Abstract

BACKGROUND: In babies born after ICSI procedures, an increase of de-novo sex chromosome abnormalities has been observed. Several hypotheses have been proposed to explain these findings: an increased rate of sex chromosome aneuploidy in sperm of oligozoospermic men, or a preferential location of the sex chromosomes in the sub-acrosomal region of the sperm nucleus which leads to a reduced DNA decondensation of this region. In order to investigate which theory may be more reliable, we studied the localization of sex chromosomes and their aneuploidy rate in sperm in men undergoing ICSI. METHODS: Using fluorescent in-situ hybridization we studied sex chromosome localization and the aneuploidy rate for sex chromosomes and chromosome 18 in 20 oligospermic men undergoing ICSI and in 10 controls. RESULTS: In 40.94 and 52.92% of cases, the X and Y chromosomes respectively were localized in the sub-acrosomal region of the sperm nucleus compared with only 14.29% of cases of chromosome 18 (P < 0.001). An increase of sex chromosome aneuploidy in sperm of oligospermic men was observed; 2.91 versus 0.69% of controls (P < 0.001). CONCLUSIONS: Sex chromosomes are localized preferentially in the sub-acrosomal region of sperm and sex chromosome aneuploidy rate in the sperm of oligozoospermic men is higher than in controls.

Introduction

The development of ICSI has permitted the achievement of pregnancy and reproduction even in cases of infertility due to severe male factor (Palermo et al., 1992; Van Steirteghem, 1993). The possibility to obtain oocyte fertilization with sperm collected from very poor samples or directly sampled from testis has raised some concerns because of the chance of injecting sperm carrying chromosomal anomalies due to the lack of sperm selection through the fertilization process (Martin, 1996). Follow-up data on babies born after the ICSI procedure seem to confirm this concern, since an increase has been reported in the incidence of sex chromosomal aneuploidy of paternal origin and structural de-novo chromosomal abnormalities when compared with the general population (In't Veld et al., 1995; Liebaers et al., 1995; Van Opstal et al., 1997; Bonduelle et al., 1998).

The possibility of visualizing the chromosomes by techniques such as fluorescence in-situ hybridization (FISH) has focused attention on the frequency of numerical chromosomal anomalies in spermatozoa of assisted reproduction patients (Trask, 1991).

An evaluation of the sperm aneuploidy rate has been obtained with the use of multi-colour FISH (Martin et al., 1993). Several studies have shown that the frequency of disomy and nullisomy for the sex chromosome is up to 0.6% (Pfeffer et al., 1999) whereas the total aneuploidy rate is up to 7.7% (Pang et al., 1999) in normozoospermic samples. However, the wide range of results reported in the literature is probably due to the number of chromosome probes tested, the number of sperm analysed and the decondensation technique used (Downie et al., 1997). All data seem to show an increased rate of chromosomal aneuploidy, nullisomy, disomy and diploidy in the sperm of oligozoospermic men who are candidates for ICSI, but with a wide variation in the figures, from 5–38% depending on the author (Moosani et al., 1995; Yurov et al., 1996; Guttenbach et al., 1997; Storeng et al., 1998; Aran et al., 1999; Colombero et al., 1999; Van Dyck et al., 2000; Vegetti et al., 2000; Ohashi et al., 2001).

Recently, it has been shown that sex chromosomes in sperm are preferentially located in the sub-acrosomal region (Luetjens et al., 1999). It has been suggested that in the case of ICSI, the introduction of sperm which did not undergo acrosomal reaction in oocyte cytoplasm, and with an intact sperm perinuclear theca, may lead to an impaired decondensation of chromatin located in the sub-acrosomal region, especially for sex chromosomes, located there more frequently (Terada et al., 2000). This may explain the increased incidence of sex chromosomal abnormalities observed in babies conceived with the ICSI procedure.

