Human Reproduction, Vol.30, No.11 pp. 2493– 2500, 2015
Advanced Access publication on September 23, 2015 doi:10.1093/humrep/dev202
ORIGINAL ARTICLE Andrology
Molecular karyotyping of single sperm
with nuclear vacuoles identifies more
chromosomal abnormalities in patients
with testiculopathy than fertile controls:
implications for ICSI
Andrea Garolla, Barbara Sartini, Ilaria Cosci, Damiano Pizzol,
Marco Ghezzi, Alessandro Bertoldo, Massimo Menegazzo,
Elena Speltra, Alberto Ferlin, and Carlo Foresta*
Department of Medicine, Unit of Andrology and Reproductive Medicine, University of Padova, Via Giustiniani 2, Padova 35121, Italy
*Correspondence address. Tel: +39-049-8218518; Fax: +39-049 8218520; E-mail: [email protected]
Submitted on March 10, 2015; resubmitted on July 27, 2015; accepted on July 30, 2015
study question: Is there a difference between molecular karyotype of single sperm selected by high-magnification microscopy from infertile patients with testicular damage and from proven fertile controls?
summary answer: The molecular karyotype of single sperm from patients with testiculopathy had a significantly higher percentage of
chromosomal alterations than fertile controls.
what is known already: Infertile patients with testicular impairment have many sperm with aneuploidies and/or increased structural
chromosome alterations. In these patients, sperm use by ICSI has poor outcome and raises concerns about the possible impact on pregnancy loss
and transmission of genes abnormalities in offspring. High-magnification microscopy has been recently introduced to select morphologically better
sperm aimed at improving ICSI outcome. However, there are no studies evaluating the molecular karyotype of sperm selected by this method.
study design, size, duration: Three consecutive infertile patients with oligozoospermia due to testicular damage and three agematched proven fertile men attending a tertiary care center, were enrolled in the study from September to November 2014. Inclusion criteria of
patients were age ≥30 ≤35 years, at least 2 years of infertility, oligozoospermia (sperm count below 10 million), reduced testicular volumes high
FSH plasma levels and absence of altered karyotype, Y chromosome microdeletions, cystic fibrosis transmembrane conductance regulator gene
mutations, sperm infections, cigarette smoking, varicocele, obesity.
participants/materials, setting, methods: Participants were evaluated for sperm parameters, sex hormones and testicular color-doppler ultrasound. From each semen sample, 20 sperm with large vacuoles (LVs), 20 with small vacuoles (SVs) and 20 with no vacuoles
(NVs) were retrieved individually by a micromanipulator system. Each cell was further analyzed by whole genome amplification and array comparative genomic hybridization (aCGH).
main results and the role of chance: The aCGH allowed us to detect chromosomal aneuploidies, unbalanced translocations
and complex abnormalities. Sperm selected from infertile patients showed a higher percentage of abnormal molecular karyotypes than controls
(19.4 versus 7.7%, respectively, P , 0.001). In particular, sperm with LV and SV showed 38.3 and 20.0% abnormal karyotype in infertile men
versus 18.3 and 5.0% in controls, respectively (both P , 0.01). Complex abnormalities were found only in the LV category. An abnormal karyotype was never found in NV sperm from both patients and controls.
limitations reasons for caution: The main limitation of this study is the low number of included subjects. Moreover, a time of
writing we have no data regarding the ICSI outcome using LV, SV or NV sperm. This is the first study evaluating the molecular karyotype of single
sperm selected by high-magnification microscopy and further confirmation of the data is needed.
wider implications of the findings: Our data showed that sperm from infertile patients with testicular impairment have a higher
& The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: [email protected]
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Garolla et al.
percentage of abnormal molecular karyotypes than sperm from fertile controls. Therefore, if confirmed, our data suggest that the use of individually retrieved NV sperm may improve ICSI outcome in infertile men with testicular damage.
study funding/competing interest(s): No external funding was sought for this study, and the authors have no conflict of interest to declare.
trial registration number: N. 2401P.
