Human Reproduction Vol.17, No.12 pp. 3201–3207, 2002
Possible interchromosomal effect in embryos generated by
gametes from translocation carriers
L.Gianaroli1,3, M.C.Magli1, A.P.Ferraretti1, S.Munné2, B.Balicchia1, T.Escudero2 and
A.Crippa1
1S.I.S.ME.R., Reproductive Medicine Unit, Via Mazzini 12, 40138 Bologna, Italy and 2St.Barnabas Medical Center, West Orange,
NJ, USA
3To
whom correspondence should be addressed. E-mail: [email protected]
BACKGROUND: The incidence of abnormal pregnancies in carriers of balanced translocations depends strictly
on the chromosomes involved in the translocations. The aim of this study was to verify whether conventional
aneuploidy screening could be advantageously combined with preimplantation genetic diagnosis (PGD) for
translocations. METHODS: Twenty-eight carriers of Robertsonian and reciprocal translocations underwent 43
PGD cycles; specific probes were used to screen the translocation in 172 embryos generated by 35 cycles; most of
these embryos were also screened for chromosomes 13, 16, 18, 21, 22 (n ⍧ 166), XY (n ⍧ 107), 1 (n ⍧ 17) and 15
(n ⍧ 88). For the remaining eight cycles (carriers of reciprocal translocations) only the chromosomes involved in
common aneuploidy screening were investigated on the 40 embryos generated in vitro. RESULTS: In Robertsonian
translocations, the proportion of embryos with abnormalities due to the translocation was 21%, common aneuploidies
contributed 31% of total abnormalities, whereas the remaining 36% of embryos had abnormalities due to both
types of chromosome. For reciprocal translocations, the chromosomes involved in the translocation were responsible
for 65% of total abnormalities; only 6% of the embryos were abnormal for common aneuploidies and 16% carried
abnormalities due to both the chromosomes involved in the translocation and those not related to the translocation.
CONCLUSIONS: An interchromosomal effect seems to play a role in the case of Robertsonian translocations,
where the relevant contribution of aneuploidy exposes the couple to an additional risk of abnormal pregnancy.
Key words: aneuploidy/interchromosomal effect/preimplantation genetic diagnosis/translocations
Introduction
Chromosomal translocations originate from chromosome partition followed by reunion in a different configuration. Clinical
effects are very severe in the case of unbalanced rearrangements
due to the altered amount of chromosomal material. Conversely,
no loss or gain of genetic material occurs in balanced rearrangements and no consequences for the phenotype occur unless
the breakpoints affect a functional gene. However, the production of a high proportion of gametes with an unbalanced
genetic complement is strictly related to translocations and
causes an increased risk of spontaneous abortions and abnormal
offspring.
A linkage has been reported between infertility and chromosomal translocations which occur at a rate of 0.6% among
infertile couples compared with an incidence of 0.2% in the
general population (Hook and Hamerton, 1977). This figure is
significantly higher in couples with multiple IVF failures or
recurrent miscarriages (3.2 and 9.2% respectively) (Stern
et al., 1999).
In recent years, preimplantation genetic diagnosis (PGD)
has been offered to carriers of balanced translocations with
© European Society of Human Reproduction and Embryology
the aim of improving the clinical outcome by selecting for
transfer those embryos with a normal or balanced chromosomal
complement. Different approaches have been proposed.
Chromosome painting probes applied to first polar body and
second polar body after metaphase conversion have been used
for the diagnosis of translocations of maternal origin (Munné
et al., 1998a; Verlinsky and Evsikov, 1999). For the analysis
of interphase chromosomes in blastomeres, specific spanning
probes can be developed for both Robertsonian (Rob.T) and
reciprocal translocations (Rec.T); however, their preparation
is expensive and time consuming (Munné et al., 1998b;
Pierce et al., 1998). More recently, the simultaneous use of
commercially available telomeric probes in combination with
centromeric probes has provided an alternative approach for
the detection of the abnormal segregation in Rec.T (Scriven
et al., 1998; Munné et al., 2000). Similarly, enumerator
α-satellite or locus-specific probes enable the detection of
aneuploid embryos in the case of Rob.T (Conn et al., 1998).
