2. ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY
Despite the promising results reported with the use of
modern chromosome screening technologies, it remains the
case that even the transfer of a morphologically perfect, chro-mosomally
normal embryo can not guarantee that a viable
pregnancy will be established. Studies published in the past
12 months indicate that implantation failure rates for euploid
blastocysts range from 25% to 50% (4–8). It is clear that
aspects of preimplantation embryo biology that are poorly
defined or invisible with the use of current methods of
embryo evaluation must also play an important role in
determining the potential of an embryo to produce a child.
One aspect of embryo biology of potential relevance to
viability is mitochondrial DNA (mtDNA) content. Mitochon-dria
are responsible for the production of most of the energy
needed by the cell for biosynthetic, metabolic, and physiologic
processes. A number of clinical and animal studies have high-lighted
the essential role of mitochondria in oogenesis and pre-implantation
development (9, 10). Decreased amounts of
mtDNA and diminished bioenergetic capacity have been
linked to fertilization failure, impaired oocyte quality, and
abnormal organization and function of the meiotic spindle
(11–14). Furthermore, bioenergetic capacity has been
correlated with embryo developmental potential and IVF
outcome (15, 16).
In response to the need for improved methods of embryo
selection, this study sought to create a new microarray plat-formcapable
of extending genetic evaluation beyond chromo-some
analysis. This microarray includes probes for aneuploidy
detection but also uses mitochondria-specific probes for the
relative quantification of mtDNA. In addition, the option of
including probes for the analysis of single-nucleotide poly-morphisms
(SNPs) was investigated, an approach that poten-tially
allows DNA fingerprinting and may provide other
information of diagnostic relevance.
MATERIALS AND METHODS
Cell Culture
Single cells derived from a variety of aneuploid fibroblast cell
lines were used during the optimization and validation of the
microarray. The cell lines had the following karyotypes:
47,XY,þ13; 47,XY,þ15; 47,XY,þ18; 47,XY,þ21; 45,X; and
48,XXY,þ18.
Cell lines were cultured under standard conditions. Single
cells were isolated in 1 mL phosphate-buffered saline solution
(PBS with 0.1% polyvinyl alcohol) and transferred to sterile
0.2-mL microcentrifuge tubes.
Selection ofWhole-genomeAmplification Method
and Development of a Customized Microarray
Development of the customized microarray involved the
testing of different whole-genome amplification (WGA)
methods, selection of the appropriate oligonucleotide probes
and array format, and identification of the best method for
labeling the amplified products. The different WGA methods
assessed were Picoplex (Rubicon Genomics), Genomeplex
(Sigma-Aldrich), and multiple displacement amplification
(MDA). Picoplex and Genomeplex were performed on isolated
single cells according to the manufacturers' instructions. For
MDA, the Repli-g Midi kit (Qiagen) was used following the
manufacturer's protocol with the use of a modified alkaline
lysis method. This MDA protocol involved incubation of the
samples in a thermocycler at 30C for 2 hours followed by
enzyme inactivation at 65C for 5 minutes.
Ultimately, the optimized microarray contained 14,334
unique probes for aneuploidy detection and 261 unique
probes for mtDNA analysis. In contrast to the most widely
used microarray for the purpose of preimplantation aneu-ploidy
screening, which uses probes composed of bacterial
artificial chromosomes, the new microarray used synthetic
oligonucleotides probes of 60 nucleotides in length.
The final protocol involved WGA of the samples and con-trol
male DNA with the use of Picoplex. Control DNA was
prepared by amplifying 1 mL male genomic DNA (1 ng/mL).
Picoplex-amplified DNA samples and control samples (8 mL)
were labeled with the use of the Cytosure labeling kit, which
incorporates Cy-dCTP using exonuclease-free Klenow
enzyme (Oxford Gene Technology [OGT]). After labeling,
the DNA was purified with the use of either Cytosure purifica-tion
columns (OGT) or Cytosure purification plates (OGT). The
sample and reference DNA were pooled together, dried, and
then resuspended in hybridization mix (Agilent). The com-bined
sample was applied on the microarray slide and incu-bated
overnight in a rotating incubator at 65C and 0.054 g.
