Effect of vitrification procedure on chromosomal status of embryos achieved from vitrified and fresh oocytes

doi:10.4236/health.2011.37077

Paper Menu >>

Journal Menu >>

Vol.3, No.7, 467-476 (2011)

doi:10.4236/health.2011.37077

Health

Effect of vitrification procedure on chromosomal s tatus

of embryos achieved from vitrified and fresh oocytes

Javier Israel García*, Luis Nori ega-Portel la, Luis Noriega-Ho ces

Laboratory of Assisted Reproduction, PRANOR Group of Assisted Reproduction, Concebir Clinic, Lima, Peru.

*Corresponding Author: jgarciaf@hotmail.com

Received 27 April 2011; revised 9 June 2011; accepted 28 June 2011.

ABSTRACT

background: In order to assess the chromoso-

mal status in embryos obtained from vitrified

and fresh donated oocytes, preimplantational

genetic diagnostic (PGD) was performed after

biopsy of one blastomere at day 3. METHODS: A

total of 249 oocytes were obtained from 23 oo-

cyte donors, 80 oocytes were used in the vitri-

fied group and 151 oocytes were used in the

fresh group. Nine chromosomes (13, 15, 16, 17,

18, 21, 22, X and Y) were investigated by fluo-

rescence in situ hybridization (FISH) analysis in

56 and 121 embryos from vitrified and fresh

group respectively. Fertilization, cleavage rate,

embryo quality and chromosomal abnormality

rate were compared between evaluated groups.

Results: Vit rified o ocytes sho wed a su rviv al rate

of 97.5%. There was no significant difference in

the fertilization rate (82.7% and 91.4%), Day 2

cleavage rate (90.3% and 87.7%) or blastocyst

formation ra te ( 31.1% and 44.6% ) for the v i trifie d

and fresh groups respectively. Chromosomal

abnormality rate (66.1% versus 71.9%), per-

centage of abnormal blastocysts (61.1% versus

64.8%) and percen tage o f abnormali ties f or each

analyzed chromosome were similar for the vitri-

fied group compared with the control group.

Conclusions: The rates of chromosomal ab-

normalities in embryos from vitrified oocytes

are similar to those published previously; and

comparable to those observed in embryos from

fresh oocytes. These results confirm that the

developmental competence and chromosomal

status of embryos obtained from vitrified oo-

cytes is not affected by the vitrification proce-

dure, and they preserve the potential to be fer-

tilized and to develop in to blastocyst stage

similar to embryos from fresh oocy tes.

Keywords: Oocytes; Vitrification; PGD; FISH; ART

1. INTRODUCTION

Human oocytes cryopreservation is an attractive

choice to the range of fertility treatments presently of-

fered. Since the very first frozen oocyte pregnancy was

achieved in humans by Chen [1], the slow cooling me-

thod has been applied with varying success [2-7]. There

are many reports about the cryopreservation of human

oocytes with good fertilization and cleavage rates [1,8]

but with variable results on pregnancy rates per cycle

[9-13]. A recent meta-analysis on slow freezing revealed

that the clinical pregnancy rate per transfer with this

method was 20.6%, and 2.3% per thawed oocyte [14].

As an alternative to slow freezing, the vitrification

procedure has been recently applied for human oocytes

and embryos [15,16] allowing to improve the percent-

ages of oocyte survival, fertilization, embryo develop-

ment rates and clinical outcomes [17-26]. Vitrification is

a non-equilibrium method and may be regarded as a

radical approach in which ice crystal formation is totally

eliminated. Nevertheless, it requires an extremely high

cooling rates and high concentrations of cryoprotectants,

compared to slow freezing [16], in which a solution/

specimen solidifies to form a glass-like, or vitreous,

structure without any ice crystal formation during cool-

ing. This state is maintained throughout the whole

warming procedure [22].

The difficulties associated with oocyte cryopreserva-

tion are mostly related to the special structure and sensi-

tivity of this cell, the exposure time to the cryoprotec-

tants solutions, the concentration of the cryoprotectants,

the equilibrium temperature and the extra and intracellu-

lar ice formation factors that affect the viability [10] and

physiology of oocytes [27]. Additionally, other biologi-

cal characteristics of human oocytes that may be suscep-

tible to damage during the cryopreservation procedure

are precocious oocyte activation induced by cryoprotec-

tants exposures that may disturb future development

J. I. Garc ía et al. / Health 3 (2011) 467-476

468

[27,28]; loss of high mitochondrial polarity associated

with a significantly reduced capacity to up-regulate the

levels of intracellular free calcium after thawing [29];

microvacuolization in the ooplasm and ultrastructural

alterations in specific oocyte microdomains linked to the

reduced developmental potential of mature cryopre-

served oocytes [30].

Proper organization of the oocyte cytoskeleton, par-

ticularly of its microtubular fraction, is essential for

normal spindle formation and chromosome segregation

[30]. The meiotic spindles are crucial for the events fol-

lowing fertilization in the completion of meiosis, second

polar body formation, migration of pronuclei, and for-

mation of the first mitotic spindle [31]. The meiotic

spindle has been shown to be extremely sensitive to

temperature variation [32-41], and to depolymerize at

low temperatures [42,43]. Subsequent impaired repoly-

merization on rewarming may lead to scattering of

chromosomes or lesions in the reformed spindle, result-

ing in misaggregation of chromatids following resump-

tion of meiosis and an increase of aneuploidies incidence

in oocytes [44] and later in embryos [45,46].

On the other hand, several studies evaluating the ef-

fect of vitrification and slow-freezing on the meiotic

spindle integrity and chromosome alignment [47-51],

showed less damage in vitrified oocytes (as observed in

fresh oocytes without cryopreservation) compared with

cryopreserved oocytes by slow-freezing. However, stud-

ies of Coticchio et al. [52]; Martinez-Burgos et al. [53]

showed that the vitrified-warmed oocytes maintain a

metaphase II (MII) spindle with a bipolar organization,

but the chromosomal alignment appears to be partly

compromised. Additionally, studies of Cobo et al. [19],

Chen and Yang, [36] and Ciotti et al. [54] reported that

spindle recovery was faster in vitrification than in slow

freezing (1-2 hours after thaw procedure). However, most

of the available information about of chromosomal status

comes from studies performed on cryopreserved oocytes,

but not on the embryos derived from those oocytes.

The introduction of preimplantation genetic diagnosis

(PGD) to the field of reproductive medicine and the

analysis of a single blastomere from a day 3 embryo

using multicolor fluorescence in-situ hybridization

(FISH), permit to detect chromosomal abnormalities and

inherited diseases without adversely affecting the de-

velopmental [55] or implantation potential of the embryo

[56,57]. Routinely, up to 9 to 12 chromosomes can be

analyzed on a single fixed nucleus by FISH, in which

DNA probes labeled with fluorochromes hybridize to

their respective chromosome, allowing for the identifica-

tion of ploidy status. The indications for PGD during an

IVF cycle are advanced maternal age, repeated implant-

tation failure, recurrent pregnancy loss and, previous

trisomic conception [58].

