Polyploidy in large-headed sperm: FISH study of three cases

F. Devillard1, C. Metzler-Guillemain2, R. Pelletier3, C. DeRobertis1, U. Bergues1, S. Hennebicq1,3, M. Guichaoua2, B. Sèle1,3 and S. Rousseaux1,3,4

1 Laboratoire de Cytogénétique, Biologie de la Reproduction et CECOS, Centre Hospitalo-Universitaire de Grenoble, 38043 Grenoble cedex 09, 2 Laboratoire de Biologie de la Reproduction et du Développement, Faculté de Médecine Timone, 27 Bd Jean Moulin, 13 385 Marseille cedex 05, 3 Unite INSERM U309, UJF Grenoble, Institut Albert Bonniot, Domaine de la Merci, 38706 Grenoble, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Acknowledgements
 References
 
BACKGROUND: Macrocephalic or large headed sperm with multiflagella is a rare abnormality often associated with infertility. Sperm chromosomal abnormalities could be associated with this specific morphological abnormality. METHODS: The cytogenetic content of large-headed sperm was assessed by dual and three-colour fluorescence in-situ hybridization in three patients carrying this specific morphological abnormality. RESULTS: In all patients nearly all sperm contained at least one copy of each sex chromosome, and in more than half of them at least two copies of either chromosome 1 or 18 were identified. In some sperm a tetraploidy was found. CONCLUSIONS: These observations suggested that both meiotic I and II divisions were affected by incomplete partition of homologous chromosomes during meiosis I and of sister chromatids during meiosis II associated with a failure of nuclear cleavage. Furthermore, they provide evidence for a clear relationship between a specific morphological abnormality of the sperm and their abnormal cytogenetic content. The treatment of infertility using ICSI would probably be unsuccessful and have a high genetic risk in these cases.

Key words: aneuploidy/FISH/ICSI/morphological abnormalities/sperm chromosomes


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Acknowledgements
 References
 
With the introduction of ICSI, infertility can often be overcome even in cases of severe male factors (Palermo et al., 1992Go; Van Steirteghem et al., 1993Go). Since this technique has now become widely used, its safety is a very important issue, and is still under investigation (Bonduelle et al., 1996Go, 1999Go). These concerns over safety also include the possibility of an abnormal genetic content of the sperm used for the injection.

In fertile males, the aneuploidy frequencies in sperm evaluated by sperm karyotyping range between 1–4% (Hassold et al., 1996Go; Guttenbach et al., 1997aGo). With this technique, only sperm able to fuse with hamster oocytes could be analysed. Nowadays, sperm cytogenetic content can also be detected by fluorescence in-situ hybridization (FISH) and numerical chromosomal abnormalities can be evaluated in large populations of human sperm. The FISH method can be performed even when spermatogenesis is impaired and allows the analysis of several thousands of sperm per patient. The simultaneous hybridization of two or three chromosome specific DNA probes enables an accurate estimation of disomy and diploidy rates.

Numerous studies analysing these rates in fertile and infertile men have been reported in recent years. According to the data of several studies reviewed by Downie et al. (1997) and Egozcue et al. (1997), in normozoospermic men disomy (hyperhaploidy) rates would be of the rate of 0.05–0.2% per chromosome (Downie et al., 1997Go; Egozcue et al., 1997Go). The overall disomy frequency in sperm from fertile men would be ~3% and the incidence of all numerical chromosomal abnormalities would be ~6.3% (including 0.3% diploidies) (Downie et al., 1997Go). The results obtained in infertile men are heterogeneous and to date there is no clear evidence of major increases in the frequencies of sperm chromosomal abnormalities in infertile men. Some studies showed elevated rates of disomy and diploidy in patients with severe oligoasthenoteratozoospermia (OAT) (Lahdetie et al., 1997Go; Bernardini et al., 1998Go; Colombero et al., 1999Go; Pang et al., 1999Go; Rives et al., 1999Go). Other studies do not show clearly any difference in aneuploidy rates between fertile and infertile men (Guttenbach, 1997b). In some studies, the difference in aneuploidy rates between men with OAT and controls, although statistically significant, was not important (Ushijima et al., 2000Go). Most data show significantly higher rates of diploidy and disomy in sperm or sperm fractions containing high numbers of morphologically abnormal sperm (Lee et al., 1996Go; In't Veld et al., 1997; Bernardini et al., 1998Go; Obasaju et al., 1999Go; Pfeffer et al., 1999Go). However there is still no clear evidence of a link between the morphology of each individual spermatozoon and its chromosome content.