Post-hybridization washes were carried out with the use of
Agilent Oligo microarray comparative genomic hybridization
(aCGH) Wash Buffer 1 for 5 minutes at room temperature and
Agilent Oligo aCGH Wash Buffer 2 for 1 minute at 37C. The
microarray slide was then scanned at 5-mm resolution on an
Agilent DNA microarray scanner. Feature extraction was
undertaken with the use of Agilent's Feature Extraction Soft-ware
(v. 10.7.3.1) and data analysis carried out with the use of
a customized version of Cytosure Interpret Software (OGT).
The whole process from DNA amplification to results could
be completed in 16 hours and at a cost similar to existing
aCGH methods.
Validation of Aneuploidy Detection with the Use
of Clinical Material
Ninety-seven Picoplex-amplified clinical samples, previously
screened for aneuploidies with the use of an alternative mi-croarray
CGH platform (24sure; Bluegnome), were reanalyzed
with the use of the new microarray and the results compared
for concordance. These samples were derived from actual
clinical cases performed previously for aneuploidy screening
of polar bodies and biopsied embryonic cells. These samples
consisted of 27 polar bodies (PBs), 50 blastomeres, and 20
trophectoderm samples. Diagnosis of aneuploidy was made
in a blinded fashion, without any knowledge of the original
screening results. An independent scientist was responsible
for assessing the two sets of data for concordance.
A subset of 20 embryos (19 cleavage stage and one blas-tocyst)
was also analyzed with the use of a third, independent
cytogenetic method, fluorescent in situ hybridization (FISH).
These whole embryos were donated to research, and all of
their cells were spread on microscope slides for FISH analysis.
2 VOL. - NO. - / - 2014
3. The methods used for cell fixation and FISH were carried out
as described previously (17, 18). Chromosomes 8, 13, 14, 15,
16, 17, 18, 20, 21, 22, X, and Y were tested in three rounds
of hybridization applied to all cells. An extra hybridization
was performed to test specific embryos for any additional
chromosomes found to be abnormal via microarray analysis
but not included in the first three rounds of FISH.
Initial evaluation of the new microarray revealed prob-lems
in detecting losses and gains of chromosome 19. New
probes for chromosome 19 were selected from a large pool
on a separate microarray platform. Probes were chosen on
the basis of consistently displaying excellent signal intensity
and providing reliable copy number analysis for chromosome
19 across multiple single-cell samples of known chromo-somal
status. The selected probes were added to the microar-ray,
this new version was tested against five new samples
(single blastomeres) derived from embryos known to be aneu-ploid
for chromosome 19, and correct results were obtained in
all cases.
Evaluation of mtDNA Quantification
To assess whether the new microarray was able to accurately
measure the relative quantity of mtDNA in a sample, four cell
lines were used (A549, Red, B2342B, and 206F). Picoplex-amplified
DNA and unamplified genomic DNA from each of
the cell lines were applied to the microarray to determine
whether WGA introduced any distortions in the relative
quantities of mtDNA. Additionally, the amount of mtDNA
in each of these DNA samples was quantified with the use
of real-time polymerase chain reaction (PCR, using standard
techniques), confirming whether the results obtained from
the microarray were accurate.
Assessment of SNP Probes for Inclusion on the
Microarray
As a proof of principle, a number of SNP probes were evaluated
for potential inclusion on the microarray. SNP probes were
selected from the commercially available International Stan-dard
Cytogenomic Array uniparental disomy array v. 1.0
(OGT) on the basis of a preliminary experiment during which
Picoplex-amplified products were applied to the microarray. A
total of 1,282 different SNPs were selected from an initial pool
of 6,186. The SNP probes were placed on the array in triplicate.
To test the new microarray, aliquots from surplus Pico-plex
products, amplified from 14 single blastomeres from
two previous clinical cases (preimplantation genetic diagnosis
for single-gene disorders) were used. The embryo samples,
along with genomic DNA extracted from the parents, were
analyzed with the use of the same microarray protocol as
outlined above.
Statistical Analysis
Fisher exact test (Graphpad) was used to make comparisons
regarding aneuploidy detection between the different sample
types. The same test was also used for analysis of SNP results.
The Mann-Whitney U test and independent-samples t test
Fertility and Sterility®
(SPSS v. 19.0.0) were used for analysis of data obtained
from relative quantitation of mtDNA in PB/embryo samples.
