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Sperm segregation analysis of a (13;22) Robertsoniantranslocation carrier by FISH: a comparison of locus-specific probe and whole chromosome painting
T.Anahory1, S.Hamamah1, B.Andreo2, B.Hedon3, M.Claustres4, P.Sarda5 and F.Pellestor2,6
1Service de Medecine et de Biologie de la Reproduction B, Ho pital Arnaud de Villeneuve, 34295 Montpellier Cedex 5, 2CNRS-UPR
1142, Institut de Genetique Humaine, 141 rue de la Cardonille, F-34396 Montpellier Cedex 5, 3Service de Gynecologie Obstetrique,
Hopital Arnaud de Villeneuve, 34295 Montpellier Cedex 5, 4Service de Genetique Moleculaire et Chromosomique, Hopital Arnaud de
Villeneuve, 34295 Montpellier Cedex 5 and 5Service de Genetique Clinique, Hopital Arnaud de Villeneuve, 34295 Montpellier
Cedex 5, France
6To whom correspondence should be addressed. E-mail: [email protected]
BACKGROUND: The t(13;22) Robertsonian translocation constitutes a rare form of rearrangement between acro-
centric human chromosomes. Most of the meiotic segregation studies of human Robertsonian translocations have
been performed on common t(13;14) and t(14;21) translocations. Analysis of the chromosomal constitution in
sperm of Robertsonian translocation carriers is of great interest for assessing the risk of unbalanced forms and
adapting genetic counselling. In the present study, we present the first meiotic segregation study of a t(13;22)
Robertsonian translocation in human sperm. METHODS: A total of 11 787 sperm nuclei were scored using two
distinct FISH labelling techniques, i.e. the locus-specific probes (LSI) method and the whole chromosome painting
(WCP) technique. RESULTS: The frequency of normal or balanced sperm resulting from alternate meiotic segre-
gation was 86%. Incidences of unbalanced complements resulting from adjacent segregation modes were 12.79%
and 14.36% in LSI and WCP assays, respectively. No significant excess of nullisomy or disomy for the affected
chromosomes was observed. CONCLUSIONS: Similar results in segregation were obtained with the two tech-
niques, demonstrating the efficiency of the two strategies for the direct segregation analysis of Roberstsonian trans-
locations. The results obtained indicated a moderate meiotic production of imbalance. This study shows that the
rare Robertsonian translocation (13;22) displays a similar distribution of balanced and unbalanced sperm patterns
as the common Robertsonian translocations previously studied. This suggests that the behaviour of acrocentric
chromosomes was similar in all cases of centric fusion.
Key words: FISH/meiotic segregation/Robertsonian translocation/sperm
Introduction
With an incidence of 1.20 per 1000 livebirths, Robertsonian
translocation is the most common structural chromosomal
aberration found in human (Evans et al., 1978; Nielsen and
Wohlert, 1991). The great majority of Robertsonian trans-
locations involve two non-homologous chromosomes and
occur between chromosomes 13 and 14 or chromosomes 14
and 21 (73% and 10% of all Robertsonian translocations,
respectively) (Therman et al., 1989).
Men carrying Robertsonian translocation have a normal
phenotype. However, they may have spermatogenesis altera-
tions expressed by oligozoospermia or azoospermia, and they
may be affected by reproductive failure owing to imbalances
in chromosome meiotic segregation (Scriven et al., 2001).
The direct analysis of sperm chromosomal constitution
can be used to determine meiotic segregation patterns of
translocated chromosomes, and to predict the risk of having
unbalanced conceptuses. Direct data were first obtained using
the human sperm–hamster egg fertilization system. To date,
six men heterozygous for Robertsonian translocation have
been investigated using this method (Balkan and Martin,
1983; Pellestor et al., 1987; Martin, 1988; Pellestor, 1990;
Martin et al., 1992; Syme and Martin, 1992). Although this
approach allowed the direct karyotyping of individual human
spermatozoa, the procedure remained time-consuming, labour
intensive and did not result in the chromosome analysis of
large numbers of sperm nuclei (from 24 to 149) (Guttenbach
et al., 1997).
