1 s2.0-s0929664613000958-main

Journal of the Formosan Medical Association (2013) 112, 537e544
Available online at www.sciencedirect.com
journal homepage: www. jfma-online.com
ORIGINAL ARTICLE
A feasible strategy of preimplantation
genetic diagnosis for carriers with
chromosomal translocation: Using blastocyst
biopsy and array comparative genomic
hybridization
Chu-Chun Huang a,d, Li-Jung Chang a,d, Yi-Yi Tsai a,
Chia-Cheng Hung b, Mei-Ya Fang b, Yi-Ning Su a,b,c,
Hsin-Fu Chen a,*, Shee-Uan Chen a,*
a Department of Obstetrics and Gynecology, National Taiwan University Hospital and College of
Medicine, Taipei, Taiwan
b Department of Medical Genetics, National Taiwan University Hospital and College of Medicine,
Taipei, Taiwan
c Graduate Institute of Clinical Genomics, National Taiwan University Hospital and College of Medicine,
Taipei, Taiwan
Received 28 September 2012; received in revised form 23 January 2013; accepted 20 February 2013
KEYWORDS
array comparative
genomic
hybridization
(aCGH);
blastocyst biopsy;
chromosomal
translocation;
infertility;
preimplantation
genetic diagnosis
(PGD)
Background/Purpose: Patients with chromosomal translocation are highly vulnerable to pro-duce
unbalanced gametes that result in recurrent miscarriages, affected offspring, or infer-tility.
Preimplantation genetic diagnosis (PGD) with blastomere biopsy and fluorescent
in-situ hybridization (FISH) has been used to select normal/balanced embryos for transfer.
However, FISH is inherent with some technical difficulties such as cell fixation and signal
reading. Here we introduce a strategy of PGD using blastocyst biopsy and array comparative
genomic hybridization (aCGH) for reproductive problems of patients with chromosomal trans-location.
Methods: Twelve patients diagnosed as having chromosomal translocation who underwent PGD
cycles were included in this single-center observational study. Blastocyst biopsy was per-formed
and biopsied blastocysts were cryopreserved individually. Testing was performed with
aCGH, and the euploid embryos were transferred in the following thawing cycles.
* Corresponding authors. Department of Obstetrics and Gynecology, National Taiwan University Hospital, 7 Chung-Shan South Road,
Taipei, Taiwan.
E-mail addresses: hfchen@ntu.edu.tw (H.-F. Chen), sheeuan@ntu.edu.tw (S.-U. Chen).
d These authors contributed equally to this study.
0929-6646/$ - see front matter Copyright ª 2013, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.
http://dx.doi.org/10.1016/j.jfma.2013.02.010

538 C.-C. Huang et al.
Results: The overall diagnostic efficiency was 90.2% (55/61) and the euploidy rate was 32.7%
(18/55). Ten cycles of thawed embryo transfer (ET) were carried out, resulting in three live
births and another three ongoing pregnancies with an ongoing pregnancy rate of 60%/transfer
cycle. The prenatal diagnosis with chorionic villi sampling confirmed the results of PGD/aCGH
in all six pregnant women. No miscarriage happened in our case series.
Conclusion: Our study demonstrates an effective PGD strategy with promising outcomes. Blas-tocyst
biopsy can retrieve more genetic material and may provide more reliable results, and
aCGH offers not only detection of chromosomal translocation but also more comprehensive
analysis of 24 chromosomes than traditional FISH. More cases are needed to verify our results
and this strategy might be considered in general clinical practice.
Copyright ª 2013, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.
Introduction
Chromosomal rearrangements are the most common struc-tural
chromosome abnormalities, with an estimated 0.19%
prevalence in the general population, and even higher in
certain populations, such as 5% in patients who underwent
in vitro fertilization (IVF) treatments and experienced
recurrent miscarriages and repeated IVF failure.1 Carriers of
balanced chromosomal rearrangements usually have normal
phenotype, but have a high risk (19e77%) of producing
chromosomally unbalanced gametes following abnormal
meiotic segregation, depending on the type of chromosome
aberration, the chromosomes involved, and the number and
location of breakpoints.2 This may result in infertility,
recurrent miscarriages (spontaneous or induced after pre-natal
diagnosis), or delivery of an affected offspring with
mental retardation or other congenital abnormalities.1,3
In the past, these patients could only have prenatal
diagnosis to confirm the karyotype of the fetus after
considerable time and effort to get pregnant, and they may
even suffer more if the results show an unbalanced trans-location.