The relationship between chromosome abnormalities,

embryo morphology and maternal age has been studied

extensively [50-62]. Approximately 30% of human em-

bryos generated from IVF treatments have an abnormal

chromosome constitution [63,64], and this percentage

may increase to 60% - 70% in embryos that come from

poor prognosis IVF patients such as low responders,

older women [65-67], IVF failure [56,68,69], or women

with a history of recurrent miscarriage [63,69-73]. In a

recent study, involving an examination of nine chromo-

somes at the cleavage stage via FISH, 60% of all ana-

lyzed embryos derived from women younger than 35

years were found to carry cytogenetic anomalies. The

observed abnormality rate increased to 80% for embryos

of women 41 years of age and older [74].

On the other hand, young egg donors are traditionally

assumed to have low rates chromosome abnormalities,

and young donors less than infertile patients, even those

with comparable ages. The scanty published PGD data

on embryos derived from oocytes donors indicate unex-

pectedly high rates of chromosome anomalies (56% -

57%) [50,75]. According to Reis Soares et al. [75] the

reason for the observed abnormalities, could be that do-

nors are frequently subject to more aggressive stimula-

tion, compared with other women of similar age, in order

to guarantee the production of a large cohort of oocytes.

Nevertheless, the effect of cryopreservation proce-

dures on chromosomal status of embryos achieved from

cryopreserved oocytes has been little studied. Cobo et al.

[76] analyzed the effect the cryopreservation by slow

freezing on the chromosomal status of embryos obtained

from frozen-thawed oocytes. They showed 28.6% and

26% of aneuploidies in embryos obtained from cryopre-

served and fresh oocytes respectively.

This study was designed to compare the rates of chro-

mosome abnormalities observed in embryos obtained

from vitrified and fresh donated oocytes and to evaluate

whether the vitrification procedure increase significantly

more chromosome abnormalities.

2. MATERIALS AND METHODS

2.1. Patients

This study is based on secondary analysis of data ob-

tained from procedure of Preimplantational Genetic Di-

agnostic (PGD) program in the Laboratory of Assisted

Reproduction at the Concebir Clinic (Lima, Peru) carried

out over 2009. This study was approved by the Institu-

tional Review Board (IRB) and the corresponding Ethics

Committee. Written informed consent forms were ob-

tained from donors, recipients and their partners in-

cluded in this study. The PGD indications were because

J. I. Garc ía et al. / Health 3 (2011) 467-476

469

all patients wish to avoid a pregnancy with trisomic dis-

ease.

The age of the oocyte donors ranged from 20 - 29-ye-

ar-old in both evaluated groups. They were subjected a

physical, gynecological and psychological examination

and there were no family history of hereditary or chro-

mosomal diseases. All participants had a normal karyo-

type and tested negative in a screening for sexually

transmitted diseases.

From the 23 oocyte donors included, 8 were assigned

to the vitrified oocytes group and; 15, to the fresh oo-

cytes group. A total of 82 and 167 oocytes were obtained

from vitrified and fresh oocytes groups respectively.

Recruitment of oocyte donors was done based on

recommendations given by other donors and the dona-

tion of their gametes was merely by altruistic reasons.

The donors and their oocytes were random and consecu-

tively assigned each evaluated group. They were matched

with their recipients according to phenotype and blood

groups; and the recipients were not aware not of the ori-

gin of oocyte received (vitrified or fresh).

2.2. Controlled Ovarian Stimulation and

Oocyte Collection

The menstrual cycles of oocyte donors were stimu-

lated using recombinant FSH (rFSH) (Puregon®, Or-

ganon laboratories, Peru) and GnRH antagonist (Or-

galutran® Organon Laboratories, Peru) according to the

stimulation protocols previously established and starting

on Day 2 of the menstrual cycle until when at least three

follicles reached ~18 mm in diameter. The oocyte re-

trieval was performed by vaginal ultrasound 36 h after

the i.m application of Human Chorionic Gonadotropin,

hCG (Ovidrel® 250 µg, Serono Laboratories, Peru). For

the procedure, the patient was under general anesthesia

with 200 mg of Propofol iv (Diprivan® 1% P/V; Astra-

Zeneca Laboratories, UK).

During follicular aspiration procedure the oocytes

were recovered in Global®-HEPES-buffered medium

(IVFonline, Canada) supplemented with 10% vol/vol

Serum Substitute Supplement (SSS; Irvine Scientific,

USA). After retrieval, cumulus-oocyte complexes were

trimmed of excess cumulus cells and maintained in ~200

µL drops of Global®-Fertilization medium (IVFonline,

Canada) plus 10% SSS under oil at 37˚C and an atmos-

phere containing 5.6% CO2, 5% O2 and 89.4% N2, for 2

hours until when the oocytes recovered were cryopre-

served (vitrified group) or 5 hours until when the oo-

cytes recovered were inseminated (fresh group).

All collected oocytes were denuded enzymatically of

cumulus cells with hyaluronidase (80 IU/mL; IVFonline,

Canada) to assess nuclear maturity. Only metaphase II

(MII) oocytes were vitrified with Cryotop minimum

volume vitrification methods [21,48,77] using comer-

cial cryoprotectant solutions (Cecolfes, Colombia) and

the Cryolock device (Biodiseño-Colombia), for a maxi-

mum period of 3 hours after of retrieval.

2.3. Oocyte Vitrification

A total of 80 MII oocytes were vitrified which corre-

sponding to the vitrified group by minimum volume

methods, which minimize the volume of vitrification

solution containing oocytes increasing the cooling and

warming rates and decreasing the chance of ice crystal

nucleation/formation in the small sample [16,78]. The

vitrification procedure was performed at room tempera-

ture (24˚C). The oocytes were equilibrated in 7.5%

vol/vol ethylene glycol (EG) plus 7.5% dimethylsulfox-

ide (DMSO) in TCM199 medium for 15 minutes (equi-

librium solution, ES) and then placed in 15% EG plus

15% DMSO plus 0.5 mmol/L sucrose (vitrification solu-

tion, VS) for a maximum 1 minute. The oocytes were

loaded quickly onto the Cryolock in a minimum drop

size of <0.1 µL of VS and immediately immersed di-

rectly in liquid nitrogen, at a cooling rate of approxi-

mately –23,000˚C /min [21]. A maximum three oocytes

were placed by Cryolock. All cryoprotectant solutions

were supplemented with 20% vol/vol SSS.