In order to determine whether the increased rate of sex chromosome abnormalities in ICSI babies may be due to the location of sex chromosomes in the sub-acrosomal region or to an increased rate of sex chromosomes aneuploidy in sperm, we evaluated, with triple-colour FISH, the semen samples of oligozoospermic patients who were candidates for ICSI.

Materials and methods

Fresh semen samples were obtained from 20 male candidates for ICSI, with oligoasthenozoospermia according to published criteria (World Health Organization, 1992) and strict criteria (Kruger et al., 1986). These were used for assessment of morphology, and 10 samples obtained from 10 normozoospermic men were used as controls. Semen samples were analysed for concentration, motility and morphology before being processed for FISH (Table I).

Sperm samples were washed twice in phosphate-buffered saline (PBS) pH 7.2, centrifuged at 280 g for 10 min, and the pellet was then re-suspended in PBS at the concentration of 10×106/ml. A drop of solution was smeared on a glass slide and allowed to dry. The slides were fixed with 99% ethanol or methanol/acid acetic 3:1 solution, and stored at –20°C.

Decondensation treatment

The slides were washed in 2× standard saline citrate solution (SSC) and incubated for 5 min in 1 mol/l Tris buffer, pH 9.5, containing 25mmol/l dithiothreitol (DTT) (Martini et al., 1995). After decondensation, the slides were washed once in 2×SSC, once in 1×PBS and finally dehydrated through an ethanol series (70–90–90–100–100%) and air-dried. In order to validate the technique used for decondensation, two other systems of nuclear decondensation, incubation in 3 mol/l NaOH for 5 min or 6 mmol/l EDTA plus 2 mmol/l DTT for 45 min, were also used in five sperm samples. No differences in sex chromosome localization were observed as a result of the variation of decondensation techniques (data not shown).

Triple-colour FISH

Three-colour FISH was performed to determine the frequency of disomy/nullisomy for sex chromosomes and their position compared with an autosome, chromosome 18, using three direct-labelled probes: CEP 18 SpectrumAquaTM, CEP X SpectrumGreenTM, and CEP Y SpectrumOrangeTM (Vysis, Downers Grove, IL, USA).

The hybridization solution, 10 μl of each probe mixture, was applied to a glass slide containing the fixed sperm and covered with a coverslip. The slide was sealed with rubber cement and placed in the Hybrita machine. The Hybrita hybridization system consists of a programmed hot plate where the slides can be co-denatured with the DNA probes and hybridized: the slides are placed on the plate and denatured for 3 min at 75°C, followed by hybridization at 37°C overnight. After hybridization, each slide was washed individually with a solution of 0.4×SSC/0.3% NP40 (Vysis) at room temperature for 2 min. The slides were then mounted with 10 μl of 4′,6-diamino-2-phenylindole (DAPI; Vysis Downers Grove, IL, USA) counterstained in anti-fade solution.

Scoring of sperm nuclei

Only slides showing hybridization efficiencies >95% were evaluated. Sperm slides were scored according to previous recommendations (Williams et al., 1993). Sperm nuclei were scored when morphologically preserved, not clumping or overlapping, with a well-defined outline tail and the sperm head decondensed to no more than twice the size of the normal non-decondensed spermatozoa. The presence of the tail was considered essential for a reliable evaluation. FISH preparations were evaluated with a fluorescent microscope (Leica DM interfaced with a computer using the Leica Q-FISH package) at ×1000 magnification. The filter used was the VysisTM Aqua, Green, Orange single filter and the VysisTM DAPI/Green/Orange triple band-pass filter set. A minimum of 1000 sperm nuclei per patient for a total of 36 768 cells were examined (24 234 from ICSI men and 12 634 from normozoospermic controls). We evaluated the cells with disomy, two distinct signals for the same chromosome each equal in intensity and size to the single signal found in normal monosomic nuclei, for each chromosome tested 18, X and Y. Spermatozoa were scored as nullisomic for a chromosome if they did not show any signal for that chromosome while a signal for a second tested chromosome was present. A spermatozoon was considered diploid if it exhibited two signals for each tested chromosome and if the tail was evident as well as the normal oval shape of the head. To determine the chromosome position in the sperm nuclei, the nucleus was divided into three regions, one sub-acrosomal, one equatorial and one basal or tail zone, and, in each sperm scored, the position of sex and chromosome 18 was analysed.

