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Hum. Reprod. Advance Access published November 11, 2012
Human Reproduction, Vol.0, No.0 pp. 1– 10, 2012
doi:10.1093/humrep/des394
ORIGINAL ARTICLE Reproductive genetics
Antonio Capalbo 1,*, Sara Bono 2, Letizia Spizzichino 2, Anil Biricik 2,
Marina Baldi 2, Silvia Colamaria 1, Filippo Maria Ubaldi 1, Laura Rienzi 1,
and Francesco Fiorentino 2
1
Centre for Reproductive Medicine, GENERA, Clinica Valle Giulia, Via G. De Notaris 2b, Rome 00197, Italy 2Molecular Genetics Laboratory,
GENOMA, Via Po, Rome 102 00198, Italy
*Correspondence address. Tel: +39 063269791; E-mail: [email protected]
Submitted on June 16, 2012; resubmitted on September 28, 2012; accepted on October 12, 2012
study question: What is the optimal stage from oocyte through preimplantation embryo development for biopsy and preimplantation genetic screening (PGS) to detect abnormal chromosome segregation patterns in eggs or embryos from advanced maternal age (AMA)
patients?
summary answer: Testing at the polar body (PB) stage was the least accurate mainly due to the high incidence of post-zygotic events.
This suggests that postponing the time of biopsy to the blastocyst stage of preimplantation embryo development may provide the most
reliable results for PGS.
what is known already: In the PGS field there is an ongoing debate about the optimal biopsy stage for PGS. This is a result of
the lack of understanding of how aneuploidy arises in the human embryo. To date, most of the cytogenetic data obtained during PGS investigations have been derived through the analysis of cells at isolated points in the preimplantation window, thus potentially missing critical
information on chromosomal segregation. Understanding the chromosome segregation patterns during preimplantation development
holds the potential to significantly increase the success rates of IVF. In this study, a sequential comprehensive chromosome analysis of
both the PBs and the corresponding embryos at both the cleavage and the blastocyst stages is presented.
study design, size, duration: This is a prospective longitudinal cohort study performed between October 2009 and August
2011 involving 9 infertile couples and 21 sets of complete comprehensive chromosomal screening data, including PB1, PB2, corresponding
blastomeres and trophectoderm (TE) samples.
participants/materials, setting, methods: Infertile couples undergoing IVF cycles with PGS where the female partner
was older than 40 years and with a good response to controlled ovarian stimulation (.10 MII oocytes retrieved) were enrolled into the
study. The exclusion criteria were (i) patients presenting with abnormal karyotype; (ii) specific ovarian pathologies including polycystic
ovary syndrome, endometriosis grade III or higher and premature ovarian failure and (iii) severe male factor infertility (motile sperm
count of ,500 000/ml after preparation of a fresh ejaculate). The PBs, blastomere and TE samples were sequentially biopsied and analyzed
by array comparative genomic hybridization (aCGH). The analysis of chromosome segregation patterns was performed to infer the origin of
aneuploidy and to investigate the diagnostic accuracy of both PB and cleavage-stage PGS strategies.
& The Author 2012. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Sequential comprehensive
chromosome analysis on polar bodies,
blastomeres and trophoblast: insights
into female meiotic errors and
chromosomal segregation in the
preimplantation window of embryo
development
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Capalbo et al.
main results and the role of chance: Twenty-one sets of complete data (PB1/PB2/blastomere/TE) including 84 aCGH
limitations, reasons for caution: The study was limited to the analysis of oocytes and embryos from AMA patients. Thus,
these findings apply only to this patient group. Comparisons with other patient populations including patients with different indications for
PGS should be made in future research. In addition, higher resolution and/or more accurate chromosomal screening tests could be used in
future studies to corroborate the current findings.
wider implications of the findings: These findings provide critical insights into the mechanisms causing errors during
female meiosis and the preimplantation embryo development period to improve the design and treatment outcome of PGS.
study funding/competing interest(s): No external funding was obtained for this study.
trial registration number: Not applicable.
Key words: preimplantation genetic screening / PGS / polar bodies / aneuploidies / blastocyst
Introduction
The prevalence of aneuploidy in human embryos provides a likely explanation for the relatively low success and the high abortion rate
observed during assisted reproductive treatment cycles in humans
(Spandorfer et al., 2004; Menasha et al., 2005). It is believed that, in
most cases, aneuploidy causes embryos to either fail to implant
after transfer or spontaneously abort early in gestation (see Macklon
et al., 2002). It has been shown that normal preimplantation
embryo development does not correlate with euploidy. Both blastocysts of good and poor morphology have almost the same probability
of carrying chromosomal abnormalities (Fragouli et al., 2008; Alfarawati et al., 2011).
