Molecular Human Reproduction vol.3 no.9 pp. 815–819, 1997
Estimation of aneuploidy for chromosomes 3, 7, 16, X and Y in
spermatozoa from 10 normospermic men using fluorescence in-situ
hybridization
Sarah E.Downie, Sean P.Flaherty1, Nicholas J.Swann and Colin D.Matthews
Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital,
Woodville, SA 5011, Australia
1To
whom correspondence should be addressed
Fluorescence in-situ hybridization (FISH) is a fast and efficient method of estimating aneuploidy in human
spermatozoa. In this study, we have estimated baseline disomy frequencies in spermatozoa from a group of
10 normospermic men, using stringent scoring criteria. A triple-probe FISH procedure was used for chromosomes 3, X and Y, while a double-probe FISH method was used for chromosomes 7 and 16. A total of 101 273
spermatozoa were scored for chromosomes 3, X and Y, resulting in 97.83% haploidy (3X or 3Y), 0.39% disomy
(33X, 33Y, 3XX, 3YY or 3XY) and 0.35% diploidy (33XX, 33YY or 33XY). A total of 100 760 spermatozoa were
scored for chromosomes 7 and 16, giving 98.9% haploidy (716), 0.11% disomy (7716 or 71616) and 0.27% diploidy
(771616). Disomy frequencies for individual chromosomes differed (chromosome 3, 0.20%; chromosome 7,
0.05%, chromosome 16, 0.06%; X1Y, 0.19%). The frequency of disomy 3 was significantly higher than disomy
7 (P J 0.019) and disomy 16 (P J 0.022), while the frequency of sex chromosome disomy was significantly
higher than disomy 7 (P J 0.0058) and disomy 16 (P J 0.0067), but not disomy 3 (P J 0.73). The disomy and
diploidy (0.27–0.35%) estimates obtained for this normospermic population were generally low and were
similar to other recent reports.
Key words: aneuploidy/chromosome/FISH/fluorescence in-situ hybridization/spermatozoa
Introduction
Chromosomal abnormalities affect the number (aneuploidy) or
structure of chromosomes. Aneuploidy is the condition in
which a cell possesses fewer or more chromosomes than an
exact multiple of the haploid number (Bond and Chandley,
1983). It can lead to infertility, pregnancy loss, infant death,
congenital malformations, mental retardation and behavioural
abnormalities (Epstein, 1986). The incidence of aneuploidy in
spontaneous abortions (26%) is higher than in liveborns
(0.3%), indicating that many aneuploid products are lost during
gestation (Jacobs, 1992). Hence, to estimate the true incidence
of aneuploidy, one should study gametes so that aneuploid
products lost prior to implantation are not overlooked.
Human spermatozoa are haploid cells which contain 22
autosomes and one sex chromosome, either 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 chromosome is missing (n – 1). Disomy
and nullisomy are collectively called aneuploidy. Diploid
spermatozoa have 44 autosomes and two sex chromosomes
(XX, YY or XY). The introduction of fluorescence in-situ
hybridization (FISH) has made it possible to study aneuploidy
in large numbers of human spermatozoa (Downie et al.,
1997). FISH is a technique whereby a specific DNA probe is
hybridized to complementary sequences on a target chromosome, then visualized using fluorescent haptens. It can be used
to detect aneuploidy in human spermatozoa using two or three
probes simultaneously to control for scoring errors and biases
© European Society for Human Reproduction and Embryology
(Williams et al., 1993; Martin et al., 1995; Robbins et al.,
1995) and it is a reliable method for estimating diploidy
(Rademaker et al., 1997). To establish reliable baseline aneuploidy frequencies in human spermatozoa, it is important to
assess a variety of chromosomes in spermatozoa from a range
of normospermic men. This has been performed in several
studies (Williams et al., 1993; Bischoff et al., 1994; Lu et al.,
1994; Martin et al., 1995; Robbins et al., 1995; Martin et al.,
1996; Spriggs et al., 1996; Van Hummelen et al., 1996),
although variable methodology, and in particular scoring criteria, has limited the value and reliability of some of the results
(Downie et al., 1997). Chromosomes 1, 12, 18, X and Y have
been extensively studied (Williams et al., 1993; Martin et al.,
1995) but, to date, many of the other autosomes such as 3, 7
and 16 have not been thoroughly studied in human spermatozoa
(Williams et al., 1993; Bischoff et al., 1994; Lu et al., 1994).