Ethics
Necessary ethical approvals were obtained for usage of
research samples. Chromosomal and mitochondrial analyses
involved informed patient consent and were carried out after
appropriate licencing and with approval from the local Ethics
Committee [National Research Ethics Service Committee
South Central].
RESULTS
Development of Customized Array and Selection
of Best WGA Method
Forty-eight WGA products were produced with the use of
three different methods (MDA, Genomeplex, Picoplex)
applied to isolated single cells. The most successful WGA
method was determined to be Picoplex, and consequently
this was the method used during all subsequent work. Anal-ysis
of Picoplex products provided a correct diagnosis for
16/16 cells, with detection of 16/16 individual aneuploidies.
In contrast, only a minority of aneuploidies was detected in
MDA samples and none in the Genomeplex products. In addi-tion,
artefactual losses and gains of some chromosomes were
observed for MDA and Genomeplex samples.
Validation of the New Oligonucleotide Microarray
for Aneuploidy Screening
Samples affected by 164 aneuploidies, including examples of
losses and gains for all 24 types of chromosome, were
assessed with the use of the first iteration of the new microar-ray.
Thirty aneuploidies were tested in PBs, 118 in single blas-tomeres,
and 16 in trophectoderm samples (Supplemental
Table 1, available online at www.fertstert.org). Scoring of
samples was carried out in a blinded fashion (i.e., no informa-tion
was given to personnel carrying out the test regarding the
original diagnosis that these samples had previously
received). The microarray successfully detected 156/164 an-euploidies
(95.1% detection rate) and detected three extra
aneuploidies not detected with the use of the 24sure microar-rays.
There was no significant difference in the degree of
concordance for the three different types of samples analyzed.
Four of the eight aneuploidies not observed with the use
of the new microarray and two of the three extra aneuploidies
detected involved chromosome 19, suggesting a potential
problem for the accurate determination of the copy number
of this chromosome. It is well known that interpretation of
chromosome 19 aneuploidies can be problematic when using
CGH, as noted in many earlier studies (19–21).
Although the microarray was developed primarily for
detection of whole chromosome aneuploidies, 14 of the 164
aneuploidies tested were segmental abnormalities. The size
of deleted/duplicated chromosomal segments ranged from
19.3 Mb to 133.5 Mb. The new microarray successfully de-tected
12/14 of these aneuploidies. The only two segmental
abnormalities not detected both affected chromosome 19
(size of segments not detected: 21.6 Mb and 23.3 Mb), again
VOL. - NO. - / - 2014 3
4. ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY
highlighting a problem with the diagnosis of aneuploidy
affecting that chromosome.
All of the embryos tested during the course of this study
underwent a single biopsy. The cells sampled were then sub-jected
to WGA and tested with the use of the new microarray
as well as the 24sure microarray. Additionally, 20 of the em-bryos
were later disaggregated and all of their remaining cells
reassessed with the use of FISH. These embryos ultimately
yielded three pieces of data: 1) aCGH of biopsied material
with the use of the new microarray; 2) aCGH of biopsied
material with the use of the 24sure microarray; and 3) FISH
analysis of multiple cells from the embryo. Included in this
cohort were the embryos for which the new microarray had
detected three extra aneuploidies not detected with the use
of the 24sure microarray.
Overall, 26 aneuploidies were detected with the use of
FISH. The 24sure microarray platform succeeded in detecting
all of these aneuploidies, and it detected one extra aneuploidy
(Supplemental Table 1). The new microarray detected 25 of
the aneuploidies detected with the use of FISH and four extra
aneuploidies. One of the extra aneuploidies was the one also
detected with the use of the 24sure microarray. The rest of the
extra aneuploidies detected by the novel microarray were not
detected with the use of FISH or the 24sure microarray, indi-cating
that they were likely to have been artefacts, although
the possibility that they were caused by low-level mosaicism
can not be entirely ruled out. Again, analysis of chromosome
19 was shown to be the site of most discrepancies.
Because chromosome 19 was responsible for more than
one-half of the discordant results (Supplemental Table 1),
new probes for this chromosome were selected and added to
the microarray. The improved version of the microarray was
tested against five additional samples derived from embryos
known to be affected by chromosome 19 aneuploidies. The
abnormalities were successfully detected in all cases (Fig. 1).