Fluorescence in-situ hybridization (FISH) with chromo-
some-specific DNA probes has offered a new strategy for
investigating the meiotic segregation of translocations.
Several studies have been carried out, essentially focusing on
Human Reproduction Vol.20, No.7 pp. 1850–1854, 2005 doi:10.1093/humrep/deh886
Advance Access publication April 21, 2005
1850 q The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.For Permissions, please email: [email protected]
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the most frequent Robertsonian translocations, i.e. the
t(13;14) and t(14;21) (Rousseaux et al., 1995; Escudero et al.,
2000; Honda et al., 2000; Morel et al., 2001; Frydman et al.,
2001; Anton et al., 2004). Only one case of Robertsonian
translocation between two homologous chromosomes, a
t(21q;21q), has been investigated by sperm FISH analysis
(Acar et al., 2002).
In this report we present the first analysis of sperm
chromosome segregation in a man heterozygous for an
uncommon (13;22) Robertsonian translocation. Both locus-
specific probes and whole chromosome painting probes were
used in parallel, in order to compare the efficiency and the
accuracy of the two procedures, and to provide complemen-
tary data on the male meiotic segregation of this rare
chromosomal rearrangement.
Materials and methods
Patient
The patient, aged 38 years, was diagnosed with a (13;22)
Robertsonian translocation after 9 years of sexual intercourse with-
out conception associated with oligoteratozoospermia (concentration
3 £ 106 /ml; 36% normal morphology; 40% progressive motility).
Physical examination and sexual development were normal. His
wife was healthy with a normal karyotype.
The patient gave his informed consent prior to participation in the
study, which was approved by the Ethical Board of the Montpellier
Hospital.
Sperm from a fertile, 35-year-old man with normal sperm
parameters and a normal karyotype was used as control.
Sperm preparation
Sperm samples were collected in sterile containers after 3 days of
sexual abstinence. After liquifying at room temperature, the samples
were washed three times in 1 £ phosphate-buffered saline by cen-
trifugation (5 min at 2000 rpm). The final pellets were fixed for 1 h
in fresh fixative (methanol:glacial acetic acid 3:1) at 2208C. The
sperm suspensions were then dropped onto clean microscopic slides
and air-dried. Slides were kept for 2 days at room temperature
before use in FISH reactions.
Before in-situ labelling reactions, the sperm nuclei decondensa-
tion was performed by slide incubation in 25 mmol/l dithiothreitol
(Sigma, St Louis, MO, USA), in 1 mol/l Tris – HCl solution at room
temperature for 5 min. Slides were then washed twice in 2 £ SSC,
dehydrated through an ethanol series and air-dried. The deconden-
sation of sperm nuclei was checked under a phase-contrast
microscope.
FISH procedure
Two distinct probe mixtures were used in this study. The first mix-
ture consisted of Vysis locus-specific probes (LSI), for chromosome
13 (LSI 13, spanning the 13q14 region and labelled in Spectrum-
Orange) and chromosome 22 (LSI 22, spanning the 22q13.3 region
and labelled in SpectrumGreen). The second probe mixture was
composed of two Vysis whole chromosome painting (WCP) probes,
i.e. a chromosome 13 painting probe (WCP 13) labelled in Spec-
trumGreen, and a chromosome 22 painting probe (WCP 22) labelled
in SpectrumOrange (Vysis, Downers Grove, IL, USA). The probes
were prepared according to the manufacturer’s instructions.
The labelling efficiency of both LSI probes and WCP probes was
determined by scoring the proportion of labelled nuclei on samples
of 200 metaphasic and interphasic lymphocytes from the patient
and the control subject, in order to assess the efficiency of probe
binding and characterisation of the derivative chromosomes. There
was no significant difference (P . 0.05) in hybridization efficiency
(from 99.60% to 99.88%) between the patient and the control, and
the detection of segregation patterns was efficient in both meta-
phases and interphase nuclei.
