1. RBMOnline - Vol 4. No 3. 106–115 Reproductive BioMedicine Online; www.rbmonline.com/Article/467 on web 25 February 2002
Articles
6
Blastomere fixation techniques and risk of
misdiagnosis for preimplantation genetic
diagnosis of aneuploidy
Esther Velilla began her studies in biology in 1990 at Universitat Autonoma de Barcelona
(Spain) and was awarded her Bachelor degree in 1995. In 1998 she obtained her M.Sc.
degree in Cellular Biology in the Science Faculty at the same University. She then moved
to the Veterinary Faculty to develop her Ph.D. thesis on ‘in-vitro maturation and fertilization
studies on prepubertal and adults goat oocytes’. During the same period she began her
work in human reproduction at the clinic Instituto de reproduccion CEFER (Barcelona). In
2001 she began working at The Institute for Reproductive Medicine and Science of Saint
Barnabas (Livingston, NJ, USA) in the field of preimplantation genetic diagnosis under the
direction of Santiago Munné.
Esther Velilla, Tomas Escudero, Santiago Munné1
Institute for Reproductive Medicine and Science of Saint Barnabas Medical Centre, 101 Old Short Hills Road, Suite
501, West Orange, NJ-07052, USA
1Correspondence: Tel. +1-973–3226236, Fax. +1-973–3226235, e-mail: santi.munne@embryos.net
Introduction
Preimplantation genetic diagnosis (PGD) for gender selection
(Griffin et al., 1992; Munné et al., 1993a), aneuploidy (Munné
et al., 1993b, 1995, 1999; Verlinsky et al., 1995; Gianaroli et
al., 1999), and structural abnormalities (Munné et al., 1996,
1998; Verlinsky and Evsikov, 1999) involves the biopsy of one
or both polar bodies or the biopsy of one or two blastomeres,
fixation to glass slides, followed by fluorescence in-situ
hybridization (FISH) analysis. One of the most critical steps in
this process is cell fixation, because ideally not a single cell
should be lost, and each cell should be informative in order to
have the best possible results for each embryo.
The traditional fixation method, first developed by Tarkowski
et al. (1966) and later modified in different ways by many
authors, involves the use of acetic acid/methanol (Carnoy)
solution applied to a small drop of hypotonic solution
containing the blastomere to be fixed. This process requires
considerable practice and skill, and for this reason at least, two
other cell fixation methods have been developed, one using
Tween 20 solution (Coonen et al., 1994) and the other a
combination of Tween 20 and Carnoy solution (Dozortsev and
McGinnis, 2001). These two last methods require less skill and
seem to be less prone to cell loss during fixation (Xu et al.,
1998; Dozortsev and McGinnis, 2001). However, because the
two newer methods rely on total or partial drying of the cell, a
large nuclear diameter is hard to achieve (Hliscs et al., 1997);
large diameters are desirable because they have been inversely
correlated to signal overlaps and FISH errors (Munné et al.,
1996). To date, no study has yet analysed the FISH error rate
of the two last methods; the traditional Carnoy’s method
produces about 10–15% errors (Munné and Weier, 1996;
Munné et al., 1998, 1999).
The purpose of this study was to compare the three fixation
methods based on number of cells lost after fixation, average
rate of informative cells, rate of signal overlaps and FISH errors.
Esther Velilla
Abstract
One of the most critical steps in preimplantation genetic diagnosis (PGD) studies is the fixation required to obtain good
fluorescence in-situ hybridization (FISH) nuclear quality without losing any of the cells analysed. Different fixation
techniques have been described. The aim of this study was to compare three fixation methods (1, acetic acid/methanol; 2,
Tween 20; 3, Tween 20 and acetic acid/methanol) based on number of cells lost after fixation, average rate of informative
cells, rate of signal overlaps and FISH errors. A total of 100, 106 and 114 blastomeres were fixed using techniques 1, 2 and
3 respectively. Technique 2 gave the poorest nuclear quality with higher cytoplasm, number of overlaps and FISH errors.
