Human Reproduction vol.15 no.4 pp.

853856, 2000
Detection of chromosomal abnormalities by fluorescent
in-situ hybridization in immotile viable spermatozoa
determined by hypo-osmotic sperm swelling test*
Hulusi Bulent Zeyneloglu1,3, Volkan Baltaci2,
immature forms of sperm cells from the testis may be obtained
Sevinc Ege1, Ali Haberal1 and S.Batioglu1
with varying success.
Poor or absent fertilization rates are obtained in cases of
1Department of Obstetrics/Gynaecology, and 2Department of
ICSI using immotile ejaculated spermatozoa of unknown
Medical Genetics, Baskent University, Ankara, Turkey
viability (Nagy et al. , 1995). Total immotility may be caused
3To whom correspondence should be addressed at: Mesa Koru
by necrozoospermia, ultrastructural defects or metabolic immaSitesi Manolya Blok 24, Cayyolu, 0630, Turkey
turity of the spermatozoa.
If randomly selected immotile spermatozoa are used for
In cases of total necrozoospermia in the ejaculate, motile
intracytoplasmic sperm injection (ICSI), pregnancy rates
spermatozoa can sometimes be recovered from a testicular
are significantly decreased. The hypo-osmotic swelling test
biopsy (Tournaye et al. , 1996). However, when selecting
(HOST) is the only method available to detect the viable,
spermatozoa to inject into an oocyte, it is always preferable
but immotile spermatozoa for ICSI. However, evidence is
to use the one that has shown some sign of motility and with
still lacking for the chromosomal abnormalities for the
best morphology available.
normal-looking, but immotile spermatozoa positive for
The hypo-osmotic swelling test (HOST) or cell staining
HOST. Sperm samples from 20 infertile men with normal
methods are often used to identify sperm viability. The mechanchromosomal constitution were obtained. After Percoll
isms of HOST and eosin-Y staining are mainly related to the
separation, morphologically normal but immotile spermafunctional integrity of sperm membrane and normal functional
tozoa were transported individually into HOST solution
activity of human spermatozoa (Jeyendran et al. , 1984). Since
for 1 min using micropipettes. Cells that showed tail curling
there is no available cell-staining technique that does not
with swelling in HOST were then transferred back into
impair the viability of the cell for future clinical use, HOST
human tubal fluid solution to allow reversal of swelling.
is the most useful method of selection for the viable spermatoThese sperm cells were fixed and processed for the multi-

zoa in an asthenozoospermic population. Successful use of

colour fluorescence in-situ hybridization (FISH) for chroimmotile spermatozoa
has been reported in ICSI (Casper
mosomes X, Y and 18. The same FISH procedure was
et al. , 1996). Although such cells have been successfully used
applied for the motile spermatozoa from the same cohort,
in ICSI, controversies exist over whether sperm phenotype
which formed the control group. The average aneuploidy
abnormalities are indicative of genotype abnormalities (Engel
rates were 1.70 and 1.54% in 1000 HOST positive immotile
et al. , 1996). The multicolour fluorescence in-situ hybridization
and motile spermatozoa respectively detected by FISH for
(FISH) may be one approach to give an insight into phenotypeeach patient. Our results indicate that morphologically
genotype interaction of sperm cells, although not the most
normal, immotile but viable spermatozoa have an aneuplosuitable technique.
idy rate similar to that of normal motile spermatozoa.
This study aims to show the chromosomal status of the
Key words: chromosome abnormality/hypo-osmotic swelling
immotile spermatozoa which show HOST positivity compared
test/male infertility/oligozoospermia/spermatozoa
to motile spermatozoa.
Materials and methods
Introduction
Sperm samples from 20 infertile men who were referred to Baskent
Intracytoplasmic sperm injection (ICSI) is the assisted reproUniversity IVF Unit were obtained by masturbation. All patients had
ductive technology procedure of choice in cases of severe
sperm analysis showing oligozoospermia according to WHO criteria
(WHO, 1999), 4% normal morphology according to Tygerberg strict
male-factor infertility and unexplained fertilization failure after
criteria (Kruger et al. , 1988) and normal chromosomal constitution in
conventional in-vitro fertilization (IVF) (Palermo et al. , 1992).
the peripheral lymphocyte metaphases. All patients had total motile
The advantages of ICSI are (i) the need of only one spermatosperm ratios that were 50% (according to WHO criteria: type A
zoon per oocyte and (ii) the ability to use not only the
type B 50%).
ejaculated spermatozoa, but also spermatozoa from testis and
The semen was washed using two-gradient (95 and 47.5%) Percoll
(Pharmacia AB, Uppsala, Sweden) separation, the pellet was diluted
in 500 l human tubal fluid solution (HTF) (IVF 50, Scandinavian
*Presented in part at the 16th World Congress on Fertility and Sterility
Scientific, Gothenburg, Sweden). The morphologically normal but

held conjointly with the 54th Annual Meeting of the American Society
immotile spermatozoa were transported individually into HOST
for Reproduction, October 49, 1998, San Francisco, CA, USA.
solution (Hypo-10, Scandinavian Scientific, Gothenburg, Sweden)
European Society of Human Reproduction and Embryology
853