Before hybridization, both probe and slide preparations were
denatured separately. The probe mixture were denatured for 5 min at
738C in a water bath, whereas the slides were denatured by immer-
sion in 70% formamide/2 £ SSC at 728C for 3 min, dehydrated
again and air-dried.
Each hybridization mixture was applied to the denatured sperm
nuclei preparations and slides were covered with coverslips, sealed
with rubber cement and hybridized overnight in a dark, moist
chamber. Coverslips were then gently removed and the slides were
washed for 10 min in a 50% formamide/50% 2 £ SSC solution at
468C, followed by a 10 min wash in 2 £ SSC at 468C, and a 5 min
wash in 2 £ SSC/0.5% Tween 20 solution, and finally mounted
with DAPI (100 ng/ml) in antifade solution.
The slides were examined by two independent observers using a
Leitz fluorescence microscope DMRB (Leica SA, Rueil-Malmaison,
France), equipped with a DAPI single band-pass, a fluorescein
single band-pass filter, a rhodamine single band-pass filter, a fluores-
cein/rhodamine double band-pass filter, and a triple filter set for sim-
ultaneous observation of fluorescein, rhodamine and DAPI signals.
Only individual and well-delineated sperm nuclei were scored.
Previously described standard assessment criteria were followed
for the analysis of in-situ sperm labelling (Pellestor et al., 2001).
The scoring criteria were similar for LSI and WCP probes. Briefly,
overlapping sperm nuclei, disrupted nuclei or large nuclei with
diffuse signals were not considered. Sperm nuclei were scored as
having two identical signals when the two spots were of equal size
and intensity and were separated by at least the diameter of one
hybridization domain. In painting assays, nuclei with two signals of
different colours clearly coupled one with the other, were considered
as displaying a balanced chromosomal pattern.
Data analysis
The x2-test was used to statistically analyse the segregation patterns
observed in the patient and compare the fluorescent phenotypes
between the translocation carrier and the control subject. Compari-
son of data between the two procedures was also performed using
x2-test. Differences were considered to be significant when
P , 0.05.
Results
A total of 11 787 sperm nuclei from the translocation carrier
and 10 332 from the control subject were scored. The hybri-
dization efficiencies of the two probe sets reached 99.40%
and 99.70% in sperm of patient and donor, respectively.
Among the 11 787 sperm nuclei analysed in the transloca-
tion carrier, 4735 nuclei were scored using the WCP probe
set (Figure 1) and 7052 nuclei using the LSI probe set
(Figure 2). The results are summarized in Table I. In the
two assays, similar frequencies of normal and balanced
spermatozoa resulting from alternate segregation were found
(86.70% and 85.19%, respectively). Unbalanced fluorescent
patterns, resulting from adjacent segregation modes, were
observed in 12.79% of nuclei scored with LSI probes
and 14.36% of nuclei from the WCP assay. There was no
Sperm segregation of a 13; 22 Robertsonian translocation
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significant difference (P . 0.05) between these values. In the
two assays, the distribution of the different unbalanced pat-
terns (nullisomies and disomies 13 or 22) was similar, with
rates of imbalances ranging from 2.66% to 4.24% (Table I).
In control sperm, the proportion of nuclei with normal fluor-
escent patterns for chromosomes 13 and 22 was 98.9%. Diso-
mies and nullisomies 13 and 22 found in this control sperm
ranged from 0.08% to 0.36%. These values were significantly
Figure 1. Labelling of sperm nuclei using WCP probes specific for chromosomes 13 and 22 (chromosome 13 in green and chromosome 22in orange). Various segregation patterns (alternate and adjacent) are observed according to the fluorescent signal distribution.
Figure 2. Labelling of sperm nuclei using LSI probes specific for chromosome 13 (LSI 13q14 in orange and LSI 22q13.3 in green). Onenucleus displays a normal or balanced fluorescent pattern whereas the other shows an adjacent unbalanced pattern 23,X or 23,Y, 213,þ t(13;22).
