Molecular Human Reproduction vol.3 no.7 pp.585–598, 1997
REVIEW
Detection of chromosomes and estimation of aneuploidy in human
spermatozoa using fluorescence in-situ hybridization
Sarah E.Downie, Sean P.Flaherty1 and Colin D.Matthews
Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital, Woodville, South
Australia 5011, Australia
1To
whom correspondence should be addressed
The development and application of fluorescence in-situ hybridization (FISH) has opened the way for
comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly
applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa
after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosomespecific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent
detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly
used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date
have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are
beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi-probe FISH studies
have generally reported disomy (hyperhaploidy) estimates of 0.05–0.2% per chromosome. There is preliminary
evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction
during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for
specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful
technique that has many applications in reproductive medicine, it must also be realized that it does have
limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.
Key words: aneuploidy/chromosome/FISH/human spermatozoa/in-situ hybridization
Definitions
Mammalian spermatozoa are haploid cells (n 5 23) which
contain 22 autosomes and one sex chromosome, either the X
or Y. Disomy (hyperhaploidy) is the condition in which a
spermatozoon has an extra chromosome (n 1 1) while nullisomy (hypohaploidy) indicates that a spermatozoon is missing
a chromosome (n – 1). Disomy and nullisomy are examples
of aneuploidy, the condition in which a cell does not have an
exact multiple of the haploid number. Diploid spermatozoa
have 44 autosomes and two sex chromosomes (XX, YY or XY).
Introduction
Historically, the first technique used to study chromosomes in
human spermatozoa was differential staining of specific regions
of chromosomes. Pearson and Bobrow (1970) used fluorescent
quinacrine to stain the distal two thirds of the long arm of
the Y chromosome (Y body) and estimated that 1.4% of
spermatozoa were aneuploid for the sex chromosomes. Subsequently, the incidence of two Y bodies in human spermatozoa
was reported to be 1.3% (Sumner et al., 1971) and 5% (Klasen
and Schmid, 1981). Autosomes have also been studied using
a Giemsa stain for the secondary constriction of chromosome
© European Society for Human Reproduction and Embryology
9 (Bobrow et al., 1972; Pearson, 1972) and a Leishman’s stain
for chromosome 1 (Geraedts and Pearson, 1973). An average
aneuploidy rate of ~2% per chromosome was reported, giving
a total aneuploidy rate of 38% if all chromosomes were
considered together (Pawlowitski and Pearson, 1972). These
estimates were excessive and unreliable due to non-specific
staining of chromosomes.
The direct visualization of human sperm chromosomes in
the ooplasm of zona-free hamster oocytes was introduced by
Yanagimachi et al. (1976) and subsequently applied by Rudak
et al. (1978). Since then, many studies have been conducted
on the spermatozoa of men with normal and abnormal karyotypes, and .20 000 sperm chromosome complements have
been examined. The majority of these studies were on small
sample sizes (Martin, 1993). It was frequently found that the
number of nullisomic spermatozoa was twice that of disomic
spermatozoa. This discrepancy was generally attributed to loss
of chromosomes during fixation, so a conservative estimate of
aneuploidy was derived by doubling the disomy rate, and this
yielded total aneuploidy frequencies of 0.9–4.0% (Kamiguchi
and Mikamo, 1986; Estop et al., 1991; Benet et al., 1992).
Sperm karyotypes were only recorded in four of the larger
studies, in which disomy frequencies of 0.7, 0.98, 0.6 and
585
S.E.Downie et al.
0.6% were found and the conservative estimates of aneuploidy
ranged from 1.2–1.9% (Brandriff et al., 1985; Brandriff and
Gordon, 1990; Martin, 1990; Mikamo et al., 1990). This
method yielded valuable data because it examines the entire
chromosome complement of each spermatozoon, and detects
structural and numerical abnormalities. However, sperm karyotyping is labour-intensive and time-consuming (Jacobs, 1992;
Martin, 1993), and the results are potentially biased in that
only those human spermatozoa which can fertilize hamster
oocytes are karyotyped; this may eliminate spermatozoa with
genetic mutations or morphological disadvantages that preclude
them from fusing with oocytes. Nevertheless, it provides useful
baseline data with which to compare results obtained using its
successor, fluorescence in-situ hybridization (FISH) (Martin
et al., 1993, 1996; Robbins et al., 1993; Spriggs et al., 1996;
Van Hummelen et al., 1996).
Fluorescence in-situ hybridization
In-situ hybridization (ISH) involves hybridization of a chromosome-specific DNA probe to complementary sequences on a
target chromosome followed by detection of the bound probe
(and hence the chromosome). Isotopic detection methods were
originally employed but are limited by long autoradiography
times and the generation of broad, indistinct signals. Hence,
they have been largely replaced by non-isotopic methods, in
particular FISH, a technique in which the probes are detected
using fluorochromes (red, green or blue) and indirect or direct
detection procedures (Trask, 1991). Indirect FISH utilizes a
DNA probe which contains a hapten such as digoxigenin
(DIG) or biotin. After hybridization of the probe to the target
DNA, the hapten is detected using a fluorochrome-conjugated
binding protein such as avidin (for biotinylated probes) or
a fluorochrome-conjugated antibody (for DIG). The main
advantages of indirect FISH are high sensitivity, the ability
to intensify the signal using sandwich techniques in which
consecutive amplifications of the signal are achieved using
antibodies, and the extensive range of detection reagents.
Disadvantages are cost, extended staining times and higher
background labelling (reduced signal-to-noise ratio). In the
direct FISH procedure, the fluorochrome is incorporated directly into the probe so that the DNA-probe complex can
be visualized by fluorescence microscopy without additional
detection steps. Probes labelled with fluorescein isothiocyanate
(FITC), tetramethyl rhodamine isothiocyanate (TRITC), aminomethyl coumarin acetic acid (AMCA), Texas Red (TR) and
cyanine dyes (Cy3 and Cy5) have been used (Trask, 1991;
Yurov et al., 1996). Vysis (Framingham, MA, USA) supplies
probes which are directly labelled with variants of these
fluorochromes called Spectrum Orange®, Spectrum Green® and
Spectrum Aqua®. Direct FISH eliminates the time-consuming
post-hybridization detection steps and reduces non-specific
labelling. The only disadvantage is reduced sensitivity (Reid
et al., 1992a), although this is not usually a serious limitation.
Localization of one chromosome using single-probe FISH
provides only limited detection of aneuploidy, so it is preferable
to simultaneously localize several chromosomes to increase
the sensitivity of detection (multi-probe FISH). In practice,
586
this is often limited to the detection of up to four chromosomes.
In this review, double-probe FISH will refer to the simultaneous
hybridization of two probes, while triple-probe FISH will
indicate hybridization of three probes. There are several
approaches that can be used for multi-probe FISH. First, up
to three chromosomes can be detected simultaneously by direct
and/or indirect FISH using three different probes and the three
basic fluorochrome emission colours: green, red and blue. The
only drawback is that a nuclear counterstain is also required,
and since this is usually either 49,6’-diamidino-2-phenylindole
(DAPI) which fluoresces blue or propidium iodide which
fluoresces orange, it restricts detection to only two chromosomes. In the second approach, each probe is labelled with up
to three different haptens (or fluorochromes), so that the probe
will produce up to three different signals in situ, either as
separate coloured signals in the same location if single bandpass
filters are used or as a signal of composite colour if a double
or triple bandpass filter is employed (Nederlof et al., 1990).
Up to seven different probes have been visualized simultaneously on human metaphase chromosomes using a combination
of three single-labelled probes, three double-labelled probes
and one triple-labelled probe (Reid et al., 1992b). The third
method is called ratio labelling. Aliquots of a probe are directly
labelled with different fluorochromes and the aliquots are then
mixed in varying ratios prior to hybridization so as to produce
a different coloured composite signal. This method can produce
up to 12 different colours from the three primary colours
(Dauwerse et al., 1992).
Three types of DNA probes are available for FISH: (i)
centromeric probes recognize repetitive DNA sequences in the
centromeric region and have been developed for most human
chromosomes (Willard and Waye, 1987). They produce a small
signal (spot) in the centromere and are routinely used to detect
aneuploidy. They are not very useful for detecting structural
abnormalities which, with the exception of Robertsonian translocations, occur on the p or q arms of chromosomes; (ii)
sequence-specific probes can be used to detect unique single
copy genes on chromosomes (Pinkel et al., 1988; Reid et al.,
1992a). These sequences may be unique to a chromosome, in
which case the probes can be used to detect aneuploidy.
Moreover, chromosome-specific centromeric probes are not
available for some chromosomes (13, 14, 21, 22) and, therefore,
sequence-specific probes must be used to detect these chromosomes. Chromosome-specific telomeric probes have recently
been used to estimate structural anomalies in human sperm
chromosomes (Van Hummelen et al., 1996); (iii) whole chromosome painting (WCP) probes are chromosome-specific
DNA libraries which label whole chromosomes and can
be used to detect structural rearrangements in metaphase
chromosomes (Pinkel et al., 1988; Dauwerse et al., 1992;
Kearns and Pearson, 1994) and study the organization of
chromosomes and chromatin in interphase nuclei (Pinkel et al.,
1988; Brandriff and Gordon, 1992). Dauwerse et al. (1992)
applied this technique to bone marrow spreads and showed
that half of the chromosomes could be painted in 12 different
colours using WCP probes carrying three distinct labels mixed
in multiple ratios. This implies that, in theory, two separate
Aneuploidy in human spermatozoa
hybridizations would allow the full complement of metaphase
chromosomes to be analysed.
Application of FISH to human spermatozoa
FISH has become the standard technique for assessing sperm
aneuploidy. However, the small size of the human sperm
nucleus restricts the number of signals that can reliably be
differentiated and therefore the simultaneous detection of more
than three chromosomes is impractical.
