Human Reproduction vol.15 no.7 pp.1529–1536, 2000
Incidence of aneuploid spermatozoa from subfertile
men: selected with motility versus hemizona-bound*
Q.Van Dyk1, S.Lanzendorf2, P.Kolm3, G.D.Hodgen2
and M.C.Mahony2,4
Key words: chromosomal aneuploidy/FISH/hemizona assay/
human spermatozoa/ICSI
1Reproductive
Medicine and Infertility Associates, P.A., 360
Sherman Street, St Paul, MN, 2The Jones Institute for Reproductive
Medicine, Department of Obstetrics and Gynecology, Eastern
Virginia Medical School, Norfolk, VA, and 3Biostatistics, Eastern
Virginia Medical School, Norfolk, VA, USA
4To
whom correspondence should be addressed at: The Jones
Institute for Reproductive Medicine, Department of Obstetrics and
Gynecology, Eastern Virginia Medical School, 601 Colley Avenue,
Norfolk, Virginia 23507, USA. E-mail: [email protected]
Spermatozoa–zona pellucida binding selects for human
spermatozoa with progressive motility, normal morphology
and functional competency. We postulated that this gamete
interaction would also act to select against spermatozoa
with chromosomal numerical aberrations. Spermatozoa
from 41 men participating in the intracytoplasmic sperm
injection (ICSI) programme were evaluated for the incidence of aneuploidy of chromosomes 18, X and Y. The
hemizona assay was utilized to determine whether zonabound spermatozoa from these patients have a reduced
incidence of aneuploidy compared with those selected by
motility only in a standard swim-up procedure. Using
multicolour fluorescence in-situ hybridization (FISH) with
DNA probes specific for chromosomes 18, X and Y, the
disomy rates for chromosomes 18, X, Y and XY were found
to be 0.31, 0.27, 0.29 and 0.14% respectively in the swimup motile fraction, and 0.31, 0.33, 0.32 and 0.19% respectively in the pellet fraction. Analysing the zona-bound
spermatozoa, the disomy rates for chromosome 18, X, Y
and XY were found to be 0.02, 0.15, 0.12 and 0.07%
respectively. The zona-bound spermatozoa had a significantly lower frequency of aneuploidy than the swim-up
motile fraction or the pellet fraction (P < 0.0001). The
incidence of chromosome 18 aneuploidy, including both
chromosome 18 disomy and nullisomy, in the swim-up
motile fractions was significantly increased in patients with
an abnormal or borderline hemizona index compared with
those with a normal hemizona index (P < 0.05). We also
found that a high incidence of sperm aneuploidy was
associated to a certain extent with low fertilization rate,
and with failure to achieve pregnancy through ICSI. This
study suggests that the human zona pellucida has the
capacity to select against aneuploid spermatozoa by an as
yet undetermined mechanism.
*Presented at the 23rd Annual Meeting of the American Society of
Andrology, Long Beach, California, USA, March 1998
© European Society of Human Reproduction and Embryology
Introduction
Intracytoplasmic sperm injection (ICSI) is the most invasive,
yet the most successful, assisted reproduction technique
currently offered to patients with male factor infertility
(Palermo et al., 1992). Concerns over the potential genetic
risks of this procedure surfaced soon after the first clinical
application of ICSI (Cummins and Jequier, 1995; Meschede
et al., 1995; Persson et al., 1996). Depending upon the
study, the percentages of abnormal karyotypes among ICSI
pregnancies range from 1% to a strikingly high incidence of
40% (Govaerts et al., 1995; In’t Veld et al., 1995; Liebaers
et al., 1995). More recently, a follow-up study of 1082 ICSI
pregnancies and children revealed a rate of 0.83% each for
sex-chromosome aberrations and autosomal aberrations, which
is significantly higher than the incidence of sex-chromosome
aberrations in the general population (Bonduelle et al., 1998).
These sex chromosome anomalies among ICSI pregnancies
are likely to be of paternal origin (Van Opstal et al., 1997).
Patients participating in ICSI programmes have on average
more severe fertility problems than patients in regular IVF
programmes. There is an increased incidence of chromosomal
structural aberrations among subfertile and especially severely
subfertile men. Men with low sperm counts have been reported
with a higher incidence of reciprocally balanced translocations
(Baschat et al., 1996). Chromosomal abnormalities can be
present in ~15% of the azoospermic men and in ~6–7% of
men with a low sperm count with or without other abnormal
semen characteristics (Bourrouillou et al., 1992). The situation
is further complicated by the fact that even chromosomally
normal individuals can produce aneuploid spermatozoa (Rudak
et al., 1978; Martin et al., 1987; Han et al., 1993; Holmes and
Martin, 1993). A significantly higher incidence of aneuploidy
was noted in semen from oligoasthenoteratozoospermic men
(infertility due to semen with low sperm concentration, poor
sperm motility and abnormal sperm morphology) compared
with those from normal men (Pang et al., 1995; Lahdetie et al.,
1997). Thus, the higher incidence of chromosomal abnormality
in the patient group, coupled with the probability of increased
chromosomal abnormalities in their spermatozoa, complicate
the risk factor analysis of ICSI.
No significant deviation in the incidence of newborns with
severe disorders born after IVF (1.5%) from the general
population (~1–2%) has been observed (American Fertility
Society, 1993). While IVF does not appear to result in increased
1529
Q.Van Dyk et al.
genetic defects, ICSI circumvents several steps necessary in
the natural fertilization process, such as spermatozoa–zona
pellucida binding and penetration, and fusion of the spermatozoon–oocyte cell membrane. The possible role of these steps
to function as a barrier against defective spermatozoa still
awaits investigation.
