Oocyte

Cryopreservation

Prof. Aboubakr

ElnasharBenha University Hospital,

Egypt

ABOUBAKR ELNASHAR

CONTENTS

1. HISTORY

2. PRINCIPLE

3. TECHNIQUES

1. Slow freezing

2. Vitrification

4. OUTCOME

5. CLINICAL APPLICATIONS

Conclusion

ABOUBAKR ELNASHAR

INTRODUCTION

Embryo and sperm cryopreservation

well-established procedures

Oocyte cryopreservation

has become recently available

preferable in many situations

{challenges the ethical, legal and religious of

embryo cryopreservation}

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1. HISTORY

First birth from a frozen oocyt

in Australia in 1986 (Chen, 1986)

The first live birth following vitrification

1999 (Kuleshova et al., 1999)

Now

1. Many advances have been made

2. Success rates:

application of oocyte cryopreservation for many

different indications

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2 advances:

1. The introduction of vitrification

as an alternative to slow freezing: reduced

damage to internal structures: superior success

rates (Antinori et al., 2007; Cao et al., 2009; Fadini et al., 2009;Smith et al.,

2010).

2. Use of ICSI

resolve fertilization issues due to zona pellucida

hardening (Kazem et al., 1995; Porcu et al., 1997; Fabbri et al., 1998).

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The Human Fertilisation and Embryology Authority

(HFEA)Allowed

use of frozen oocytes for infertility treatment in 2000(Wise, 2000).

storage of gametes for a standard 10-year period, which

can be extended under certain circumstances

(HFEA, 2015).

In 2013, ASRM lifted the experimental label applied to oocyte freezing,

following 4RCT

(Cobo et al., 2008, 2010a, Rienzi et al., 2010; Parmegiani et al., 2011)

IVF using vitrified/warmed oocytes could produce similar

fertilization and PR to IVF with fresh oocytes(ASRM, 2013).

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2. PRINCIPLES

Oocytes are notoriously difficult cells to cryopreserve

1. Their low surface area to volume ratio:

high susceptibility to intracellular ice formation(Paynter et al., 1999).

2. The negative effects of cryopreservation on the

stability of microtubules and microfilaments(Pickering and Johnson, 1987; Sathananthan et al., 1988; Vincent et

al., 1989; Pickering et al., 1990; Joly et al., 1992)

which are vital for normal chromosomal segregation (Glenister et al.,1987).

3. Hardening of the zona pellucida: low fertilization

rates(Johnson et al., 1988; Vincent et al., 1990).

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• It’s the largest cell in the body

• It has a lot of water content

• Its cell membrane does not allow water to pass easily

• Chromosomes are spread on a spindle

A special expertise is required for oocytecryopreservation

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Cryopreservation

Preservation of cells and tissues for

extended periods of time

At sub-zero temperatures.

Cryoprotective additives (CPAs)

Reduce cryo damage by preventing ice formation.

Classified acc to their ability to cross the cell

membrane. Permeating:

dimethyl sulfoxide (DMSO), glycerol,1,2 propanediol (PrOH)

Non-permeating: sucrose, glucose, fructose, trehalose

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3. TECHNIQUES

1. Slow freezing

Used in early protocols

Controlled slow freezing

Results in a liquid changing to a solid state

2. Vitrification

Now well established

Ultrarapid cooling

Results in a non-crystalline amorphous solid.

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1. Slow freezing

Cells are exposed to

low concentration of CPAs and

slow decreases in temperature.

1. Oocytes are first cooled to temperature -5◦C to -7 ◦ C, at

which equilibration and seeding take place.

2. Oocytes are then cooled at a slow rate of 0.3–0.5 ◦

C/minute, until a temperature of between -30 ◦ C and -

65 ◦ C has been reached, before being added to liquid

nitrogen for storage (Saragusty and Arav, 2011).

Slow frozen vs fresh oocytes

Poorer outcomes(Levi Setti et al., 2006; Borini et al., 2010; Magli et al., 2010).

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2. Vitrification

Requires

Higher concentrations of CPAs, lowering the risk of

ice nucleation and crystallization

Cooling rates of100s to10 000◦ C per minute before

submersion into liquid nitrogen (Saragusty and Arav, 2011).

Cryotop’ vitrification method

Developed in 2005

widely used method (Kuwayama et al., 2005).

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Slow

cooling

- 0.3°C/min

Vitrifikation

- 50.000°C/min

1 sec.

Vitrification

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Vitrification protocols:

1. Open

direct contact between oocytes and liquid

nitrogen

using low volume devices such as capillary glasses,

cooper devices,

pulled straws, and

loops (Glujovsky et al., 2014).