It was shown that in some cases of teratozoospermia affecting all sperm with 100% abnormal head morphology, ICSI could be the only therapeutic approach to overcome the infertility, although the implantation and ongoing pregnancy rates remained very low (Tasdemir et al., 1997Go). However it is not yet established whether ICSI can be performed successfully and safely in these situations.

The presence of macrocephalic or large headed sperm with multiflagella in an ejaculate is rare and is often associated with infertility. Sperm chromosomal abnormalities could be associated with this specific morphological abnormality. To explore this hypothesis, a FISH analysis of large headed sperm from three patients was performed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Acknowledgements
 References
 
Patients
Patient A was a 46 year old man with primary infertility associated with severe OAT. His sperm count was 3.2x106/ml, 75% of the sperm were immotile and 25% had reduced motility. All of them were morphologically abnormal with a large head of irregular shape and in >30% of the cells more than one flagella was found.

Patient B was a 38 year old man with secondary infertility. A 5 year old girl had been born from a previous union and three miscarriages had occurred with his current wife. His sperm count was 30x106/ml with reduced motility. A large head with an indistinguishable acrosome was observed in >95% of the sperm. All of the large sperm also had thick and rigid tails.

Patient C was a 34 year old man with primary infertility. His sperm count was 10x106/ml with reduced motility. All sperm were morphologically abnormal with large heads and/or multiple flagella.

FISH on sperm
Dual colour FISH experiments with specific probes for chromosomes Y and 1, as well as for chromosomes X and Y were successively performed. Three-colour FISH experiments were performed with probes specific for chromosomes X, Y and 18.

Sperm preparation
Cryopreserved semen samples from the patients as well as from the control subjects were thawed and washed twice in 0.01 mol/l Tris, pH 8 for 5 min at x600 g. The in-vitro decondensation procedure was performed as previously described (Rousseaux and Chevret, 1995Go). Briefly, the sperm nuclei were incubated for 3–10 min with 10 mmol/l dithiothreitol (Sigma-Aldrich, St Quentin Fallavier, France) at room temperature, then dropped onto clean dry slides and fixed with 3:1 ethanol/acetic acid. Slides were kept frozen at –20°C until use.

DNA probes
Three probes, pXBR2, pHY2.1 and pUC1.77, specific for X, Y and 1 respectively, were used. X probe is an alpha satellite probe, the Y probe is specific for a repeated sequence on the long arm of the Y (Yq12) and pUC1.77 hybridizes to the pericentromeric heterochromatin in 1q12. All probes were labelled by nick-translation with digoxigenin-11-dUTP or biotin-16-dUTP (both from Roche Molecular Biochemicals, Meylan, France) and hybridized on the sperm preparations in dual FISH experiments.

For the three-colour FISH experiments a mixture of labelled probes specific for chromosomes X, Y and 18 was purchased from Vysis (Voissin le Bretonneux, France).

