Human Reproduction vol.15 no.2 pp.440–444, 2000
Fluorescence in-situ hybridization of sex chromosomes
in spermatozoa and spare preimplantation embryos of a
Klinefelter 46,XY/47,XXY male
Magdalena Bielanska1, Seang Lin Tan1 and
Asangla Ao1,2,3
Departments of 1Obstetrics and Gynecology and 2Human Genetics,
Royal Victoria Hospital, Women’s Pavilion, McGill University,
Montreal, QC H3A 1A1, Canada
3To
whom correspondence should be addressed
It has been suggested recently that 47,XXY germ cells are
able to progress through meiosis to produce hyperhaploid
spermatozoa. We report on a 46,XY/47,XXY Klinefelter
patient whose spermatozoa were recovered from the ejaculate and used for intracytoplasmic sperm injection (ICSI).
Fluorescence in-situ hybridization (FISH) analysis of the
patient’s spermatozoa and of spare preimplantation
embryos with DNA probes specific for chromosomes X, Y
and 18 revealed sex chromosome hyperploidy in 3.9% of
the sperm nuclei analysed (2.23% XY18, 1.12% XX18,
0.56% YY18), while only three out of 10 spare embryos
analysed were normal for chromosomes tested. The abnormalities included two diploid mosaic embryos with the
majority of the blastomeres normal for the chromosomes
tested, and five embryos with mostly abnormal blastomeres
and chaotic chromosome X, Y and 18 patterns. None of
the embryos analysed showed a XXY1818 or XXX1818
chromosome complement. The frequency of sex chromosome hyperploidy in the spermatozoa of the mosaic Klinefelter patient was higher than the mean reported for
karyotypically normal males, supporting the hypothesis
that 47,XXY germ cells are able to complete meiosis and
produce aneuploid spermatozoa. However, most of the
spermatozoa analysed were normal for sex chromosomes,
and ICSI of the patient’s spermatozoa did not result in a
spare embryo with a uniform 47,XXY or 47,XXX chromosome complement. Instead, fertilization produced a high
percentage of mosaic embryos with chaotic chromosome
arrangements.
Key words: fluorescence in-situ hybridization/intracytoplasmic
sperm injection/Klinefelter syndrome/preimplantation embryos/
sex chromosomes
tile males, accounting for 4.6% of oligozoospermic or azoospermic men (De Braekeleer and Dao, 1991).
Klinefelter patients present with a somatic 47,XXY, or less
commonly with a mosaic 46,XY/47,XXY karyotype. Although
the clinical features are variable, the majority of patients are
infertile due to primary testicular failure. Very few cases of
naturally conceived offspring of proven paternity have been
reported (Laron et al., 1982; Terzoli et al., 1992). The retrieval
of spermatozoa from severely oligozoospermic ejaculates or
from testicular biopsies in Klinefelter patients is possible.
With recent developments in reproductive technologies, the
recovered spermatozoa have been used for intracytoplasmic
sperm injection (ICSI), with successful fertilization of oocytes,
and biochemical pregnancies and births have been achieved
(Harari et al., 1995; Staessen et al., 1996; Bourne et al., 1997;
Hinney et al., 1997; Tournaye et al., 1997; Nodar et al.,
1998; Palermo et al., 1998; Reubinoff et al., 1998; Ron-El
et al., 1999).
Recent chromosome studies have reported that Klinefelter
males have an increased incidence of 24,XY sperm cells
compared with that found in karyotypically normal men,
demonstrating that 47,XXY spermatogonia may undergo
meiosis to produce hyperhaploid spermatozoa (Cozzi et al.,
1994; Chevret et al., 1996; Martini et al., 1996; Guttenbach
et al., 1997; Hinney et al., 1997; Estop et al., 1998; Kruse
et al., 1998). An increase in the frequency of sex chromosome
hyperhaploidy in this population of patients may present a risk
of retrieving sperm cells with an abnormal sex chromosome
complement for ICSI protocols. This, in turn, may increase
the risk of chromosomally imbalanced embryos.
In the present study, to determine whether a 46,XY/47,XXY
Klinefelter patient had an increased risk of a chromosomally
abnormal conceptus, we used three-colour fluorescence in-situ
hybridization (FISH) with probes specific to chromosomes X,
Y and 18 to examine the sex chromosomes in the patient’s
spermatozoa and spare preimplantation embryos produced by
ICSI. To our knowledge, this is the first study which reports
on the sex chromosomes constitution of the sperm cells and
embryos from the same Klinefelter patient.