This poor correlation of conventional embryo selection methods
and chromosomal complement led to the introduction of preimplantation genetic screening (PGS) as a complementary treatment during
IVF cycles to avoid the transfer of aneuploid embryos. It was
thought that implementing PGS would improve delivery rates especially for patients with advanced maternal age (AMA). However, even
though the premise behind PGS is widely accepted, its benefits with
regard to live birth rate per started cycle has not yet been consistently
demonstrated (Harper et al., 2010; Mastenbroek et al., 2011). Two
main reasons have been put forward to explain this: (i) the cytogenetic
investigation methods used, such as fluorescence in situ hybridization
(FISH), are able to detect only copy number variation of a few chromosomes and have a considerable per chromosome diagnostic error
rate when applied on single cells (Munné et al., 2002; Baart et al.,
2004; Li et al., 2005; Colls et al., 2007; Magli et al., 2007; Harper
and Harton, 2010, b; Gutiérrez-Mateo et al., 2011); (ii) the stage of
analysis and the cell type used to infer the chromosome copy
number of the embryo do not always correlate with the embryo as
a whole (Vanneste et al., 2009a).
The application of comprehensive chromosome screening (CCS)
technologies in the field of PGS has revealed that segregation errors
occur at a notable frequency even for those chromosomes that
normally were not tested by conventional 9-chromosomes FISH
(Voullaire et al., 2000; Wells and Delhanty, 2000; Fishel et al., 2010;
Treff et al., 2010a,b; Schoolcraft et al., 2010; Fiorentino et al., 2011;
Gutiérrez-Mateo et al., 2011; Fiorentino, 2012). It is thus hypothesized
that high-throughput molecular karyotyping methods have to be used
to gain the desired diagnostic accuracy.
The second major issue for the outcome of PGS programs relates
to the type of cell biopsied. The biopsied sample should be representative of the embryos’ chromosomal constitution and viability after
transfer. There are different possible sources of genetic material for
testing in the preimplantation window in patients undergoing an IVF
cycle: (i) the first and second polar bodies (PBs) (PB approach); (ii)
one or two cells biopsied from 5- to 10-cell cleavage-stage embryos
on Day 3 and (3) several trophoblast cells (usually 5–10) sampled
from the blastocyst. Each of these stages presents with specific diagnostic advantages as well as critical limitations that relate to aneuploidy
genesis during both meiosis and the preimplantation period of embryo
development (Angell, 1991; Hassold and Hunt, 2001; Delhanty, 2005;
Vanneste et al., 2009b; Northrop et al., 2010; Handyside et al., 2012;
van Echten-Arends et al., 2011). In the PGS field there is an ongoing
debate about the optimal stage for PGS as a consequence of the
lack of a complete understanding of the genesis of human embryo aneuploidy. Indeed, most of the cytogenetic data obtained during PGS
investigations have been derived through the analysis of cells at isolated points in the preimplantation window, thus potentially missing
critical previous or subsequent information on chromosomal segregation. Understanding the chromosome segregation patterns during preimplantation development holds the potential to significantly increase
the success rates of IVF.
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experiments showed a pattern of multiple meiotic errors typically caused by sister chromatid separation errors and predominantly arising in
the second meiotic division. Twenty-two of the 24 (91.7%) errors in the first meiotic division arose as a consequence of premature sister
chromatid predivision. In half of these cases, the second meiotic division resulted in a balancing chromosome segregation event producing a
normal female complement for that chromosome in the resulting embryo. Overall, only 62 out of 78 (79.5%) of the abnormal meiotic segregations had errors in the either one or both PBs consistent with the aneuploidies observed in their resulting embryos. Ten of the 21
(47.6%) embryos had aneuploidies other than female meiotic-derived ones, most of which detected on Day 3 and confirmed on Day 5
or 6 of embryo development (20/25) with chromosomal loss being three times more frequent than gains. Notably, as high as 20% of
female-derived aneuploidies detected on PBs and confirmed on Day 3 were rescued at the blastocyst stage, mainly as a result of diploidization of trisomic chromosomes. On a per chromosome basis, the sensitivity in predicting blastocyst chromosomal complement was significantly lower for PB approach, 61.7%, compared with blastomeres analysis, 86.4% (P , 0.01).
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Chromosome analysis on polar bodies and embryos
Materials and Methods
Study design
This is a prospective longitudinal cohort study. Nine infertile couples
undergoing IVF cycles with PGS where the female partner was older
than 40 years and with a good response to controlled ovarian stimulation
(.10 MII oocytes retrieved) were enrolled into the study. The exclusion
criteria were (i) patients presenting with abnormal karyotype; (ii) specific
ovarian pathologies including polycystic ovary syndrome, endometriosis
grade III or higher and premature ovarian failure and (iii) severe male
factor infertility (motile sperm count of ,500 000/ml after preparation
of a fresh ejaculate).
