RBMOnline - Vol 19. No 2. 2009 228-237 Reproductive BioMedicine Online; www.rbmonline.com/Article/3798 on web 17 June 2009
Article
Comparative genomic hybridization of oocytes
and first polar bodies from young donors
After obtaining her BSc in Molecular Biology from the University of Surrey, UK and her
MSc and PhD in human genetics from University College London, Elpida Fragouli worked
at the UCL Centre for PGD and then took up a position at Yale University Medical School’s
Department of Obstetrics and Gynecology. In 2007 she returned to the UK, to the Nuffield
Department of Obstetrics and Gynaecology, University of Oxford. Her research interests
focus on the incidence of chromosome abnormality in human oocytes and embryos,
and the mechanisms leading to aneuploidy. Dr Fragouli also serves as a consultant for
Reprogenetics-UK, involved in the PGD of chromosome abnormalities affecting oocytes
and embryos.
Dr Elpida Fragouli
E Fragouli1,2, A Escalona3, C Gutiérrez-Mateo4, S Tormasi4, S Alfarawati1, S Sepulveda5, L Noriega5, J Garcia5, D
Wells1,2, S Munné4,6
1
Reprogenetics-UK, Oxford, UK; 2Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford,
UK; 3Unitat de Biologia Cel·lular i Genètica Mèdica, Facultat de Medicina, Universitat Autònoma de Barcelona, Spain;
4
Reprogenetics, Livingston, NJ, USA; 5Clinica Concebir, Lima, Peru
6
Correspondence: e-mail: munne@reprogenetics.com
Abstract
Chromosome abnormalities are common in oocytes derived from patients undergoing IVF treatment. The proportion of oocytes
displaying aneuploidy is closely related to maternal age and may exceed 60% in patients over 40 years old. However, little
information currently exists concerning the incidence of such anomalies in oocytes derived from young fertile women. A total
of 121 metaphase II oocytes and their corresponding first polar bodies (PB) were analysed with the use of a comprehensive
cytogenetic method, comparative genomic hybridization (CGH). The oocytes were donated from 13 young women (average
age 22 years) without any known fertility problems. All oocytes were mature at the time of retrieval and were unexposed
to spermatozoa. A low aneuploidy rate (3%) was detected. These results clearly indicate that meiosis I segregation errors
are not frequent in oocytes of young fertile women. The higher aneuploidy rates reported in embryos derived from donor
oocytes could be due to aggressive hormonal stimulation, in combination with male factors. However a definite contributing
factor remains to be elucidated. The data obtained during this study also illustrate that CGH accurately and efficiently detects
aneuploidy, confirming that it is suitable for application in a clinical setting for the assessment of oocytes, via PB analysis.
Keywords: chromosome, comparative genomic hybridization, oocyte, oocyte donor, polar body, preimplantation genetic diagnosis
Introduction
Many of the embryos generated using IVF techniques are
chromosomally abnormal (Munné et al., 1995; Márquez et
al., 2000; Magli et al., 2001; Bielanska et al., 2002; Munné et
al., 2007). In a recent study, involving an examination of nine
chromosomes at the cleavage stage via fluorescence in-situ
hybridization (FISH), 60% of all analysed 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 (Munné et al.,
2007). Considering only good-morphology embryos, the rate
of chromosome errors seen ranged from 56–67%, depending on
maternal age (<35 or ≥40 years).
Most of the chromosome errors detected are incompatible
with implantation or birth, and are therefore predicted to
negatively affect assisted reproductive treatments. The high
prevalence of aneuploid embryos, and the lethality of most
chromosome abnormalities, has led many IVF centres to adopt
the use of preimplantation genetic diagnosis (PGD; also named
preimplantation genetic screening) in infertility treatment. The
purpose of PGD in this context is to identify euploid embryos
that may have a higher chance of producing a healthy clinical
pregnancy and insure that they are prioritized for transfer to the
uterus. Chromosome screening using PGD techniques is usually
offered to patients with poor prognosis such as advanced maternal
228
© 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK
Article - Comparative genomic hybridization with young donors - E Fragouli et al.
age (Munné et al., 1993, 1999, 2003, 2006a; Gianaroli et al., 1999;
Staessen et al., 2004; Colls et al., 2007), recurrent pregnancy loss
(Vidal et al., 1998; Pellicer et al., 1999; Rubio et al., 2003; Werlin
et al., 2003; Munné et al., 2005; Platteau et al., 2005; Garrisi et
al., 2008), previous aneuploid conception (Munné et al., 2004),
repeated implantation failure (Gianaroli et al., 1999, 2001, 2003;
Kahraman et al., 2000; Pehlivan et al., 2002; Wilton et al., 2003)
and/or extreme male factor (Silber et al., 2003).