2.4. Oocyte Warming

Oocytes were warmed at fast warming rates of

–12,000˚C/min [21]. The Cryolock was taken out of

liquid nitrogen and quickly placed in 1.0 mol/L sucrose

in TCM199 medium (thawing solution-TS) for 1 minute

at 37˚C. The oocytes were then placed in 0.5 mol/l su-

crose in TCM199 medium (diluent solution-DS) for 3

minutes and at room temperature (24˚C), followed by

two consecutive 5-min flushes in TCM199 medium (wa-

shing solution-WS). Oocytes were then immediately pla-

ced in Global®-Fertilization medium plus 10% SSS un-

der oil at 37˚C and an atmosphere containing 5.6% CO2,

5% O2 and 89.4% N2 for 2 hours before ICSI. All warm

solutions were supplemented with 20% vol/vol SSS.

2.5. Insemination, Fertilization and Embryo

Culture

In the vitrified group, 2 h after warming the viability

of oocytes was evaluated microscopically based on the

morphology of the oocyte membrane integrity. All viable

oocytes were inseminated by intracytoplasmic sperm

injection (ICSI) with spermatozoa from recipient’s hus-

band. After ICSI procedure (Day 0), all injected oocytes

were cultures at 37˚C in an atmosphere of 5.6% CO2,

5% O2 and 89.4% N2.

In the fresh group, ~5 h after of oocyte retrieval, all

J. I. Garc ía et al. / Health 3 (2011) 467-476

470

MII oocytes were inseminated or injected, depending

about seminal characteristics, with spermatozoa from

recipient’s husband.

In both groups, the fertilization was evaluated 16 - 18

hours after insemination by presence of two pronuclei

(Day 1). The zygotes were individually cultured under

mineral oil, in 10 µL droplets of Global® medium (IV-

Fonline, Canada) supplemented with 10% vol/vol SSS

from Day 1 to Day 3. On Day 3, the embryos were biop-

sied when they had seven-eight blastomeres. After em-

bryo biopsy, the embryos were moved to fresh 10 µl

droplets of Global® medium +10% SSS.

On Day 2 and 3 the embryos were evaluated for cell

number and fragmentation; and on Day 5 for develop-

ment to blastocyst and expansion.

2.6. Embryo Biopsy, Fixation, and FISH

Analysis

Embryo biopsies were performed on the third day af-

ter insemination, at the 7 - 8 blastomeres stage following

a protocol described elsewhere [57]. Individual embryos

were placed into calcium/magnesium-free media (PGD

Biopsy Medium; IVFonline, Canada). The hole of the

zone pellucida was opened with Tyrode's acid solution

and one nucleated blastomere was removed mechanic-

cally. Blastomeres were fixed individually following a

protocol to minimize signal overlap and loss of micro-

nuclei [79]. After biopsy all biopsied embryos were

rinsed thoroughly and individually cultured under oil in

10-µl droplets of Global® medium +10% SSS at 37˚C

and an atmosphere containing 5.6% CO2, 5% O2 and

89.4% N2.

PGD analysis was performed by FISH using probes

specific for nine chromosome types X, Y, 13, 15, 16, 17,

18, 21 and 22. These probes were used because in the

past their use has shown improvements in implantation

rates after PGD (56, 57). The FISH analysis consisted of

two consecutive hybridizations following previously

published protocols [80]. The first hybridization was

performed with probes for chromosomes 13, 16, 18, 21

and 22 (Multivysion PB; Vysis, Downer’Grove, IL,

USA). The second hybridization consisted of a home-

made combination of probes for chromosomes X, Y, 15

and 17 [80]. Scoring was performed by eye without the

need of any software. If the specific signals for a chro-

mosome were not clearly diagnosable, a third hybridiza-

tion using a probe binding to a different locus for that

chromosome was used [81].

2.7. Embryo Quality

On Day 2 and 3 the embryos were evaluated for cell

number, fragmentation, and multinucleation. Good qual-

ity Day 2 embryos were defined as those with 2 - 4 cells

and ≤10% of fragmentation and absence of multinuclea-

tion. Good-quality Day 3 embryos were defined as those

with 6 - 8 cells and ≤10% of fragmentation. Good-quality

blastocysts were defined as having an inner cell mass

(ICM) and trophoectoderm type A or B [82]. The ICM

score was evaluated as follow: type A = compact area,

many cells present; type B = cells are loosely grouped.

The trophoectoderm was scored as follows: type A =

many cells forming a tight epithelial network of cells;

type B = few cells forming a loose network of cells.

2.8. Sperm Collection

The semen samples were collected by masturbation

from the recipients’ partners. Motile spermatozoa were

separated from the seminal plasma by centrifugation

through 1.0 mL 95% and 45% Isolate gradients (Irvine

Scientific, USA). For oligospermic samples the sperm

were washed and resuspended in varying amounts of

sperm washing medium depending on initial concentra-

tion and motility and then placed into 10 µL drops of

HEPES-buffered Global medium + 10% SSS for ICSI.

2.9. Statistical Analysis

Data were statistically analyzed using the χ2 test and

Student’s t-test as appropriate and differences were con-

sidered to be significant at P < 0.05. All statistical analy-

sis was carried out using the statistic package Stata 10

(StataCorp, College Station, TX, USA).

3. RESULTS

The ages of oocyte donors were similar in both evalu-

ated groups (24.6 ± 3.5 vs. 24.9 ± 2.7 years; P:NS). The

mean days of stimulation (9.4 ± 0.9 vs. 9.1 ± 0.8), mean

days of antag-GnRH (1.6 ± 0.7 vs. 1.5 ± 0.7) and rFSH

treatment (1825 ± 291.6 vs. 1801.7 ± 261.1 IU/L) were

similar between vitrified and fresh groups (data no

shown).

Results obtained from vitrified and fresh group are

shown in Table 1. A total of 82 and 167 oocytes were

collected from donors of vitrified and fresh groups, re-

spectively. In the vitrified group, there were 80 MII oo-

cytes vitrified, resulting in the survival of 78 oocytes

(97.5%). Seventy five and 151 oocytes from vitrified and

fresh groups, respectively, were inseminated. The normal

fertilization (2PN) was similar in both evaluated groups

(Vitrified group: 82.7% vs. Fresh group: 91.4%). In the

vitrified group, 90.3% of zygotes underwent cleavage on

Day 2, whereas for zygotes from fresh group, the cleav-

age rate was 87.7%. Mean cell numbers at Days 2 and 3

were similar in both groups. Embryo quality from vitri-

fied oocytes was similar to those derived from fresh oo-

J. I. Garc ía et al. / Health 3 (2011) 467-476

471

Table 1. Laboratory results in the vitrified and the fresh

group.

