Human Reproduction Vol.18, No.11 pp. 2302±2308, 2003
DOI: 10.1093/humrep/deg460
10, 15 reciprocal translocation in an infertile man:
ultrastructural and ¯uorescence in-situ hybridization sperm
study: Case report
B.Baccetti1,2,4, E.Bruni1, G.Collodel1, L.Gambera1,2, E.Moretti1,2, R.Marzella3 and
P.Piomboni1,2
1
Department of Paediatrics, Obstetrics and Reproductive Medicine, Section of Biology, University of Siena, 2Regional Referral
Center for Male Infertility, Azienda Ospedaliera Senese, Siena and 3Department of Pathological Anatomy and Genetics D.A.P.E.G,
University of Bari, Bari, Italy
4
To whom correspondence should be addressed at: Department of Paediatrics, Obstetrics and Reproductive Medicine,
Section of Biology, via T. Pendola 62, 53100 Siena, Italy. E-mail: [email protected]
BACKGROUND: Peculiar sperm defects are described in a sterile man heterozygous for a balanced translocation
t(10;15) (q26;q12). As this structural reorganization was absent in the parents, the translocation must have
appeared de novo in the present patient. METHODS: Spermatozoa were analysed under light and transmission electron microscopy (TEM). Fluorescence in-situ hybridization (FISH) was performed on the lymphocyte karyotype.
Aneuploidy frequencies of chromosomes 18, X and Y in sperm nuclei, not involved in the translocation, were investigated using three-colour FISH. Dual- colour FISH was used to evaluate segregation of chromosomes 10, 15 in decondensed sperm nuclei. Moreover, three-colour FISH, using telomeric probes for chromosomes 10, 15 was performed
in order to distinguish balanced and unbalanced gametes. RESULTS AND CONCLUSIONS: Overall, structural
characteristics indicate general immaturity of the germinal cells. FISH sperm analysis detected an increase in
chromosome 18 disomy (0.81%) suggesting an interchromosomal effect. A high frequency of diploidies, particularly
18,18,X,X and 18,18,X,Y, was also found. FISH segregation analysis for chromosomes 10, 15 indicated that 32.8%
were balanced gametes, whereas 68.2% were unbalanced. Taken together, these data demonstrate in a male carrier
of a reciprocal translocation t(10;15) the presence of diffuse ultrastructural sperm alterations and a high frequency
of sperm aneuploidies. The existence of a correlation among these factors is proposed.
Key words: electron microscopy/¯uorescence in-situ hybridization/male infertility/reciprocal translocation/spermatozoa
Introduction
Human male infertility and chromosomal anomalies are often
closely related. In particular, reciprocal translocations are the
most frequent (1 in 600) structural chromosomal anomalies in
humans (Estop et al., 1997), and among infertile males
chromosomal reorganizations are about 10 times more frequent
than in the general population (Van Assche et al., 1996). The
frequency of chromosomally unbalanced spermatozoa in cases
of reciprocal translocations is 50% on average, and depends
heavily on the chromosomes involved in the translocation (Shi
and Martin, 2001). The risk of miscarriage or birth defects
resulting from unbalanced gamete formation in balanced
translocation carriers is very high.
The possible correlation between human male infertility due
to impaired spermatogenesis and chromosome anomalies
(mainly translocations) was postulated almost 30 years ago
(Chandley, 1975; 1976). Likewise, others (Plymate et al.,
1976; Leonard et al., 1979) found a correlation between
2302
derangement of spermatogenesis and autosomal translocations.
Recently, the correlation between chromosomal anomalies and
human male infertility has become more evident. One group
(Pellestor et al., 2001) reported oligoteratozoospermia in
carriers of reciprocal or Robertsonian chromosomal translocation; nevertheless, they found normal sperm number and
motility in males with different structural chromosomal
anomalies. Other authors (Marmor et al., 1980; Matsuda
et al., 1991; Testart et al., 1996), when examining the
morphology and motility of human spermatozoa with light
microscopy, did not ®nd any correlation between sperm quality
and chromosome translocations or inversions.