The PB, blastomere and trophectoderm (TE) samples were sequentially
biopsied and processed for CCS. The analysis of chromosome segregation
patterns was performed to infer the origin of aneuploidy. Concordance
between the sequential biopsies was also analyzed to determine the fate
of aneuploid chromosomes in the preimplantation window.
The Institutional Review Board of both the Valle Giulia clinic, from
which the couples were recruited, and the GENOMA center approved
the study and a written informed consent was obtained from each
couple after counselling.
Ovarian stimulation and laboratory
procedures
Controlled ovarian stimulation was performed using a GnRH-agonist long
protocol as described previously (Ubaldi et al., 2010) and oocyte collection was performed at 35 h post-hCG administration. Denudation of the
oocyte from the cumulus oophorus was performed by a brief exposure
to 40 IU/ml hyaluronidase solution in fertilization media (Sage In-Vitro
Fertilization, Inc., Trumbull, CT, USA), followed by mechanical removal
of all the corona radiata with the use of plastic pipettes of defined
diameters (denuding pipette; COOK Ireland Ltd, Limerick, Ireland). The
denudation procedure was performed in a controlled (6% CO2 at 378) environment (IncuChamber L-323, Ksystems, Birkerod, Denmark) between
37 and 40 h post-hCG administration. Particular attention was paid to the
removal of all adhering cumulus and coronal cells with the aim of avoiding
maternal DNA contamination during the amplification steps. MII oocytes
were then subjected to ICSI, between 36 and 38 h post– hCG administration, using previously described techniques and instrumentation (Rienzi
et al., 1998). 16 –18 h post-ICSI, oocytes were assessed for the presence
of pronuclei. Those displaying two pronuclei and a second PB were cultured further. All embryos were cultured separately in 35 ml microdrops
of cleavage medium under mineral oil (Sage) up to Day 3 of embryo
development followed by blastocyst medium (Sage) up to Day 5 or 6, in
MINC incubators (COOK) (hypoxic atmosphere containing 6% CO2 at
378). Vitrification of blastocysts on Day 5 or 6 was performed according
to Rienzi et al., 2010.
Biopsy procedures
All the biopsy procedures were performed on the heated stage of a Nikon
IX-70 microscope, equipped with micromanipulation tools, in dishes prepared with three droplets of 10 ml of HEPES-buffered medium (Sage)
overlaid with pre-equilibrated mineral oil. A diode laser was used to
assist the opening of a 10 – 20 mm hole in the zona pellucida by 2 – 4
laser shots. At all the biopsy stages, an attempt was made to use the
initial zona breach to extract the target cells. A PB biopsy was performed
sequentially by aspiration with a PB aspiration pipette (Research instruments, Cornwall TR11 4TA, UK). PB1 was biopsied immediately before
ICSI and PB2 was biopsied 16– 18 h after ICSI, allowing the distinction
between the PBs in all cases and avoiding the degradation of the genetic
material. Occasionally, a second hole was required for sampling the
second PB. A cleavage-stage embryo biopsy was performed on Day 3 of
embryo development by blastomere extrusion of one blastomere from
embryos reaching at least the six-cell stage as described by Tarı́n and Handyside, 1993. The cleavage-stage biopsy was performed in calcium –
magnesium-free HEPES buffered medium (Sage) using a blastomere
aspiration pipette (Research instruments). On Day 5 or 6 of embryo development expanding and expanded blastocysts with or without herniating
cells underwent TE biopsy. Five to 10 TE cells were aspirated into the TE
biopsy pipette (Research instruments) followed by laser-assisted removal
of the target cells from the body of the embryo. All embryos on Day 5,
which did not reach the expanding blastocyst stage, were transferred to
fresh individual 35 mL drop of blastocyst medium (Sage) and a biopsy
was performed on Day 6 if the appropriate stage of development was
reached. aCGH was performed only on the material obtained from
oocytes/embryos where at least further biopsy was performed on Day
3. All viable blastocysts were cryopreserved by means of vitrification in
line with the Italian legislation.
Array CGH and microsatellite analysis
All biopsied samples obtained during the study were washed in sterile
phosphate-buffered saline (PBS) solution, in a laminar flow cabinet in
order to avoid any contamination of the sample, placed in microcentrifuge
tubes containing 2 ml of PBS and then processed for aCGH analysis
according to the 24 sure protocol (Blugnome) (Fiorentino et al., 2011).
Visualization and reporting of aneuploidy were performed using Bluefuse
Software (BlueGnome) on a per chromosome basis. Only whole chromosome aneuploidies (gains and losses) were scored. The PBs, blastomeres
and TE samples with amplification but no diagnosis were determined inconclusive. Structural chromosome defects have been ignored for this
analysis. Only whole chromosome aneuploidies (gains and losses) were
scored. The PB, blastomere and TE samples with amplification but no diagnosis were determined to be inconclusive. Microsatellite analysis was
performed according to the PCR-based PGD protocol described elsewhere (Fiorentino et al., 2010).