Although technique 1 showed better nuclear quality in terms of greater nuclear diameter, fewer overlaps and FISH errors, it
is difficult to perform correctly. However, technique 3 shows reasonably good nuclear quality and is both easier to learn and
use for PGD studies than the others.
Keywords: cell fixation, FISH, fluorescence in-situ hybridization, preimplantation genetic diagnosis, signal overlap

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Materials and methods
Source of embryos
Embryos were obtained from patients undergoing IVF at The
Institute for Reproductive Medicine and Science at Saint
Barnabas Medical Centre (Livingston, NJ, USA) in
accordance with guidelines approved by the internal review
board. Written patient consent for donated embryo research
and for PGD was provided for all embryos.
There were two sources of embryos. The first source was
supernumerary embryos with compromised morphology
and/or development not used in embryo replacement or
cryopreservation. Severely compromised development
included embryos with fewer than four cells on day 3, or with
a normal number of cells but with >35% fragmentation or
multinucleation on day 2 of development (Alikani et al., 1999,
2000). The other source was embryos that after PGD were
found to be chromosomally abnormal.
Only those embryos with three or more cells with observed
nuclei after fixation were included in this study.
Blastomere fixation and FISH
The PGD embryos had one cell biopsied on day 3 of
development (Grifo, 1992). Those embryos considered to be
chromosomally, morphologically and developmentally normal
were replaced. Many of the embryos classified as normal by
PGD were transferred to the patient on the same day as the
analysis. The non-transferred embryos, either chromosomally
normal or abnormal, had their zonae pellucida removed by
exposure to Pronase solution (3 mg/ml, Sigma), were
transferred to Ca/Mg-free media for 10 min, and were then
fixed each cell individually as described below. Infrequently,
some chromosomally and morphologically normal embryos, in
excess of the ones transferred, were disaggregated and
analysed, because cryopreservation of biopsied embryos is
considered inefficient (Magli et al., 1999).
Only one person performed the fixation for all three methods.
This person had no previous experience in fixation, and started
the study once she felt proficient in all three methods.
Embryos were randomly assigned to one of three fixation
methods: (1) acetic acid/methanol (Tarkowski, 1966, modified
by Munné et al., 1998); (2) Tween 20HCl (Coonen et al.,
1994); or (3) Tween 20-HCL and acetic acid/methanol
(Dozortev et al., 2001).
Method 1
This method was described by Tarkowski (1966) and modified
for single blastomere fixation by Munné et al. (1998). The
whole process was performed using a stereoscope (Leica
MZ9.5, Leica Wild MZ8) with a base having a mirror that
could move from vertical to horizontal position; for this
process, the mirror was at a 30° angle. The blastomere was
exposed to hypotonic solution [0.075 mol/l KCl supplemented
with 0.6% BSA (w/v)] for 2 min. Then the blastomere was
placed onto the microscope slide in a small hypotonic drop
(1–2 μl) using a 0.16 mm inner-diameter microneedle. After
that, 1 drop of methanol:acetic acid (3:1) fixative was added
over the blastomere. Often, the blastomere moved after the
first fixative drop, but was easily detectable if the slide was
clean of dust because the position of the stereoscope mirror
produced a three-dimensional effect that allowed localization
of objects protruding from the glass slide (Figure 1).
Blastomeres before cytoplasm breakdown appear refringent
and with smooth circular edges. After the blastomere settled,
but before the cytoplasm burst, a second drop of hypotonic
was added. While the second drop was drying, humidity was
added by breathing over the blastomere to facilitate cytoplasm
Table 1. Analysable cells depending on different fixation methods and studies.
Method Fixed Cells Found No Nucleated Analysable (%)
lost (%) nuclei
Xu et al. (1998)
1 121 26 (21.5)a 95 ND ND 76 (62.8)c
2 131 8 (6.1)b 123 ND ND 60 (45.8)d
Dozortsev
et al. (2001)
1 16 2 (13.0) ND ND ND 13 (81.0)
2 16 1 (16.0) ND ND ND 14 (87.0)
3 18 0 (0.0) ND ND ND 18 (100.0)
Present study
1 110 4 (3.6) 106 15 91 89 (84.0)e
2 106 3 (2.8) 103 22 81 71 (68.9)f
3 114 3 (2.6) 111 10 101 92 (82.9)
a versus b, c versus d: P < 0.001; e versus f: P < 0.025.
