Molecular Human Reproduction vol.6 no.6 pp. 566–570, 2000
Segregation of sex chromosomes in spermatozoa of 46,XY/47,XXY
men by multicolour fluorescence in-situ hybridization
Frédéric Morel1, Christophe Roux and Jean-Luc Bresson
Service de Cytogénétique-Immunocytologie-Biologie du Développement et de la Reproduction, CECOS Besançon
Franche Comté, Hôpital Saint Jacques and CNRS 6025 Faculté de Médecine, 25030 Besançon, France
1To
whom correspondence should be addressed
The sex chromosome disomy and diploidy rates on ejaculated spermatozoa from two patients with mosaic
Klinefelter’s syndrome were estimated, using X/Y/15 multicolour fluorescence in-situ hybridization (FISH). A
8/18 dual fluorescence in-situ hybridization analysis was also carried out. In triple FISH, a total of 1691
(patient 1) and 811 (patient 2) spermatozoa were analysed. Frequencies of cells with hyperhaploidies for sex
chromosomes were 2.01% and 3.45% for patients 1 and 2 respectively, with both patients showing a
significantly increased incidence of 24,XY and 24,XX disomies and only patient 2 showing a significantly
increased incidence of 24,YY disomy in comparison to the control (P < 0.001). The 46,XX diploidy rate in
patient 1 was also significantly higher than the control (P < 0.01). The ratio of X-bearing to Y-bearing
spermatozoa differed from the expected 1:1 ratio for only patient 1 (1.18:1). There was no significant difference
for chromosomes 8, 15 or 18 disomy frequencies in comparison to those estimated in the control population.
These results support the hypothesis that some 47,XXY cells are able to go through meiosis and form
spermatozoa with an abnormal gonosomal complement. Thus, there is an increased risk, for these 46,XY/
47,XXY men, of producing offspring with a gonosomal abnormality.
Key words: aneuploidy/fluorescence in-situ hybridization/Klinefelter’s syndrome/spermatozoa
Introduction
Klinefelter’s syndrome is one of the most frequent sex chromosome abnormalities in human males, affecting ~1/1000 newborns. Patients with Klinefelter’s syndrome present with a
47,XXY karyotype in 80% of cases, whereas higher grade
aneuploidy (48,XXXY; 48,XXYY; 49,XXXXY) or mosaicism
(46,XY/47,XXY) distinguishes the remaining 20%.
Generally, mosaic Klinefelter’s syndrome patients are less
affected than non-mosaics and the severity of the syndrome is
thought to increase with the proportion of the abnormal cell
population. This syndrome is often associated with azoospermia
or severe oligozoospermia. Only a few cases of fertility (Kaplan
et al., 1963) with proven paternity have been reported (Terzoli
et al., 1992). Recently the application of artificial reproductive
techniques has enabled these patients to reproduce through
intracytoplasmic sperm injection (ICSI) performed with spermatozoa recovered from oligozoospermic semen or from
testicular tissue (Harari et al., 1995; Tournaye et al., 1996;
Hinney et al., 1997; Nodar et al., 1998; Ron-El et al., 1999).
However, the question remains: is there an increased risk for
men with Klinefelter’s syndrome of producing offspring with
a gonosomal abnormality?
Previous meiotic studies support the hypothesis that, in
patients with mosaic 46,XY/47,XXY, only these germ cells
with normal sex chromosomes can go through meiosis
(Kjessler, 1966; Luciani et al., 1970; Laurent et al., 1973).
Consequently, there should be no increased risk for chromosomal aberrations in spermatozoa. These results were confirmed
in an analysis of diakinesis preparations from a mosaic XY/
566
XXY individual, showing no evidence for the presence of
XXY germ cells, therefore suggesting that they were eliminated
from the meiotic process (Rajendra et al., 1981). However, a
synaptonemal complex study by light and electron microscopy
contradicted this opinion (Vidal et al., 1984). It has been
proposed that a few 47,XXY germ cells could initiate the
meiotic process (Skakkebaek et al., 1969; Vidal et al., 1984).
Using the technique of in-vitro penetration of zona free
hamster eggs, a study of 543 sperm karyotypes from an XY/
XXY mosaic patient found a significantly increased incidence
of hyperhaploid 24,XY spermatozoa (Cozzi et al., 1994).
These results support the hypothesis that some 47,XXY cells
are able to go through meiosis and form spermatozoa. Thus,
until recently, contradictory results and opinions have been
reported on the behaviour of 47,XXY cells.