Various studies have suggested that women 35 years of age or
younger, who are undergoing IVF in combination with PGD
due to recurrent pregnancy loss or repeated implantation failure,
could have a predisposition to aneuploidy in their oocytes and/
or embryos (Rubio et al., 2003; Munné et al., 2004; Fragouli
et al., 2006a). In other words, the high frequency of abnormal
embryos generated by this group may not be representative of
younger patients as a whole. If this is indeed the case, then one
might predict that young, fertile egg donors produce oocytes
and embryos, which in their great majority are chromosomally
euploid. Unfortunately, it is difficult to confirm or disprove this
hypothesis, since oocytes and embryos produced by egg donors
are rarely subjected to chromosome screening and consequently
data from such patients is sparse.
Occasionally, oocyte recipients request PGD as they may wish to
minimize the risk of a trisomic conception. The scanty published
PGD data on embryos derived from oocyte donors indicate
unexpectedly high rates of chromosome anomalies (56–57%)
(Reis Soares et al., 2003; Munné et al., 2006b). According to Reis
Soares et al. (2003) the reason for the observed abnormalities,
could be that donors are frequently subject to more aggressive
stimulation, compared with other women of similar age, in order
to guarantee the production of a large cohort of oocytes. In their
study, they compared the rate of chromosome abnormalities in
embryos derived from donor oocytes with those from young
patients undergoing PGD for X-linked diseases. They found
a significant difference in the number of oocytes retrieved (25
and 15, respectively), chromosome abnormalities scored (56%
and 37%) and implantation rates (25% to 36.4%). The increased
aneuploidy rate in the donor group is particularly striking given
that the mean age of the donors was younger than the X-linked
PGD patients (27 versus 31 years). They concluded that larger
cohorts of embryos could show higher aneuploidy rates (Reis
Soares et al., 2003).
Munné et al. (2006b) could not confirm that the oocyte cohort
size was the issue. They analysed over 1800 embryos from 124
oocyte donation cycles (average age 25 years) and compared
them to a control group of poor prognosis infertile PGD patients
younger than 35 (average age 31 years). They observed that 57%
of embryos from donor cycles were abnormal compared with
66% of control embryos, and that the mean number of embryos
produced was 16 and 11, respectively. This study also found
high variability between cycles, with more than a quarter (27%)
producing 30% or less euploid embryos. Additionally, the use of
oocytes from the same donor for different recipients demonstrated
the influence of male-derived factors, which caused intra-donor
variations in chromosome abnormalities between cycles (Munné
et al., 2006b).
The data obtained from the aforementioned studies suggest that
the high aneuploidy rates seen in embryos generated during oocyte
donation cycles could be due to aggressive stimulation, male
factor or other issues, but a definite contributing cause remains
to be defined. A better approach for examining the incidence of
aneuploidy in young fertile women is to analyse oocytes directly,
removing male-derived confounding factors.
The objectives of this investigation were two-fold. The first
aim was to investigate the first meiotic division in young
women with no known fertility problems by analysing mature
metaphase II (MII) oocytes, unexposed to spermatozoa, and their
corresponding first polar bodies (PB). To achieve this, comparative
genomic hybridization (CGH) was employed, which is a more
comprehensive method than alternative FISH techniques and
reveals aneuploidy affecting any chromosome. The second aim
was to evaluate the efficiency of CGH analysis of polar bodies
prior to future clinical application for the purpose of PGD.
Although previous studies have performed CGH analysis of the
female gamete, all have focused on examination of oocytes and
PB derived from infertile patients of various ages. Additionally,
most of the material used in these studies have been unsuitable
for IVF (for example, metaphase I (MI) oocytes and germinal
Table 1. Summary of published reports of full-karyotype analysis on first polar body and confirmation of results by
oocyte metaphase II analysis.