Key words: high-magnification microscopy / male infertility / molecular karyotype / oligozoospermia / sperm vacuoles / whole genome
amplification / array comparative genomic hybridization
Introduction
Many conditions may lead to primary testicular failure and infertility including cryptorchidism, orchitis, testis trauma, torsions and iatrogenic
forms such as gonadotoxic medications, chemo/radiotherapy, inguinal
surgery and so on. It is well known that testicular impairment is frequently
associated with alterations of sperm chromosomes in terms of numerical
or structural abnormalities (Pang et al., 1999; Bonduelle et al., 2002). Infertile couples with male factor infertility are usually treated by ICSI, but in
these cases the outcome is very poor (Krausz, 2011). Moreover, concerns have been raised in these patients about the possible transmission
to progeny of ICSI of chromosomal alterations leading to congenital malformations, developmental abnormalities and a higher risk of imprinting
diseases (Erenpreiss et al., 2006). The standard semen analysis is of fundamental importance to diagnose and define the severity of the male
factor infertility, but it is unable to detect alterations of the sperm nucleus.
In recent years, more attention has been focused on the genomic
quality of the male gamete aimed at improving ICSI outcome and reducing the possible risk to transmit genetic alterations. However, current
methods evaluating the sperm DNA status, such as fragmentation,
nuclear condensation, aneuploidies or chromosomal structural rearrangements, are invasive for cells and thus they cannot be used to select
the sperm for ICSI use. Bartoov et al. (2002) suggested a non-invasive
technique of sperm selection aimed to predict the genetic normalcy of
the male gamete. This technique is based on the evaluation of single
sperm by high-magnification microscopy to detect the presence of
minor anomalies and nuclear vacuoles, which are not visible at the standard magnification used for ICSI. Human sperm vacuoles were first
described as ‘nuclear holes’ when examined by electron microscopy
and 2D imaging (Zamboni, 1987). Thanks to higher resolution techniques and technical progress in microscopic imaging, it was recently
shown that vacuoles are not nuclear holes but concavities extending
from the surface of the sperm head to the nucleus (Watanabe et al.,
2009, Boitrelle et al., 2011; Perdrix and Rives, 2013). The origin and consequences of sperm head vacuoles are still subject to controversy
because they have been suggested to originate from spermatogenesis impairment or abnormal maturation during male genital tract transit or
acrosome modification during the acrosome reaction (Perdrix et al.,
2011). The hypothesis that sperm head vacuoles originated from acrosomes has been explored by assessing vacuole parameters after induction of the acrosome reaction. A significantly decreased presence of
vacuoles was observed after induction of the acrosome reaction
(Kacem et al., 2010; Montjean et al., 2012). Independent of their size,
vacuoles seem relatively common in the sperm heads from fertile and infertile men with normal or abnormal semen parameters (Perdrix and
Rives, 2013). However, some authors observed a strong relation
between the presence of large nuclear vacuoles and the impairment of
sperm chromatin condensation (Franco et al., 2008; Garolla et al.,
2008), a fundamental process involved in protection of the paternal
genome before fertilization and in the early phases of embryonic development (Ward, 2010). Interestingly, Claussen (2005) and Finch et al.
(2008) demonstrated that chromosome position in the sperm nucleus
also reflects the status of chromatin integrity. Moreover, Perdrix et al.
(2013) showed that nuclear position of chromosomes is different in
spermatozoa with large nuclear vacuoles compared with spermatozoa
with no vacuoles. These observations supported the hypothesis of
an association between the presence of large nuclear vacuoles at
sperm head and concomitant alterations of sperm nucleus. On
this basis, some authors performed ICSI using a selection of sperm
with no large vacuoles (LVs) and observed a higher fertilization,
reduced abortion rate and better pregnancy outcome (Berkovitz et al.,
2005; Figueira et al., 2011). However, other studies failed to observe
any correlation between the presence and size of nuclear vacuoles
and nuclear status. Even a recent Cochrane review comparing the ICSI
and intracytoplasmic morphologically selected sperm injection (IMSI)
techniques showed no significant difference in the pregnancy outcome
(Teixeira et al., 2013).
The aim of the present study was to evaluate the entire genome,
including aneuploidies and chromosomal alterations, of single sperm
selected by high-magnification microscopy. To achieve this aim, we
used array comparative genomic hybridization (aCGH), a new method
able to evaluate numerical and structural alterations of all 23,X/Y
sperm chromosomes (Patassini et al., 2013). This analysis was performed on 360 single sperm with LV, small (SV) or no nuclear vacuoles
obtained in semen samples from infertile patients with oligozoospermia
due to testicular impairment and from normozoospermic, proven
fertile men.