In this study, we estimated the incidence of disomy in
spermatozoa from 10 normospermic men using a triple-probe
FISH protocol for chromosomes 3, X and Y, and a doubleprobe FISH protocol for chromosomes 7 and 16. The use of
double- and triple-probe FISH protocols enabled us to distinguish between disomy and diploidy, and between nullisomy
and hybridization failure. Stringent scoring criteria were used
and a minimum of 10 000 spermatozoa were scored for each
sample. The specific aims were: (i) to estimate the incidence
of disomy for these five chromosomes in spermatozoa from a
reference population of normospermic men, (ii) to determine
whether the disomy frequencies differed for the three
815
S.E.Downie et al.
autosomes studied (chromosomes 3, 7, 16), and (iii) to compare
disomy frequencies for autosomes and the sex chromosomes.
Materials and methods
Subjects
Semen samples were obtained from 10 healthy donors who regularly
attended the Andrology Laboratory at The Queen Elizabeth Hospital,
Woodville, Australia. Eight of the donors were of proven fertility.
Their mean age (6 SD) was 34.7 6 7.0 years.
Collection and processing of semen samples
Semen samples were produced by masturbation, allowed to liquefy
at room temperature and then analysed using standard procedures
(World Health Organization, 1992). One sample was used from each
of five donors, two samples were used from each of four donors, and
three samples were used from the remaining donor. The values (mean
6 SD) for the 16 samples were: volume 3.7 6 0.9 ml; sperm
concentration 95 6 503106/ml; progressive motility 54 6 5%; sperm
morphology 33 6 10% normal. The donors all routinely produced
samples with .20% normal morphology, which is in the normal
range for our laboratory (Duncan et al., 1993).
Spermatozoa were washed in HEPES-buffered human tubal fluid
(HTF) medium (Quinn et al., 1985) three times for 10 min each. The
pellet was resuspended in 1–4 ml of HEPES–HTF medium and
20–50 µl of the sperm suspension was smeared onto clean glass
microscope slides and air-dried. Slides were stored with desiccant
at –20°C.
Pretreatment of spermatozoa
A modification of Williams et al. (1993) was used. Slides were
incubated for 30 min in 10 mM dithiothreitol (DTT; Sigma Chemical
Co, St Louis, MO, USA) in 0.1 M Tris, pH 8.0, and then for 2.7 6
1.6 h (mean 6 SD) in 1 mM DTT and 5–10 mM lithium
3,5-diiodosalicylic acid (LIS; Sigma) in 0.1 M Tris, pH 8.0. After
pretreatment, slides were washed in 0.1 M Tris, pH 8.0, rinsed in
Milli Q H2O (Millipore Corporation, Bedford, MA, USA) and
air-dried.
Mitotic chromosome spreads
Mitotic chromosome spreads from human male and female lymphocytes (Han et al., 1993) were used as positive controls for each
hybridization procedure.
DNA probes
Centromeric probes specific for chromosomes X, 3, 7 and 16 and a
probe specific for the long arm of the Y chromosome were used. X
and Y chromosome probes labelled with Spectrum Orange® and
Spectrum Green® respectively were purchased from Vysis (Framingham, MA, USA). A biotinylated chromosome 3 probe (D3Z1),
fluorescein isothiocyanate (FITC)-labelled chromosome 7 probe
(D7Z1), and biotinylated chromosome 16 probe (D16Z1) were purchased from Oncor (Gaithersburg, MD, USA).
Fluorescence in-situ hybridization (FISH)
Slides were treated with 100 µg/ml of RNase A in 300 mM NaCl,
30 mM sodium citrate, pH 7.0 [23 sodium chloride/sodium citrate
(SSC)] for 60 min at 37°C, rinsed three times in Milli Q H2O,
dehydrated through a series of ethanol solutions (70–100%) and airdried. For the triple-probe FISH protocol, a mixture of the X, Y and
3 probes was prepared in Vysis hybridization buffer. Hybridization
mix (~10 µl) was applied to each slide and a coverslip was sealed in
816
place using rubber cement. The slides were treated at 72–75°C for
10 min, and then hybridization proceeded at 37°C for 16–18 h. The
slides were washed three times in 15 mM NaCl, 1.5 mM sodium
citrate, pH 7.0 (0.13 SSC) at 60°C. The biotinylated chromosome 3
probe required a three-step detection procedure. The slides were first
incubated for 30 min in phosphate-buffered saline (PBS), pH 7.4
containing 1% blocking reagent (Boehringer Mannheim, Castle Hill,
NSW, Australia). They were incubated in a mixture of Texas Red–
avidin and FITC–avidin (Vector Laboratories, Burlingame, CA, USA)
in PBS containing 0.5% bovine serum albumin and 1% blocking
reagent for 1 h at 37°C for the first and third labelling steps. For the
second labelling step, slides were incubated in biotinylated antiavidin (Vector Laboratories) for 1 h at 37°C. The slides were washed
in 0.1% Tween 20 in PBS between each labelling step. After the final
wash, the slides were dehydrated through a series of ethanol solutions
(80 to 100%) and air-dried. They were mounted with a glycerolbased solution containing 0.1 µg/ml 4,6-diamidino-2-phenylindole
(DAPI; Sigma) as a nuclear counterstain and 20 mg/ml 1,4-diazobicyclo[2,2,2]octane (DABCO; Sigma) as an antifade.