H.B.Zeyneloglu et al.
Figure 2. Photographs of immotile spermatozoa after FISH analysis
with centromeric probes for the chromosomes 18 (blue), X (green)
Figure 1. The spermatozoa incubated in hypo-osmotic solution, the
and Y (orange) in a three-colour hybridization protocol. All probes
HOST () spermatozoa demonstrate curling and swelling of the
were directly labelled with fluorochromes and obtained
tails. Scale bar 5 m.
commercially. Scale bar 5 m.
using micropipettes (Hunter Scientific, Saffron Walden, Essex UK)
of a microinjection device (Nikon, Narishige, Japan); if the final

Table I. Comparison of chromosomal status of motile and immotile sperm

washout did not produce sufficient immotile spermatozoa, then the
cells from 20 infertile men after FISH analysis
spermatozoa from the interface between the gradients of Percoll were
Immotile HOST
Motile
used. The spermatozoa were incubated in hypo-osmotic solution for
positive cells (%)
sperm cells (%)
10 min. The cells which showed tail curling with swelling in HOST
(viable cells) (Figure 1) were then transferred back into HTF solution
X chromosome aneuploidy
0.46
0.43
to allow reversal of swelling. Then the cells were transferred to a
Y chromosome aneuploidy
0.33
0.25
glass microscope slide with a small amount of HTF solution. The
18 chromosome aneuploidy
0.27
0.23
cells were allowed to dry in the drop of HTF solution on the slide
Any combinationn
0.81
0.72
Average incidence of
1.70 4.48
1.54 4.13
with constant viewing under the inverted microscope. After complete
spermatozoa with abnormal
(0.92.6) range
(0.82.5) range
drying of the HTF solution, 1 drop (5 l) of the Carnoys fixative (1
chromosomal status
ml acetic acid, 3 ml methanol) was added to the spermatozoa. The
cells were constantly observed under the inverted microscope and
The two types of sperm cell were not significantly different.
another drop of fixative was added before complete drying and this
step repeated 5 times.
Decondensations of the spermatozoa were performed in 25 nmol/l
Results
dithiothreitol (DTT) in 0.1% trypsin (Biochrom KG, D-1000 Berlin,
The mean sperm concentration of the patients was 7.3
Germany). After incubation with DTT for 15 min, slides were rinsed
2.7106/ml and median was 7.5106/ml (range 2.312). Mean

twice in 2SSC (sodium chloride and sodium citrate) and dehydrated

percentage total motile (type A type B type C) spermatothrough ethanol series (70, 85, 90%) and air dried (Coonen et al. ,
zoa was 18.8 7.6% (median: 19%; range: 735). The mean
1991). Dehydrated samples were then denatured at 73C in 70%
percentage normal morphology according to Tygerberg strict
formamide/2SSC and hybridized overnight at 37C, using probes
criteria was 1.8 1.0% (median: 2.0; range: 03). Average
for the chromosomes 18, X and Y in a three-colour hybridization
protocol (Bischoff et al. , 1994; Blanco et al. , 1996). All probes were X chromosome, Y
chromosome and 18 chromosome anomaly
directly labelled with fluorophores and obtained commercially (Vysis
rates individually were 0.46, 0.33 and 0.27% respectively.
Inc., Downers Grove, IL, USA). The post-hybridization wash was
Overall, the average incidence of spermatozoa with abnormal
performed in 0.4SSC/0.3% NP40 at 73C for 2 min. The same
chromosomal status was 1.70 4.48% (0.92.6%) in immotile
fixation and FISH procedure were applied for the motile sperm from
HOST positive sperm cells detected by FISH for each patient.
the same cohort, which served as the control group. Slides were
These data are summarized in Table I.
examined using a DAPI/FITC/Texas Red triple-band pass filterblock
On the other hand, FISH analysis of motile spermatozoa
(Nikon, Japan) allowing the simultaneous visualization of orange,
yielded the chromosome, Y chromosome and 18 chromogreen and blue fluorophores, and photographed with a camera (Nikon
some anomaly rates as 0.43, 0.25 and 0.23% respectively
F-601 QD) (Figure 2). Hybridization efficiency was 95% in both
(Table I). Overall, the average incidence of spermatozoa with
motile and immotile sperm groups. An autosomal probe (chromosome
abnormal chromosomal status in motile HOST positive sperm
18 probe) served as internal control. Only intact cells and cells which
did not overlap were scored. Slides were scored blindly by two
cells detected by FISH for each patient was 1.54 4.13%
independent investigators. The statistical analysis included 2-ana(0.82.5%). The statistical analysis showed that the difference
lysis, and t-test. The mean values were given with their standard
in aneuploidy rate between the motile and immotile spermatodeviations (SD) in addition to median values and the range.
zoa was not significant ( P 0.23).
854
Chromosomal abnormalities in immotile spermatozoa
Discussion
McInnes et al. investigated sperm samples from infertile men
There is no described test with non-invasive properties to
using FISH with probes for chromosomes 13 and 21 and found