FISH
FISH was performed simultaneously on sperm slides from patients A and B and from the control subjects. The method has already been described (Pinkel et al., 1986Go; Rousseaux and Chevret, 1995Go). Before hybridization, sperm DNA slides were treated with RNAase A (100 µg/ml) for 1 h at 37°C, dehydrated in ethanol (70/90/100%), and denatured in 70% formamide (2 min at 70°C), dehydrated again and air dried. Twenty microlitres of the hybridization mixture [50% formamide, 10% dextran sulphate, 0.5xsodium saline citrate (SSC), and 0.5xsodium saline phosphate EDTA (SSPE)] containing 100 ng of each probe and 10 µg of sonicated salmon sperm DNA were heated for 5 min at 75°C to denature the probes. The mixture was applied to each sperm nuclei preparation. The slides were covered with a 324 mm2 coverslip and hybridized at 37°C for 20 h. They were then washed three times (5 min each) in 50% formamide/2xSSC at 45°C, three times in 2xSSC at 45°C and once in 0.1xSSC at 60°C. Biotinylated and/or digoxigenin-labelled probes were simultaneously visualized with, respectively, avidin-fluorescein isothiocyanate (FITC) (1/300, Vector Laboratories, AbCys, Valiobiotech, Paris, France) and anti-digoxigenin-rhodamine Fab fragments (1/200, Roche Molecular Biochemicals).

In the three-colour FISH experiments on the sperm of patient C, the FISH was performed with a few modifications as recommended by the manufacturer. Briefly, 8 µl of the probe mixture was used on each slide. Both probes and slides were simultaneously denatured at 75°C for 5 min and then hybridized at 42°C overnight. It was followed by the rapid wash procedure recommended by Vysis.

Nuclei were counter-stained with 4', 6-diamino-2-phenyl-indole dihydrochloride (DAPI) (200 ng/ml) in an antifade solution (Vector Laboratories). Slides were screened using a 100x objective on a Zeiss Axiophot microscope equipped with a FITC/rhodamine/DAPI triple band-pass filter.

Only individual and well-delineated sperm were scored. Two signals of the same colour were scored as two copies of the corresponding chromosome when they were the same intensity and size and when the distance between them (from edge to edge) was at least equal to the diameter of one single signal.

Results
Totals of 2948, 1645 and 258 sperm nuclei were scored from patients A, B and C respectively after hybridization with probes specific for chromosomes Y and 1 (Table IGo). As a first estimate of the number of abnormal sperm, the total percentages of sperm containing more than one chromosome 1 and/or more than one chromosome Y were calculated. They were 60, 47.3 and 50.8% in patients A, B and C respectively. There were <1% sperm with no Y chromosome in patient A and none in patients B or C. In patient C, 72 sperm were analysed with probes specific for chromosomes X and 1 (Table IIGo), 51.4% of which contained more than one chromosome 1 and/or more than one chromosome X. Totals of 1333 and 1203 sperm nuclei were analysed with X and Y specific probes in patients A and B respectively. The detection of X and Y chromosomes showed that the majority of the sperm contained one of each gonosomes (X/Y), 80.4% in patient A and 71.3% in patient B (Table IIIGo, Figure 1Go). Most of the other sperm contained three or four gonosomes. There were very few sperm containing one chromosome X and no chromosome Y (0.4%) in patient A, and no X/0 or Y/0 sperm from patient B.


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Table I. Chromosomal constitution of sperm from patients A, B and C detected by FISH with chromosomes 1 and Y probes
 

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Table II. Chromosomal constitution of sperm from patient C detected by FISH with chromosomes 1 and X probes
 

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Table III. Chromosomal constitution of sperm from patients A and B detected by FISH with chromosomes X and Y probes
 


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Figure 1. Detection of chromosomes X (red) and Y (green) on large-headed sperm showing from left to right: an XY, an XYY and an XXYY tetraploid sperm.

 
A total of 473 sperm from patient C were analysed using three-colour FISH with X, Y and chromosome 18 probes, of which 62.6% contained one of each gonosome (X/Y), with nearly all the rest of them containing three or four gonosomes (Table IVGo, Figure 2Go). A single sperm (0.2%) was found containing just one gonosome (Y).