Segregation pattern analysis and embryo
classification data analysis
Following aCGH, each PB, blastomere and TE sample was analyzed for the
chromosome copy number and the ploidy status of each of the corresponding oocyte/embryos was predicted in a blinded fashion (Fig. 1).
The per chromosome aneuploidy at each biopsy stage is displayed as
gain (G), loss (L) or normal (N) copy number as previously reported by
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By sequential biopsy and array comparative genomic hybridization
(a-CGH) analysis of both PBs, blastomeres and TE of the same
embryo, this study achieved a comprehensive analysis of the chromosomal segregation patterns from female meiosis through to the blastocyst stage of preimplantation embryo development. This sequential
chromosome analysis on individual embryos has allowed inference
of mechanisms causing errors during meiosis leading to a better understanding of the aetiology of aneuploidy in a population of AMA
patients. This approach has also allowed the concordance studies
between molecular karyotyping results at three different points of analysis. Moreover, the study design has allowed the direct evaluation of
the potential correction mechanisms of meiotic aneuploidies acting
later in preimplantation development as well as an unbiased estimation
of the incidence of male and post-zygotic-derived errors in preimplantation human embryos.
4
Capalbo et al.
Handyside et al. (2012) with the addition of a follow-up at the blastocyst
stage of embryo development (detailed in Fig. 1). Specifically, (i) the segregation pattern N/N/G/N or N/N/L/N was interpreted as an early
mitotic error occurring during the first cleavage divisions leading to mosaicism on Day 3 blastomere analysis but not evident at the blastocyst stage
and (ii) the segregation pattern N/N/N/G or N/N/N/L was considered
to have originated by a de novo mitotic error early in the development, not
detected at the cleavage stage or having arisen during the final stage of preimplantation development during blastocyst formation.
Using the TE biopsy result as a reference, per chromosome copy
number concordance of both the PB and the blastomere samples was
analyzed. The results were divided into two subsets, either ‘consistent’
or ‘inconsistent’, to investigate the diagnostic accuracy of both PB and
cleavage-stage PGS strategies. This analysis was based on the 21 sets of
complete CCS data including a total of 21 × 23 ¼ 483 chromosome segregation patterns. In this study it was not possible to systematically distinguish between whole chromosomes versus single chromatids copy
number variations by interrogation of the aCGH profile of PB1. Thus,
MI errors balanced at MII were considered inconsistent when a normal
chromosome copy number was observed in the embryo.
Statistical analysis
Continuous data and categorical variables are reported as the mean and
percentage frequency, respectively, with 95% confidence interval (CI)
and range. Fisher’s exact test was used to test the categorical variables.
Diagnostic sensitivity and specificity of PBs and blastomere comprehensive
chromosomal analysis against blastocysts molecular karyotype were measured as the proportion of actual positives, which are correctly identified
as such, and the proportion of negatives, which are correctly identified.
Alpha was set at 0.05.
Results
Nine couples undergoing IVF/ICSI cycles with a median age of 43
years (range 40– 45) were included in the study. A total of 96
cumulus –oocyte complexes were retrieved. Of the 82 MII oocytes
injected by ICSI, 64 fertilized (78%; 95% CI: 67.5 –86.4). Of the 49,
.6 cell embryos obtained on Day 3, 34 reached the blastocyst
stage on Day 5/6 (69.4%; 95% CI: 54.6 –81.7). Successful biopsy
and DNA amplification were obtained in 45 of 49 (91.8%; 95% CI:
80.4 –97.7) PB1, 41 of 49 (83.7%; 95% CI: 70.3 –92.7) PB2, 93.9%
(46/49; 95% CI: 83.1– 98.7) of blastomeres and 100% (34/34, 95%
CI: 89.7 –100) of TE samples. There was a significantly higher diagnostic failure rate in the PB approach when considering both PBs together
(22.4%, 11/49, 95% CI: 11.8 –36.7) when compared with the blastocyst stage approach (0%, 0/34, 95% CI: 0–10.3%; P ¼ 0.002). In total,
21 (21/34; 61.8%; 95% CI: 43.6 –77.8) sets of complete aCGH data,
including PB1, PB2, corresponding blastomeres and TE samples, were
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Figure 1 Study design and copy number segregation pattern analysis. (A) Sequential biopsy and comprehensive chromosome analysis of both polar
bodies before and after fertilization was performed along with a single blastomere and TE sample in the resulting embryos. In (B) a diagrammatic
representation of the main abnormal copy number segregation patterns resulting from non-disjunction of whole chromosomes and premature predivision of sister chromatids during female meiosis or from mitotic errors during preimplantation development is illustrated. Note that not all possible
segregation patterns are represented. G, chromosome copy number gain; L, chromosome copy number loss; N, normal chromosome copy number;
PB1, polar body one; PB2, polar body 2.
