live birth of a healthy baby from slow-freezing cryopreserved pronuclear stage embryos warmed using...

7/28/2019 Live birth of a healthy baby from slow-freezing cryopreserved pronuclear stage embryos warmed using a standar

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embryos cryopreserved through the slow-freezig

method, stored for ie years, ad warmed through a

stadard devitrificatio protocol.

CASE REPORT

Patient consent

Before a ew embryo trasfer attempt was performed,

both the patiet ad her husbad were fully iformed

o the circumstaces ivolvig the procedure, ad

coseted to havig their embryos warmed through

the stadard devitrificatio procedure.

Slow-freezing cryopreservation

A 36-year-old patiet bega ifertility treatmet i

2003 due to male factor. The stimulatio cycle was

iitiated with 300 UI/day of GRH aalog (Syarel

Zodiac, So Paulo, Brazil) durig 21 days, followed by

150 UI of hMG (Merioal Mezler, So Paulo, Brazil).Serial trasvagial ultrasoud was performed to moi-

tor ad cotrol follicular growth ad edometrial thick-

ess. Recombiat hCG (Ovidrel Merk, So Paulo,Brazil) was admiistered o the ight of the eleveth

day of the cycle, followed by trasvagial ultrasoud-

-guided oocyte retrieval 35 to 36 hours later. After

oocyte aspiratio, cumulus cells were removed from

all oocytes ad twelve mature metaphase II (MII)

oocytes were selected out of twety three. Itracyto-

plasmic sperm ijectio (ICSI) was performed i all

twelve MII oocytes, ad after 16h i cotrolled culture

(37oC ad 6% CO2 atmosphere), they were checked

for fertilizatio.

Two pro-uclei were observed i 10 out of the 12 ijec-

ted oocytes. Six prouclear stage embryos were cryo-

preserved while four embryos were kept i culture util

day 3, whe they were loaded ito a Sydey IVF embryotrasfer catheter (Cook IVF Brisbae, Australia), ad

trasferred ito the uterie lume uder tras-abdomial

ultrasound guidance. -hCG serum levels were measured

12 days after embryo trasfer to determie biochemical

pregacy. Trasvagial ultrasoud was performed at 6

weeks of gestation to conrm clinical pregnancy, which

cosisted of two gestatioal sacs ad oe heartbeat. The

result was the birth of a healthy boy.

Recebido em 04-11-2012

Aceito em 14-11-2012

Copyright - Todos os direitos reservados a

SBRA - Sociedade Brasileira de Reproduo Assistida

Case Report

Live birth of a healthy baby from slow-freezig cryopreserved

prouclear stage embryos warmed usig a stadard

devitrication protocol: Case report

Reata F. Erberelli1, Reato M. Salgado1, Fracisco J. Lopes2; Carlos G. Almodi3,*, Paula M. Almodi3,

ad Philip Wolff1,2,4

1 Geics Reproductive Medicie ad Geomics 04063-000 So Paulo, Brazil2 Cliimater Huma Reproductio 11045-301 Satos, Brazil3 Materbaby Huma Reproductio 87.013-230 Marig, Brazil4 Ivitrogeese Developmet Biology ad Assisted Reproductio 04063-000 So Paulo, Brazil

Running Title: Live birth from devitried slow-frozen embryos

ABSTRACTThis paper reports o the birth of a healthy child afterthe trasfer of prouclear stage embryos cryopreser-

ved through slow-freezig method, stored for ieyears, ad warmed through a stadard devitrificatioprotocol. Survivig embryos were trasferred to a45-year-old patiet, resultig i cliical pregacy adthe birth of a live child. This case report demostra-ted that log-term-stored prouclear stage embryoscryopreserved through the slow-freezig method maysuccessfully preset cleavage, implatatio ad deve-lopmetal viability after beig warmed usig a sta-dard devitrificatio protocol.keyords: Case report; Cryopreservatio; Devitrifi-catio; Slow-freezig.