Alternatively, they might decide to choose a
sperm donor or oocyte donor after repeated failure to
achieve a normal pregnancy. Sometimes this may cause
infertility and usually the couples experience repeated IVF
failures and enormous psychological stress when they seek
help from assisted reproductive technologies (ART).3,4
Preimplantation genetic diagnosis (PGD), combined with
ART, can identify and select only chromosomally normal or
balanced embryos to be transferred to the uterus.5e7 It has
been shown that PGD can significantly shorten the time to
achieve a successful live birth from 4e6 years to <4 months,
and decrease the incidence of miscarriage from >90% to
<15% in the translocation carriers.4,8 This offers an alter-native
for the carriers in that they do not need to suffer from
the tremendous grief of repeated early pregnancy losses or
to terminate the affected pregnancy once revealed by the
prenatal diagnosis.4
The most widespread method of PGD for translocation is
eight-cell stage embryo biopsy with fluorescence in situ hy-bridization
(FISH) followed by Day 4 or Day 5 embryo transfer
(ET) in a fresh IVF cycle.9e11 However, the FISH method has
some drawbacks including difficulties in fixation of blasto-meres,
hybridization failure, signal overlap, and signal split-ting
that can affect the accuracy of the interpretation.12,13
Newer techniques have been proposed to increase the
diagnostic accuracy and efficiency, such as the application of
polymerase chain reaction (PCR)-based PGD protocol, meta-phase
comparative genomic hybridization (CGH), and array
CGH (aCGH).1,3 However, FISH and PCR-based methods only
allow identification for a limited number of chromosomes,
whereas CGH and aCGH can detect all 24 chromosomes.
Although FISH methods are cheaper and can usually provide
translocation screening, they usually need workup to deter-mine
the appropriate probes to be utilized for the often de-novo
rearrangements specific to the individual. Also, they
do not allow for comprehensive aneuploidy screening.1,3,12
Furthermore, in some cases commercial FISH probes are not
available for the chromosome of interest due to cross hy-bridization
with other regions. However, these patients can
now benefit from aCGH as it is genome wide and does not
require a specific test to be developed.1,3,11,12
The most suitable methodological design in the practice
of PGD has not been well established. Polar body biopsy can
be used for the genetic analysis of the oocyte but is not
feasible for detecting male genetic problems. In contrast to
eight-cell embryo biopsy, blastocyst biopsy provides more
cells than the cleavage-stage biopsy (5e10 cells vs. 1 or 2
cells) for genetic analysis and may potentially increase the
diagnostic accuracy. Both of them allow analysis of chro-mosomes
from maternal and paternal origin.6,7,14 Here we
report a strategy of PGD using blastocyst biopsy, aCGH, and
thawed ET for patients with chromosomal translocation.
We analyzed the data of clinical treatments. The pre-liminary
results indicate that this strategy is an important
option for these patients.
Materials and methods
Study population
This was a single-center observational study. Patients who
were diagnosed as having chromosomal translocations and
requested PGD at National Taiwan University Hospital from
January 2011 to April 2012 were included. A comprehensive
counseling was provided by an experienced clinical genet-icist
and reproductive endocrinologist to each couple prior
to starting the treatment. The participants were well
informed on the procedures of IVF and PGD with aCGH, its
efficacy, and limitations. Data on the parental age, family
and reproductive history, reasons for karyotyping, and their

PGD with aCGH for chromosomal rearrangement 539
specific types of chromosomal aberration were recorded.
Written informed consent was obtained from each couple.
This study was approved by the Ethics Committee of Na-tional
Taiwan University Hospital.
For each PGD cycle the following data were recorded:
the number of retrieved oocytes; number of mature oo-cytes
(metaphase II oocytes); number of fertilized oocytes;
number of biopsied embryos; location and size of chromo-somal
aberrations in each diagnosed embryos; and preg-nancy
outcomes after ET. The primary outcome
measurements were diagnostic rate, euploidy rate, and
ongoing pregnancy rate.
Blastocyst biopsy and cryopreservation of biopsied
embryos
Ovarian stimulation was performed with either
gonadotropin-releasing hormone (GnRH) antagonist proto-col
or GnRH agonist short protocol as described pre-viously.
15e17 Retrieval of oocytes was performed vaginally.
In order to avoid contamination from sperm or surrounding
cumulus cells, intracytoplasmic sperm injection was per-formed
in every case. Embryos that had reached blastocyst
stage were biopsied at trophectoderm on either Day 5 or
Day 6, based on the method described by Chen et al.18 Five
to 10 trophoblasts were aspirated from each blastocyst and
were transferred to a separate PCR tube. The biopsied
blastocyst was treated with cryoprotectants and trans-ferred
onto a Cryotop (Kitazato Supply Co., Fujinomiya,
Japan) individually.19
Whole genome amplification and aCGH
The multiple displacement amplification method using
SurePlex Whole Genome Amplification (WGA) Kit (Blue-
Gnome, Cambridge, UK) was performed for WGA, according
to the manufacturer’s instruction. This technology is based
on random fragmentation of genomic DNA and subsequent
amplification by PCR utilizing flanking universal priming
sites. Amplified sample DNA and control DNA were then
labeled with Cy3 and Cy5 fluorophores respectively and co-hybridized
competitively to a bacterial artificial chromo-some
microarray platform (24sure; BlueGnome). The times
for labeling and hybridization were 16 hours and 3 hours,
respectively. The resolution of the microarray was esti-mated
at 2e5 Mbp depending on the specific type and
location of a genetic aberration. The hybridized 24sure
slides were then washed to remove unhybridized DNA with
Hybex Microarray Incubation System (BlueGnome). Micro-array
slides were dried in the vacuum centrifuge and then
scanned. The hybridized fluorophores were excited and the
resulting images of the hybridization read and stored by a
two-channel laser scanner. Finally the results were
analyzed and the whole chromosomal loss or gain reported
with BlueFuse Multi software (BlueGnome).