The aim of the present study was to verify whether conventional aneuploidy screening, as normally performed to increase
the take-home baby rate after IVF (Gianaroli et al., 1999a;
Munné et al., 1999), could be combined with PGD for
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L.Gianaroli et al.
Table I. Patients’ history
No. patients
No. patients with previous abortions (%)
No. abortions (mean ⫾ SD)
Spontaneous
After ICSI
Rob.T carriers
Rec.T carriers
Total
15
3
6 (2.0 ⫾ 1.5)
4
2
13
6
17 (2.8 ⫾ 1.7)
17
0
28
9 (32)
23 (2.5 ⫾ 1.7)
21
2
Rob.T ⫽ Robertsonian translocation; Rec.T ⫽ reciprocal translocation.
translocations. The analysis of the results was especially
focused towards the detection of an interchromosomal effect.
The possibility that a chromosomal rearrangement could affect
the behaviour at meiosis of other chromosomes has always
been a controversial issue in human genetics. The use of
several probes specific for different positions along the chromosomes has shown pairing defects in translocation heterozygotes
not only in the chromosomes affected by the translocation but
in other chromosomes as well. The resulting interchromosomal
effect could be a consequence of small breaks in the formation
of homologue synapses or could depend on defects in the
alignment or chromosome condensation (McKim and
Hawley, 1995).
The final goal was the development of a system, based on
multiple round hybridization, that could maximize the chances
of pregnancy in carriers of translocations by avoiding the
transfer of aneuploid embryos.
Materials and methods
Patients
Twenty-eight patients, carriers of a balanced translocation, attended
the S.I.S.ME.R. Reproductive Medicine Unit to undergo IVF treatment
cycles for infertility in combination with the screening of aneuploidy
on the in-vitro-generated embryos. Indication for PGD was the
presence of a Robertsonian or a reciprocal translocation in one of the
partners. Fifteen couples entered the Rob.T group and 13 the Rec.T
group. Nine of the 28 couples had a history of repeated abortions:
three with Rob.T and six with Rec.T which accounted for a total of
23 abortion events (Table I).
Five couples with a Rec.T did not have their embryos screened
for the translocation due to the impossibility of having the correct
spanning probes available in a short time; therefore they decided to
perform eight conventional PGD cycles with the screening for the
chromosomes XY, 1, 13, 15, 16, 18, 21 and 22.
Between September 1996 and May 2001, 43 PGD cycles for
translocation were performed. Induction of multiple follicular growth
was accomplished by the administration of exogenous gonadotrophins
after a long desensitization protocol with long-acting GnRH analogues
(Ferraretti et al., 1996). Ovulation was induced by hCG administration;
34–36 h later, oocytes were transvaginally collected via ultrasound
guidance, and cultured in Earle’s balanced salt solution (EBSS)
supplemented with 10% heat-inactivated maternal serum (MS), in a
5% CO2 moist gas atmosphere at 37°C. Oocyte insemination was
performed by ICSI or conventional IVF depending on semen
sample indices.
Assessment of fertilization and embryo development
At 14–18 h after insemination, oocytes were checked for the presence
of pronuclei and polar bodies. Regularly fertilized oocytes were
3202
cultured individually in EBSS 15% MS and scored at 40, 62 and
88 h post-insemination. Number and morphology of nuclei and
blastomeres, and percentage of fragmentation were recorded. After
embryo biopsy, embryos were transferred to blastocyst growing
medium (Vitrolife Sweden AB, Gothenburg, Sweden). Transfers
were performed into the uterine cavity; only embryos diagnosed
as chromosomally normal or balanced were transferred. Clinical
pregnancies were assessed by ultrasound analysis as the presence of
a gestational sac with fetal heartbeat. The implantation rate (IR)
defined the ratio between the number of gestational sacs with
fetal heartbeat and the total number of embryos transferred. The
implantation rate per pregnant patient (IRPP) was calculated as the
number of gestational sacs with fetal heartbeat divided by the total
number of embryos transferred in the pregnant patients.