Materials and Methods
Patients and controls
This study was approved by the Institutional Ethics Committee of the Hospital of Padova, Italy, Protocol number 2401P. Three patients, oligozoospermic due to testicular impairment, attending our Andrology Unit between
September and November 2014 were included in the study. Three agematched proven fertile volunteers with normal sperm parameters served
as controls for sperm parameters, sex hormones, testicular volumes and
aCGH analysis. Inclusion criteria of patients were: age ≥30 ≤35 years, at
least 2 years of infertility, sperm count below 10 million observed at two different semen analyses performed at least 3 months apart according to World
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Molecular karyotype of single sperm and nuclear vacuoles
Health Organization guidelines (WHO, 2010), reduced testicular volumes at
testicular color-doppler ultrasound and plasma FSH levels higher than 8 IU/l.
All criteria had to be fulfilled in patients for inclusion. Exclusion criteria were
altered karyotype, Y chromosome microdeletions, cystic fibrosis transmembrane conductance regulator gene mutations and confounding factors such as
presence of sperm infections, cigarette smoking, varicocele and obesity. Inclusion criteria of controls were: age ≥30 ≤35 years, fatherhood during
the last year and normal sperm parameters.
The study flow is shown in Fig. 1. Semen samples of severe oligozoospermic patients and normozoospermic fertile controls were analyzed by
high-magnification microscopy. From semen samples of patients, 68
sperm with LVs, 68 sperm with SVs and 67 without vacuoles (NVs), were
retrieved individually by a micromanipulator system. Analogously, from
semen samples of controls, for a total of 404 sperm. Each cell was further
analyzed by whole genome amplification (WGA) and aCGH to obtain its
molecular karyotype.
Semen sample collection and preparation
Human semen samples were obtained by masturbation in sterile containers
after 2 –5 days of sexual abstinence. Samples were allowed to liquefy for
30 min and were examined for seminal parameters according to the
World Health Organization criteria (WHO, 2010). All samples had normal
viscosity, pH and semen volume. Sperm infections were excluded by
sperm culture. Whole samples were washed twice by centrifugation at
300 g for 10 min in sterile phosphate-buffered saline (PBS) and a 1 ml pellet
was incubated in 10-ml microdrops of polyvinylpyrrolidone solution 7%
(Origio Medicult Media, Måløv, Denmark) in a Petri dish, covered with
liquid paraffin (Origio Medicult Media) at room temperature for 15 min
and immediately used for further analyses.
Analysis of sperm at high magnification
The total calculated magnification for motile sperm organelle morphology
evaluation (MSOME) analysis was obtained with an Uplan Apo × 100/1.40
objective lens and a 0.52 numerical aperture condenser lens. The images
were captured by a DS-5M-L1 digital color video camera (Nikon) with a
1.7-cm aperture containing 5 240 000 effective picture elements (pixels)
for high-quality image production, and a color video monitor (Hansol
AMW-M196A liquid crystal display). The morphologic assessment was conducted on the monitor screen, which, under the above configuration,
reached a real magnification of ×13 161, as determined by a 0.01-mm glass micrometer, as previously described (Garolla et al., 2008). The criteria for the
evaluation of the MSOME analysis were defined according to Bartoov et al.
(2002), observing sperm at higher magnification. The analysis took 30 min
per patient. According to MSOME criteria, we identified three categories of
sperm cells: with normal morphology and: (i) no nuclear vacuoles (NVs), (ii)
at least one SV (,4% of total nuclear area) and (iii) at least one LV (.4% of
total nuclear area).
A total of 404 single sperm, selected one by one with a micromanipulator
system (Nikon Eclipse TE2000-U equipped with Nomarski optics, HMC
MC2 620/0.40 objective lens), were individually placed into a 0.2 ml PCR
tube with 2 ml PBS for further aCGH analysis. Only cells showing satisfactory
parameters of amplification and hybridization (see below) were considered,
up to 20 of each type from each subject. The final number of cells included in
the study was 360.