Materials and methods
Introduction
With an incidence of approximately 1 in 600 in live births,
and 1 in 300 in spontaneous abortions, Klinefelter syndrome
is the most common sex chromosome abnormality in humans
(Hassold and Jacobs, 1984; Nielsen and Wolhert, 1991). It is
also the most frequent chromosomal abnormality among infer440
Patients
The couple, a 36-year-old female and her 35-year-old husband, were
referred to the McGill Reproductive Centre for infertility arising from
primary testicular failure associated with a karyotype consistent with
Klinefelter syndrome. Two peripheral blood chromosome analyses
had been performed. One revealed a 47,XXY karyotype in all 50
cells analysed. The second investigation, at a different medical
© European Society of Human Reproduction and Embryology
Hyperploidy in spermatozoa and embryos in a Klinefelter male
laboratory, disclosed the probable presence of low-level mosaicism,
with one cell in 60 showing a 46,XY karyotype. The patient had an
elevated concentration of follicle stimulating hormone (FSH). Past
seminal analysis demonstrated severe oligozoospermia or azoospermia. Analysis of semen used for ICSI showed severe oligozoospermia,
with a sperm count of 0.1⫻106 per ml of ejaculate and motility of
60%. The wife had no evidence of fertility problems. The couple
opted for preimplantation genetic diagnosis (PGD). However, due to
technical difficulties, FISH results could not be obtained on the
biopsied blastomeres. After consultation with the patients, three
embryos of unknown sex chromosome constitution were transferred,
and resulted in an ongoing twin pregnancy.
Preparation of sperm nuclei
Supernumerary spermatozoa suspended in sperm washing medium
(Medicult, Hopkington, USA) were obtained after ICSI had been
performed according to standard protocols (Palermo et al., 1998).
Spermatozoa were prepared for FISH as described previously (Martin
and Ko, 1995). Spermatozoa were washed three times with 10 mmol/
l Tris–0.9% NaCl. An aliquot (2–5 µl) of sperm suspension was
smeared on glass slides and air-dried. Slides were aged at room
temperature for 2 days. Sperm heads were decondensed by a 30 min
immersion in dithiothreitol (DTT)/0.1 mol/l Tris, followed by a 3 h
immersion in a 1 mmol/l DTT/10 mmol/l lithium diiosalicyalte/0.1
mol/l Tris solution at room temperature. Slides were rinsed in 2⫻
SSC solution for 5 min, and air-dried.
Preparation of blastomeres
Fifteen out of 19 normally fertilized embryos were cultured under
oil in Gardner’s sequential media (Scandinavian IVF Science,
Gothenburg, Sweden) to day 4. Ten spare, monospermic embryos,
ranging from the 6- to 11-cell stages of development, were washed
in phosphate-buffered saline (PBS). Each embryo was transferred in
a microdroplet to a poly-L-lysine-treated slide. Blastomeres were
desegregated and nuclei spread with 0.01 NHCl, 0.1% Tween 20
(Coonen et al., 1994). The slides were air-dried, washed in PBS for
5 min, and dehydrated through an ethanol series. The location of the
nuclei was marked using a diamond pen.
FISH
Immediately before FISH analysis, slides with embryonic or sperm
nuclei were pretreated with pepsin (100 µg/ml) in 0.01 mol/l HCl for
20 min at 37°C, rinsed in PBS, and dehydrated in an ethanol series.
Three-colour FISH was performed using directly labelled alpha
satellite DNA probes, specific for chromosomes X (spectrum green),
Y (spectrum orange) and 18 (spectrum aqua) (Vysis, Downers Grove,
IL, USA).
Sperm nuclei FISH
Slides with sperm nuclei were denatured in a Coplin jar containing
70% formamide/2⫻ saline sodium citrate (SSC) at 72°C, for 2 min.
Slides were immediately dehydrated in a cold ethanol series, and airdried. The probe mixture was denatured at 75°C for 5 min, and
immediately applied to the specimen. The probe was hybridized to
sperm DNA by incubation in a moist chamber at 37°C for 4 h. Slides
were washed in 0.04⫻ SSC/0.3% Tween 20 solution for 3 min,
followed by 2⫻ SSC, at room temperature, for 1 min. Slides were
rinsed in PBS and counterstained with 2 ng/ml of 4⬘,6-diamidino-2phenylindole (DAPI) for 20 s, rinsed and mounted in anti-fade
medium (Vector, Burlingame, CA, USA).