Embryo biopsy
Day 3 embryos with 艌4 regular blastomeres and a percentage of
fragmentation 艋50% were selected to undergo embryo biopsy for
fluorescent in-situ hybridization (FISH) analysis. Embryos were
manipulated individually in HEPES-buffered medium overlaid with
36 h pre-equilibrated mineral oil. One blastomere was aspirated by
using a polished glass needle, which was introduced into the perivitelline space after opening a breach of ~20 µm in the zona pellucida
with acidic Tyrode’s solution. The biopsied cell was transferred to
hypotonic solution, fixed in methanol:acetic acid on a glass slide, and
dehydrated in increasing ethanol series.
Fluorescent probes and FISH
Table II represents the scheme of the study, and Table III the
translocations and corresponding probes. In all, 212 embryos were
analysed by two-, three- or four-round FISH. In the first round (or
first two rounds in the case of Rec.T), the chromosomes involved in
the translocation were tested; in the following round, chromosomes
13, 16, 18, 21 and 22 were tested, followed by the last round involving
the probes for chromosomes XY, 1 and 15. A total of 111 embryos
was generated by the 23 cycles of the Rob.T carriers and screened
for the chromosomes involved in the translocations as well as, in 105
of them, for chromosomes 13, 16, 18, 21 and 22; 95 were also
screened for chromosomes XY, 76 for chromosome 15 and 10 for
chromosome 1. A total of 101 embryos was analysed in the case of
Rec.T; five of the patients had 40 embryos, generated by eight cycles,
analysed only for chromosomes 13, 16, 18, 21, 22 in the first round
FISH, and chromosomes XY, 1 and 15 in the second round; whereas
61 embryos from the remaining 12 cycles were diagnosed for the
chromosomes involved in the translocation (first round FISH) as well
as for the chromosomes 13, 16, 18, 21 and 22; in addition, chromosomes XY, 1 and 15 were investigated in 12 embryos and chromosome
1 in seven embryos.
For Robertsonian translocations, enumerator probes for interphase
nuclei were used: in cases of t(13;14) and t(14;21) the Multivysion
PB panel from Vysis (Vysis Inc., Downers Grove, IL, USA) was used
Interchromosomal effect and translocation carriers
Table II. Number of embryos analysed for each chromosome panel
Total no.
embryos
analysed
Rob.T
Rec.T
111
101
For the chromosomes involved
in the translocation
For chromosomes involved in conventional
aneuploidy screening
111
0 (8 cycles)
61 (12 cycles)
13, 16, 18, 21, 22
XY
1
15
105
40
61
95
40
12
10
40
7
76
40
12
Rob.T ⫽ Robertsonian translocation; Rec.T ⫽ reciprocal translocation.
Table III. Robertsonian (Rob.T) and reciprocal (Rec.T) translocations included in the study with the
corresponding probes used for the diagnosis of the chromosomes involved in the translocation
No. patients
Rob.T
9
3
2
1
Rec.T
1
1
1
1
1
1
1
1
1
1
1
1
1
Translocation
No. cycles
Probes
45,XY,der(13;14)(q10;q10)
45,XX,der(13;14)(q10;q10)
45,XY,der(14;21)(q10;q10)
45,XY,der(13;21)(q10;q10)
16
4
2
1
LSI
LSI
LSI
LSI
46,XY,t(12;13)(p13;q32)
46,XY,t(4;17)(p16.1;q11.2)
46,XY,t(4;15)(q21;q15)
46,XY,t(18;20)(q11.2;p11.1)
46,XY,t(2;8)(p16;q21)
46,XY,t(1;11)(p12;q13)
46,XY,t(1;22)(p10;q10)
46,XY,t(3;12)(q28;q12)
46,XX,t(1;3)(q32.1;q27.3)
46,XX,t(4;20)(p10;p10)
46,XX,t(11;22)(q25;q13)
46,XY,t(14;20)(p11.2;q11.2)
46,XY,t(16;17)(q24;q22)
1
1
1
2
1
1
1
1
1
1
1
2
1
in the first round for the detection of chromosomes 13, 16, 18, 21
and 22, followed by a second round with a probe specific for
chromosome 14 (binding to the 14q11.2 region) labelled with Spectrum Green™ (Vysis). The screening for t(13;21) was achieved by a
single round hybridization with the Multivysion PB panel from Vysis.
