Human Reproduction Vol.17, No.3 pp. 752–759, 2002
DNA fingerprinting of sister blastomeres from human IVF
embryos
M.G.Katz1,2, A.O.Trounson1 and D.S.Cram1,2
1Centre
for Early Human Development, Monash Institute of Reproduction and Development, Monash University, Clayton,
Victoria and 2Monash IVF, Melbourne, Australia
3To
whom correspondence should be addressed at: Centre for Early Human Development, Monash Institute of Reproduction and
Development, Level 3, 27–31 Wright St, Clayton 3168, Victoria, Australia. E-mail: [email protected]
BACKGROUND: Previously published single cell DNA fingerprinting systems have been plagued by high rates of
allele drop-out (ADO) and preferential amplification (PA) preventing clinical application in preimplantation genetic
diagnosis. METHODS: Tetranucleotide microsatellite markers with high heterozygosity, known allelic size ranges
and minimal PCR stutter artefacts were selected for chromosomes X, 13, 18 and 21 and optimized in a multiplex
fluorescent (FL)-PCR format. FL-PCR products were analysed using the ABI Prism 377 DNA sequenator and
Genescan software. Validation of the DNA fingerprinting system was performed on single diploid (n ⍧ 50) and
aneuploid (n ⍧ 25) buccal cells and embryonic blastomeres (n ⍧ 21). RESULTS: The optimized pentaplex PCR
DNA fingerprinting system displayed a high proportion of successful amplifications (>91%) and low ADO and PA
(<6%) when assessed on 50 human buccal cells. DNA fingerprints of single cells from a subject with Down’s
syndrome detected the expected tri-allelic pattern for the chromosome 21 marker, confirming trisomy 21. In a blind
study on 21 single blastomeres, all embryos were identifiable by their unique DNA fingerprints and shared parental
alleles. CONCLUSIONS: A highly specific multiplex FL-PCR based on the amplification of five highly polymorphic
microsatellite markers was developed for single cells. This finding paves the way for the development of a more
complex PCR DNA fingerprinting system to assess aneuploidy and single gene mutations in IVF embryos from
couples at genetic risk.
Key words: aneuploidy/DNA fingerprinting/embryonic blastomeres/single cell PCR
Introduction
Chromosomal abnormalities such as aneuploidies are associated with human reproductive failure and early embryonic loss
(Munné et al., 1993, 1995). Aneuploidies originate from
meiotic non-disjunction, predominantly observed in the first
meiotic division of the oocyte or sperm (Antonarakis et al.,
1992) and increase with advanced maternal age (Verlinsky
et al., 1998). Aneuploidies can also arise post-fertilization
from mitotic non-disjunction in the second and third cleavage
divisions that culminate in chromosomal mosaicism and a
mixture of normal, monosomic and trisomic cells (Delhanty and
Handyside, 1995). Various aneuploidies have been observed in
cleavage stage embryos as well as the inner cell mass cells of
IVF blastocysts (Evsikov and Verlinsky, 1998; Magli et al.,
2000). The most common aneuploidies seen in spontaneous
abortions are trisomies involving chromosomes 13, 16, 18, 21
and 22, and monosomy X (Boué and Boué, 1976; Eiben
et al., 1990; Vidal et al., 1998). Although these chromosomal
abnormalities are generally associated with implantation failure
or miscarriage in the first trimester, a very small proportion
can develop to term. The remaining possible trisomies or
monosomies occurring in the preimplantation stage almost
752
always result in embyronic lethality (Boué et al., 1985;
Sandalinas et al., 2000).