The double-probe FISH protocol for chromosomes 7 and 16 was
as described above with three modifications: (i) oncor hybridization
buffer was used; (ii) the post-hybridization wash was 0.13 SSC at
60°C for 5 min; and (iii) the post-hybridization labelling procedure
for the chromosome 16 probe involved incubations for 30 min at
37°C in Texas Red–avidin for steps 1 and 3 and biotinylated antiavidin for step 2.
Scoring criteria
Slides were examined at a magnification of 31250 using a microscope
equipped with epifluorescence and a triple band-pass filter block
(Chroma Technology Corporation, Brattleboro, VT, USA). This
enabled simultaneous observation of the blue nuclear counterstain
and the red (X, 16), green (Y, 7) and yellow (3) hybridization signals.
Slides were scored only if the hybridization efficiency was ù98%,
and ~13104 spermatozoa were scored from each slide by one
observer (S.D).
The following scoring criteria were used. Only nuclei with an
attached tail were scored to eliminate non-sperm cells; the proportion
of nuclei without tails was ,0.06%. Overlapping or clumped sperm
nuclei were not scored, nor were nuclei which were over decondensed
or had indistinct boundaries. Two signals were scored as disomic if
they were of similar size and intensity and were separated by at least
one signal domain, otherwise they were considered to be split signals
and were scored as one. It has become well accepted that such
stringent criteria are required to ensure accurate aneuploidy estimates
(Downie et al., 1997). In the triple-probe FISH procedure, haploid
spermatozoa exhibited one yellow signal (3) and a red (X) or green
signal (Y). In the double-probe FISH procedure, haploid spermatozoa
exhibited one red signal (7) and one green signal (16).
Statistical analysis
Statistical analyses were performed using Excel 5.0 (Microsoft
Corporation, Redmond, WA, USA). Differences in disomy and
diploidy frequencies were analysed using a single factor analysis of
variance and paired t-tests. P , 0.05 was considered to be statistically
significant.
Results
Overall results
A total of 101 273 spermatozoa were scored for chromosomes
3, X and Y with an overall hybridization efficiency of 99.6%.
Aneuploidy in human spermatozoa
Table I. Disomy and diploidy estimates in spermatozoa from 10
normospermic men
Table II. Studies of sex chromosome disomy in human sperm using tripleprobe fluorescent in-situ hynbridization (FISH)
Donor no.
Study
Hybridization (%)
XX
YY
XY
Griffin et al. (1995)
Martin et al. (1995)
Robbins et al. (1995)
Martin et al. (1996)
Spriggs et al. (1995)
This study
–
ù98
–
ù98
ù98
ù99.6
0.02
0.07
0.031
0.07
0.07
0.03
0.03
0.18
0.031
0.18
0.21
0.03
0.10
0.16
0.095
0.16
0.15
0.13
1
2
3
4
5
6
7
8
9
10
Mean
SD
Disomy for chromosome:
Diploidy
3
7
16
X1Y
7,16
3,X,Y
0.39
0.52
0.31
0.22
0.08
0.15
0.17
0.04
0.06
0.07
0.20
0.15
0.02
0.05
0.07
0.03
0.05
0.04
0.09
0.06
0.04
0.06
0.05
0.02
0.05
0.07
0.03
0.05
0.00
0.08
0.14
0.07
0.04
0.06
0.06
0.03
0.11
0.42
0.23
0.26
0.05
0.30
0.11
0.09
0.17
0.13
0.19
0.11
0.06
0.25
0.13
0.27
0.33
0.25
0.43
0.31
0.37
0.29
0.27
0.10
0.13
0.42
0.35
0.40
0.25
0.42
0.63
0.39
0.37
0.15
0.35
0.14
Of the spermatozoa scored, 97.83% had a normal haploid
complement (3X or 3Y), 0.35% were diploid (33XX, 33YY
or 33XY) and 1.42% were aneuploid. Of the aneuploid
spermatozoa, 0.39% were disomic (33X, 33Y, 3XX, 3YY or
3XY) and 1.03% were nullisomic (30, 0X or 0Y).
