nline - Vot 14. No 2. 2007208-213 Reproductive BloMedicine Online: ww\\.rbmonllne.comlArticle!2620on web 8 January 2007

Article

Cryoloop vitrification of human day 3 cleavage-stage embryos: post-vitrification development,pregnancy outcomes and live births

Dr Nina Desai received her doctorate in microbiology/cell biology from the University ofMedicine and Dentistry of New Jersey, USA and then completed a post-doctoral fellowshipin infertility at The Ohio State University, Columbus. She joined the faculty of Case WesternReserve University, Cleveland, Chio in 1993 and was subsequently appointed Directorof the IVF/Andrology Laboratories at University MacDonald Women's Hospital. In 2000,she became Director of IVF and Clinical Research at the Cleveland Clinic Foundation anddesigned a new state-of-the-art IVF laboratory there. Her research has focused on growthfactors, extracellular matrices, co-culture systems and the study of embryonic growth andapoptosis, and ooeyte and embryo vitrification.

Dr Nina Desai

Nina Desai'•^ Heather Blackmon^ Julia Szeptycki'. James Goldfarb''Cleveland Clinic Fertility Centre/Cleveland Clinic Foundation, Beachwood, OH; ̂ Carolina Conceptions, Raleigh, NC,USA^Correspondence: Cleveland Clinic Fertility Centre, 26900 Cedar Road, Beachwood, OH 44122, USA. Tel: +1 2168392907; e-mail: desain@ccf.org

Abstract

Vitrification technology has shown great promise for cryopreservation of human embryos. The majority of this work has beenwith blasiocysl-stage embryos. This report describes initial clinical results following vitrification of human embryos on day3 of culture at Ihe 6- to 8-cell stage. A total of 236 embryos were cryopreserved on cryokxtps using a vitrilicalion protocol,Wanned embryos were cultured until day 5 before transfer to the patient. Tbe post-warming survival rate was 85%. Theclinical pregnancy rate was 44% (34/77). and the implantation rate was 207c (40/201). [n transfers where at least one warmedembryo reached ihe blastocyst stage by the day of transfer, tbe clinical pregnancy rate was 58% (28/48). The cryoloop was anexcellent vessel for ultra-rapid cryopreservation of embryos. This study supports ihe effectiveness of a dimethylsulphoxide/ethylene glycol cryoprotectant combination for vitrification of human embryos at the 6- to 8-cell stage.

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Keywords: cryoloop, cryopreservation., IVF, post-vitrification survival., pregnancy outcome, vitrification

Introduction

Clinical pregnancy rates associated witb IVF have increasedtremendously since the birth of tbe first IVF baby in 1978.Wilh Ihis increase has come a movement to reduce the numberof embryos being transferred to avoid high order multiplepregnancies. Effective techniques for cryopreservation ofsupernumerary embryos for a future cycle are vital to avoidembryo wastage and to augment the pregnancy rate from asingle ooeyte retrieval.

Cryopreservation results have been quite variable amongstclinics depending on embryo stage and cryopreservationprotocol used. Based on tbe 2004 national registry data for IVFclinics (see US Department of Health and Human Servicesand the Center for Disease Control and Prevention. 2006).cryopreservation cycles resulted in a live birtb rate of 28-30%

for patients under 38 years of age. Slow freeze protocols usingcontrolled rate freezers that slowly lower tbe temperature tobelow -3O''C bave traditionally been used forembryo freezingin the clinical laboratory. Different cryoprotectant solutionshave been utilized for embryo dehydration depending oncell stage. Propanedioi/sucrose-based protocols have beenthe most commonly used methodology for slow freezing ofpronuclear and early cleavage (2- to 8-cell) stage embryos(Lassalleeffl/. 1985; Testart i-ra/. 1986; Fugger t'/a/. 1988).Gtyeerol-based cryoprotectant solutions have been favouredfor blastocyst cryopreservation (Coben et al., 1985).

Application of vitrification technology to human embryoeryopreservation has received much attention in reeent years.Vitrification is an ultra-rapid metbod of cryopreservation

© 2007 Published by Reproduciive Hcalihcare Lid. Duck End Farm, Dry Drayltin, Cambridge CB3 8DB. UK

Article - Cryoloop vitrification and pregnancy - N Desai et al.

whereby the embryo is transitioned from 37°C to -I96°Cin <l s. High concentrations of cryoprotectants and highcooling rates are necessary to get the embryos into a 'glass-like state' (Vajta and Kuwayama, 2006). Direct exposure ofembryos to liquid nitrogen and Introduction of novel carriersystems that minimize vitrihcation solution volumes havebeen instrLuncnlal in achieving the rapid temperature shiftnecessary for this technique to be successful (Park et ai,2000).