Embryonic nuclei FISH
The probe mixture was applied to the slide with embryonic nuclei
and covered with a plastic coverslip. Probe and target nuclear
Table I. Results from fluorescence in-situ hybridization of patient’s
spermatozoa with probes specific for chromosomes X, Y and 18
FISH
results
Presumed
karyotype
No. of
spermatozoa
% of spermatozoa
X18
Y18
XX18
YY18
XY18
XY1818
XXY18
18
Total
23,X
23,Y
24,XX
24,YY
24,XY
46,XY
24,XXY,-18
22, -X or -Y
180
158
4
2
8
3
1
2
358
50.28
44.13
1.12
0.56
2.23
0.84
0.28
0.56
100
DNA were co-denatured by heating the slide to 78°C for 6 min.
Hybridization was carried out in a moist chamber at 37°C for 2 h.
Following hybridization, the slides were washed using 0.04⫻ SSC/
0.3% Tween 20 solution, at 73°C, for 3 min. The slides were airdried, and mounted in DAPI (II) counterstain (Vysis).
Analysis
Following FISH, the nuclei and fluorescence signals were viewed
using a fluorescence Olympus DX 60 microscope equipped with
appropriate filters: FITC/Texas red/DAPI (Applied Imaging, Santa
Clara, CA, USA) and Aqua (Vysis). Only intact, non-overlapping
embryonic nuclei and sperm heads, with clear fluorescence signals
were scored. Two signals of the same colour were considered to
represent two individual chromosomes only when the same-coloured
signals were a minimum of one diameter apart. Images were captured
using a CCD camera and Cytovision software (Applied Imaging).
Results
Spermatozoa
FISH results from a total of 358 spermatozoa available for
analysis are presented in Table I. In total, 338 (94.4%) sperm
cells were normal with an X18 (50.28%) or a Y18 (44.13%)
chromosome pattern. The difference in 23Y- and 23X-bearing
spermatozoa was not statistically significant. An abnormal
chromosome complement was detected in 5.6% of spermatozoa. Sex chromosome disomy was detected in 3.9% of nuclei
and included cells with XY18 (2.23%), XX18 (1.12%) and
YY18 (0.56%) signals (Figure 1a). Other abnormal chromosome patterns detected included XY1818 (0.84%), XXY18
(0.28%) and 018 (0.56%).
Embryos
A total of 80 nuclei, obtained from 75 blastomeres, were
spread from ten day 4 embryos. Upon analysis, 67 (84%)
yielded intact nuclear material with large, bright fluorescence
signals. The results of FISH using probes specific for chromosomes X, Y and 18 are shown in Table II. Of ten normally
fertilized spare embryos analysed, three were normal for
chromosomes tested, one with an XX1818, and two with an
XY1818 pattern. Two embryos were diploid mosaic, with
the majority of blastomeres uniformly normal for the three
chromosomes tested (Figure 1b). Five embryos consisted of
mostly abnormal blastomeres, with a variable number of XY18
441
M.Bielanska, S.L.Tan and A.Ao
Figure 1. Fluorescence in-situ hybridization of Klinefelter syndrome patient’s spermatozoa and spare preimplantation embryo with CEP®
alpha satellite DNA probes specific for chromosomes X (green), Y (red) and 18 (aqua). (a) An abnormal sperm nucleus showing an XY18
chromosome complement. (b) Normal nucleus of a blastomere from a mosaic embryo showing XY1818 chromosome signals. (c–f)
Abnormal nuclei from four blastomeres of a chaotic embryo showing varied number of XY18 signals, (c) Y181818, (d) XXYY1818, (e)
X1818, (f) Y18. (Original magnification,⫻1000.)
signals in the nuclei, consistent with chaotic chromosome
segregation (Figure 1c–f). None of the seven spare embryos
with chromosomally abnormal blastomeres showed a uniform
XXY1818 or XXX1818 complement. Three embryos of
unknown sex chromosome constitution which had been transferred resulted in an ongoing twin pregnancy. Amniocentesis
showed both fetal karyotypes to be 46,XX.
Discussion
Most Klinefelter patients with a pure 47,XXY somatic karyotype are azoospermic, and spermatozoa for ICSI are retrieved
via testicular biopsy (Staessen et al., 1996; Tournaye et al.,
1996; Nodar et al., 1998; Palermo et al., 1998; Reubinoff
et al., 1998; Ron-El et al., 1999). In contrast, in cases with a
46,XY/47,XXY mosaic karyotype, spermatozoa can usually
be recovered from the ejaculate (Harari et al., 1995; Martini
et al., 1996; Guttenbach et al., 1997; Hinney et al., 1997;
Kruse et al., 1998; Okada et al., 1999). Our patient’s cytogenetic analysis had shown a dominantly 47,XXY karyotype,
with only one 46,XY cell in 60. The proband was diagnosed
with primary testicular failure; however, his ejaculate contained
enough spermatozoa for ICSI and for subsequent FISH analysis.