2. Closed

indirect contact between oocytes and liquid

nitrogen using tubing systems (Glujovsky et al., 2014).

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Open:

similar success rates with fresh oocytes(Cobo et al., 2008, 2010; Rienzi et al., 2010; Parmegiani et al., 2011)

Concerns:

sterility {potential cross-contamination between the vitrification

sample and liquid nitrogen}.

bacteria were present in 45%

To overcome:

1. Liquid nitrogen sterilization using ultraviolet light

(Parmegiani et al., 2010) or

2. Oocyte storage in vapour phase liquid nitrogen,

which contains a lower density of environmental air

borne contaminants(Cobo et al., 2010).

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Closed

offered a solution to contamination concerns(Vajta et al., 2015).

bacteria free(Criado et al.2011)

Concerns:

efficiency of oocyte vitrification

{decreased cooling rates}.

±not preserve the oocyte ultrastructure as well as in open

systems (Bonetti et al., 2011).

reduced fertilization, cleavage, and CPR(Paffoni et al., 2011).

Outcome: excellent (Smith et al., 2010; Stoopetal.,2012;Papatheodorouet al.,2013).

UK clinics:

75% used closed containers(Brison et al., 2012).

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Warming rate

more important than the cooling rate for oocyte

survival (Seki and Mazur, 2009; Leibo and Pool, 2011; Mazur and Seki,2011).

Optimal outcomes if

vitrification and warming are carried out in the

same clinic or

by matched protocols (Brison et al., 2012).

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Vitrification Vs slow freeze protocols.

better survival, fertilization, and PR

(18.2 vs 7.6%). (Oktay et al., 2006, Cao et al., 2009; Fadini et al., 2009; Smith et al., 2010)

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Vitrified oocytes Vs fresh oocytes

similar outcomes(Nagy et al., 2009; Almodin et al., 2010; Ubaldi et al.,2010; Rienzi et al.,

2012).

Fertilization, embryo cleavage, high quality

embryos, CPR did not differ (Cobo and Diaz, SR 2011).

Oocyte survival rates of over 84%

CPR per transfer

between 35.5 and 65.2% (Cobo et al., 2008, 2010; Rienzi et al., 2010; Parmegiani et al.,

2011).

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Many IVF programmes now

favour vitrification as a technique to cryopreserve

oocytes(Rudick et al., 2010; Brison et al., 2012; Glujovsky et al.,2014),

NICE guidelines states:

In cryopreservation of oocytes and embryos, use

vitrification instead of controlled-rate freezing if the

necessary equipment and expertise is available’ (NICE, 2013).

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4. OUTCOMES

Birth weight and incidence of congenital abnormalities

2.5%

similar to those born from

spontaneous conceptions or

through regular IVF

(Chian et al., 2008, Noyes et al., 2009).

Cobo et al. (2014)

vitrification does not increase adverse obstetric and

perinatal outcomes.

No long-term follow-up of these children has yet

been published.

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5. CLINICAL APPLICATIONS

1. Fertility preservation in cancer patients

2. Social egg freezing

3. Other indications

1. Medical indications

2. Donation programmes

3. Storage of ‘spare’ gametes during IVF.

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1. Fertility preservation in cancer patientsCancer treatment regimes

can have a detrimental effect on female fertility

{removal of reproductive organs or

use of radiation therapy and cytotoxic agents}.

The extent of damage depends on

Follicular reserve

patient age

type and dose of treatment

with alkylating agents being particularly gonadotoxic(Knopman et al., 2010; Fleischer et al., 2011).

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Around 10% of cancers

occur in women under the age of 45 (Donnez and Dolmans, 2013)

demand for fertility preservation amongst these

patients has increased with improving cancer survival

rates(Fleischer et al., 2011; Noyes et al., 2011).

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Embryo cryopreservation

has been an option for some time (ASRM, 2005; Lobo, 2005; Lee et al., 2006; Kim, 2006)

Disadvantages

Risk of dispute if couples separate.

Not appropriate for

single women or

young patients without a stable partner (Noyes et al., 2011).

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Oocyte Vs embryo cryopreservation

Embryo cryopreservation

an established ART technique

Advantages.

The ability to store surplus embryos:

reduce the number of embryos transferred

during a fresh cycle:

minimize the risk of multiple pregnancy

reduce the need for repeated stimulation

cycles,

increase cumulative pregnancy rates.

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Disadvantages:

Not an option for all couples

personal religious or

moral objections, or

restrictive legislation in certain countries.