Over the last few years, in-situ hybridization has made it
possible to directly detect the sex chromosomes in ejaculated
spermatozoa. As a powerful alternative technique to sperm
karyotyping it is less laborious and time-consuming, relatively
simple and enables the study of more cells; it therefore sheds
new light on the behaviour of 47,XXY cells from Klinefelter’s
syndrome males.
To our knowledge, since 1996, nine studies (Chevret et al.,
1996; Martini et al., 1996; Guttenbach et al., 1997; Foresta
et al., 1998; Estop et al., 1998; Kruse et al., 1998; Foresta
et al., 1999; Lim et al., 1999; Okada et al., 1999) have used
in-situ hybridization to analyse the results of segregation of sex
chromosomes in spermatozoa from subjects with Klinefelter’s
© European Society of Human Reproduction and Embryology
FISH analysis in spermatozoa of 46,XY/47,XXY men
Table I. Semen parameters of the two Klinefelter patients
Patient No.
Volume (ml)
Sperm concentration
(⫻ 106/ml)
Motility
grades a ⫹ b (%)
Normal morphology (%)
1
2.5
0.55
45
2
1.5
0.30
6
rare spermatozoa
generally abnormal
13
syndrome. This study reports the sex chromosome meiotic
distribution, using multicolour fluorescence in-situ hybridization (FISH) on ejaculated spermatozoa, from two patients with
46,XY/47,XXY karyotypes.
Materials and methods
Patients and sperm characteristics
Cytogenetic analysis of 90 (patient 1) and 50 (patient 2) peripheral
blood cells showed that the two patients had a mosaic 46,XY/47,XXY
with 70% (patient 1) and 78% (patient 2) of 47,XXY lymphocytes.
Analysis of semen showed severe oligoteratozoospermia for patient
1 and severe oligoasthenoteratozoospermia for patient 2 (Table I). A
testicular biopsy was not performed on either patient. A fertile man
with a normal 46,XY karyotype and normal sperm characteristics
(World Health Organization, 1999), represented the control spermatozoa population.
Prior to this study, patients were informed of the investigations
and gave their consent. The study was reviewed by the ethics
committee of the Besançon University Hospital.
Analysis of aneuploidy
Hybridization procedure
The hybridization procedure was performed as previously described
(Mercier et al., 1996; Morel et al., 1999). Briefly, in dual FISH,
digoxigenin-labelled hybrids were detected using tetra-methyl1-rhodamine-β-isothiocyanate conjugate (TRITC)-labelled antidigoxigenin (Boehringer Mannheim), and biotinylated labelled hybrids
were detected with fluorescein isothiocyanate (FITC)-labelled avidin
(Vector Laboratories, Biosys Compiègne, France) and then biotinylated goat anti-avidin (Vector Laboratories) and FITC-labelled avidin.
In triple FISH, biotinylated labelled hybrids were detected with
amino-methyl-coumarin–acetic acid (AMCA)-labelled avidin (Vector
Laboratories) and then biotinylated goat anti-avidin (Vector
Laboratories) and AMCA-labelled avidin.
Finally, the slides were counterstained with a medium containing
glycerol, an antifading reagent (Vector Laboratories), and propidium
iodide (Sigma Aldrich, Saint-Quentin, France).
Data collection and analysis
The slides were analysed using a Zeiss Axiophot epifluorescence
microscope. Sperm nuclei were counted for each sample using strict
selection criteria (Mercier et al., 1998; Morel et al., 1998). Only
nuclei with tails were analysed. A few immature germ cells or somatic
cells were present in the sperm preparations but these were not
included in the analysis. Subsequent image acquisition using a CCD
camera with digital image enhancement was performed (Cytovision,
Applied Imaging).
Sperm preparation
The procedure has been described elsewhere (Morel et al., 1997).
Briefly, each semen sample was washed twice in phosphate-buffered
saline, and 100 µl of sperm suspension was dropped and fixed onto
a slide. Sperm nuclei were partially decondensed for 2–15 min with
a decondensation solution of 0.2 mol/l Tris–HCl (pH 8.6) containing
1.25% papain, 0.16% dithioerythritol and 0.5% dimethylsulphoxide
under a phase-contrast microscope. The variable decondensation times
are due to different sperm sensitivity to the decondensation.
Statistical analysis
An independent χ2-test was used to compare the results obtained for
the two 46,XY/47,XXY men and for the control patient.
Molecular DNA probes
The gonosomal equipment in the spermatozoa of these two patients
and the control patient was analysed by triple FISH with two DNA
probes for the centomeric regions of chromosome 15 (biotinylatedprobe D15Z, Oncor; Illkirch, France) of chromosome X (DXZ1;
M.Morris, Geneva, Switzerland) and one DNA probe for the heterochromatin region of chromosome Y (pHY2.1; J.H. Cooke, Edinburgh,
UK) (Cooke et al., 1982).