Figure 1. Study design. Semen samples of three oligozoospermic patients and three normozoospermic fertile controls were analyzed by highmagnification microscopy. From semen samples of patients and controls, 60 sperm with LVs, 60 with SVs and 60 without vacuoles (NVs) were retrieved
individually by a micromanipulator system (20 of each type from each subject) giving a total of 360 sperm. Each sperm was further analyzed by WGA and
aCGH to obtain its molecular karyotype.
2496
Lysis and extraction of sperm DNA and sperm
decondensation
This procedure was performed according to the previous study of Patassini
et al. (2013). Briefly, the DNA from single sperm was extracted and amplified
by the SurePlex DNA Amplification System (SurePlex WGA, BlueGnome,
Cambridge, UK). The protocol was formed by a lysis phase and two
rounds of PCR amplification for WGA SurePlex using a WGA procedure
based on three steps: extraction and random fragmentation of genomic
DNA, a pre-amplification reaction and a subsequent amplification reaction
by PCR performed with flanking universal priming sites. After centrifugation,
cell extraction buffer was added to each sample. Then samples were incubated in a PCR thermocycler at 758C for 10 min and at 958C for 4 min. In
the second step, SurePlex Pre-Amplification mix was added to each 10 ml
of sample. Decondensation was assessed with proteinase K (700 nM) and
dithiothreitol (1 mM).
WGA and aCGH
After the decondensation, in order to improve the sensitivity and the accuracy of the amplification, the last PCR-based cycling step in SurePlex has been
developed in real-time, adding SYBR Green at the final concentration of
0.125×. Data analysis was performed by subtracting the baseline fluorescence levels at the end of each cycle, and the difference in fluorescence
was plotted against the cycle number. This analysis enabled us to define a
threshold of 10 cycles above which subsequent hybridization fails.
The aCGH procedure was divided into four steps: labeling, combination
and centrifugal evaporation, hybridization and washing. In the first step,
equal amounts of sperm DNA and reference DNA (SureRef. Reference
male DNA) were differentially labeled with Cyanine 3 and Cyanine 5.
These samples were denatured in a thermalcycler for 5 min at 948C and
transferred immediately to ice for 5 min. Then, 1 ml of Klenow enzyme
was added to each PCR tube to proceed with the labeling reaction. Subsequently, labeled sperm DNA and control DNA were combined, then
co-precipitated adding 25 ml of COT Human DNA and concentrating by a
centrifugal evaporator for 1 h at 608C, until around 3 ml remained in each
tube. In the third step, labeled DNA was resuspended in dextran sulfate hybridization buffer, denatured at 758C for 10 min and hybridized under cover
slips to 24sure BACarrays (BlueGnome). Then 24sure slides were placed in
prepared hybridization chamber (slide box, tissue saturated with 6 ml 26
SSC/50% formamide) and incubated in a non-circulating lidded water bath
for 16– 20 h at 47 mC.
In the last step, hybridized 24sure slides were washed to remove
un-hybridized DNA. The cover slip was removed from each slide by manually
agitating in 26 SSC/0.05% Tween 20 at room temperature. Afterwards each
slide was washed according these conditions: first wash (on stirrer) in buffer
26 SSC/0.05% Tween 20 at room temperature for 10 min; second wash (on
stirrer) in buffer 16 SSC at room temperature for 10 min; third wash in buffer
0.16 SSC at 60 mC for 5 min; fourth wash (on stirrer) in buffer 0.16 SSC at
room temperature for 1 min. Finally, each slide was dried by centrifugation
at 170 g for 6 min. A laser scanner (G2565CA microarray scanner, Agilent,
USA) was used to read the resulting images of the hybridization. Scanned
images were then analyzed and quantified by BlueFuse Multi Software (BlueGnome). The difference in fluorescence between sample DNA and reference DNA was plotted against all chromosomes and signals above or
below the fluorescence thresholds indicate, respectively, gains and losses
of the DNA content. We considered for this analysis only the clones characterized by a low signal of background noise, an hybridization value of 1500 –
2000, a percentage of included clones of at least 90%, a Mean Spot Amplitude
Ch1/Ch2 .1200 (hybridization index) and the SBR Ch1/Ch2 .4 (index of
the background noise).
Garolla et al.