5
Chromosome analysis on polar bodies and embryos
cases of non-complementary meiotic segregations were observed.
One loss in PB1 with normal PB2 and three normal PB1 with losses
in PB2 resulted in disomy for the relevant chromosomes in the corresponding embryos observed either in the blastomere or TE samples.
Meiotic chromosomal errors and PBs
genetic screening outcome
Chromosomal segregation at the cleavage
and at the blastocyst stage of development
All the analyzed oocytes had chromosomal abnormalities. One of 21
oocytes contained only meiosis I errors, 10/21 oocytes contained
only meiosis II errors with 10/21 containing errors from both
meiotic divisions. Twelve oocytes had multiple (more than 2)
meiotic aneuploidies with only nine oocytes showing either a single
(four oocytes) or double aneuploidy (five oocytes). A significantly
higher incidence of copy number errors were detected in the PB2
(69/91) compared with the PB1 (22/91; P , 0.001), with 54 chromatids non-disjunction errors arising at meiosis II (Table I). Copy number
gains and losses were approximately equally represented in both PBs
and all chromosomes, with the exception of chromosomes 3 and 4,
were involved in female-derived errors. The most frequent aneuploidies were for the smaller and acrocentric chromosomes, with 22,
15, 16 and 17 being the most prevalent. Overall, only 62 out of 78
(79.5%; 95% CI: 68.8 –87.8) abnormal meiotic segregations had
copy number gains or losses in either one or both PBs consistent
with the aneuploidies observed in the resulting embryos (Table I).
As shown in Table I, the occurrence of chromatid errors in meiosis
I with a combined reciprocal gain or loss during the second meiosis
mainly explained the inconsistency between PB screening and
embryo chromosomes copy number (Table I). Furthermore, four
In this data set it was observed that the number of aneuploidies
increased during embryo development to Day 3. A total of 87
copy number gains (37) or losses (50) were recorded on Day
3. However, between the cleavage stage and blastocyst development
the aneuploidy frequency was lowered to 81 copy number gains (30)
and losses (51). A total of 10 of 21 (47.6%; 95% CI: 25.7 –70.2)
embryos had aneuploidies other than those inferred through PB analysis. At the cleavage stage of embryo development, 25 de novo mitotic
errors and/or male-derived aneuploidies were recorded, 20 of which
were confirmed at the blastocyst stage (80.0%; 95% CI: 59.3 –93.2;
Table II), with a skewed ratio towards the losses (18) compared
with gains (2). Overall mosaic chromosomal segregations were
detected in 6 out of 21 embryos. The five aneuploidies detected
only on Day 3, occurred in two single embryos. It is suggested that
this may have occurred as a result of mitotic errors during early embryogenesis leading to mosaicism on Day 3. Eleven de novo mitotic
aneuploidies were recorded only at the blastocyst stage, with a
normal copy number detected in both PBs and blastomeres
(Table II). These errors may have equally arisen during early embryo
development and not detected on Day 3 blastomere analyses or as
a consequence of a new mitotic error after Day 3.
Table I Chromosomal segregation patterns of copy number gains (G) and loss (L) in the first (PB1), second polar bodies
(PB2) and resulting embryos associated with errors in first and/or second meiosis.
Segregation pattern (PB1/PB2/blastomere)
N (% of total meiotic errors)
% MI errors
Interpretation
.............................................................................................................................................................................................
First meiosis errors
G/L/L
1 (1.3)
4.3
G/L/N
7 (9.0)
30.4
G/N/L
3 (3.8)
13.0
L/G/G
0
0
L/G/N
5 (6.4)
21.7
L/G/L
1 (1.3)
4.3
L/N/G
5 (6.4)
21.7
L/N/N
1 (1.3)
4.3
MI non-disjunction
MI chromatid predivision balanced at MII
MI chromatid predivision unbalanced at MII
MI non-disjunction
MI chromatid predivision balanced at MII
MI chromatid predivision unbalanced at MII
MI chromatid predivision unbalanced at MII
MI anaphase lag or monosomic oogonia
Second meiosis errors
N/L/N
3 (3.8)
N/L/G
28 (35.9)
MII chromatids non-disjunction
N/G/L
24 (30.8)
MII chromatids non-disjunction
Total number of abnormal meiotic segregations
78
Female meiotic errors in the embryos
62
% of chromatid errors
MII anaphase lag
97.4 (76/78)
On the right column is reported the interpretation of meiotic mechanism causing error based on the chromosomal segregation pattern analysis. N, normal chromosome copy number.