This finding demonstrates the presence of limited spermatogenesis in this patient. Furthermore, it indicates testicular
mosaicism, and suggests that the one 46,XY cell observed was
not an artefact. Perhaps the mosaicism also extended to, and
442
was present in various percentages in, other non-investigated
tissues.
Multi-colour FISH using chromosome-specific DNA probes
has been successfully used to determine the frequencies of
numerical chromosome abnormalities in the spermatozoa of
both normal and infertile men (Moosani et al., 1995; Martin
et al., 1996; Rademaker et al., 1997; Finkelstein et al., 1998;
Storeng et al., 1998; Pang et al., 1999). Despite the scarcity
of spermatozoa in many Klinefelter syndrome males, a number
of studies were able to analyse from 24 to 27 097 spermatozoa
per patient (Martini et al., 1996; Hinney et al., 1997; Estop
et al., 1998; Kruse et al., 1998). In the present study, we were
able to screen chromosomes X, Y and 18 in a total of 358
available spermatozoa.
The total incidence of sex chromosome disomy in our
patient’s spermatozoa was 3.9%. This value is higher than that
determined by FISH (0.14% to 0.43%) for karyotypically
normal fertile men (Williams et al., 1993; Wyrobek et al.,
1994; Martin et al., 1996; Spriggs et al., 1996; Rademaker
et al., 1997; Storeng et al., 1998). It is also higher than the
reported mean values of sex chromosome disomy for males
with abnormal seminal parameters (0.33% to 2.95%) (Moosani
et al., 1995; Storeng et al., 1998; Pang et al., 1999). The total
frequency of sex chromosome hyperploidy for our patient falls
in the range ascertained in other mosaic (0.92% to 5.0%) and
non-mosaic (1.36% to 25%) Klinefelter syndrome individuals
Hyperploidy in spermatozoa and embryos in a Klinefelter male
Table II. Results of fluorescence in-situ hybridization of patients’ spare embryos with probes specific for
chromosomes X, Y and 18
Embryo
number
No. of
blastomeres
No. of
nuclei
1
2
8
6
8
6
3
4
5
6
7
6
11
7
7
8
11
7
7
7
7
8
8
10
9
10
10
10
Total
5
75
6
80
FISH results (No. of nuclei)
Embryo diagnosis
XX1818 (5)
XY1818(1)/Y(1)/XXYY(1)/X(1)/
XY18 (1)/Y1818 (1)
XX 1818(2) / X1818(3) / X18(2)
XX1818(3) / 18 (1)
XY1818(8)
XX1818(2)/XXX18181818(3)/
XXXX18181818 (1)/XX18(1)
XY1818(2)/XXY181818 (2)/
XXXY18181818(2)/18(1)
XY1818(6)/XXYY181818(1)/
XX1818(1)/X(1)
XY1818(1)/ X1818(3)/Y18(1) Y(1)/
Y181818(1)/XXYY18181818(1)/
XXYY1818(1)
XY1818(5)
(67)
Diploid
Chaotic mosaic
(Cozzi et al., 1994; Chevret et al., 1996; Martini et al., 1996;
Estop et al., 1998; Kruse et al., 1998).
Non-disjunction in normal 46,XY spermatogonia in meiosis I
should produce the same number of 24,XY and 22,-X,-Y
sperm cells. Non-disjunction in meiosis II should produce
equal frequencies of spermatozoa with 24,XX, 24,YY and
sex chromosome hypohaploidy. In contrast to the increased
frequency of spermatozoa with sex chromosome disomy
(3.9%), only 0.56% of nuclei from our patient’s spermatozoa
did not contain gonosomes. The lack of corresponding sperm
cells nullsomic for sex chromosomes in our patient suggests
that the 24,XY and 24,XX spermatozoa probably arose from
the abnormal 47,XXY spermatogonia, thus supporting the
evidence from previous studies that these germ cells can
complete meiosis.