Publication
Population Age
Average Number of Aneuploidy Not detectable
type category age MII oocytes (%) by 9-probe
(years) (years) or PB1
FISH
Gutiérrez-Mateo et al., 2004a
Infertile
Gutiérrez-Mateo et al., 2004b
Infertile
Gutiérrez-Mateo et al., 2005
Infertile
Fragouli et al., 2006b
Infertile
All the above
Infertile
This study
Donor
All
<35
All
<35
All
<35
All
<35
<35
<35
33.2
27.8
35.8
30.6 32.9
30.3
32.5
28.0 28.5
22.5
FISH = fluorescence in-situ hybridization; MII = metaphase II; PB1 = first polar body.
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25
13
42
13
16
10
79
66
102
106
11 (44.0)
2 (15.4)
20 (47.6)
7 (53.9)
4 (25.0)
1 (10.0)
8 (10.1)
5 (7.6)
15 (14.7)
3 (2.8)
6 (55)
1 (50)
12 (60)
3 (43)
1 (25)
0 (0)
3 (38)
1 (25)
5 (33)
2 (67)
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Article - Comparative genomic hybridization with young donors - E Fragouli et al.
vesicles matured to MII stage, failed fertilization) (GutiérrezMateo et al., 2004a,b, 2005; Fragouli et al., 2006a,b) The data
obtained from these studies are summarized in Table 1.
Materials and methods
Oocyte donors
A total of 121 MII oocyte–PB pairs was examined during the
course of this study. These were donated by 13 women (average
age 22.2 years, age range 19–27 years) who were recruited at
Clinica Concebir (Grupo Pranor, Lima, Peru) as oocyte donors
but were not used for donation for various reasons (eight for
poor donor response, three for scheduling issues of recipients
and two for problems in producing/providing sperm samples)
(Table 1). The patients had consented that if the oocytes were
not to be used for oocyte donation they could be used for
research. The study took place under signed informed consent
and after approval from the institutional review board of the
IVF centre.
The ovarian stimulation protocol consisted of pituitary downregulation using gonadotrophin-releasing hormone antagonist
Ovidrel (Merck Serono). Follicular development was
achieved by administering daily injections of 150–225 IU of
gonadotrophin (Gonal F; Merck Serono). Follicular growth was
observed by transvaginal ultrasonography, and a dose of 10,000
IU of human chorionic gonadotrophin (Orgalutran; Schering
Plough) was administered between 7 and 11 days (Table
2). Collection of oocytes took place 36 h later by ultrasound
transvaginal aspiration.
Cells were processed as outlined in Fragouli et al. (2006a). The
single cells (PB and eggs) were placed in microcentrifuge tubes
containing 0.5 µl of phosphate-buffered saline (0.1% polyvinyl
alcohol), frozen at –20°C and sent to the Reprogenetics
laboratory in Livingston, NJ, USA. Cell lysis took place
by incubating the samples in 2 µl proteinase K (125 µg/ml)
(Roche, USA) and 1 µl sodium dodecyl sulphate (17 mmol/l)
(Sigma), at 37°C for 1 h, followed by an incubation at 95°C for
15 min to inactivate the proteinase K.
Comparative genomic hybridization
The CGH methodology used for the detailed cytogenetic
examination of the MII oocyte–PB pairs took place as described
previously (Fragouli et al., 2006b). The reference DNA against
which the oocytes and PB were hybridized and compared was
extracted from the blood of a normal female individual (46,XX),
and diluted to a concentration of 0.5–1 ng/µl.
Whole genome amplification of test (oocytes and PB) and
reference (46,XX) DNA was achieved with the use of the
degenerate oligonucleotide-primed PCR with the modification
of Wells et al. (1999). Nick translation was then employed in
order to fluorescently label the test oocyte and PB DNA in
green and the reference 46,XX DNA in red (Spectrum GreendUTP, Spectrum Red-dUTP, nick translation kit; Abbott,
Illinois, USA). Co-precipitation of test and reference DNA,
their denaturation, along with that of the slides, and the posthybridization washes all took place as described previously in
Fragouli et al. (2006a). The hybridization time was 72 h.
Oocyte and PB processing
Microscopy, image analysis and
interpretation
PB were biopsied mechanically by opening a double slit in the
zona pellucida (Verlinsky and Kuliev, 2005). Oocytes were
removed from the zona pellucida using Tyrode’s acidified
solution (Sigma, USA).