Statistical analysis
All results are expressed as the mean value + SD, and categorical variables
are expressed as a percentage. Comparison between groups was performed
by Student’s t test for continuous data after acceptance of normality with the
Kolmogorov– Smirnov test and by the x 2 test for categorical data. P-values of
,0.05 were considered to be statistically significant. Data analysis was performed using the Statistical Package for the Social Sciences version 13.0
(SPSS, Inc., Chicago, IL, USA).
Results
Table I shows clinical characteristics of the three patients with testiculopathy and three age-matched proven fertile normozoospermic subjects.
Patients had significantly lower sperm number, motility and morphology,
higher plasma FSH and LH levels and reduced right and left testicular
volume (all P , 0.01 versus controls). No significant difference in plasma
testosterone levels was found in patients compared with controls.
The incidence of sperm showing LV, SV and NV in patients and controls
was 39.7, 57.1, 3.2% and 22.1, 69.4, 8.5%, respectively (data not shown).
Figure 2 shows some examples of molecular karyotypes obtained by
aCGH from sperm with LV, SV or no vacuole (NV) selected by highmagnification microscopy. The LV sperm has a 23/X complex molecular
karyotype (upper). The SV sperm has a 23/Y karyotype with loss of
whole chromosome 14 (medium) and the NV sperm has a 23/X normal
karyotype (lower). Results of array CGH are reported in Table II. Considering sperm from patients with oligozoospermia as a whole, they showed a
higher mean percentage of sperm with abnormal karyotype than controls
(19.4 versus 7.7%, respectively, P , 0.001), independently from the presence or absence of nuclear vacuoles. In particular, infertile patients had
38.3 and 20.0% of abnormal karyotype in sperm with LV and SV, respectively. Control subjects, had 18.3 and 5.0% abnormal karyotype in LV and
SV sperm, respectively. No abnormal karyotype was found in NV sperm
of both patients and controls. Comparing abnormal karyotype in oligospermic and normozoospermic patients for each group of vacuoles, statistical
analysis showed significant differences between LV and NV, and between
SV and NV sperm in oligozoospermic patients (P , 0.001 and P , 0.01, respectively) and between LV and NV sperm in fertile controls (P , 0.01).
Table III shows the type and number of alterations observed by molecular
karyotype in LV, SV and NV sperm from infertile patients and fertile controls.
In oligozoospermic patients, the aCGH showed that among the 35 sperm
with abnormal karyotype, 9.4% had partial loss or gain of chromosomes,
4.4% had XY or XX disomy, 2.8% had loss or gain of single entire chromosomes, 2.2% had complex alterations involving more chromosomes
and 0.6% had sex chromosome nullisomy. In control subjects, the analysis
showed 14 abnormal sperm with the following alterations: 3.8% had
partial loss or gain of chromosomes, 2.2% had XY or XX disomy, 1.1%
had loss or gain of single entire chromosomes and 0.6% had sex chromosome nullisomy. Interestingly, all sperm alterations were more represented
in infertile patients and no complex abnormality was found in control
subjects, even when LV sperm were analyzed. The most frequent abnormality observed in both groups was the partial loss or gain of chromosomes
(24/360 sperm, respectively, 9.4 and 3.8% in patients and controls).
Discussion
Over the last few years, it has been found that the injection of selected,
morphologically normal spermatozoa into the oocyte is associated with
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Molecular karyotype of single sperm and nuclear vacuoles
Table I Clinical parameters of oligozoospermic infertile patients and normozoospermic fertile controls.
Age
(years)
Sperm
concentration
(million/ml)
Sperm
count
(million)
Progressive
motility (%)
Sperm
morphology
(%)
FSH
(U/l)
LH
(U/l)
T
(nmol/l)
Right
testicular
volume
(cc)
Left
testicular
volume
(cc)
.............................................................................................................................................................................................