Overall, 100 760 spermatozoa were scored for chromosomes
7 and 16, and the hybridization efficiency was 99.8%. Of the
spermatozoa scored, 98.9% were haploid (716), 0.27% were
diploid (771 616) and 0.64% were aneuploid. Of the aneuploid
spermatozoa, 0.11% were disomic (7716 or 71 616) while
0.53 % were nullisomic (70 or 016).
Inter-chromosomal disomy differences
Disomy results for each donor are presented in Table I. Using
single-factor analysis of variance, it was found that there were
significant inter-chromosomal differences in the frequency of
disomy (F 5 6.40, P 5 0.0014). Paired t-tests confirmed that
the frequency of disomy 3 was significantly higher than disomy
7 (t 5 2.844, P 5 0.019) and disomy 16 (t 5 2.765, P 5
0.022). Comparison of disomy for the sex chromosomes
(0.19%) and the autosomes revealed that sex chromosome
disomy was significantly higher than disomy 7 (t 5 0.186,
P 5 0.006) and disomy 16 (t 5 3.500, P 5 0.007), but not
disomy 3 (t 5 0.355, P 5 0.73).
Inter-donor disomy differences
The frequencies of disomy for chromosomes 3, X and Y varied
between individual donors, ranging from 0.04 to 0.52% for
chromosome 3, and 0.05 to 0.42% for the sex chromosomes
(Table I). In contrast, the disomy frequencies for chromosomes
7 (0.02 to 0.09%) and 16 (0 to 0.14%) showed less variation.
Two donors (#5,8) showed low disomy frequencies for all
five chromosomes, while donor #2 had markedly elevated
frequencies of disomy 3 and disomy X1Y, but normal values
for disomy 7 and disomy 16.
Diploidy estimates
The diploidy estimates (0.27, 0.35%) obtained from the two
FISH procedures were not significantly different (single-factor
analysis of variance, F 5 2.039, P 5 0.17). Marked interdonor differences in diploidy were noted (see Table I), with
estimates ranging from 0.13 to 0.63% with the triple-probe
FISH and from 0.06 to 0.43% with the double-probe FISH
procedure. Donors #1 and 7 showed very low and very high
diploidy levels respectively.
Discussion
In this study, we have estimated baseline disomy and diploidy
frequencies for chromosomes 3, 7, 16, X and Y in spermatozoa
from 10 normospermic men using double- and triple-probe
FISH procedures. The two FISH methods yielded clear signals
and ù98% hybridization efficiency for all the samples studied.
The overall percentages of haploid and diploid spermatozoa
were similar for the two protocols, however, the incidences of
aneuploid spermatozoa differed. The triple-probe protocol
resulted in 1.42% aneuploidy whereas only 0.64% of the
spermatozoa were scored as aneuploid using the double-probe
procedure. Nullisomy estimates in spermatozoa can be biased
by localized hybridization failure of one or more probes, so it
is routine practice to tabulate disomy and nullisomy separately
(Downie et al., 1997). In this study, the incidence of nullisomy
was 1.03% using three probes, over 2.5 times higher than
disomy using the same protocol. For the double-probe FISH
protocol, 0.53% of the spermatozoa were scored as nullisomic,
nearly five times higher than the incidence of disomy. Since
an equal number of nullisomic and disomic spermatozoa would
be expected from meiosis, it seems likely that nullisomy was
overestimated. This may have been due to spermatozoa which
failed to hybridize with one probe due to inadequate pretreatment, uneven nuclear decondensation or localized failure of
hybridization. Errors in the detection of biotinylated probes
using avidin and anti-avidin reagents may also have contributed
to this bias. In this study, 0.39% of spermatozoa were disomic
using the triple-probe method while 0.11% were disomic using
a double-probe method. We would expect these values to differ
due to differences in the rates of non-disjunction for individual
chromosomes.
We found that the incidence of sex chromosome disomy
(0.19%) was significantly higher than disomy 7 (0.05%) and
disomy 16 (0.06%), but not disomy 3 (0.20%). The incidence
of disomy 3 was also significantly higher than disomy 7 and
disomy 16. Chromosome 3 disomy in human spermatozoa has
only been studied in one other published study (Bischoff et al.,
1994), and they reported incidences of 0.41 and 0.27% in
spermatozoa from two donors. While these values are high,
as in the present study, only 1000 spermatozoa per sample
were scored which is insufficient for accurately estimating
817
S.E.Downie et al.
aneuploidy and therefore makes valid comparisons impossible.