To improve outcomes of IVF patients with a poor prognosis
for pregnancy due to advanced maternal age (⬎35 years), a
history of unexplained recurrent miscarriages and repeated
failed IVF (more than three cycles), fluorescent in-situ hybridization (FISH) has been performed to identify euploidy in
oocyte polar bodies or embryonic blastomeres using up to nine
different chromosomal probes on one fixed nucleus (Gianaroli
et al., 1997a,b; Magli et al., 1998; Munné et al., 1998;
Verlinsky et al., 1998). FISH analysis of embryos from these
patient groups consistently reveals aneuploidy and mosaicism
rates of up to 50% (Munné et al., 1994; Harper et al., 1995;
Kuo et al., 1998; Magli et al., 1998; Verlinsky et al., 1998;
Gianaroli et al., 1999; Bielanska et al., 2000). In most clinical
preimplantation genetic diagnosis (PGD) programmes, the
selection of euploid embryos for transfer has resulted in higher
implantation and ongoing pregnancy rates for poor prognosis
patients, in particular for women of advanced maternal age
(Magli et al., 1998; Damario et al., 1999; Giarnaroli et al.,
1999; Rubio et al., 2000). More recently, efforts have focused
on development of new methods to determine the numeracy
© European Society of Human Reproduction and Embryology
DNA fingerprinting of embryonic blastomeres
Figure 1. Single cell DNA fingerprinting. (A) DNA fingerprint of a single human female buccal cell. Homozygous loci (D21S1413 and
D13S631) and heterozygous loci (DXS8377, D13S258 and D18S51). (B) DNA fingerprint of a single human buccal cell from a male
subject with Down’s syndrome. Homozygous loci (DXS8377 and D18S51), heterozygous loci (D13S258 and D13S631) and tri-allelic locus
(D21S1413). Fluorochromes are as follows: green (TET), blue (6-FAM) and black (HEX). Red (TAMRA) peaks indicate internal mol. wt
markers.
of all 23 pairs of human chromosomes, including comparative
genomic hybridization (CGH) (Wells and Delhanty, 2000;
Vouillaire et al., 2000), spectral karyotyping (Marquez et al.,
1998) and nuclear conversion (Verlinsky and Evsikov, 1999).
However, any added clinical benefit of these methods over
FISH has yet to be assessed in a large randomized trial.
Conventional and fluorescent polymerase chain reaction (FLPCR) incorporating various microsatellite markers (Mansfield,
1993; Muggleton-Harris et al., 1993; Pickering et al., 1994;
Pickering and Muggleton-Harris, 1995; Pertl et al., 1994) has
been investigated for potential application in PGD. It has been
shown that single cell multiplex FL-PCR can simultaneously
provide information on multiple loci including identity of sex,
an individual DNA fingerprint and genetic status for inherited
conditions such as cystic fibrosis (Findlay et al., 1995; Findlay
and Quirke, 1996). FL-PCR DNA fingerprinting using
chromosome-specific microsatellite markers has also been
used in prenatal diagnosis for the detection of chromosomal
aneuploidies (Mansfield, 1993; Pertl et al., 1994, 1996) and
on single cells (Sherlock et al., 1998; Findlay et al., 1999).
Theoretically, the amount of DNA produced in FL-PCR
amplification is proportional to the quantity of the initial target
sequence when strict experimental conditions are adhered to,
and thus allelic ratios for any particular locus can be calculated
from the final fluorescent yield (Ferre, 1992; Wells and
Sherlock, 1998). Accordingly, disomy can be defined by an
allelic ratio of 1:1, whereas a trisomy can either be defined as
a tri-allelic pattern with an allelic ratio of 1:1:1 or a double
dosage di-allelic pattern with an allelic ratio of 2:1. Previously
published DNA fingerprinting systems developed for single
cells, where the target DNA is in the order of 6 pg, have been
plagued with several problems, including high rates of either
preferential allelic amplification (PA) or allelic drop-out (ADO)
(Sherlock et al., 1998; Findlay et al., 1998, 1999). ADO is
defined as the total amplification failure of one allele at a
heterozygous locus so that only one allele is detectable after
the analysis of the PCR product, whereas PA is the underrepresentation of one of the two heterozygous alleles resulting
in a distortion from the expected 1:1 allelic ratio. The effect
of ADO, PA or both reduces the degree of reliability for
quantitation of FL-PCR products at the single cell level.
A reliable single cell PCR DNA fingerprinting system would
be a very powerful tool in PGD for unique identification of a
DNA sample and a means to simultaneously detect specific
gene defects and chromosomal aneuploidy. No other current
technique has the potential for such a multitude of diagnoses.
To this end, we developed a new single cell PCR DNA
fingerprinting system based on multiplex FL-PCR amplification
of five highly polymorphic microsatellite markers located on
four different chromosomes and evaluated its performance on
both buccal cells and blastomeres from cleavage stage IVF
embryos.
