Similar to Development and validation of an accurate quantitative real time polymerase chain reaction-based assay for human blastocyst comprehensive chromosoma
Similar to Development and validation of an accurate quantitative real time polymerase chain reaction-based assay for human blastocyst comprehensive chromosoma (20)
2. SEMINAL CONTRIBUTION
bodies from the oocyte and zygote, blastomeres from the
cleavage-stage embryo, and trophectoderm from the blasto-cyst
(3). Although there are advantages and disadvantages
to each of these components of aneuploidy screening, the
amount of time required to perform comprehensive chromo-some
screening (CCS) by any of these methodologies typically
exceeds 12 hours. Although this is less critical when applied to
polar bodies or blastomeres, analysis of aneuploidy in the tro-phectoderm
would likely require cryopreservation of the blas-tocyst
to provide sufficient time for analysis while also
preserving the appropriate synchrony between embryo and
endometrial development (4). Although methods of blastocyst
cryopreservation have improved, some risk remains, and
many patients prefer to avoid the additional expense and
time associated with a frozen embryo transfer cycle. To rou-tinely
perform comprehensive aneuploidy screening of the
blastocyst without cryopreservation, more rapid methodolo-gies
need to be developed.
Indeed, a more rapid method of preimplantation-stage
DNA analysis, polymerase chain reaction (PCR), has been in
clinical practice to manage patients with risk of transmitting
monogenic disorders for20 years (5). However, the ability to
reliably diagnose aneuploidy of all 24 chromosomes with the
use of PCR has not been established. Because of the initial
success with PCR-based preimplantation genetic diagnosis
(PGD), many improvements in the application of PCR have
been made, including the incorporation of multiplexing, nest-ing,
and fluorescence detection (6). Particularly noteworthy is
the development of quantitative real-time PCR (qPCR) (7). Al-though
qPCR has typically been applied to gene expression
studies where relative transcript quantities are determined,
there is also potential to evaluate the quantity of DNA in
a given sample (8). Nevertheless, the ability of qPCR to quan-tify
chromosomes in limited numbers of cells has yet to be
demonstrated. The present study characterizes the accuracy
of a novel method of 24 chromosome quantificatation in lim-ited
starting material as a preclinical step toward the applica-tion
of a rapid CCS method in the diagnosis of human
embryonic aneuploidy at the blastocyst stage of development.
MATERIALS AND METHODS
Experimental Design
This study was conducted in two phases with emphasis on eval-uating
the technical variation of qPCR-based 24-chromosome
copy number assignments by avoiding analysis of samples
with potential biologic variation. In phase I, only cell lines
with little to no evidence of mosaicism (biologic variation)
for the previously well characterized whole-chromosome aneu-ploidies
were used. In a similar attempt to avoid the impact of
mosaicism in embryos in phase 2, only blastocysts with two
consistent SNP microarray–based diagnoses were reevaluated
by qPCR. Randomized and blinded evaluation of consistency
of qPCR with cell line karyotypes and embryo SNP microarray
diagnoses were used as a measure of accuracy.
Phase 1: Cell Lines
Nine established and stable cell lines (fibroblasts and lympho-cytes)
were purchased from the Coriell Cell Repository (Cam-den,
NJ) and cultured as recommended by the supplier.