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Table IV. Chromosomal constitution of sperm from patient C detected by FISH with chromosomes 18, X and Y probes
 


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Figure 2. Detection of chromosomes X (green), Y (red) and 18 (blue) on large-headed sperm: sperm containing X/Y/18 (upper left), X/X/Y/Y/18/18 (upper right), X/X/Y/18/18/18 (lower left) and X/X/Y/Y/18/18 (lower right).

 
Discussion
Dual-colour FISH experiments with specific probes for chromosomes 1, X and Y were performed on semen samples from two patients with large headed-multiflagella sperm. Moreover three-colour FISH experiments were also performed on patient C to simultaneously detect chromosomes X, Y and 18.

The consistency of the different FISH experiments can be appreciated by comparisons of the rates of occurrence of Y and Y/Y sperm, between the two experiments, in each patient. The number of sperm carrying one Y chromosome in the first experiment (added rates of 1/Y, 1/1/Y, 1/1/1/Y and 1/1/1/1/Y sperm: 80.7, 81 and 73.6% in patients A, B and C respectively, see Table IGo) was not very different from the number of sperm carrying one Y in the X/Y or X/Y/18 experiments (added rates of the X/Y and X/X/Y sperm: 89.8, 84.0 and 69.6% in patients A, B and C respectively, see Tables III, IVGoGo). However, in patient A, the rate of YY was higher in the Y/1 experiment than in the X/Y experiment (18.6 versus 9.8%, see Tables I, IIIGoGo). This discrepancy could be explained by a lower hybridization efficiency of the Y probe in the X/Y experiment than in the 1/Y experiment, or by a difficulty in differentiating some of the YY signals in the sperm of the X/Y experiment. Also, in patient C, the rates of X or XX sperm were close in both dual-colour 1/X and three-colour X/Y/18 experiments (see Tables II and IVGoGo).

When analysing the aneuploidy rate, it was found that it affected the gonosomes as well as the autosomes. The X/Y FISH experiment showed that the majority of sperm contained both X and Y chromosomes. The 1/Y experiment showed that more than half the sperm contained more than one chromosome 1. Therefore the combined results of the two double-colour FISH experiments X/Y and Y/1 suggested that most of the sperm carried several chromosomal abnormalities. The presence of many sperm with more than two gonosomes and/or more than one chromosome 1 suggests that there is a high rate of tri- and tetraploid sperm. Taken together, these results suggest that all sperm in both patients A and B were aneuploid, either showing a polyploidy or a hyperhaploid content with an extra sex chromosome. This was further confirmed by the three-colour FISH study of the third patient, although the precise numbers of chromosomes in each sperm were difficult to assess when the nuclei contained five or more signals.

Using a rapid FISH method with several autosomal probes (chromosomes 1, 3, 11, 12, 17 and 18), Yurov et al. (1996) analysed the sperm of an infertile man from which 40% were large-headed (Yurov et al., 1996Go). Their results suggested that most large-headed sperm were diploid, whereas the majority of normal sized sperm were haploid and chromosomally normal. In a case report published by In't Veld et al., (1997), 1000 sperm from an ICSI patient with 100% large-headed sperm were analysed by three-colour FISH. This study showed that <2% of the patient's sperm were haploid, 40% were diploid, 24% were triploid and 22% were hyperhaploid with an extra sex chromosome (In't Veld et al., 1997). Although the proportions of each category of sperm are different, these results are compatible with the present study, when both dual-colour and three-colour experiments are combined. More recently, the cytogenetic analysis by FISH of the sperm from four patients with severe teratozoospermia has been reported (Viville et al., 2000Go). Of the four patients, the only one with macrocephalic sperm showed a highly elevated aneuploidy rate.