Materials and methods
Isolation of single human buccal cells
Buccal cell samples were collected by twirling a cytology brush
(EndoScanPlus; Medico, USA) on the inner cheek for 30 s. Cells
were collected into an 1.5 ml Eppendorf tube containing 750 µl of
phosphate buffer solution (PBS) (Gibco Life Technologies, Australia),
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M.G.Katz, A.O.Trounson and D.S.Cram
Figure 2. Evaluation of the single cell pentaplex DNA fingerprinting system. The proportion of successful amplifications, amplification
failure, accuracy, allele drop-out (ADO) and preferential amplification (PA) are shown for each individual tri- and tetranucleotide
microsatellite marker. No significant difference was observed between the individual loci for any of the above parameters, P ⬎ 0.05.
washed twice with PBS, and resuspended in 500 µl of PBS. A 10 µl
aliquot was examined for single nucleated cells under an inverted
microscope (Leica MS5) and several intact single cells aspirated into
finely pulled 22.9 cm long glass Pasteur pipettes (Becton Dickinson,
USA). Single cells were successively washed through three further
5 µl drops of PBS buffer and transferred together with 1–2 µl of
PBS buffer into a sterile 0.2 ml PCR tube on ice. Using the same
pipette, 1–2 µl of PBS buffer from the last wash droplet was
transferred into a second sterile 0.2 ml PCR tube on ice to serve as
a negative PCR control. Tubes were immediately frozen at –80°C
prior to PCR analysis.
Embryo biopsy and FISH
Couples on the PGD programme at Monash IVF underwent standard
IVF treatment including ovulation induction, surgical aspiration of
the oocytes and sperm collection. Oocytes were fertilized by ICSI.
On day 3 of embryonic development, cleavage stage embryos with
5–8 cells were considered suitable for biopsy. Embryos were incubated
in Ca2⫹/Mg2⫹ free medium prior to zona drilling using acid Tyrode’s
solution and one or two cells were biopsied.
Isolation of human blastomeres from aneuploid embryos
Aneuploid embryos diagnosed by FISH at day 3 are regarded as
genetically abnormal. Nine aneuploid embryos were obtained from
patients with either advanced maternal age (⬎36 years) or repeated
IVF failure (⬎3 cycles) who had a history of infertility. Under
guidelines established by the Infertility Treatment Authority in
Victoria, aneuploid embryos deemed to be unsuitable for transfer
must be left to ‘succumb’ on the bench for 24 h before being available
for research. Succumbed embryos were treated with pronase
(2 mg/ml in HEPES buffered human tubal fluid culture medium) for
1 min to dissolve the zona pellucida and transferred into Ca2⫹/Mg2⫹free medium to dissociate the blastomeres. Single blastomeres were
carefully washed through three 5 µl drops of PBS buffer and
transferred together with 1–2 µl of PBS buffer into a sterile 0.2 ml
PCR tube on ice.
Microsatellite markers
Five microsatellite markers were used in the pentaplex single cell
DNA fingerprinting: D21S1413 (Findlay et al., 1998), D18S51 (Straub
et al., 1993), D13S258 (Toth et al., 1998), D13S631 (Sherlock et al.,
1998) and DXS8377 (Hu et al., 1996). Each microsatellite marker
was selected for high heterozygosity (average 0.91). Based on
754
available published allelic size ranges, appropriate fluorochrome tags
(6-FAM, HEX and TET) were selected, where possible, to avoid
overlapping profiles. Primers were synthesized and fluorescently
labelled by Applied Biosystems, Australia. All primer pairs were
diluted in molecular biology grade H2O (Sigma, Melbourne, Australia)
to 200 pmol/µl stock solutions under sterile conditions and stored in
aliquots of 100 pmol/µl at –20°C until use. For development of an
octaplex single cell DNA fingerprinting system, primers for an
additional three microsatellite markers were added to the pentaplex
system.