Included were GM09286 (47,XY,þ9), GM02948 (47,XY,þ13),
GM04610 (47,XX,þ8[75]/46,XX,þ8,dic(14;21)(14qter 14p
13::21p13 21qter)[25]), GM04435 (48,XY,þ16,þ21[45]/
47,XY,þ21[5]), GM00323 (46,XY), AG16777 (47,XX,þ21
[21]/47,XX,þ21,t(21;22)(q22;q13)[29]), AG16778 (46,XX),
AG16782 (46,XY), and GM01454 (47,XY,þ12[48]/47,XY,
þ12,add(13)(q34)[52]). Earlier studies have indicated that the
typical trophectoderm biopsy contains about five cells (9). To
model this in evaluating cell lines, 5-cell samples were prepared
by placement offive individual cells into a PCRtube under a dis-secting
microscope, as previously described (10). Lymphocyte
lines were prepared directly and fibroblast lines after trypsin
EDTA treatment. Seven 5-cell samples from cell line
GM00323 were used to serve as a reference dataset to interpret
results from 42 randomized and blinded 5-cell test samples
(GM00323, n ¼ 10; GM09286, n ¼ 4; AG16777, n ¼ 5;
AG16778, n ¼ 3; AG16782; n ¼ 3; GM01454, n ¼ 5;
GM02948, n ¼ 5; GM04610, n ¼ 5; and GM04435, n ¼ 2), as
described subsequently. Randomization was performed using
Microsoft Excel to avoid potential bias fromsequential analysis
ofmultiple samples fromthe same cell line. The identification of
the origins of each samplewas blinded by using decoded sample
names created in Microsoft Excel. The amount of time to com-plete
the procedure was recorded for each sample.
Phase 2: Embryos
Seventy-one embryos were included in this study. All em-bryos
had two consistent SNP microarray–based aneuploidy
screening results of trophectoderm biopsies (from days 5
and 6), as previously described (9, 11, 12). Thirty-seven of
the 71 embryos included in this study had arrested by day 6
and were subsequently found to be euploid by SNP microar-ray
analysis. The remaining 34, despite developmental nor-malcy
on day 6, were found to possess aneuploidy by SNP
microarray analysis. A third biopsy of each of the 71 day 6
embryos was randomized, blinded, and evaluated by qPCR
with the same seven 5-cell reference sample set used in the
cell line study described above. Again, randomization and
blinding was performed in Microsoft Excel to avoid interpre-tation
bias. The amount of time to complete the procedure was
recorded for each embryo.
qPCR
Cell line 5-cell samples and embryo biopsies were processed by
alkaline lysis as previously described (13). Multiplex amplifi-cation
of 96 loci (four for each chromosome, as previously de-scribed
[14]) was performed with the use of TaqMan Copy
Number Assays and TaqMan Preamplification Master Mix as
recommended by the supplier (Applied Biosystems), and in
a 50-mL reaction volume for 18 cycles using an Applied Bio-systems
2720 thermocycler. Real-time PCR was performed in
quadruplicate for each of the individual 96 loci using TaqMan
Gene Expression Master Mix (Applied Biosystems), a 5-mL
reaction volume, a 384-well plate, and a 7900 HT sequence
detection system, as recommended by the supplier (Applied
Biosystems). A unique method of the standard delta delta
threshold cycle (ΔΔCt) method of relative quantitation (15)
820 VOL. 97 NO. 4 / APRIL 2012
3. was applied. First, a chromosome-specific ΔCt was calculated
from the average Ct of the 16 reactions targeting a specific
chromosome (four replicates of four loci) minus the average
Ct of all of the 336 reactions targeting all of the remaining au-tosomes
(four replicates of four loci of 21 remaining auto-somes).
The same process was used to individually determine
the ΔCt for each of the 24 chromosomes in the test sample.
Each chromosome-specific ΔCT was then normalized to the
average chromosome specific ΔCt values derived from the
same evaluation of seven normal male (GM00323) 5-cell sam-ples
(reference set). The resulting chromosome-specific ΔΔCt
values were used to calculate fold change by considering the
ΔΔCt values as the negative exponent of 2, as previously de-scribed
(15). All autosome fold changes were then multiplied
by 2, whereas the sex chromosome fold changes were used
as is, to determine the 24-chromosome copy number in each
sample. This methodology was designed to specifically iden-tify
whole-chromosome but not segmental aneuploidy.
Statistics
Sample specific concurrence. To evaluate the utility of
a previously established strategy for identifying poor-quality
data independent of knowing its accuracy (12), the
overall concurrence was calculated for each sample. In this
analysis, it is first assumed that the qPCR assay can assess
only whole-chromosome aneuploidy, such that the four
copy number assignments within each chromosome should
always agree. Therefore, the standard deviation of the four
measurements of copy number for each chromosome was cal-culated.