In dual FISH, two DNA probes for the centomeric regions of
chromosome 8 (pJM128; American Type Culture Collection,
Rockville, MD, USA) and chromosome 18 (L1.84; American Type
Culture Collection) were used.
The probes were labelled by nick translation with biotin-7-deoxyadenosine triphosphate (Gibco BRL; Cergy Pontoise, France) (L1.84
probe), with digoxigenin-11-deoxyuridine triphosphate (Boehringer
Mannheim, Meylan, France) (pJM128 and DXZ1 probes), or with
fluorescein-12-deoxyuridine triphosphate (Boehringer Mannheim)
(pHY2.1 probe) according to the kit instructions (Boehringer
Mannheim). The probes were tested for specificity on non-sperm cell
populations as previously described (Mercier and Bresson, 1995).
Frequencies of sperm sex chromosome set in the
two 46,XY/47,XXY males and control patient (Table II)
A total of 1691 (patient 1), 811 (patient 2) and 10 091
(control patient) spermatozoa were analysed. The hybridization
efficiency rates were 95.97, 95.92 and 95.66% respectively.
The frequencies of X- and Y-bearing spermatozoa was 50.5/
42.81%, 47.1/44.88%, 48.23/46.95% for patients 1, 2 and
control patient respectively. Only the X/Y ratio estimated from
spermatozoa of patient 1 was different from the expected 1:1
proportion (1.18:1) with an excess of X-bearing cells. In
patient 1, the total frequency of cells with hyperhaploidies for
sex chromosomes was 2.01% (24,XY: 1.3%; 24,XX: 0.71%;
24,YY: 0%). Diploidy was estimated at 0.24% (0.12% of
46,XY; 0.12% of 46,XX) and 0.06% of observed sperm cells
were tetraploid. In patient 2, the total frequency of cells with
hyperhaploidies for sex chromosomes was 3.45% (24,XY:
1.73%; 24,XX: 0.86%; 24,YY: 0.86%) and 0.25% of diploid
cells. In the control patient, the frequency of disomic spermato-
Results
567
F.Morel, C.Roux and J.-L.Bresson
Table II. Frequencies of sperm sex chromosome set in two 46,XY/47,XXY males and control patient
Presumed
Karyotype
Patient 1
Patient 2
Control
23,
24,
46,
X
Y
YY
XX
XY
50.5
47.1
48.23
42.81
44.88
46.95
0.86
0.05
0.71
0.86
0.04
1.3
1.73
0.11
Table III. Disomy 8, 18 and diploidy levels in two 46,XY/47,XXY males
and control patient
Patient 1
Patient 2
Control
8 Disomy
18 Disomy
Diploidy
0.32
0.20
0.25
0.26
0.10
0.21
0.21
0.20
0.12
zoa was 0.20% (24,XY: 0.11%; 24,XX: 0.04%; 24,YY: 0.05%)
and diploid rate was 0.09%.
There was an increased incidence of XY and XX disomies
in the spermatozoa of the two 46,XY/47,XXY men in comparison to those estimated in the control patient (P ⬍ 0.001); the
YY disomy rate in patient 2 was also higher than that of the
control patient (P ⬍ 0.001). The rate of 46,XX diploid cells
was also significantly higher in patient 1 than that of the
control patient (P ⬍ 0.01).
There was no significant difference for either the XY, XX
disomy frequencies or diploidies or tetraploid rate between the
spermatozoa of the two 46,XY/47,XXY men. However, there
was a significant difference between the YY disomic sperm
rate in patient 1 and in patient 2 (0% versus 0.86%).
There was no significant difference for 15 disomy frequencies between the spermatozoa of the two 46,XY/47,XXY men
and the control patient.
Disomy 8, 18 and diploidy levels in the two 46,XY/
47,XXY males and control patient (Table III)
The hybridization efficiency rates were 96.4, 97.36 and 97.61%
with respectively a total of 1890 (patient 1), 1020 (patient 2)
and 10 106 (control patient) spermatozoa analysed.
The 8 disomy frequencies were 0.32, 0.20 and 0.25 for
patients 1, 2 and control patient respectively and the 18 disomy
frequencies were 0.26, 0.10 and 0.21 for patients 1, 2 and the
control patient respectively. The diploid rates were 0.21%
(patient 1), 0.20% (patient 2) and 0.12% (control patient).
There was no significant difference for either the 8 and 18
disomy frequencies or diploidies between the spermatozoa of
the two 46,XY/47,XXY men and the control patient.