Thawed ET
When clear results of aCGH were seen, euploid blastocysts
were thawed and transferred with two different protocols
of endometrial preparation. One was natural ovulation
cycle that no additional gonadotropin or synthesized
ovarian steroids were given prior to ovulation and the em-bryos
were thawed 6 days after observing serum luteinizing
hormone surge. Another endometrial preparation consisted
of estradiol valerate (Estrade; Synmosa, Taipei, Taiwan)
and 8% intravaginal progesterone gel (Crinone; Merck
Serono, Geneva, Switzerland) supplement and the embryos
were thawed on the sixth day of progesterone administra-tion.
20 Patients with a positive pregnancy test continued
with estrogen and progesterone supplementation until 12
weeks’ gestation.
ET was performed 3e4 hours after warming, as described
previously.20 One or two embryos were transferred/cycle.
Biochemical pregnancy was confirmed with serum b-human
chorionic gonadotropin test 12 days after ET. Implantation
was determined by the presence of a gestational sac ac-cording
to transvaginal ultrasound at a gestational age of 5
weeks. The ongoing pregnancy was defined as presence of
fetal heart beat until 12 weeks of gestation.
Confirmation of karyotype of fetus with
conventional prenatal diagnosis
In cases where pregnancies were achieved, conventional
prenatal diagnosis with chorionic villi sampling (CVS) or
amniocentesis was offered to confirm the karyotype of the
fetus.
Statistical analyses
Statistical analysis was performed using SPSS version 17.0
software (SPSS Inc., Chicago, IL, USA). Categorical data are
presented as number of events/cases and percentages for
each group of interest. Continuous data are presented as
mean and standard error of the mean.
Results
A total of 12 patients diagnosed with a variety of chromo-somal
translocation, including 11 with reciprocal trans-location
and one with Robertsonian translocation, were
enrolled. The age, karyotype, the reason for karyotyping,
pregnancy history of each couple are listed in Table 1 and
Table 2. The reasons for karyotyping included six cases of
repeated miscarriages, three cases of oligospermia, two
cases of previously affected child, and one case of repeated
IVF failure. The case with repeated IVF failure underwent a
total of eight IVF cycles due to endometriosis factor prior to
karyotyping and all failed. The karyotypes of the spouses
were all normal.
Previous pregnancy history of 12 couples showed a total
of 24 previous pregnancies. These resulted in 20 cases of
spontaneous abortions prior to 12 weeks of gestational age,
one case of intrauterine fetal demise at second trimester,
and one case of elective termination of pregnancy at sec-ond
trimester due to abnormal karyotyping by prenatal
diagnosis. There was one normal live birth, which was
conceived via conventional IVF treatment. The other live
birth was affected with unbalanced autosomal trans-location,
showing congenital hypotonia and developmental
delay.

The characteristics and outcomes of PGD cycles are
shown in Table 3. A total of 12 PGD cycles were performed
with blastocyst biopsy and aCGH, followed by cryopreser-vation
and thawed ET in the next menstrual cycle. A total of
201 oocytes were retrieved (average 16.8 oocytes/cycle,
range 4e33) and 172 of them were at the metaphase II
stage. Of these, 137 were fertilized normally under intra-cytoplasmic
sperm injection procedure, achieving a 79.7%
fertilization rate. Sixty-one (44.5%) embryos developed into
blastocysts and underwent blastocyst biopsy either on Day 5
(26 embryos, 42.6%) or Day 6 (35 embryos, 57.4%),
depending on the speed of development.
WGA was processed successfully on 55 samples and the
results of aCGH were clearly obtained on all samples with
successful amplification. The smallest detected
chromosomal aberration is 1.6 Mbp. Among the diagnosed
embryos, 18 of them (32.7%) were unaffected or balanced
without other aneuploidy; 16 (29.1%) were unbalanced
translocation without other aneuploidy; 15 (27.3%) were
unbalanced translocation with other unrelated aneu-ploidy;
and six (10.9%) were unaffected or balanced
translocation but with aneuploidy affecting other unre-lated
chromosomes. Overall, the percentage of abnormal
embryos was 67.3% (37/55) in our cohort. The overall
Table 1 The characteristics of the patients in this cohort.
