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Aseptic vitrification of blastocysts from infertile

patients, egg donors and after IVM

Dr Pierre Vanderzwalmen

After graduating in biotechnology and biochemistry, Pierre Vanderzwalmen entered the field

of embryology in 1978 in the Veterinary Faculty of the University of Liege, Belgium. He then

joined the team of Dr Massip, developing vitrification techniques. In 1989, he moved to the

Schoysman Infertility Management Foundation where he spent 14 years, during which time

the laboratory obtained the first TESE baby. At present, he is coordinating the scientific IVF

activities in the Institute of Professor Zech in Bregenz, Austria and of Professor Lejeune in

CHIREC, Belgium. His current research interests focus on vitrification of oocytes and

embryos, on selection of spermatozoa and embryo culture techniques.

P Vanderzwalmen1,2,3,5,9, F Ectors2, L Grobet3, Y Prapas4, Y Panagiotidis4, S Vanderzwalmen5, A Stecher1,

P Frias6, J Liebermann7, NH Zech1,8

1IVF Centers Prof. Zech, Bregenz, Austria; 2FNRS, GIGA Transgenic Platform, University of Lie` ge, Lie` ge, Belgium;

3GIGA-Research, University of Lie` ge, Lie` ge, Belgium; 4Iakentro IVF Centre, Thessaloniki, Greece; 5Centre Hospitalier

Inter Re´ gional Cavell (CHIREC), Braine l’Alleud – Bruxelles, Belgium; 6Fertility and Sterility National Center

(CENALFES), Cochabamba, Bolivia; 7Fertility Centers of Illinois, Chicago, IL, USA; 8Unit of Gynecological

Endocrinology and Reproductive Medicine, University of Graz, Austria;

9Correspondence: e-mail:


During embryo vitrification, it is advisable that cooling and storage should occur in a carrier device in which there is

complete separation of the embryos from liquid nitrogen to ensure asepsis. The consequence of a reduction in the

cooling rate resulting from the heat-insulating barrier aseptic devices has to be counteracted by gradually increasing

intracellular concentrations of cryoprotectants without inducing a toxic effect. Blastocysts originating from couples

with male and/or female factor infertility (group 1) or from oocyte donors (group 2) or from in-vitro matured

oocytes (group 3) were gradually exposed to increasing concentrations of dimethylsulphoxide/ethylene glycol

(5/5%, 10/10% and 20/20%) before aseptic vitrification using a specially designed carrier (VitriSafe), a modification

of the open hemi-straw plug device. A total of 120 aseptic vitrification/warming cycles were performed in group 1, 91

in group 2 and 22 in group 3. Survival rates before embryo transfer, ongoing pregnancy and implantation rates were

as follows: for group 1, 73, 43 and 26%; for group 2, 88, 53 and 34%; and for group 3, 69, 50 and 38%, respectively.

In spite of reduced cooling rates due to aseptic vitrification conditions, a three-step exposure to cryoprotectant solutions

protects the embryos effectively from cryo-injuries and guaranties high survival rates.

Keywords: egg donation, embryo carrier device, human blastocyst, in-vitro maturation


Vitrification is a cryopreservation procedure by which solutions

are converted into a glass-like amorphous solid, free

of any crystalline structures. After the initial application

of vitrification to cleaving embryos (Mukaida et al., 1998)

or blastocysts in closed 0.25 ml insemination straws (Yokota

et al., 2001; Vanderzwalmen et al., 2002), the trend was

to increase significantly the cooling and warming rates from

<2000C/min to >20,000C/min in order to reduce the likelihood

of lethal ice-crystal formation in the crystalline

phase (Lane et al., 1999). Several embryo carrier devices

(open systems) were therefore designed (Vajta and Nagy,

2006) in a way to allow a direct contact of the biological

sample with liquid nitrogen in order to permit cooling of

embryos instantaneously below the glass transition temperature

where cells are captured in an amorphous state.

Under such ultra-rapid cooling conditions, a vitrified state

is obtained even if embryos are exposed for only a very

short period of time to high concentrations of the cryoprotectant

solutions. This allows the extraction of intracellular

water while limiting the amount of cryoprotectant permeating

into the cells. During the warming process, the open

devices allow for instantaneous release of cryopreserved

RBMOnline - Vol 19. No 5. 2009 700–707 Reproductive BioMedicine Online; on web 28 September 2009

 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK


Author's personal copy

embryos into a diluting solution. In this way, extremely

high warming rates (>20,000C/min) can be achieved and

the recrystallization phenomenon avoided. Most publications

on the clinical application of ultra-rapid vitrification

have mainly focused on the blastocyst stage (Lane et al.,

1999; Son et al., 2002; Mukaida et al., 2003; Vanderzwalmen

et al., 2003; Hiraoka et al., 2004; Huang et al., 2005;

Kuwayama et al., 2005; Stehlik et al., 2005; Takahashi et al.,

2005; Liebermann and Tucker, 2006; Balaban et al., 2008).