The standard deviations of each of the 24 chromo-somes
were then averaged for each sample. Outliers
(nonconcurrent samples) were defined as samples found out-side
an interquartile range of 1.5 from the overall distribution
of average sample-specific standard deviations for each sam-ple
type as determined with the use of Analyse-It software for
Microsoft Excel. Means and variations of the rates of concur-rence
in cell lines and embryos were evaluated for signifi-cance
with a Student t test and an F test, respectively.
Consistency of diagnosis. Consistency of the cell line 5-cell
samples’ qPCR-based 24-chromosome copy number predic-tions
with the cell lines’ karyotype (previously established
by the Coriell Cell Repository by conventional karyotyping)
was evaluated at the level of individual chromosome copy
numbers and for the entire 24 chromosomes of each sample
tested. Consistency of embryo qPCR-based 24-chromosome
copy number assignments with previously established SNP
microarray–based diagnoses was also evaluated at the level
of individual chromosome copy numbers for the entire 24
chromosomes of each sample tested and for the overall diag-nosis
of aneuploidy or euploidy. Results were evaluated with
and without the application of a threshold of concurrence as
described above.
RESULTS
Phase 1: Cell Lines
Forty-two randomized blinded samples were evaluated for
24-chromosome copy number and compared for consistency
FIGURE 1
Fertility and Sterility®
Examples of qPCR-based 24-chromosome copy number results from
5-cell samples derived from nine cell lines with previously well
characterized karyotypes.
Treff. 4-hour qPCR-based 24-chromosome CCS. Fertil Steril 2012.
with the cell lines’ karyotype previously determined by con-ventional
g-banding at the commercial provider’s laboratory.
Examples of qPCR results for 5-cell samples from the cell lines
are shown in Figure 1. One of the samples (GM00323; 46,XY)
produced a false positive trisomy 18, giving an overall consis-tency
of chromosome copy number assignment of 99.90%
(1,007/1,008) and an overall 24-chromosome diagnosis con-sistency
of 97.6% (41/42). There were no false negative
VOL. 97 NO. 4 / APRIL 2012 821
4. SEMINAL CONTRIBUTION
diagnoses for aneuploid chromosomes or inaccurate predic-tions
of gender. Analysis of concurrence identified the only
discordant cell line sample as the only outlier (i.e., nonconcur-rent;
Fig. 2). Therefore, by applying a threshold of concur-rence,
the cell line study resulted in 97.6% reliability of
obtaining a diagnosis and a 100% level of consistency of
chromosome-specific (n ¼ 984) and 24-chromosome copy
number (n ¼ 41) assignments. The amount of time taken to
complete the procedure for each sample was 4 hours.
Phase 2: Embryos
Seventy-one embryos with consistent SNP microarray–based
24 chromosome aneuploidy screening results from 2 biopsies
were rebiopsied, randomized, and blinded for analysis of con-sistency
of qPCR-based diagnoses. These were selected to re-duce
the risk of mosaicism. Examples of embryo biopsy qPCR
results are shown in Figure 3, and the details of karyotype pre-dictions
are included in Supplemental Table 1 (available on-line
at www.fertstert.org). In one embryo, consistently
diagnosed as 45,XY,13,14,þ18 by SNP microarray analy-sis
of two biopsies, qPCR failed to detect monosomy 14. All of
the remaining chromosomes for all of the remaining samples
were consistent between qPCR and microarray, giving an
overall chromosome-specific consistency of 99.94% (1,703/
1,704) and an overall 24-chromosome diagnosis consistency
of 98.6% (70/71). There were no false positive aneuploid chro-mosomes
observed or inaccurate predictions of sex. The over-all
rate of concurrence in cell lines from phase 1 was
equivalent to the rate of concurrence in embryos in phase 2
(P¼.96). The variation in concurrence rates within cell lines
and embryos was also equivalent (P¼.34). Analysis of con-currence
identified only one embryo sample as an outlier
(i.e., nonconcurrent; Fig. 2). However, this was not the sample
with the false negative monosomy 14, and therefore the
consistency of the embryo results was the same with or with-out
applying a threshold for concurrence. Because the only
false negative aneuploidy diagnosis occurred in an embryo
with other consistently diagnosed aneuploidies (monosomy
13 and trisomy 18), the overall qPCR-based diagnosis of
aneuploidy or euploidy was 100% consistent with SNP
FIGURE 2
Box-whisker plots representing the distribution of average 24-
chromosome four-loci copy number standard deviations for each of
the 42 cell line samples and 71 blastocyst biopsies. For each sample
type, one outlier was identified, including the only cell line sample
with an inconsistent qPCR diagnosis.