Table I. Segregation of sperm chromosomes for a 13;22 Robertsonian translocation
Segregationtypes
Chromosomalpatterns
In-situfluorescent phenotypes
FISH results Proportion (%) of fluorescent phenotypes
LSI assay WCP assay in control
n % n %
Alternate 13q/22q or der (13q;22q) Normal 6114 86.70 4034 85.19 98.90Adjacent 13q Nullisomy 22 222 3.15 161 3.40 0.16
22q/der(13q;22q) Disomy 22 243 3.44 176 3.71 0.0822q Nullisomy 3a 188 2.66 142 2.99 0.3613q/der (13q;22q) Disomy 13a 249 3.53 201 4.24 0.13
902 12.79 680 14.3603:00 or diploid 13q/22q/der (13q;22q) or
13q/13q/22q/22qDiploidy 36 0.51 21 0.44 0.37
Total 7052 4735
aIn both LSI and WCP assays, the frequency of 13 disomic nuclei exceed the frequency of complementary 13 nullosomies. However, the values don’t display astatistically significant difference (P . 0.05).
T. Anahory et al.
1852
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lower than the rates of imbalances found in the patient. The
incidence of double hybridization pattern, corresponding to
either diploid sperm nuclei or 3:0 segregation pattern was not
significantly different (P . 0.05) between the translocation
carrier (mean 0.47%) and the control subject (0.37%).
Discussion
This is the first report of the study of a (13;22) Robertsonian
translocation. In humans, the (13;22) Robertsonian transloca-
tion remains a rare event, with an occurrence estimated at
1% of all Robertsonian translocations (Therman et al., 1989).
The few cases reported were ascertained through trisomy 13
or various population surveys. Thus, in the European colla-
borative study on prenatal diagnosis, Boue and Gallano
(1984) found only three cases of Robertsonian translocation
(13;22) among 262 Robertsonian prenatal diagnoses not
involving chromosome 21.
To carry out the present FISH study, we utilized two
different procedures, i.e. LSI and WCP probes. The LSI
method has been used in the majority of previous FISH
sperm studies performed in Robertsonian translocation car-
riers (Rousseaux et al., 1995; Escudero et al., 2000; Honda
et al., 2000; Frydman et al., 2001; Acar et al., 2002; Anton
et al., 2004), whereas WCPs have been used only once for
sperm analysis in three Robertsonian translocation (13;14)
carriers (Morel et al., 2001). The use of LSI on sperm can be
limited by the DNA compaction and inefficient decondensa-
tion of sperm nuclei, or the small size of fluorescent signals
usually obtained with these probes. Signals generated by
WCP appeared easier to detect in situ on sperm nuclei
because of their size and intensity. Previous uses of painting
probes on human sperm have demonstrated that this type of
probe possessed both the specificity and the sensitivity
required for the in-situ scoring of chromosomal imbalances
in human sperm (Rives et al., 1998; 1999; Morel et al.,
2001). In addition, the use of WCP requires only a moderate
sperm decondensation to provide a high hybridization effi-
ciency (Rives et al., 1998).
The combined use of these two labelling procedures
allowed us to compare the efficiency of the two techniques on
human sperm, and provided an internal control for the analysis
of meiotic segregation. Both procedures gave similar results
in terms of balanced and unbalanced nuclei. This indicates
that FISH with WCP is an accurate approach for sperm study
in Robertsonian translocation carriers, and so can be used to
complement the LSI technique. Morel et al. (2001) reported
that the WCP method allowed the in-situ differentiation of
both normal spermatozoa (with two signals of different colour
separated by at least one diameter) and balanced spermatozoa
(with two signals of different colour coupled one with the
other). In our study, although such a distinction was possible
in numerous sperm nuclei, a significant proportion of nuclei
also displayed partially overlapping signals. This observation
lead us to consider that the WCP approach was not efficient
enough to precisely estimate the proportion of normal and
balanced nuclei resulting from the alternate segregation mode.
Consequently, no distinct classification was made between
normal and balanced sperm nuclei.