Case Maternal
age
Paternal
age
Karyotype Reason for karyotyping Previous
miscarriage
Previous
affected
offspring
Previous
IVF/ICSI
cycles prior
to PGD
1 33 33 46XY, t(6;14)(q26;q31) Repeated miscarriage 3 0 0
2 43 45 46XX, t(2;7)(q36.2;p15.1) Affected childa 1 1 0
3 34 36 46XY, t(20;21)(p10;q10) Oligospermia 0 0 0
4 31 35 46XY, t(5;21)(q11.2;q11.2) Repeated miscarriage 2 0 0
5 34 36 46XY, t(9;17)(q34.3;p13.3) Repeated miscarriage 2 0 2
6 35 36 46XY, t(7;10)(q34;q26.12) Affected child 0 2b 0
7 36 35 46XY, t(2;10)(p10;q10) Repeated IVF failure 3 0 8
8 36 42 46XX, t(6;10)(q15;p11.2) Repeated miscarriage 3 0 0
9 32 36 46XY, t(2;14)(p10;p10) Oligospermia 1 0 3
10 35 25 45XY, rob(13;14)(q11;q11) Oligospermia 0 0 0
11 22 25 46XX, t(1;14)(q31;q31) Repeated miscarriage 2 0 0
12 31 32 46XY, t(9;12)(q11;q13) Repeated miscarriage 3 0 0
PGD Z preimplantation genetic diagnosis; rob Z Robertsonian translocation.
a This affected offspring showed limb defects associated with abnormal karyotype inherited paternally and was terminated at 21
weeks of gestation.
b Their first pregnancy resulted in live birth but was affected with unbalanced autosomal translocation, showing congenital hypotonia
and developmental delay. Their second pregnancy also showed unbalanced translocation and they decided to terminate it.
Table 2 Characteristics of the patients.
Number of couples 12
Maternal age 33.8 1.2 (25e43)
Paternal age 34.7 1.7 (25e45)
Number of previous miscarriages 1.7 0.4 (0e3)
Number of previous live births 0.2 0.1 (0e1)
Number of IVF/ICSI cycles prior
1.2 0.7 (0e8)
to PGD
Reasons for karyotyping
Oligospermia 3 (25%)
Repeated miscarriage 6 (50%)
Affected child 2 (17%)
Repeated IVF failure 1 (8%)
Values are expressed as mean standard error of the mean
(range) or n (%).
IVF Z in vitro fertilization; ICSI Z intracytoplasmic sperm in-jection;
PGD Z preimplantation genetic diagnosis.
Table 3 Characteristics and outcomes of OPU and PGD
cycles.a
Total number of OPUþPGD cycles 12
Total number of oocytes retrieved 201
Mean number of oocytes retrieved 16.8 2.5 (4e33)
Total number of mature oocytes (MII) 172 (85.6%)
Mean number of mature oocytes (MII) 14.3 2.5 (4e31)
Total number of fertilized embryos 137 (79.7%)
Mean number of fertilized embryos 11.4 2.1 (1e23)
Total number of biopsied embryos 61 (44.5%)
Mean number of biopsied embryos 5.1 0.9 (1e11)
Day of biopsy
Day 5 26 (42.6%)
Day 6 35 (57.4%)
Total number of embryos diagnosed 55 (90.2%)
Normal or balanced translocation 18 (32.7%)
Unbalanced, without other aneuploidy 16 (29.1%)
Unbalanced, with other aneuploidy 15 (27.3%)
Balanced, with other aneuploidy 6 (10.9%)
Total number of embryos undiagnosed 6 (9.8%)
MII Z metaphase II; OPU Z oocyte pick-up;
PGD Z preimplantation genetic diagnosis.
a Values are expressed as mean standard error of the mean
(range) or n (%).

diagnostic efficacy was 90.2% (55 results from a total of 61
samples) and the main reason for nondiagnosis was
amplification failure.
Thawed ET was performed in nine couples following PGD
cycles as in Table 4. Three couples did not have normal/
balanced embryos. In total 15 normal or balanced embryos
were thawed and 14 survived with a survival rate of 93.3%.
Seven of 14 transferred embryos (50%) implanted, resulting
in three live births and another three ongoing pregnancies.
The mean number of transferred embryos was 1.4. The
ongoing pregnancy rate was 60%/transfer cycle and 50%/
PGD cycle. The prenatal diagnosis with CVS reconfirmed the
results of PGD/aCGH in all six pregnant women. There were
three euploid embryos still cryopreserved for three cou-ples,
which could be transferred in the future. The detailed
karyotypic information of each diagnosed embryo is listed
in Table 5.
Discussion
Our study demonstrates an effective strategy of PGD
treatment for the reproductive problems of carriers of
chromosomal translocations using blastocyst biopsy, aCGH,
and thawed ET. Reviewing the literature, we found two
other studies using aCGH to perform PGD for the carriers of
chromosomal translocations.1,3 The diagnostic efficacy
ranged from 90% to 93.5% in our study and the other two
studies, which were comparable with those studies using
FISH.1e3,21,22 However, the clinical pregnancy rate seemed
to be higher in the studies using aCGH, which were 42.9%/
PGD cycle by Fiorentino et al,3 and 50% in our study. The
reported clinical pregnancy rate in those studies using FISH
ranged from 16% to 33%.1e3,9,21e25 More interestingly, no
case of miscarriage took place in our study and the other
two studies using aCGH, whereas the reported miscarriage
rates of the studies using FISH were 2e37.5% in the liter-ature.