Treff. 4-hour qPCR-based 24-chromosome CCS. Fertil Steril 2012.
FIGURE 3
Examples of (gray) single-nucleotide polymorphism microarray– and
(white) qPCR-based 24-chromosome copy number results from
blastocyst-stage embryo biopsies.
Treff. 4-hour qPCR-based 24-chromosome CCS. Fertil Steril 2012.
822 VOL. 97 NO. 4 / APRIL 2012
5. microarray based predictions. The amount of time taken to
complete the procedure for each sample was 4 hours.
DISCUSSION
Results of the present study have demonstrated the validity of
a new 4-hour method for CCS in human blastocysts. The tech-nical
accuracy was measured in two phases. The first phase
involved the use of cell lines with previously well character-ized
karyotypes. Although it is possible for biologic variation
of cell line karyotypes to exist as a result of extended culture
(16, 17) or from unidentified low-level mosaicism in the orig-inal
sample used to create the cell line, the potential impact of
these biologic artifacts can be avoided by the use of early pas-sages
of cell lines that show little to no evidence of mosaicism
by conventional karyotyping. With this strategy we demon-strated
a consistency of qPCR-based CCS of concurrent
5-cell samples of 100%.
To evaluate a more relevant tissue type, the second phase
of the study involved the evaluation of discarded human em-bryos.
Because the presence of mosaicism in embryos as a re-sult
of postzygotic mitotic aneuploidy development
represents a well documented phenomenon that could con-tribute
to biologic variation in blastocysts (11, 18), we
selected embryos which specifically demonstrated
consistent SNP microarray diagnoses from 2 biopsies. This
approach may help reduce the impact of mosaicism and
biologic variation on evaluating the technical accuracy of
new methods such as qPCR. Indeed, analysis of these well
controlled blastocysts by qPCR demonstrated 98.6% 24-
chromosome consistency with the highly validated method
of SNP microarray–based aneuploidy screening (12). Impor-tantly,
all SNP microarray–based euploid embryos were diag-nosed
as euploid and all SNP microarray–based aneuploid
embryos as aneuploid by qPCR (100% laboratory diagnostic
consistency). Furthermore, because trophectoderm biopsies
may not all possess five cells as modeled in phase I, the results
of evaluating actual trophectoderm biopsies in phase II pro-vides
additional evidence of validity to samples with variable
and potentially fewer numbers of cells.
Another important observation regarding the perfor-mance
of this qPCR methodology was the equivalent levels
of concurrence measured in cell lines and embryos (Fig. 2).
It has been suggested that PGD-based assays typically per-form
differently on different cell types (i.e., lymphocytes, fi-broblasts,
and embryonic cells) (19). Given the high degree
of similarity in performance between cell lines and embryos
in the present study, qPCR-based aneuploidy screening ap-pears
to be a robust methodology independent of the cell
type. This may be in part due to the use of locus-specific mul-tiplex
PCR rather than whole-genome amplification for the
initial processing of the sample. It is also possible that the
use of trophectoderm biopsies, which may possess more
than five cells, provided an advantage compared with the
use of five lymphocytes or fibroblasts for providing consistent
copy number assignments across each chromosome. The same
advantage might be expected when comparing concurrence
of trophectoderm with either blastomeres or polar bodies
where less template DNA is present. Although this method
Fertility and Sterility®
was not applied to blastomeres or polar bodies (single cells),
it is theoretically possible. In addition, this methodology
could also be applicable to evaluating segmental aneuploidies
associated with inheritance of unbalanced translocations by
simply adding specific assays targeting positions on either
side of the breakpoints of the chromosomes involved. Finally,
one important challenge that should be considered is the need
to process multiple embryos in parallel. Although this cer-tainly
involves an additional expense (a limitation on its
own), the procurement of multiple thermal cyclers and the
use of standard laboratory automation solutions can be used
to completely circumvent this challenge.