Vitrified

group Fresh

group

No. total oocytes

No. total MII oocytes

No. total vitrified oocytes

No. total oocytes survival (%)

No. total injected oocytes

No. total fertilized oocytes (2PN) (%)

Cleavage rate by embryo at Day 2 (%)

No. cell/embryo at Day 2 (Mean ± SD)

Good quality embryos at Day 2 (%)

No. cell/embryo at Day 3 (Mean ± SD)

Good quality embryos at Day 3 (%)

No. total blastocyst (%)

Good quality blastocyst (%)

Early blastocyst (%)

Full blastocyst (%)

Expanded blastocyst (%)

Hatching blastocyst (%)

78 (97.5)

62 (82.7)

90.3

3.63 ± 0.40

91.1

7.11 ± 1.01

80.4

18 (32.1)

66.7

33.3

44.4

16.7

5.6

167

151

138 (91.4)

87.7

3.89 ± 0.46

92.6

7.43 ± 0.97

86.8

54 (44.6)

75.9

24.1

59.3

14.8

1.8

P:NS

cytes on Day 2 (91.1% vs. 92.6%) and Day 3 (80.4% vs.

86.8%). Blastocyst development rates were similar

(32.1% and 44.6%, respectively) for the vitrified and the

fresh groups. In addition, embryos reaching the blasto-

cyst stage were morphologically similar in both groups.

Table 2 shows the results obtained after FISH analysis.

A total of 61 (98.4%) and 129 (93.5%) embryos were

biopsied from vitrified and fresh groups respectively. A

hundred percent of blastomeres biopsied were fixed in

both evaluated groups. 91.8% and 93.8% of fixed blas-

tomeres were analyzed for both the vitrified and the

fresh groups. The proportion of abnormal embryos ob-

tained from vitrified oocytes was similar to those ob-

served embryos obtained from fresh oocytes (66.1% vs.

71.9%; respectively). Additionally, from those embryos

that reached the blastocyst stage, 61.1% and 64.8% in

the vitrified and fresh groups respectively, showed

chromosomal abnormalities to nine analyzed chromo-

somes.

The percentage of abnormalities for each chromosome

analyzed in both analyzed groups is shown in Table 3.

The proportion of abnormalities according to the differ-

ent chromosomes analyzed was similar between em-

bryos obtained from vitrified and fresh oocytes.

4. DISCUSSION

Since the first pregnancy achieved with a vitri-

fied/warmed human oocyte [83] and the first birth of a

healthy baby [84], vitrification results have improved

Table 2. Results of fluorescence in situ hybridization analy-

sis in embryos obtained from vitrified and fresh oocytes.

Vitrified

group Fresh

group

No. total zygotes (2PN)

No. total biopsied embryos (%)

No. total fixed embryos (%)

No. total analyzed embryos (%)

No. total abnormal embryos (%)

No. total blastocyst

No. total abnormal blastocysts (%)

61 (98.4)

61 (100)

56 (91.8)

37 (66.1)

11 (61.1)

138

129 (93.5)

129 (100)

121 (93.8)

87 (71.9)

35 (64.8)

P:NS.

Table 3. Percentage of abnormalities for each chromosome

analyzed in the vitrified and the fresh group.

Chromosome

analyzed Vitrified Group Fresh Group

34.8%

28.3%

21.7%

19.6%

28.3%

45.7%

19.6%

26.1%

36.2%

40.8%

27.7%

21.5%

29.2%

33.1%

31.5%

23.1%

P:NS.

significantly during the last decade [17-26,85-88].

Vitrification is the glass-like solidification of a solu-

tion at low temperature, without ice crystal formation.

This is possible through the extreme elevation in viscos-

ity during freezing. This can be achieved by increasing

the freezing and warming rates and/or increasing the

concentration of the cryoprotectants [89]. Unlike slow

freezing, vitrification results in the total elimination of

ice crystal formation, within the cells being vitrified and

outside the cells in the surrounding solution [78]. Oocyte

vitrification avoids meiotic spindle damage and has re-

sulted in survival rates of over 80% [19-21,23,48,90].

Additionally, pronuclear zygotes [91], cleavage-stage

embryos [82], and blastocysts [92-94] have been suc-

cessfully vitrified.

During the present study has shown the feasibility and

efficiency of oocyte vitrification. We have showed high

recovery and post warm survival in vitrified oocytes, and

similar fertilization rates and preimplantational devel-

opment embryo with vitrified and fresh oocytes, results

that confirm data showed previously by others investi-

gators [19,20,23]. In the present study, the vitrification

procedures were all carried out using the Cryolock de-

vice, which has a similar design and handle to those of

Cryotop. The Cryolock has been designed and developed

specifically for use in vitrification procedures with slots

J. I. Garc ía et al. / Health 3 (2011) 467-476

472

in the ends and top for better manipulation, avoiding the

risk that these loosen or fall during their use or storage

(Biodiseño-Colombia). Moreover, the vitrification pro-

cedure, cryoprotectants and warming media (Cecolfes,

Colombia) and the Cryolock device (Biodiseño, Colom-

bia) used during oocytes vitrification procedure has been

showed previously to be efficient, safety and don’t affect

the viability and physiology of oocytes [20].

In recent years PGD technology has been increasingly

used by infertile patients undergoing IVF treatment, in

order to screen their embryos for chromosomal abnor-

malities. Aneuploidy is extremely common in human

embryos and leads to developmental arrest, implantation

failure and spontaneous abortion. The inadvertent trans-

fer of chromosomally abnormal embryos is believed to

explain a significant proportion of failed IVF cycles. By

screening for aneuploidy and ensuring the transfer of

chromosomally normal embryos it has been suggested

that a variety of IVF outcomes (including implantation

and pregnancy rates) can be improved.

Advances in vitrification methods have led to egg sur-

vival rate over 85% and the pregnancy rates comparable

to those achieved with autogenous and donated fresh

oocytes [17,19-21,23,25,26,48,89,95,96]. Additionally,

has been demonstrated that vitrification of in-vitro ma-

turated oocytes results in high survival rates, normal

meiotic spindle and chromosome alignment and no in-

creased incidence of aneuploidy compared to those

cryopreserved by slow freezing [97]. However, no infor-

mation is available about the chromosomal status of hu-

man embryos resulting from vitrification and warming

procedure, fertilization and preimplantational develop-

ment embryo.

In the present study, hasn’t been observed deleterious

effect of vitrification procedure on the oocyte viability

from fertilization to blastocyst stage and principally the

morphological characteristics of blastocyst at day 5,

similar results to observed by García et al. [20].

On the other hand, has been showed similar aneup-

loidy rates in embryos at day 3 (66.1% vs. 71.9%) and in

those embryos that achieved the blastocyst stages

(66.1% vs. 64.8%) from vitrified and fresh oocytes re-

spectively. Similar results were obtained by Baart et al.