INTRODUCTIONRecetly, a ew ultra-rapid cryopreservatio techique,

known as vitrication, has emerged as a more effecti-ve alterative to more traditioal methods used i thepreservatio of huma oocytes ad embryos (Lieber-ma et al., 2002; Stehlik et al., 2005; Vatja et al.2006; Stachecki, 2008; Almodi et al, 2010). Thus,there has bee a icreasig tred for the use of vitri-cation in cryopreservation processes in replacement

to slow-freezig methods (Al-Hasai et al., 2007; Sara-gusty et al., 2011; de Moraes et al., 2009). Due tothis shift towards vitrication, companies that produce

media used i the cryopreservatio of huma cells arelosig iterest i producig solutios for slow-freezigprotocols. As a result, it is possible that, i the ear

future, warmig kits for the slow-freezig method maydisappear from the market altogether. Besides, ot olythe techique, but also the type ad the cocetra-

tions of cryoprotectants used in vitrication are totally

differet from those used i the slow-freezig methods(Rall et al.; 1985; Park et al., 2000). These have raisedsome cocer i may huma reproductio ceters allover the world o how to warm huma cells that havebee cryopreserved through the slow-freezig methodad bee kept stored for log periods of time.This paper reports o the birth of a healthy child afterthe trasfer of log-term-stored prouclear stage


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i which the water remaiig iside ad aroud the

cells is coverted ito a amorphous crystal-free solidresemblig glass (Vatja et al., 2006). This is achieved

by the combiatio of high cocetratios of cryopro-tectats ad ultra-rapid coolig (Almodi et al., 2010).The fact that there is o ice-crystal formatio, which is

oe of the mai problems foud with more traditioalcryopreservatio techiques, has made vitrificatio a

very attractive techique.Although there is o ice crystal formatio durig

the vitrificatio process, this may take place durigwarmig. Seki ad Mazur (2009) demostrated thatsamples of mouse oocytes submitted to the vitrificatio

procedure, whe warmed at high rates had a survivalrate higher tha 80%. However, whe samples were

warmed at low rates survival was close to 0%, regar-

dless the freezig rate used. They iterpreted thelethality of slow warmig as a cosequece of allowig

the growth of small itracellular ice crystals.Accordig to Mishima ad Staley (1998) threepheomea occur durig warmig: i) the coversio

of amorphous ice ito ultra-viscous water; ii) devitrifi-catio, which is the coversio of ultra-viscous water

ito clear ice; ad iii) by icreasig the temperature,

ice goes through a third pheomeo, recrystalliza-tio. Hece, warmig is also a critical process due to

the occurrece of crystallizatio ad recrystallizatio.Two factors are importat i this process, warmig

rates ad the cocetratio of solutes. Rapid warmigis essetial to miimize both the formatio of itra-cellular ice crystals ad their growth to lethal size by

recrystallizatio (Seki et al., 2008). Therefore, withboth the slow-freezig as well as vitrificatio protocols

the formatio of ice crystals durig warmig is possi-ble, a problem that may be avoided by high warmigrates. This is the reaso why the straws removed from

liquid itroge were submitted to a water bath at 37Cbefore beig added to the DV- I solutio, as ormally

performed i the divitrificatio process.

The evets that follow are the exit of cryoprotec-tats from the cell, as well as its rehydratio. High

sucrose levels are eeded durig warmig to dimiishthe harmful effects of permeable cryoprotectats,

by reducig osmotic stress ad cotrollig cell wateriflux (Fabbri et al., 2000), ad thus reducig the timeof exposure of cells to the toxic cryoprotective agets.

I geeral, the solutios used i slow-freezig metho-ds are composed of 10% (1-2M) of permeable cryo-protectats, whereas the solutios used i vitrificatio

protocols cotai 30% (5-8M) (Shaw et al., 2003). Asthe cocetratio of cryoprotectats used i vitrifica-

tio is much higher tha that used i the slow-freezigmethod, it is fair to ifer that the warmig protocolused i vitrificatio has bee stadardized i such a

way as to avoid cell osmotic chock eve more tha ithe slow-freezig protocol. Thus the warmig proto-

col used i vitrificatio could be safely used for thewarmig of cells cryopreserved with the slow-freezigprotocol, a cocept that has bee fully demostrated

by this report.This case report, therefore, demostrated that log-

-term-stored prouclear stage embryos cryopreservedthrough the slow-freezig method may successfullypreset cleavage, implatatio ad developmetal

viability after beig warmed usig a stadard devitri-ficatio protocol. Prospective radomized comparative

studies should be carried out to certify the viabilityad safety of devitrifcatio of embryos cryopreservedwith the slow-freezig methods.