ND = not determined

3. Articles - Fixation techniques for PGD of aneuploidy - E Velilla et al.
Figure 1. An expanded blastomere before cytoplasm burst,
showing a three-dimensional effect.
Figure 2. Blastomeres were analysed by FISH with probes for
chromosomes 13 (red), 16 (pale blue), 18 (dark blue), 21
(green), 22 (gold). Blastomeres (nucleus diameter = 62 μm)
were fixed using method 1.
Figure 3. Blastomere (nucleus diameter = 17 μm) shows
overlaps between chromosomes 16 and 13, 16 and 21, 21 and
22, and 22 and 18. Blastomeres were analysed by FISH with
probes for chromosomes 13 (red), 16 (pale blue), 18 (dark
blue), 21 (green), 22 (gold) and fixed using method 2.
Figure 4. A binucleated blastomere (nucleus diameter = 20
and 10 μm) shows overlaps in the top nucleus between
chromosomes 13 and 18, and 21 and 22. In addition excess
cytoplasm can be seen. Blastomeres were analysed by FISH
with probes for chromosomes 13 (red), 16 (pale blue), 18
(dark blue), 21 (green), 22 (gold) and fixed using method 2.
Table 2. Number of signal overlaps and FISH errors in the present study.
Method Analysablea Diameter Nuclei with No. total FISH (%)
±SD (μm) errors (%) overlaps
1 89 58.5 ±20.7 12 (13.5)a 16d 9 (10.1)g
2 71 30.8 ±12.9 41 (57.7)b 88e 21 (29.6)h
3 92 46.0 ±18.7 36 (39.1)c 45f 16 (17.4)
a versus b, a versus c, d versus e, d versus f: P <0.001. g versus h: P < 0.005.
aSee differences between techniques in Table 1 for “Analysable”. 8

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Figure 5. Nucleus (diameter = 21 μm) with overlaps between
chromosomes 16, 18 and 21 and between 16 and 18, with
considerable cytoplasm. Blastomeres were analysed by FISH
with probes for chromosomes 13 (red), 16 (pale blue), 18 (dark
blue), 21 (green), 22 (gold) and fixed using method 2.
membrane breakdown. Once cytoplasm breakdown occurred,
the nucleus could be seen under the stereoscope for a few
seconds; but not once the fixative dried completely.
Method 2
This method was first described by Coonen et al. (1994). The
blastomere was placed (5–10 s) into hypotonic solution (1%
sodium citrate in 0.2 mg/ml BSA) and transferred onto a Petri
dish containing Tween 20 solution for 2 min. After that, it was
transferred onto a glass slide within approximately 3 μl of
Tween 20 solution. Tween 20 solution was continuously added
to the drop until cell membrane breakdown under stereoscope
observation. After membrane breakdown, the slide was
allowed to dry completely and the slide was treated with 1%
pepsin to digest the remaining cytoplasm.
Figure 6. Another binucleated blastomere (nucleus diameter =
24 and 27 μm) with overlaps between chromosomes 13, 16
and 18 and between 13 and 16 in the top nucleus, and overlaps
between chromosomes 13, 16 and 21 and between 13 and 16
in the bottom nucleus. Blastomeres were analysed by FISH
with probes for chromosomes 13 (red), 16 (pale blue), 18 (dark
blue), 21 (green), 22 (gold) and fixed using method 2.
Method 3
This method was described by Dozortsev and McGinnis
(2001). The blastomere was placed (5–10 s) in hypotonic
solution (1% sodium citrate in 0.2 mg/ml BSA) and then
washed in Tween 20 solution (1% of Tween 20 in 0.01 N HCL,
1% in 0.01 N HCL) for 40 s. After that, it was placed onto a
glass slide with 3–4 μl of Tween 20 under a stereoscope
microscope and allowed to dry completely. In order to remove
the remaining cytoplasm, several drops of methanol: acetic
(3:1) fixative were added.