Discussion
Using multicolour FISH, the sex chromosomal equipment in
ejaculated spermatozoa from two men with a 46,XY/47,XXY
karyotype were analysed. The use of three colour FISH (X
and Y probes and one autosomal probe) was needed to
distinguish between the gonosomal disomy spermatozoa and
the diploid spermatozoa.
568
YY
92,
24,
XY
XX
XXYY
X/Y⫹15
0.12
0.25
0.09
0.12
0.06
0.35
0.25
0.19
Non-hybridization
or nullisomies
4.03
4.08
4.34
This study showed that the gonosomal chromosomal complements of the two patients were almost identical; in the main,
there was a significantly increased incidence of XY and XX
disomies (also YY disomy for patient 2) in comparison with
the control patient, twice as many XY- (1.3% and 1.73%) as
XX- (0.71% and 0.86%) bearing spermatozoa for patients 1
and 2 respectively.
If this increase is only due to 46,XY cells, meiotic I and II
non-disjunctions in the 46,XY germ cell line should result in
the same proportions of hyperhaploidies (24,XY ⫹ 24,XX ⫹
24,YY) and hypohaploidies (22,0 sperm cells). However, no
increase in the percentage of hypohaploidies was observed in
the spermatozoa of the 46,XY/47,XXY men in comparison to
that estimated in the control. Thus, this increase could be due
to the 47,XXY cells.
The results of this study seem to indicate that some 47,XXY
germ cells are able to complete the meiotic process by
producing spermatozoa with abnormal gonosomal equipment.
A theoretical meiotic distribution of the three gonosomes in
XXY cells would result in an increase of XX and XY
spermatozoa, with twice as many XY- as XX-bearing spermatozoa but without an increase in hypohaploidies. For the haploid
spermatozoa there would be an excess of X-bearing spermatozoa, namely the comparable deviations with those observed in
our results (Figure 1).
Few studies (Chevret et al., 1996; Martini et al., 1996;
Guttenbach et al., 1997; Estop et al., 1998; Foresta et al.,
1998, 1999; Kruse et al., 1998; Lim et al., 1999; Okada et al.,
1999) have analysed the meiotic segregation of gonosomes
from 46,XY/47,XXY or 46,XY/47,XXY/48,XXXY or 47,XXY
men by in-situ hybridization (Table IV).
Only four studies (Chevret et al., 1996; Martini et al., 1996;
Lim et al., 1999; Okada et al., 1999) have analysed the
gonosomal constitution on ejaculate spermatozoa from 46,XY/
47,XXY men with respectively 2.5% (Martini et al., 1996),
10% (Chevret et al., 1996), 30% (Lim et al., 1999) of
47,XXY cells.
Martini et al. (1996) found 2.5% of sex chromosome
aneuploidies with 1.3%, 0.5% and 0.7% of XY, XX and YY
cells respectively; the sex ratio was not significantly different
from the expected 1:1. However, using dual in-situ hybridization (ISH), Martini et al. (1996) could not distinguish nuclei
that had two signals for gonosome as disomic (24,XY, 24,XX
or 24,YY) or diploid (normal 46,XY, 46,XX or 46,YY
respectively).
Chevret et al. (1996), Lim et al. (1999) and Okada et al.
(1999), using triple FISH, found 2.203%, 1.23% and 2.40%
of sex hyperhaploidies respectively. The diploid rates were
FISH analysis in spermatozoa of 46,XY/47,XXY men
Figure 1. Theoretical distribution of the segregation of the three gonosomes in a 47,XXY spermatogonia.
Table IV. Summary of different results concerning the presumed karyotype of gametes in 46,XY/47,XXY men
Presumed
karyotype
46,XY/47,XXY
Chevret et al. (1996)
Martini et al. (1996)
Lim et al. (1999)
Okada et al. (1999)
46,XY/47,XXY/48,XXXY
Kruse et al. (1998)
47,XXY
Guttenbach et al. (1997)
Foresta et al. (1998)
Estop et al. (1998)
Okada et al. (1999)
Foresta et al. (1999)
a22,X or Y and ambiguous.
bSum of diploid cells.