Metaphase spreads were observed with the use of an Olympus
BX 61 fluorescence microscope with a cooled charge-coupled
device system and filters for the fluorochromes used. On
average, 10 metaphases were captured per hybridization.
Table 2. Donor and stimulation protocol details.
Ovulation
Donor Age
Start of
Duration of No. of
No. of
induction no.
(years) stimulation stimulation oocytes mature
(day) (days) retrieved oocytes
Antagonist, HMG, rHCG
Antagonist, rFSH, rHCG
230
1
2
3
4
5
6
7
8
9
10
11
12
13
23
27
19
26
23
27
23
19
23
21
19
23
20
4
2
3
3
2
2
2
2
2
4
2
1
2
9
10
10
9
10
9
7
9
9
9
11
9
10
10
7
14
9
22
14
13
17
10
6
9
5
25
6
7
13
8
16
11
8
13
5
6
7
3
18
HMG = human menopausal gonadotrophin, rHCG = recombinant human chorionic gonadotrophin; rFSH = recombinant FSH.
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Article - Comparative genomic hybridization with young donors - E Fragouli et al.
Analysis and interpretation of the captured images took
place with the use of Cytovision CGH software (version 3.9,
Applied Imaging, San Jose, USA) that converted fluorescent
intensities into a red:green ratio for each chromosome. Equal
sequence copy number between the test and reference DNA
was seen as no fluctuation of the ratio profile from 1:1. Test
sample under-representation was seen as fluctuation of the ratio
profile in favour of the red colouration (below 0.80), whilst
test sample over-representation was seen as fluctuation of the
ratio profile towards the green colouration (above 1.20). Such
fluctuations were respectively scored as losses or gains in the
test sample, compared with the reference sample. Distinction
of chromosome and chromatid errors took place as described
previously (Fragouli et al., 2006a,b).
Results
The women who donated the MII oocyte–PB pairs that were
examined during the course of this study were assumed to be
of normal karyotype and were without any known fertility
problems. No information concerning additional aspects of the
women’s medical histories were available. The average age of
these women was 22.2 years and their ages ranged between 19–
27 years. All investigated oocytes were at the metaphase II stage
of meiosis at the time of retrieval, appeared morphologically
normal and were unexposed to spermatozoa.
In total, 121 of MII oocytes-PB pairs were separated and
had their genomes amplified. Successful amplification was
achieved for 155 cells (64%), 67 oocytes and 88 PB (49 pairs),
as demonstrated by visualization of smears of DNA fragments
upon agarose gel analysis of the amplified products, with sizes
varying between 200 and 4000 base pairs (bp). CGH yielded
results for all 155 successfully amplified cells. Results were
obtained from both the oocyte and the corresponding PB for 49
MII oocyte–PB pairs. Additionally, CGH was successful for 39
first PB and 18 MII oocytes without data from the corresponding
oocyte or PB.
The efficiency of the CGH methodology was 64%,
significantly lower than achieved in previous studies using this
technique (Gutiérrez-Mateo et al., 2004a,b, 2005; Fragouli et
al., 2006a,b). CGH success rates were somewhat higher for
first PB (88/121, 73%) compared with MII oocytes (67/121,
55%). The reduced CGH efficiency observed during the
current study appears to have been a consequence of logistical
problems reported by the laboratory preparing the cells, since
the first three samples were delayed in customs. Thereafter,
CGH efficiency increased with each additional donor cycle
processed, demonstrating a learning curve with regard to
sample preparation: From the last six cycles, 85% of polar
bodies yielded analysable CGH results.
Table 3 lists the results obtained after CGH analysis of the MII
oocytes and their corresponding PB. The ages of the oocyte
donors are also included. Out of the 106 MII oocyte–PB
complexes that yielded results (49 pairs, 18 MII oocytes, 39 PB),
103 were characterized as haploid normal, 23,X. Abnormalities
were scored for two different MII oocyte–PB pairs, with
reciprocal results obtained for the oocyte and corresponding PB.