For all three methods, the temperature and humidity conditions
were the same (22ºC and 30–45% respectively), based on
previous observations that nuclear diameters are a function of
temperature and humidity (Spurbeck et al., 1996).
FISH analysis was performed using probes for chromosomes
X, Y, 13, 15, 16, 17, 18, 21 and 22 following the previously
published protocol (Munné et al., 1998), except that instead of
a locus-specific probe for chromosome 14, a centromeric one
was used for chromosome 17, also labelled in Spectrum
Orange (Vysis).
Data evaluation and scoring criteria
Classification of chromosomal abnormalities in cleavage-stage
embryos, usually with 2–12 cells, requires scoring criteria
based on the analysis of as many cells as possible to
differentiate mosaicism (30% of cleavage-stage embryos;
Munné et al., 1995) from FISH errors (10% of single cells
analysed; Munné et al., 1998). In this study, the previously
described criteria distinguishing mosaics from FISH errors
were used without modification (Munné et al., 1994; Munné
and Cohen, 1998). Previous criteria were also used to
differentiate close signals from split signals when analysing
Figure 7.Anucleus with split signals for chromosomes 21 and
22. Blastomeres (nucleus diameter = 85 μm) were fixed using
method 1 and analysed by FISH with probes for chromosomes
13 (red), 16 (pale blue), 18 (dark blue), 21 (green), 22 (gold).

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Table 3. Example of 20 embryos with FISH errors.
Embryo No. cells 13 16 18 21 22 XY 15 17 Embryo diagnosis
1 1 2 2 2 2 2 xy 1a 2 Normal
2 2 2 2 2 2 xy 2 2
1 2 2 2 2 2 xy 2 1a
2 1 1a 1a 2 2 2 xy 2 2 Normal
7 2 2 2 2 2 xy 2 2
3 6 2 2 2 2 2 XY 2 2 Mosaic 2N/4N
1 3a 4 4 4 4 XXYY4 4
1 2 2 2 nr 1a XY 2 2
4 2 3 3 3 3 3 xxx 3 3 Triploid
3 3 2a 3 3 3 xxx 3 3
5 1 2 nr 2 2 2 xy 2 3a Normal
3 2 2 2 2 2 xy 2 2
6 3 2 2 3 2 2 xx nr 1 Trisomy 18, and
1 2 3a 4 2 2 xx nr 3 Aneuploid mosaic (18,17)b
1 2 2 2 2 2 xx 2 2
7 3 2 2 2 2 2 xx 2 2 Normal
1 2 nr 2 2 2 xx 1a 2
8 1 2 2 2 3 2 xy 0a 1a Trisomy 21
2 2 2 2 3 2 xy 2 2
9 1 2 2 2 2 2a xx 2 2 Trisomy 22
3 2 2 2 2 3 xx 2 2
10 3 2 2 2 2 3 xx 2 2 Trisomy 22
1 2 nr 2 2 2a xx 2 2
11 1 2 2 nr 3 2 xx 1 2 Monosomy 15, and
4 2 2 2 2 2 xx 1 2 Aneuploid mosaic (21)b
1 1a 2 2 1 2 xx 1 1a
1 2 3a 2 2 2 xx 2a 2
12 1 2 nr 2 2 2 xy 1a 2 Normal
5 2 2 2 2 2 xy 2 2
13 1 2 3a 2 2 2 xy 2 2 Normal
3 2 2 2 2 2 xy 2 2
14 1 2 1a 2 2 1 XY 2 2 Monosomy 22
3 2 2 2 2 1 XY 2 2
15 2 2 nr 2 2 2 xy 2 2 Trisomy 16
4 2 3 2 2 2 xy 1a 2
5 2 3 2 2 2 xy 2 2
1 2 3 2 2 2 xy 2 2
16 3 2 2 2 2 1 xy 3 2 Monosomy 22
1 2 2 2 2 1 xy 2a 2
1 2 1a 2 2 1 xy 3 2
17 1 4 4 4 4 4 xxyy 4 4 Mosaic 2N/4N
7 2 2 2 2 2 xy 2 2
1 3a 4 4 4 4 xxyy 4 4
18 2 2 2 2 2 2 xx 2 2 Normal
1 2 nr 2 2 nr xx 2 2
1 2 nr 2 nr 2 xx 1a 2
19 1 3a nr 2 2 2 XX 2 2 Normal
6 2 2 2 2 2 XY 2 2
1 2 2 2 2 2 XY 1a 2
20 1 1 1 1 2a 1 xx 2 2 Complex mosaic
3 2 2 2 2 nr 4x 4 4
aConsidered errors.