We propose a mechanism which could explain our findings and those reported earlier (Figure 3Go). It is known that just before entering meiosis, each diploid germ cell undergoes DNA synthesis and results in a cell containing 2n duplicated chromosomes or 4c chromatids, which will then go through two successive meiotic divisions, and produce four haploid cells. Each meiotic division includes (i) a separation of the homologous chromosomes (MI) or the sister chromatids (MII) followed by (ii) nuclear cleavage. The presence of both X and Y chromosomes in almost all sperm nuclei suggests that these chromosomes failed to separate during the first meiotic division. Moreover, the high number of sperm containing two of the same sex chromosome (XX or YY) suggested that the meiotic II separation of the gonosomes was also affected. These could be non-disjunctions specifically affecting the gonosomal pair. However, in many sperm more than one chromosome 1 and more than one chromosome 18 were observed, suggesting that the autosome separation was also abnormal. Therefore, the analysed sperm could all result from a failure of the first and second meiotic divisions, affecting the separation of all chromosome pairs, including the gonosomes and the autosomes. After this abnormal meiotic separation of the chromosomes there would then be two possibilities: cleavage of the nucleus or no nuclear cleavage. Sperm resulting from an abnormal chromosome segregation followed by nuclear cleavage would contain abnormal numbers of each chromosome pair, with either an excess or an absence of the chromosomes of each pair. The fact that no sperm were seen where one given chromosome was absent suggested that there was no nuclear cleavage. Indeed the presence of both chromosomes X and Y in nearly all sperm suggests that the nuclear cleavage did not occur at the end of the first meiotic division. Similarly, there were no sperm where either chromosome 1 or chromosome 18 were missing, but many with three or four chromosomes of the same pair, suggesting that nuclear cleavage did not occur during meiosis II.



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Figure 3. Illustrations of the possible mechanism of sperm aneuploidies in large headed sperm. None of these cells was able to undergo nuclear or cell cleavage during meiosis. (A) Duplicated chromosomes remain as they are before meiosis I (no cleavage of homologous chromosomes or of sister chromatids). (B) Homologous chromosomes have separated (MI) but sister chromatids remain associated. (C) Homologous chromosomes have separated (MI) and sister chromatids have cleaved (MII).

 
This hypothesis is further supported by earlier observations (Escalier, 1985Go), where meiosis was studied by electronic microscopy in a man with macrocephalic sperm. In the meiotic cells, the centrioles were centrally located and there was no nuclear cleavage. Therefore, during the first meiotic division, some pairs of homologues would separate but there would be no nuclear cleavage. During the second meiotic division, again, there would be a partition of some of the sister chromatids but no cleavage. Hence, due to the absence of nuclear cleavage, all sperm would be, in fact, cells containing a set of four chromatids of each chromosome. Each pair of homologues would be identified by FISH as one, two, three or four signals depending on the degree of association of these four chromatids.

The origin of this meiotic abnormality could be genetic or environmental. The history of patient B (secondary infertility, father of a 5 year old girl), as well as a similar previously reported case (In't Velt et al., 1997), suggests that there could be an increase in the sperm morphological abnormality rate over the years, which could be associated with a gradual deterioration of the meiotic divisions. The factors involved in this potential deterioration are unknown. Also, many genes involved in the control of meiosis and chromosome segregation remain unknown and further studies of these pathologies are of great interest in understanding meiosis.

In conclusion, in three patients with large-headed sperm we clearly demonstrate a correlation between the phenotype of the sperm (large headed/multiflagella) and a polyploid chromosomal constitution. In such circumstances, the use of ICSI should not be recommended, not only because of its low chances of success but also because of its high genetic risk.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Acknowledgements
 References
 
We thank Dr Saadi Khochbin (INSERM U309, UJF, Grenoble) for critical reading of this manuscript. This work was partly supported by Delegation à la Recherche Clinique (DRC, CHU de Grenoble).


    Notes
 
4 To whom correspondence should be addressed. E-mail: sophie.rousseaux{at}ujf-grenoble.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Acknowledgements
 References
 
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Submitted on August 21, 2001; resubmitted on October 15, 2001; accepted on January 11, 2002.