For reciprocal translocations, telomeric and centromeric probes
were used in a combination that properly characterized the translocation (Munné et al., 2000). Depending on the availability of
fluorochromes, one- or two-round FISH were used. In the preparatory
phase to PGD, the probes were tested on metaphases obtained from
the carrier’s lymphocytes where one of the telomeric probes was
located in the same chromosome labelled with the centromeric probe.
The probe mixture was evaluated on lymphocytes in interphase as
well to estimate the efficiency of the procedure.
The hybridizing solution was added to the fixed nuclei and codenaturation of nuclear DNA and probes was achieved by heating at
73°C for 5 min; hybridization followed for at least 3 h at 37°C.
The conventional aneuploidy screening used multicolour FISH in
a two step protocol for the simultaneous detection of the chromosomes
13, 16, 18, 21 and 22 (Multivysion PB; Vysis) in the first round
followed by an additional round including the probes for the chromosomes XY, 1 and 15. The scoring criteria adopted to classify the
FISH signals have been previously described (Munné et al., 1998c).
The whole procedure required 8–16 h depending on the number
of FISH rounds required to complete the analysis programmed for
⫹ 2 aneuploidy
⫹ 2 aneuploidy
⫹ 1 aneuploidy
Tel
Tel
Tel
Tel
Tel
Tel
Tel
Tel
Tel
Tel
Tel
13q ⫹ Tel12p, CEP 12
17q ⫹Tel 4p, CEP 4
4q ⫹ CEP 4, CEP 15
18q ⫹ Tel 20p, CEP 18
2q ⫹ Tel 2p, CEP 8
1p ⫹ Tel 11q, CEP 1
1p ⫹ Tel 22q, CEP 1
12q, Tel 3q, CEP 3
1q ⫹ CEP 3, CEP 1
4p ⫹Tel 20q, CEP 4
11q ⫹ CEP 11, Tel 22q
aneuploidy
aneuploidy
each case; consequently embryo transfer was scheduled on day 4
(Gianaroli et al., 1999b).
Embryo spreading
The cells from 84 non-transferable embryos were spread after a short
incubation with acidic Tyrode’s solution to digest the zona pellucida.
The nuclei obtained (n ⫽ 475) were fixed and hybridized with the
same probes used for PGD according to the same protocol already
described. The results obtained were compared with the 1-cell
diagnosis in order to verify the efficiency of the technique (Gianaroli
et al., 2001).
Statistical analysis
Data were analysed by Student’s t-test and χ2-analysis applying the
Yates correction, 2⫻2 contingency tables.
Results
The overall FISH and clinical results of Rob.T are shown in
Table IV. A total of 131 embryos was generated from 23
treatment cycles in carriers of Rob.T whose mean maternal
age was 35.5 ⫾ 3.6 years; 111 of them showed regular
development and were selected for FISH analysis. Following
FISH, 26 were diagnosed as normal or balanced (23%) whereas
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L.Gianaroli et al.
Table IV. Preimplantation genetic diagnosis of Robertsonian translocations
No. cycles
Age (mean ⫾ SD)
No. generated embryos
No. analysed embryos
FISH normal (%)
FISH abnormal (%)
No. transferred cycles (%)
No. transferred embryos (mean ⫾ SD)
No. clinical pregnancies (%)
Term deliveries
No. abortions
Implantation rate (%)
Implantation rate per pregnant patient (%)
23
35.5 ⫾ 3.6
131
111
26 (23)
85 (77)
13 (57)
1.8 ⫾ 0.8
8 (62)
6
2a
43.5
62.5
aOne after amniocentesis (fetal karyotype was normal).