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available for analysis. A total of 1932 chromosomes were investigated
in 84 array CGH experiments.
Among the chromosomal segregations analyzed, 355 of 483 (73.5%;
95% CI: 69.3 –77.4) had a normal pattern (N/N/N/N). All the
blastocysts analyzed were aneuploid and no embryo transfer was
performed in this cohort of patients during the course of the trial.
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Capalbo et al.
Table II Male and/or mitotic-derived copy number aneuploidies in the embryo following a normal chromosomal
segregation during female meiosis.
Segregation pattern (PB1/PB2/blastomere/TE)
No. of events
% Among post-zygotic errors
Interpretation
.............................................................................................................................................................................................
N/N/G/G
2
5.6
N/N/L/L
18
50.0
Male or early mitotic error
N/N/G/N
2
5.6
Early mitotic error
Early mitotic error
Male or early mitotic error
3
8.3
4
11.1
Early or late mitotic error
N/N/N/L
7
19.4
Early or late mitotic error
Total number of post-zygotic errors
36
% of post-zygotic losses
77.8 (28/36)
% of embryos with errors other than female meiotic-derived ones
47.6 (10/21)
On the right column is reported the interpretation of the mechanism causing errors based on the chromosomal segregation analysis at cleavage and blastocyst stage of preimplantation
embryo development. PB1, polar body one; PB2, polar body 2; TE, trophectoderm; G, chromosome copy number gain; L, chromosome copy number loss; N, normal chromosome
copy number.
Table III Trisomic and monosomic zygote chromosome rescues at the blastocyst stage.
Segregation pattern (PB1/PB2/
blastomere/TE)
No. of events per segregation
type (%)
% of meiotic
errors
Predicted parental origin of the rescued
chromosomes
.............................................................................................................................................................................................
N/L/G/N
7/28 (25.0)
11.3 (7/62)
N/G/L/N
2/24 (8.3)
3.2 (2/62)
mat ID
L/G/L/N
1/1 (100)
1.6 (1/62)
mat ID
L/N/G/N
2/5 (40.0)
3.2 (2/62)
mat ID, mat HD or BPD
Total number of correction events
Total correction rate (%)
No. of embryos with at least one
correction event (%)
mat ID or BPD
12
12/62 (19.4)
7/21 (33.3)
In the right column is reported the predicted parental origin of the rescued chromosomes. PB1, polar body one; PB2, polar body 2; TE, trophectoderm; G, chromosome copy number
gain; L, chromosome copy number loss; N, normal chromosome copy number; ID, Isodisomy; HD, Heterodisomy; BPD, biparental disomy.
Female-derived meiotic aneuploidy rescue at
the blastocyst stage
A total of 12 of 62 (19.4%; 95% CI: 10.4 –31.4) female-derived aneuploidies detected on PBs and confirmed on Day 3 were not detected
at the blastocyst stage (Table III). Most of these were the result of a
trisomic rescue event. In nine cases a trisomic chromosomes arising
from a female meiotic error and confirmed on Day 3 lost a chromosome returning then to a disomic chromosome configuration at the
blastocyst stage. In two of these cases where the parental DNA
was accessible to STR analysis was performed on blastocyst DNA
to test for loss of heterozygosity of trisomic rescued chromosomes.
As shown in Fig. 2, in both cases the trisomies observed in these
embryos originated following a chromatid predivision error in MI
unbalanced in MII. The microsatellite analysis confirmed a disomic
bi-parental configuration with the presence of both chromosomes
13 from the male and female partner in one embryo and a maternal
uniparental disomy for chromosome 10 in the other.
Diagnostic accuracy of PBs and Day 3
chromosomal screening in predicting the
resulting blastocyst karyotype
The diagnostic accuracy of the PB and blastomere stage PGS was computed against the molecular karyotype of the resulting blastocysts. On
a per chromosome basis, the sensitivity in predicting blastocyst
chromosomal complement was significantly lower for the PB approach
61.7% (50/81, 95% CI: 50.3 –72.3) compared with the cleavage stage
blastomere analysis, 86.4% (70/81; 95% CI: 77.0 –93.0; P , 0.001).
The specificity was 93.0% (374/402; 95% CI: 90.1–95.3) and 95.8%
(385/402; 95% CI: 93.3–97.5) for PB approach and cleavage stage
blastomere analysis, respectively (P ¼ 0.09). The overall accuracy was
also significantly lower for PB approach (87.8%, 424/483; 95% CI:
84.5–90.6) compared with cleavage stage analysis (94.2%, 455/483;
95% CI: 91.5–95.9; P , 0.001). Female meiotic-derived aneuploidies
were correctly predictive for all chromosomes by PB analysis in only 12
out of 21 (57.1%; 95%; CI 34.0–78.2) blastocyst. Moreover, a full
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N/N/L/N
N/N/N/G
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Chromosome analysis on polar bodies and embryos
concordance between PB analysis and blastocyst chromosomal complement was observed only in 8 of 21 (38.1%; 95%; CI: 18.1–61.6)
blastocysts.