Electron microscopy enables more detailed evaluation of
sperm alterations (Baccetti, 1984; Zamboni, 1987; Bartoov
et al., 1994; Chemes et al., 1998), and permits a distinction to
be made between phenotypic and genotypic defects (Baccetti,
2000; Baccetti et al., 2001). Hereditary male sterility due to
genotypic sperm defects is a possibility. The original idea of
Human Reproduction 18(11) ã European Society of Human Reproduction and Embryology 2003; all rights reserved
Infertile male 10, 15 reciprocal translocation carrier
generic human male infertility correlated to chromosome
anomalies could give way to the concept that male infertility is
due to particular defects, caused by chromosome anomalies,
heralding research into the genes responsible for hereditary
sperm characteristics and their mutations. Recently, ®brous
sheath dysplasia of `stump' spermatozoa was associated with
pericentric inversion of chromosome 9 (Baccetti et al., 1997).
However, the clearest connection between a sterilizing human
sperm defect and a speci®c chromosome alteration was the
discovery of mutations in the DNAI1 gene, mapping to 9 p13p21, in three Kartagener's syndrome patients (Blouin et al.,
2000; Guichard et al., 2001). In studying the association
between chromosome mutations and sperm defects in sterile
men, immature sperm were found in a human carrier of
Robertsonian translocation 14;22 (Baccetti et al., 2002). This
anomaly was most likely in a chromosome region involved in
spermatogenesis.
Herein, peculiar ultrastructural sperm defects are described
in a sterile male carrier of a balanced translocation t(10;15)
(q26;q12). Fluorescence in-situ hybridization (FISH) was used
in decondensed sperm nuclei to evaluate chromosomes 10, 15
segregation and possible interchromosomal effects, detected
through an increase in aneuploidy frequencies of chromosomes
18, X, Y that were not involved in the translocation.
Materials and methods
A 32-year-old man, an only child, was referred to the authors'
laboratory for semen analysis after 3 years of sexual intercourse
without conception. His wife, who was aged 30 years, had no apparent
fertility problem. A testicular echo-colour-Doppler examination did
not reveal the presence of any functional alterations such as varicocele
or hydrocele; testicle size was small bilaterally. The subject had never
smoked, drunk alcohol or been addicted to drugs, and was not exposed
to chemical contaminants or radiation. Baseline plasma concentrations
of FSH, LH, estradiol, cortisol, prolactin, thyroid-stimulating hormone
(TSH) and free thyroxine were normal, but the testosterone level was
low. The patient had never received hormone therapy.
Karyotype
Conventional cytogenetic analysis of 24±48 h cultures of blood
lymphocytes of the patient and his parents was performed using
standard techniques and evaluated by Giemsa-Trypsin-Giemsa (GTG)
banding at about the 400 band level according to the 1995
International System for Human Cytogenetic Nomenclature (ISCN,
1995).
Karyotype FISH analysis was performed with a painting probe for
chromosome 15, obtained from human±hamster hybrid (GM11418)
retaining chromosome 15 as the only human contribution (a kind gift
from the Coriell Institute for Medical Research, NJ, USA).
DNA from the hybrid was dual Alu-PCR ampli®ed according to
published methods (Liu et al., 1993). The PCR products were biotinlabelled by nick translation and used as probe for FISH experiments on
chromosome metaphases of phytohaemagglutinin (PHA)-stimulated
peripheral blood lymphocytes of the patient. Chromosome preparations were hybridized in situ essentially as described previously
(Lichter et al., 1990), albeit with minor modi®cations (Marzella et al.,
1997). In FISH experiments, chromosomes were identi®ed by
diamino-phenylindole (DAPI) counterstaining, and digital images
obtained using a Leika epi¯uorescence microscope equipped with a
cooled charge-coupled device (CCD) camera. Cy3 (Amersham) and
DAPI ¯uorescence signals were recorded separately as grey-scale
images. Pseudocolouring and merging were performed using commercial Adobe Photoshop software.
Light and electron microscopy
Semen samples were obtained by masturbation after 4 days of sexual
abstinence and examined after liquefaction for 30 min at 37°C.
Volume, pH, concentration and motility were evaluated following
published guidelines (World Health Organization, 1999). Semen
analysis was performed three times at 6-month intervals.
