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: Franck.Pellestor@igh.cnrs.fr

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: journals.permissions@oupjournals.org

 

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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

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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

T. Anahory et al.

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