Human embryo vitrification has been attempted with a varietyof vessels such as electron microscope grids (Park et aL. 2000;Son et al.. 2002). open pulled and hemi-straws (El-Danasouriand Selman. 2001; Vanderzwalmen et al.. 2002. 2003). theflexipipet (Liebermann ci at,. 2002). the cryotop (Kuyania.2006) and the cryoloop (Lane et al., 1999; Mukaida ei al,,2001. 2OO3a.b; Reed et at.. 2002; Rama Raju et at., 2005).

To date, clinical reports using vitrification have been limitedand mainly involve ooeyte (Yoon el al,, 2003; Kuwayamaet at.. 200Sb: Kuwayama, 2006) or blastocyst-stagecryopreservation (Hiraoka et al,, 2004; Huang ei at.. 2005;Kuwayama et al.. 2t)05a; Stehlik ei at., 2005; Takahashi etat., 2005; Zech et at., 2005; Kuwayama. 2006; Liebermannand Tucker, 2006). Cryopreservation of human embryos atthe 6- to 8-cell stage using vitrification methodology has notbeen widely reported (Mukaida ('/a/.. 1998; Saitoc/c//.. 2000;El-Danasouri and Selman. 2001; Rama Raju et at.. 2005; Zhuetat..2O(i5).

This report presents initial clinical results followingvitrification of human embryos on day 3 of culture at the 6-to 8-cell stage. Post-warming embryo morphology and itsrelationship to pregnancy outcome were assessed. The paperalso describes e.xperience with the cryoloop as a carrier forthe vitrification procedure.

Materials and methods

Patients

Vitrification was introduced into the authors' clinical practicein November 2002 for cryopreservation of cleavage-stageembryos at the 6- to 8-cell stage, immediately followingfresh transfer. Seventy-seven couples undergoing initial IVFtreatment between November 2(K)2 and April 2006, relumed fora subsequent warming-cycle. All vitrification-warming cycleswere retrospectively analysed. No patient selection or exclusioncriteria were used,

Ovarian stimulation

Ovulation induction was carried out after down-regulation withIcuprolide acetate (Lupron; TAP Pharmaceuticals. Lake Forest.lL, USA). FSH (Follistim; Organon. USA or Gonal F; Serono.Italy) was initiated at a dose of 225 lU per day. unless previousstimulations indicated otherwise. When at least two follicleswere of 18 mm mean diameter, lO.O(X) IU of human chorionicgonadolrophin (HCG) was administered 36 h before scheduledooeyte retrieval. Oocytes were recovered by transvaginalaspiration of follicles under ultrasound guidance.

Embryo culture

Patients" oocytes were fertilized by intracytoplasmic sperminjection 3 ^ h after retrieval. Fertilization check was performedlhe following moming.Zygoles were cultured individually in 20/(I media drops under an oil overlay. Human lubal lluiil medium(HTF; Life Global. Ontario. Canada) supplemented with 10%synthetic serum substitute (SSS; Irvine Scientific. USA) wasused for embryo culture until day 3. All culture was perfomiedat 37"C with 5.5% CO, and air. Fresh embryo transfers wereperformed on day 3. Good quality embryos not selected fortransfer were considered for vitrification on day 3 if they werebetween 6- and 8-cells with <2i.W( fragmentation. Laboratorypractice has been to vitrify 2-6 good quality embryos on clay 3and culture all remaining embryos to the blastocyst stage priorto cryopreservation.