For electron microscopy, a single sperm sample was ®xed in cold
Karnovsky ®xative and maintained at 4°C for 2 h. Fixed semen was
washed in 0.1 mol/l cacodylate buffer (pH 7.2) for 12 h, post®xed for 1
h at 4°C in 1% buffered osmium tetroxide, dehydrated, and embedded
in Epon Araldite. Ultra-thin sections were cut with a Supernova
ultramicrotome (Reickert Jung, Vienna, Austria), mounted onto
copper grids, stained with uranyl acetate and lead citrate, observed
and photographed with a Philips CM 10 transmission electron
microscope (TEM; Philips Scienti®cs, Eindhoven, The Netherlands).
A total of 300 ultra-thin sperm sections was examined for major
submicroscopic characteristics. TEM data were evaluated using a
mathematical formula (Baccetti et al., 1995).
An aliquot from the same sperm sample was also processed for
scanning electron microscopy (SEM), ®xing the spermatozoa as
described above and smearing them onto poly-lysine (1%) -coated
coverslips. After dehydration, specimens were dried using the critical
point technique, coated in gold and examined using a Philips CM 515
scanning electron microscope (Philips Scienti®cs).
FISH analysis of spermatozoa
An aliquot from the same sperm sample as used for TEM and SEM
analyses was washed with 150 mmol/l NaCl and 10 mmol/l Tris±HCl
(pH 8), smeared onto glass slides and air-dried. Slides were then ®xed
in methanol:acetic acid (3:1) for 10 min, dehydrated in 70, 80 and
100% cold ethanol, and air-dried. Samples were swell-treated with
0.01 mol/l dithiothreitol (Biorad) in 0.1 mol/l Tris±HCl (pH 8),
followed by 20 mmol/l 3,5-diiodosalicylic acid, lithium salt (Sigma)
in the same buffer, checking sperm head swelling. The slides, rinsed in
23 standard saline citrate (SSC), pH 7, air-dried and then dehydrated
and denatured in 70% formamide (Aldrich) 23 SSC at 73° for 4 min.
Slides were then quickly dehydrated in a graded ethanol series at 0°C
and air-dried. During this last step, chromosome enumeration probes
(Vysis, IL, USA), a-satellite DNA probes for chromosomes X, Y, 10,
15 and 18 directly labelled with different ¯uorochromes, were used.
Moreover, self-made telomeric probes RP11-10D13 for 10p, RP11108K14 for 10q and RP11-9B21 for 15q, were used to distinguish
alternate and adjacent-1 segregations.
The probe mix was denatured for 5 min at 73°C in a water bath.
Hybridization was carried out at 37°C in a moist chamber for 12 h. The
slides were then washed with 0.43 SSC±0.3% Nonidet P40 (NP40)
for 2 min at 73°C, quickly in 23 SSC±0.1% NP40 at room
temperature, and ®nally mounted with DAPI 125 ng/ml in antifade
solution (Vysis). In total, 3023 spermatozoa were analysed using
triple-colour FISH (18, X, Y), 2606 were scored by dual-colour FISH
(10, 15), and 2100 sperm cells were examined with telomeric probes.
Scoring criteria
The overall hybridization ef®ciency was >99%. Sperm nuclei were
scored according to published criteria (Martin and Rademaker, 1995).
According to these criteria, sperm nuclei are scored only if they are
intact, non-overlapping and have a clearly de®ned border. In the case
of aneuploidy, the presence of a sperm tail was con®rmed. A
spermatozoon was considered disomic if the two ¯uorescent spots
2303
B.Baccetti et al.
Figure 1. Fluorescence in-situ hybridization (FISH) analysis of lymphocyte karyotype performed with speci®c painting probes for
chromosome 15, showing reciprocal translocation t(10;15) (10q26;15q12). Chromosome 15 shows a breakpoint at 15q12 and chromosome 10
at 10q26, giving rise to der(10)t(10;15) and der(15)t(10;15). Note normal chromosome 10, normal chromosome 15, derivative chromosome
10, fused with a fragment of chromosome 15 devoid of centromere, and fragment of derivative chromosome 15 including centromere, fused
with telomeric region of chromosome 10.
were of the same colour, comparable in size, shape and intensity and
positioned inside the edge of the sperm head at least one domain apart.