The zona pellucida is an extremely stringent tool to select
live and functional spermatozoa. Zona-bound spermatozoa can
be generally characterized as having good motility, normal
morphology, ability to bind tightly to the zona pellucida,
hyperactivate and to acrosome react (Coddington et al., 1990;
Menkveld et al., 1991; Oehninger et al., 1997). Sperm performance in the hemizona assay, a zona pellucida binding assay,
was found to be a valuable parameter of predicting the outcome
of in-vitro fertilization (Coddington et al., 1994). Thus, the
zona pellucida has proven to be an important barrier against
physiologically and functionally abnormal spermatozoa.
We postulate that bypassing the zona pellucida in the
fertilization process poses an increased risk of the oocyte being
fertilized by a spermatozoon with a genetic defect. We therefore
focused this study on determining whether zona pellucidabound spermatozoa exhibited lower aneuploidy rates than
spermatozoa selected by motility alone. At the Jones Institute
for Reproductive Medicine, ICSI is offered to patients with
male factor infertility and to patients who have previously
failed to achieve pregnancy through conventional IVF. The
hemizona assay is used for the identification and diagnosis of
male infertility, and to guide the selection of therapy. In this
study, we determined the aneuploidy incidences for chromosomes X, Y and 18 in spermatozoa from patients participating
in the ICSI programme. The incidences of aneuploidy for the
three chromosomes in the patients’ spermatozoa isolated in
swim-up supernatants and the pellet were compared with that
in hemizona-bound spermatozoa. In addition, the relationships
among different sperm characteristics [including concentration,
motility, morphology, hemizona assay (HZA) index, hypoosmotic swelling test index], fertilization index, pregnancy
status and the incidence of chromosome aneuploidy in spermatozoa from ICSI patients were also analysed.
Materials and methods
This study was conducted with the approval of the Institutional
Review Board of the Eastern Virginia Medical School. Forty-one
patients (age range 30 to 54 years) participating in the ICSI programme
were included in this study during the period from November 1996
to June 1997.
Semen preparation and evaluation
Semen samples were obtained by masturbation after 2–4 days of
sexual abstinence, and analysed after complete liquefaction within 1
h of collection. Sperm concentration and motility were evaluated
using the HTM-IVOS Motility Analyser (Hamilton-Thorne Research
Inc., Danvers, MA, USA). A 3 µl fraction of the sample was loaded
onto a 4-chamber microcell slide (Fertility Tech, Rockaway, NJ,
USA) and then transferred to the HTM-IVOS. Data were collected
on randomly selected fields along the length of the microcell chamber
until at least 100 motile spermatozoa were analysed. The analysis
was performed at 37°C. Sperm morphology was analysed using the
1530
strict criteria method. Morphology slides were prepared by making a
thin smear of semen on clean slides, and stained by the Diff-Quick
(Baxter Health Corp., McGaw Park, IL, USA) staining technique
(Kruger et al., 1986). From each specimen, the morphology of at
least 200 spermatozoa was determined at ⫻1000 magnification.
The motile sperm fractions were separated by swim-up. A 0.5 ml
fraction of semen was diluted with 1.5 ml of Ham’s F-10 medium
(GIBCO Lab., Grand Island, NY, USA) supplemented with 0.3%
human serum albumin (HSA) (Irvine Sci., Santa Anna, CA, USA),
and centrifuged at 300 g for 7 min. The sperm pellet was suspended
in 0.5 ml medium and re-centrifuged for 5 min. A 0.5 ml aliquot of
the medium was then layered gently over the pellet, and the specimen
was incubated at 37°C and 5% CO2 in water-saturated air. At the end
of the 1 h incubation, the supernatant was gently harvested without
disturbing the pellet. The sperm concentration and motility in the
swim-up supernatant was also evaluated using the HTM-IVOS
Motility Analyser.
Hemizona assay (HZA)
The HZA was performed using a protocol described previously
(Oehninger et al., 1991). Briefly, salt-stored human oocytes were
microbisected into matching hemizonae using Narishige micromanipulators (Narishige, Tokyo, Japan) mounted on a phase-contrast
inverted microscope (Nikon Diaphot, Garden City, NY, USA). Hemizonae were transferred into Ham’s F-10 medium with 0.3% HSA
droplets one day before the initiation of HZA, and stored at 4°C.
Droplets of each patient’s sperm swim-up suspension (500 000 motile
sperm/100 µl) were placed in a Petri dish, along with control (from
known fertile donors) sperm droplets of the same motile sperm
concentration. One hemizona of a pair was placed in the droplet of
the patient’s spermatozoa, while the other matching hemizona was
placed in the control sperm droplet. The droplets were covered with
mineral oil and incubated for 4 h (37°C, 5% CO2 in water-saturated
air). At the end of the incubation, each hemizona was removed and
rinsed through multiple droplets of fresh medium to dislodge loosely
attached spermatozoa. The number of spermatozoa tightly attached
to the outer surface of each hemizona was counted. For each hemizona
pair, the HZA index was calculated as follows: (number of patient
spermatozoa bound to the hemizona/number of control spermatozoa
bound to the hemizona)⫻100. The final HZA index was the average
of the three individual HZA indices. A final HZA index 艌35 was
considered normal, an index between 14 and 35 was considered
borderline, and an index ⬍14 was considered abnormal.