Taking into consideration all results obtained from vali-dation
of the revised microarray, the aneuploidy concordance
rate was determined to be 97.7% at the level of the individual
chromosome (171/175 aneuploidies). In terms of clinical
diagnosis (i.e., normal or abnormal), the second iteration of
the microarray agreed with the established array in 99% of
samples (101/102).
Validation of Microarray-based mtDNA
Quantification
The novel microarray was assessed also for its ability to
correctly determine the relative quantity of mtDNA in the
samples tested. Unamplified genomic DNA samples from
four cell lines were applied to microarrays, and the average
fluorescence intensity for the 261 probes specific for the mito-chondrial
genome was calculated. This process was repeated
with the use of DNA derived from the same cell lines and
amplified with the use of Picoplex. Results for Picoplex-amplified
DNA were concordant with those obtained from
unamplified DNA, indicating that WGA did not introduce
distortions in relative amount of mtDNA. The cell lines were
placed in the following order according to their mtDNA
content, regardless of whether WGA was used:
A549 Red B2342B 206F. This order was also confirmed
by comparing Ct values after amplification of a mitochondrial
DNA sequence with the use of quantitative real-time PCR.
Analysis of mtDNA Quantity in Clinical Samples
After confirmation that relative mtDNA quantity can be
determined with the use of the microarray approach, 89 sam-ples
(27 PBs, 42 blastomeres, and 20 trophectoderm samples)
were analyzed. Not surprisingly, given the large difference in
cytoplasmic volume, blastomeres were found to contain
significantly more mtDNA than PBs and trophectoderm
samples (P.001), and trophectoderm samples had more
mtDNA than PBs (P.001). Results were analyzed according
to maternal age (Fig. 2). Two groups were considered for
each sample type: those derived from younger patients
(30–37 years old), and samples from patients of advanced
reproductive age (ARA; 38–45 years old). In PB samples,
mtDNA quantity in the ARA group was found to be signifi-cantly
lower than in the younger age group (P¼.04). In
contrast, single-blastomere and trophectoderm samples of
the ARA group had a tendency toward larger quantities of
mtDNA than the samples derived from younger patients.
However, differences observed for blastomeres and trophec-toderm
samples did not reach statistical significance.
Assessment of SNP Probes for Inclusion on the
Developed Microarray
Figure 3A shows a comparison of the SNP calls obtained from
an embryo and its parents. When all chromosomes were
examined, the informative SNPs giving a correct call (i.e., a
genotype consistent with inheritance of parental alleles)
were found to represent 40.1%. The SNPs giving mendelian
inconsistencies (i.e., SNPs showing genotypes incompatible
with inheritance from the specific parents because of geno-typing
errors) constituted 13.6% of the overall informative
SNPs. The rest of the informative SNPs displayed homozygos-ity,
indicative of phenomena such as allele dropout.
It was clear that the number of SNPs giving accurate data
was insufficient to allow reliable determination of genotypes
at individual loci. However, when taken together, the data
were adequate to confirmwhether or not an embryowas derived
from a specific couple. To verify this, results fromembryos were
compared with data from mismatched parents as well as their
true parents. Figure 3B shows results of SNP analysis when
embryo DNA was compared with nonmatching parents. Of
the informative SNPs tested, the proportion with a genotype
compatible with inheritance from the nonmatched couple was
fourfold fewer (9.8%) than seen when embryo genotypes were
compared with the correct parents (P.001). Additionally, the
proportion of SNPs displaying mendelian inconsistencies
almost doubled (to 25.1%) for mismatched couples and embryos
(P.01). Results from all 14 samples tested with the use of the
microarray were similar to those illustrated in Figure 3.
DISCUSSION
There is growing evidence that aneuploidy is one of the most
important causes of embryo implantation failure and that
4 VOL. - NO. - / - 2014
5. FIGURE 1
Fertility and Sterility®
Chromosome profile of an aneuploid blastomere processed with two microarray comparative genomic hybridization platforms. (A) Red circles
indicate gain and loss of chromosomal material as detected by the 24sure microarray (v. 2). The same microarray indicated the embryo to be
female (relative excess of X chromosome material and deficiency of Y chromosome compared with a normal male reference sample). (B) Results
from the customized microarray were entirely concordant. Error bars represent the minimum and maximum values obtained from the
fluorescence intensity of the probes attached on the microarray after applying the whole-genome amplification samples and the reference sample.