Diploidy was recognized by the presence of two double ¯uorescent
spots, following the above criteria. Observation and scoring was
performed on a Leitz Aristoplan Optic Microscope equipped with
¯uorescence apparatus, with a triple bandpass ®lter for Aqua, Orange,
Green Fluorochromes (Vysis) and a monochrome ®lter for DAPI.
Controls
Semen samples from seven healthy normal men (age range 26±39
years) were collected to constitute a control group. All subjects were
of proven fertility and showed normal seminal parameters, according
to published guidelines (World Health Organization, 1999).
Ultrastructural analysis evaluated by the mathematical formula
(Baccetti et al., 1995) con®rmed that sperm quality was in the range
of natural fertility.
Results
Lymphocyte karyotyping performed with conventional GTGbanding revealed balanced reciprocal translocation 10;15
(10q26;15q12). The karyotypes of the patient's parents were
normal.
FISH analysis on chromosomal metaphases from blood
lymphocytes using speci®c painting probes for chromosome 15
2304
Table I. Semen analysisa
Parameter
Valueb
Volume (ml)
pH
Sperm concentration (3106/ml)
Motility (%; 30 min)
Absent
In situ
Slow progressive
Rapid progressive
1.5 6 0.5
7.6
42.0 6 4.7
94.0 6 0.5
0
2.0 6 1.1
4.0 6 2.6
aBaseline
bValues
seminal parameters evaluated 30 min after ejaculation.
are mean 6 SEM of three examinations.
showed a reciprocal translocation involving chromosomes 10
and 15 (Figure 1). Chromosome 15 showed the breakpoint at
15q12 and chromosome 10 at 10q26, giving rise to
der(10)t(10;15) and der(15)t(10;15).
Semen analysis data are reported in Table I. The characteristic feature was a very poor progressive motility of only 6% in
the three samples analysed.
Sperm ultrastructure was evaluated by transmission electron
microscopy as suggested previously (Baccetti et al., 1995). The
percentage of sperm probably devoid of ultrastructural defects
was 0%. The main defects were in the head region, where the
Infertile male 10, 15 reciprocal translocation carrier
Figure 2. TEM micrograph of an immature sperm: a large
cytoplasmic residue (CR) is present around the head showing
deformed acrosome (A) and altered nucleus (N), with uncondensed
chromatin and intranuclear vacuole (iv). Coiled tail shows
disorganized axoneme (AX). Mitochondria (M) are swollen and not
assembled in regular helix. See also translucent vesicle (V) in
cytoplasmic residue. Scale bar = 0.87 mm.
Figure 3. TEM micrograph of sperm: the nucleus (N) has regular
shape, but the acrosome (A) is altered and an intranuclear vacuole
(iv) is present. A large cytoplasmic residue (CR) embeds the neck
region and the middle piece with displaced mitochondria (M). Scale
bar = 0.85 mm.
acrosome was often absent and never normal, having reduced
dimensions, altered position, sparse content, abnormal shape
and often being far from the nucleus (Figures 2 and 3). The
nucleus showed similar defects (Figures 2 and 3); shape was
generally abnormal, spherical or irregular, with uncondensed
chromatin in half of the sections examined, and 10% of sperm
showed double nuclei (Figure 4). Taken together, these
characteristics indicate immaturity of most head structures,
with apoptosis (marginated chromatin) or necrosis (disrupted
chromatin) affecting 20±25% of the total sperm population,
including immature sperm, which represented 70% of the
sperm population. Cytoplasmic residuesÐanother index of
Figure 4. TEM micrograph of a binucleated germinal cell showing
altered acrosome (A) and nuclei (N), with intranuclear vacuoles (iv),
all embedded in a large cytoplasmic residue (CR). Scale bar = 0.87
mm.