Single cell multiplex FL-PCR
The optimized single cell multiplex FL-PCR developed for the five
microsatellite markers consisted of the following: 2.5 µl of 10⫻Taq
PCR Buffer (500 mmol/l KCl, 100 mmol/l Tris–HCl, pH 9.0 and
15 mmol/l MgCl2), 0.5 µl of 10 mmol/l dNTP (200 µmol/l), 0.3 µl
of Taq polymerase (5 U/µl) (Amersham Pharmacia Biotech, Sydney,
Australia), 11.20 µl molecular biology grade H2O and 10.5 µl of
primer mix making a final volume of 25 µl. Multiplex FL-PCR was
performed using a Hotstart on the 9700 Thermocycler PCR machine
(Applied Biosystems). Reactions were subjected to 35 thermal cycles
consisting of denaturation for 45 s at 94°C, annealing for 45 s at
60°C, and extension for 1 min at 72°C. With each single cell multiplex
FL-PCR, positive and negative controls were always included to
ensure that the PCR reaction mix was functional and none of the
reagents were contaminated. Positive control tubes contained 10–20
cells in 1–2 µl of PBS buffer, whereas negative control tubes contained
either 1–2 µl of PBS buffer from the last wash droplet and no cell.
Genescan analysis of DNA fingerprints
All PCR products were analysed using the ABI Prism 377 DNA
Sequencer and associated Genescan 672 software (Applied Biosystems). PCR product (0.5–1.0 µl) was mixed with 1.54 µl of formamide,
0.15 µl loading buffer and 0.31 µl of Genescan TAMRA internal
standard (Applied Biosystems). Samples were denatured at 95°C for
3 min, placed on ice and 2.5 µl loaded into the pre-formed wells of
a 6% denaturing polyacrylamide gel. Samples were electrophoresed
in 1⫻Tris/borate/EDTA (TBE) buffer for 3.5 h at 3000 V and
fragments automatically sized by Genescan software using the internal
standard and a local Southern sizing alogrithm. Fluorescent product
yield was calculated from integration of the peak area. Genescan
profiles were generated showing the PCR products as coloured peaks
DNA fingerprinting of embryonic blastomeres
Table I. Analysis of sister blastomeres from aneuploid embryos by fluorescent (FL)-PCR
Cohort no.
Embryo no. (E)
Aneuploid diagnosis
DNA fingerprints: allelic status
Cohort 1
E1
E5
B1 ⫽ monosomy 16
B2 ⫽ monosomy 16
B1 ⫽ monosomy X
E6
B1 ⫽ monosomy 13
E8
B1 ⫽ monosomy 21
B2 ⫽ monosomy 21
B1 ⫽ euploid
B2 ⫽ monosomy 16
B1 ⫽ monosomy X
and 22
B2 ⫽ monosomy 22
B1 ⫽ trisomy 16
and monosomy 21
B2 ⫽ failed FISH
B1 ⫽ trisomy 13
and monosomy 18
B3
B4
B2
B3
B2
B3
B3
B4
B3
B4
B3
E14
Cohort 2
E6
E8
Cohort 3
E6
E20
B1 ⫽ trisomy 21,
monosomy 13 and 18
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
(X,X), (13,13)a, (13,13)b, (18,18), (21,21)
(13,13)a, (13,13)b, (18,18), (21,21)
(X,X), (13,13)a, (13,13)b, (18,18)
(X,X), (13,13)a, (13,13)b, (18,18)
(X,X), (21,21)
(X,X), (13,13)a, (13,13)b, (18,18), (21,21)
(X,X), (13,13)a, (13,13)b, (18,18)*, (21)
(X,X), (13,13)a, (13,13)b, (18,18), (21)
(X,X), (13,13)a, (13,13)b, (18,18), (21,21)
(13, 13)b, (21,21)
(X), (18,18)
B4 ⫽ (13,13)a, (18,18)
B3 ⫽ (X), (13,13)*a, (13,13)*b, (18,18), (21)
B4
B2
B3
B4
B5
B2
B3
B4
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
(13,13)*a, (13,13)*b, (18,18)
(X), (13,13)*a, (13,13)*b, (18,18)*, (21,21)
(X), (13,13)*a, (13,13)*b, (18,18), (21,21)
(X), (13,13)*a, (13,13)*b, (18,18)
(X)
(X,X), (13)a, (13)b, (18), (21,21)*
(X,X), (13)a, (13)b, (18)
(X,X), (13)a, (13)b, (21,21)*
*Indicates a double dosage di-allelic pattern representative of a trisomy.
aD13S631, bD13S258.
dependent on the fluorescent dye used: TET (green), HEX (black)
and 6-FAM (blue).