Konstantinidis. Comprehensive assessment of human embryos. Fertil Steril 2014.
VOL. - NO. - / - 2014 5
6. ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY
ongoing pregnancy rates can be improved in assisted
reproductive cycles if chromosomally normal embryos are
identified and selected for transfer (4–7). The new
microarray described in the present study demonstrated a
concordance rate of 97.7% compared with the most widely
used method for the comprehensive cytogenetic analysis of
preimplantation embryos. Given that many embryos
contain more than one aneuploidy, the clinical diagnoses
(i.e., normal or abnormal) showed even closer agreement
(99%) between the two platforms.
Earlier aCGH studies of polar bodies, blastomeres, and
trophectoderm biopsies have revealed that diagnostic errors
can occur, although in most cases the incidence is 5%
(22–24). A combination of biological and technical
challenges means that no aneuploidy screening method
applied to embryos can be considered to be infallible, a fact
that makes it difficult to place an absolute value on
accuracy. What can be said of the new microarray is that its
performance appears to be similar to existing methods of
aCGH in current use for the purpose of embryo analysis.
As well as providing information about aneuploidy, the
new microarray was shown to be capable of determining
the relative amount of mtDNA. Although a single organelle
can contain more than one copy of the mitochondrial
genome, it is likely that the quantity of mtDNA is reflective
of the relative number of mitochondria in the sample (i.e.,
when comparing samples of the same type, more mtDNA
generally means a larger number of mitochondria per cell).
The number of mitochondria may provide an insight into
the bioenergetic capacity of the cells analyzed and therefore
of the oocyte/embryo from which they were derived, although
ultimately experiments measuring metabolic activity would
be needed for confirmation.
Results obtained during this study revealed that PBs
derived from ARA women tend to have lower quantities of
mtDNA compared with those derived from younger women.
Other studies carried out on human oocytes have also indi-cated
that mtDNA copy number decreases with advancing
maternal age (25, 26). Although much of the decline in
oocyte competence seen with age can be attributed to
increasing aneuploidy rates (27), it is conceivable that
mitochondrial abnormalities might also play a role (28).
Interestingly, at the blastocyst stage a nonsignificant
trend in the opposite direction was seen: embryos from older
mothers tending to have increased levels of mtDNA. This is
concordant with a recent study that measures the amount
of mtDNA in blastocysts with the use of an alternate method
(quantitative PCR) (10). That study also demonstrated that
25% of chromosomally normal blastocysts that fail to implant
after transfer to the uterus contain an excessive quantity of
mtDNA, confirming that measurement of this feature has
the potential to provide clinically relevant information
additional to that provided by aneuploidy testing (10).
In addition to aneuploidy screening and relative quanti-fication
of mtDNA, the possibility of including SNP probes
on the microarray was examined. SNPs have previously
been assessed in human embryos with the use of microarrays,
but never in combination with aCGH (29–32). As with earlier
studies that have used microarrays to assess SNPs, a large
number of genotyping errors were observed, attributable to
problems such as allele dropout. Nevertheless, it was clear
that when results from an embryo were cross-referenced
with those of its parents, the number of SNP loci with plau-sible
genotypes (i.e., consistent with inheritance of one allele
from each parent) was significantly higher than when SNP
results from an embryo were compared with those of an un-related
couple (P.001). These results indicate that although
assessment of polymorphisms cannot avoid samples being
mixed up in the IVF laboratory, it does have the potential to
allow detection of such errors before embryo transfer.
Aswell as confirmation of parental origin, analysis ofSNPs
has the potential to reveal which of the transferred embryos (in
cases of multiple embryo transfer) actually resulted in birth,
providing a powerful tool for research studies assessing factors
that might affect the implantation potential of embryos (3, 31,
33). Analysis of SNPs also has the potential to indicate samples
contaminated with extraneous DNA (e.g., an excessive number
of alleles might be detected that are not present in either
parent). Reducing the risk of errors caused by contamination
with extraneous genetic material is a major consideration for
any genetic diagnosis requiring DNA amplification.