FISH ⫽ fluorescence in-situ hybridization.
Table V. Chromosomal abnormalities in embryos generated by Robertsonian
(Rob.T) and reciprocal (Rec.T) translocation carriers
No. diagnosed embryos
No. FISH abnormal (%)
For chromosomes translocation-related
For chromosomes translocation-related plus
chromosomes translocation-non related
For chromosomes translocation-non related (%)
Haploidy/polyploidy (%)
Rob.T
Rec.T
111
85 (77)
18 (21)a
31 (36)b
61
54 (89)
35 (65)a
9 (16)b
26 (31)c
10 (12)
3 (6)c
7 (13)
acP ⬍ 0.001.
bP ⬍ 0.025.
FISH ⫽ fluorescence in-situ hybridization.
chromosomal abnormalities were detected in the remaining 85
embryos (77%). Embryo transfer was performed in the 13
cycles where euploid or balanced embryos were available
(57% of the oocyte retrievals); an average of 1.8 ⫾ 0.8
embryos was transferred per cycle which generated eight
clinical pregnancies with a pregnancy rate of 62% per transferred cycle and 35% per oocyte retrieval. The IR was 43.5%
and the IRPP was 62.5%. All the pregnant patients underwent,
as recommended, conventional prenatal diagnosis: PGD was
confirmed for nine of the 10 gestational sacs, with five of
them balanced carriers of the inherited translocation; in the
remaining case from a couple screened for a translocation 13;
14, trisomy 21 was diagnosed and the pregnancy was terminated. Six of the eight pregnancies delivered seven healthy
babies and two ended in abortion (one therapeutic and the
other lost after amniocentesis with a 46,XY fetal karyotype).
The analysis of the detected chromosomal abnormalities
revealed that the chromosomes involved in the translocation
were present in an aneuploid condition in 49 embryos (58%
of total abnormalities): in 18 of them they were the only
aneuploid chromosome, while in the remaining 31 the aneuploid condition was associated with the aneuploidy of another
chromosome (Table V). In 26 embryos (31% of total abnormalities) the condition of abnormality was due to the aneuploidy
of chromosomes different from those involved in the translocation and the remaining 10 embryos were either haploid or
polyploid (12%).
3204
Table VI. Reciprocal translocations: fluorescence in-situ hybridization
(FISH) and clinical results after conventional aneuploidy screening (group
1) and after the analysis of the chromosomes involved in the translocation
followed by conventional aneuploidy screening (group 2)
No. cycles
Age (years) (mean ⫾ SD)
No. generated embryos
No. diagnosed embryos
FISH normal (%)
FISH abnormal (%)
No. transferred cycles (%)
No. transferred embryos (mean ⫾ SD)
No. clinical pregnancies (%)
Term deliveries
No. abortions
Implantation rate (%)
Group 1
Group 2
8
34.6 ⫾ 5.1
45
40
17 (43)a
23 (57)b
7 (89)
2.4 ⫾ 0.5c
1 (14)
1
0
5.9
12
34.1 ⫾ 4.9
73
61
7 (11)a
54 (89)b
4 (33)
1.2 ⫾ 0.5c
1 (25)
0
1
20.0
a,bP ⬍ 0.001.
cP ⫽ 0.006.
FISH ⫽ fluorescence in-situ hybridization.
The results of the Rec.T cycles are reported in Table VI;
they were divided into two groups depending on the type of
analysis: in the first group, conventional aneuploidy screening
was performed (five patients which underwent eight cycles,
mean maternal age 34.6 ⫾ 5.1 years), and in the second group,
PGD of translocation was followed by conventional aneuploidy
screening (11 patients and 12 cycles, mean maternal age
34.1 ⫾ 4.9 years).
In the first group, 17 of the 40 embryos analysed were
euploid for the eight chromosomes tested (43%); embryo
transfer was performed in seven cycles by replacing 2.4 ⫾ 0.5
chromosomally normal embryos which generated one clinical
pregnancy and an implantation rate of 5.9%. Thirteen of
the detected 23 abnormalities (56%) involved one of the
chromosomes related to the translocation which were diagnosed
as part of the conventional aneuploidy screening (see Table
III), seven were independent from the chromosomes involved
in the translocation (30%) and three were haploid or polyploid (13%).