Decisions over the fate of stored embryos: major

disagreements or legal disputes, particularly in the

case of divorce or separation(Pennings,2000; Lee et al., 2006).

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Oocyte cryopreservation

a feasible alternative to embryo cryopreservation for

many couples undergoing IVF(ASRM, 2013).

1. less moral sensitivity surrounding the

storage and fate of gametes

2. fewer issues surrounding ownership

{genetic material originates from one individual}

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Oocyte cryopreservation

can now be offered routinely for fertility preservation.

71.6% of cancer patients enquiring about fertility

preservation underwent oocyte cryopreservation

(Garcia- Velasco et al., 2013).

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The disadvantage of cryopreserving mature,

metaphase II (MII), oocytes in cancer patients:

Need for ovarian stimulation.

This can delay cancer treatment by several

weeks, and may carry particular risk for those

patients with a hormone receptive cancer (Kim et al., 2011).

These problems can be reduced by

1. anytime start’ protocols

(Cakmak et al., 2013)

2. use of antiestrogens during stimulation for

women with breast cancer (Reddy and Oktay, 2012).

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Alternatives include

1. Cryopreservation of oocytes at the germinal

vesicle(GV) stage (Toth et al., 1994) or

2. Retrieval of immature oocytes followed by in-vitro

maturation (IVM)

(Rao et al., 2004; Huang et al., 2008; Oktay et al., 2010)

neither of which require ovarian stimulation.

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3. Ovarian tissue banking.

does not delay cancer treatment

An ovarian cortical biopsy or oophorectomy is

performed as a day-case surgical procedure,

independent of the menstrual cycle.

Disadvantages

1. Patient must be fit for surgery

2. Tissue must subsequently be regrafted, with

potential risk of reintroducing malignant cells (Hoekman et al., 2015).

3. Few births have been reported after

regrafting frozen ovarian tissue (Donnez and Dolmans, 2015).

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Fertility preservation cycles and chemotherapy in

breast cancer patients

could be started earlier before breast surgery

9 of 35 patients referred before surgery were able to

undergo two fertility preservation cycles, compared to

only 1 of 58 patients referred after surgery. (Lee et al., 2010)

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2. Elective oocyte cryopreservationSocial egg freezing

Oocyte cryopreservation for age-related fertility decline

In almost all countries, an increasing number of

women are postponing motherhood: rising numbers

experiencing childlessness which they had not

necessarily intended (Kneale and Joshi, 2008).

considered acceptable (ESHRE Task Force on Ethics andLaw, 2012)

a popular subject within the media

increased rapidly (Garcia-Velasco et al., 2013).

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Advantages:

1. The use of younger oocytes can reduce the risk of

fetal loss and aneuploidies associated with ageing

oocytes(Goold and Savulescu, 2009).

2. The use of cryopreserved, autologous oocytes also

allows the mother to have a genetic relation to her

child that could not be achieved through oocyte

donation(Dondorp and DeWert, 2009; Goold and Savulescu, 2009)

3. Higher chance of pregnancy than the use of

standard IVF at an older age (Sauer et al., 1990).

PR: age-dependent

optimal number of stored MII oocytes: at least 8–10 (Cobo et al., 2015).

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Age associted infertility=Age-related fertility decline

The fertility decline experienced by women, which

accelerates after the age of 35, is a well-known

phenomenon (Dunson et al., 2004; Sozou and Hartshorne, 2012).

Causes

1. Decrease in follicular quality

2. Decrease in follicular number

(Faddy et al., 1992).

3. Significantly higher risk of fetal chromosomal

abnormalities and fetal loss (Hook, 1981; Nybo Anderson et al., 2000).

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Social and ethical implications

Reasons for which women may wish to delay

motherhood

to focus on their career

to find a suitable partner or

because they simply do not feel ‘ready’ (Goold and Savulescu, 2009).

There is an obvious gender inequality in

reproduction, since men are able to reproduce at

much older ages than women (Dondorp and De Wert, 2009).

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Oocyte cryopreservation

a ‘breakthrough for reproductive autonomy’ (Harwood, 2009)

an ‘emancipation’ for women (Homburg et al., 2009).

give women the ability to make more reproductive

choices

to decide when and with whom they wish to have

children.

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Concerns in response to oocyte cryopreservation.

1. Risks in

ovarian stimulation

oocyte retrieval(Goold and Savulescu, 2009; Bayliss, 2015).

2. When older women come back to use oocytes:

more likely to experience pregnancy complications:

pre-eclampsia, hypertension and gestational diabetes

CS(Ziadeh and Yahaya, 2001; Joseph et al., 2005).