Sperm nuclear decondensation
Mammalian spermatozoa are haploid interphase cells which
have a unique packaging and arrangement of DNA that differs
significantly from somatic cells (Ward and Coffey, 1991;
Barone et al., 1994). The linear, side-by-side arrays of DNA,
cross-linked by disulphide bonds between adjacent protamines,
create a condensed, genomically-inert nucleus (Bedford and
Calvin, 1974; Balhorn, 1982) which is inaccessible to DNA
probes. Consequently, early ISH studies on human spermatozoa
were problematical and unsuccessful. For example, Joseph et al.
(1984) used probes that were specific for the Y chromosome and
chromosome 1, but achieved no hybridization in ejaculated
spermatozoa and variable results in testicular spermatozoa.
They used untreated spermatozoa and ejaculated spermatozoa
which had been pretreated with 1% trypsin for 30–60 s
followed by 0.01% dithiothreitol (DTT) for 60–90 s. Seuanez
et al. (1976) reported that hybridization occurred in immature
sperm cells, but the hybridization efficiency decreased as
spermatozoa matured.
It is now well recognized that to achieve efficient hybridization, the sperm nucleus must be swollen and made accessible
to probes by reducing disulphide bonds between protamine
molecules. This has been achieved using a variety of protocols
which are outlined in Table I. The earliest successful reports
on non-isotopic ISH with ejaculated spermatozoa were Pieters
et al. (1990) and Coonen et al. (1991) who found that
pretreatment of spermatozoa with 25 mM DTT and 0.1%
trypsin for 5–20 min promoted nuclear decondensation. However, this only resulted in approximately half of the sperm
nuclei being accessible to DNA probes. Most researchers have
subsequently used trypsin, lithium diidosalicylic acid (LIS),
ethylene diaminetetraacetic acid (EDTA), or cetyl trimethylammonium bromide (CTAB) in combination with a disulphide
reducing agent, DTT, to release the disulphide bonds between
adjacent protamine molecules and thereby induce swelling of
the sperm head (Table I). Wyrobek et al. (1990) sought a
reproducible pretreatment procedure for human spermatozoa
and found that pretreatment of sperm nuclei with 10 mM LIS
and 1 mM DTT induced uniform swelling of the nucleus from
1.5 to 2.5 times its normal area and maintained the characteristic
oval shape of the human sperm nucleus. This pretreatment
procedure, or a modification of it, has subsequently been used
in many studies and has proven to be a reliable pretreatment
procedure (Robbins et al., 1993, 1997; Williams et al., 1993;
Wyrobek et al., 1993a, 1994; Miharu et al., 1994; Spriggs and
Martin, 1994; Spriggs et al., 1995; Martin et al., 1996, 1997;
Van Hummelen et al., 1996).
Two other approaches have also been used. Chevret et al.
(1994) and Martini et al. (1995) used DTT alone to disrupt
the disulphide bonds without excessive swelling of the sperm
head, thus retaining sperm morphology while ensuring efficient
hybridization. In contrast, Guttenbach and co-workers did not
pretreat human spermatozoa in any way prior to hybridization,
relying instead on an extended denaturation in formamide, or
denaturation in 3 M NaOH to swell the sperm heads
(Guttenbach and Schmid, 1990; 1991; Guttenbach et al.
1994a,b). However, the hybridization efficiencies were considerably lower (.80%) than those obtained using DTT (95–
99%) (Table I).
Irrespective of which pretreatment method is used, there are
several requirements. Firstly, it should enable hybridization of
probes to a very high proportion of the spermatozoa, to
minimize scoring biases. In practice, researchers often use a
cut-off of 95–98%. Secondly, it must not result in excessive
loss of spermatozoa from the slides. Thirdly, it should maintain
the oval shape of the sperm head and not result in excessive
distortion of the nucleus which might lead to disruption of
DNA integrity and hence signal splitting. Finally, the sperm
tail should remain attached to the sperm head so that spermatozoa can be readily distinguished from non-sperm cells such as
leukocytes and immature germ cells which are also present
in semen.
Single-probe versus multi-probe FISH
Single-probe FISH has been used to estimate aneuploidy in
spermatozoa (Table II); however, it is now recognized that this
method has several technical limitations which reduce its
efficacy (Figure 1). First, it is impossible to accurately differentiate between disomy and diploidy when only a single probe
is used. Some researchers have attempted to distinguish these
conditions on the basis of nuclear size by assuming that diploid
spermatozoa have a large nucleus and two hybridization
signals, whereas disomic spermatozoa have a normal size
nucleus and two signals (Coonen et al., 1991; Han et al.,
1992, 1993a,b; Guttenbach et al., 1994a,b; Wang et al., 1994).
This association has never been proven, and it would therefore
seem prudent to avoid using nuclear size to estimate the ploidy
status, especially in view of the fact that Williams et al. (1993)
found that the rate of diploidy for many donors was higher
than the disomy rate. Second, the absence of a hybridization
signal is impossible to interpret when only a single probe has
been used; it could indicate nullisomy or failure of hybridization, but it is impossible to differentiate between these two
situations. This is an even greater problem when a single sex
chromosome probe is used because only half of the spermatozoa
should exhibit a hybridization signal. Multi-probe FISH overcomes these limitations, and enables reliable distinction
between diploid and disomic spermatozoa, and between nullisomy and failed hybridization (Figures 2–4). More accurate
estimates of autosomal disomy can be obtained using doubleprobe FISH because each spermatozoon should generate two
signals, one for each of the autosomes. Thus, spermatozoa
with two signals for each chromosome are diploid, whereas
those with a single signal for one probe and two signals for
the other probe are disomic for the latter autosome. Similarly,
587
S.E.Downie et al.
Table I. Pretreatment (nuclear decondensation) of human spermatozoa for in-situ hybridization (ISH) and fluorescence in-situ hybridization (FISH)
Method and studies
Method*
Comments
DTT
Rousseaux and Chevret (1995)
Martini et al. (1995)
10 mM DTT in 0.05 M Tris, pH 8, 10–50 min
25 mM DTT in 1 M Tris, pH 9.5, 5 min
Adequate nuclear swelling, intact tail morphology.
Trypsin 1 DTT
Coonen et al. (1991)
0.1% trypsin 1 25 mM DTT, 5–20 min
Goldman et al. (1993)
Bischoff et al. (1994)
0.1% trypsin 1 25 mM DTT, 12 min, RT
0.1% trypsin 1 25 mM DTT, 2 min, 37°C
LIS 1 DTT
Wyrobek et al. (1990)
Robbins et al. (1993)
Williams et al. (1993)
Miharu et al. (1994)
(1) Sperm nuclei isolated with MATAB and DTT;
(2) 10 mM LIS 1 1 mM DTT, 3 h
(1)
(2)
(1)
(2)
(1)
(2)
10 mM DTT, 30 min on ice;
4 mM LIS, 90 min, RT
10 mM DTT in 0.1 M Tris, pH 8, 30 min, RT;
10 mM LIS 1 1 mM DTT, 1–3 h
5 mM DTT, 10 min;
10 mM LIS 1 0.5 mM DTT, 70 min
Many tails lost. Optimal time (5–15 min) determined
for each sample.
Concentrations as low as 1 mM LIS induced swelling
of isolated sperm nuclei. Swelling was uniform and
the oval shape was maintained.
No fixation. Sperm air dried onto slides before
decondensation.
Modification of Wyrobek et al. (1990)
Modification of Wyrobek et al. (1990). Sperm nuclei
were swollen to 1.5 times the original nuclear
diameter.
EDTA 1 DTT
Han et al. (1992)
Pang et al. (1994)
Wang et al. (1994)
(1) 6 mM EDTA; (2) 2 mM DTT, 45 min
6 mM EDTA 1 2 mM DTT, 45 min, 37°C
(1) 6 mM EDTA; (2) 2–4 mM DTT, 45 min
Preferable to trypsin 1 DTT and SDS 1 DTT.
CTAB 1 DTT
Holmes and Martin (1993)
(1) 10 mM DTT in 0.05 M Tris, pH 8;
Modification of Balhorn et al. (1977). Swelling to 1.5
times nuclear area.
Tail and nuclear membrane were removed by CTAB
and DTT.
(2) sonication, 4°C; (3) 1% CTAB, 30 min at 4°C.
No pretreatment
Guttenbach and Schmid (1990)
Denatured with probe in formamide, 10 min, 72°C
Guttenbach et al. (1994a)
3 M NaOH, 3–10 min, RT
Modification of Han et al. (1992).
Denaturation prior to hybridization caused all sperm
nuclei to swell.
3 M NaOH allowed decondensation to be controlled.
DTT 5 dithiothreitol; LIS 5 lithium diidosalicylic acid; MATAB 5 mixed alkyltrimethylammonium bromide; CTAB 5 cetyl trimethylammonium bromide;
RT 5 room temperature.
*Numbers in parentheses indicate the order of sequential treatments.
Figure 1. Single-probe fluorescence in-situ hybridization (FISH)
using a X chromosome-specific probe. Only 50% of the
spermatozoa exhibit a signal, and the gender of the unlabelled
spermatozoon is uncertain. Furthermore, it is impossible to
distinguish disomy from diploidy.
spermatozoa with only one signal are nullisomic for the other
autosome, whereas those with no signals have failed to
hybridize (Figure 2).
Estimation of sex chromosome aneuploidy engenders further
difficulties. Double-probe FISH with X and Y probes has been
588
used to estimate sex chromosome aneuploidy (Goldman et al.,
1993; Han et al., 1993a,b; Chevret et al., 1994; Wang et al.,
1994; Wyrobek et al., 1994), however, this approach cannot
differentiate between disomic (22 XX, 22 YY, 22 XY) and
diploid (44 XX, 44 YY, 44 XY) spermatozoa (Figure 3).