Statistical analysis was performed using Student's t-test for continuous variables and χ2-test or Fisher's exact test for discontinuous variables and χ2 for trend test to determine the correlation between the position of chromosomes. Statistical significance was defined as P < 0.05. The data of the frequency of chromosomal abnormalities are reported as percentage and 95% confidence interval (CI).

Results

A total of 36798 cells of 30 patients were analysed after staining with X, Y and chromosome 18 probes at the same time. Chromosome X was localized in the sub-acrosomal region of the sperm in 40.94% of cells, in the central region in 50.29% of cells and in the tail region in 8.77% of cells. Chromosome Y was localized in the sub-acrosomal region of the sperm in 52.92% of cells, in the central region in 38.14% of cells and in the tail region in 8.93% of cells. Chromosome 18 was localized in the sub-acrosomal region of the sperm in 14.49% of cells, in the central region in 31.35% of cells and in the tail region in 54.36% of cells. There was a significant statistical difference between the distribution of sex chromosomes and chromosome 18 (P < 0.0001) (Table II). No differences in the distribution of chromosomes were observed between normozoospermic and oligozoospermic samples.

Chromosomal abnormalities for sex chromosomes (X, Y) were 2.16% in the oligozoospermic samples, 1.01% were 18/0 and 1.15% were 18/XY, whereas in normospermic patients they were 0.28%, 0.15% were 18/0 and 0.13% were 18/XY (P < 0.001). Chromosomal abnormalities for chromosome 18 in oligozoospermic samples were 0.55%, 0.34% of the sperm were disomic and 0.21% were nullisomic, whereas in normozoospermic patients they were 0.34%, 0.18% of the sperm were disomic and 0.16% were nullisomic (Figure 1). The data are summarized in Table III.

No correlations were observed between the rate of chromosomal abnormalities and the rate of pathological sperm morphology, nor between the rate of chromosomal abnormalities and chromosome position.

Discussion

Some authors have reported that the X chromosome is preferentially located in the sub-acrosomal region of sperm nuclei (Luetjens et al., 1999). Our study, in which we analysed >36 000 cells hybridized with X, Y and 18 chromosome probes, confirmed these data for both sex chromosomes. The X and Y chromosomes were located in the sub-acrosomal region 41–53% of the time, in the equatorial region 31–50% of the time and in the basal region 8–9%, whereas chromosome 18 was located 14% of the time in the sub-acrosomal region, 31% in the equatorial region and 54% close to the tail. The use of DTT to decondensate sperm nuclei is the technique, among the several used for DNA decondensation, which allows the maintenance of the cell structure without disrupting the tail and with low cell swelling, as has also been reported (Downie et al., 1997). DTT breaks the disulphide bonds of protamines and makes the DNA accessible for chromosome painting, and it does not change the distribution and the tridimensional position of chromosomes. Validation of the decondensation technique in our system was performed on five sperm samples. Probably during the meiosis process there is a strict organization of the spindle and in the chromosome migration (Gazvani et al., 2000; Hazzouri et al., 2000). It has been suggested (Terada et al., 2000) that sperm injected in oocytes may generate embryos with sex chromosome aneuploidy, since the preferential localization of sex chromosomes in the sub-acrosomal region of the nucleus and the intact acrosome status may lead to an impairment of sperm sex chromatin decondensation.