Oligozoospermic infertile patients
1
30
1.8
4.5
15
7
10.6
9.2
10.7
8
10
2
32
2.5
7.1
8
5
15.1
7.7
12.5
5
9
3
35
1.1
2.2
18
4
14.3
6.9
13.9
5
6
Mean
32.3
1.8*
4.6*
13.6*
5.3*
13.3*
7.9*
12.3
6.0*
8.3*
2.5
0.7
2.4
5.1
1.5
2.4
1.1
1.6
1.7
2.1
SD
Normozoospermic fertile controls
1
32
36.9
110.7
41
11
4.1
3.1
13.2
14
15
2
32
72.6
211.5
38
13
3.3
2.5
15.6
17
19
3
33
59.1
195.1
44
16
3.7
4.0
16.8
16
17
Mean
32.3
56.2
172.4
41.0
13.3
3.7
3.2
15.2
15.6
17.0
0.6
18.0
54.1
3.0
2.5
0.4
0.7
1.8
1.5
2.0
SD
Comparison between groups was performed by Student’s t test for continuous data and by the x 2 test for categorical data.
T, testosterone.
*P , 0.01 versus controls.
Figure 2. Examples of molecular karyotype obtained by aCGH in sperm with LV, SV, or no vacuoles selected individually at high-magnification microscopy (×13 161) from semen samples of patients with oligozoospermia. Panel LV: sperm with LVs (left) and a 23,X,21,+8,+12,+13,+14,+16,+17,+21
complex molecular karyotype (right); panel SV: sperm with SVs (left) and a 23,Y karyotype with loss of whole chromosome 14 (right); panel NV: sperm with
no vacuoles (left) with a 23,X normal molecular karyotype (right). Final magnification used for the images shown here was ×13 161.
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Garolla et al.
higher blastocyst implantation and ongoing pregnancy rate and/or lower
miscarriage rate (Bartoov et al., 2001, 2002; Berkovitz et al., 2005, 2006;
Antinori et al., 2008; Vanderzwalmen et al., 2008; Balaban et al., 2011;
Knez et al., 2011; Klement et al., 2013). Other studies have supported
similar findings and showed that IMSI improves fertilization and blastocyst
formation rates (Cassuto et al., 2009; Knez et al., 2011). Moreover, in a
previous study from our group, we showed that sperm cells containing
head vacuoles had a higher incidence of aneuploidies, mitochondrial
impairments and DNA fragmentation (Garolla et al., 2008). The study
of sperm chromosomes seems to be particularly important in those subjects with testicular damage and in particular in those patients with
constitutional chromosome alterations, such as in Klinefelter syndrome,
and subjects with translocations (Foresta et al., 2005; Patassini et al.,
Table II Results of aCGH in single sperm with LVs, SVs
and no vacuoles from oligozoospermic infertile patients
and fertile normozoospermic controls.
Sperm with abnormal karyotype
...............................................
n
%
........................................................................................
Oligozoospermic infertile patients
LV
23/60
38.3*
SV
12/60
20.0**
NV
0/60
Total
0
19.4#
35/180
Normozoospermic fertile controls
LV
11/60
18.3**
SV
3/60
5.0
NV
0/60
0
Total
14/180
7.7
Comparison between groups was performed by the x 2 test for categorical data.
LV, large vacuoles; SV, small vacuoles; NV, no vacuoles.
*P , 0.001 versus NV of the same group.
**P , 0.01 versus NV of the same group.
#
P , 0.001 versus fertile controls.
2013). In fact, the high aneuploidy rate frequently reported in these subjects (above 35%) compared with controls (below 8%) (Patassini et al.,
2013), raises important concerns regarding the potential role in ICSI
failure and the transmission of genetic diseases (In’t Veld et al.,
1995a,b; Liebaers et al., 1995; Loft et al., 1999; Foresta et al., 2005).
The sperm vacuole is a concavity extending from the surface of the
head to the nucleus through the acrosome, but its nature and significance
remain still unclear. In the 1950s, by transmission electron microscopy of
an ultrathin cross-section of the sperm head, it was shown that the
human sperm nucleus frequently contains one or more vacuoles at
different locations (Schnall, 1952; Schultz-Larsen, 1958). More recently,
sperm vacuoles have been suggested to originate from spermatogenesis
impairment or abnormal maturation during male genital tract transit
or acrosome modification during the acrosome reaction (Perdrix et al.,
2011). Most researchers suggested that nuclear vacuoles should
not be considered as degenerative structures, but instead as normal
features of the sperm head with no pathological significance (Fawcett,
1958; Chrzanowski, 1966; Pedersen, 1969). In contrast, some authors
suggested that vacuoles are not just a polymorphism, but pose a risk
for sperm abnormality at chromosome level (Bartoov et al., 2001;
Vanderzwalmen et al., 2008). This hypothesis was strengthened by the
demonstration that the presence of nuclear vacuoles is related to
chromosome position and to chromatin integrity (Claussen et al.,
2005; Finch et al., 2008; Perdrix et al., 2013).