Hypo-osmotic swelling test (HOST)
HOST was carried out on spermatozoa in both the swim-up motile
fractions and the pellet fractions. The test was performed according
to a previously published method (Jeyendran et al., 1984), but with
some modifications. Briefly, an aliquot of 10 µl sample (either swimup supernatant or resuspended swim-up pellet) was added to a droplet
containing 100 µl hypo-osmotic solution (7.35 g sodium citrate and
13.5 g fructose in 1 l distilled water). The droplet was partially
covered with mineral oil and incubated (37°C, 5% CO2 in watersaturated air) for 30 min. Spermatozoa with thickening or curling
tails were scored as positive, while spermatozoa with straight tails
were scored as negative. A total of 100 spermatozoa were counted, and
the HOST index expressed as the percentage of positive spermatozoa in
the sample following the incubation.
Fixation and decondensation of the sperm nuclei
Spermatozoa in the swim-up supernatant fraction, pellet fraction, and
hemizona–spermatozoa complexes were washed separately in three
changes of phosphate-buffered saline (PBS; 0.15 mol/l NaCl, 10
Analysis of zona-bound and motility-selected spermatozoa using FISH
Table I. Sperm parameters of 41 ICSI patients evaluated for the incidence
of aneuploidy of chromosomes 18, X, and Y
Sperm parameter
Mean ⫾ SE
Semen concentration (⫻106/ml)
Semen motility (%)
Morphology (%)
Swim-up concentration (⫻106/ml)
Swim-up motility (%)
Swim-up HOST (%)
Hemizona index (%)
Fertilization rate (%)
Pregnancy rate (%)
90.6
43.2
5.6
32.9
85.1
66.0
80.2
74.6
33.3
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
13.8
3.1
0.5
7.1
2.1
2.2
12.7
2.9
Range
3.2–398
5–90
0.5–14.7
0.2–293.6
41–99
33–87
2–150
33–100
HOST ⫽ hypo-osmotic swelling test.
Figure 1. Hemizona-bound sperm nuclei hybridized with alphasatellite centromeric probes (⫻600 magnification) specific for
chromosomes X (green), Y (orange) and 18 (aqua) and were
counterstained with DAPI II.
mmol/l sodium phosphate, pH 7.2), and fixed on a clean glass slide
with freshly prepared fixative (methanol:acetic acid; 3:1). The slides
were air-dried and stored at –20°C until evaluation. For FISH analysis,
fixed spermatozoa were washed in 2⫻ SSC (0.3 mol/l NaCl, 30
mmol/l sodium citrate, pH 7.5) to remove any residual fixative and
incubated in 0.1 mol/l Tris–HCl buffer, pH 8.0, containing 50 mmol/
l dithiothreitol. After sperm nuclei decondensation, the slides were
washed once in 2⫻ SSC, dehydrated through an ethanol series (70–
90–100%), and air-dried (Jones et al., 1987).
Fluorescence in-situ hybridization (FISH) analysis
The DNA probes (Vysis, Downers Grove, IL, USA) used in this
study recognize the alpha satellite DNA of the centromeric region of
human chromosomes X (Xp11.1-q11.1), Y (Yp11.1-q11.1) and 18
(18p11.1-q11.1). The probes detecting chromosome X, Y and 18
were labelled with fluorescent haptens CEP (chromosome enumeration
probe) SpectrumGreen, CEP SpectrumOrange and CEP SpectrumAqua respectively (Figure 1). The process of FISH was carried
out according to the manufacturer’s recommendation (Vysis). Briefly,
slides were denatured at 75°C for 5 min in 70% formamide/2⫻ SSC,
followed by dehydration through an ethanol series (70–85–100%).
Slides were air-dried, and warmed to 45–50°C before the denatured
probe mixture was applied. The area with the probe mixture was
covered with a coverslip, sealed with rubber cement, and the slides
were placed in a humidified box in a 37°C incubator. Post-hybridization washes were carried out after overnight hybridization incubation.
Slides were first immersed in three changes of 50% formamide/2⫻
SSC for 10 min each, and then washed with 2⫻ SSC for another 10
min. Slides were finally washed for 5 min in 2⫻ SSC/0.1% NP-40.
All the washing buffers were incubated at 45°C. Slides were then airdried in the dark, and the counterstain, 1,4-diamidino-2-phenylindole
(DAPI II) was added just before viewing.
Microscopy
FISH signals were analysed with a Nikon epifluorescence microscope equipped with an appropriate triple bandpass filter set for
SpectrumOrange™, SpectrumGreen™, SpectrumAqua™ and DAPI
(Vysis). A sperm nucleus was scored only if it was intact and not
overlapped. An X or Y chromosome in a sperm nucleus was
recognized by a green or an orange fluorescent spot respectively.
Chromosome 18 was recognized by the presence of an aqua fluorescent
spot in the sperm nucleus. Sperm nuclei were scored as disomic for
sex chromosomes when an extra X or Y signal and a single
aqua fluorescent spot were clearly visible within the nucleus, were
comparable in brightness and size, and were at least one domain
apart. Sperm nuclei were scored as nullisomic for sex chromosome
when only a single chromosome 18 signal was visible. Sperm nuclei
were considered diploid when an extra X or Y chromosome signal
and two chromosome 18 signals were present. At least 500 spermatozoa were evaluated in each swim-up and pellet sample, and all
hemizona-bound spermatozoa were evaluated.
Statistical analysis
Sperm parameters such as concentration, motility, morphology and
HOST index were analysed as continuous variables, and Spearman’s
rank order correlation was used to interpret the relationship between
those parameters and the incidence of sperm aneuploidy. HZA index,
ICSI indications and pregnancy status were analysed as categorical
data, and the Wilcoxon rank-sum test was used to interpret the
relationship between these categories and the incidence of sperm
aneuploidy. The significance level chosen was P ⬍ 0.05.