[98] and Munné et al. [50] when analyzed 196 and 1800

embryos from oocyte donation cycles respectively. They

observed that 66% and 57% of embryos were abnormal

for 10 and 8 chromosomes analyzed including X, Y, 1, 7,

13, 15, 16, 18, 21 and 22. These results are direct evi-

dence that oocytes conserve intact their capability of

fertilization and potential the reach the blastocyst stage

after being vitrified similar to that observed with fresh

oocytes. This study shown that a correct repolymeriza-

tion of the spindle may occur during the restoration of

the physiological conditions after warming, leading to

the right sequence of events involved in the completion

of meiosis and fertilization., Finally, our results suggest

that vitrification procedure has a lower impact on oocyte

physiology, does not affect the normal preimplantational

embryo development or increase the aneuploidies inci-

dences compared to those observed in embryos from

fresh oocytes. Additionally, Chian et al. [18] analyzed

the obstetric and perinatal outcomes in 165 pregnancies

and 200 infants conceived following oocytes vitrification

cycles in three reproductive centers. The results indicate

that the mean birth weight and incidence of congenital

anomalies are comparable to that of spontaneous con-

ceptions in fertile women or infertile women undergoing

in-vitro fertilization treatment. These results confirm that

oocytes conserve their capability of fertilization and po-

tential to reach the blastocyst stage after being vitrified,

similar to the data obtained with fresh oocytes.

On the other hand, the vitrification of human oocytes

solves the legal restrictions and ethical problems [99]

associated with the cryopreservation of embryos in pa-

tients undergoing in vitro fertilization procedures. Also,

it may offer the possibility of extending the reproductive

capability of young women with malignant diseases in

cases where the treatment by surgery, chemotherapy or

radiotherapy may compromise the ovarian reserve

[100-103]. Moreover, it also provides alternatives for

infertile patients who are subject to ovarian hyperstimu-

lation syndrome, poor responder to ovarian stimulation,

premature ovarian failure or who require oocyte dona-

tion [100].

In conclusion, our observations demonstrate that vitri-

fication is an efficient procedure for oocyte cryopreser-

vation, the developmental competence of vitrified MII

oocytes to rich the blastocyst stage and the chromosomal

status are similar to those observed with fresh oocytes.

In conclusion, the vitrification procedure represents the

best current choice for creation of reliable egg banks.

REFERENCES

[1] Chen, C. (1986) Pregnancy after human oocytes cryopre-

servation. Lancet, 1, 884-886.

doi:10.1016/S0140-6736(86)90989-X

[2] Allan J. (2004) Pregnancy from intracytoplasmic injec-

tion of a frozen-thawed oocyte. The Australian and New

Zealand Journal of Obstetrics and Gynaecology, 44, 588.

doi:10.1111/j.1479-828X.2004.00317.x

[3] Levi Setti, P.E., Albani, E., Novara, P.V., Cesana, A. and

Morreale, G. (2006) Cryopreservation of supernumerary

oocytes in IVF/ ICSI cycles. Human Reproduction, 21,

370-375. doi:10.1093/humrep/dei347

[4] Nawroth, F. and Kissing, K. (1998) Pregnancy after in-

tracytoplasmic sperm injection (ICSI) of cryopreserved

human oocytes. Acta Obstet Gynecol Scand, 77, 462-463.

J. I. Garc ía et al. / Health 3 (2011) 467-476

473

[5] Porçu, E., Fabbri, R. and Seracchioli, R. (1997) Birth of

a healthy female after intracytoplasmic sperm injection

of cryopreserved human oocytes. Fertility and Sterility,

68, 724-726. doi:10.1016/S0015-0282(97)00268-9

[6] Van Uem, J.F., Siebzehnrubl, E.R., Schuh, B., Koch, R.,

Trotnow, S. and Lang, N. (1987) Birth after cryopreser-

vation of unfertileized oocytes. Lancet, 1, 752-753.

doi:10.1016/S0140-6736(87)90398-9

[7] Young, E., Kenny, A., et al. (1998) Triplet pregnancy

after intracyto-plasmic sperm injection of cryopreserved

oocytes: Case report. Fertility and Sterility, 70, 360-361.

doi:10.1016/S0015-0282(98)00150-2

[8] Gook, D., Schiewe, M.C., et al. (1995) Intracytoplasmic

sperm injection and embryo development of human oo-

cytes cryopreserved using 1, 2-propanediol. Human Re-

production, 10, 2637-2641.

[9] Borini, A., Bonu, M.A., et al. (2004) Pregnancies and

births after oocyte cryopreservation. Fertility and Steril-

ity, 82, 601-605. doi:10.1016/j.fertnstert.2004.04.025

[10] Fabbri, R., Porçu, E., et al. (2001) Human oocytes cryo-

preservation: New perspectives regarding oocytes sur-

vival. Human Reproduction, 16, 411-416.

doi:10.1093/humrep/16.3.411

[11] Porçu, E., Fabbri, R. and Damiano, G. (2000) Clinical o

experience and applications of oocytes cryopreservation.

Molecular and Cell u l a r E n d o c ri n o l o g y, 69, 33-37.

[12] Tucker, M.J., Morton, P.C., et al. (1998) Clinical applica-

tion of human egg cryopreservation. Human Reproduc-

tion, 13, 3156-3159. doi:10.1093/humrep/13.11.3156

[13] Winslow, K.L., et al. (2001) Oocyte cryopreservation a

three year follow-up of six- teen births. Fertility and Ste-

rility, 76, 120. doi:10.1016/S0015-0282(01)02354-8

[14] Oktay, K., Cil, A.P. and Bang, H. (2006) Efficiency of

oocyte cryopreservation: A meta-analysis. Fertility and

Sterility, 86, 70-80. doi:10.1016/j.fertnstert.2006.03.017

[15] Al-Hasani, S., Butuhan, O. and Koutlaki, N. (2007)

Three years of routine vitrification of human zygotes: Is

it still fair to advocate slow-rate freezing? Reproductive

BioMedicine Online, 14, 288-293.

doi:10.1016/S1472-6483(10)60869-3

[16] Vajta, G. and Nagy, Z.P. (2006) Are programmable freez-

ers still needed in the embryo laboratory? Review on vit-

rification. Reproductive BioMedicine Online, 12, 779-

796. doi:10.1016/S1472-6483(10)61091-7

[17] Antinori, M., Licata, E., et al. (2007) Cryotop vitrifica-

tion of human oocytes results in high survival rate and

healthy deliveries. Reproductive BioMedicine Online, 14,

72-79. doi:10.1016/S1472-6483(10)60766-3

[18] Chian, R.C., Huang, J.Y.H., et al. (2008) Obstetric and

perinatal outcome in 200 infants conceived from vitrified

oocytes. Reproductive BioMedicine Online, 16, 608-610.

doi:10.1016/S1472-6483(10)60471-3

[19] Cobo, A., et al. (2008) Comparison of concomitant out-

come achieved with fresh and cryopreserved donor oo-

cytes vitrified by the Cryotop method. Fertility and Ste-

rility, 89, 1657-1664.

doi:10.1016/j.fertnstert.2007.05.050

[20] García, J.I., et al. (2011) Efficacy of oocyte vitrification

combined with blastocyst stage transfer in an egg dona-

tion program. Human Reproduction, 26, 782-790.