Although the number of Klinefelter syndrome cases with a
known chromosomal constitution of the zygote following ICSI
is limited, no affected progeny have been reported to date. A
normal karyotype, following fertilization with a Klinefelter
patient’s spermatozoon, was detected as a 9-week fetus (Hinney
et al., 1997). To date, the 11 liveborn in-vitro-conceived
Klinefelter patients’ offspring have also been found to be
chromosomally normal (Bourne et al., 1997; Tournaye et al.,
1997; Nodar et al., 1998; Palermo et al., 1998; Reubinoff
et al., 1998; Ron-El et al., 1999).
Two recent studies used FISH to analyse preimplantation
embryos following ICSI with a Klinefelter patient’s spermatozoa. In the first (Staessen et al., 1996), PGD was performed
with probes for chromosomes X,Y on five embryos, from a
total of three patients. All five embryos were diagnosed with
a normal chromosome content. The second study (Reubinoff
et al., 1998) reported on PGD with probes for chromosomes
X, Y and 18 in three embryos from two patients. One embryo
was diagnosed as normal, was transferred, and resulted in
the birth of a karyotypically normal neonate. The biopsied
blastomere from the second embryo had an abnormal XX18
constitution. The blastomere from the third embryo was found
Chaotic mosaic
Diploid mosaic
Diploid
Chaotic mosaic
Chaotic mosaic
Diploid mosaic
Chaotic mosaic
Diploid
also to contain an abnormal pattern, XXY1818. Spreading of
the remainder of the cells from the above two embryos revealed
nuclei with various X and Y patterns, consistent with chaotic
cell division. Therefore, among all the abnormal embryos
combined, no uniform XXY or XXX chromosome complement
had been detected. Our observations in 10 spare preimplantation
embryos are very similar. Although only three (33%) of
the spare embryos produced by ICSI using our patient’s
spermatozoa were uniformly normal, none of the abnormal
embryos analysed was uniformly aneuploid for the sex chromosomes. Instead, as in the above reports, the abnormal embryos
were mosaics with chaotic chromosome segregation patterns.
Intracytoplasmic fertilization of oocytes with a Klinefelter
male’s 24,XY- or 24,XX-bearing spermatozoa, could theoretically produce a 47,XXY or a 47,XXX embryo. This chromosomal status of ICSI-induced conceptions cannot be excluded
from the three transferred embryos among the six reported
(Reubinoff et al., 1998), though chromosome analysis was not
performed. Similarly, the gonosome constitution of one of the
three embryos that had been transferred to our patient remains
unknown. Furthermore, diagnosis of six embryos reported
earlier by one group (Reubinoff et al., 1998) and four embryos
reported by another group (Staessen et al., 1996) was based
on only single blastomeres. The incidence of chromosomal
mosaicism in in-vitro-fertilized human preimplantation
embryos is substantial (Coonen et al., 1994; Munné et al.,
1994; Harper et al., 1995; Handyside, 1996; Delhanty et al.,
1997). Among the seven abnormal embryos produced from
our patient’s spermatozoa, all contained at least one normal
XX1818 or XY1818 blastomere in addition to those which were
chromosomally abnormal. Therefore, mitotic non-disjunction
and/or sex chromosome loss in one blastomere of a 47,XXY
or 47,XXX embryo, followed by a biopsy of this now ‘rescued’
blastomere in the embryos diagnosed by PGD is possible.
Such an event would lead to a misdiagnosis. It is therefore
important to advise PGD patients to undergo prenatal diagnosis
to ensure the normality of the fetus.
443
M.Bielanska, S.L.Tan and A.Ao
In conclusion, despite an increased frequency of sperm
hyperhaploid for sex chromosomes, FISH analysis of our
patient’s spermatozoa indicated that the majority of spermatozoa (94.4%) were normal for the chromosomes tested. This
agrees with findings in other Klinefelter cases, and confirms
that the karyotype in peripheral blood in this population of
patients is not indicative of the chromosome constitution of
other tissues such as the testis. Furthermore, ICSI of the
patient’s spermatozoa induced fertilization, produced a high
percentage of cleavage-stage embryos, and led to implantation
and ongoing pregnancy. None of the spare embryos analysed
had a Klinefelter syndrome karyotype. In order to have a clear
understanding of the risk of affected progeny in Klinefelter
males procreating via ICSI, further screening for gonosomes
in spermatozoa, preimplantation spare embryos, concepti and
neonates of this population of patients is necessary.
Acknowledgements
The authors wish to thank the couple for the donation of the
spermatozoa and spare preimplantation embryos for the study, and
the embryologists and clinicians at the McGill Reproductive Centre
for their assistance and cooperation.
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Received on July 2, 1999; accepted on November 4, 1999