Discussion
The design of this study has allowed a comprehensive description of
the chromosomal segregation events taking place from female
meiosis to the blastocyst stage of preimplantation embryo development. It has thus been possible to accurately describe the origin of
female-derived meiotic aneuploidies based on the confirmation of
PB data in both blastomere and TE samples. This study is not
biased by the uncertainty-related PB morphology because of sequential biopsy of the PBs. It has recently been shown that distinction
between PB1 and PB2 by morphology alone is only 63% consistent
when biopsied simultaneously (Treff et al., 2012).
Our observations in AMA patients has revealed a pattern of multiple meiotic errors, typically caused by chromatid mal-segregation
and arising predominantly at meiosis II as a cause of chromatid nondisjunction. Most of the first meiotic aneuploidies arose as a consequence of premature sister chromatid predivision, leading to balancing
events in about half the case during the second meiosis. Notably, all
cases of MI errors balanced at MII had a normal mitotic chromosomal
segregation until the blastocyst stage, suggesting no downstream effect
of premature sister chromatid errors during preimplantation embryo
development.
Only one segregation pattern consistent with monosomic oogonia
was detected in our data set of IVF AMA patients, suggesting that
mitotic errors before entry into meiosis are not a common phenomenon and are not related to a preferential recruitment with increasing
reproductive age. This observation is strictly in contrast to the recent
theory of the oocyte mosaicism selection model, suggesting that aneuploid oocytes in primordial follicles are preferentially recruited with
increased maternal age (Hultén et al., 2010; Obradors et al., 2010).
In line with recent findings obtained in the same study population
reported by Handyside et al., 2012 and the molecular evidence
reported by Tachibana-Konwalski et al., 2010, it is reasonable to conclude from this study that chromatid errors during both female meiotic
divisions are the main pathogenic mechanisms responsible for the
female age effect on human aneuploidy. This is likely to be as a
result of the prolonged arrest of the chromosomes at the dictyotene
stage of the first meiosis.
Furthermore, abnormal meiotic segregation patterns leading to noncomplementary oocyte and PBs chromosome copy number were
observed (Fig. 3). In this data set there are several examples of one PB
being nullisomic with the corresponding oocyte normal haploid for the
same chromosome. For these cases contamination issues can be confidentially excluded since the other embryo aneuploidies were concordant with the original PB profile. These abnormal segregations may have
originated from the compensation of aneuploidy by the sperm or more
likely from meiotic chromosome anaphase lag events, leaving behind
some chromosomes in the oocyte that would then be degraded to
Downloaded from http://humrep.oxfordjournals.org/ at Istanbul Memorial Hastanesi Tup Bebek ve Genetik Merkezi, on November 14, 2012
Figure 2 Microsatellite analysis performed on blastocyst DNA to study the parental origin of the trisomic rescued chromosomes. The aCGH ratio
plots in (A) and (B) shown the two sets of complete sequential segregation patterns, characterized by a gain in the PB1, a normal copy number in the
PB2, a trisomy in the corresponding blastomeres and a final disomy for the same chromosome in the TE. Both chromosomes 13 from the male and
female partner were observed in one case (A) and a maternal uniparental disomy for chromosome 10 in the other one (B). mat UPD, maternal uniparental disomy; BPD, biparental disomy.
8
Capalbo et al.
make the resulting embryo disomic for those chromosomes. Displacements of chromosomes outside the meiotic spindle have been extensively observed by electron microscopy analysis of mature eggs
(Bromfield et al., 2009; Coticchio et al., 2009). Even if more data are
needed to get an accurate estimation of the clinical meaning of noncomplementary aneuploidies between PBs and corresponding oocyte,
this segregation pattern deserves future investigation being at risk of misdiagnosis based on PB screening.
These data also revealed a large proportion of embryos with aneuploidies other than those originating during female meiosis. Most of
these were chromosomal loss detected on Day 3 and confirmed on
Day 5/6. Even if it was not possible to distinguish between male or
mitotic origin, these data are well matched with both the increased
level of sperm monosomies compared with trisomy observed in
human cleavage stage embryos (Rabinowitz et al., 2012) and with previous data showing anaphase lag (AL) as the main mitotic mechanism
causing error during preimplantation development (Delhanty, 2005).