In conclusion, with these measures of accuracy in place
and the fact that this protocol can be accomplished within 4
hours of receiving a biopsy, this qPCR-based methodology
provides the first opportunity for same-day trophectoderm bi-opsy
24-chromosome aneuploidy screening and fresh blasto-cyst
transfer. Given the level of consistency with an
established method of aneuploidy screening that has also
demonstrated excellent predictive value for clinical outcome
(20), this qPCR method can now be justifiably evaluated for
clinical efficacy in a randomized controlled trial (RCT). In-deed,
preliminary RCT results of 24-chromosome aneuploidy
screening with qPCR on trophectoderm biopsies and subse-quent
fresh euploid blastocyst transfer indicate a significant
increase in the success of IVF (21).
REFERENCES
1. Harper JC, Harton G. The use of arrays in preimplantation genetic diagnosis
and screening. Fertil Steril 2010;94:1173–7.
2. Treff NR, Su J, Tao X, Northrop LE, Scott RT Jr. Single-cell whole-genome am-plification
technique impacts the accuracy of SNP microarray-based geno-typing
and copy number analyses. Mol Hum Reprod 2011;17:335–43.
3. Delhanty JD. Is the polar body approach best for pre-implantation genetic
screening? Placenta 2011;32(Suppl 3):S268–70.
4. van Voorhis BJ, Dokras A. Delayed blastocyst transfer: is the window shut-ting?
Fertil Steril 2008;89:31–2.
5. Handyside AH, Kontogianni EH, Hardy K, Winston RM. Pregnancies from bi-opsied
human preimplantation embryos sexed by Y-specific DNA amplifica-tion.
Nature 1990;344:768–70.
6. Harton GL, De Rycke M, Fiorentino F, Moutou C, SenGupta S, Traeger-
Synodinos J, et al. ESHRE PGD consortium best practice guidelines for
amplification-based PGD. Hum Reprod 2010;26:33–40.
7. Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR analysis: real-time
monitoring of DNA amplification reactions. Biotechnology 1993;11:1026–30.
8. D’Haene B, Vandesompele J, Hellemans J. Accurate and objective copy num-ber
profiling using real-time quantitative PCR. Methods 2010;50:262–70.
9. Schoolcraft WB, Treff NR, Stevens JM, Ferry K, Katz-Jaffe M, Scott RT Jr. Live
birth outcome with trophectoderm biopsy, blastocyst vitrification, and sin-gle-
nucleotide polymorphism microarray-based comprehensive chromo-some
screening in infertile patients. Fertil Steril 2011;96:638–40.
10. Treff NR, Su J, Tao X, Miller KA, Levy B, Scott RT Jr. A novel single-cell DNA
fingerprinting method successfully distinguishes sibling human embryos.
Fertil Steril 2009;94:477–84.
11. Northrop LE, Treff NR, Levy B, Scott RT Jr. SNP microarray-based 24 chromo-some
aneuploidy screening demonstrates that cleavage-stage FISH poorly
predicts aneuploidy in embryos that develop to morphologically normal blas-tocysts.
Mol Hum Reprod 2010;16:590–600.
12. Treff NR, Su J, Tao X, Levy B, Scott RT Jr. Accurate single cell 24 chromosome
aneuploidy screening using whole genome amplification and single nucleo-tide
polymorphism microarrays. Fertil Steril 2010;94:2017–21.
13. Cui XF, Li HH,Goradia TM, Lange K, Kasasian HH Jr,Galas D, et al. Single-sperm
typing: determination of genetic distance between the G gamma-globin and
VOL. 97 NO. 4 / APRIL 2012 823
6. SEMINAL CONTRIBUTION
parathyroid hormone loci by using the polymerase chain reaction and allele-specific
oligomers. Proc Natl Acad Sci U S A 1989;86:9389–93.