Figure 5. TEM micrograph of sperm tail cross-section. A
cytoplasmic residue (CR) embeds a coiled tail. The mitochondria
(M) are swollen and not assembled in a regular helix. The axonemal
pattern (AX) and accessory ®bres (AF) are disorganized; the ®brous
sheath (FS) is scanty and disrupted. Scale bar = 0.19 mm.
immaturityÐwere observed in 25% of sperm, embedding the
head or the midpiece regions (Figures 2±5). Mitochondria were
often swollen and assembled in a very irregular helix (Figure 5).
The tail had normal shape (50%), but the axoneme was
generally anomalous due to missing or abnormal doublets,
dynein arms (99%), accessory ®bres (90%, usually reduced in
size) or ®brous sheath (80%, generally scanty and disrupted)
(Figures 5 and 6). Some of these characteristics may be due to
necrosis, which evidently also occurred in immature spermatozoa.
2305
B.Baccetti et al.
Table II. FISH analysis for chromosomes 18, X and Y
Patient
Controlsa
Normal spermatozoa (%)
Disomy frequency (%)
Diploidy frequency (%)
X-bearing
Y-bearing
18,18
X,X
Y,Y
X,Y
18,18,X,X
18,18,Y,Y
18,18,X,Y
49.60
49.80 6 0.30
46.80
49.58 6 0.22
0.81
0.11 6 0.003
0.17
0.06 6 0.001
0.14
0.08 6 0.001
0.17
0.09 6 0.002
0.80
0.09 6 0.001
0.17
0.11 6 0.002
0.47
0.08 6 0.003
In the patient, 3023 cells were scored by triple-colour FISH analysis.
Frequencies of nullisomies were: 18/0 = 0.47%; X/0 = 0.21%; Y/0 = 0.05%.
Multiple ¯uorescent signals (>4) were detected in 0.14% of cells.
aMean percentages (6 SD) of disomies and diploidies for the examined chromosomes in seven healthy men of proven fertility (controls). The mean number of
sperm scored by triple-FISH analysis was 5600 6 260.
Table III. FISH analysis for chromosomes 10, 15
Disomy frequency
Diploidy frequency
10,10
10,10,15
15,15
15,15,10
10,10,15,15
2.61%
0.90%
0.30%
0.30%
1.15%
In the patient, 2602 sperm were scored by dual-colour FISH analysis.
Frequencies of nullisomies were: 10/0 = 0.70%; 15/0 = 0.50.
Triple sets of chromosomes were detected in 0.20% of cells.
Multiple ¯uorescent signals (>4) were detected in 0.10% of cells.
Figure 6. TEM micrograph of a 9+2 axonemal pattern, but with
some microtubular doublets displaced. Dynein arms (arrows) are
present only in some doublets. Scale bar = 0.03 mm.
SEM analysis of a large cell sample con®rmed the presence
of spermatozoa with immature features as the presence of
spheroid heads, surrounded by large cytoplasmic residues,
detected also in the tail region.
Meiotic segregation in the t(10;15) (10q26;15q12) translocation carrier was investigated by FISH. The aneuploidy
frequencies of chromosomes 18, X and Y, not involved in the
translocation, are summarized in Table II. A very high value of
chromosome 18 disomy and diploidies (particularly 1818XX
and 1818XY) was found in comparison with values of the
control group (Table II). FISH analysis of chromosomes
involved in translocation (10;15) showed a very high percentage of chromosome 10 disomy and diploidy (Table III).
The diploidy of sperm cells could be generated by a
binucleate sperm head or by diploid nuclei. The ®rst case was
demonstrated by the presence of a membranous septum
between the two nuclei; the second was deduced by the
presence of double sets of 10, 15, and 18, X, Y chromosomes.
Almost the same frequency of diploid cells (mean 1.285 6
0.19) was detected in the scoring of double- and triple-colour
FISH.
2306
With regard to the meiotic process, by using dual-colour
FISH 93.4% of sperm were found with both 10, 15 chromosome signals, indicating an alternate or adjacent-1 segregation
pattern. The products of adjacent-2 segregation were 2.8%, and
those of 3:1 segregation 2.4%, of total products. By using FISH
with telomeric probes for chromosomes 10, 15 it was possible
to distinguish alternate segregation (producing balanced
gametes) in 32.8% of sperm from other segregation patterns,
all of which generated unbalanced gametes in 68.2% of sperm.