The exceedig embryos were cryopreserved through

a slow-freezig method usig FREEZE-KIT (Vitrolife Sa Diego, USA), which cosists of three solutios:Cryo-PBS (phosphate-buffered salie), EFS 1 (1.5M

1,2-propaediol) ad EFS 2 (1.5M 1,2-propaediol ad0.1M Sucrose). Embryos were washed for 5 miutesi Cryo-PBS, icubated for 20 miutes i EFS 1 adwashed quickly i EFS 2. They were the loaded ito

straws (two per straw), which were sealed ad placed ia programmable freezer. The temperature was program-

med to decrease to -7oC at 2oC/mi rate, held at -7oCto allow maual seedig, ad subsequetly dropped to-30oC at 0.3oC/mi rate. Samples were the plugedito liquid itroge for storage util warmig.

wARmINGI Jauary 2012, the couple retured to the cliic totry to achieve a ew pregacy usig the embryos that

had bee kept stored i liquid itroge for the past ieyears. The patiet, ow 45 years old, had her edo-metrium prepared for ET through the admiistratio

of estradiol valerate (2mg/day), durig 16 cosecutivedays, util the edometrial liig reached 10 mm. The

vitrication-warming system developed by Ingamed(Marig, Brazil) was used i this case (Vitrig-Kit).

The warmig kit is costituted of three solutios: DV-I(warmig), DV-II (diluet) ad DV-III (risig). Theirchemical compositio is based o differet cocetra-tios of sucrose (Almodi et al., 2010).

The straws cotaiig the embryos were removed fromLn2 ad left at room temperature for 30 secods adthe icubated i water bath at 37oC for 30 secods.

After the straws were wiped with sterile gauze, botheds were opeed, the cotets expelled i a petridish ad aalyzed uder a stereomicroscope. Embryos

were collected ad icubated i DV-I for 1 mi at 37oC,ad subsequetly moved to DV-II, i which they wereicubated for 3 mi. Fially, the embryos were washed

i DV-III twice for 5 mi each, ad the moved itopre-equilibrated G-1 Plus medium (Vitrolife SaDiego, USA) at 37oC, with a survival rate of 75%.The zygotes were kept i culture for 24 hours ad

three embryos were trasferred to the uterus o day 2,each oe grade A with four cells (symmetric blastome-res with no fragmentation). -hCG serum levels were

measured 12 days after embryo trasfer to determie

biochemical pregacy. Luteal phase was supportedby 600 mg of itravagial progesteroe per day util12 weeks of gestatio. Trasvagial ultrasoud was

performed at 6 weeks of gestatio to cofirm cliicalpregacy, which cosisted of a gestatioal sac adoe heartbeat. nie moths later the result was thelive birth of a healthy baby boy.

DISCUSSIONThis case report is iterestig as it reports o a IVF

live birth of a healthy boy resultig from the trasferof prouclear stage embryos cryopreserved througha slow-freezig method, stored for ie years, adwarmed usig a stadard devitrificatio protocol.

Several studies have bee performed i order to mii-mize cryostorage duratio ad the risk of itracellu-lar ice crystal formatio, ad thus optimizig embryo

survival (Trad et al., 1999). The developmet of well--established ultra-rapid protocols (vitrificatio) wasa practical solutio to the matter (Lieberma et al.,2002). Vitrificatio is a cryopreservatio strategy


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Live birth from devitried slow-frozen embryos - Erberelli R.F. et cols.

AUThORS ROLESRFE, RMS ad PW were resposible for embryowarmig ad culture. FJL was resposible for embryotrasfer ad luteal phase support. RFE, ad PMA wereresposible for draftig the article, ad CGA for revi-sig the article critically for importat itellectualcotet. CGA ad PW were resposible for the fialapproval of the versio to be published.

ACkNOwLEDGEmENTSThe authors would like to thak Mr. Atoio CarlosCorrea for his cotributio i revisig the Eglishversio of the mauscript.

FUNDINGno specific fudig was obtaied for this study

CONFLICT OF INTERESTnoe declared

* Corresponding Autor:

Carlos Gilberto Almodi, MD, MSc, PhDAv. XV de novembro, 1232

87.013-230 - Marig PR - BrazilPhoe: (55 44) 3224-3992Fax: (55 44) 225-1162E-mail: [email protected]

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2. Almodi CG, Miguetti-Cmara VC, Paixo CL, Pereira PC.Embryo developmet ad gestatio usig fresh ad vitrifiedoocytes. Hum Reprod 2010; 25: 1192-1198.

3. de Moraes LAM, de Aguiar LPT, Lamaita RM, MarihoRM, Caetao JPJ. Comparao de resultados obtidos com

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