐Nature
Is a frozen oocyte equal to a fresh one?
Oocyte cryopreserva%on: KPIs
Alpha Consensus., 2012
All oocytes are equal, but some oocytes are
“more equal” than the others
• Modifica%ons in protocols and handling should be needed based on the
“nature” of the oocytes (high or poor quality)
• Cryopreserva%on media should be improved in order to minimize the stress (an%oxidants, free radical chelators)
“Towards a tailor-‐made oocyte cryopreserva%on”
Oocyte cryopreserva%on: Indica%ons
• Fer%lity preserva%on in cancer pa%ents
• Oocyte cryopreserva%on for medical reasons
• Endometriosis, autoimmune diseases etc.
• Oocyte Dona%on
• Elec%ve oocyte cryopreserva%on (social freezing)
• Age-‐related fer6lity decline
• “Emergency" oocyte cryopreserva%on
Argyle et al., 2016
Social egg freezing
• 1468 women undergoing elec%ve oocyte cryopreserva%on for non-‐oncologic reason
• 137 returned to use them – pregnancy rates were found to be age-‐dependent
• Op%mal number of stored MII oocytes should be at least 8 – 10.
Cobo et al., 2015 Concerns
• Procedure-‐associated risks?
• Proper consen%ng?
Oocyte dona%on from vitrified oocytes
Cobo et al., 2015
Follow up on children born a`er cryopreserva%on of oocytes
• To date, there are no long-‐term follow up studies for children from cryopreserved oocytes and most data are from case reports and retrospec%ve studies.
• Mean birthweight and incidence of congenital abnormali%es were similar in infants born through regular IVF (n=200 infants)
(Chian et al. 2008)
• Incidence of congenital abnormali%es were similar a`er slow freezing or vitrifica%on (n=936 infants)
(Noyes et al. 2009)
Human Oocyte Cryopreserva%on
Noyes, et al. Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. RBM Online
18:769-776, 2009.
Parameter Slow-Freeze Vitrification Both Total
# Embryo transfers 2003 844 19 2866
# live born
babies 532 392 12 936
Birth Defects (Incidence)
6 (one in 89)
6
(one in 65)
0 12
(one in 78)
All birth anomalies*
Approximate incidence in natural conception
births
Incidence in total of 936 oocyte cryopreservation
births (n)
One in 33 One in 78 (12)
* Skin haemangioma, cardiac defects, neural tube defects, cleft lip/palate, clubfoot, Arnold-Chiari syndrome, chroanal atresia, biliary atresia,
Rubinstein-Taybi syndrome
Conclusions: Where are we now?
• Oocyte cryopreserva%on has now been an established technology with a wide range of indica%ons.
• Vitrifica%on has now been the method of choice.
• However, there is an urgent need to monitor the medical indica%ons and technical approaches on clinical outcome and long term follow up of children a`er oocyte cryopreserva%on/
warming.
• More and extensive research is needed to monitor the possible effects of cryopreserva%on on DNA (gene%c & epigene%c changes)