Results
Semen specimens from 41 patients who participated in the
ICSI programme were included in this study. A summary
of semen parameters including sperm concentration, percent
motility, percentage of spermatozoa with normal morphology
according to strict criteria, hypo-osmotic swelling index and
hemizona index, as well as fertilization and pregnancy rates
are presented in Table I. In total, 21 839 sperm nuclei from
the motile fractions, 21 936 sperm nuclei from the pellet
fractions, and 4081 sperm nuclei from the hemizona-bound
fractions were analysed. The mean number of sperm nuclei
per patient evaluated was 533 ⫾ 13 for the motile fractions,
535 ⫾ 14 for the pellet fractions, and 117 ⫾ 14 for the
hemizona-bound fractions. Hybridization efficiency was 97%.
No difference in hybridization efficiency among the three
sperm groups was observed.
In addition to examining aneuploidy rates in the patient
population, testing was completed for donors whose specimens
were utilized as controls in the hemizona assay. The rates of
sex-chromosome aneuploidy were 0.38% in the swim-up motile
fractions, 0.17% in the pellet fraction, and 0.22% in the
spermatozoa–hemizona complexes. The rate of chromosome
1531
Q.Van Dyk et al.
Table II. The incidence of sperm sex-chromosome nullisomy and disomy in the swim-up motile fractions (SU), the pellet fractions (Pellet), and the
spermatozoa–hemizona complexes (S-HZ) in 41 ICSI patients
Total
spermatozoa
SU
Pellet
S-HZ
a,bComparisons
21 839
21 936
4081
Nullisomy (%)
0.70
0.85
0.27
Disomy (%)
Sex-chromosome
aneuploidy (%)
XX
YY
XY
Total
0.27
0.33
0.15
0.29
0.32
0.12
0.14
0.19
0.07
0.70
0.84
0.34
1.41a
1.69a
0.61b
between a and b showed significant difference (P ⬍ 0.0001).
Table III. The incidence of sperm chromosome 18 nullisomy and disomy in the swim-up motile fractions
(SU), the pellet fractions (Pellet), and the spermatozoa–hemizona complexes (S-HZ) in 41 ICSI patients
SU
Pellet
S-HZ
a,bComparisons
Total
spermatozoa
Nullisomy
(%)
Disomy
(%)
Chromosome 18
aneuploidy (%)
21 839
21 936
4081
0.29
0.41
0.07
0.31
0.31
0.02
0.60a
0.72a
0.10b
between a and b showed significant difference (P ⬍ 0.0001).
18 aneuploidy was 0.14% in both the swim-up motile fractions
and the pellet fraction, and 0.02% in the spermatozoa–hemizona
complexes. Rates of total diploidy, a summary of 46,XX,
46,YY and 46,XY, were 0.17% in the swim-up motile fractions,
0.14% in the pellet fraction, and 0.04% in the spermatozoa–
hemizona complexes.
Overall assessment of aneuploidy incidence in spermatozoa
from ICSI patients
The incidence of sperm sex-chromosome nullisomy and disomy
in the swim-up motile fractions, in the pellet fractions and in
the spermatozoa–hemizona (HZ-bound) complexes is presented
in Table II. The pellet samples had a greater percentage of sexchromosome aneuploidy (1.69%) than the swim-up samples
(1.41%), but this difference was not statistically significant.
Both the swim-up and the pellet sperm samples had a significantly greater percentage of sex-chromosome aneuploidy than
the HZ-bound sperm samples (0.61%, P ⬍ 0.0001 for both
comparisons). The incidence of chromosome 18 nullisomy and
disomy in the swim-up motile fractions, in the pellet fractions,
and in the HZ-bound complexes are presented in Table III.
No difference in incidence of chromosome 18 aneuploidy
between the swim-up samples (0.60%) and the pellet samples
(0.72%) was observed, but both were significantly greater than
the incidence in HZ-bound spermatozoa (0.10%, P ⬍ 0.0001
for both comparisons). We also observed that the incidence of
sex-chromosome aneuploidy was found to be significantly
greater than the incidence of chromosome 18 aneuploidy in
the swim-up motile fractions, the pellet fractions and the
HZ-bound fractions (P ⬍ 0.0001 for all three comparisons)
(Table IV).
The incidence of diploid spermatozoa in the swim-up motile
and pellet fractions and in the HZ-bound complexes is presented
1532
Table IV. The incidence of sex-chromosome aneuploidy and the incidence
of chromosome 18 aneuploidy in the swim-up motile fractions (SU), in the
pellet fractions (Pellet), and in the spermatozoa–hemizona complexes (SHZ) in 41 ICSI patients
Sample
Sex-chromosome
(%)
Chromosome 18
(%)
P
SU
Pellet
S-HZ
1.41
1.69
0.61
0.60
0.72
0.10
⬍0.0001
⬍0.0001
⬍0.0001
in Table V. The incidence of diploid spermatozoa in the pellet
samples (0.35%) was significantly greater than that in the
HZ-bound spermatozoa (0.07%, P ⬍ 0.0001). This incidence
in the swim-up samples (0.26%) was not significantly different
from either the pellet samples or the HZ-bound spermatozoa.
Correlation between the incidence of aneuploidy in spermatozoa and semen parameters
Semen concentration
There was a moderate but significant correlation between the
incidence of sex chromosome nullisomy in the swim-up
samples and the semen concentration (r ⫽ 0.33, P ⫽ 0.0332).
Similarly, the correlation between the incidence of sex-chromosome aneuploidy in the swim-up samples and the semen
concentration was moderate, but significant (r ⫽ 0.31, P ⫽
0.0481).
