Oocyte cryopreservation. Prof Aboubakr Elnashar

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Oocyte Cryopreservation Prof. Aboubakr Elnashar Benha University Hospital, Egypt ABOUBAKR ELNASHAR

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  • 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

    ABOUBAKR ELNASHAR

  • 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).

    ABOUBAKR ELNASHAR

  • 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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  • Its 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

    ABOUBAKR ELNASHAR

  • 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

    ABOUBAKR ELNASHAR

  • 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.

    ABOUBAKR ELNASHAR

  • 1. Slow freezing

    Cells are exposed to

    low concentration of CPAs and

    slow decreases in temperature.

    1. Oocytes are first cooled to temperature -5C to -7 C, at which equilibration and seeding take place.

    2. Oocytes are then cooled at a slow rate of 0.30.5

    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).

    ABOUBAKR ELNASHAR

  • 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

    - 0.3C/min

    Vitrifikation

    - 50.000C/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).

    ABOUBAKR ELNASHAR

  • 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).

    ABOUBAKR ELNASHAR

  • 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).

    ABOUBAKR ELNASHAR

  • 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)

    ABOUBAKR ELNASHAR

  • 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).

    ABOUBAKR ELNASHAR

  • 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).

    ABOUBAKR ELNASHAR

  • 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).

    ABOUBAKR ELNASHAR

  • ABOUBAKR ELNASHAR

  • 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.

    ABOUBAKR ELNASHAR

  • 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).

    ABOUBAKR ELNASHAR

  • 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}

    ABOUBAKR ELNASHAR

  • 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).

    ABOUBAKR ELNASHAR

  • 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.

    ABOUBAKR ELNASHAR

  • 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).

    ABOUBAKR ELNASHAR

  • 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)

    ABOUBAKR ELNASHAR

  • 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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  • ABOUBAKR ELNASHAR

  • 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 810 (Cobo et al., 2015).

    ABOUBAKR ELNASHAR

  • 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).

    ABOUBAKR ELNASHAR

  • 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).

    ABOUBAKR ELNASHAR

  • 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.

    ABOUBAKR ELNASHAR

  • 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).

    ABOUBAKR ELNASHAR

  • 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).

    ABOUBAKR ELNASHAR

  • 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)

    ABOUBAKR ELNASHAR

  • 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)

    ABOUBAKR ELNASHAR

  • 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).

    ABOUBAKR ELNASHAR

  • 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

    ABOUBAKR ELNASHAR

  • 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. Emergencyoocyte 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).

    ABOUBAKR ELNASHAR

  • 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.

    ABOUBAKR ELNASHAR