Results
Development of a single cell pentaplex DNA fingerprinting
system
A single cell DNA fingerprinting system based on the amplification of two chromosome 13 markers (D13S631 and
D13S258), one chromosome 21 marker (D21S1413), one
chromosome 18 marker (D18S51) and one X chromosome
marker (DXS8377) was developed and optimized on buccal
cells from a female subject. This subject was homozygous for
D13S631 and D21S1413, and heterozygous for D13S258,
DXS8377 and D18S51 (Figure 1A). The reliability and accuracy of this fingerprinting system was assessed on 50 single
buccal cells. Since homozygous loci display a single peak
following PCR, the two possible alleles are indistinguishable.
Consequently, using these five microsatellite markers on 50
single buccal cells, only 400 of the theoretically possible 500
peaks could be analysed. Of these, 362 specific amplifications
were observed establishing a 91% reliability rate (defined as
the proportion of successful allelic amplifications). ADO and
PA were assessed on the three heterozygous loci (300 alleles).
There were 12 incidences of ADO (4%) and 18 incidences of
PA (6%) observed (calculated as the area under the smaller
peak divided by the area under the larger peak, ⬍0.5). Based
on the percentage of correct allelic amplifications (taking into
account the incidence of ADO), an overall accuracy rate for
this pentaplex DNA fingerprinting system was calculated at
95%. There was no significant difference (P ⬎ 0.05) observed
between the individual loci for either the proportion of success-
ful amplifications, total amplification failure, accuracy, ADO
or PA (Figure 2). The vast majority of the DNA fingerprints
(94%) were informative (amplification of at least three microsatellite markers, capable of identifying an individual), with
78% fully comprehensive (amplification of all five microsatellite markers).
The accuracy of this single cell DNA fingerprinting system
for aneuploidy detection was also blindly assessed on single
buccal cells collected from a male subject with Down’s
syndrome and a healthy female subject. Normal buccal cells
(diploid) (n ⫽ 15) and trisomy 21 buccal cells (n ⫽ 25) were
coded by an independent person, DNA fingerprinted, analysed
and decoded. In 22 of the known Down’s cells, a tri-allelic
pattern for the chromosome 21 marker D21S1413 was
observed. In the remaining three cells, two demonstrated ADO
and one exhibited total amplification failure of the D21S1413
locus. The fingerprint from the subject with Down’s syndrome
showed one allele each for DXS8377 (male) and D18S51
(homozygous), two alleles each for D13S258 and D13S631
(heterozygous), and three alleles for D21S1413 diagnostic of
trisomy 21 (Figure 1B). Based on the percentage of correct
tri-allelic amplifications, taking into account the incidence of
ADO, the overall accuracy for detection of the known trisomy
by this pentaplex DNA fingerprinting system with only one
chromosome 21 microsatellite marker was 92%.
DNA fingerprinting of blastomeres from aneuploid embryos
To determine if this system could also reliably and accurately
DNA fingerprint embryonic cells, blastomeres from aneuploid
embryos identified by FISH were subjected to PCR DNA
fingerprinting. Nine slow developing aneuploid embryos were
obtained, and following dissociation using pronase, a total of
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M.G.Katz, A.O.Trounson and D.S.Cram
Figure 3. PCR DNA fingerprinting of sibling embryos from cohort 1 embryos (A–E). ADO was observed at locus D18S51 for embryo E
with the presence of only one parental allele, while preferential amplification was observed at locus D18S51 for embryo B with a calculated
allelic ratio of 0.32. Fluorochromes are as follows: green (TET), blue (6-FAM) and black (HEX). Red (TAMRA) peaks indicate internal
mol. wt markers.
21 blastomeres were isolated intact and transferred into PCR
tubes (some additional blastomeres lysed or fragmented during
dissociation). From all embryos, at least two sister blastomeres
were available for PCR DNA fingerprinting. The aneuploid
status of these embryos determined by independent FISH
analysis (Table I) was unknown prior to fingerprinting analysis.