Semen motility
There was a moderate, but significant, correlation between the
incidence of chromosome X disomy in the swim-up motile
fractions and the semen motility (r ⫽ 0.31, P ⫽ 0.0451),
Analysis of zona-bound and motility-selected spermatozoa using FISH
Table V. The incidence of diploid spermatozoa in the swim-up motile fractions (SU), in the pellet fractions
(Pellet), and in the spermatozoa–hemizona complexes (S-HZ) in 41 ICSI patients
Total
spermatozoa
SU
Pellet
S-HZ
a,bComparisons
21 839
21 936
4081
Diploid (%)
Total diploid (%)
46,XX
46,YY
46,XY
0.10
0.12
0.05
0.07
0.09
0.02
0.08
0.15
0.00
0.26
0.35a
0.07b
between a and b showed significant difference (P ⬍ 0.0001).
between the incidence of sex chromosome nullisomy in the
swim-up motile fractions and the semen motility (r ⫽ 0.31,
P ⫽ 0.0494), and between the incidence of chromosome 18
disomy in the swim-up motile fractions and the semen motility
(r ⫽ 0.38, P ⫽ 0.0140). Both the incidence of sex-chromosome
aneuploidy and the incidence of chromosome 18 aneuploidy
in the swim-up motile fractions correlated with the semen
motility (r ⫽ 0.33, P ⫽ 0.0345 and r ⫽ 0.032, P ⫽ 0.0381
respectively). The incidence of disomy in the swim-up motile
fractions was significantly correlated with the semen motility
(r ⫽ 0.37, P ⫽ 0.0171), and the incidence of nullisomy in the
pellet fractions was significantly correlated with semen motility
(r ⫽ 0.32, P ⫽ 0.0404).
Semen morphology
There was moderate, significant correlation between the incidence of sex-chromosome nullisomy in the swim-up motile
fractions and semen morphology (r ⫽ 0.38, P ⫽ 0.0150). The
incidence of 46,XY diploidy was not significantly correlated
with semen morphology (r ⫽ –0.30, P ⫽ 0.536).
HOST index
The incidence of chromosome 18 nullisomy in the swim-up
fractions was negatively and significantly correlated with the
HOST index of the pellet fractions (r ⫽ –0.38, P ⫽ 0.0429).
The incidence of sex-chromosome disomy XY was also
negatively and significantly correlated with the HOST index
of the pellet fractions (r ⫽ –0.41, P ⫽ 0.0280). Interestingly,
the higher the incidence of chromosome 18 aneuploidy in the
motile fraction, the lower was the percentage of live spermatozoa found in the pellet fraction, according to HOST.
HZA index
The incidence of chromosome 18 disomy in the swim-up
motile fractions was significantly greater in patients with
abnormal HZA index (⬍14) than in patients with borderline
HZA index (14 to ⬍35) or normal HZA index (艌35) (P ⫽
0.0247). The incidence of chromosome 18 aneuploidy (including both chromosome 18 disomy and nullisomy) in the swim-up
motile fractions was also significantly greater in patients with
abnormal HZA index than in patients with borderline HZA
index or normal HZA index (P ⫽ 0.0356). In other words,
samples with higher chromosome 18 aneuploidy rate in the
motile fraction had a poorer binding capacity for human zona
pellucida.
Fertilization
There were significant negative correlations between the incidence of sex-chromosome disomy 24,XY in the swim-up
samples and the rate of fertilization (r ⫽ –0.45, P ⫽ 0.0033),
between the incidence of diploid 46,XY in the swim-up
samples and the rate of fertilization (r ⫽ –0.41, P ⫽ 0.0074),
and between the incidence of diploid 46,XY in the pellet
samples and the rate of fertilization (r ⫽ –0.46, P ⫽ 0.0022).
Thus, we found that the presence of diploid 46,XY spermatozoa
or sex-chromosome disomy 24,XY spermatozoa in the semen
sample indicated a lower fertilization rate using ICSI.
Pregnancy
The incidence of diploid 46,YY in swim-up motile fractions
was significantly greater in non-pregnant patients (0.71%) than
in pregnant patients (0.56%, P ⫽ 0.0174). The total rate of
diploidy (including diploidy 46,XX, 46,YY and 46XY) in
swim-up motile fractions in non-pregnant patients (1.81%)
was also significantly greater than that in pregnant patients
(0.50%, P ⫽ 0.0107). Thus, the increased presence of diploid
spermatozoa in the semen sample was related to a reduced
pregnancy rate.
Age of the patients
There were no significant correlations existing between the
age of the patients and the aneuploidy incidences in spermatozoa from the swim-up motile fractions (P ⬎ 0.07) or the pellet
fractions (P ⬎ 0.14).
Discussion
The introduction of ICSI technology has brought revolutionary
change to modern assisted reproductive medicine, and has
contributed greatly to the overall success of infertility treatment.
However, since spermatozoa used in ICSI frequently are
obtained from semen of extremely poor quality, there is concern
over the achieved genetic normalcy rate of ICSI pregnancy.
In this study, we investigated whether the zona pellucida can
function as a barrier against spermatozoa with numerical
chromosome error.
Data presented were collected from 41 ICSI patients. The
analysis of aneuploidy was performed on swim-up motile
fractions, pellet fractions and hemizona-bound spermatozoa.
The emphasis of our study was to examine the capacity of
human zona pellucida to select against aneuploid spermatozoa
by examining differences in rates among the three groups.
Since our objective was not to assess the relationship between
peripheral and sperm aneuploidy, we did not complete either
karyotypes or FISH analysis of peripheral blood specimens.