In the second group, 61 embryos were screened for the
chromosomes involved in the translocation and conventional
aneuploidy. The overall results demonstrated only seven
embryos with a normal or balanced chromosomal condition
(11%), whereas the remaining 54 were diagnosed as abnormal
(89%). Consequently, only four cycles were transferred (33%
of the oocyte retrievals) with a single pregnancy that miscarried
at 7 weeks gestation (no information on the fetal karyotype
was available).
The observed chromosomal abnormalities were: unbalanced
arrangement of the chromosomes involved in the translocation
in 44 embryos (81% over total abnormalities): in 35 of them
they were the only abnormality detected whereas in the
remaining nine aneuploidy for another chromosome was also
found; three embryos carried aneuploidy for chromosomes
different from those involved in the translocation (6%) and
seven were haploid or polyploid (13%).
Finally, Figure 1 represents the cellular stage of the embryos
at 62 h post insemination before embryos biopsy was per-
Interchromosomal effect and translocation carriers
were chaotic mosaic embryos that had been diagnosed as
monosomic by PGD from three patients with translocations
13;14, 18;20 and 1;22 respectively. Finally, two embryos
classified as monosomy 13 generated by two patients with
a 13;14 and a 13;21 translocation each, turned out to be
chromosomally normal giving a misdiagnosis rate of 2%.
Figure 1. Cellular stage of the embryos evaluated at 62 h post
insemination in Robertsonian and reciprocal translocations. The
percentage of 4-cell embryos was significantly higher in the case of
reciprocal translocations compared with Robertsonian translocations
(a,bP ⬍ 0.025). Conversely, embryos at the 7–8-cell stage were
more frequent in Robertsonian translocations (a,bP ⬍ 0.025).
formed. The percentage of embryos at the 4-cell stage was
significantly higher in the case of Rec.T (35 versus 17%,
P ⬍ 0.025), while embryos with 艌7 cells were more frequent
in Rob.T (43 versus 24%, P ⬍ 0.025).
The main differences between Rob.T and Rec.T are summarized in Table VI. The data of Rec.T only refer to group 2
where both the chromosomes involved in the translocation
and those independent from the translocation were screened.
Although the percentage of chromosomally abnormal embryos
did not vary significantly between the two categories, the
distribution of the abnormalities detected was totally different.
Most of the defects were due to the chromosomes related to
the translocation with the highest incidence in Rec.T (65%)
compared with Rob.T (21%; P ⬍ 0.001). Conversely, a more
relevant contribution to aneuploidy from chromosomes not
related to the translocation was detected in the case of Rob.T
(31%), compared with Rec.T (6%, P ⬍ 0.001); this difference
did not seem to be related to maternal age, which was similar
in both Rob.T and Rec.T couples (P ⫽ 0.35, not significant).
The simultaneous presence of numerical variations derived
from chromosomes related to the translocation and from other
chromosomes contributed 36% of total abnormalities in Rob.T
versus 16% in Rec.T (P ⬍ 0.025). Similar results occurred
in the proportion of aneuploid embryos not related to the
translocation when group 1 was also taken into consideration: 67% in Rob.T (31 ⫹ 26/85) and 40% in Rec.T
(9 ⫹ 3 ⫹ 20/77; P ⬍ 0.005).
The results obtained from the FISH analysis of 84 nontransferable embryos after spreading of their blastomeres
revealed 93% overall confirmation of PGD results (78/84).