The observed high frequency (89%) of sperm nuclei result-
ing from alternate segregation is in good agreement with
results from previous analysis of sperm in Robertsonian trans-
location carriers, using either the human–hamster fertilization
system or FISH procedure (Pellestor, 1990; Honda et al.,
2000; Anton et al., 2004). Similar high incidences of alternate
meiotic segregation in sperm of Robertsonian translocation
carriers were also observed in other mamalian species such as
mice and bulls (Gropp and Winking, 1981; Tateno et al.,
1994). All these data support the existence of a similar meiotic
behaviour of rearranged chromosomes in Robertsonian trans-
locations. The predominance of alternate segregation over
other segregation types does not appear to be influenced by
the difference in chromosomes involved in the translocation.
Meiotic analyses of trivalent synaptonemal complexes in
Robertsonian translocations have shown the predominant pair-
ing of the acrocentric elements in cis-configuration. Such con-
figuration favours alternate meiotic segregation (Vidal et al.,
1982; Luciani et al., 1984; Navarro et al., 1991).
The overall frequency of unbalanced spermatozoa resulting
from adjacent segregation modes is 12%. This is also consist-
ent with the results of previous FISH studies (results ranging
from 10.8% to 22.6%). The similarity of meiotic configur-
ations in Robertsonian translocation could explain the rela-
tively homogeneous rates of imbalances. In addition, both the
number and the location of chiasmata could contribute to
produce similar proportions of imbalances in all Robertsonian
translocations. Because of the correlation between the line of
chromosomal segregation and the chiasmata line, there is a
strong prevalence of alternate segregation, resulting in a low
rate of unbalanced spermatozoa, and consequently in a low
risk of imbalance in progeny of male carriers. However, the
incidences of imbalances in sperm of Robertsonian transloca-
tion carriers are always higher than the incidences of imbal-
ances drawn from studies of fetuses (Boue and Gallano,
1984) or newborns (Daniel et al., 1989). This finding indi-
cates that there is a strong in-utero selection against unba-
lanced conceptuses. In this way, it is interesting to note that
studies of female carriers of Robertsonian translocation per-
formed by polar body FISH analysis (Munne et al., 2000;
Durban et al., 2001) reported significantly higher rates of
unbalanced oocytes (32–36%). Such variations emphasize
the difference in the outcome of adjacent segregation in male
and female meiosis, probably linked to the weakness of the
female meiosis checkpoint mechanisms (LeMaire-Adkins
et al., 1997). Also, some reports of preimplantation genetic
diagnosis for Robertsonian translocation carriers have indi-
cated elevated rates of imbalanced chromosomal constitution
(Conn et al., 1998; Iwarsson et al., 2000; Alves et al., 2002).
The observed abnormalities were essentially mosaicism and
chaotic chromosomal constitution. However, these data are
still in discussion, since other PGD reports with no evidence
for high frequency of unbalanced embryo do not support
the contention that Robertsonian translocations could predis-
pose to malsegregation, and suggest that the observed
chromosomal abnormalities could essentially result from
Sperm segregation of a 13; 22 Robertsonian translocation
1853
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culture conditions (Scriven et al., 2001). Post-zygotic events
affecting the chromosomal segregation during early cleavage
stages could also influence the occurrence of unbalanced con-
ceptuses, and this could be a patient-related phenomenon.
The production of unbalanced gametes renders difficult the
investigation of meiotic segregation in Robertsonian translo-
cation based on data from live birth or prenatal diagnoses.
The sperm analysis of Robertsonian translocation carriers
constitutes a unique and efficient approach for predicting the
meiotic behaviour of these chromosomal rearrangements and
estimating the risk of occurrence of unbalanced embryo in
translocation carrier couples. The present study has shown
that the rare Robertsonian translocation (13;22) displayed a
similar distribution of balanced and unbalanced sperm pat-
terns as the common Robertsonian translocations previously
studied, thus suggesting that the behaviour of acrocentric
chromosomes was similar in all cases of centric fusion.
Further investigations of other rare Robertsonian transloca-
tions are now required to confirm these data.
Acknowledgements
This study was supported by a french research project PHRC (No.7732) from the CHU of Montpellier.
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Submitted on November 5, 2004; resubmitted on January 5, 2005; accepted on January 21, 2005
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