doi:10.1093/humrep/der008

[21] Kuwayama, M., et al. (2005) Highly efficient vitrifica-

tion method for cryopreservation of human oocytes. Re-

productive BioMedicine Online, 11, 300-308.

doi:10.1016/S1472-6483(10)60837-1

[22] Kuwayama, M. (2007) Highly efficient vitrification for

cryopreservation of human oocytes and embryos: Cryo-

top method. Theriogenology, 67, 73-80.

doi:10.1016/j.theriogenology.2006.09.014

[23] Lucena, E., et al. (2006) Successful ongoing pregnancies

after vitrification of oocytes. Fertility and Sterility, 85,

108-111. doi:10.1016/j.fertnstert.2005.09.013

[24] Nagy, Z.P., et al. (2009) Clinical evaluation of the effi-

cacy of an oocyte donation program using egg cryo-ban-

king. Fertility and Sterility, 92, 520-526.

doi:10.1016/j.fertnstert.2008.06.005

[25] Selman, H., et al. (2006) Ongoing pregnancies after vit-

rification of human oocytes using a combined solution of

ethylene glycol and dimethyl sulfoxide. Fertility and Ste-

rility, 86, 997-1000.

doi:10.1016/j.fertnstert.2006.02.117

[26] Yoon, T.K., Lee, D.R. and Cha, S.K. (2007) Survival rate

of human oocytes and pregnancy outcome after vitrifica-

tion using slush nitrogen in assisted reproductive tech-

nologies. Fertility and Sterility, 88 , 925-926.

doi:10.1016/j.fertnstert.2006.12.071

[27] Gardner, D.K., et al. (2007) Analysis of oocyte physiol-

ogy to improve cryopreservation procedures. Therio-

genology, 67, 64-72.

doi:10.1016/j.theriogenology.2006.09.012

[28] Larman, M.G., Sheehan, C.B. and Gardner, D.K. (2006)

Vitrification of mouse pronuclear oocytes with no direct

liquid nitrogen contact. Reproductive BioMedicine On-

line, 12, 66-69. doi:10.1016/S1472-6483(10)60982-0

[29] Jones, A., Van Blerkom, J., Davis, P. and Toledo, A.

(2004) Cryopreservation of metaphase II human oocytes

effects mitochondrial membrane potential: Implications

for developmental competence. Human Reproduction, 19,

1861-1866. doi:10.1093/humrep/deh313

[30] Nottola, S.A., et al. (2007) Ultrastructure of human ma-

ture oocytes after slow cooling cryopreservation using

different sucrose concentrations. Human Reproduction,

22, 1123-1133. doi:10.1093/humrep/del463

[31] Schatten, G., Simerly, C. and Schatten, H. (1985) Micro-

tubule configuration during fertilization, mitosis, and

early development in the mouse and the requirement for

egg microtubule-mediated motility during mammalian

fertilization. Proceedings of the National Academy of

Sciences, 82, 4152-4156. doi:10.1073/pnas.82.12.4152

[32] Bianchi, V., et al. (2005) Meiotic spindle imaging in

human oocytes frozen with a slow freezing procedure

involving high sucrose concentration. Human Reproduc-

tion, 20, 1078-1083. doi:10.1093/humrep/deh736

[33] Chen, S.U., et al. (2000) Cryopreservation of mature

human oocytes by vitrification with ethylene glycol in

straws. Fertility and Sterility, 74, 804-808.

doi:10.1016/S0015-0282(00)01516-8

[34] Chen, S.U., et al. (2001) Vitrification of mouse oocytes

using closed pulled straws (CPS) achieves a high sur-

vival and preserves good patterns of meiotic spindles,

compared with conventional straws, open pulled straws

(OPS), and grids. Human Reprod, 16, 2350-2356.

[35] Chen, C.K., et al. (2004) Evaluation of meiotic spindles

in thawed oocytes after vitrification using polarized light

J. I. Garc ía et al. / Health 3 (2011) 467-476

474

microscopy. Fertility and Sterility, 82, 666-672.

doi:10.1016/j.fertnstert.2003.12.053

[36] Chen, S.U. and Yang, Y.S. (2009) Slow freezing or vitri-

fication of oocytes: Their effects on survival and meiotic

spindles, and the time schedule for clinical practice.

Taiwanese Journal of Obstetrics and Gynecology, 48, 15-

22. doi:10.1016/S1028-4559(09)60030-9

[37] Coticchio, G., et al. (2005) Criteria to assess human oo-

cyte quality after cryopreservation. Reproductive Bio-

Medicine Online, 11, 421-427.

doi:10.1016/S1472-6483(10)61133-9

[38] De Santis, L., et al. (2007) Objective evaluation of the

viability of cryopreserved oocytes. Reprod Biomed

Online, 15, 338-345.

doi:10.1016/S1472-6483(10)60348-3

[39] Eroglu, A., et al. (1998) Alterations of the cytoskeleton

and polyploidy induced by cryopreservation of meta-

phase II mouse oocytes. Fertility and Sterility, 69, 944-

ocytes.

00.

957.

[40] Larman, M.G., et al. (2007) Maintenance of the meiotic

spindle during vitrification in human and mouse o

Reproductive BioMedicine Online, 15, 692-7

doi:10.1016/S1472-6483(10)60537-8

[41] Rienzi, L., et al. (2004) Polscope analysis of meiotic

spindle changes in living metaphase II oocytes during the

freezing and thawing procedures. Human Reproduction,

19, 655-659. doi:10.1093/humrep/deh101

[42] Magistrini, M. and Szollosi, D. (1980) Effects of cold

and of isopropyl-N-phenylcarbamate on the second mei-

otic spindle of mouse oocytes. European Journal of Cell

ion and

Biology, 22, 699-707.

[43] Sathananthan, A.H., et al. (1992) The effects of cooling

mouse oocytes. Journal of Assisted Reproduct

Genetics, 9, 139-148. doi:10.1007/BF01203754

[44] Wang, W.H., et al. (2001) Limited recovery of meiotic

spindle in living human oocytes after cooling-rewarming

observed using polarized light microscopy. Human Re-

oling

of the

ility, 92,

production, 16, 2374-2378.

[45] Sathananthan, A.H., et al. (1988) The effects of co

human oocytes. Human Reproduction, 3, 968-977.

[46] Van der Elst, J., et al. (1988) Effect of 1,2-propanediol

and di- methylsulphoxide on the meiotic spindle

mouse oocyte. Human Reproduction, 3, 960-967.

[47] Cao, Y.X., et al. (2009) Comparison of survival and em-

bryonic development in human oocytes cryopreserved by

slowfreezing and vitrification. Fertility and Ster

1306-1311. doi:10.1016/j.fertnstert.2008.08.069

[48] Katayama, P., et al. (2003) High survival rate of vitrified

human oocytes results in

Sterility, 80, 223-224.

clinical pregnancy. Fertility and

doi:10.1016/S0015-0282(03)00551-X

[49] Kuwayama, M. (2001) Vitrification of human oocytes

and embryos. In: Suzuki, S. Ed., IVF Update, Medical

ng egg donors. Re-

View Co., Tokyo, 230-234.

