Vol.3, No.7, 467-476 (2011)
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/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.
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
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
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
[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.
2.1. 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
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
all patients wish to avoid a pregnancy with trisomic dis-
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
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
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
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
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).
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
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
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Table 1. Laboratory results in the vitrified and the fresh
group Fresh
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)
3.63 ± 0.40
7.11 ± 1.01
18 (32.1)
138 (91.4)
3.89 ± 0.46
7.43 ± 0.97
54 (44.6)
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-
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.
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.
group Fresh
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)
129 (93.5)
129 (100)
121 (93.8)
87 (71.9)
35 (64.8)
Table 3. Percentage of abnormalities for each chromosome
analyzed in the vitrified and the fresh group.
analyzed Vitrified Group Fresh Group
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
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
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.
[1] Chen, C. (1986) Pregnancy after human oocytes cryopre-
servation. Lancet, 1, 884-886.
[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.
[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
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
[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.
[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.
[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.
[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.
[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.
[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.
[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.
[21] Kuwayama, M., et al. (2005) Highly efficient vitrifica-
tion method for cryopreservation of human oocytes. Re-
productive BioMedicine Online, 11, 300-308.
[22] Kuwayama, M. (2007) Highly efficient vitrification for
cryopreservation of human oocytes and embryos: Cryo-
top method. Theriogenology, 67, 73-80.
[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.
[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.
[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.
[27] Gardner, D.K., et al. (2007) Analysis of oocyte physiol-
ogy to improve cryopreservation procedures. Therio-
genology, 67, 64-72.
[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.
[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
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
microscopy. Fertility and Sterility, 82, 666-672.
[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.
[38] De Santis, L., et al. (2007) Objective evaluation of the
viability of cryopreserved oocytes. Reprod Biomed
Online, 15, 338-345.
[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-
[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
[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-
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
[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.
[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-
[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
[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
[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.
[59] Gianaroli, L., et al. (2007) Oocyte aneuploidy, pronu-
clear zygote morphology and embryo chr
complement. Human Reproduomosomal
ction, 22, 241-249.
[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.
[61] Márquez, C., et al. (2000) Chromosome abnormalities in
1255 cleavage-stage human e
Medicine Online, 1, 17-27.
mbryos. Reproductive Bio-
[62] Munné, S., et al. (2004a) Differences in chromosome
susceptibility to aneuploidy and survival to fir
Reproductive BioMedicine Online, 8, 8
st trimester.
[63] Rubio, C., et al. (2003) Chromosomal abnormalities and
embryo development in recurrent m
Human Reproduction, 18, 182
iscarriage couples.
[64] Rubio, C., et al. (2005) FISH screening of aneuploidies
in preimplantation embryos to improve IVF ou
Reproductive BioMedicine Online, 11, 497-506.
[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.
[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-
[67] Munné, S., et al. (2002) Preimplantation genetic diagno-
J. I. Garc ía et al. / Health 3 (2011) 467-476
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
er indications. Fer-sis for advanced maternal age and oth
tility and Sterility, 78, 234-236.
[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.
[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.
[75] Reis Soares, S., et al. (2003) High frequency of chro
somal abnormalities in embryo obta
ined from oocyte
donation cycle. Fertility and Sterility, 80, 656-657.
[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-
[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.
[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.
[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.
[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.
[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.
[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.
[89] Fahy, G.M., et al. (1984) Vitrification as an approach to
cryopreservation. Cryobiology, 21, 407-426.
[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.
[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.
[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.
[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.
[97] Huang, J.Y., et al. (2007) Comparison of spindle and
J. I. Garc ía et al. / Health 3 (2011) 467-476
Copyright © 2011 SciRes. http://www.scirp.org/journal/HEALTH/Openly accessible at
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.
[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-
[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.
[103] Yang, D., et al. (2007) Live birth after the transfer of
human embryos developed from cryopreserved oocytes