Interestingly, as high as 20% of female-derived aneuploidies
detected on PBs and confirmed on Day 3 were not present at the
blastocyst stage. As expected, all these events were mostly the
result of a trisomic rescue because both non-dijunction and AL contribute to reduce the hyperploidy. If loss of one of the three chromosomes from a trisomy occurs randomly, then uniparental disomy
(UPD) would be expected in the derivative diploid lineage 1/3 of
the time. By microsatellite analysis we have been able to report the
first direct observation of a biparental disomy rescue of a meiotic
trisomy between the cleavage and the blastocyst stage of preimplantation development. For another chromosome a maternal UPD was
confirmed. Unfortunately, it was not possible to assess whether the
corrected chromosomes were in a mosaic configuration or constitutional within the embryo. In humans, constitutional and mosaic UPD
are thought to mainly originate as a consequence of trisomic zygote
rescue (Purvis-Smith et al., 1992; Kotzot, 1999; Robinson, 2000)
and are involved in clinical conditions by producing either aberrant patterns of imprinting and also homozygosity for recessive mutations in
the case of isodisomy. Whole chromosome UPDs have been also extensively detected in products of conception and during prenatal
testing (Engel, 1999; Fritz et al., 2001; Tsukishiro, 2005), with a preponderance of maternal versus paternal origin. Due to the high correction rate observed here and considering the dramatic increase in
aneuploidies with female age, maternal UPD may be particularly significant during PGS cycles of patients with AMA and deserves further
investigation.
From a clinical perspective, important information can be drawn out of
this data set. Primarily, testing the first PB alone is definitively not effective
as a screening approach for oocyte aneuploidies as previously reported
(Sher et al., 2007). According to our results the screening of PB1 alone
should not be offered to patients when balancing events and new
errors during the second meiosis cannot be concomitantly assessed by
the analysis of PB2. With the addition of the PB2 data, more accurate information inferring oocyte chromosome copy number can be obtained.
However, concerns related to whether the accuracy achievable using PB
screening is good enough to improve the IVF clinical outcome still
remains (Angell, 1994; Scriven et al., 2012). This study showed that
the accuracy of PB approach was singificantly lower compared to the
cleavage stage single blastomere analysis when results were correlated
with blastocyst karyotype. These findings can be manly explained by (i)
the high false-positive rate obtained by PB approach because of the inability to consistently identify the MI errors balanced at MII (Fig. 3); (ii)
the considerable proportion of female meiotic aneuploidy correction
at the blastocyst stage and (iii) the high rate of male and/or mitoticderived aneuploidies observed in the embryo. These intrinsic limitations
of PB analysis may result in one case discarding viable embryos, while in
the other transferring abnormal ones.
Downloaded from http://humrep.oxfordjournals.org/ at Istanbul Memorial Hastanesi Tup Bebek ve Genetik Merkezi, on November 14, 2012
Figure 3 Normal female meiosis (A) and abnormal (B) meiotic segregation patterns resulting in misdiagnosis based on PB analysis when the genetic
test is unable to discriminate between whole chromosome versus chromatid copy number gains or losses. Green coloured PBs indicate chromosome
gains. Red coloured PBs indicate chromosome losses. AL, anaphase lag.
9
Chromosome analysis on polar bodies and embryos
Authors’ roles
L.R. and A.C. participate in the study design and discussion. A.C. and
L.R. performed the embryo laboratory procedures and the oocyte and
embryo biopsies. A.C. performed the data analysis and wrote the
manuscript. F.F. conceived the study, validated array-CGH results
and was involved in critical discussion of the manuscript. S.L. and
B.S. performed the array-CGH experiments and interpreted the
results. B.A. performed the array-CGH experiments, carried out
PCR analysis and interpreted the results. F.F., M.B., L.R. and F.U. provided a critical discussion of the manuscript and improved it.
Funding
No external funding was either sought or obtained for this study.
Conflict of interest
All authors have completed the ICMJE conflict of interest disclosure
form, and have no conflicts to declare.
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It should be noted that in our data set all the embryos with a correction
event remained abnormal due to the presence of additional aneuploidies.
Even if our study was based on a per-chromosome analysis in a severe
poor prognosis population of patients and no euploid embryos have
been obtained at the blastocyst stage, there is no reason to assume that
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European Society of Human Reproduction and Embryology PGS Task
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Another major problem related to PB biopsy is the paucity of material that is available. In our hands as well as in the practice of other
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obtained from all embryos tested. Moreover, the economic and logistic issues cannot be disregarded, with PB screening being the most
time-consuming and least cost-effective approach among the PGS
strategies. A further advantage of postponing the time of biopsy is
to not affect the blastocyst development rate by extra-manipulations
prior to blastocyst development (Scott et al., 2012a).
All this evidence points to postponing the time of biopsy as late as
possible in the preimplantation window, possibly with the use of technologies able to determine not only the chromosomal count in a cell
but also parental origin of each chromosome, i.e. whether a disomy is
euploid versus UPD.
To conclude, these new findings provide critical information to
improve the design of clinical PGS strategies as well as the genetic preconception counseling of IVF patients.
10
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