Additionally, one single MII oocyte was found to be abnormal,
with CGH failure being observed for its corresponding PB. The
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abnormal samples were produced by donors 9 and 10 (aged 23
and 21, respectively). Out of the anomalies scored, two oocyte–
PB pairs involved imbalance affecting a single chromosome
(chromosomes 3 and 13), whereas a partial aneuploidy
(affecting the entire long arm of chromosome 1, 1q21—1q43)
was observed in the other pair. The aneuploidy rate at the end
of the first meiotic division was calculated to be 2.8%. Figure 1
shows the hybridization and profiles from a normal oocyte–PB
pair and an abnormal oocyte.
Discussion
CGH efficacy
In previous studies that used CGH to examine the female
gamete, the vast majority of oocytes and corresponding polar
bodies yielded reciprocal results (e.g. if a chromosome was
lost in a polar body, the same chromosome showed a gain in
the corresponding oocyte). This, is precisely as theory would
predict and clearly demonstrates the accuracy and sensitivity of
the CGH method (Gutiérrez-Mateo et al., 2004a,b; Fragouli et
al., 2006a,b). In the current study, there was 100% concordance
between oocytes and associated first PB.
The CGH methodology described here is identical to that
previously employed in studies of oocytes derived from older
IVF patients. If CGH is capable of accurately identifying
chromosomal abnormalities, a much higher aneuploidy rate
is expected for oocytes from older women, due to the wellestablished relationship between maternal age and aneuploidy.
Again, this is precisely what has been observed. The 3%
aneuploidy rate detected using CGH in the current study (MII
oocytes from donors with a mean age of 22 years) compares
with a 22% aneuploidy rate observed for IVF patients of mean
maternal age of 31 years (Fragouli et al., 2006b) and a 43%
rate after CGH analysis of oocytes from IVF patients of mean
age of 41 (unpublished data). Each of these figures represents
the aneuploidy rate at the end of meiosis I. The ultimate oocyte
aneuploidy rate upon completion of the second meiotic division
is anticipated to be somewhat higher. In the case of the donor
samples reported here, ~5% of oocytes are predicted to be
abnormal after completion of both meiotic divisions. This
estimate is based upon the relative incidence of chromosome
imbalance detected in first and second polar bodies using FISH
(Kuliev et al., 2003).
Polar body CGH as a clinical test
The concept of using CGH clinically for assessing oocytes (via polar
body analysis) is an attractive one. CGH is able to simultaneously
screen all 23 chromosomes present in the polar body rather than
the highly restricted set (usually five chromosomes) scored via
FISH (Verlinsky et al., 2005). Additionally, CGH avoids the
need to spread polar bodies on a microscope slide, a notoriously
difficult technique that frequently leads to errors due to artifactual
loss of chromosomes. However, the screening method chosen for
assessing polar bodies and oocytes must have a high efficiency as
well as accuracy. In the current study, the proportion of PB that
yielded a result (73%) was lower than expected: a poorer diagnostic
efficiency than observed during previous CGH studies (Fragouli
et al. 2006b; Gutiérrez-Mateo et al. 2004a) and unacceptably low
for clinical application.
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Table 3. Comparative genomic hybridization (CGH) analysis
results of egg-polar body (PB) pairs.
232
Donor no.
(Age in years)
Egg CGH results
Classification
no. Oocyte First PB
1 (23)
2 (27)
3 (19)
4 (26)
5 (23)
6 (27)
7 (23)
1
2
3
4
5
1
2
3
4
1
2
3
4
5
6
7
8
9
10
11
1
2
3
4
5
6
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1
2
3
4
5
6
7
8
9
10
11
1
2
3
4
5
23,X
23,X
23,X
–
–
23,X
23,X
23,X
–
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
–
23,X
23,X
–
–
–
–
23,X
23,X
23,X
23,X
23,X
–
–
–
–
–
–
–
–
–
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
–
23,X
23,X
23,X
23,X
23,X
23,X
23,X
–
23,X
23,X
23,X
–
–
23,X
23,X
23,X
23,X
–
–
–
–
–
–
–
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
lost
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
23,X
–
–
23,X
23,X
23,X
23,X
23,X
23,X
Pair normal
Pair normal
Egg normal
PB normal
PB normal
Pair normal
Egg normal
Egg normal
PB normal
Pair normal
Pair normal
Pair normal
Egg normal
Egg normal
Egg normal
Egg normal
Egg normal
Egg normal
Egg normal
PB normal
Pair normal
Pair normal
PB normal
PB normal
PB normal
PB normal
Pair normal
Pair normal
Pair normal
Pair normal
Egg normal
PB normal
PB normal
PB normal
PB normal
PB normal
PB normal
PB normal
PB normal
PB normal
Pair normal
Pair normal
Pair normal
Pair normal
Pair normal
Pair normal
Pair normal
Pair normal
Egg normal
Egg normal
PB normal
Pair normal
Pair normal
Pair normal
Pair normal
Pair normal
Continued on page 233
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Table 3. Continued from page 232
Donor no.