bAneuploid mosaic for the chromosomes in brackets.
nr = no result

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single cells in PGD cases (Munné and Weier, 1996). Embryos
were classified as normal, aneuploid, polyploid, haploid
and/or mosaic according to guidelines described elsewhere
(Munné et al., 1995; Munné and Cohen, 1998).
Parameters to be evaluated
Cells lost during fixation
These were cells that in method 1 were lost after adding the
fixative, and where the refringent protrusion was not observed.
For methods 2 and 3, the blastomere seldom moved but the cell
could burst, and these cells were also counted as lost.
Analysable cells
A cell was considered analysable when it had at least five
informative chromosomes out of the eight analysed.
Nuclear diameter
This was measured in microns under phase contrast
observation before FISH analysis.
Nuclei with overlaps
Overlaps between the two-homologue chromosomes cannot
be precisely quantified in a single cell and are one source of
misdiagnosis (see below, FISH errors). However, any other
overlap between non-homologue chromosomes can be easily
measured if these chromosomes were labelled in different
colours, as is the case here. Thus the parameter ”nuclei with
overlaps” indicated the presence of overlaps between non-homologous
chromosomes in a specific nucleus.
Total number of overlaps
This is the measure of the total number of overlaps between
non-homologous chromosomes in a specific nucleus.
FISH errors
To differentiate between FISH errors and mosaicism in this
study, previously described criteria were used (Munné et al.,
1994).
AChi-square test using the algorithm GENSTAT (1988 version)
was used to evaluate statistical differences between proportions.
The significance chosen for the test was P < 0.025.
Results
The three fixation techniques were evaluated according to the
following parameters: cells lost during fixation, nucleated
cells with no result, analysable cells, nuclear diameter, nuclei
with overlaps, and total number of overlaps, and FISH errors,
as defined in the materials and methods (Tables 1 and 2).
These parameters were also compared with other previously
published studies (Table 1), although FISH errors and
overlaps had not been previously evaluated in relation to
blastomere fixation methods.
The present results indicate that similar rates of lost cells were
observed for the three methods evaluated (Table 1), ranging
from 2.8 to 3.6%. This contrasts markedly with data published
by others reporting lost cells ranging from 0 to 21.5% (Table
1). The fraction of nucleated cells that was analysable after
FISH was significantly higher for method 1 than for method 2,
in this study as well as in that of Xu et al. (1998) (Table 1).
As far as is known, no other study has yet evaluated the
number of signal overlaps and FISH errors according to
fixation technique. The present results indicate a significant
differences in nuclear diameter after fixation, with an average
of 58 μm for method 1, 31 for method 2, and 46 for method 3
(P < 0.001) (Table 2), which translates to higher rates of
nuclei with signal overlaps (from 14% for method 1 to up to
58% for method 2, P < 0.001), total number of overlaps, and
FISH errors (from 10% for method 1 to 30% for method 2, P
< 0.005) (Table 2).
Examples of embryos with FISH errors are shown in Table 3
and examples of overlaps in Figures 3–7. The correlation
between overlaps and FISH errors and small nuclear diameter
was also observed irrespective of the type of fixation. For
instance, taking all embryos together and grouping them by
diameters, those cells of <30 μm in diameter had more
overlaps and FISH errors than those cells of >60 μm in
diameter (P < 0.005) (Table 4).