Furthermore, one cannot determine whether unlabelled spermatozoa are nullisomic for the sex chromosomes or failed to
hybridize, and disomy and diploidy estimates are indirectly
reduced if nullisomic spermatozoa are incorrectly classified
as unlabelled. Triple-probe FISH using probes for the sex
chromosomes and one autosome can be used to accurately
determine sex chromosome aneuploidy in spermatozoa and
differentiate between unlabelled spermatozoa which are nullisomic for the sex chromosomes and spermatozoa which are
unlabelled because the probes did not hybridize (Figure 4). With
the inclusion of the third autosomal probe, each spermatozoon
should exhibit one autosomal signal and one sex chromosome
signal (X or Y). Sex chromosome disomy is characterized by
one autosomal signal and two sex chromosome signals, whereas
a diploid spermatozoon has two autosomal signals and two
sex chromosome signals. Sex chromosome nullisomy is indicated by the presence of only a single autosomal signal in the
spermatozoon, whereas spermatozoa that are unlabelled due
to hybridization failure exhibit no signals at all. It is also
Aneuploidy in human spermatozoa
Table II. Frequency of two signals (disomy or diploidy) in human spermatozoa using single-probe in-situ hybridization (ISH) or fluorescence in-situ
hybridization (FISH)
Study
Hybridization
(%)*
Frequency of two signals for chromosome no.
1
Joseph et al. (1984)
West et al. (1989)
Guttenbach and Schmid (1990)
Pieters et al. (1990)
Coonen et al. (1991)
Guttenbach and Schmid (1991)
Jackson-Cook and Haller (1991)
Han et al. (1992)
Holmes and Martin (1993)
Martin et al. (1993)
Robbins et al. (1993)
Guttenbach et al. (1994a)
Guttenbach et al. (1994b)
Miharu et al. (1994)
Martini et al. (1995)
80/48
47
49
40–60
40–90
–
–
96/48
98/99/49
98–99
97.5/50.3
.80
.80
.95
98.7
3
7
10
11
12
13/21
15
16
17
18
X
0.35
Y
0.18
0.03
0.27
0.8
0.67
0.41
0.5
0.06
0.6
0.1
0.33
0.6
0.29
0.03
0.04
0.14
0.17
0.11
0.056
0.14
0.31
0.31
0.32
0.34
0.2
0.31
0.34
0.36
0.14
0.69
0.17
0.13
0.08
*Ranges or separate values for each of the chromosomes studied are given.
Figure 3. Double-probe fluorescence in-situ hybridization (FISH)
using X- and Y-specific probes. While XX, YY and XY
spermatozoa can be identified, it is impossible to distinguish
disomic from diploid spermatozoa, and the sex chromosome status
of unlabelled spermatozoa is unclear.
Figure 2. Double-probe fluorescence in-situ hybridization (FISH)
using autosomal probes (chromosomes 1 and 8). All spermatozoa
should exhibit at least one signal unless hybridization failure has
occurred (unlabelled). Disomy, nullisomy and diploidy can be
distinguished by the number and colours of signals.
possible to distinguish between sex chromosome disomy and
diploidy of meiosis I and II origin; non-disjunction at meiosis
II results in only one disomic spermatozoon (XX or YY)
whereas non-disjunction at meiosis I results in two disomic
XY spermatozoa. Rademaker et al. (1997) recently demonstrated that comparable diploidy frequencies are obtained
using double-probe FISH and triple-probe FISH on the same
sperm samples.
Estimation of aneuploidy in human spermatozoa
using FISH
Since 1989, there have been over 50 reports on the estimation
of aneuploidy in human spermatozoa using ISH and FISH.
The results of many of these studies are summarized in Tables
II, III and IV. For ease of comparison, single- and multi-probe
studies have been grouped together.
Single-probe FISH
While isotopic ISH was used in the earliest studies (Joseph
et al., 1984; West et al., 1989), most of the more recent studies
have used FISH (Table II). Nevertheless, Guttenbach and
Schmid (1990, 1991) and Guttenbach et al. (1994a,b) used a
589
S.E.Downie et al.
Figure 4. Triple-probe fluorescence in-situ hybridization (FISH) using sex chromosome probes and an autosomal probe (chromosome 8)
enables accurate determination of sex chromosome aneuploidy in human spermatozoa. Haploid spermatozoa can be distinguished from
disomic, nullisomic and diploid spermatozoa, and the status of spermatozoa can be determined.
Table III. Studies on disomy in human spermatozoa using double-probe fluorescence in-situ hybridization (FISH)
Study
Hyb.
(%)
Disomy frequency for chromosome no.
1
Goldman et al. (1993)
Han et al. (1993a)
Han et al. (1993b)
Schattman et al. (1993)
Williams et al. (1993)
Bischoff et al. (1994)*
Bischoff et al. (1994)*
Chevret et al. (1994)
Lu et al. (1994)
Martin et al. (1994)
Wyrobek et al. (1994)
Rousseaux & Chevret
(1995) **
Spriggs et al. (1995)
Blanco et al. (1996)
Martin et al., (1996)
Spriggs et al. (1996)
99.8
96
95
99.1
96–97
99.9
99.9
99
–
92–95
99.8
97–99
ù98
99
.98
ù98
2
3
4
6
7
8
9
10
12
15
16
17
18
20
21
0.13
0.08
0.28 0.29 0.23 0.54 0.19 0.39
0.09 0.32 0.17 0.24 0.0 0.0
0.41 0.30 0.15 0.09 0.04
0.27 0.18 0.0 0.0 0.18
X
Y
XY
0.08
0.28
0.25
0.04
0.08
0.1
0.21
0.23
0.09
0.11
0.23
0.21
0.15
0.17
0.05 0.05 0.42
0.16
0.17 0.16
0.15
0.8
0.4
0.04 0.04 0.06
0.2
0.08
0.1
0.10
0.16 0.11
0.11
0.14
0.38
0.11
0.16
0.08
0.11
0.14
0.11
0.12 0.29
*The two sets of data are for two different donors.
**Disomy frequencies of 0.09% for chromosome 11 and 0.17% for chromosome 14 were also obtained.
non-isotopic ISH method which involved hybridization of
biotinylated DNA probes followed by detection of the probes
with streptavidin-peroxidase and diaminobenzidine. They
reported that the brown signals were easy to distinguish from
the Giemsa-stained chromatin, making it easier to differentiate
two adjacent signals. The disomy frequencies (0.27–0.41%)
were much higher than those obtained by Robbins et al. (1993)
590
and Miharu et al. (1994) using LIS/DTT pretreatment and
single-probe FISH. Martini et al. (1995) also presented an ISH
method which uses enzyme immunocytochemical detection of
probes. The advantages of this method are that sperm morphology is well preserved, slides are scored using brightfield
microscopy, and the preparations are permanent. They used
single-probe ISH and recorded a chromosome 1 disomy rate
Aneuploidy in human spermatozoa
Table IV. Studies of disomy in human spermatozoa using triple-probe fluorescence in-situ hybridization (FISH)
Study
Hybridization
(%)
Disomy frequency for chromosome no.
1
Schattman et al. (1993)
Williams et al. (1993)
Wyrobek et al. (1993a)
Wyrobek et al. (1993b)
Bischoff et al. (1994)
Lu et al. (1994)
Martin (1994)
Chevret et al. (1995)
Martin et al. (1995)
Griffin et al. (1995)
Robbins et al. (1995)
Spriggs et al. (1995)
Abruzzo et al. (1996)
Martin et al. (1996)
Van Hummelen et al. (1996)
98
95
–
–
99.9
–
–
ù99
ù98
–
ù98
ù98
–
ù98
ù99
0.142
8
12
18
X
Y
XY
0.0
0.1
0.04
0.055
0.038
0.375
0.25 *
0.07
0.04
0.07
0.02
0.031
0.07
0.018
0.07
0.2
0.055
0.061
0.042
0.086
0.25 *
0.12
0.009
0.18
0.03
0.031
0.21
0.027
0.18
0.39
0.09
0.089
0.091
0.12
0.25 *
0.16
0.34
0.16
0.10
0.095
0.15
0.094
0.16
0.074
0.068
0.12
0.17
0.08
0.2
0.11
0.16
0.04
0.065
0.033
0.017
0.019
*The combined sex chromosome (X 1 Y) disomy frequency was 0.25%.
of 0.69%. This is higher than in most published studies on
chromosome 1 disomy, except for Coonen et al. (1991) who
reported a chromosome 1 disomy rate of 0.67% (Table II).
Robbins et al. (1993) analysed samples from donors whose
spermatozoa had previously been karyotyped using the hamster
oocyte technique. Disomy frequencies for chromosome 1
(0.14%) and chromosome Y (0.057%) were not significantly
different from those obtained for the same donors by karyotyping, which demonstrated the efficacy of FISH. Miharu et al.
(1994) studied spermatozoa from 21 donors and found that
chromosome 1 disomy varied from 0.07–0.20% for individual
donors. In contrast, Holmes and Martin (1993) examined
10 000 spermatozoa from only a single donor and reported
a chromosome 1 disomy frequency of only 0.06%, which
illustrates the importance of studying spermatozoa from more
than one donor, and suggests that some of the variation in
Table II is due to inter-donor variation.
In general, single-probe FISH and ISH has yielded higher
(and less reliable) estimates of aneuploidy than multi-probe
FISH (Tables II, III and IV), and there has been considerable
variability in the estimates for specific chromosomes. This is
probably due to the fact that many of the single-probe studies
were undertaken during the formative years of this technology
when probes, pretreatment procedures, hybridization protocols,
sample sizes and scoring criteria were less well developed and
less standardized than they are now. Furthermore, one cannot
differentiate between disomy and diploidy using single-probe
FISH, so the disomy estimates undoubtedly include diploid
spermatozoa.
Multi-probe FISH
The simultaneous hybridization of two or three probes enables
more accurate estimates of sperm aneuploidy. Disomy estimates
range up to 0.8% per chromosome, although the majority are
0.02–0.2% (Tables III, IV). Less variation between studies and
more consistent results have been reported than using singleprobe FISH. One exception was the study by Martin et al.
(1994), which yielded higher disomy frequencies for chromo-
somes 1 and 12. The results of Bischoff et al. (1994) were
also divergent, although this is probably because they only
scored 1000 spermatozoa for each chromosome.
The results of triple-probe FISH are summarized in Table
IV. Most of these studies concentrated on sex chromosome
aneuploidy in spermatozoa because double-probe FISH is
adequate for estimating autosomal aneuploidy and a third
probe is only needed to determine sex chromosome aneuploidy.