14. Treff NR, Tao X, Su J, Lonczak A, Northrop LE, Ruiz A, et al. Tracking embryo
implantation using cell-free fetal DNA enriched from maternal circulation at
9 weeks gestation. Mol Hum Reprod 2011;17:434–8.
15. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative
CT method. Nat Protoc 2008;3:1101–8.
16. Maitra A, Arking DE, Shivapurkar N, Ikeda M, Stastny V, Kassauei K, et al.
Genomic alterations in cultured human embryonic stem cells. Nat Genet
2005;37:1099–103.
17. Draper JS, Smith K, Gokhale P, Moore HD, Maltby E, Johnson J, et al. Recur-rent
gain of chromosomes 17q and 12 in cultured human embryonic stem
cells. Nat Biotechnol 2004;22:53–4.
18. Fragouli E, Wells D. Aneuploidy in the human blastocyst. Cytogenet Ge-nome
Res 2011.
19. Glentis S, SenGupta S, Thornhill A, Wang R, Craft I, Harper JC. Molecular
comparison of single cell MDA products derived from different cell types. Re-prod
Biomed online 2009;19:89–98.
20. Scott RT Jr, Ferry K, Su J, Tao X, Scott K, Treff NR. Comprehensive chromo-some
screening is highly predictive of the reproductive potential of human
embryos: a prospective, blinded, nonselection study. Fertil Steril 2012. doi:
10.1016/j.fertnstert.2012.01.104.
21. Scott RT Jr, Tao X, Taylor D, Ferry K, Treff N. A prospective randomized con-trolled
trial demonstrating significantly increased clinical pregnancy rates
following 24 chromosome aneuploidy screening: biopsy and analysis on
day 5 with fresh transfer. Fertil Steril 2010;94:S2.
824 VOL. 97 NO. 4 / APRIL 2012
![SEMINAL CONTRIBUTION
bodies from the oocyte and zygote, blastomeres from the
cleavage-stage embryo, and trophectoderm from the blasto-cyst
(3). Although there are advantages and disadvantages
to each of these components of aneuploidy screening, the
amount of time required to perform comprehensive chromo-some
screening (CCS) by any of these methodologies typically
exceeds 12 hours. Although this is less critical when applied to
polar bodies or blastomeres, analysis of aneuploidy in the tro-phectoderm
would likely require cryopreservation of the blas-tocyst
to provide sufficient time for analysis while also
preserving the appropriate synchrony between embryo and
endometrial development (4). Although methods of blastocyst
cryopreservation have improved, some risk remains, and
many patients prefer to avoid the additional expense and
time associated with a frozen embryo transfer cycle. To rou-tinely
perform comprehensive aneuploidy screening of the
blastocyst without cryopreservation, more rapid methodolo-gies
need to be developed.
Indeed, a more rapid method of preimplantation-stage
DNA analysis, polymerase chain reaction (PCR), has been in
clinical practice to manage patients with risk of transmitting
monogenic disorders for20 years (5). However, the ability to
reliably diagnose aneuploidy of all 24 chromosomes with the
use of PCR has not been established. Because of the initial
success with PCR-based preimplantation genetic diagnosis
(PGD), many improvements in the application of PCR have
been made, including the incorporation of multiplexing, nest-ing,
and fluorescence detection (6). Particularly noteworthy is
the development of quantitative real-time PCR (qPCR) (7). Al-though
qPCR has typically been applied to gene expression
studies where relative transcript quantities are determined,
there is also potential to evaluate the quantity of DNA in
a given sample (8). Nevertheless, the ability of qPCR to quan-tify
chromosomes in limited numbers of cells has yet to be
demonstrated. The present study characterizes the accuracy
of a novel method of 24 chromosome quantificatation in lim-ited
starting material as a preclinical step toward the applica-tion
of a rapid CCS method in the diagnosis of human
embryonic aneuploidy at the blastocyst stage of development.
