Human Reproduction, Vol. 16, No. 11, 2274-2277,
November 2001
© 2001 European Society of Human Reproduction and Embryology
Assisting reproduction of infertile men carrying a Robertsonian translocation
1 Service de Génétique et Reproduction, Hôpital Antoine Béclère, Clamart, 2 Département de Génétique, U393, Hôpital Necker Enfants Malades, Paris and 3 Service de Gynécologie Obstétrique, Hôpital Antoine Béclère, Clamart, France.
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Abstract |
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BACKGROUND: In order to provide better genetic counselling for Robertsonian translocation carriers, the meiotic segregation of chromosomes 13, 14 and 21 from six infertile (13;14) and (14;21) Robertsonian translocation carriers was examined. METHODS: Dual-colour fluorescence in-situ hybridizaion analysis using locus-specific probes was carried out on spermatozoa of translocation carriers. Spermatozoa from six proven fertile subjects were analysed using the same probes as controls. RESULTS: We observed that the frequencies of unbalanced spermatozoa were similar in the (13;14) translocation carriers (9.0, 10.0 and 12.9%) and in the (14;21) translocation carriers (8.7, 7.2 and 7.0%). These frequencies were significantly increased compared with the control population (P < 0.05). CONCLUSIONS: This high frequency might justify the use of preimplantation genetic diagnosis in these patients where the translocation is usually associated with infertility, requiring intracytoplasmic sperm injection, as it might improve the outcome of the assisted reproduction technique.
Key words: FISH/meiotic segregation/Robertsonian translocation/spermatozoa
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Robertsonian translocations are the most common structural chromosomal abnormalities observed in humans with a total frequency of 1.23 per thousand (Nielsen and Wohlert, 1991
). Among them translocations (13q;14q) and the translocation (14q;21q) are the most common, with an estimated frequency of 0.97 and 0.20 respectively.
It is well known that in infertile males, the frequency of chromosomal aberrations is increased and varies from 1.9–4.0% (Montag et al., 1997; Meschede et al., 1998; Tuerlings et al., 1998; Van Der Ven et al., 1998). Among them, Robertsonian translocations and numerical sex chromosomal aberrations are the most frequent.
Until now genetic counselling of Robertsonian translocations carriers has been based on outcomes observed during prenatal diagnosis or at birth (Boué and Gallano, 1984). In addition, direct investigations performed on spermatozoa (Martin, 1988) have provided a better understanding of the meiotic process. Therefore, more accurate genetic counselling can be given to Robertsonian translocation carriers. Originally, these studies were carried out using heterospecific IVF with zona-free hamster or mouse oocytes. This technique is time consuming and labour intensive, and only a small number of metaphases can be studied. Furthermore, translocation carriers with oligoasthenozoospermia have a poor experimental fertilization rate and could not be explored. More recently, the fluorescence in-situ hybridization (FISH) technique has been applied to spermatozoa (Han et al., 1992; Holmes and Martin, 1993). This technique does not need fertilization to proceed and allows the analysis of a large number of spermatozoa. For example, FISH analysis on spermatozoa were realized for two (13;14) translocation carriers (Escudero et al., 2000) and for two (14;21) translocation carriers (Rousseaux et al., 1995; Honda et al., 2000).
In the present work, the meiotic segregation of six infertile men bearing a Robertsonian translocation [three t(13;14) and three t(14;21)] has been assessed using FISH on spermatozoa. On the basis of the unbalanced spermatozoa rate found, the interest of preimplantation genetic diagnosis (PGD) is discussed.
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Materials and methods |
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Patients
Six unrelated and infertile Robertsonian translocation carriers were included in the study. Their karyotypes were 45,XY,der(13;14)(q10;q10) in three cases (P1, P2, P3) and 45,XY,der (14;21)(q10;q10) in the other three cases (P4, P5, P6). The patients' ages ranged from 29–38 years. Sperm counts ranged between 4–22x106 spermatozoa/ml. The proportion of spermatozoa with progressive motility ranged from 4–30%, and 3–36 % of spermatozoa had a normal morphology according to the David classification (David et al., 1975
) (Table I). Six semen samples with normal parameters from proven fertile subjects were used as controls.
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Sperm preparation and sperm head swelling
After liquifaction for 20 min at 37°C, each semen sample was prepared using the Puresperm (JCD, Lyon, France) migration technique. The pellet was then washed in Ferticult (JCD). Following centrifugation at 500 g, the pellet was incubated for 10 min at room temperature with a hypotonic solution (natrium citrate 0.5%). After further centrifugation, the pellet was resuspended in Carnoy's solution (methanol/acetic acid 3:1). The sperm pellets were dropped onto slides, air dried and stored at –20°C until their use.
The sperm nuclei decondensation was performed using NaOH solution (1 mol/l) for 2 min at room temperature.