In our study, a statistically significant increase of sperm with sex chromosome abnormalities was found, whereas no statistically significant difference in the chromosome 18 aneuploidy was observed. Many studies have shown that oligozoospermic samples have an increase of aneuploidy in sperm both for sex and autosomic chromosomes (Moosani et al., 1995; In't Veld et al., 1997; Lahdetie et al., 1997; Storeng et al., 1998; Aran et al., 1999; Colombero et al., 1999; Pang et al., 1999; Pfeffer et al., 1999; Vegetti et al., 2000; Ohashi et al., 2001). This discrepancy may be due to different patient selection, different methods of nucleus fixation and DNA decondensation and different chromosomal probes or hybridization techniques used (Downie et al., 1997). The presence in the semen samples of oligozoospermic men of 1–5% disomic or nullisomic sperm for sex chromosomes, which in turn may be randomly injected in the cytoplasm of oocytes, and originate embryos with sex chromosome aneuploidy, may explain the higher incidence of sex chromosome abnormalities in offspring born after ICSI procedures (Bonduelle et al., 1998). The embryos with sex chromosome abnormalities are potentially viable, since most sex chromosome numerical abnormalities are compatible with life. The rate of sperm with sex chromosome numerical abnormalities may well resume the expected risk of having babies with sex chromosome abnormalities. This seems a more convincing explanation for the increased rate in sex chromosome aneuploidy in ICSI infants, than the lack of sex chromosome decondensation in intact acrosome sperm injected inside the oocyte.

The examination of sperm with FISH may be recommended in order to substantiate the risk for sex aneuploidy in embryos. The non-random localization of sex chromosomes in sperm nuclei may also have a role in the determination of sex chromosome aneuploidy, but this theory needs to be scrutinized with further studies, even though it seems to play a marginal role.

Table I.

Characteristics of semen samples of oligozoospermic patients and normozoospermic controls

 Oligospermic (n = 20) Controls (n = 10) 
Age 34.2 ± 2.8 33.1 ± 2.1 
Sperm concentration (×106/ml) 14.3 ± 1.8 39.6 ± 3.5 
Sperm motility (%) 21.2 ± 4.5 52.8 ± 5.7 
Normal sperm morphology (%) 3.1 ± 0.7 16.3 ± 1.9 
Leukocyte concentration (×106/ml) 1.6 ± 0.1 0.8 ± 0.2 
Germ cells (×106/ml) 3.0 ± 0.4 0.3 ± 0.1 
 Oligospermic (n = 20) Controls (n = 10) 
Age 34.2 ± 2.8 33.1 ± 2.1 
Sperm concentration (×106/ml) 14.3 ± 1.8 39.6 ± 3.5 
Sperm motility (%) 21.2 ± 4.5 52.8 ± 5.7 
Normal sperm morphology (%) 3.1 ± 0.7 16.3 ± 1.9 
Leukocyte concentration (×106/ml) 1.6 ± 0.1 0.8 ± 0.2 
Germ cells (×106/ml) 3.0 ± 0.4 0.3 ± 0.1 
Table II.

Localization of sex chromosomes (X,Y) and the chromosome 18 used as control in 36 798 spermatozoa of 20 oligoasthenozoospermic and 10 normozoospermic samples

Localization Chromosomes P-value 
 X (%) Y (%) 18 (%)  
Sub-acrosome 15 065 (40.94) 19 473 (52.92) 5332 (14.49) < 0.0001 
Equatorial 18 506 (50.29) 14 034 (38.14) 11 536 (31.35) < 0.0001 
Tail 3227 (8.77) 3286 (8.93) 20 003 (54.36) < 0.0001 
Localization Chromosomes P-value 
 X (%) Y (%) 18 (%)  
Sub-acrosome 15 065 (40.94) 19 473 (52.92) 5332 (14.49) < 0.0001 
Equatorial 18 506 (50.29) 14 034 (38.14) 11 536 (31.35) < 0.0001 
Tail 3227 (8.77) 3286 (8.93) 20 003 (54.36) < 0.0001 
Table III.