Vitrification procedure

Vitrification of embryos was carried oul by the two-stepprotocol of Mukaida el al, (2001). This technique usesdimethylsulphoxide (DMSO). ethylene glyeol and sucroseas lhe cryoprolectant agents. The basal medium was GlobalBlastocyst Medium (Life Global) with 20% SSS. All stepswere performed on a heated laminar (low hood at 37°C.Vitrification Solution #i consisted of 7.5% DMSO and 7.5%ethylene glycol. Following a 2-min incubation, the embryoswere moved to Viirificalion Solution #2 containing 15%DMSO, 15% ethylene glycol. 10 mg/ml Ficoll and 0.65mol/1 sucrose for 35 s. Using a fine micropipette and theaid of a dissecting microscope, the embryos were quicklyloaded onto a cryoloop (Hampton Research, Laguna. CA.USA) covered with a thin lilm of the same cryoprotectani(Figure la.b). The cryoloop with embryos wasimmediately plunged into a vial filled with liquid nitrogen.The liny amouni of fluid used for cryoloop vitrilication (<1//I) necessitated careful handling of the viirilicd sample loprevent inadvertent warming during transfer lo and from thelarge nitrogen storage tank. Each vial was snapped onto aseparate cane to facilitate quick transfer of samples. Allhandling of vials containing viirified embryos was donewhile keeping the samples immersed in liquid nitrogen orvery briefly (<l min) in the vapour phase. Canes were placedin a liquid nitrogen storage tank that was dedicated exclusivelylo vitrified embryos.

Warming procedure

Warming of vitrified embryos was also performed al 37''C.The cryoloop was directly immersed in a warming solutioncontaining 0.25 mol/l sucrose. The embryos that fell offthe eryoloop were visualized using the dissecting microscope.Following a 2-min rinse, the recovered embryos were movedto the second warming solution, which contained 0.125mol/1 sucrose. The embryos were incubated for 3 min.The final rinse was in culuire media for 5 min beforeplacement in an incubator at 37''C with 5.5% CO,. Thewarmed embryos were cultured in Global Blastocyst Mediumwith 10% SSS until transfer. Cell damage was assessedimmediately upon warming. Embryos were considered to bedamaged if fewer than half of their blaslomeres were intaciupon warming. The authors' policy has been to transfer all

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Article - Cryoloop vitrilication and pregnancy - A' Desai et al.

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Figure 1. (a) Loading of an embryo onto a cryoloop during the vitrification procedure, (b) Embryo on a cryoloop viewed under aninverted microscope. Original magnification xlOO.

warmed embryos with any sign of vitality regardless ofmorphology. Only completely degenerated embryos wereexcluded from transfer.

Embryo replacement

Patients were prepared for vitrified-warmed embryo transferusing hormone replacement therapy consisting of increasingdoses of estrace (2-6 mg p.o. daily) and progesterone {50-100 mg i.m.) starting on day 13. The only monitoring inthe replacement cycle was the evaluation of endometriumprior to progesterone administration. Warming cycles werecancelled if the endometrial thickness was <7 mm. Vitrifiedcleavage-stage embryos were warmed and cultured for 48 hbefore transfer on day 5 of progesterone administration. Post-warming survival and daily development were monitored.All embryos were photographed at three time points: atvitrification, immediately upon warming and just prior totransfer. Embryo transfer was performed under ultrasoundguidance using a Wallace Sure View catheter (IrvineScientific). Pregnancy testing was performed 15 days afterthe embryo transfer. Clinical pregnancy was confirmed byIhe presence of a fetal heart beat on ultrasound examinationat 6-8 weeks. The implantation rate was derived from thenumber of fetuses with a heart beat divided by the totalnumber of embryos transferred.

Results

Table 1 presents results for human embryos vitrified atlhe 6- to 8-cell stage. The data set represents consecutivevitrification-warming cycles performed over a 2.5-yearinterval. A total of 236 embryos were warmed. The averagenumber of embryos transferred per patient was 2.66 ± 0.86.All warming cycles resulted in a transfer.

The clinical pregnancy rate was 44% (34/77) and theimplantation rate was 20̂ /r (40/201). The embryos wereevaluated for blastomere number and cytoplasmic integrity.The post-warming survival rate was 85% (201 /236).Only 4.5% of surviving embryos had outward signsof injury and considerable hlastomere cell loss on thawing.Figure 2 shows the excellent morphology typically seenfollowing warming of vitrified human embryos.

It was found thai 78% (184/236) of warmed embryosshowed signs of embryonic compaction and/orblastulation by the time of transfer. The bUistulationrate from warmed cleavage-stage embryos was 44% after48 h in culture. Predominantly early stage blastocysts werenoted during lhe morning observation but by lateafternoon expansion was clearly evident in high qualityembryos. The timely post-warming development ofvitrified embryos appeared to be an excellent indicator ofsubsequent developmental potential. Morphology on theday of viirified-warmed embryo transfer was associatedwith pregnancy outcome. In transfers where at least onewarmed embryo reached the blastocyst stage by the day oftransfer, the pregnancy rate was 58% (28/48). In contrast,when warmed embryos developed only lo the morula/compacting stage by ihe transfer day. lhe pregnancy rate wasnotably lower, at 26% (6/23). No pregnancies occurred whenthe embryos failed to show signs of compaction by the dayof transfer (0/6).