[50] Munné, S., et al. (2006) Wide range of chromosome

abnormalities in the embryos of you

prod Biomed Online, 12, 340-346.

doi:10.1016/S1472-6483(10)61007-3

[51] Silber, S., et al. (2003) Chromosomal abnormalities in

embryos derived from TESE. Fertility and Sterility, 79,

30-38. doi:10.1016/S0015-0282(02)04407-2

[52] Coticchio, G., et al. (2009) Vitrification may increase the

rate chromosome misalignment in the metaphase II spin-

dle of human mature oocytes. Reproductive BioMedicine

Online, 19, 29-34. doi:10.1016/S1472-6483(10)60281-7

[53] Martínez-Burgos, M., et al. (2011) Vitrification versus

slow freezing of oocytes: effects on morphologic ap-

pearance, meiotic spindle configuration, a

age. Fertility and Sterility, 95, 374-377

nd DNA dam-

doi:10.1016/j.fertnstert.2010.07.1089

[54] Ciotti, P.M., et al. (2009) Meiotic spindle recovery is

faster in vitrification of human oocytes compared to slow

freezing. Fertility and St erility, 91, 23

99-2407.

doi:10.1016/j.fertnstert.2008.03.013

[55] Hardy, K., et al. (1990) Human preimplantation devel-

opment in vitro is not adversely affected by biopsy at the

rility, 72,

8-cell stage. Human Reproduction, 5,708-714.

[56] Gianaroli, L., et al. (1999) Preimplantation diagnosis for

aneuploidies in patients undergoing in vitro fertilization

with poor prognosis: Identification of the categories for

which it should be proposed. Fertility and Ste

837-844. doi:10.1016/S0015-0282(99)00377-5

[57] Munné, S., et al. (2003) Improved implantation after

Preimplantation genetic of an

BioMedicine Online, 7, 91-97.

euploidy. Reproductive

doi:10.1016/S1472-6483(10)61735-X

[58] Munné, S., et al. (2004b) Increased rate of aneuploid

embryos in young women with previous ane

ceptions. Prenatal Duploid con-

iagnosis, 24, 638-647.

doi:10.1002/pd.957

[59] Gianaroli, L., et al. (2007) Oocyte aneuploidy, pronu-

clear zygote morphology and embryo chr

complement. Human Reproduomosomal

ction, 22, 241-249.

doi:10.1093/humrep/del334

[60] Magli, M.C., et al. (2001) Double locus analysis of

chromosome 21 for Preimplantation genetic diag

aneuploidy. Prenatal nosis of

Diagnosis, 21, 1080-1085.

doi:10.1002/pd.248

[61] Márquez, C., et al. (2000) Chromosome abnormalities in

1255 cleavage-stage human e

Medicine Online, 1, 17-27.

mbryos. Reproductive Bio-

doi:10.1016/S1472-6483(10)61988-8

[62] Munné, S., et al. (2004a) Differences in chromosome

susceptibility to aneuploidy and survival to fir

Reproductive BioMedicine Online, 8, 8

st trimester.

1-90.

doi:10.1016/S1472-6483(10)60501-9

[63] Rubio, C., et al. (2003) Chromosomal abnormalities and

embryo development in recurrent m

Human Reproduction, 18, 182

iscarriage couples.

-188.

doi:10.1093/humrep/deg015

[64] Rubio, C., et al. (2005) FISH screening of aneuploidies

in preimplantation embryos to improve IVF ou

Reproductive BioMedicine Online, 11, 497-506.

tcome.

doi:10.1016/S1472-6483(10)61146-7

[65] Kahraman, S., et al. (2000) Healthy births and ongoing

pregnancies by preimplantation genetic diagnosis in pa-

tients with advanced maternal age and recurrent impl

tation failure. Human Reproductan-

ion, 15, 2003-2007.

doi:10.1093/humrep/15.9.2003

[66] Munné, S., et al. (1995) Embryo morphology, develop-

mental rates, and maternal age are correlated with chro-

mosomal abnormalities. Fertility and Sterility, 64, 382-

391.

[67] Munné, S., et al. (2002) Preimplantation genetic diagno-

J. I. Garc ía et al. / Health 3 (2011) 467-476

475

er indications. Fer-sis for advanced maternal age and oth

tility and Sterility, 78, 234-236.

doi:10.1016/S0015-0282(02)03239-9

[68] Pehlivan, T., et al. (2003) Impact of preimplantation

genetic diagnosis on IVF outcome in implanttation fail-

ure patients. Reproductive BioMedicine Online, 6, 232-

237. doi:10.1016/S1472-6483(10)61715-4

[69] Wilding, M., et al. (2004) Preimplantation genetic diag-

nosis for treatment of failed in vitro fertilization embryo

transfer and habitual abortion. Fertility and Sterility, 81,

1302-1307. doi:10.1016/j.fertnstert.2003.10.028

[70] Pellicer, A., et al. (1999) In vitro fertilization plus preim-

plantation genetic diagnosis in patients with recurrent

miscarriage: an analysis of chromosome abnormalities in

human preimplantation embryos. Fertility and Sterility,

71, 1033-1039. doi:10.1016/S0015-0282(99)00143-0

[71] Simón, C., et al. (1998) Increased chromosome abnor-

malities in human Preimplantation embryo after in vitro

fertilization in patients with recurrent miscarriage

production, Fertility a. Re-

nd Development, 10, 87-92.

doi:10.1071/R98030

[72] Vidal, F., et al. (1998) FISH preimplantation diagnosis of

chromosome aneuploidy in recurrent pregnancy wastage.

Journal of Assisted Reproduction and Genetics, 15, 309-

312. doi:10.1023/A:1022552713015

[73] Werlin, L., et al. (2003) Preimplantation genetic diagno-

sis as both a therapeutic and diagnosis tool in assisted re-

productive technology. Fertility and Sterility, 80, 467-

468. doi:10.1016/S0015-0282(03)00605-8

[74] Munné, S., et al. (2007) Maternal age, morphology, de-

velopment and chromosome abnormalities in over 6000

cleavage-stage embryo. Reproductive BioMedicine

Online, 14, 628-634.

doi:10.1016/S1472-6483(10)61057-7

[75] Reis Soares, S., et al. (2003) High frequency of chro

somal abnormalities in embryo obta

mo-

ined from oocyte

donation cycle. Fertility and Sterility, 80, 656-657.