select a live spermatozoon for microinjection purposes, since

a highly significant increase in the frequency of spermatozoa
the spermatozoa identified by supravital stains such as eosindisomic for chromosome 13 in infertile patients (0.28%)
Y or acridine orange cannot be used for further clinical
compared to control donors (0.13%) and for chromosome 21
study. Desmet et al. reported that HOST might be useful in
(0.48% in infertile men versus 0.37% in controls) (McInnes
distinguishing viable from non-viable immotile spermatozoa
et al. , 1998).
for ICSI. A 30% fertilization rate was obtained with the
It has been shown (Bischoff et al. , 1994) that using single
injection of selected spermatozoa into 1-day-old oocytes, which
colour FISH was not sufficient to distinguish failed hybridizahad failed to fertilize after conventional IVF (Desmet et al. ,
tion from nullisomy, or disomy from diploidy, but a multicolour
1994). Casper et al. reported three pregnancies in eight clinical
FISH strategy provides an internal control. Three colour
probes (sex chromosomes plus one autosomal chromosome)
ICSI cycles after a fertilization rate of 43% of the oocytes
are sufficient to provide an internal control. For this reason,
injected with spermatozoa selected using HOST, compared to
one of the probes for chromosomes 13, 18 or 21 is frequently
the fertilization rate 26% using random sperm injection; i.e.
used for the demonstration of autosomal chromosomal status
the fertilization rate was almost doubled (Casper et al. , 1996).
(Bischoff et al. , 1994).
Vandervorst et al. used immotile spermatozoa intracytoplasmic
Immotility or non-progressive motility in spermatozoa may
injection in 11 patients without HOST and they had a 34%
result from centriolar or tubular abnormalities or absence of
fertilization rate, but no pregnancies ensued and they postulated
centrioles or microtubules such as supernumerary microtubules,
that they were able to find motile spermatozoa and succeeded
imperfect organization of the microtubules (Sauvalle et al. ,
in four pregnancies in the subsequent cycles (Vandervorst
1983), absence of the central pair of microtubules or the
et al. , 1997).
complete axoneme (Neugebauer et al. , 1990) or ultrastrucVerheyen et al. compared three hypo-osmotic solutions,
tural abnormalities such as the absence of dynein arms
namely, Jeyendran solution (Jeyendran et al. , 1984) containing
(Gopalkrishnan et al. , 1995). These abnormalities were comsodium citrate and fructose; milli-Q water; and a mixture of
monly detected in many variants of immotile cilia syndromes.
50% culture medium and 50% milli-Q water (Verheyen et al. ,

Despite these abnormalities, pregnancies were reported after

1997). They found that the solution composed of 50% culture
intracytoplasmic injections of immotile spermatozoa from
medium and 50% water was the most appropriate for selection
ejaculate or testicular extract of men with immotile cilia
of viable immotile spermatozoa for ICSI. We used a commercial
syndrome (Kahraman et al. , 1997; Olmedo et al. , 1997).
HOST solution in this study which is mainly based on
However, anti-sperm antibodies, infection of the genital tract
Jeyendran solution and we did not detect any toxicity, since a
and prolonged periods of anejaculation are most common
considerable number of the spermatozoa lived up to 24 h after
causes of total immotility (Vandervorst et al. , 1997). In
reversal of the hypo-osmotic swelling conditions.
addition, the low energy production in the spermatozoa or
Chromosomal abnormalities are detected in ~15% of
exposure to radical oxygen species may cause inadequate use
azoospermic subjects and 67% of subjects with 10106
of the energy sources in the spermatozoa. Coenzyme Q10,
spermatozoa/ml, either alone or in combination with other
being an antioxidant and energy promoter, is located in the
abnormalities of the semen (Bourrouillou et al. , 1992). Even
mitochondria of the midpiece. Men treated with oral CoQ-10
in subfertile patients with normozoospermia, an increased rate
did better in the next ICSI cycle after a failed cycle (Lewin
of chromosomal aberrations was detected (Matsuda et al. ,
and Lavon, 1997).
1989). Sex and autosomal chromosomes were found to be
It is a common procedure to use morphologically normal
abnormal in 4.2 and 1.5% of infertile men respectively.
spermatozoa for ICSI, if available, and it has recently been
While sex chromosome anomalies were predominant in nonconfirmed in an ESHRE abstract that decreased fertilization
obstructive azoospermic patients, autosomal chromosomal
rates result with the injection of spermatozoa with abnormal
anomalies were more common in patients with obstructive
morphology (De Vos et al. , 1999). In addition, our collaborators
azoospermia (Van Assche et al. , 1996). The most common
have already shown a relationship between diminished sperm
abnormality involving sex chromosomes is XXY and the most
maturity, increased cytoplasmic retention/head size and
common molecular defect is microdeletions in the AZF region
increased rate of chromosomal abnormalities as detected by
of the long arm of the Y chromosome (Reijo et al. , 1995).
multicolour FISH (Moretti et al. , 1997). A case report has
Bernardini investigated the cytogenetic constitution of infertile