Four of the six non-confirmed embryos had a different abnormality (5%): one was monosomy 16 instead of monosomy 18
and belonged to a patient with 13;14 translocation; and three
Discussion
As already reported, carriers of Rob.T derive a notable benefit
from FISH analysis (Munné et al., 1998d, 2000; Evsikov et al.,
2000; Scriven et al., 2001). According to the current results,
the high percentage of chromosomally abnormal embryos
detected in these couples, (77%; Table IV) made the selection
by PGD a useful tool to achieve pregnancy in 35% of the
cycles undergoing oocyte retrieval. This outcome is especially
relevant when considering that even in the case of embryos at
the 7–8-cell stage on the morning of day 3, chromosomal
abnormalities were detected in 65% of them, implying that in
these categories of patients normal morphology and development, or blastocyst growth are not sufficient criteria for
euploidy (Evsikov et al., 2000). As repeated pregnancy losses
are a frequent consequence of unbalanced rearrangements, it
is hardly sustainable, despite some reports (Ménézo et al.,
1997), that transferring at the blastocyst stage prevents abnormal implantation.
The analysis of chromosomal abnormalities revealed that
58% (82/139) of them were due to the chromosomes directly
involved in the translocation, either alone or in combination
with other chromosomes (Table V). The concomitant study of
other chromosomes was essentially aimed at maximizing the
chances of pregnancy in these patients by avoiding the transfer
of aneuploid embryos. Actually, 26 of the 111 embryos
diagnosed by FISH carried abnormalities due to chromosomes
different from those involved in the translocation: nine were
monosomic, 10 trisomic, one showed simultaneous monosomy
and trisomy and six carried complex abnormalities. Interestingly, five of these trisomies involved chromosome 21 and
were detected in embryos generated by carriers of the translocation 13;14. In other words, almost a fourth of the screened
embryos were removed from the possibility of being transferred
by the screening for conventional aneuploidy. Unfortunately,
one misdiagnosis occurred and a trisomic 21 embryo was
transferred to a patient 37 years whose husband carried a 13;
14 translocation; this was due to partial overlapping of two
signals which, according to the scoring criteria adopted, were
diagnosed as one. Since then, scoring criteria has been revised,
and, in the presence of a split signal, a specific telomeric probe
is used to confirm the diagnosis (Magli et al., 2001). In
consideration of the clinical implications related to trisomy
21, a new strategy using two different probes for this chromosome has been designed: one probe is locus specific whereas
the other, used in a second-round hybridization, targets the
telomere. Under these conditions, the risk of missing a trisomy
21 is close to zero (Magli et al., 2001).
Patients with Rec.T entered this study following two different
approaches depending upon the type of probes used. The
results from group 1 were included since the FISH results
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L.Gianaroli et al.
were different when the chromosomes related to the translocation were not fully characterized with specific probes, confirming that carriers of Rec.T are highly exposed to unbalanced
products (Table VI).
In Table V, data from group 2 only were considered in order
to distinguish the contribution to total abnormalities from the
chromosomes related to the translocation from those not
involved in the translocation. As reported, 81% of the abnormalities in Rec.T were due to the chromosomes involved in
the translocations (either alone or in combination with others)
and this was significantly higher compared with the situation
found in Rob.T (58%; P ⬍ 0.01). This could be explained by
the differences in the meiotic behaviour of these two types of
translocations (Scriven et al., 1998). During meiosis, the
chromosomes involved in the Rob.T pair their homologous
segments forming a trivalent figure, which only can segregate
in an alternate way (producing balanced or normal gametes)
or in an adjacent way (producing unbalanced gametes). If the
chromosomes involved in the translocation are of the same
size (D;D or G;G), the translocation is more prone to segregate
in an alternate way than if the chromosomes are of different
size (D;G). Rec.T pair their homologous segments forming a
tetravalent figure, which can segregate in five different ways:
alternate (producing balanced or normal gametes), adjacent I
and II (producing unbalanced gametes), 3:1 or 4:0 (also
producing unbalanced gametes). Depending on the position of
the breakpoints in the chromosomes involved in the translocation and the size of these chromosomes, the translocation
would be more prone to segregate in a 2:2 way (alternate and
adjacent segregations) or a 3:1 or 4:0 way (Boue et al., 1984;
Pellestor et al., 1987; Martin and Hultén, 1993).