As a result, we are presently unable to determine whether the
1533
Q.Van Dyk et al.
aneuploidy rates observed are due to inherited or to de-novo
chromosomal aberrations. The original semen specimens were
not available as the clinical procedures were prioritized. FISH
analysis of sperm chromosome aneuploidy was carried out
independently of other assays or procedures; hence, the results
of sperm aneuploidy incidence were not biased by the information from other tests.
The incidence for sex-chromosome aneuploidy and chromosome 18 aneuploidy in hemizona-bound spermatozoa (0.61
and 0.10% respectively) was significantly lower than that in
both the swim-up motile fractions (1.41 and 0.60% respectively) and in the pellet fractions (1.69 and 0.72% respectively).
These data are in agreement with previous reports on sexchromosome aneuploidy frequency in ICSI patients (Storeng
et al., 1998). The frequency of sex-chromosome disomy in
the swim-up motile and pellet fractions was much higher than
that reported for donors or randomly selected men of couples
seeking infertility treatment (Martin et al., 1996; MartinezPasarell et al., 1997a; Morel et al., 1997). Conversely, the sex
chromosome disomy incidence was much lower than that
reported for severe oligoasthenoteratozoospermic patients
(Pang et al., 1995). This was not unexpected, as patients
included in this study had a wider range of semen parameters.
Compared with samples selected based on motility only,
there was a reduced frequency of spermatozoa with chromosome aneuploidy and diploidy bound to hemizona. Clearly,
zona selection did not completely block the fertilization by
spermatozoa with numerical chromosomal aberration. However, the selectivity of the zona pellucida might be the
single significant mechanism at the gamete level of blocking
fertilization by an abnormal spermatozoon. Using zona-free
hamster oocytes, it was demonstrated (Martinez-Pasarell et al.,
1997b) that sperm capacity to fuse with the oocyte membrane
and subsequently form pronuclei did not reduce the incidence
of either aneuploidy or diploidy.
As expected, no significant difference was observed between
the rates of nullisomy and disomy. Sex-chromosome aneuploidy was significantly greater than the incidence of chromosome
18 aneuploidy. Subfertile men have been reported to exhibit
decreased metaphase II/metaphase I ratios in their testicular
biopsy specimens (Lamont et al., 1981). Since a decreased
number of cells reaching metaphase II correlates with an
increased percentage of cells with unpaired sex-chromosomes
(Chandley et al., 1976), our results are consistent with those
of microscopic studies of testicular specimens. Similarly, a
study of fertile donors showed sex-chromosome aneuploidy
frequencies to be greater than those of autosomal aneuploidy
(Martin et al., 1996).
We noted a negative correlation between chromosome 18
aneuploidy, but not sex-chromosome aneuploidy, and the HZA
index. Although it is premature to speculate that aneuploid
spermatozoa are defective in spermatozoa–zona binding capacity, there is obviously an inferred association between the high
frequency of aneuploidy and reduced zona-binding capacity in
human spermatozoa. Interestingly, earlier studies reported a
significant association between HZA index and fertilization
rate in vitro (Coddington et al., 1994). The human hemizona
has been demonstrated previously to select not only for motile
1534
spermatozoa, but also for functionally normal spermatozoa
(Oehninger et al., 1997). Although the incidence of chromosome 18 aneuploidy is less than that of sex-chromosome
aneuploidy in human spermatozoa, it is apparently associated
to some degree with impaired sperm function, namely zonabinding.
This study and those of others have strongly proven that
the incidence of sperm aneuploidy among ICSI patients is
significantly higher. For example, sex-chromosome aneuploidy
1.41% (swim-up) and 1.69% (pellet) in ICSI patients versus
0.38% reported here compared well with 0.42% for normal
fertile donors reported previously (Martin et al., 1996). Similarly, chromosome 18 aneuploidy at 0.60% (swim-up) and
0.72% (pellet) in ICSI patients was significantly greater than
0.14% reported here, and compared with 0.11% for normal
fertile donors (Spriggs et al., 1995). Cytogenetic analysis of
the largest number of ICSI pregnancies also appears to support
our finding, since the incidences of de-novo and inherited
autosomal aberrations are higher than those reported for the
general population (Nielsen and Wohlert, 1991; Bonduelle
et al., 1998). It has been well established that the incidence
of chromosomal aberrations among children born through
conventional IVF is not significantly different from that of
the general population. Selection at the fertilization level is
supported by the fact that pregnancies with sperm-derived
aneuploidy are less common than expected from analysis of
sperm chromosomes in men with and without constitutional
chromosome rearrangements (Brandiff et al., 1986; Martin
et al., 1987; Martin, 1988a,b). Thus, the potentially higher
incidence of aneuploidy in pregnancies through ICSI could be
the result of two factors: (i) increased incidence of aneuploidy
in spermatozoa from participating patients; and (ii) the
bypassing of zona pellucida selection against aneuploid spermatozoa.
It is demonstrated here that the zona pellucida selects against
spermatozoa with chromosome numerical disorder. However,
this selectivity of the zona pellucida cannot yet be generalized,
as its mechanism is unclear and it is also unknown whether this
selectivity extends to spermatozoa with structural chromosomal
disorders. Also, it is understood that the zona pellucida does
not select against spermatozoa carrying single gene defects,
otherwise the Mendelian laws of inheritance would not be true.