Abruzzo et al. (1996) used triple-probe FISH to assess whether
there is a relationship between the size of the centromere and
the propensity for non-disjunction; they found no association
between Y chromosome α satellite array length and disomy
YY in 14 normal donors. Williams et al. (1993) used doubleprobe FISH (18,Y or 18,X) to evaluate meiosis II sex chromosome disomy, and chromosome 16 and 18 probes to study
autosomal disomy (Table III). To study meiosis I sex chromosome disomy, they used triple-probe FISH for chromosomes
X, Y and 8 (Table IV). To account for different rates of nondisjunction at meiosis I and meiosis II, they corrected the
disomy estimates to 0.055% for the Y chromosome and 0.04%
for the X chromosome. Bischoff et al. (1994) demonstrated
the importance of using sex chromosome probes and an
autosomal probe to estimate sex chromosome aneuploidy. The
frequency of unlabelled spermatozoa was 2.1 and 3.9% when
double-probe FISH with X and Y probes was used, whereas
it was only 0.19 and 0.76% when a chromosome 12 probe
was included in a triple-probe procedure.
Several key aneuploidy issues have recently started to
be investigated using multi-probe FISH: inter-chromosomal
differences, inter-donor variability and paternal age effects.
Inter-chromosomal differences
Spriggs et al. (1995) reported that sex chromosome disomy
(XX 1 YY 1 XY; 0.43%) was significantly higher than
disomy for chromosomes 1, 12, 15 and 18 (0.12%). This
confirmed the results of Williams et al. (1993) in which sex
chromosome disomy (0.19%) was higher than disomy for
chromosomes 16 (0.13%) and 18 (0.08%). Spriggs et al. (1996)
591
S.E.Downie et al.
reported that the sex chromosomes and chromosome 21 had a
significantly higher frequency of disomy than the other
autosomes tested, while Blanco et al. (1996) published a higher
incidence of disomy 21 compared with disomy 6. Taken
together, these results suggest that during male meiosis, the
sex chromosomes and chromosome 21 may be more susceptible
to non-disjunction than the other autosomes. Data from sperm
karyotyping, spontaneous abortions and live births support this
contention (Martin et al., 1991; Jacobs, 1992; Templado et al.,
1996), and there is evidence that the sex chromosomes are
more susceptible to pairing and first meiotic segregation errors
(Armstrong et al., 1994).
Inter-donor variation
Valid comparisons between studies are difficult at present
because of differences in donor selection and FISH techniques,
but several groups have examined inter-donor variation. Martin
(1994) analysed spermatozoa from five donors and found
consistent disomy frequencies for chromosomes 12, Y and
XY; however, there were significant inter-donor differences in
the frequencies of diploidy and disomy for chromosomes 1
and X. Spriggs et al. (1995) studied spermatozoa using doubleprobe FISH and probes to chromosomes 1, 12, 15 and 18.
They reported significant inter-donor variation for disomy 1
and disomy 15, but not disomy 12 and 18. The same group
used triple-probe FISH for chromosomes X, Y and 1 and
showed significant inter-donor variation for YY and XY
disomy in spermatozoa from five normal men (Spriggs et al.,
1996). These results confirm that inter-donor variability is an
important consideration when making intra- and inter-study
comparisons of disomy for specific chromosomes in spermatozoa, but clearly additional, well-designed studies are needed
to clarify the extent of this variation in normospermic men.
Paternal age effects on sperm aneuploidy
Several studies have used multi-probe FISH to examine
whether paternal age influences the frequency of aneuploidy
in human spermatozoa. Martin et al. (1995b) reported a
significant age-related increase in disomy for chromosomes Y
and 1, and Wyrobek et al. (1994) also found a paternal age
effect on the incidence of disomy Y. Robbins et al. (1995)
reported increased frequencies of sex chromosome disomy in
older men, but suggested that a study involving more men of
diverse age distribution would provide a definitive answer
about the influence of paternal age on sperm aneuploidy.
Griffin et al. (1995) studied spermatozoa from 24 men aged
18–60 and found that there was no relationship between age
and disomy 18, but the incidences of disomy XX, YY and
XY were elevated in older men. These results correlate well
with a study which demonstrated an increased incidence of
sex chromosome aneuploidy in spermatozoa from aged mice
(Lowe et al., 1995). Further investigations are needed.
Total aneuploidy estimates for spermatozoa
Assuming that a higher rate of non-disjunction exists for the
sex chromosomes but the autosomes all have a similar nondisjunction rate, the overall risk of aneuploidy in human
spermatozoa can be roughly estimated. For instance, using
592
mean autosomal and sex chromosome disomy rates of 0.12
and 0.31% respectively (Williams et al., 1993; Spriggs et al.,
1995), the overall disomy frequency in spermatozoa from a
normospermic man would be ~3%. If the incidence of
aneuploidy is assumed to be twice the disomy rate, then the
total aneuploidy rate would be 6%. Given a mean diploidy
rate of 0.32% (Williams et al., 1993; Spriggs et al., 1995), the
overall incidence of numerical chromosomal anomalies in
spermatozoa would be about 6.3%. This is a conservative
estimate, however, because we have only assumed one error
per spermatozoa and we have assumed uniform disomy frequencies for the various autosomes, but it is similar to, or
slightly higher than results obtained by karyotyping spermatozoa (Martin, 1993). As more well-designed studies on a wide
range of chromosomes and men with well defined semen
characteristics are published, it should be possible to more
accurately estimate overall aneuploidy levels in spermatozoa.
FISH studies on structural chromosome anomalies in spermatozoa
Van Hummelen et al. (1996) recently used FISH for the first
time to detect both structural and numerical chromosome
anomalies in human spermatozoa. They used a triple-probe,
four colour FISH procedure, centromeric probes for chromosomes 1 and 8, and a midi satellite p36.3 probe for chromosome
1. They studied 120 686 spermatozoa from three donors and
recorded frequencies of 0.029% for telomeric deletions of 1p,
0.032% for telomeric duplications of 1p, 0.017% for disomy
1, 0.019% for disomy 8, and 0.066% diploidy. The incidence
of telomeric deletions and duplications correlated closely with
the results of earlier sperm karyotyping studies. This is an
important advance in sperm FISH which requires further
investigation.
FISH studies on spermatozoa from infertile and
subfertile men
The introduction of intracytoplasmic sperm injection (ICSI)
has revolutionized the treatment of male infertility (Van
Steirteghem et al., 1993; Payne and Matthews, 1995). Even
men with numerical or structural chromosome anomalies that
affect spermatogenesis can be treated (Testart et al., 1996;
Tournaye et al., 1996). With this advance, however, has come
an enhanced recognition of the potential risks of transmission
of genetic abnormalities from subfertile spermatozoa to
embryos and offspring (Engel et al., 1996), especially given
the association between infertility and chromosomal anomalies
(De Brackeleer and Dao, 1991). Hence, studies have recently
been undertaken to clarify the risks.
Estimation of aneuploidy in spermatozoa from infertile men
To date, there have only been a few FISH studies on aneuploidy
in spermatozoa from infertile men. Miharu et al. (1994) found
no significant differences between fertile and infertile men in
disomy rates (0.08–0.17%) for chromosomes 1, 16, X or Y.
Their results for fertile men compared favourably with Robbins
Aneuploidy in human spermatozoa
et al. (1993) which may reflect the use of similar scoring
criteria. Guttenbach et al. (1997) recently reported similar
findings. They studied 45 infertile men and six fertile men
using single- and double-probe ISH and FISH and estimated
similar disomy rates for chromosomes 1, 7, 10, 17, X and Y
in the two groups. In contrast, Pang et al. (1994) reported a
10-fold increase in chromosome 1 disomy and a significantly
higher frequency of nullisomy for chromosome 1 in spermatozoa from oligoasthenoteratozoospermic men compared with
normospermic men (0.96 versus 16% respectively). In a study
using sperm karyotyping in hamster oocytes and FISH for
chromosomes 1, 12, X and Y, Moosani et al. (1995) found
that men with idiopathic infertility exhibited an increased
frequency of chromosomal abnormalities. Of five men, three
had an increased disomy 1 frequency, one had an increased
disomy 12 frequency, and four had an increased XY disomy
frequency. The XX and YY disomy rates were normal. Yurov
et al. (1996) studied spermatozoa from a man with a 40%
incidence of macrocephalic spermatozoa using a rapid,
rehybridization FISH procedure and found that many of the
large headed spermatozoa were diploid.
Collectively, these studies do not demonstrate a clear correlation between male infertility and numerical chromosome abnormalities in spermatozoa, although the results of Pang et al.
(1994) and Moosani et al. (1995) certainly warrant further
investigation. This is not surprising given the multi-factorial
nature of male infertility and the fact that most of the studies
to date involved scoring small numbers of spermatozoa with the
attendant inaccuracies and potential biases (see next section). It
will be interesting to see if trends emerge from controlled
studies which utilize clinically-relevant patient selection criteria, stringent scoring criteria and large sperm sample sizes.
While it may be difficult to obtain sufficient spermatozoa from
oligozoospermic men, it must be recognized that aneuploidy
estimates will not be accurate unless well designed studies
are undertaken in which at least 10 000 spermatozoa per
chromosome per man are assessed.
Sperm aneuploidy in men with structural or numerical chromosome anomalies
Lu et al. (1994) used double-probe FISH on spermatozoa from
33 normal donors and a man carrying a 46 X,Y,t(2;4;8)(q23;
q27;p21) translocation to test the hypothesis that an increase
in aneuploidy would be found for those chromosomes involved
in the translocation due to an increased likelihood of nondisjunction. Approximately 900 spermatozoa from the translocation carrier were scored and disomy frequencies of 2.0
and 2.7% were estimated for chromosomes 4 and 8 respectively.
These values were higher than normal, suggesting that
translocated chromosomes are more susceptible to meiotic
errors. A study by Spriggs and Martin (1994) also showed
increased disomy rates for the translocated chromosomes in
a male reciprocal translocation carrier, t(1:11)(p36.3;q13.1).