Thirty singleton pregnancies and two sets of twins andtriplets were conceived. To date, there have been 11 deliveriesof healthy infants and two miscarriages. No congenitalanomalies have been reported.

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Article - Cryoloop vitrification and pregnancy - N Desai et al.

Table I. Results of vitrification-warming cycles of 236 humanembryos vitrified at the 6-8-cell stage.

Parameter

No. of warming cyclesNo. of transfersPatient age (years)"Post-warming survival rate (%)No. of embryos warmed per cycle"No. of embryos transferred per patient"Clinical pregnancy rate (%)Implantation rate (%)

Value

111134.1 ±4.5201/236(85)3,1 ± 1.02.7 ±0.934/77 (44)40/201 (20)

'Mean ± SD.

Figure 2. (a) Vitrified human embryos Immediately after warming, (b) Wanned embryos after 24 h in culture. In a and b, originalmagnification x300.

Discussion

The present series, achieved exclusively with cryoioopvitrification, represents the largest clinical series to date showingsuccessful vitrification of day 3 cleavage-stage embryos.

Vitrification technology has been shown to be a very promisingalternative to slow-freezing mcthtxlology for cryopreservation ofchill-sensitive cells such as ootytes (Hong et at,. 1999; Chung etat., 2000; Kuleshova and Lopata. 2002; Liebermann et at., 2003;Yoon et al., 2003; Chian et al., 2004; Liebermann and Tucker,2004; Kuwayama et aL, 2005b; Kuwayama. 2006) and even forbiastocyst-stage embryos with large fluid volumes (Lane et ai.

1999; Reed et al., 2002; Mukaida et ai. 2003b; Vanderzwalmenet al,, 2003; Liebermann and Tucker. 2004, 2(K)6; Walker etaL, 2004; Kuwayama et ai, 2005a. 2(X)6; Stehlik et al,, 2(H)5).Numerous publications describe blastocyst-stage vitrification andclinical outcomes (Mukaida et ai. 2001.2(K)3a; Reed et al.. 2(X)2;Vander7.walmen et ai. 2002. 2(X)3; Hiraoka et ai. 2004; Huang etal., 2005; Kuwayama et ai. 2()O5a; .Stehlik et al,, 2005; Takahashiet ai. 2005; Liebermann and Tucker. 2006) using a variety ofdifferent techniques. Pregnancy outcomes in these early trialswith vitrified human blastocysts have ranged from 23 to 53%.

Clinical application of vitrification technology to human cleavageembryos at the 6- to 8-cell stage has nol been widely explored.Mukaida and colleagues (Mukaida ei ai. 1998) reported high

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Article - Cr_yoloop vitrification and pregnancy - A* Desai et ai

survival of 8-cell embryos vitrified with 40% ethyiene glycolcombined with Ficolt and sucrose, but pregnancy rates wereonly 5.5%. Saito et ai (2000) obtained three clinical pregnanciesand two deliveries in 31 transfers with vitrified cleavage-stageembryos. Two subsequent studies (El-Danasouri and Selman,2001; Rama Raju et ai, 2(X)5) used a similar two-step prot(K;olwith 10^ ethylene glycol, followed by a 40% ethyiene glycol-sucrose step for vitrification, but omitted Ficoll from theirvitrification solution. Pregnancy rates were 31 (11/36) and 35%(14/40) using the open straw method (El-Danasouri and Selman.2001) and a nylon loop (Rama Raju ei al., 2005) respectively.The implantation rate per warmed embryo transferred was 10 and15% respectively.

The present clinical trial, in contrast to the aforementionedstudies, used a combination of DMSO and ethylene glycol.It was thus possible to use lower concentrations of individualcryoprotectants. thereby possibly reducing any cytotoxic effects(Kasai et ai. 1992; Zhu et ai. 1993; Archer et ai. 2003). Thecurrent two-step protocol adapted from Mukaida et ai (2001.2002) involved exposing embryos to 7.5% DMSO/ethyleneglycol for 2 min followed by 35 s in the final vitrification solutioncontaining 15% DMSO/ethylene giycol. sucrose and Ficoil.This fonnulation was originally developed for biastocyst-stagecryopreservation. and to date there have been no clinical reportson day 3 cleavage-stage embryos using this protocol.