Although only a few cases were studied, it is very clear that
the two categories of balanced translocations have a completely
different molecular basis, and this leads to a diverging clinical
outcome after PGD. This diversity is already perceived at the
morphological analysis of in-vitro-generated embryos, whose
development is significantly retarded in the case of Rec.T
(Figure 1), although unbalanced embryos can develop to
blastocysts (Evsikov et al., 2000). The reason for this behaviour
is not clear, especially when considering that the majority of
the translocation cases presented here were paternal in origin,
and that until the 4–8-cell stage the embryo relies upon the
oocyte machinery for the first cleavage divisions (Braude et al.,
1988). Similarly, the high rates of mosaicism detected in
translocation cases do not have a clear explanation (Conn
et al., 1998; Iwarsson et al., 2000).
The FISH results obtained in this study basically suggest
two considerations. First, the high proportion of abnormalities
due to the chromosomes involved in the translocations is
significantly higher in carriers of Rec.T. This reduces markedly
the number of embryos available for transfer and makes the
quality of the response to hormonal stimulation critical for
these patients’ clinical outcome. Second, an interchromosomal
effect could be postulated in the case of Rob.T due to the
relevant contribution of aneuploidy involving chromosomes
unrelated to the translocation. The analysis of the aneuploid
events associated with chromosomes different from those
involved in the translocation revealed that, as already men3206
tioned, five of the detected trisomies resulted from chromosome
21; in total, six of the monosomies and eight of the trisomies
were due to one chromosome from the D group (13, 14, 15)
or the G group (21, 22) which are the chromosomes entering
Rob.T. In order to evaluate a putative interchromosomal effect,
the contribution of aneuploidy in age-matched patients with a
normal karyotype was estimated; 17% of 235 embryos diagnosed in 46 ICSI cycles, which were treated during the same
period, were monosomic or trisomic. This value is similar to
the aneuploidy rate found in embryos generated by Rec.T.
carriers, but is significantly lower when compared with Rob.T
(P ⬍ 0.025).
Contradictory data have been reported on the analysis of
sperm obtained from carriers of translocations, with some
reports in favour of an interchromosomal effect both in
Robertsonian and reciprocal translocations (Rousseaux et al.,
1995; Mercier et al., 1998; Blanco et al., 2000; Estop et al.,
2000; Morel et al., 2001; Oliver-Bonet et al., 2001; Shi and
Martin, 2001) and others demonstrating no evidence of this
phenomenon (Martin, 1988; Pellestor, 1990; Martin et al.,
1992; Syme and Martin, 1992; Blanco et al., 1998). According
to these reports, the occurrence of an interchromosomal effect
seems to depend both on the type of chromosomes and the
chromosomal regions involved, resulting in a particular meiotic
configuration that could determine an increased sperm disomy
(Estop et al., 2000). In addition, a direct correlation between
poor quality sperm and alteration in the recombination frequency of other chromosomes has been reported, suggesting
that infertile carriers are at higher risk for interchromosomal
effects (Pellestor et al., 2001). Furthermore, a follow-up study
of the genetics and epidemiology of Down’s syndrome in
⬎2⫻105 births postulated an interchromosomal effect in seven
infants out of the 391 Down’s infants which were karyotyped
(Stoll et al., 1998).
Taken altogether, these observations suggest that the high
incidence of aneuploidy detected by PGD in this study for
chromosomes unrelated to the translocation, could be due to
the fact that most of these embryos were generated by infertile
patients. The use of ICSI, which was necessary in 80% of the
cases, could have contributed an additional element since the
possibility of a natural selection against aneuploid sperm
following conventional insemination (or natural conception)
was completely bypassed.
Additional data are certainly required to support the
hypothesis of an interchromosomal effect associated with
carriers of translocations; particularly important would be to
promote the inclusion in the aneuploidy screening of other
chromosomes, reassured by the finding that multiple-round
hybridization (at least four, as used in this study) does not
seem to affect FISH efficiency.
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Submitted on November 13, 2001; resubmitted on July 25, 2002; accepted on
August 15, 2002
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