Discussion
The two most important aspects of cell fixation are to ensure
that each single cell is fixed and that the fixed nucleus is
informative. One of the steps in method 1 involves the mixture
of fixative with the drop of hypotonic solution containing the
blastomere. This act produces turbulence, during which the
cell may be lost, and the risk is about 3% in expert hands; but
could be higher for technicians using method 1 only
occasionally (Xu et al., 1998; Dozortsev and McGinnis,
2001). In contrast, methods 2 and 3 overcome the turbulence
step and are easier to learn, but they have other problems.
For instance, the presence of cytoplasm interferes with probe
binding to the nucleus especially with locus specific probes.
These probes are longer than the repetitive ones and easily
attach to the cytoplasm debris, increasing the background
signal and limiting the attachment of the probes to their target.
Moreover, cytoplasm is refringent by itself, masking the
signals. In short, cytoplasm can increase misdiagnosis or
render the nucleus non-informative (Figures 4 and 6). This is
a considerable problem in method 2, although modifications
made by Xu et al. (1998) to this method reduced the number
Table 4. Nuclear diameter in relation to overlaps and FISH
errors.
Diameter Analysable Average No. nuclei Total no. No. FISH
(μm) diameter with overlaps errors
(μm) overlaps (%) (%)
<30 55 21.63 28 (50.9)a 62 14 (25.5)c
30–60 139 42.97 45 (32.4) 71 29 (20.9)
>60 58 78.72 16(27.6)b 16 3 (5.2)d
a versus b: P < 0.025, c versus d: P < 0.005.

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of nuclei with cytoplasm to <5%. Removal of cytoplasm by
pepsin may also be detrimental because overexposure to
pepsin may degrade the DNA (Xu et al., 1998; Dozortsev and
McGinnis, 2001).
Another source of background interference found in this study
was poly-L-lysine, used in method 2 to avoid nuclear loss in
attaching the cell to the slide. This substance produces a mask
along the slide, which interferes with the analysis of the
signals. In methods 1 and 3, the cells are fixed to the slide by
methanol:acetic acid instead of poly-L-lysine. In consequence,
these fixation methods show clearer nuclei for FISH analysis
(Figure 7).
Another factor in PGD analysis is the nuclei diameter. Small
nuclei preserve their three-dimensional structure, and as a
consequence the signals lie on different focus planes, making
the analysis more prone to misdiagnosis (Figures 3 and 6). In
addition, two signals close to each other or overlapping could
be misdiagnosed as a single signal (Munné et al., 1996).
Methods 1 and 3 provide flat nuclei with all the signals in the
same focus plane, thereby reducing the frequency of overlaps.
It was observed in this study that the ideal diameter is 60 μm
and above (Figure 2). However, nuclei >80 μm show
excessively decondensed chromatin, as the signals are spread
more widely and are weaker than regular sized nuclei.
Sometimes they are almost imperceptible, which leads to
misdiagnosis of false nullisomies, monosomies or disomies. It
is important to mention that nuclear diameter is not only
dependent on the fixation technique, but also on temperature
and humidity (Spurbeck et al., 1996; Hliscs et al., 1997).
Previous observations that nuclear diameter is linked to FISH
errors (Munné et al., 1996) were confirmed.
In this respect, method 1, which produced the largest nuclei,
produced the fewest number of signal overlaps and the fewest
errors after FISH (Figure 2). In contrast, method 2 (Coonen et
al., 1994) produced the highest number of overlaps and three
times more FISH errors than method 1 (Figures 2–5).
Method 2 has been previously used in PGD for gender
determination (Harper et al., 1994, 1995; Coonen et al., 1996;
Soussis et al., 1996) and PGD diagnosis on translocation
carriers (VanAsche et al., 1999; Coonen et al., 2000; Iwarsson
et al., 2000; Scriven et al., 2001) using probes for one or two
chromosome pairs. The probes used for gender determination
bind to repetitive sequences in the satellite regions (centromere
X and 18, or most of the q arm of chromosome Y), producing
a large signal, usually even under the worst fixation
conditions. In addition, misdiagnosis of gender using FISH can
be improved because the mere presence of different colours
(irrespective of the number of signals) can identify the sex,
even though signal overlaps could still misdiagnose the ploidy.