Rousseaux et al. (1995a,b) reported slightly increased disomy
1 frequencies in spermatozoa from two sibling reciprocal
translocation carriers, t(6:11)(q14;p14), a reciprocal translocation carrier, t(2:14) (p23.1;q31), and a Robertsonian carrier,
t(14q,21q), suggesting that inter-chromosomal effects on non-
disjunction may occur during spermatogenesis. In a recent
study, Mercier and Bresson (1997) studied spermatozoa from
a 46,XY/47,XY18 man using double- and triple-probe FISH
and probes to chromosomes 8, 18, X and Y. Relative to
controls, they found elevated frequencies of disomy 8 (1.59
versus 0.17 and 0.21%) and diploidy (0.21 versus 0.03 and
0.09%). These studies all suggest that there are increased risks
of aneuploidy in spermatozoa from men with constitutional
chromosome anomalies.
Spermatozoa from XYY and XXY males
Sex chromosome anomalies are the most common mitotic
anomaly associated with male infertility (De Brackeleer and
Dao, 1991). Wong et al. (1987) postulated that the extra Y
chromosome in 47,XYY males is the result of non-disjunction
at the second meiotic division in paternal germ cells, and this
suggests that the incidence of YY disomy in spermatozoa from
47,XYY males might be higher than in 46,XY males. Han
et al. (1994) tested this hypothesis using single-probe FISH
to detect chromosome 17 and double-probe FISH to detect the
X and Y chromosomes in spermatozoa from a 47,XYY male,
but did not find an elevated frequency of YY spermatozoa.
This supports the results of an earlier study using sperm
karyotyping (Benet and Martin, 1988) and suggests that
the YY cells are probably lost from the germ line during
spermatogenesis (Melnyk et al., 1969; Burgoyne, 1979).
Several other groups have subsequently studied spermatozoa
from 47,XYY males using FISH, although the results are
somewhat contradictory. Martini et al. (1996) reported that
spermatozoa from two 47,XYY men had a 1:1 ratio of X- to
Y-bearing spermatozoa, however, the incidence of aneuploidy
was very high (15%). Mercier et al. (1996) examined spermatozoa from one 47,XYY male and found a slight excess of Y
spermatozoa (X:Y ratio 0.78:1), but also recorded an increased
incidence of sex chromosome aneuploidy (14.6%) relative to
controls. Blanco et al. (1997) used a triple-probe FISH procedure and probes to chromosomes 18, X and Y. They reported
a 1:1 ratio of X- to Y-bearing spermatozoa but an elevated
incidence of sex chromosome aneuploidy relative to controls
(1.76 versus 0.61%). Chevret et al. (1997) compared two
47,XYY males with five 46,XY males and found that the X:Y
ratio in spermatozoa from the 47,XYY males differed from
1:1 with an excess of Y-bearing spermatozoa, but compared
with the other studies described above, they found a relatively
low incidence of sex chromosome aneuploidy. These data
indicate that the 47,XYY condition and its consequences in
spermatozoa may be quite heterogeneous, that most mature
germ cells will have a XY complement, but some aneuploid
germ cells can produce aneuploid spermatozoa.
Martini et al. (1996) reported that spermatozoa from a
46,XY/47,XXY male (Klinefelter’s mosaic) had an equal
proportion of X- to Y-bearing spermatozoa, but increased
levels of aneuploidy. Chevret et al. (1996) also studied spermatozoa from a Klinefelter’s mosaic (46,XY/47,XXY) but
reported an increased frequency of XY disomy relative to
controls (2.09 versus 0.36%) and an excess of X-bearing
spermatozoa (X:Y ratio 52.8:43.9). These data suggest that
593
S.E.Downie et al.
47,XXY germ cells can give rise to mature XY disomic
spermatozoa.
Chemotherapy and sperm aneuploidy
Using sperm karyotyping after penetration of zona-free hamster
oocytes, it was shown that cancer patients have higher frequencies of chromosomal abnormalities in their spermatozoa after
treatment (Martin et al., 1986; Genesca et al., 1990). Martin
et al. (1995a) used sperm karyotyping and FISH for chromosomes 1, 12, X and Y to examine spermatozoa from a man
who had undergone MACOP-B (methotrexate, leucovorin,
doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin) chemotherapy, and found that the incidence of chromosomal abnormalities was not elevated. Disomy frequencies
were 0.1, 0.11, 0.04, 0.05 and 0.18% for 1, 12, XX, YY and
XY respectively, which were all within the normal range.
Martin et al. (1997) subsequently studied spermatozoa from
men with testicular cancer, both before and after BEP (bleomycin, etoposide, cisplatin) chemotherapy, and found similar preand post-treatment (respectively) rates for disomy 1 (0.11 and
0.06%), disomy 12 (0.18 and 0.15%), disomy XX (0.1 and
0.9%), disomy YY (0.13 and 0.1%), and disomy XY (0.25
and 0.2%). Robbins et al. (1997) found significant increases
in hyperhaploidy in spermatozoa from men with Hodgkin’s
lymphoma who had undergone NOVP (novantrone, vincristine,
vinblastine, prednisone) chemotherapy. The increases were
2.3-fold for XX, 4.7-fold for XY, no increase for YY, 3.3 fold
for disomy 8, and overall a 5-fold increase in hyperhaploidies.
However, these effects were temporary and sperm aneuploidy
levels decreased to pretreatment levels by 100 days postchemotherapy. These studies suggest that men who have had
chemotherapy are not at higher risk of aneuploidy in their
spermatozoa, although further studies are needed to investigate
the incidence of structural chromosome anomalies.
Technical limitations of using FISH to estimate
sperm aneuploidy
While multi-probe FISH has opened the way for extensive
studies of aneuploidy in human spermatozoa, there are technical
considerations and certain limitations and pitfalls which must
be addressed. Further work is required to fully validate this
emerging methodology.
Pretreatment procedures, probes and hybridization
conditions
As discussed above, a wide variety of pretreatment procedures
have been employed in FISH studies on human spermatozoa.
These methods differ significantly in their propensity to decondense sperm nuclei and thereby disrupt and alter the conformation of probe binding sites. As such, they may not yield
comparable aneuploidy estimates. Over-swelling of nuclei
generates split signals which can lead to over-estimates of
aneuploidy (see below), so it is important that sperm pretreatment is standardized to minimize this bias. Swelling sperm
nuclei to 1.5 to 1.7 times their original size minimizes
signal splitting without compromising hybridization efficiency
(Holmes and Martin, 1993; Robbins et al., 1993; Wyrobek
594
et al., 1994; Robbins et al., 1995). The choice of probes,
hybridization conditions and post-hybridization washing procedures also affects the generation of signals, and at this stage,
we do not fully understand the contribution of these variables
to: (i) differences in aneuploidy estimates for the same chromosome in different studies; and (ii) differences in the frequency
of aneuploidy for different chromosomes. The impact of
these technical factors on the estimation of aneuploidy in
spermatozoa requires careful evaluation.
Storage of sperm slides
Sperm smears or fixed sperm nuclei can be stored at –20°C
for extended periods prior to hybridization. Martin et al. (1994)
compared the aneuploidy rate for chromosome 1 in fresh and
frozen human spermatozoa, and reported that the hybridization
efficiency was significantly reduced in frozen spermatozoa
(93.2%) compared with fresh spermatozoa (97.1%). They
found that storage of sperm nuclei in fixative at –20°C for
prolonged periods (.2 years) influenced the size of the
sperm nucleus, which in turn led to decreased hybridization
efficiencies and an increased estimation of disomy.
Signals and scoring criteria
In FISH, an assumption is made that each spot indicates the
presence of a chromosome, so the presence of two spots
represents two chromosomes. However, under certain conditions this assumption may be invalid. First, two signals can
arise from one chromosome due to signal splitting, and this
can be a problem when centromeric probes are used because
of the distribution of repetitive satellite DNA sequences in the
centromeric region and the tendency of this region to fragment.
Retention of chromatin integrity and the morphology of probebinding domains is therefore an important issue. Martin and
Rademaker (1995) tested the effect of different scoring stringencies on aneuploidy estimates in spermatozoa. They used
multi-probe FISH, probes to chromosomes 1, 12, X and Y,
and two criteria for disomic signals, one half a signal domain
apart and one signal domain apart. There was a significant
decrease in the disomy frequency for all chromosomes except
chromosome 12 when one signal domain was used as the
minimum separation distance between two signals. This suggests that the signals for chromosomes 1, X and Y can split
into two signals, thus giving the appearance of disomy and
resulting in over-estimates of aneuploidy. Moreover, this reinforces the importance of standardizing the definition of two
signals to enable valid comparisons between studies. Martin
and Rademaker (1995) also suggested that excessive decondensation of human sperm nuclei induces signal splitting,
hence, they only score sperm nuclei which are decondensed
up to twice the size of an undecondensed nucleus. Wyrobek
et al. (1993b) showed that in human spermatozoa, fluorescent
signals from repetitive DNA probes to chromosomes Y and 8
can split into several signals. Optimized sperm pretreatment
procedures and stringent scoring criteria reduce the impact of
split signals on aneuploidy estimates, but this phenomenon
should not be overlooked.
A second problem relates to the arrangement of signals.
Sperm nuclei are three dimensional structures and the
Aneuploidy in human spermatozoa
centromeric regions of different chromosomes, and hence the
FISH signals generated, are not always separate and clearly
defined. Signals from different chromosomes, or from two
copies of the same, can be very close together or completely
overlapping and therefore cannot be distinguished. This will
lead to incorrect estimates of aneuploidy and/or diploidy. The
impact of this bias increases as more chromosomes are
simultaneously studied in multi-probe FISH. A third problem
is that we assume that if a chromosome is present, the probe
will always bind and we will always see a signal. However,
localized hybridization failure of the probe to one or more
chromosomes could lead to an incorrect assessment of the
ploidy status of spermatozoa. For example, if double-probe
FISH with two autosomal probes (chromosomes 1, 8) were
used and hybridization failure occurred with the chromosome
8 probe, a diploid spermatozoa (1,1,8,8) would be misclassified
as disomic (1,8,8), a haploid spermatozoa (1,8) would be
misclassified as nullisomic (1), and a disomic spermatozoa
(1,8,8) would be misclassified as haploid (1,8). Given the
difficulties involved in decondensing human sperm nuclei, this
should not be ignored.