Although satisfactory results were reported, one major

drawback to the ultra-rapid cooling process is the possible

risk of bacterial as well as viral contamination of the biological

sample during the cooling process as well as during

long-term storage (Bielanski, 2005; Morris, 2005). However,

the question of contamination during storage in liquid

nitrogen remains debatable (Kyuwa et al., 2003). Nevertheless,

a European Parliament directive on tissues and cells

(European Union, 2004), and the revised version (European

Union, 2006), has defined medical safety requirements for

the cryopreservation of human cells. In view of the issues

raised by this directive, it was imperative to revise this study

centre’s ultra-rapid vitrification procedure (Vanderzwalmen

et al., 2002, 2003) and to develop a new vitrification technique

ensuring total protection and isolation of the sample

from the liquid nitrogen during the cooling procedure as

well as during long-term storage.

Hermetically closed containers (closed systems) have already

been developed to vitrify mice and human oocytes and

embryos (Kuleshova and Shaw, 2000; Walker et al., 2004;

Isachenko et al., 2005, 2007; Kuwayama et al., 2005; Larman

et al., 2006; Stachecki et al., 2008). Kuwayama et al. (2005)

developed the Cryotip device, allowing isolation of

biological material from liquid nitrogen. In the first report,

Kuwayama et al. (2005) showed similar pregnancy rates

after vitrification of blastocysts in an open (53% with the

Cryotop device) and in a closed system (51% with

the Cryotip device). Nevertheless, in a more recent paper,

the superiority of the open Cryotop system was recognized

(Kuwayama, 2007). At present, the clinical application of

the Cryotip device remains sporadic and ultra-rapid vitrification

with the Cryotop device is still the method of choice

in the majority of assisted reproduction laboratories.

Recently, Stachecki et al. (2008) reported encouraging

results after vitrification of human blastocysts in closed

0.25 ml straws. After warming, a survival rate of 89% was

obtained. Out of 43 transfers, clinical pregnancy and implantation

rates of 60% and 45% were recorded, respectively.

Therefore, to achieve maximal survival rates with hermetically

sealed and thus aseptic embryo carriers, the problem

relating to the heat-insulating barrier, which dramatically

reduces the speed of cooling from >20,000C/min to

<2000C/min, has to be solved.

Since the probability of fixing the intracellular parts into a

glass-like state depends on the cooling–rewarming speeds

and the concentration of cryoprotectant (Yavin and Arav,

2007), it is obvious that the risk of ice-crystal formation will

increase if the cells have not been exposed long enough to

cryoprotectant solutions. Consequently, the decrease in

the speed of cooling and rewarming, as observed with aseptic

devices, has to be compensated by higher intracellular

concentrations of cryoprotectant. As a consequence, aseptic

cooling, by which the embryos are shielded from liquid

nitrogen, may have an adverse effect on embryo survival

after warming if the embryos are not exposed sufficiently

to the cryoprotectants at the beginning of the procedure.

The initial aim of this study was to construct an aseptic

embryo carrier device easy to handle and guaranteeing

medical safety of vitrified human embryos. With the aim

of achieving adequate intracellular vitrified conditions

before aseptic cooling without inducing cell toxicity, two

parameters regarding the optimum cryoprotectant concentration

and the adequate exposure time to the different cryoprotectants

were studied. Finally, this study reports

further on the results of the clinical application of aseptic

vitrification of blastocysts originating from couples with

male and/or female factor infertility or from oocyte donors

and also after in-vitro maturation (IVM).

Materials and methods

The development of an aseptic vitrification technique was

performed after informed consent on supernumary human

blastocysts that were not eligible for a fresh embryo transfer

or for ultra-rapid vitrification.

The embryo carrier device: the VitriSafe


The VitriSafe (VitriMed, Austria) is an aseptic embryo carrier

device specifically designed for the study centre, based

on a modification of the open hemi-straw plug device (Vanderzwalmen

et al., 2003). It consists of a large gutter in

which a small quantity of cryoprotectant (<1 ll) containing

the blastocysts can be deposited (Figure 1). Before plunging

the biological material into liquid nitrogen, the VitriSafe is

inserted into a high-security 0.3 ml straw (Cryo Bio System,

France). Welding of both edges of the outer protective straw

took place before being plunged into liquid nitrogen. This

ensures a hermetic isolation of the sample. Under these conditions,

the cooling rate decreased from above 20,000C/

min to less than 1300C/min. Measurement of the cooling

rate was carried out between 20 and 120C with a very

thin thermocouple (Digital thermometer GMH 3230; Greisinger

Electronic, Germany) introduced in a small drop

located in a 0.3 ml straw (Cryo Bio System).

For warming, the top of the outer straw is cut and the Vitri-

Safe device is removed without contact with the liquid

nitrogen. In order to achieve an ultra-rapid warming rate

of >20,000C/min, the tip of the gutter containing the biological

material is instantaneously immersed into the dilution


Solutions used for vitrification and dilution

after warming

Three non-vitrifying solutions (NVS), 2.5/2.5, 5/5 and

10/10 NVS and one vitrification solution (20/20 VS) were

Article - Aseptic vitrification of blastocysts - P Vanderzwalmen et al.