For translocation carriers, only the chromosomes involved in
the translocation are diagnosed, usually involving only a
centromeric probe and a pair of distal probes instead of the five
probes simultaneously used for the first aneuploidy
hybridization. In addition, for PGD of translocations, either
telomeric or home-made locus-specific (LSI) probes are used,
while the LSI probes used for PGD of aneuploidy (for
chromosomes 13, 21, and 22) are different. The three fixation
methods were evaluated for their use in PGD of aneuploidy
and method 2 may still be used for gender determination based
on FISH errors or translocation PGD cases, but it is certainly
not recommended for PGD of aneuploidy.
The FISH error rate observed for method 1, 10%, was
comparable to that found in previous studies using the same
fixation method (Munné et al., 1996). Although in this study
PGD was not performed using the three techniques, if the error
rates observed here for methods 2 and 3 are maintained when
performing PGD, as for method 1, a 30% error rate for method
3 would render it useless for PGD of aneuploidy.
In conclusion, method 1 gives the best nuclear quality for PGD
analysis, even though it is the hardest to accomplish. Method
3 gives reasonably good nuclear quality, and is easy to learn.
Method 2 is also easy to learn, but under the conditions of this
study, the quality of the results was not sufficiently good for
PGD of aneuploidy.
References
Alikani M, Cohen J, Tomkin G et al. 1999 Human embryo
fragmentation in vitro and its implications for pregnancy
implantation. Fertility and Sterility 71, 836–842.
Alikani M, Calderon G, Tomkin G et al. 2000 Cleavage anomalies in
human embryos and survival after prolonged culture in-vitro.
Human Reproduction 15, 2634–2643.
Coonen E, Dumoulin JCM, Ramackers FCS, Hopman AHN 1994
Optimal preparation of preimplantation embryo interphase nuclei
for analysis by fluorescence in-situ hybridization. Human
Reproduction 9, 533–537.
Coonen E, Dumoulin JCM, Dreesen JCFM et al. 1996 Clinical
application of FISH for sex determination of embryos in
preimplantation diagnosis of X-linked diseases. Journal of
Assisted Reproduction and Genetics 13, 133–136.
Coonen E, Martini E, Dumoulin JCM et al. 2000 Preimplantation
genetic diagnosis of a reciprocal translocation
t(3;11)(q27.3;q24.3) in siblings. Molecular Human Reproduction
6, 199–206.
Dozortsev DI, McGinnis KT 2001 An improved fixation technique
for fluorescence in-situ hybridization for preimplantation genetic
diagnosis. Fertility and Sterility 76, 186–188.
Gianaroli L, Magli C, Ferraretti AP, Munné S 1999 Preimplantation
diagnosis for aneuploidies in patients undergoing in vitro
fertilization with a poor prognosis: identification of the categories
for which it should be proposed. Fertility and Sterility 72,
837–844.
Griffin DK, Wilton LJ, Handyside AH et al. 1992 Dual fluorescent
in-situ hybridization for simultaneous detection of X and Y
chromosome-specific probes for the sexing of human
preimplantation embryonic nuclei. Human Genetics 89, 18–22.
Grifo JA 1992 Preconception and preimplantation genetic diagnosis:
polar body, blastomere, and trophectoderm biopsy. In Cohen J,
Malter HE, Talansky BE, Grifo JA (eds) Micromanipulation of
Gametes and Embryos. Raven Press, NY, pp. 223–249.
Harper JC, Coonen E, Ramaekers FCS et al. 1994 Identification of
the sex of human preimplantation embryos in two hours using an
improved spreading method and fluorescent in-situ hybridization
(FISH) using directly labelled probes. Human Reproduction 9,
721–724.
Harper JC, Drawson K, Delhanty JDA, Winston RML 1995 The use
of fluorescent in-situ hybridization (FISH) for the analysis of in-vitro
fertilization embryos: a diagnostic tool for the infertile
couple. Human Reproduction 10, 3255–3258.
Hliscs R, Muhlig P, Claussen U 1997 the spreading of metaphases is
a slow process, which leads to a stretching of chromosomes.
Cytogenetics and Cell Genetics 76, 167–171.