1e3,9,21e25 The elevation of clinical pregnancy rate
and elimination of miscarriage in our study and Fiorentino
et al’s study might be due to the advantage of the
comprehensive 24 chromosomal aneuploidy screening pro-vided
by aCGH. More studies will be needed to verify our
preliminary result.
aCGH using 24sure technology was performed for genetic
analysis in our study. It provides 24-chromosome analysis to
screen both the translocation and all the other aneu-ploidies,
instead of a set of usually 5e12 chromosomal
probes used by traditional FISH method. It can detect
spontaneous aneuploidy other than specific translocation
derived from carriers.26,27 In our study, 10.9% (6/55) em-bryos
were normal or balanced at the chromosomes
involved in the translocation but were found to have other
de novo aneuploidies. They will be misdiagnosed as normal
embryos by the conventional FISH method and be trans-ferred,
which might end with poor pregnancy outcomes,
such as miscarriage or an affected offspring.
Array CGH also avoids many technical drawbacks
inherent in the FISH method. One critical but difficult step
is the fixation of a single cell onto a microscopic
slide.13,26,27 Also, the misdiagnosis may happen due to hy-bridization
failure, suboptimal signal interpretation, such
as overlapping signals or split signals.13 Moreover, aCGH
allows automation in data reading through computerized
calculation of signal intensities, instead of interpreting the
signals by eye in the FISH method, which may be subject to
interoperator error.13 Another advantage of aCGH is that it
does not require preclinical validation prior to each IVF
cycle, which is required for FISH. This avoids postponement
of treatment.3,7,28
Both allele dropout (ADO) and preferential amplifica-tion
(PA) can decrease the reliability of PGD, especially
for the autosomal dominant single gene disorder.29
Because amplification failure of the affected allele will
cause serious consequence, one solution is to analyze
more loci near the targeted allele.11,29 From the previous
studies published on single-cell multiple displacement
amplification, the ADO rates and PA rates could reach as
high as 25%.29 If more loci are analyzed at the same time,
ADO rates can be decreased to less than 10% and PA rates
even lower.29 Fiorentino et al also reported that when
three short tandem repeat markers are put in either side
of the translocation breakpoints, the failure rate of the
test is about 0.002%, which happens only when ADO oc-curs
in all markers at the same time.12 For carriers of
chromosomal translocation, the aberrant chromosomal
segments of the unbalanced gametes are much larger,
which usually involve more than four or five continuous
probes on the microarray platform. Therefore the inci-dence
of co-amplification failure of all four or five probes
should also be low.
The smallest aberration detected in our study was a
1.6 Mbp deletion, which is better than FISH or even the
traditional metaphase CGH (5e10 Mbp),1 providing a con-vincible
and satisfactory screening resolution with this
technique. The cost of FISH is US$2167 in our center, no
matter how many embryos are analyzed. The cost of aCGH
depends on the number of analyzed embryos and is US$600/
embryo. The average number of analyzed embryos is 5.1,
giving an average cost of around US$3000 in our study.
Therefore it is often more expensive to perform aCGH than
FISH and both the cost and the benefits should be discussed
with the patients in clinical practice.
Table 4 Pregnancy outcomes of thawed embryo transfer.
Total number of embryo transfer cycles 10
Total number of embryos thawed 15
Number of embryos surviving after thawing 14 (93.3%)
Total number of embryos transferred 14
Mean number of embryos transferred 1.4 0.2
(1 or 2)
Total number of embryos implanted (sac) 7
Implantation rate/embryo transferred 50% (7/14)
Total number of FHB 7
Pregnancy rate (positive b-hCG)/transfer
cycle
80% (8/10)
Ongoing pregnancy rate/transfer cyclea 60% (6/10)
Ongoing pregnancy rate/PGD cyclea 50% (6/12)
b-hCG Z b-human chorionic gonadotropin; FHB Z fetal heart
beat.
a Ongoing pregnancy was defined as the presence of fetal
heart beat until 12 weeks of gestation.

Table 5 Detailed information of microarray platform in each diagnosed embryo.
Case Karyotype of the
affected couple
Embryo
no.