(Age in years)
Egg CGH results
Classification
no. Oocyte First PB
8 (19)
9 (23)
10 (21)
11 (19)
12 (23)
13 (20)
6
7
8
1
2
3
4
5
6
7
8
9
10
11
12
13
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
6
7
1
2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
23,X
–
Egg normal
23,X
–
Egg normal
–
23,X
PB normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
–
23,X
PB normal
23,X,+1q23,X,–1q Pair abnormal
22,X,-3 –
Egg abnormal
–
23,X
PB normal
–
23,X
PB normal
–
23,X
PB normal
22,X,-13 24,X,+13 Pair abnormal
23,X
23,X
Pair normal
–
23,X
PB normal
–
23,X
PB normal
–
23,X
PB normal
23,X
23,X
Pair normal
–
23,X
PB normal
–
23,X
PB normal
–
23,X
PB normal
–
23,X
PB normal
–
23,X
PB normal
–
23,X
PB normal
23,X
23,X
Pair normal
23,X
–
Egg normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
23,X
23,X
Pair normal
23,X
–
Egg normal
–
23,X
PB normal
–
23,X
PB normal
–
23,X
PB normal
–
23,X
PB normal
–
23,X
PB normal
–
23,X
PB normal
–
23,X
PB normal
–: No result.
In 15 cases, neither the oocyte nor the corresponding polar body yielded a result. These pairs
have been excluded from this table.
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Article - Comparative genomic hybridization with young donors - E Fragouli et al.
Figure 1. Comparative genomic hybridization (CGH) analysis. All chromosomes for oocyte 1 (a) and polar body 1 (b) from donor
1 display a yellow coloration, indicating equal hybridization of red (46,XX reference) and green (test) DNA samples, leading to
a diagnosis of 23,X for both cells. Oocyte 2 from donor 9 display an excess of red fluorescence on chromosome 3 (c), indicating
that this oocyte has lost a copy of this chromosome. This aneuploidy was confirmed by computer-assisted visualization of the
red:green fluorescence ratio along the length of each chromosome (d), in which chromosome 3 displays an obvious shift away from
the central black line that denotes a 1:1 red:green ratio. Deviations from a 1:1 ratio seen at the telomeres and centromeres are not
scored during CGH analysis, as these regions are blocked from hybridizing DNA. Additionally, the Y-chromosome is not present
in the oocyte, polar body or reference DNA, thus any fluorescence observed on this chromosome is attributable to background
fluorescence.
It seems that the poor CGH success rate in the current study
was attributable to two factors. Firstly, the diagnostic efficiency
increased as the study progressed, from 38% in the first three
donor cycles, which were affected by logistical problems
regarding its transport from Lima, Peru to New Jersey, USA, to
a much more acceptable 85% in the last six donor cycles. This
improvement in performance is indicative of a learning curve
regarding the processing of polar bodies for CGH analysis and
highlights the need for training and evaluation of the biopsy/
processing technique prior to clinical application. The second
factor affecting diagnostic efficiency was the lack of a –80°C
freezer in the IVF laboratory. Previous studies at the study centre
have found that storage at –20°C is insufficient for maintaining
polar body DNA in a suitable condition for subsequent CGH
analysis, leading to reduced DNA amplification and poor
fluorescence (Fragouli et al., 2006a). With optimal methods,
experienced staff and ultralow refrigeration, CGH success
rates of over 90% can be achieved for polar bodies, which is a
suitable efficiency for clinical application.
234
It must be acknowledged that CGH requires extensive
experience with a range of cytogenetic and molecular genetic
techniques. For this reason, CGH is likely to remain focused
in specialist PGD reference laboratories. However, while
the genetic analysis component of CGH is challenging, the
component performed in the IVF laboratory (cell preparation)
is actually more straightforward than the methods used for
FISH-based analyses. For CGH, biopsied cells are simply
placed into microcentrifuge tubes rather than being subjected to
the more challenging and laborious technique of spreading on a
microscope slide. Thus, paradoxically, embryologists are likely
to find it easier to send cells for complex CGH testing than for
more simplistic FISH analysis.