8. 13
Articles - Fixation techniques for PGD of aneuploidy - E Velilla et al.
Iwarsson E, Malmgren H, Inzunza J et al. 2000 Highly abnormal
cleavage divisions in preimplantation embryos from translocation
carriers. Prenatal Diagnosis 20, 1038–1047.
Magli MC, Gianaroli L, Fortini D et al. 1999 Impact of blastomere
biopsy and cryopreservation techniques on human embryo
viability. Human Reproduction 14, 770–773.
Munné S, Cohen J 1998 Chromosome abnormalities in human
embryos. Human Reproduction Update 4, 842–855.
Munné S, Weier U 1996 Simultaneous enumeration of chromosomes
13, 18, 21, X and Y in interphase cells for preimplantation
genetic diagnosis of aneuploidy. Cytogenetics and Cell Genetics
75, 263–270.
Munné S, Weier HUG, Stein J et al. 1993a A fast and efficient
method for simultaneous X and Y in-situ hybridization of human
blastomeres. Journal of Assisted Reproduction and Genetics 10,
82–90.
Munné S, Lee A, Rosenwaks Z et al. 1993b Diagnosis of major
chromosome aneuploidies in human preimplantation embryos.
Human Reproduction 8, 2185–2191.
Munné S, Grifo J, Cohen J, Weier HUG 1994 Chromosome
abnormalities in arrested human preimplantation embryos: a
multiple probe fluorescence in-situ hybridization (FISH) study.
American Journal of Human Genetics 55, 150–159.
Munné S, Alikani M, Tomkin G et al. 1995 Embryo morphology,
developmental rates and maternal age are correlated with
chromosome abnormalities. Fertility and Sterility 64, 382–391.
Munné S, Dailey T, Finkelstein M, Weier HUG 1996 Reduction in
signal overlap results in increased fish efficiency: implications
for preimplantation genetic diagnosis Journal of Assisted
Reproduction and Genetics 13, 149–156.
Munné S, Magli C, Bahçe M et al. 1998 Preimplantation diagnosis
of the aneuploidies most commonly found in spontaneous
abortions and live births: XY, 13, 14, 15, 16, 18, 21, 22. Prenatal
Diagnosis 18, 1459–1466.
Munné S, Magli C, Cohen J et al. 1999 Positive outcome after
preimplantation diagnosis of aneuploidy in human embryos.
Human Reproduction 14, 2191–2199.
Scriven PN, Flinter FA, Braude PR, Ogilvie CM 2001 Robertsonian
translocations: reproductive risks and indications for
preimplantation genetic diagnosis. Human Reproduction 16,
2267–2273.
Soussis I, Harper JC, Kontogianni E et al. 1996 Pregnancies
resulting from embryo biopsied for preimplantation diagnosis of
genetic disease: biochemical and ultrasonic studies in the first
trimester of pregnancy. Journal of Assisted Reproduction and
Genetics 13, 254–258.
Spurbeck JL, Zinsmeister AR, Meyer KJ, Jalal SM 1996 Dynamics
of chromosome spreading. American Journal of Medical Genetics
61, 387–393.
Tarkowski AK 1966 An air-drying method for chromosome
preparation mouse eggs. Cytogenetics 5, 394–400.
Van Asche E, Staessen C, Vegetti W et al. 1999 Preimplantation
genetic diagnosis and sperm analysis by fluorescence in-situ
hybridization for the most common reciprocal translocation
t(11;22). Molecular Human Reproduction 5, 682–690.
Verlinsky Y, Evsikov S 1999 Karyotyping of human oocytes by
chromosomal analysis of the second polar body. Molecular and
Human Reproduction 5, 89–95.
Verlinsky Y, Cieslak J, Frieidine M et al. 1995 Pregnancies following
pre-conception diagnosis of common aneuploidies by
fluorescence in-situ hybridization. Human Reproduction 10,
1923–1927.
Xu K, Huang T, Tiezheng L et al. 1998 Improve the fixation method
for preimplantation genetic diagnosis for fluorescent in-situ
hydridization. Journal of Assisted Reproduction and Genetics 13,
570–574.