Sixteen of the 21 blastomeres produced informative DNA
fingerprints (amplification of at least three microsatelite
markers) with 13 of the 16 informative DNA fingerprints being
fully comprehensive (amplification of all five microsatellite
markers). The remaining five blastomeres produced unacceptable DNA fingerprints (amplification of two or less microsatellite markers). Due to the aneuploid status of these embryos,
calculating the number of possible allelic amplifications would
be misleading. Consequently, the total number of microsatellite
marker amplifications was employed to establish the rate of
reliability. Using these five microsatellite markers on 21
blastomeres, a total of 81 microsatellite marker amplifications
were observed out of a possible 105 (Table I), establishing a
77% reliability rate. There were five incidences of ADO (6.5%)
and five incidences of PA (6.5%). Based on the percentage of
correct microsatellite marker amplifications (taking into
account the incidence of ADO), an overall accuracy rate for
this pentaplex DNA fingerprinting system on blastomeres was
calculated at 94%.
This pentaplex DNA fingerprinting system has a high power
of discrimination, making it possible to distinguish between
different embryo cohorts. The allelic contributions of each
couple were recognizable even in the absence of parental DNA
756
fingerprints and confirmed that no DNA contamination had
occurred during the dissociation and isolation of these cells.
In addition, each individual sibling embryo in a couple’s cohort
was uniquely identifiable by its own individual DNA fingerprint
derived from the contribution of one paternal and one maternal
allele at each microsatellite locus, even with the infrequent
occurrence of amplification failure and ADO (Figure 3).
From the analysis of chromosomal numeracy, no tri-allelic
patterns were observed in any of the blastomere DNA fingerprints. However, several double dosage di-allelic patterns (with
a ratio calculated as the amount of fluorescent yield under the
peak from the first allele correlated with the amount of fluorescent yield of the second allele, 1.7–2.3:1) were observed in
some sister blastomeres, suggesting trisomy of the involved
chromosomes (Table I). For example, in three blastomeres from
E6 of cohort 3, all DNA fingerprints indicated a third copy of
chromosome 13 which would be concordant with the original
diagnosis of trisomy 13 by FISH. Single peaks or mono-allelic
patterns were also observed in some sister blastomeres, suggesting monosomy of the chromosomes involved (Table I). For
example, in two blastomeres from E8 of cohort 1, both DNA
fingerprints indicated the presence of only one copy of chromosome 21 and was concordant with monosomy 21 diagnosed by
FISH in two other sister blastomeres. However, in most cases,
it was not possible with the analysis of only one or two microsatellite markers per chromosome to differentiate between a double
dosage di-allelic pattern and the occurrence of PA or between a
mono-allelic pattern and the occurrence of ADO/parental homozygosity.
DNA fingerprinting of embryonic blastomeres
Figure 4. Octaplex single cell DNA fingerprinting of a single human female buccal cell. Fluorochromes are as follows: green (TET), blue
(6-FAM) and black (HEX). Red (TAMRA) peaks indicate internal mol. wt markers
Discussion
A reliable and accurate single cell PCR DNA fingerprinting
system based on the fluorescent amplification of five highly
polymorphic microsatellite markers was successfully
developed and used to DNA fingerprint IVF embryos. DNA
fingerprints obtained from single buccal cells and blastomeres
showed strong and accurate allelic amplification with virtually
no non-specific background interference to confound interpretation. However, the reliability of DNA fingerprints obtained
from blastomeres (as assessed by the proportion of successful
microsatellite marker amplifications) was lower than that
observed from buccal cells (as assessed by the proportion
of successful allelic amplifications). The smaller number of
blastomeres and/or differences in cell quality may account for
the higher proportion of reliable and comprehensive DNA
fingerprints derived from buccal cells compared to blastomeres.
Buccal cell samples were taken directly from the cheek lining
and single cells frozen at –80°C within an hour of isolation.
In contrast, blastomeres were dissociated from morphologically
low grade aneuploid embryos that were left to succumb on
the bench for up to 24 h. It has been reported that the reliability
of PCR amplification decreases as the embryo quality decreases
(Findlay et al., 1999). In addition, blastomeres from arrested
or fragmented embryos have also been shown to yield much
lower amplification efficiencies (Ray et al., 1998) and this is
presumably due to partial or total nuclear DNA degeneration
in some cells (Cui and Matthews, 1996). On this basis, we
would expect the pentaplex DNA fingerprinting system to
exhibit higher amplification efficiency on fresh biopsied blastomeres from PGD cases. Nevertheless, this pentaplex single
cell PCR DNA fingerprinting system performed significantly
better than other previously published systems (Sherlock et al.,
1998; Findlay et al., 1998, 1999) that have reported ADO
rates as high as 56%, PA rates as high as 25%, and reliability
rates (total number of successful amplifications) in the order
of only 75% on several cell types.