Results of microarray platform Final characterization
Chromosomal gain Chromosomal loss
1 46XY, t(6;14)(q26;q31) 1 14q32.12e14q32.33 d Abnormal
2 d 14q32.12e14q32.33 Abnormal
3 Euploid Normal/balanced
4 1q42.2e1q44 Chr. 2 Abnormal
5 No signal Undiagnosed
7 14q32.12e14q32.33 d Abnormal
9 9q21.33e9q34.3;
14q32.12e14q32.33
Chr. 16 Abnormal
11 d Chr. 14 Abnormal
2 46XX, t(2;7)(q36.2;p15.1) 1 d Chr. 2; 7p22.3e7p15.3 Abnormal
3 46XY, t(20;21)(p10;q10) 1 Euploid Normal/balanced
2 Chr. 1, 3, 5, 9, 13, Y Chr. 4, 8, 18, 21, 22, X Abnormal
3 d 9q31.3e9q34.3; Abnormal
11p15.5e11p15.4;
20p13e20p11.21
5 8q23.3e8q24.3;
10q26.2e10q26.3;
11q13.1e11q25;
20p13e20p11.21
d Abnormal
4 46XY, t(5;21)(q11.2;q11.2) 1 21q21.1eq22.3 5q13.2eq35.3 Abnormal
3 5p15.33eq13.1 21q11.2eq21.1 Abnormal
4 5q13.2eq35.3;
11p15.5ep15.1
5p15.33eq13.1;
21q21.1eq22.3
Abnormal
5 21q21.1eq22.3 5q13.2eq35.3 Abnormal
6 1p12eq44; 6q22.31eq27;
21q11.2eq22.3
5q13.2eq35.3;
9p24.3e21.3
Abnormal
7 21q11.2eq21.1 5p15.33eq13.1 Abnormal
9 22q11.1eq13.31 d Abnormal
11 5q13.2eq35.3 21q21.1eq22.3 Abnormal
5 46XY, t(9;17)(q34.3;p13.3) 1 9q33.2eq34.3 17p13.3ep12 Abnormal
6 46XY, t(7;10)(q34;q26.12) 1 Euploid Normal/balanced
2 10q26.13eq26.3 Chr. 2, 8; 7q36.1eq36.3 Abnormal
5 7q36.1eq36.3 Chr. 22; 10q26.13eq26.3 Abnormal
6 Chr. 22; 7q36.1eq36.3 8p23.3ep12;
10q26.13eq26.3
Abnormal
7 46XY, t(2;10)(p10;q10) 1 Chr. 10, 15, 20 Chr. 14, 17, 21 Abnormal
2 10p15.3eq21.1 2p11.12eq37.3 Abnormal
3 Chr. 1, 3, 4, 6, 8, 10, 12,
14, 16, 17, 20, 22
Chr. 2, 5, 7, 9, 11, 13,
15, 18, 19, 21
Abnormal
8 46XX, t(6;10)(q15;p11.2) 1 Euploid Normal/balanced
2 10p15.1ep15.1 6q16.1eq27 Abnormal
3 10p14ep15.3 6q16.1eq27 Abnormal
4 6q21eq27 10p15.3ep13 Abnormal
5 6q21eq27; Chr. 16 10p15.3ep13 Abnormal

The best timing for embryo biopsy is still controversial. It
has been well documented that as many as 66e87% of
cleavage stage embryos exhibit mosaicism,26,30 which
means there are at least two different cell lines in one
embryo. Therefore sampling only one blastomere at an
early developmental stage may not represent the final ge-netic
composition and predict the real destiny of the em-bryo.
26,30 By contrast, sampling several cells (usually 5e10
cells) during blastocyst biopsy can provide more genetic
material for analysis, decreasing the risk of misdiagnosis
due to mosaicism, amplification failure, and allele drop-out
phenomenon.9 It is assumed that performing biopsy on the
trophectoderm, which is extraembryonic and develops into
placental tissue, is less harmful to embryo viability than
Day 3 blastomere biopsy.9,13,14,31 Moreover, it is also highly
cost-effective that only competent embryos capable of
developing to blastocysts and with highest implantation
potential are biopsied.9,18
With the advance of modern laboratory techniques, the
successful rate of blastocyst culture and cryopreservation
has yielded improving and satisfying results.5,31,32 Embryo
cryopreservation and thawed ET has become the new trend
of ART with many advantages. First, it can decrease the risk
of ovarian hyperstimulation syndrome.33 Second, because
PGD is usually performed at a limited number of specialized
genetic laboratories, thawed ET provided ample time to
transport the biopsied samples from the remote infertility
clinic and to perform genetic diagnosis.7 Third, it has been
shown that the endocrionological status and the uterine
receptivity are altered by ovarian stimulation. That may
decrease the implantation potential in a fresh transfer
cycle, especially in a high responder.34e36 ET in a subsequent
thawed cycle can provide a more natural and synchronized
endometrial environment for embryonic implantation.13,31
Our data also showed a high implantation rate of 50%.
In summary, for carriers with chromosomal trans-locations,
our study describes an feasible PGD strategy,
applying blastocyst biopsy, aCGH, embryo cryopreserva-tion,
and thawed ET. aCGH can provide comprehensive 24-
chromosome analysis and the ongoing pregnancy rate was
50%/PGD cycle in our study, which seems to be higher than
previous studies using FISH methods. There was no case of
miscarriage. Although the number of the patients in our
study is too small to draw robust conclusions, the pre-liminary
results may support a promising choice of future
PGD treatment for these patients.
Acknowledgments
The authors would like to thank Ms Chia-Jen Shieh and Ms
Yi-Lin Yao for their technical assistance. Chu-Chun Huang
analyzed the data and wrote the article. Li-Jung Chang, Yi-
Yi Tsai, Chia-Cheng Hung, and Mei-Ya Fang analyzed the
data and provided essential technical support. Yi-Ning Su,
Hong-Jiang Chang, Hsin-Fu Chen, and Shee-Uan Chen per-formed
the research and designed the study. This study was
supported in part by grants from the National Science
Council (100-2314-B-002-022-MY3) and National Taiwan
University Hospital (NTUH 100-S1555), Taipei, Taiwan.
Table 5 (continued)
Case
Karyotype of the
affected couple
Embryo
no.