The aCGH analysis involves the screening of the entire chromosome
complement by DNA microarray and was used in this study to evaluate
the molecular karyotype of single sperm selected by high-magnification microscopy. By this procedure, we analyzed the ‘molecular karyotype’ at
higher resolution than a traditional karyotype, overcoming the diagnostic
limit of fluorescence in situ hybridization on sperm. The development
and clinical application of aCGH in recent years has revolutionized the diagnostic process in several diseases, and aCGH is gradually joining or replacing standard cytogenetic techniques in a growing number of genetic
and molecular laboratories (Vissers et al., 2003; Fiorentino et al., 2011).
Data from the present study showed that sperm karyotype is dependent on the presence and size of nuclear vacuoles. Moreover, infertile
patients with non-obstructive oligozoospermia had a higher percentage
of chromosomal alterations than controls. Finally, sperm with a normal
Table III Type and number of alterations at aCGH analysis observed in LV, SV and NV sperm singularly retrieved from
semen of oligozoospermic infertile patients and fertile normozoospermic controls.
XY or XX
disomy, n (%)
Sex chromosome
nullisomy, n (%)
Loss/gain of single entire
chromosomes, n (%)
Loss/gain of part of
chromosomes, n (%)
Complex abnormal
karyotype, n (%)
.............................................................................................................................................................................................
Oligozoospermic infertile patients
LV (n ¼ 60)
5 (8.3)
1 (1.7)
3 (5.0)
10 (16.6)
SV (n ¼ 60)
3 (5.0)
0
2 (3.3)
7 (11.7)
NV (n ¼ 60)
0
0
0
0
Total 180
8 (4.4)
1 (0.6)
5 (2.8)
17 (9.4)
4 (6.7)
0
0
4 (2.2)
Normozoospermic fertile controls
LV (n ¼ 60)
3 (5.0)
1 (1.7)
2 (3.3)
5 (8.3)
0
SV (n ¼ 60)
1 (1.7)
0
0
2 (3.3)
0
NV (n ¼ 60)
0
0
0
0
0
Total 180
4 (2.2)
1 (0.6)
2 (1.1)
7 (3.8)
0
Molecular karyotype of single sperm and nuclear vacuoles
morphology and no vacuole always showed a normal molecular karyotype both in patients and controls. It is worthy of mention that just
some of all sperm retrieved from patients had an abnormal karyotype
(19.4%). However, we have to consider that chromosomal alterations
represent only a mirror of more subtle sperm DNA alterations, such
as chromatin mispackaging and DNA fragmentation.
Future application of aCGH on sperm might give important information
on the biology and pathophysiology of spermatogenesis and sperm
chromosome aberrations in healthy fertile subjects, as well as in patients
at higher risk of producing unbalanced sperm, such as infertile men, carriers
of karyotype anomalies, men with advanced age, subjects treated with
chemotherapy and partners of couples with repeated miscarriage and
repeated failure of assisted reproduction techniques. In these patients,
pre-implantation genetic screening and PGD could also be considered
to reduce the impact of paternal abnormalities on embryo aneuploidy. In
conclusion, our data demonstrate that molecular karyotypes of sperm
from infertile patients show a higher incidence of abnormalities and these
alterations are related to the presence of nuclear vacuoles. Further and
larger studies performed in patients with severe testicular failure are
needed to understand if sperm selection for ICSI by high-magnification microscopy could represent a reliable method to improve the ICSI outcome
and to reduce the transmission of genes abnormalities to offspring.
Authors’ roles
A.G. study design, data interpretation and writing text; B.S. data collection and writing text; I.C. semen analysis, data collection, writing text;
D.P. enrolling subjects and statistical analysis; M.G. enrolling subjects
and statistical analysis; A.B. generation of tables and figures; M.M.
semen and molecular analyses; E.S. molecular analyses; A.F. study
design and data interpretation; C.F. study design and data interpretation.
Funding
No external funding was sought for this study.
Conflict of interest
All authors have no conflict of interest to declare regarding this manuscript.
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