A diploidy status at any particular locus was determined by
the presence of two alleles with an expected allelic ratio of
1:1, a monosomy by the presence of only one allele and a
trisomy by the presence of three alleles with the expected ratio
of 1:1:1 (tri-allelic) or two alleles with an expected allelic ratio
of 2:1 (double dosage di-allelic). There were five incidences
of double dosage di-allelic patterns observed in the DNA
fingerprints of blastomeres in this study. Even though it is not
possible to differentiate between double dosage di-allelic and
PA, this single cell DNA fingerprinting system displayed a PA
rate of only 6.5% and therefore it was more than likely that a
third copy of the chromosome was present. There were also
five incidences of discordance between sister blastomeres
(Table I). This could either be explained by embryonic
mosaicism resulting from mitotic cell division errors, or by
the occurrence of PA at some of these loci, distorting the allelic
ratios. Overall, due to the possibility of parental homozygosity,
ADO and PA, it is essential that three or more microsatellite
markers per chromosome under analysis are included in a
single cell DNA fingerprinting system for confident aneuploidy
detection.
PCR DNA fingerprinting has many unique advantages
including confirmation of parental allelic contribution to the
embryo, the identification of extraneous DNA contamination
that could cause a misdiagnosis and detection of uniparental
disomy in an embryo if parental DNA fingerprints were
available. The newly developed pentaplex DNA fingerprinting
system has high discriminating power for identification with
a 1 in 630 probability that any two siblings will share
an identical DNA fingerprint. Using this pentaplex DNA
fingerprinting system it was possible to separate each sibling
embryo in the cohorts by their unique allelic fingerprints.
Thus, this system could potentially be used to track IVF
embryos from the time of transfer to term and identify the
viable embryo that produced the pregnancy in a multiple
transfer. It is also possible to combine mutation detection for
PGD for single gene disorders with the amplification of
microsatellite markers for aneuploidy detection (Blake et al.,
1999). Many couples presenting for PGD for single gene
disorders have either experienced the birth of an affected child
or undergone prenatal testing resulting in termination of
pregnancy or several pregnancies. Hence, these women are
usually of advanced maternal age and have a greater chance
of producing embryos with chromosomal aneuploidies. It
would be most unfortunate if a single gene disorder-free
pregnancy resulted in a chromosomally aneuploid fetus, for
example Down’s syndrome. Consequently, it would be possible to combine a pentaplex chromosome 21 specific DNA
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M.G.Katz, A.O.Trounson and D.S.Cram
fingerprinting system with mutation detection and offer simultaneous mutation and chromosome 21 screening for couples
of advanced maternal age presenting primarily for PGD of a
single gene disorder.
The incorporation of additional microsatellite markers on
clinically relevant chromosomes to this pentaplex DNA
fingerprinting system is a feasible prospect and is currently
under development. To date, we have successfully added a
further three microsatellite marker primers to produce an
octaplex DNA fingerprinting system (Figure 4). Initial studies
on 15 single buccal cells showed high reliability and accuracy
(⬎90%) and low ADO and PA (⬍10%) and produced DNA
fingerprints that were easily interpretable with virtually no
background interference or non-specific amplification. This
octaplex DNA fingerprinting system (two markers for chromosomes 13, 18, 21 and X) is therefore exceptionally robust. The
physical limits of single cell DNA fingerprinting are unknown,
although the further addition of four microsatellite markers
could be possible in view of the successful development of
this octaplex system. A more complex and comprehensive
single cell PCR DNA fingerprinting system based on compatible microsatellite marker primers for clinically relevant chromosomes (three microsatellite markers per chromosome) could
potentially offer PGD couples the possibility of a combined
detection system for single gene disorders and chromosomal
aneuploidy. The availability of more comprehensive fingerprinting systems would also allow a larger study to be
undertaken to assess the origin, nature and incidence of
chromosomal mosaicism in IVF embryos from different
patient groups.
Acknowledgements
We thank Lyn Gras and Jennifer Mansfield for performing embryo
biopsy and FISH analysis. This study was supported by a research
grant from Monash IVF Pty Ltd, Melbourne, Australia.
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Submitted on August 24, 2001; accepted on October 24, 2001
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