One positive attribute following vitrification-wanning withthis protocol was the remarkably low percentage of embryoswith considerable cellular damage (4.5%). Historically, with theconventional slow freezing protocol using cryovials. almost aquarter of human embryos surviving the freeze-thaw pr(K:esshad greater than 50% damage, resulting in blastomere toss anddegeneration (Desai et aL. 2(X)3). Higher damage rates withconventional slow freezing compared with vitrification have beenobserved by others (Walker el ai. 2004). Cell loss on thawing hasalso been related to reduced pregnancy rates (Archer ei aL. 2(K)3).

Another advantage of the vitrification procedure was that itwas extremely efficient, taking <5 min as compared with themoi-e lengthy programmed slow freezing protixols, whichrequired a minimum of 2 h per run and several freezer units toaccommodate cryopreservation for all patients. Introductionof cryoloop vitrification to the clinical laboratory did.however, require extensive technician training with mouseembryos. The embryologist's speed and skill in performing thevitrification-warming procedure were paramount to success.

This series was also unique in that wanned cleavage-stageembryos were cultured until the morula/early blastocyst stageprior to transfer. Culture for this additional 48-h interval allowedbetter assessment of post-warming damage and embryonicpotential after vitrification. This work demonstrates a clearrelationship between einbryt>nic compaction after warming andsubsequent developmental potential. Lieberman and Tucker(2002) using embryos derived from abnormally fertilized zygotescultured to day 3. found that only 39% of vitrified-warmedembryos were capabie of compaction. The high compaction rate(78%') observed in the present study was reflective of overallembryo quality on wanning. It also attests to the suitability ofthis vitrification procedure for human embryo cryopreservation.Vitrification at the cleavage .stage allowed more embryos to bevitrified per patient and idleviated the need for extended culture

of large numbers of spare embryos. The patient also benefited byhaving embryos frozen at both early and late stages.

The pregnancy and implantation rates achieved in this seriesare indicative of the tremendous potential of vitrification in theciinicai setting. The ciinicai pregnancy rate of 44%' and the embryoimplantation rate of 20% were higher Ihan historical results wilhslow progriuiimed freezing (Desai et ai, 2003). Introduction ofthis technology into the laboratory allowed for more efficientcryopreservation of palient embryos with good success rates.

One issue with vitrification on cryoloops is that it involves directexposure to liquid nitrogen. The risk of cross-contamination inliquid nitrogen storage containers even at -I96"°C has beenwidely debated (Bicianski el aL. 2000. 2(X)3). This concern wasaddressed in part by keeping all vitrified embryos in a separate,newly purchased tank. With cryoloops. the voiume of fluidin each vial was iess than 1 /d and at its maximum capacitythe liquid nitrogen storage Dewar couki handle 320 vials (onevial per cane). With less ihan 320 //I of fluid in the entire 79 Itank, it vvas felt that the risk of possible cross-contaminationwas minimal. Other 'closed' systems for vitrification are beingconsidered, but most involve heat-sealing ofthe can'ier vessel. Ifheal is transfen-ed to a sample, it could have a negative impact,especially with vitrification, since fluid volumes are so small.

In conclusion, initial experiences with vitrification of humanembryos were positive. The vitrification prcvedure has beenadapted successfully to the authors" IVF laboratory after a |ieri(Klof employee training, and the clinical pregnancy and implantationrates were promising. Moreover, it was found that the cryoloop wasan excellent carrier for ultra-rapid cryopreservation of embryos.This study suppoiis the effectiveness of a DMSO/ethylene glycolcryoprotectant combination for viunfication of human embryos atthe 6- to 8-cell stage. Moreover, ethylene glyco! could be usedat far iower concentrations than has previously been rt'poiiedfor cieavage-stage vitrification. Il remains to be determined ifthis same protocol can be applied to other cell stages with equalsuccess.

Acknowledgements

The authors thank the Cleveland Clinic Fertility Centre team ofdoctors, nurses and embryologists. We are also grateful to DrFaten Abdel-Hafez for her assistance in manuscript preparation.

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Presented at tbe Conjoint Annual Meeting of the Atnerican Societyof Reproductive Medicine and tbe Canadian Fenility Association,Montreal. Canada October 15-19, 2005.

Received II October 2006: refereed 6 November 2006; accepted 27November 2006,

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