Discussion
In this case of male sterility, the patient was heterozygous for a
balanced reciprocal translocation t(10;15) (10q26;15q12)
which was absent in his parents. The translocation therefore
appeared de novo in the present patient. It has been suggested
previously (Olson and Magenis, 1988) that chromosome
rearrangements are of preferential paternal origin.
In infertile men, structural chromosomal anomalies occur
more frequently than in the general population (Van Assche
et al., 1996; Kalantari et al., 2001). Semen analysis carried out
in a large population of infertile males (Bourrouillou et al.,
1997; Pandiyan and Jequier, 1996) revealed a strong correlation
between the rate of chromosomal anomalies and the severity of
oligozoospermia. Other authors (Pellestor et al., 2001)
observed oligoteratozoospermia in carriers of structural
chromosomal alterations. Nevertheless, morphological sperm
quality is always evaluated with optical microscopy, sometimes
applying Kruger's strict criteria. Structural sperm anomalies
can be highlighted only by the use of electron microscopy,
which allows detailed evaluation of inner sperm organelles. By
using this method, one group (Quintana de la Rosa et al., 2001)
reported a generalized ¯agellar abnormality in an infertile man
carrier of t (3;22), while others (Baccetti et al., 2002) observed
Infertile male 10, 15 reciprocal translocation carrier
an unusual structural sperm immaturity in a sterile carrier of
t(14;22). In the present report, the electron microscopic analysis
of ejaculated spermatozoa from an infertile male carrier of
reciprocal translocation 10;15, demonstrated severe ultrastructural sperm alterations, indicating diffuse sperm immaturity.
Nevertheless, no particular monomorphic sperm defect was
observed which affected the entire sperm population, as
reviewed previously (Baccetti et al., 2001).
Derangement of meiotic segregation is indicated by the
abnormal chromosomal constitution of spermatozoa detected
using FISH analysis. The high frequency of chromosome
18 disomy could be considered as a positive interchromosomal effect. This effect depends on the type of structural
reorganization, the chromosomes involved and the chromosome breakpoints (Blanco et al., 2000; Anton et al., 2002),
and results in a particular meiotic behaviour, namely unsynapsed regions or preferential meiotic con®gurations that could
lead to the observed increase in chromosome 18 disomies.
Moreover, the high frequency of sperm diploidies detected
by dual- and triple-colour FISH, using centromeric probes,
indicates incomplete meiosis process leading to immature
sperm cells with double nuclei, as observed by TEM, or with a
double chromosome set. Dual-colour FISH performed in sperm
nuclei highlighted the segregation pattern of chromosomes 10
and 15 involved in the translocation. The detection of both
signals in 93.4% of sperm indicated alternate or adjacent-1
segregation patterns, producing normal, balanced or unbalanced gametes. The products of adjacent-2 segregation (2.8%)
and 3:1 segregation (2.4%) were certainly unbalanced gametes.
By using triple-colour FISH and telomeric probes it was shown
that 32.8% of sperm were balanced, but 68.2% were unbalanced. These data were in agreement with previous reports of
between 19 and 77% of the spermatozoa of reciprocal
translocation carriers being chromosomally unbalanced,
while about 50% were chromosomally abnormal (Martin and
Spriggs, 1995; Shi and Martin, 2001).
In conclusion, in this male carrier of a reciprocal translocation t(10;15), the existence of a correlation is proposed
between these diffuse ultrastructural sperm alterations and the
high frequency of sperm aneuploidies. Ultrastructural ®ndings
indicated very poor sperm quality incompatible with natural
fertility and, as in all cases of immature sperm, the only
possibility for fertilization is ICSI. However, the results
obtained with karyotyping and FISH analysis in the present
patient contraindicate the use of arti®cial reproduction techniques due to the high risk of imbalances in zygotes or fetus in
reciprocal translocation carriers.
Acknowledgements
The authors thank Professor Mariano Rocchi from DAPEG of Bari
University (Italy), for his collaboration. This study was supported by
University of Siena, P.A.R., grant 2002.
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Submitted on March 13, 2003; resubmitted on June 16, 2003; accepted on
July 25, 2003