Stringent scoring criteria are therefore needed to ensure
accurate aneuploidy estimates and enable meaningful comparisons between chromosomes, donors and studies (Martin
and Rademaker, 1995). Some of the approaches that have
been used are as follows. To ensure that they differentiated
split signals from disomy and diploidy, Williams et al.
(1993) only scored signals of equal size and intensity,
counted as two signals only those which were separated
from each other by at least one signal domain (to avoid
including split signals), and only scored signals which were
positioned well within the sperm head. Goldman et al.
(1993) through-focussed each spermatozoon, checked that
spermatozoa with more than one signal were not two
overlapping cells, and differentiated between split signals
and two separate signals by their distance and size. To do
this, they took ~1000 photographs of each sample and the
four authors reviewed them independently, then they defined
aneuploid and diploid nuclei by group discussion. Cells with
unclassifiable signals were excluded (134 spermatozoa out
of 60 000). Schattman et al. (1993) concentrated on three
aspects of scoring. First, only sperm heads with attached
tails were scored to eliminate scoring somatic cells as
spermatozoa, which would otherwise have elevated the
frequency of XY spermatozoa. Second, overlapping sperm
nuclei were excluded, and finally, non-specific crosshybridization of the probes to other chromosomes was
accounted for in the scoring criteria so that true signals
were distinguished from cross-hybridization signals. Spriggs
et al. (1995) only scored slides in which the hybridization
efficiency was ù98%. Disomy was classified as two signals
of the same colour, well within the nucleus, comparable in
size and intensity, and at least one signal domain apart.
They eliminated nuclei that were overlapping, disrupted,
diffuse or devoid of signals.
tozoa must be evaluated to ensure that reliable aneuploidy
estimates are obtained. Williams et al. (1993) stated that scoring
5000 spermatozoa to determine the aneuploidy frequency
for each chromosome was insufficient for comparisons of
chromosome-specific disomy rates between donors, however
this sample size was adequate for comparisons between chromosomes if the results from a group of donors were pooled.
They recommended instead that a minimum of 10 000 spermatozoa should be scored from each sample to provide an
accurate estimate for each chromosome. Many researchers
have employed this recommendation (Robbins et al., 1993,
1995; Wyrobek et al., 1994; Griffin et al., 1995; Martin and
Rademaker, 1995; Martin et al., 1995b, 1996; Moosani et al.,
1995; Spriggs et al., 1995, 1996; Van Hummelen et al.,
1996), however, others have scored much lower numbers of
spermatozoa with the attendant limitations (Schattman et al.,
1993; Bischoff et al., 1994; Lu et al., 1994; Pang et al., 1994).
The statistical pitfalls associated with scoring small numbers
of spermatozoa are obvious. For example, if the true disomy
rate were 0.2%, then this would equate to only two disomic
spermatozoa per 1000 scored but 20 per 10 000 scored. If the
true disomy rate were 0.1%, then this would equate to only
one disomic spermatozoon per 1000 scored or 10 per 10 000
scored. However, if the true disomy rate were only 0.05%,
which has been found in many studies (see Tables III, IV)
then this would equate to fewer than one disomic spermatozoa
per 1000 scored or only five per 10 000 scored. Clearly, there
is great potential for error if only 1000 spermatozoa are scored
for each chromosome because the disomy rate will depend on
how many disomic spermatozoa are in the cluster of 1000
spermatozoa scored. Scoring one or two more or less spermatozoa would change the disomy rate significantly in this situation.
Sample sizes
Aneuploidy for a given chromosome occurs at a very low
frequency in human spermatozoa, so large numbers of sperma-
The introduction of FISH has facilitated the routine screening
of large numbers of spermatozoa for numerical chromosomal
abnormalities (aneuploidy), and has paved the way more
Inter- and intra-technician scoring variation
The importance of inter- and intra-technician variation should
not be underestimated when scoring a phenomenon, aneuploidy,
which is encountered at such very low frequencies in human
spermatozoa. In some laboratories, efforts are being made to
standardize scoring procedures so that inter- and intra-technician
variation is minimized and meaningful intra- and inter-donor
variations can therefore be compiled. Van Hummelen et al.
(1996) scored a total of 10 000 cells per slide, and all slides were
scored in two steps. Slides were coded, 5000 spermatozoa were
scored, then the slides were re-coded and a second group of 5000
spermatozoa in a different area of the slide were scored. Robbins
et al. (1997) used a similar system in which two researchers each
scored 5000 spermatozoa per specimen; they were blinded to
the identity and treatment status of the samples, as well as to
each others scoring results. In the study of Chevret et al. (1997),
slides were scored by two independent observers who each
counted about 3000 spermatozoa per slide. No significant differences were detected between the results for the two observers.
Conclusions and future directions
595
S.E.Downie et al.
recently for studies on structural anomalies as well. Multiprobe FISH has removed the uncertainties associated with
estimating aneuploidy using single-probe FISH, and should
always be used in preference to single-probe FISH. The
availability now of direct-labelled probes ensures that procedures can be completed in one day, and improved signal-tonoise ratios are obtained. If necessary, however, indirect FISH
techniques can be used to increase signal intensity or provide
greater flexibility. It has been suggested that multi-probe FISH
in spermatozoa will create problems unless computerized
image analysis is introduced to detect multiple signals (Reid
et al., 1992a; Schattman et al., 1993). However, this does not
appear to be the case. Double-probe FISH provides the
means for obtaining accurate and clinically significant data on
autosomal aneuploidy frequencies in human spermatozoa,
while triple-probe FISH should be used to study sex chromosome aneuploidy.
However, the technological evolution must continue so that
the limitations of FISH are resolved. Particular attention should
be given to the establishment of optimal pretreatment and
hybridization conditions, the influence of different probes, and
meaningful scoring criteria should be established and verified.
It is also important to determine the extent of inter-donor
variability, and while some researchers have attempted to
address this issue, it will only be achieved using standardized
methodology and large numbers of men. Providing that
adequate attention is given to these technical aspects and to
experimental design, FISH will provide useful estimates of
aneuploidy in human spermatozoa under a variety of clinical
conditions.
References
Abruzzo, M.A., Griffin, D.K., Millie, E.A. et al. (1996) The effect of Ychromosome alpha-satellite array length on the rate of sex chromosome
disomy in human sperm. Hum. Genet., 97, 819–823.
Armstrong, S.J., Kirkham, A.J. and Hulten, M.A. (1994) XY chromosome
behaviour in the germ-line of the human male: a FISH analysis of spatial
orientation, chromatin condensation and pairing. Chromosome Res., 2,
445–452.
Balhorn, R. (1982) A model for the structure of chromatin in mammalian
sperm. J. Cell Biol., 93, 298–305.
Barone, J.G., de Lara, J., Cummings, K.B. and Ward, D.S. (1994) DNA
organization in human spermatozoa. J. Androl., 15, 139–144.
Bedford, J.M. and Calvin, H.I. (1974) The occurrence and possible functional
significance of -S-S- crosslinks in sperm heads, with particular reference
to eutherian mammals. J. Exp. Zool., 188, 137–156.
Benet, J. and Martin, R.H. (1988) Sperm chromosome complements in a
47,XYY man. Hum. Genet., 78, 313–315.
Benet, J., Genesca, A., Navarro, J. et al. (1992) Cytogenetic studies in motile
sperm from normal men. Hum. Genet., 89, 176–180.
Bischoff, F.Z., Nguyen, D.D., Burt, K.J. and Shaffer, L.G. (1994) Estimates
of aneuploidy using multicolor fluorescence in situ hybridization on human
sperm. Cytogenet. Cell Genet., 66, 237–243.
Blanco, J., Egozcue, J. and Vidal, F. (1996) Incidence of chromosome
21 disomy in human spermatozoa as determined by fluorescent in-situ
hybridization. Hum. Reprod., 11, 722–726.
Blanco, J., Rubio, C., Simón, C. et al. (1997) Increased incidence of disomic
sperm nuclei in a 47,XYY male assessed by fluorescent in situ hybridization
(FISH). Hum. Genet., 99, 413–416.
Bobrow, M., Madan, K. and Pearson, P.L. (1972) Staining of some specific
regions of human chromosomes, particularly the secondary constriction of
number 9. Nature New Biol., 238, 122–124.
Brandriff, B.F. and Gordon, L.A. (1990) Human sperm cytogenetics and the
one-cell zygote. In Banbury Report 34: Biology of Mammalian Germ Cell
596
Mutagenesis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
pp. 183.
Brandriff, B.F. and Gordon, L.A. (1992) Spatial distribution of sperm-derived
chromatin in zygotes determined by fluorescence in situ hybridization.
Mutat. Res., 296, 33–42.
Brandriff, B., Gordon, L., Ashworth, L. et al. (1985) Chromosomes of human
sperm: variability among normal individuals. Hum. Genet., 70, 18–24.
Burgoyne, P.S. (1979) Evidence for an association between univalent Y
chromosomes and spermatocyte loss in XYY mice and men. Cytogenet.
Cell Genet., 23, 84–89.
Chevret, E., Rousseaux, S., Monteil, M. et al. (1994) Male meiotic segregation
of gonosomes analysed by two colour FISH in human interphase
spermatozoa. Hum. Genet., 94, 701–704.
Chevret, E., Rousseaux, S., Monteil, M. et al. (1995) Meiotic segregation of
the X and Y chromosomes and chromosome 1 analyzed by three-colour
FISH in human interphase spermatozoa. Cytogenet. Cell Genet., 71,
126–130.
Chevret, E., Rousseaux, S., Monteil, M. et al. (1996) Increased incidence of
hyperhaploid 24,XY spermatozoa detected by three-colour FISH in a
46,XY/47,XXY male. Hum. Genet., 97, 171–175.
Chevret, E., Rousseaux, S., Monteil, M. et al. (1997) Meiotic behaviour of
sex chromosomes investigated by three-colour FISH on 35 142 sperm
nuclei from two 47, XYY males. Hum. Genet., 99, 407–412.