The relationships between the incidence of aneuploidy and
other semen parameters were analysed in an attempt to explain
whether aneuploid spermatozoa tended to be morphologically
abnormal or were motility-impaired; and hence whether zona
selection could be mediated by these two mechanisms. This
study did not establish such a relationship. We found an
association between sperm motility and the frequency of sexchromosome and chromosome 18 aneuploidy. We observed
an association between the incidence of sex-chromosome
nullisomy in the swim-up motile fraction and the morphology
index of the original semen. We found it difficult to draw a
firm conclusion, since spermatozoa in the motile fraction make
up a very small portion of the total spermatozoa in the semen
sample. Our findings are consistent with the report that all
nine de-novo sex-chromosome aberration pregnancies were
obtained using spermatozoa from severe oligoasthenoterato-
Analysis of zona-bound and motility-selected spermatozoa using FISH
zoospermic patients, while no association could be found to
link this defect with any specific semen parameters (Bonduelle
et al., 1998). In another study, no correlation between sperm
morphology and chromosomal aberrations could be reported
(Martin and Rademaker, 1988). With spermatozoa exhibiting
a low strict morphology index, a link between chromosome
disomy and specific head deformation of the sperm head was
reported (Estop et al., 1997). In analysing sperm pronuclei, it
was found (Lee et al., 1996) that head defects were associated
with chromosomal structural aberrations, but not numerical
aberrations. We did find the incidence of chromosome 18
disomy in spermatozoa to be negatively associated with the
hypo-osmotic swelling test index, suggesting that an increase
in chromosome 18 disomy is associated with increased sperm
membrane defects.
This study demonstrated that the zona pellucida did select
against spermatozoa with chromosome aberrations, and in
particular against aneuploidy. We are still unclear about the
underlying mechanism of this selection. Gathering facts from
this study and those of others, there is apparently evidence to
support the association between sperm chromosomal aneuploidy and severe oligoasthenoteratozoospermia, between sperm
chromosomal aberration and sperm head defects, between
sperm chromosomal aneuploidy and defects in sperm membrane integrity, between sperm chromosomal aneuploidy and
impaired spermatozoa–zona binding, between sperm chromosomal aneuploidy and impaired sperm pronuclei formation,
and finally, between sperm chromosomal aneuploidy and failure
to achieve pregnancy with ICSI.
We demonstrated here the importance of zona selectivity of
normal spermatozoa. At present, it may be premature to apply
these zona-selected spermatozoa in clinical practice. However,
improving the genetic normalcy of spermatozoa in an era of
the widespread use of ICSI has significant importance, and
should certainly be considered.
References
American Fertility Society (1993) Assisted reproductive technology in the
United States and Canada: 1991 results from the Society for Assisted
Reproductive Technology generated from the American Fertility Society
Registry. Fertil. Steril., 59, 956–962.
Baschat, A.A., Kupker W., al Hasani S. et al. (1996) Results of cytogenetic
analysis in men with severe subfertility prior to intracytoplasmic sperm
injection. Hum. Reprod., 11, 330–333.
Bonduelle, M., Aytoz, A., Van Assche, E. et al. (1998) Incidence of
chromosomal aberrations in children born after assisted reproduction through
intracytoplasmic sperm injection. Hum. Reprod., 13, 781–782.
Bourrouillou, G., Bujan, L. and Calvas, P. (1992) Role and contribution of
karyotyping in male infertility. Prog. Urol., 2, 189–195.
Brandiff, B., Gordon, L., Ashworth, L.K. et al. (1986) Cytogenetics of human
meiotic segregation in two translocation carriers. Am. J. Hum. Genet., 38,
197–208.
Chandley, A.C., Maclean, N. and Edmond P. (1976) Cytogenetics and infertility
in men II: testicular histology and meiosis. Ann. Hum. Genet. 40, 165–76.
Coddington, C.C., Fulgham D.L., Alexander N.J., et al. (1990) Sperm bound
to zona pellucida in hemizona assay demonstrate acrosome reaction when
stained with T-6 antibody. Fertil Steril., 54, 504–508.
Coddington, C.C., Oehninger, S.C., Olive, D.L. et al. (1994) Hemizona index
(HZI) demonstrates excellent predictability when evaluate sperm fertilizing
capacity in in vitro fertilization patients. J. Androl., 15, 250–254.
Cummins, J.M. and Jequier, A.M. (1995) Concerns and recommendations
for intracytoplasmic sperm injection (ICSI) treatment. Hum. Reprod., 10
(Suppl. 1), 138–143.
Estop, A.M., Vandermark, K.K., Munne, S. et al. (1997) Sperm morphology
and chromosome aneuploidy in men with infertility as determined by
fluorescence in situ hybridization (FISH). Fertil. Steril., 68 (1S), 208S.
Govaerts, I., Englert, Y., Vamos, E. et al. (1995) Letters to the editor: Sex
chromosome abnormalities after intracytoplasmic sperm injection. Lancet,
346, 1095–1096.
Han, T.L., Flaherty, S.P., Ford, J.H. et al. (1993) Detection of X- and Ybearing human spermatozoa after motile sperm isolation by swim up. Fertil.
Steril., 60, 1046–1051.
Holmes, J.M. and Martin, R.H. (1993) Aneuploidy detection in human sperm
nuclei using fluorescence in situ hybridization. Hum. Genet., 91, 20–24.
In’t Veld, P., Brandenburg, H., Verhoeff, A. et al. (1995) Sex chromosomal
abnormalities and intracytoplasmic sperm injection. Lancet, 346, 773.
Jeyendran, R.S., Van der Ven, H.H., Perez-Pelaez, M. et al. (1984) Development
of an assay to assess the functional integrity of the human sperm membrane
and its relationship to other semen characteristics. J. Reprod. Fertil., 70,
219–228.
Jones, K.W., Singh, L. and Edwards, R.G. (1987) The use of probes for the
Y chromosome in pre-implantation embryo cells. Hum. Reprod., 2, 439–445.
Kruger, T.F., Menkveld, R., Stander, F.S.H. et al. (1986) Sperm morphologic
features as a prognostic factor in in vitro fertilization. Fertil. Steril., 46,
1118–1123.