doi:10.1016/S0015-0282(03)00787-8

[76] Cobo, A., et al. (2001) Use of fluorescence in situ hy-

bridization to assess the chromosomal status of embryos

obtained from cryopreserved oocytes. Fertility and Ste-

rility, 75, 354-360. doi:10.1016/S0015-0282(00)01725-8

[77] Kuleshova LL, Lopata A. (2002) Vitrification can be

more fa- vorable than slow cooling. Fertility and Steril-

ity,; 78, 449-454. doi:10.1016/S0015-0282(02)03305-8

[78] Rall, W.F. (1987) Factors affecting the survival of mouse

em- bryos cryopreserved by vitrification. Cryobiology,

24, 387-402. doi:10.1016/0011-2240(87)90042-3

[79] Velilla, E., Escudero, T. and Munné, S. (2002) Blas-

tomere fixation technique and risk of misdiagnosis for

PGD of aneuploidy. Reproductive BioMedicine Online, 4,

210- 217. doi:10.1016/S1472-6483(10)61808-1

[80] Munné, S., et al. (1998) Preimplantation diagnosis of the

aneuploidies most commonly found

tions and live births: X, 13, 14, 15, 16, 18, 21, 22. Preg-

nancy Diagnosis, 18, 1

in spontaneous abor-

459-1466.

doi:10.1002/(SICI)1097-0223(199812)18:13<1459::AID

-PD514>3.0.CO;2-V

[81] Colls, P., et al. (2004) PGD analysis for aneup

patient heterozygous

loidy in a

for a polymorphism of chromosome

16 (16qh-). Prenatal Diagnosis, 24, 741-744.

doi:10.1002/pd.887

[82] El-Danasouri, I. and Selman, H. (2001) Successful preg-

nancies and deliveries after a simple vitrification protocol

for day 3 human embryos. Fertility and Sterility, 76, 400-

402. doi:10.1016/S0015-0282(01)01907-0

[83] Hong, S.W., et al. (1999) Improved human o

velopment after vitrification: A comp

ocytes de-

arison of thawing

methods. Fertility and Sterility, 72, 142-146.

doi:10.1016/S0015-0282(99)00199-5

[84] Yoon, T.K., et al. (2000) Pregnancy and delivery of

healthy infants developed from vitr

stimulated in vitro fertilization-embryo

ified oocytes in a

transfer program.

Fertility and Sterility, 74, 180-181.

doi:10.1016/S0015-0282(00)00572-0

[85] Kim, T.J., et al. (2010) Vitrification of oocy

dures high pregnancy rates when c

tes proce-

arried out in fertile

women. Fertility and Sterility, 93, 467-474.

doi:10.1016/j.fertnstert.2008.12.094

[86] Rienzi, L., et al. (2010) Embryo development of fresh

“versus” vitrified metaphase II oocytes after ICSI: A

s survival, fertilization and

oductive BioMedi-

prospective randomized sibling-oocyte study. Reproduc-

tive BioMedicine Online, 25, 66-73.

[87] Sher, G., et al. (2008) Selective vitrification of euploid

oocytes markedly improve

pregnancy-generating potential. Repr

cine Online, 17, 524-529.

doi:10.1016/S1472-6483(10)60240-4

[88] Ubaldi, F., et al. (2010) Cumulative ongoing pregnancy

rate achieved with oocyte vitrification and cleavag

transfer without embryo selec

e stage

tion in a standard infertility

program. Human Reproduction, 25, 1199-1205.

doi:10.1093/humrep/deq046

[89] Fahy, G.M., et al. (1984) Vitrification as an approach to

cryopreservation. Cryobiology, 21, 407-426.

doi:10.1016/0011-2240(84)90079-8

[90] Chian, R.C., et al. (2005) High survival rates a

nancies of human oocytes followin

nd preg-

g vitrification: Pre-

liminary report. Fertility and Sterility, 80, 36.

doi:10.1016/j.fertnstert.2005.07.086

[91] Isachenko, V., et al. (2005) Aseptic technolog

cation of human pronuclear

y of vitrifi-

oocytes using open-pulled

straws. Human Reproduction, 20, 492-496.

doi:10.1093/humrep/deh605

[92] Lane, M., et al. (1999) Containerless vitrification of

mammalian oocytes and embryos. Nat Biotechnol, 17,

1232-1236. doi:10.1038/70795

[93] Lieberman, J., et al. (2002) Blastocyst development after

vitrification of multipronuclear zygotes using the flexipet

denuding pipette. Reproductive BioMedicine Online, 4,

146-150. doi:10.1016/S1472-6483(10)61932-3

[94] Mukaida, T., et al. (2003) Vitrificatio

cysts using cryoloops: Clinic

n of human blasto-

al outcome of 223 cycles.

Human Reproduction, 18, 384-391.

doi:10.1093/humrep/deg047

[95] Cobo, A., et al. (2010) Use of cryo-banked oocytes in an

ovum donation programme: A prospective, randomized,

controlled, clinical trial. Human Reproduction, 25, 2239-

2246. doi:10.1093/humrep/deq146

[96] Yoon, T.K., et al. (2003) Live births after vitrification

oocytes in a stimulated in vitro fertiliz

ation-embryo tran-

sfer program. Fertility and Sterility, 79, 1323-1326.

doi:10.1016/S0015-0282(03)00258-9

[97] Huang, J.Y., et al. (2007) Comparison of spindle and

J. I. Garc ía et al. / Health 3 (2011) 467-476

476

red chromosome configuration in vitro and in vivo matu

mouse oocytes after vitrification. Fertility and Sterility,

90, 1424-1432. doi:10.1016/j.fertnstert.2007.07.1335

[98] Baart, E.B., et al. (2006) Preimplantation genetic

screening reveals a high incidence of a

mosaicism in embryos from

neuploidy and

young women undergoing

IVF. Human Reproduction, 21, 223-233.

doi:10.1093/humrep/dei291

[99] Parmegiani, L., et al. (2008) Freezing within 2 h from

oocyte retrieval increases the efficiency of human oocyte

cryopreservation when using slow freezing/rapid thawing

protocol with high sucrose concentration. Human Re-

production, 23, 1771-1777. doi:10.1093/humrep/den119

[100] Gidoni, Y., et al. (2008) Fert

cine Online, 16, 792-800.

ility presservation in patients

with non-oncological conditions. Reproductive BioMedi-

doi:10.1016/S1472-6483(10)60144-7

[101] Li, X.H., et al. (2005) Cryopreserved oocytes of infertile

couples undergoing assisted reproductive technology

could be an important source of oocyte donation: A clini-

cal report of successful pregnancies. Human Reproduc-

tion, 20, 3390-3394. doi:10.1093/humrep/dei262

[102] Porçu, E., et al. (2008) Healthy twins delivered after

oocyte cryopreservation and bilateral ovariectomy for

ovarian cancer. Reproductive BioMedicine Online, 17,

ed before cancer treatment. Fertility and Sterility,

87, 4.

265-267.

[103] Yang, D., et al. (2007) Live birth after the transfer of

human embryos developed from cryopreserved oocytes

harvest