Sperm nuclei with chromosomal abnormalities in the samples of oligozoospermic men (n = 20) and normozoospermic men (n = 10)

 Oligoasthenozoospermic % (95% CI) Controls % (95% CI) P-value 
NS = not significant. 
Normosomic 97.09 (96.88–98.30) 99.31 (99.21–99.41) NS 
Disomy X/Y (total) 1.15 (1.02–1.28) 0.13 (0.12–0.14) 0.01 
    XY 0.65 (0.50–0.75) 0.07 (0.06–0.08) 0.01 
    XX 0.30 (0.15–0.35) 0.03 (0.02–0.04) 0.01 
    YY 0.20 (0.15–0.22) 0.03 (0.02–0.04) 0.01 
Nullisomy X/Y 1.01 (0.90–1.12) 0.15 (0.14–0.16) 0.01 
Total X/Y aneuploidy 2.16 (2.09–2.21) 0.28 (0.22–0.35) 0.01 
Disomy 18 0.34 (0.28–0.40) 0.18 (0.16–0.20) NS 
Nullisomy 18 0.21 (0.18–0.25) 0.16 (0.13–0.18) NS 
Total 18 aneuploidy 0.55 (0.45–0.65) 0.34 (0.27–0.41) NS 
Diploidy 0.10 (0.08–0.12) 0.07 (0.05–0.09) NS 
Total aneuploidy 2.91 (1.05–3.12) 0.69 (0.59–0.79) 0.01 
 Oligoasthenozoospermic % (95% CI) Controls % (95% CI) P-value 
NS = not significant. 
Normosomic 97.09 (96.88–98.30) 99.31 (99.21–99.41) NS 
Disomy X/Y (total) 1.15 (1.02–1.28) 0.13 (0.12–0.14) 0.01 
    XY 0.65 (0.50–0.75) 0.07 (0.06–0.08) 0.01 
    XX 0.30 (0.15–0.35) 0.03 (0.02–0.04) 0.01 
    YY 0.20 (0.15–0.22) 0.03 (0.02–0.04) 0.01 
Nullisomy X/Y 1.01 (0.90–1.12) 0.15 (0.14–0.16) 0.01 
Total X/Y aneuploidy 2.16 (2.09–2.21) 0.28 (0.22–0.35) 0.01 
Disomy 18 0.34 (0.28–0.40) 0.18 (0.16–0.20) NS 
Nullisomy 18 0.21 (0.18–0.25) 0.16 (0.13–0.18) NS 
Total 18 aneuploidy 0.55 (0.45–0.65) 0.34 (0.27–0.41) NS 
Diploidy 0.10 (0.08–0.12) 0.07 (0.05–0.09) NS 
Total aneuploidy 2.91 (1.05–3.12) 0.69 (0.59–0.79) 0.01 
Figure 1.

Sperm nuclei hybridized with probes for chromosome X, Y and 18; (A) normosomic sperm nuclei with chromosome X and Y preferentially located in the sub-acrosomal region; (B) XY disomic sperm (white arrow), diploid sperm (green arrow), nullisomic sperm for sex chromosomes and disomic for chromosome 18 (red arrow); (C) nullisomic sperm for sex chromosomes (white arrows); (D) normal sperm (white bar = 5 μm).

Figure 1.

Sperm nuclei hybridized with probes for chromosome X, Y and 18; (A) normosomic sperm nuclei with chromosome X and Y preferentially located in the sub-acrosomal region; (B) XY disomic sperm (white arrow), diploid sperm (green arrow), nullisomic sperm for sex chromosomes and disomic for chromosome 18 (red arrow); (C) nullisomic sperm for sex chromosomes (white arrows); (D) normal sperm (white bar = 5 μm).

5
To whom correspondence should be addressed at: Center for Endocrinology and Reproductive Medicine, Via Carlo Porta 10, 00153, Rome, Italy. E-mail: marcandrea@hotmail.com
Submitted on April 23, 2001

We thank Dr M.Pesce (IDI, Istituto Dermopatico dell'Immacolata, Rome, Italy) for his valuable technical assistance.

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