Chromosome abnormalities in oocytes of
infertile and fertile patients
Data from clinical pregnancies indicate that approximately 85%
of aneuploidies originate from maternal meiosis I (reviewed
in Hassold et al., 2007). However, these results are based on
linkage analysis, which determines when the homologous
chromosomes disjoined or sister chromatids prematurely
divided, but not when this meiotic error was resolved, i.e. the
underlying chromosome segregation error could have occurred
in meiosis I, but ultimately led to abnormal chromosomal
segregation at MI or MII. Indeed, a large study of first and
second polar bodies using FISH indicated that the resolution
of meiotic errors takes place with a similar frequency in both
meiotic divisions, with 42% of abnormal oocytes having an
abnormal first PB, 31% an abnormal second PB and 27% both
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Article - Comparative genomic hybridization with young donors - E Fragouli et al.
PB abnormal (Kuliev et al., 2003). This finding is explained by
high rates of predivision of chromatids during meiosis I, which
can lead to the generation of chromosomal imbalance after
either meiosis I or II (Angell, 1991).
Data from FISH analysis of both the first and second PB
from zygotes of advanced maternal age, infertile women,
demonstrate very high aneuploidy rates despite only assessing
a limited number of chromosomes (52.1% aneuploidy after
testing chromosomes 13, 16, 18, 21 and 22, mean age 38.5
years) (Kuliev et al., 2003). Even higher rates of oocyte
abnormality have been recorded after CGH analysis of the first
and second PB in patients averaging 40.5 years of age (67%)
(Fragouli et al., 2009). The incidence of aneuploidy is expected
to be significantly lower for young patients. However, very few
studies have assessed oocytes from young women. As a result,
the abnormality rate for such patients has had to be inferred
from the incidence of aneuploid pregnancy seen for young
mothers. Given that most forms of chromosome abnormalities
are lethal before prenatal testing is possible, it is likely that
many aneuploid conceptions spontaneously abort without being
detected. Thus, without direct analysis of the oocyte it is not
possible to gauge the true level of chromosomal abnormality.
An aneuploidy rate of 22% has been previously reported in
unfertilized oocytes from relatively young infertile women
(mean age 31 years), analysed using comprehensive methods of
chromosomal analysis (e.g. CGH or multiplex FISH) (Table 1;
Fragouli et al., 2006b; Gutierrez-Mateo et al., 2005). However,
the oocytes assessed in these studies were mostly discarded
from IVF cycles, having failed to fertilize after sperm exposure.
A key question is whether the incidence of abnormality would
be the same in mature, fertilization-competent oocytes derived
from fertile women. The data reported here clearly indicates
that young, oocyte donors without any known fertility problems
have an extremely low rate of aneuploidy in their oocytes (~3%)
after the completion of meiosis I.
The incidence of oocyte aneuploidy observed in the current
study is lower than rates published in large FISH studies, as
well as some previous CGH reports. This can be attributed
to several factors, the most important of which is likely to be
the difference in maternal age. The mean maternal age for the
donors presented here is about 9 years younger than the infertile
patients previously assessed by CGH and more than 15 years
younger than most large FISH studies (Kuliev et al., 2003;
Gutiérrez-Mateo et al., 2004a,b, 2005; Verlinsky et al., 2005;
Fragouli et al., 2006a,b). The underlying infertility of the IVF
patients who donated oocytes to previous CGH studies might
also lead to differing aneuploidy rates, as a few of these women
appear to be predisposed to aneuploidy (Fragouli et al., 2006a).
Additionally, differences in chromosome abnormality rates
might be caused by variation in the oocyte donor populations
studied (South European, North American, South American) or
in the hormonal stimulation regimes employed: an average of
25 oocytes produced per cycle by Reis Soares et al. (2003), 16
by Munné et al. (2006b), and 9.3 oocytes in the present study.