CONCLUSION
A novel microarray was developed, capable of aneuploidy
identification in varieties of cells typically used for the pur-pose
of preimplantation diagnosis of aneuploidy. Chromo-some
abnormalities of the type detected are known to be
extremely common in human oocytes and embryos and are
likely to be one of the most significant factors influencing
embryo implantation. In addition to aneuploidy detection,
the microarray developed during this study allows relative
quantitation of mtDNA copy number; a factor of potential
clinical and scientific significance. The possibility that mea-surement
of mtDNA could assist in the selection of the most
FIGURE 2
Normalized fluorescence intensity values obtained for mtDNA from
analysis of polar bodies (PBs), blastomeres, and trophectoderm
samples for different age categories. The error bars represent the
minimum and maximum values obtained from the fluorescence
intensity of the mtDNA specific probes. The black lines inside the
box plots represent the median values.
Konstantinidis. Comprehensive assessment of human embryos. Fertil Steril 2014.
6 VOL. - NO. - / - 2014
7. appropriate embryos for transfer—and thereby enhance IVF
success rates—should be investigated further. In the present
research, links between mtDNA content and age were
observed, suggesting that mitochondria may have a role,
direct or indirect, in reproductive aging.
This study confirmed that it is technically feasible to
combine interrogation of SNP loci with aCGH on a single mi-croarray.
This offers exciting possibilities in terms of embryo
identification and avoidance of misdiagnoses due to DNA
contamination. However, further optimization and validation
are required (e.g., selection of SNP probes that consistently
provide correct calls) before such an approach can be used
for diagnosis of specific mutations. Taken together, the
unique combination of genotyping, aneuploidy detection,
and mtDNA quantification offered by this microarray pro-vides
a valuable tool for scientific research and offers new
possibilities for the clinical evaluation of embryos and
oocytes.
Fertility and Sterility®
Acknowledgments: The authors thank the staff of Reproge-netics
at New Jersey (USA) for their assistance with this work,
particularly Drs. Pere Colls and Xue Zhong Zheng and Mr.
Tomas Escudero. The authors also thank Ms. Lorna Macleod
(University of Oxford) for her help in validation performed
regarding mtDNA probes included on the developed array.
Furthermore, they thank Prof. Joaquima Navarro (Universitat
Autonoma de Barcelona) for provision of certain cell lines used
in this study. Dagan Wells is funded by the Oxford National
Institute for Health Research Biomedical Research Centre.
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![ORIGINAL ARTICLE: REPRODUCTIVE BIOLOGY
Despite the promising results reported with the use of
modern chromosome screening technologies, it remains the
case that even the transfer of a morphologically perfect, chro-mosomally
normal embryo can not guarantee that a viable
pregnancy will be established. Studies published in the past
12 months indicate that implantation failure rates for euploid
blastocysts range from 25% to 50% (4–8). It is clear that
aspects of preimplantation embryo biology that are poorly
defined or invisible with the use of current methods of
embryo evaluation must also play an important role in
determining the potential of an embryo to produce a child.
One aspect of embryo biology of potential relevance to
viability is mitochondrial DNA (mtDNA) content. Mitochon-dria
are responsible for the production of most of the energy
needed by the cell for biosynthetic, metabolic, and physiologic
processes. A number of clinical and animal studies have high-lighted
the essential role of mitochondria in oogenesis and pre-implantation
development (9, 10). Decreased amounts of
mtDNA and diminished bioenergetic capacity have been
linked to fertilization failure, impaired oocyte quality, and
abnormal organization and function of the meiotic spindle
(11–14). Furthermore, bioenergetic capacity has been
correlated with embryo developmental potential and IVF
outcome (15, 16).
In response to the need for improved methods of embryo
selection, this study sought to create a new microarray plat-formcapable
of extending genetic evaluation beyond chromo-some
analysis. This microarray includes probes for aneuploidy
detection but also uses mitochondria-specific probes for the
relative quantification of mtDNA. In addition, the option of
including probes for the analysis of single-nucleotide poly-morphisms
(SNPs) was investigated, an approach that poten-tially
allows DNA fingerprinting and may provide other
information of diagnostic relevance.