Interestingly, four of the donors we studied (#5, 8, 9 and 10)
had low frequencies of disomy 3, comparable with disomy 7
and disomy 16, so inter-donor differences may have influenced
our results. There is no evidence for a higher incidence of
chromosome 3 aneuploidy in spontaneous abortions, liveborns
or human sperm karyotypes (Martin et al., 1991; Jacobs,
1992), so further studies on the incidence of chromosome 3
disomy in spermatozoa from normospermic donors are required
to clarify the present findings.
The frequency of disomy 7 in human spermatozoa has been
estimated only in two other FISH studies to date. Bischoff
et al. (1994) reported frequencies of 0.09% and 0% in two
donors, which is similar to this study, while Lu et al. (1994)
reported a higher frequency of 0.17%. However, both of these
groups scored low numbers of spermatozoa, making it difficult
to compare our baseline frequency with theirs. Similarly,
chromosome 16 disomy in human spermatozoa has been
estimated in only a few studies. Williams et al. (1993) reported
an incidence of 0.13%, which is twice that found in this study.
However, when we compare the range of disomy 16 estimates
in our study (0.0–0.14%) with theirs (0.03–0.20%), we find
that the ranges are very similar. Spriggs et al. (1996) reported
a disomy 16 frequency of 0.11%, while Bischoff et al. (1994)
reported much higher frequencies of 0.24 and 0.54% in two
men. Further detailed studies of chromosomes 7 and 16 are
required to establish the true range of disomy values for these
chromosomes in human spermatozoa.
Sex chromosome aneuploidy in spermatozoa has been
reported many times in the recent literature (Williams et al.,
1993; Chevret et al., 1995; Griffin et al., 1995; Martin et al.,
1995; Robbins et al., 1995; Spriggs et al., 1995; Martin et al.,
1996), and the results of some of these studies are outlined in
Table II. The frequencies in this study (disomy XX, 0.03%;
disomy YY, 0.03%; disomy XY, 0.13%) were similar to two
other studies which scored 10 000 spermatozoa per sample
for at least 10 different donors and adhered to strict scoring
criteria (Griffin et al., 1995; Robbins et al., 1995). However,
other studies have estimated higher levels of disomy XX and
disomy YY (Martin et al., 1995, 1996; Spriggs et al., 1995).
In all the published studies to date, however, similar estimates
of disomy XY have been reported. The higher incidence of
disomy XY relative to disomy XX or YY is not entirely
unexpected as there is evidence to suggest that the sex
chromosomes are more susceptible to first meiotic segregation
errors (Armstrong et al., 1994).
While multi-probe FISH has enabled extensive studies of
aneuploidy in human spermatozoa, there are technical limitations which must be taken into consideration and the technology
is still developing (reviewed by Downie et al., 1997). The
reliability of aneuploidy estimation is dependent upon the
application of consistent and reliable methodology, and some
of the technical features of published studies might have
resulted in over- or under-estimates. This could account for
some of the significant differences between studies. A variety of
pretreatment procedures have been used to partially decondense
sperm nuclei and their different modes of action might have
influenced estimates of aneuploidy. For instance, insufficient
818
pretreatment of spermatozoa can result in an overestimation
of nullisomy whereas excessive pretreatment could result in
an overestimation of disomy due to excessive signal splitting.
More reliable estimates will result from further improvements
and standardization of sperm pretreatment procedures. Another
aspect which influences the results is the specificity and
reliability of the probes used and their hybridization efficiency.
However, one of the most important factors is the use of
stringent, standardized scoring criteria and scoring of an
adequate sample size. Considering the low incidence of aneuploidy (1–4 per 1000 spermatozoa scored), scoring errors and
biases are very significant if only 1000 spermatozoa are scored.
Williams et al. (1993) therefore recommended that a minimum
of 10 000 spermatozoa should be scored from each sample to
minimize this bias. The importance of using stringent scoring
criteria to differentiate between disomy and split signals was
clearly demonstrated by Martin and Rademaker (1995).
The use of multi-probe FISH, efficient chromosome-specific
probes and stringent scoring criteria facilitated estimation of
aneuploidy in this study. Our results demonstrate that the
incidence of aneuploidy for chromosomes 3, 7, 16, X and Y
is quite low in spermatozoa from normospermic men. Ongoing
studies in our laboratory aim to study aneuploidy in spermatozoa from subfertile men, the importance of which was demonstrated by In’t Veld et al. (1997).
Acknowledgements
We would like to thank the staff of the Andrology Laboratory at The
Queen Elizabeth Hospital for assisting with donor recruitment, sample
processing and semen analyses.
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Received on April 4, 1997; accepted on June 18, 1997
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