MATERIALS AND METHODS
Experimental Design
This study was conducted in two phases with emphasis on eval-uating
the technical variation of qPCR-based 24-chromosome
copy number assignments by avoiding analysis of samples
with potential biologic variation. In phase I, only cell lines
with little to no evidence of mosaicism (biologic variation)
for the previously well characterized whole-chromosome aneu-ploidies
were used. In a similar attempt to avoid the impact of
mosaicism in embryos in phase 2, only blastocysts with two
consistent SNP microarray–based diagnoses were reevaluated
by qPCR. Randomized and blinded evaluation of consistency
of qPCR with cell line karyotypes and embryo SNP microarray
diagnoses were used as a measure of accuracy.
Phase 1: Cell Lines
Nine established and stable cell lines (fibroblasts and lympho-cytes)
were purchased from the Coriell Cell Repository (Cam-den,
NJ) and cultured as recommended by the supplier.
Included were GM09286 (47,XY,þ9), GM02948 (47,XY,þ13),
GM04610 (47,XX,þ8[75]/46,XX,þ8,dic(14;21)(14qter 14p
13::21p13 21qter)[25]), GM04435 (48,XY,þ16,þ21[45]/
47,XY,þ21[5]), GM00323 (46,XY), AG16777 (47,XX,þ21
[21]/47,XX,þ21,t(21;22)(q22;q13)[29]), AG16778 (46,XX),
AG16782 (46,XY), and GM01454 (47,XY,þ12[48]/47,XY,
þ12,add(13)(q34)[52]). Earlier studies have indicated that the
typical trophectoderm biopsy contains about five cells (9). To
model this in evaluating cell lines, 5-cell samples were prepared
by placement offive individual cells into a PCRtube under a dis-secting
microscope, as previously described (10). Lymphocyte
lines were prepared directly and fibroblast lines after trypsin
EDTA treatment. Seven 5-cell samples from cell line
GM00323 were used to serve as a reference dataset to interpret
results from 42 randomized and blinded 5-cell test samples
(GM00323, n ¼ 10; GM09286, n ¼ 4; AG16777, n ¼ 5;
AG16778, n ¼ 3; AG16782; n ¼ 3; GM01454, n ¼ 5;
GM02948, n ¼ 5; GM04610, n ¼ 5; and GM04435, n ¼ 2), as
described subsequently. Randomization was performed using
Microsoft Excel to avoid potential bias fromsequential analysis
ofmultiple samples fromthe same cell line. The identification of
the origins of each samplewas blinded by using decoded sample
names created in Microsoft Excel. The amount of time to com-plete
the procedure was recorded for each sample.
Phase 2: Embryos
Seventy-one embryos were included in this study. All em-bryos
had two consistent SNP microarray–based aneuploidy
screening results of trophectoderm biopsies (from days 5
and 6), as previously described (9, 11, 12). Thirty-seven of
the 71 embryos included in this study had arrested by day 6
and were subsequently found to be euploid by SNP microar-ray
analysis. The remaining 34, despite developmental nor-malcy
on day 6, were found to possess aneuploidy by SNP
microarray analysis. A third biopsy of each of the 71 day 6
embryos was randomized, blinded, and evaluated by qPCR
with the same seven 5-cell reference sample set used in the
cell line study described above. Again, randomization and
blinding was performed in Microsoft Excel to avoid interpre-tation
bias. The amount of time to complete the procedure was
recorded for each embryo.
qPCR
Cell line 5-cell samples and embryo biopsies were processed by
alkaline lysis as previously described (13). Multiplex amplifi-cation
of 96 loci (four for each chromosome, as previously de-scribed
[14]) was performed with the use of TaqMan Copy
Number Assays and TaqMan Preamplification Master Mix as
recommended by the supplier (Applied Biosystems), and in
a 50-mL reaction volume for 18 cycles using an Applied Bio-systems
2720 thermocycler. Real-time PCR was performed in
quadruplicate for each of the individual 96 loci using TaqMan
Gene Expression Master Mix (Applied Biosystems), a 5-mL
reaction volume, a 384-well plate, and a 7900 HT sequence
detection system, as recommended by the supplier (Applied
Biosystems). A unique method of the standard delta delta
threshold cycle (ΔΔCt) method of relative quantitation (15)
820 VOL. 97 NO. 4 / APRIL 2012](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)