c22,-X or -Y and 44,-XX, -XY
d22,0
23,
24,
25,
XXY
X
Y
YY
XX
52.78
46.70
46.74
43.18
43.88
43.50
49.62
47.51
0.003
0.70
0.06
0.16
0.11
0.50
0.29
1.12
2.09
1.30
0.41
0.96
0.47
0.16
50.5
42.1
2.0
5.0
0.5
43.43
51.87
56.00
20.8
42.21
48
45
48.82
24.60
28.63
29.2
46.40
24
25
0.09
0.21
0.09
0.17
1.22
6.92
3.34
1.01
8
5
XY
1.36
14.58
10.03
25
1.34
12
20
0.09
0.17
46,
92,
24,
XXYY X/Y⫹auto
YY
XY
XX
0.02
0.28
0.03
1.70b
0.48 0.32
0.003
0.18
0.71
0.80
Non-hybridization N° SPZ
or nullisomies
0.62a
1.50
5.30
27 097
3800
1701
623
202
0.09
0.05
0.03
0.17
0.05
4.2
0.17
0.50
4.35c
1.77d
0.67
1.88d
20.8c
7.71
8d
5d
2206
10 000
10 000
24
597
25
20
or -YY.
and/or 45,X/Y.
estimated at 0.33% (Chevret et al., 1996), 1.70% (Lim et al.,
1999) and 0.80% (Okada et al., 1999).
Chevret et al. (1996) showed that the sex ratio was significantly different from the expected 1:1 with the proportions of
X- and Y-bearing spermatozoa estimated at 52.78% and 43.88%
respectively. Lim et al. (1999) and Okada et al. (1999) found
the sex ratio did not differ from the expected 1:1 ratio.
These previous results and our study agree with an incidence
of 1.23–3.45% of gonosomal abnormalities in the spermatozoa
of 46,XY/47,XXY men.
Kruse et al. (1998) reported that after dual FISH of 202
sperm nuclei from a patient with XXY[3]/XXXY[45]/XY[1]
mosaicism, the proportion of hyperhaphoid 24,XY and 25,XXY
spermatozoa was 5.0% and 0.5% respectively and of hyperhaploid 24,XX spermatozoa was 2.0%. However, as with
Martini et al. (1996), this study also did not differentiate
between the hyperhaploid and diploid cells.
Recently, five studies have presented FISH results on sex
chromosome segregation in subjects carrying a homogeneous
somatic karyotype 47,XXY (Guttenbach et al., 1997; Estop
et al., 1998; Foresta et al., 1998,1999; Okada et al., 1999).
The estimations of global frequencies of spermatozoa with an
gonosomal hyperhaploid ranged from 2.69% to 25% depending
on the patients with the disomy frequencies evaluated for XY
569
F.Morel, C.Roux and J.-L.Bresson
(1.34 to 25%), XX (0 to 8%) and YY (0 to 0.21%). Thus,
contrary to 46,XY/47,XXY men, in 47,XXY homogeneous
patients the incidence of these abnormalities is much higher
and more scattered, and a high interindividual variability seems
to appear.
All these studies, by ISH or FISH, on mosaic or homogeneous Klinefelter’s patients suggested that 47,XXY germ cells
can produce mature ejaculated spermatozoa, frequently bearing
sex chromosome aneuploidy. It would therefore appear that
the chromosomal risk might be greater for the offspring of
these men than for those of the general population.
In contrast, Mroz et al. (1998) have utilized an XXY mouse
model and suggested that ‘the meiotic abnormalities observed
in spermatozoa from XXY males are attributable to segregation
errors in XY germ cells, rather than to the survival of XXY
germ cells in the testis’.
A theoretical meiotic distribution of the chromosome in XXY
spermatogonia (Figure 1), irrespective of any hypothetical
correction, would result in the same number of normal and
abnormal spermatozoa with, among the normal spermatozoa,
twice as many X- as Y-bearing cells and, among the abnormal
spermatozoa, twice as many XY- as XX-bearing spermatozoa.
Nevertheless, five studies presenting FISH results on sex
chromosome segregation in men carrying a homogeneous
somatic karyotype 47,XXY found, contrary to theory, a minority of sperm nuclei with a gonosomal abnormality (2.69
to 25%).
Thus, a sequential study, applying FISH analysis to testicular
biopsies of the whole spermatogenic process from spermatogonia to spermatozoa in the same individual would provide
enough information to establish the true patterns of meiotic
segregation in male Klinefelter’s syndrome.
Acknowledgements
The authors wish to thank the Centre d’Etude et de Conservation des
oeufs et du Sperme humain, Besançon Franche Comté. We are also
grateful to Professor Cooke of the Western General Hospital of
Edinburgh (UK) and to Dr Morris of the General Hospital of Geneva
(Switzerland) for providing pHY 2.1 and DXZ1, respectively. They
also thank M.Gill for her help with the translation of the manuscript.
This research was supported by the Fondation pour la Recherche
Médicale (FRM) and the Association Régionale pour le Développement des Etudes Biologiques en Génétique et Reproduction Humaines,
Centre Hospitalier Universitaire, Besançon.
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Received on December 2, 1999; accepted on March 8, 2000