MATERIALS AND METHODS
Cell Culture
Single cells derived from a variety of aneuploid fibroblast cell
lines were used during the optimization and validation of the
microarray. The cell lines had the following karyotypes:
47,XY,þ13; 47,XY,þ15; 47,XY,þ18; 47,XY,þ21; 45,X; and
48,XXY,þ18.
Cell lines were cultured under standard conditions. Single
cells were isolated in 1 mL phosphate-buffered saline solution
(PBS with 0.1% polyvinyl alcohol) and transferred to sterile
0.2-mL microcentrifuge tubes.
Selection ofWhole-genomeAmplification Method
and Development of a Customized Microarray
Development of the customized microarray involved the
testing of different whole-genome amplification (WGA)
methods, selection of the appropriate oligonucleotide probes
and array format, and identification of the best method for
labeling the amplified products. The different WGA methods
assessed were Picoplex (Rubicon Genomics), Genomeplex
(Sigma-Aldrich), and multiple displacement amplification
(MDA). Picoplex and Genomeplex were performed on isolated
single cells according to the manufacturers' instructions. For
MDA, the Repli-g Midi kit (Qiagen) was used following the
manufacturer's protocol with the use of a modified alkaline
lysis method. This MDA protocol involved incubation of the
samples in a thermocycler at 30C for 2 hours followed by
enzyme inactivation at 65C for 5 minutes.
Ultimately, the optimized microarray contained 14,334
unique probes for aneuploidy detection and 261 unique
probes for mtDNA analysis. In contrast to the most widely
used microarray for the purpose of preimplantation aneu-ploidy
screening, which uses probes composed of bacterial
artificial chromosomes, the new microarray used synthetic
oligonucleotides probes of 60 nucleotides in length.
The final protocol involved WGA of the samples and con-trol
male DNA with the use of Picoplex. Control DNA was
prepared by amplifying 1 mL male genomic DNA (1 ng/mL).
Picoplex-amplified DNA samples and control samples (8 mL)
were labeled with the use of the Cytosure labeling kit, which
incorporates Cy-dCTP using exonuclease-free Klenow
enzyme (Oxford Gene Technology [OGT]). After labeling,
the DNA was purified with the use of either Cytosure purifica-tion
columns (OGT) or Cytosure purification plates (OGT). The
sample and reference DNA were pooled together, dried, and
then resuspended in hybridization mix (Agilent). The com-bined
sample was applied on the microarray slide and incu-bated
overnight in a rotating incubator at 65C and 0.054 g.
Post-hybridization washes were carried out with the use of
Agilent Oligo microarray comparative genomic hybridization
(aCGH) Wash Buffer 1 for 5 minutes at room temperature and
Agilent Oligo aCGH Wash Buffer 2 for 1 minute at 37C. The
microarray slide was then scanned at 5-mm resolution on an
Agilent DNA microarray scanner. Feature extraction was
undertaken with the use of Agilent's Feature Extraction Soft-ware
(v. 10.7.3.1) and data analysis carried out with the use of
a customized version of Cytosure Interpret Software (OGT).
The whole process from DNA amplification to results could
be completed in 16 hours and at a cost similar to existing
aCGH methods.
Validation of Aneuploidy Detection with the Use
of Clinical Material
Ninety-seven Picoplex-amplified clinical samples, previously
screened for aneuploidies with the use of an alternative mi-croarray
CGH platform (24sure; Bluegnome), were reanalyzed
with the use of the new microarray and the results compared
for concordance. These samples were derived from actual
clinical cases performed previously for aneuploidy screening
of polar bodies and biopsied embryonic cells. These samples
consisted of 27 polar bodies (PBs), 50 blastomeres, and 20
trophectoderm samples. Diagnosis of aneuploidy was made
in a blinded fashion, without any knowledge of the original
screening results. An independent scientist was responsible
for assessing the two sets of data for concordance.
A subset of 20 embryos (19 cleavage stage and one blas-tocyst)
was also analyzed with the use of a third, independent
cytogenetic method, fluorescent in situ hybridization (FISH).
These whole embryos were donated to research, and all of
their cells were spread on microscope slides for FISH analysis.
2 VOL. - NO. - / - 2014](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)