Lahdetie, J., Saari, N., Ajosenpaa-Saari, M. et al. (1997) Incidence of aneuploid
spermatozoa among infertile men studied by multicolor fluorescence in situ
hybridization. Am. J. Med. Genet., 71, 115–121.
Lamont, M.A., Faed, M.J. and Baxby, K. (1981) Comparative studies of
spermatogenesis in fertile and subfertile men. J. Clin. Pathol., 34, 145–150.
Lee, J.D., Kamiguchi, Y. and Yanagimachi, R. (1996) Analysis of chromosome
constitution of human spermatozoa with normal and aberrant head
morphologies after injection into mouse oocytes. Hum. Reprod., 11,
1942–1946.
Liebaers, I., Bonduelle, M., Van Assche, E. et al. (1995) Letters to the editor:
Sex chromosome abnormalities after intracytoplasmic sperm injection.
Lancet, 346, 1095.
Martin, R.H. (1988a) Cytogenetic analysis of sperm from a male heterozygous
for a 13; 14 Robertsonian translocation. Hum. Genet., 80, 357–361.
Martin, R.H. (1988b) Meiotic segregation of human chromosomes in
translocation heterozygotes: report of a t(9;10)(q34;q11) and a review of
the literature. Cytogenet. Cell Genet., 47, 48–51.
Martin, R.H. and Rademaker, A. (1988) The relationship between sperm
chromosomal abnormalities and sperm morphology in humans. Mutat. Res.,
207, 159–164.
Martin, R.H., Rademaker, A.W., Hildebrand, K., et al. (1987) Variation in the
frequency of sperm chromosomal abnormalities among normal men. Hum.
Genet., 77, 108–114.
Martin, R.H., Spriggs, E. and Rademaker, A.W. (1996) Multicolor fluorescence
in situ hybridization analysis of aneuploidy and diploidy frequencies in
225,846 sperm from 10 normal men. Biol. Reprod., 54, 394–398.
Martinez-Pasarell, O., Marquez, C., Coll, M.D. et al. (1997a) Analysis of
human sperm-derived pronuclei by three-color fluorescent in-situ
hybridization. Hum. Reprod., 12, 641–645.
Martinez-Pasarell, O., Vidal, F., Colls, P. et al. (1997b) Sex chromosome
aneuploidy in sperm-derived pronuclei, motile sperm and unselected sperm,
scored by three-color FISH. Cytogenet. Cell Genet., 78, 27–30.
Menkveld, R., Franken, D.R., Kruger, T.F. et al. (1991) Sperm selection
capacity of the human zona pellucida. Mol. Reprod. Dev., 30, 346–352.
Meschede, D., De Geyter, C., Nieschlag, E. et al. (1995) Genetic risk in
micromanipulative assisted reproduction. Hum. Reprod., 10, 2880–2886.
Morel, F., Mercier, S., Roux, C. et al. (1997) Estimation of aneuploidy levels
for 8, 15, 18, X and Y chromosomes in 97 human sperm samples using
fluorescence in situ hybridization. Fertil. Steril., 67, 1134–1139.
Nielsen, J. and Wohlert, M. (1991) Chromosome abnormalities found among
34,910 newborn children: results from a 13 year incidence study in Arrhus,
Denmark. Hum. Genet., 87, 81–83.
Oehninger, S., Acosta, A.A., Veeck, L. et al. (1991) Recurrent failure of
in vitro fertilization: role of the hemizona assay in the sequential diagnosis
of specific sperm-oocyte defects. Am. J. Obstet. Gynecol., 164, 1210–1215.
Oehninger, S., Mahony, M., Ozgur, K. et al. (1997) Clinical significance of
human sperm-zona pellucida binding. Fertil. Steril., 67, 1121–1127.
Palermo, G., Joris, H., Devroey, P. et al. (1992) Pregnancies after
intracytoplasmic injection of single spermatozoon into an oocyte. Lancet,
340, 17–18.
Pang, M.G., Zackowski, J.L., Hoegerman, S.F. et al. (1995) Detection by
fluorescence in situ hybridization of chromosome 7, 11, 12, 18, X and Y
1535
Q.Van Dyk et al.
abnormalities in sperm from oligoasthenospermic patients of an in vitro
fertilization program. J. Assist. Reprod. Genet., 12, 53S.
Persson, J.W., Peters, G.B. and Saunders, D.M. (1996) Genetic consequences
of ICSI. Is ICSI associated with risks of genetic disease? Implications for
counseling, practice and research. Hum. Reprod., 11, 921–932.
Rudak, E., Jacobs, P.A. and Yanagimachi, R. (1978) Direct analysis of the
chromosome constitution of human spermatozoa. Nature, 274, 911–913.
Spriggs, E.L., Rademaker, A.W. and Martin, R.H. (1995) Aneuploidy in human
sperm: results of two-and three-color fluorescence in situ hybridization using
centromeric probes for chromosomes 1, 12, 15, 18, X, and Y. Cytogenet.
Cell Genet., 71, 47–53.
Storeng, R.T., Plachot, M., Theophile, D. et al. (1998) Incidence of sex
chromosome abnormalities in spermatozoa from patients entering an IVF
or ICSI protocol. Acta Obstet. Gynecol. Scand., 77, 191–197.
Van Opstal, D., Los, F.J., Ramlakhan, S. et al. (1997) Determination of the
parent of origin in nine cases of prenatally detected chromosome aberrations
found after intracytoplasmic sperm injection. Hum. Reprod., 12, 682–686.
Received on July 30, 1999; accepted on March 28, 2000
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