Coonen, E., Pieters, M.H.E.C., Dumoulin, J.C.M. et al. (1991) Nonisotopic
in situ hybridization as a method for non-disjunction studies in human
spermatozoa. Mol. Reprod. Dev., 28, 18–22.
Dauwerse, J.G., Wiegant, J., Raap, A.K. et al. (1992) Multiple colours by
fluorescence in situ hybridization using ratio-labelled DNA probes create
a molecular karyotype. Hum. Mol. Genet., 1, 593–598.
De Brackeleer, M. and Dao, T.-N. (1991) Cytogenetic studies in male
infertility: a review. Hum. Reprod., 6, 245–250.
Engel, W., Murphy, D. and Schmid, M. (1996) Are there genetic risks
associated with microassisted reproduction? Hum. Reprod., 11, 2359–2370.
Estop, A.M., Cieply, K., Vankirk, V. et al. (1991) Cytogenetic studies in
human sperm. Hum. Genet., 87, 447–451.
Genesca, A., Benet, J., Caballin, M.R. et al. (1990) Significance of structural
chromosome aberrations in human sperm: analysis of induced aberrations.
Hum. Genet., 85, 495–499.
Geraedts, J. and Pearson, P. (1973) Specific staining of the human number 1
chromosome in spermatozoa. Humangenetik, 20, 171–173.
Goldman, A.S.H., Fomina, Z., Knights, P.A. et al. (1993) Analysis of the
primary sex ratio, sex chromosome aneuploidy and diploidy in human
sperm using dual-color fluorescence in situ hybridisation. Eur. J. Hum.
Genet., 1, 325–334.
Griffin, D.K., Abruzzo, M.A., Millie, E.A. et al. (1995) Non-disjunction in
human sperm: evidence for an effect of increasing paternal age. Hum. Mol.
Genet., 4, 2227–2232.
Guttenbach, M. and Schmid, M. (1990) Determination of Y chromosome
aneuploidy in human sperm nuclei by nonradioactive in situ hybridization.
Am. J. Hum. Genet., 46, 553–558.
Guttenbach, M. and Schmid, M. (1991) Non-isotopic detection of chromosome
1 in human meiosis and demonstration of disomic sperm nuclei. Hum.
Genet., 87, 261–265.
Guttenbach, M., Schakowski, R. and Schmid, M. (1994a) Incidence of
chromosome 3, 7, 10, 11, 17 and X disomy in mature human sperm nuclei
as determined by nonradioactive in situ hybridization. Hum. Genet., 93,
7–12.
Guttenbach, M., Schakowski, R. and Schmid, M. (1994b) Incidence of
chromosome 18 disomy in human sperm nuclei as detected by nonisotopic
in situ hybridization. Hum. Genet., 93, 421–423.
Guttenbach, M., Martinez-Exposito, M.-J., Michelmann, H.W. et al. (1997)
Incidence of diploid and disomic sperm nuclei in 45 infertile men. Hum.
Reprod., 12, 468–473.
Han, T.L., Webb, G.C., Flaherty, S.P. et al. (1992) Detection of chromosome
17- and X-bearing human spermatozoa using fluorescence in situ
hybridization. Mol. Reprod. Dev., 33, 189–194.
Han, T.L., Ford, J.H., Webb, G.C. et al. (1993a) Simultaneous detection of
X- and Y-bearing human sperm by double fluorescence in situ hybridization.
Mol. Reprod. Dev., 34, 308–313.
Han, T.L., Flaherty, S.P., Ford, J.H. and Matthews, C.D. (1993b) Detection
of X- and Y-bearing human spermatozoa after motile sperm isolation by
swim-up. Fertil. Steril., 60, 1046–1051.
Han, T.L., Ford, J.H., Flaherty, S.P. et al. (1994) A fluorescent in situ
hybridization analysis of the chromosome constitution of ejaculated sperm
in a 47,XYY male. Clin. Genet., 45, 67–70.
Aneuploidy in human spermatozoa
Holmes, J.M. and Martin, R.H. (1993) Aneuploidy detection in human sperm
nuclei using fluorescence in situ hybridization. Hum. Genet., 91, 20–24.
Jackson-Cook, C. and Haller, V.L. (1991) Estimation of aneuploidy levels in
human spermatozoa using chromosome complements and chromosome
specific probes. Am. J. Hum. Genet., 49 (Suppl.), 18.
Jacobs, P.A. (1992) The chromosome complement of human gametes. Oxford
Rev. Reprod. Biol., 14, 47–72.
Joseph, A.M., Gosden, J.R. and Chandley, A.C. (1984) Estimation of
aneuploidy levels in human spermatozoa using chromosome specific probes
and in situ hybridisation. Hum. Genet., 66, 234–238.
Kamiguchi, Y. and Mikamo, K. (1986) An improved, efficient method for
analyzing human sperm chromosomes using zona-free hamster ova. Am. J.
Hum. Genet., 38, 724–740.
Kearns, W.G. and Pearson, P.L. (1994) Fluorescent in situ hybridization using
chromosome-specific DNA libraries. Meth. Mol. Biol., 33, 15–22.
Klasen, M. and Schmid, M. (1981) An improved method for Y-body
identification and confirmation of a high incidence of YY sperm nuclei.
Hum. Genet., 58, 156–161.
Lowe, X., Collins, B., Allen, J. et al. (1995) Aneuploidies and micronuclei
in the germ cells of male mice of advanced age. Mutat. Res., 338, 59–76.
Lu, P.Y., Hammitt, D.G., Zinsmeister, A.R. and Dewald, G.W. (1994) Dual
color fluorescence in situ hybridization to investigate aneuploidy in sperm
from 33 normal males and a man with a t(2;4;8)(q23;q27;p21). Fertil.
Steril., 62, 394–399.
Martin, R.H. (1990) Chromosomal analysis of human spermatozoa.[Abstr.]
J. In Vitro Fertil. Embryo Transfer, 7, 196.
Martin, R.H. (1993) Detection of genetic damage in human sperm. Reprod.
Toxicol., 7, 47–52.
Martin, R.H. (1994) Detection of aneuploidy for chromosomes 1, 12, X
and Y in human spermatozoa using multicolour fluorescence in-situ
hybridization.[Abstr. no. 48] Hum. Reprod., 9 (Suppl.), 4.
Martin, R.H. and Rademaker, A. (1995) Reliability of aneuploidy estimates
in human sperm: results of fluorescence in situ hybridization studies using
two different scoring criteria. Mol. Reprod. Dev., 42, 89–93.
Martin, R.H., Hildebrand, K., Yamamoto, J. et al. (1986) An increased
frequency of human sperm chromosomal abnormalities after radiotherapy.
Mutat. Res., 174, 219–225.
Martin, R.H., Ko, E. and Rademaker, A.W. (1991) Distribution of aneuploidy
in human gametes: comparison between human sperm and oocytes. Am.
J. Med. Genet., 39, 321–331.
Martin, R.H., Ko, E. and Chan, K. (1993) Detection of aneuploidy in human
interphase spermatozoa by fluorescence in situ hybridization (FISH).
Cytogenet. Cell Genet., 64, 23–26.
Martin, R.H., Chan, K., Ko, E. and Rademaker, A.W. (1994) Detection of
aneuploidy in human sperm by fluorescence in situ hybridization (FISH):
different frequencies in fresh and stored sperm nuclei. Cytogenet. Cell
Genet., 65, 95–96.
Martin, R.H., Rademaker, A.W. and Leonard, N.J. (1995a) Analysis of
chromosomal abnormalities in human sperm after chemotherapy by
karyotyping and fluorescence in situ hybridization (FISH). Cancer Genet.
Cytogenet., 80, 29–32.
Martin, R.H., Spriggs, E., Ko, E. and Rademaker, A.W. (1995b) The
relationship between paternal age, sex ratios, and aneuploidy frequencies
in human sperm, as assessed by multicolor FISH. Am. J. Hum. Genet., 57,
1395–1399.
Martin, R.H., Spriggs, E., and Rademaker, A.W. (1996) Multicolor fluorescence
in situ hybridization analysis of aneuploidy and diploidy frequencies in
225846 sperm from 10 normal men. Biol. Reprod., 54, 394–398.
Martin, R.H., Ernst, S., Rademaker, A. et al. (1997) Chromosomal
abnormalities in sperm from testicular cancer patients before and after
chemotherapy. Hum. Genet., 99, 214–218
Martini, E., Speel, E.J.M., Geraedts, J.P.M. et al. (1995) Application of
different in-situ hybridization detection methods for human sperm analysis.
Hum. Reprod., 10, 855–861.
Martini, E., Geraedts, J.P.M., Liebaers, I. et al. (1996) Constitution of semen
samples from XYY and XXY males as analysed by in-situ hybridization.
Hum. Reprod., 11, 1638–1643.
Melnyk, J., Thompson, H., Rucci, A.J. et al. (1969) Failure of transmission
of the extra chromosome in subjects with 47,XYY karyotype. Lancet, ii,
797–798.
Mercier, S. and Bresson, J.L. (1997) Analysis of chromosomal equipment in
spermatozoa of a 46,XY/47,XY/18 male by means of multicolour
fluorescent in situ hybridization: confirmation of a mosaicism and evaluation
of risk for offspring. Hum. Genet., 99, 42–46.
Mercier, S., Morel, F., Roux, C. et al. (1996) Analysis of the sex chromosomal
equipment in spermatozoa of a 47,XYY male using two-colour fluorescence
in-situ hybridization. Mol. Hum. Reprod., 2, 485–488.
Miharu, N., Best, R.G. and Young, S.R. (1994) Numerical chromosome
abnormalities in spermatozoa of fertile and infertile men detected by
fluorescence in situ hybridization. Hum. Genet., 93, 502–506.
Mikamo, K., Kamiguchi, Y. and Tateno, H. (1990) Spontaneous and in vitro
radiation-induced chromosome aberrations in human spermatozoa:
application of a new method. In Mendelsohn, M.L. and Albertini, R.J.