Results of microarray platform
Chromosomal gain Chromosomal loss Final characterization
9 46XY, t(2;14)(p10;p10) 1 No signal Undiagnosed
3 2q36.1eq37.1 10q26.3eq26.3 Abnormal
10 45XY, rob(13;14)(q11;q11) 1 1p36.33e1q44;
10p15.3e10q26.3;
12p13.33e15q24.33;
19p13.3e19q13.42
5p15.33e5q35.3;
9p24.3e9q34.3;
21q11.2e21q22.3;
Yp11.31eYq12
Abnormal
11 46XX, t(1;14)(q31;q31) 1 Euploid Normal/balanced
2 1p36.33e1q44;
7p22.3e7q36.3
14q11.2e14q24.3 Abnormal
3 Euploid d Normal/balanced
12 46XY, t(9;12)(q11;q13) 1 16p13.3e16q24.3 d Abnormal
2 10p15.3e10q26.3 d Abnormal
5 e Yp11.2eYq12 Abnormal
6 1p36.33e1q44;
15q11.1e15q26.3;
18p11.32e18q23
d Abnormal
7 d 12p13.33e12q24.33 Abnormal
Chr. Z chromosome; rob Z Robertsonian translocation.

References
1. Alfarawati S, Fragouli E, Colls P, Wells D. First births after
preimplantation genetic diagnosis of structural chromosome
abnormalities using comparative genomic hybridization and
microarray analysis. Hum Rep 2011;26:1560e74.
2. Lim CK, Cho JW, Song IO, Kang IS, Yoon YD, Jun JH. Estimation
of chromosomal imbalances in preimplantation embryos from
preimplantation genetic diagnosis cycles of reciprocal trans-locations
with or without acrocentric chromosomes. Fertil
Steril 2008;90:2144e51.
3. Fiorentino F, Spizzichino L, Bono S, Biricik A, Kokkali G,
Rienzi L, et al. PGD for reciprocal and Robertsonian trans-locations
using array comparative genomic hybridization. Hum
Reprod 2011;26:1925e35.
4. Keymolen K, Staessen C, Verpoest W, Michiels A, Bonduelle M,
Haentjens P, et al. A proposal for reproductive counselling in car-riers
of Robertsonian translocations: 10 years of experience with
preimplantation genetic diagnosis.HumReprod 2009;24:2365e71.
5. Sermon K, Van Steirteghem A, Liebaers I. Preimplantation ge-netic
diagnosis. Lancet 2004;363:1633e41.
6. Simpson JL. Preimplantation genetic diagnosis at 20 years.
Prenat Diagn 2010;30:682e95.
7. Chang LJ, Chen SU, Tsai YY, Hung CC, Fang MY, Su YN, et al. An
update of preimplantation genetic diagnosis in gene diseases,
chromosomal translocation, and aneuploidy screening. Clin
Exp Reprod Med 2011;38:126e34.
8. Fischer J, Colls P, Escudero T, Munne´ S. Preimplantation ge-netic
diagnosis (PGD) improves pregnancy outcome for trans-location
carriers with a history of recurrent losses. Fertil Steril
2010;94:283e9.
9. Kokkali G, Traeger-Synodinos J, Vrettou C, Stavrou D, Jones GM,
Cram DS, et al. Blastocyst biopsy versus cleavage stage biopsy
and blastocyst transfer for preimplantation genetic diagnosis of
beta-thalassaemia: a pilot study. Hum Reprod 2007;22:1443e9.
10. El-Toukhy T, Kamal A, Wharf E, Grace J, Bolton V, Khalaf Y,
et al. Reduction of the multiple pregnancy rate in a preim-plantation
genetic diagnosis programme after introduction of
single blastocyst transfer and cryopreservation of blastocysts
biopsied on day 3. Hum Reprod 2009;24:2642e8.
11. Harper JC, Harton G. The use of arrays in preimplantation
genetic diagnosis and screening. Fertil Steril 2010;94:1173e7.
12. Fiorentino F, Kokkali G, Biricik A, Stavrou D, Ismailoglu B, De
Palma R, et al. Polymerase chain reaction-based detection of
chromosomal imbalances on embryos: the evolution of preim-plantation
genetic diagnosis for chromosomal translocations.
Fertil Steril 2010;94:2001e11.
13. Fragouli E, Lenzi M, Ross R, Katz-Jaffe M, Schoolcraft WB,
Wells D. Comprehensive molecular cytogenetic analysis of the
human blastocyst stage. Hum Reprod 2008;23:2596e608.
14. McArthur SJ, Leigh D, Marshall JT, Gee AJ, De Boer KA,
Jansen RP. Blastocyst trophectoderm biopsy and preimplanta-tion
genetic diagnosis for familial monogenic disorders and
chromosomal translocations. Prenat Diagn 2008;28:434e42.
15. Lee TH, Wu MY, Chen HF, Chen SU, Ho HN, Yang YS. Association
of a single dose of gonadotropin-releasing hormone antagonist
with nitric oxide and embryo quality in in vitro fertilization
cycles. Fertil Steril 2006;86:1020e2.
16. Ho CH, Chen SU, Peng FS, Chang CY, Lien YR, Yang YS. Pro-spective
comparison of short and long GnRH agonist protocols
using recombinant gonadotrophins for IVF/ICSI treatments.