These risks are the same as those in older women

using conventional IVF (Goold and Savulescu, 2009).

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3. Oocyte cryopreservation encourages women to

become unworried about their declining fertility (Goold and Savulescu, 2009).

women should receive the correct information

about oocyte cryopreservation

success rates

do not accept it as an ‘insurance policy’ as it is

often portrayed (Lockwood, 2011).

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4. Companies such as Apple and Facebook offer

‘social egg freezing’ to their employees: women

feeling pressured to delay childbirth undermining the

concept of reproductive autonomy.(Bayliss, 2015)

5. There are concerns that women will delay

motherhood until their late 50s or 60s.(Dondorp and De Wert, 2009)

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6. The high cost of oocyte cryopreservation.

if women were to cryopreserve their oocytes at the

age of 25 and return to them at 40, this would be a

less cost-effective strategy than simply undertaking

ART at the age of 40 if difficulties were found trying to

conceive naturally.(Hirshfeld-Cytron et al.2012)

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oocyte cryopreservation before the age of 38

reduces the cost of achieving a live birth aged 40 and

beyond(Devine et al., 2015).

oocyte cryopreservation was a more cost-effective

strategy than IVF (Van Loendersloot et al.,2011).

Women should be encouraged to have

children early,

rather than to invest in an expensive technique with

no guarantee of pregnancy.

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Attitudes surrounding ‘social’ oocyte cryopreservation

Although it is still unclear exactly how many women

will access elective oocyte cryopreservation (Stoop et al., 2014)

77.6% had been previously aware of the technique

and 31.5% of women would potentially cryopreserve

their oocytes(Stoop et al., 2011).

Medical students in Singapore

36.4% of respondents had heard of the technique

26.4% would consider it (Tan et al., 2014).

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In USA

The main reason stated for delaying childbirth (88%)

was the lack of a partner

84%: were over the age of 35. This is significant,

since age at time of oocyte cryopreservation is an

important determinant of outcome. (Hodes-Wertz et al., 2013).

Age: means of 36.7 and 36.9 years (Baldwin et al., 2015; Stoop et al., 2015).

Why?

65% as an ‘insurance’ against future infertility

49% wanted more time to find a partner (Stoop et al., 2015).

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Follow-up of ‘social-freezers’

Hodes-Wertz et al. (2013)only 6% of women (11 respondents) had used their

oocytes during the 6-year timeframe.

Of these,

3 patients reported having subsequently achieved a

pregnancy from a thaw cycle

5 were unsuccessful

3 failed to comment.

Stoop et al. (2015)50.8% of women who cryopreserved their oocytes

anticipated using them at some point.

Baldwin et al., 2015.23 women who had cryopreserved their oocytes

2 had returned to use them with one successful pregnancy

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3. Other indications

1. Medical conditions other than cancer

(Donnez and Dolmans, 2013)

1. Endometriosis who may experience reduced

ovarian reserve post surgery (Elizur et al., 2009)

2. Autoimmune diseases requiring gonadotoxic

treatment

(Elizur et al., 2008)

3. Fertility preservation in gender reassignment

surgery.

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2. Oocyte donation

Reproductive potential can be extended by the use

of donated oocytes from younger women (Sauer et al., 1990; Navot et al., 1991)

similar success rates to their younger counterparts(Sauer et al., 1990).

offspring from oocyte donation are not genetically

related, and the process can be psychologically,

emotionally, and financially challenging.

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3. Women at risk of early menopause

Genetic aberrations leading to subfertility or risk of

early menopause (Goswami and Conway, 2005).

Assessment of ovarian reserve using AFC and

AMH, FSH.

Although ovarian reserve measurement has not

been shown to have predictive value for spontaneous

pregnancy, it is a reasonable strategy for these

women to consider elective oocyte cryopreservation.

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4. low-responders

allowing the storage and accumulation of oocytes

from multiple ovarian stimulation cycles before IVF

and embryo transfer (Cobo et al., 2012).

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5. Emergency’oocyte cryopreservation

male partner fails to produce a sperm sample on the

day of oocyte retrieval for IVF (Emery et al., 2004).

sperm extraction from male partners with non-

obstructive azoospermia had failed (Song et al., 2011).

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CONCLUSION

Oocyte cryopreservation

an established technology with a wide range of

indications.

an exciting technique that will prove beneficial to

many patients in the future.

Future research.

the long-term follow-up of children born following

oocyte vitrification.

ethical questions that need to be considered,

particularly in the case of ‘social egg freezing’.

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