(eds), Mutation and the Environment. Part B. Metabolism, Testing Methods
and Chromosomes. John Wiley and Sons, New York, pp. 447–456.
Moosani, N., Pattinson, M.A., Carter, M.D. et al. (1995) Chromosomal analysis
of sperm from men with idiopathic infertility using sperm karyotyping and
fluorescence in situ hybridization. Fertil. Steril., 64, 811–817.
Nederlof, P.M., Van der Flier, S., Wiegant, J. et al. (1990) Multiple fluorescence
in situ hybridization. Cytometry, 11, 126–131.
Pang, M.G., Zackowski, J.L., Ryu, B.Y. et al. (1994) Aneuploidy detection
for chromosomes 1, X and Y by fluorescence in situ hybridization in
human sperm from oligoasthenoteratozoospermic patients. Am. J. Hum.
Genet., 55, A113.
Pawlowitski, I.H. and Pearson, P.L. (1972) Chromosomal aneuploidy in human
spermatozoa. Humangenetik, 16, 119–122.
Payne, D. and Matthews, C.D. (1995) Intracytoplasmic sperm injection clinical results from the Reproductive Medicine Unit, Adelaide. Reprod
Fertil. Dev., 7, 219–227.
Pearson, P. (1972) The use of new staining techniques for human chromosome
identification. J. Med. Genet., 9, 264–275.
Pearson, P.L. and Bobrow, M. (1970) Fluorescent staining of the Y chromosome
in meiotic stages of the human male. J. Reprod. Fertil., 22, 177–179.
Pieters, M.H.E.C., Geraedts, J.P.M., Meyer, H. et al. (1990) Human gametes
and zygotes studied by nonradioactive in situ hybridization. Cytogenet.
Cell Genet., 53, 15–19.
Pinkel, D., Landegent, J., Collins, C. et al. (1988) Fluorescence in situ
hybridization with human chromosome-specific libraries: detection of
trisomy 21 and translocations of chromosome 4. Proc. Natl. Acad. Sci.
USA, 85, 9138–9142.
Rademaker, A. Spriggs, E., Ko, E. and Martin, R. (1997) Reliability of
estimates of diploid human spermatozoa using multicolour fluorescence insitu hybridization. Hum. Reprod., 12, 77–79.
Reid, T., Landes, G., Dackowski, W. et al. (1992a) Multicolor fluorescence
in situ hybridization for the simultaneous detection of probe sets for
chromosome 13, 18, 21, X and Y in uncultured amniotic fluid cells. Hum.
Mol. Genet., 1, 307–313.
Reid, T., Baldini, A., Rand, T.C. and Ward, D.C. (1992b) Simultaneous
visualization of seven different DNA probes by in situ hybridization using
combinatorial fluorescence and digital imaging microscopy. Proc. Natl.
Acad. Sci. USA, 89, 1388–1392.
Robbins, W.A., Segraves, R., Pinkel, D. and Wyrobek, A.J. (1993) Detection
of aneuploid human sperm by fluorescence in situ hybridization: evidence
for a donor difference in frequency of sperm disomic for chromosomes 1
and Y. Am. J. Hum. Genet., 52, 799–807.
Robbins, W.A., Baulch, J.E., Moore II, D. et al. (1995) Three-probe
fluorescence in situ hybridization to assess chromosome X, Y and 8
aneuploidy in sperm of 14 men from two healthy groups: evidence for a
paternal age effect on sperm aneuploidy. Reprod. Fertil. Dev., 7, 799–809.
Robbins, W.A., Meistrich, M.L., Moore II, D. et al. (1997) Chemotherapy
induces transient sex chromosomal and autosomal aneuploidy in human
sperm. Nature Genet., 16, 74–78.
Rousseaux, S. and Chevret, E. (1995) In-vitro decondensation of human
spermatozoa for fluorescence in-situ hybridization. Mol. Hum. Reprod., 1,
see Hum. Reprod., 10, 2209–2213.
Rousseaux, S., Chevret, E., Monteil, M. et al. (1995a) Meiotic segregation in
males heterozygote for reciprocal translocations: analysis of sperm nuclei
by two and three colour fluorescence in situ hybridization. Cytogenet. Cell
Genet., 71, 240–246.
Rousseaux, S., Chevret, E., Monteil, M. et al. (1995b) Sperm nuclei analysis
of a Robertsonian t(14q21q) carrier, by FISH, using three plasmids and
two YAC probes. Hum. Genet., 96, 655–660.
Rudak, E., Jacobs, P.A. and Yanagimachi, R. (1978) Direct analysis of the
chromosome constitution of human spermatozoa. Nature, 274, 911–913.
Schattman, G.L., Munné, S., Grifó, J.G. et al. (1993) Aneuploidy in
spermatozoa using fluorescence in situ hybridization. J. Assist. Reprod.
Genet., 10, 360–365.
Seuanez, H., Mitchell, A.R. and Gosden, J.R. (1976) The chromosomal
distribution of human satellite III DNA during meiosis. Cytobios, 15, 79–84.
597
S.E.Downie et al.
Spriggs, E.L. and Martin, R.H. (1994) Analysis of segregation in a human
male reciprocal translocation carrier, t(1;11)(p36.3;q13.1), by two-colour
fluorescence in situ hybridization. Mol. Reprod. Dev., 38, 247–250.
Spriggs, E.L., Rademaker, A.W. and Martin, R.H. (1995) Aneuploidy in
human sperm: results of two- and three-colour fluorescence in situ
hybridization using centromeric probes for chromosomes 1, 12, 15, 18, X
and Y. Cytogenet. Cell Genet., 71, 47–53.
Spriggs, E.L., Rademaker, A.W. and Martin, R.H. (1996) Aneuploidy in
human sperm: the use of multicolor FISH to test various theories of
nondisjunction. Am. J. Hum. Genet., 58, 356–362.
Sumner, A.T., Robinson, J.A. and Evans, H.J. (1971) Distinguishing between
X, Y and YY-bearing human spermatozoa by fluorescence and DNA
content. Nature New Biol., 229, 231–233.
Templado, C., Marquez, C., Munné, S. et al. (1996) An analysis of human
sperm chromosome aneuploidy. Cytogenet. Cell Genet., 74, 194–200.
Testart, J., Gautier, E., Brami, C. et al. (1996) Intracytoplasmic sperm injection
in infertile patients with structural chromosome abnormalities. Hum.
Reprod., 11, 2609–2612.
Tournaye, H., Staessen, C., Liebaers, I. et al. (1996) Testicular sperm recovery
in nine 47,XXY Klinefelter patients. Hum. Reprod., 11, 1644–1649.
Trask, B.J. (1991) DNA sequence localization in metaphase and interphase
cells by fluorescence in situ hybridization. Meth. Cell Biol., 35, 3–35.
Van Hummelen, P., Lowe, X.R. and Wyrobek, A.J. (1996) Simultaneous
detection of structural and numerical chromosome abnormalities in sperm
of healthy men by multicolor fluorescence in situ hybridization. Hum.
Genet., 98, 608–615.
Van Steirteghem, A.C., Nagy, Z., Joris, H. et al. (1993) High fertilization and
implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 8,
1061–1066.
Wang, H.X., Flaherty, S.P., Swann, N.J. and Matthews, C.D. (1994) Assessment
of the separation of X- and Y-bearing sperm on albumin gradients using
double-label fluorescence in situ hybridization. Fertil. Steril., 61, 720–726.
Ward, W.S. and Coffey, D.S. (1991) DNA packaging and organization in
mammalian spermatozoa: comparison with somatic cells. Biol. Reprod.,
44, 569–574.
West, J.D., West, K.M. and Aitken, R.J. (1989) Detection of Y-bearing
spermatozoa by DNA–DNA in situ hybridisation. Mol. Reprod. Dev., 1,
201–207.
Willard, H.F. and Waye, W.S. (1987) Hierarchical order in chromosomespecific human alpha satellite DNA. Trends Genet., 3, 192–198.
Williams, B.J., Ballenger, C.A., Malter, H.E. et al. (1993) Nondisjunction in
human sperm: results of fluorescence in situ hybridization studies using
two and three probes. Hum. Mol. Genet., 2, 1929–1936.
Wong, T.W., Horvath, K.A. and Kao, N.L. (1987) Chromosome anomalies
and male infertility. In Gondos, B. and Riddick, D.H. (eds), Pathology of
Infertility. Thieme, New York, pp. 244–263.
Wyrobek, A.J., Alhborn, T., Balhorn, R. et al. (1990) Fluorescence in situ
hybridization to Y chromosomes in decondensed human sperm nuclei. Mol.
Reprod. Dev., 27, 200–208.
Wyrobek, A.J., Robbins, W.A., Weier, H.U. and Pinkel, D. (1993a) Detecting
sex-chromosomal and autosomal aneuploidy in human sperm by multicolour fluorescence in situ hybridization. [Abstr.] In 6th ICEM Satellite
Meeting, Barossa Valley, South Australia.
Wyrobek, A.J., Robbins, W.A., Tang, C. et al. (1993b) Hierarchical organization
of human sperm chromatin is a critical factor in the detection of
chromosomal aneuploidies in fluorescence in situ hybridization. Am. J.
Hum. Genet., 53 (Suppl.), 130.
Wyrobek, A.J., Robbins, W.A., Mehraein, Y. et al. (1994) Detection of sex
chromosomal aneuploidies X-X, Y-Y, and X-Y in human sperm using twochromosome fluorescence in situ hybridization. Am. J. Med. Genet., 53, 1–7.
Yanagimachi, R., Yanagimachi, H. and Rogers, B.J. (1976) The use of zonafree animal ova as a test system for the assessment of the fertilizing
capacity of human spermatozoa. Biol. Reprod., 15, 471–476.
Yurov, Y.B., Saias, M.J., Vorsanova, S.G. et al. (1996) Rapid chromosomal
analysis of germ-line cells by FISH: an investigation of an infertile male
with large-headed spermatozoa. Mol. Hum. Reprod., 2, 665–668.
Received on December 10, 1996; accepted on May 7, 1997
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