The low aneuploidy rate detected in donor oocytes in this study
is in sharp contrast to the 65% abnormalities reported by Sher et
al. (2007) in their attempt to examine donor oocytes and PB via
CGH. It is important to note that previous data, obtained using a
wide variety of cytogenetic techniques, including CGH, spectral
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karyotyping and conventional chromosome banding studies, are
all indicative of a low aneuploidy rate for donor oocytes. The
incidence of chromosome imbalance in these studies varies from
4.5–23%, with most finding <12% aneuploidy for oocytes of
women under 30 years of age (Gutiérrez-Mateo et al., 2004a,b;
Sandalinas et al., 2002; Pellestor et al., 2003; Fragouli et al.,
2006b). The extreme difference in oocyte abnormality rates
reported by this study and Sher et al. (2007) could be attributed
to various technical/methodological issues or patient-specific
factors, as has been discussed in detail previously (Fragouli et
al., 2007).
Comparison of oocyte and embryo data
generated from young donors
FISH analysis of embryos generated during oocyte donation
cycles has demonstrated an abnormality rate of approximately
60% (Reis Soares et al., 2003; Munné et al., 2006b), very
different to the 3% aneuploidy rate identified in this work. The
difference in abnormality rates between the two sets of data can
be largely explained by the fact that analysis of the first PB
only provides information on errors occurring during meiosis
I. In contrast, analysis of embryonic cells not only reveals
chromosome errors originating in meiosis I, but also meiosis II,
accounting for ~30% of abnormal oocytes (Kuliev et al., 2003),
paternally derived aneuploidies and errors of post-zygotic
origin, the latter of which affect ~30% of embryos (Munné et
al., 2007). However, this does not seem to fully explain the
difference in the data sets.
Additionally, both Reis Soares and colleagues (2003) and
Munné and colleagues (2006b) questioned whether the high
error rates seen in embryos generated by young fertile oocyte
donors could be attributed to a combination of an aggressive
hormonal stimulation and, in some cases, male factor. In the
IVF centre that participated in the current study, the hormonal
stimulation protocol employed did not differ from the one
employed for patients who are undergoing treatment for
infertility. In other words, the oocyte donation cycles were
not stimulated in an aggressive manner, and this is evident by
the fact that the donors produced on average only 9.3 mature
oocytes, compared with 25 in Reis Soares et al. (2003) and 16
in Munné et al. (2006b).
In addition to the proportion of normal eggs, the total number
obtained is also important. For example, in one of the few
publications comparing different hormonal stimulations,
Weghofer et al. (2008) reported that a long agonist plus FSH
protocol produced more embryos (average 14) and a higher
aneuploidy rate than a long agonist and human menopausal
gonadotrophin protocol (average 12). Interestingly, the number
of chromosomally normal embryos per cycle was approximately
the same for each protocol (average 3).
It is possible that aggressive stimulation increases the risk that
genetically abnormal oocytes will be recruited, or that some of
the oocytes retrieved may have progressed too rapidly through
maturation, which stresses key cellular pathways and leaves
them predisposed to chromosome malsegregation. It is also
possible that, in a perfectly functioning ovary, abnormal oocytes
are recognized and targeted for degeneration, but this qualitycontrol mechanism may be disrupted by natural processes, such
235
Article - Comparative genomic hybridization with young donors - E Fragouli et al.
as advancing age, or by artificial factors, such as aggressive
ovarian stimulation. Further research is required to confirm or
refute these hypotheses and, for the time being, they must be
considered highly speculative.
In conclusion, this is the first comprehensive cytogenetic
investigation of mature high quality MII oocytes, unexposed to
spermatozoa and generated from young oocyte donors without
any known fertility problems. It is evident from these data that
the aneuploidy rate for an average maternal age of 22 years is
~3%. Additionally, these results, coupled with those of previous
studies, suggest that CGH has the ability to accurately and
efficiently detect chromosome errors. Such an approach could
be useful in a clinical setting for the assessment of oocytes, via
first and possibly second PB analysis. Chromosomal screening
of this type permits preferential transfer of embryos derived
from normal haploid oocytes, potentially improving pregnancy
rates and reducing the incidence of spontaneous abortion by
avoiding non-viable aneuploid conceptions.
Acknowledgements
DW was funded by NIHR Biomedical Research Centre
Programme. NIH grant 5-R44-HD-044313–03.
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Declaration: The authors report no financial or commercial
conflicts of interest.
Received 13 July 2008; refereed 29 September 2008; accepted 16
March 2009.
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