recently been presented of a male with Y:16 chromosome

and normal men and found higher rates of sperm aneuploidy
translocation and FISH analysis of morphologically normal
for autosomes 1 and 17 with decreasing semen quality
spermatozoa showing 51% chromosomally normal or balanced
(5106/ml) compared to controls (1.43 versus 0.8%)
sperm cells (Giltay et al. , 1999). However, if morphologically
(Bernardini et al. , 1998). Guttenbach found a 0.310.41%
abnormal cells were also included in the FISH analysis nearly
disomy rate in individual sperm cells from 20 healthy fertile
90% of all the spermatozoa were unbalanced.
men using FISH with six different chromosome-specific DNA
Based on the above evidence, we chose to assess the
probes, and they found similar disomy rates as indicated by
aneuploidy status of the morphologically normal but immotile
two distinct hybridization signals for all chromosomes, ranging
spermatozoa in this study. Our data showed that normal but
from 0.31 to 0.34% (Guttenbach et al. , 1994a,b). In addition,
immotile spermatozoa might not differ from the chromosomal
855
H.B.Zeyneloglu et al.
status of the motile spermatozoa. The limited reports in the
abnormal sperm morphology in in vitro fertilization. Fertil. Steril. , 49, 112117.
current literature support the use of immotile, alive and normal
Lewin, A. and Lavon, H. (1997) The effect of coenzyme Q10 on sperm
spermatozoa yielded to achieve normal pregnancy.
motility and function. Mol. Aspects Med. , 18, S213219.
We suggest that any men with severe oligoasthenozoosperMatsuda, T., Nonomura, M., Okada, K. et al. (1989) Cytogenetic survey of
mia should have genetic consultation to rule out chromosomal
subfertile males in Japan. Urol. Int. , 44, 194197.
McInnes, B., Rademaker, A., Greene, C.A. et al. (1998) Abnormalities for
abnormalities or Y chromosome microdeletions. In such cases,
chromosomes 13 and 21 detected in spermatozoa from infertile men. Hum.
preimplantation diagnosis to the embryos, or prenatal diagnosis
Reprod. , 13, 27872790.
should be offered to the couple. We also call for further
Moretti, E., Gergely, A., Zeyneloglu, H.B. et al. (1997) Relationship among studies to
further the evaluation of the men with peripheral
head size, morphology and chromosome structure in human spermatozoa.
In American Society for Reproduction Medicine 53 Annual Meeting 18
chromosomal abnormality. Our results show that the normal
22 October, 1997, Cincinnati, OH. Fertil. Steril. , 68, S158.
karyotype of the immotile but morphologically normal spermaNagy, Z.P., Liu, J., Joris, H. et al. (1995) The result of intracytoplasmic sperm tozoa is
likely to decrease the risk of genetic transmission

injection is not related to any of the three basic sperm parameters. Hum.
Reprod. , 10, 11231129.
of the chromosomal abnormalities. In conclusion, immotile
Neugebauer, D.C., Neuwinger, J., Jockenhovel, F. et al. (1990) 9 0
spermatozoa from men with normal karyotype in the absence
axoneme in spermatozoa and some nasal cilia of a patient with totally
of microdeletions of the Y chromosome may be safely used
immotile spermatozoa associated with thickened sheath and short midpiece.
Hum. Reprod. , 5, 981986.
for ICSI purposes.
Olmedo, S.B., Nodar, F., Chillik, C. et al. (1997) Successful intracytoplasmic sperm
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fibrous sheath and chronic respiratory disease. Hum. Reprod. , 12, 14971499.
Palermo, G., Joris, H., Devroey, P. et al. (1992) Pregnancies after
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