Reprod Biomed Online 2008;16:632e9.
17. Wu MY, Chang LJ, Chen MJ, Chao KH, Yang YS, Ho HN. Outcomes
of assisted reproductive techniques for HIV-1-discordant couples
using thawed washed sperm in Taiwan: comparison with control
and testicular sperm extraction/microscopic epididymal sperm
aspiration groups. J Formos Med Assoc 2011;110:495e500.
18. Chen YL, Hung CC, Lin SY, Fang MY, Tsai YY, Chang LJ, et al.
Successful application of the strategy of blastocyst biopsy,
vitrification, whole genome amplification, and thawed embryo
transfer for preimplantation genetic diagnosis of neurofibro-matosis
type 1. Taiwan J Obstet Gynecol 2011;50:74e8.
19. Chen SU, Yang YS. Vitrification of oocytes: various procedures.
In: Tucker MJ, Liebermann J, editors. Vitrification in human
assisted reproduction. New York: Informa Healthcare; 2007. p.
129e44.
20. Chen SU, Lien YL, Tsai YY, Chang LJ, Ho HN, Yang YS. Cry-opreserved
sibling oocytes and intracytoplasmic sperm injec-tion
rescue unexpectedly poor fertilization in conventional
in vitro fertilization. J Assist Reprod Genet 2004;21:367e9.
21. Goossens V, Harton G, Moutou C, Traeger-Synodinos J, Van
Rij M, Harper JC. ESHRE PGD Consortium data collection IX:
cycles from January to December 2006 with pregnancy follow-up
to October 2007. Hum Reprod 2009;24:1786e810.
22. Gianaroli L, Magli MC, Fiorentino F, Baldi M, Ferraretti AP.
Clinical value of preimplantation genetic diagnosis. Placenta
2003;24(Suppl. B):S77e83.
23. Munne´ S. Preimplantation genetic diagnosis for translocations.
Hum Reprod 2006;21:839e40.
24. Verpoest W, Haentjens P, De Rycke M, Staessen C, Sermon K,
Bonduelle M, et al. Cumulative reproductive outcome after
preimplantation genetic diagnosis: a report on 1498 couples.
Hum Reprod 2009;24:2951e9.
25. Verlinsky Y, Tur-Kaspa I, Cieslak J, Bernal A, Morris R,
Taranissi M, et al. Preimplantation testing for chromosomal
disorders improves reproductive outcome of poor-prognosis
patients. Reprod Biomed Online 2005;11:219e25.
26. Johnson DS, Gemelos G, Baner J, Ryan A, Cinnioglu C,
Banjevic M, et al. Preclinical validation of a microarray method
for full molecular karyotyping of blastomeres in a 24-h proto-col.
Hum Reprod 2010;25:1066e75.
27. Gutie´rrez-Mateo C, Colls P, Sa´nchez-Garcı´a J, Escudero T,
Prates R, Ketterson K, et al. Validation of microarray
comparative genomic hybridization for comprehensive chro-mosome
analysis of embryos. Fertil Steril 2011;95:953e8.
28. Vanneste E, Melotte C, Voet T, Robberecht C, Debrock S,
Pexsters A, et al. PGD for a complex chromosomal rearrange-ment
by array comparative genomic hybridization. Hum
Reprod 2011;26:941e9.
29. Spits C, Sermon K. PGD for monogenic disorders: aspects of
molecular biology. Prenat Diagn 2009;29:50e6.
30. Wilton L. Preimplantation genetic diagnosis and chromosome
analysis of blastomeres using comparative genomic hybridiza-tion.
Hum Reprod Update 2005;11:33e41.
31. Schoolcraft WB, Fragouli E, Stevens J, Munne S, Katz-Jaffe MG,
Wells D. Clinical application of comprehensive chromosomal
screening at the blastocyst stage. Fertil Steril 2010;94:1700e6.
32. McArthur SJ, Leigh D, Marshall JT, de Boer KA, Jansen RP.
Pregnancies and live births after trophectoderm biopsy and
preimplantation genetic testing of human blastocysts. Fertil
Steril 2005;84:1628e36.
33. Devroey P, Polyzos NP, Blockeel C. An OHSS-Free Clinic by
segmentation of IVF treatment. Hum Reprod 2011;26:2593e7.
34. Arslan M, Bocca S, Arslan EO, Duran HE, Stadtmauer L,
Oehninger S. Cumulative exposure to high estradiol levels
during the follicular phase of IVF cycles negatively affects
implantation. J Assist Reprod Genet 2007;24:111e7.
35. Joo BS, Park SH, An BM, Kim KS, Moon SE, Moon HS. Serum
estradiol levels during controlled ovarian hyperstimulation in-fluence
the pregnancy outcome of in vitro fertilization in a
concentration-dependent manner. Fertil Steril 2010;93:442e6.
36. Huang CC, Lien YR, Chen HF, Chen MJ, Shieh CJ, Yao YL, et al.
The duration of pre-ovulatory serum progesterone elevation
prior to hCG administration affects the outcome of IVF/ICSI
cycles. Hum Reprod 2012;27:2036e45.

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