DNA probes
Dual colour FISH experiments were performed using DNA probes specific for chromosomes 13, 14 and 21. Probes were prepared from YAC and BAC clones: YAC 908C3 mapped on 13q34, YAC 265H12 mapped on 21q22.3 and BAC 158A2 localized on 14q32. YACs 908C3 and 265H12 were labelled by nick-translation with fluorescein 12-dUTP (Roche) and BAC 158A2 with rhodamine 6-dUTP (Roche).
Fluorescent in-situ hybridization
Before hybridization, sperm DNA slides were dehydrated in ethanol (70, 90, 100%) and air dried. The probes mixture was applied to the sperm nuclei preparation, and slide was covered with a coverslip and sealed with rubber cement. The denaturation was performed simultaneously on sperm nuclei and probes for 4 min at 75°C. The slides were then hybridized in a dark moist chamber at 37°C for 15 h. Coverslips were then removed and slides were washed for 2 min in 0.4xSSC 0.4% NP40 solution at 73°C. Nuclei were counterstained with 4,6-diamino-2-phenylindole dihydrochloride (DAPI). Slides were screened using a X-100 objective on an Olympus epi-fluorescent microscope equipped with fluorescein isothiocyanate (FITC)/ Rhodamine/DAPI triple band-pass filter. The slides of patients and controls were examined by two observers in double blind fashion and for each observer 1000 sperm nuclei were counted in dual colour. Only individual and well delineated spermatozoa were scored. A spermatozoon was scored as disomic if it showed two signals of the same colour, intensity and size. According to the scoring criteria of Martin and Rademaker, two spots separated by less than the diameter of one hybridization domain were scored as a single signal (Martin and Rademaker, 1995). The absence of signal for a single chromosome was scored as nullisomy for this chromosome only when the other probed chromosome gave a signal.
Statistical analysis
The data were analysed using the 
2 test. We compared the observed results in the translocation carriers with those observed in the control subjects. In addition, results obtained in both translocation carriers, namely (13;14) translocation and (14;21) translocation, were compared between themselves. Finally, the disomy and nullisomy rates were also compared. Differences were considered to be statistically significant when the probability value was < 0.05.
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Results |
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FISH results of balanced, disomic and nullisomic spermatozoa from the six patients and control population are indicated in the Tables II and III
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For the (13;14) translocation carriers, FISH analysis revealed that the unbalanced segregation rate was ~10% and did not vary significantly between patients. The percentage of balanced gametes was significantly decreased compared with the control population in which the mean value was 99.5% (P < 0.05). The disomy rate ranged between 1.4–3.3% for chromosome 13 and 3.5–4.4% for chromosome 14. The nullisomy rate ranged between 2.0–3.3% for chromosome 13. For chromosome 14 it ranged between 1.5–2.8%. Both rates (disomy and nullisomy) were significantly increased compared with the control population (P < 0.05) (Table II
).
For (14;21) translocation carriers the unbalanced segregation rate was ~8%. No significant difference was observed between patients. The percentages of balanced gametes were significantly decreased compared with the one observed in the control population (i.e. 99.6%, P < 0.05). The disomy rates ranged from 2.5–3.6% for chromosome 14 and from 1.1–1.4% for chromosome 21. The nullisomy rates varied 2.1–2.9% for chromosome 14 and between 0.8–1.0% for chromosome 21. Disomy and nullisomy rates were significantly increased compared with the control population (P < 0.05) (Table III).
For each chromosome, the nullisomy rate and the disomy rate were compared. No significant difference was found between these two rates, except in P2 for chromosome 14.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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In the present study, the meiotic segregation of six infertile men with a Robertsonian translocation [t(13;14) or t(14;21)] was assessed using FISH on spermatozoa.
We found an homogeneous frequency of balanced spermatozoa for (13;14) translocations (87.1–91.0%) as well as for (14;21) translocations (91.3–93.0%).
Five previous reports (Pellestor et al., 1987; Martin, 1988; Escudero et al., 2000; Ogawa et al., 2000; Morel et al., 2001) have analysed spermatozoa from (13;14) translocation carriers. The three first studies used heterospecific IVF. Therefore only a small number of spermatozoa was analysed (78, 116 and 45 respectively). They showed a high proportion of normal and balanced translocated gametes (74, 94 and 87% respectively). Recently, FISH procedure has been used to count a much larger number of spermatozoa (Escudero et al., 2000; Morel et al., 2001). Escudero et al. examined two (13;14) translocation carriers and found 26.4 and 22.6% of unbalanced spermatozoa (Escudero et al., 2000). Morel et al. observed a rate of unbalanced spermatozoa from 10.8–18.1% by using painting probes in three (13;14) translocation carriers (Morel et al., 2001). In this study, we found frequencies of 9, 10 and 12.9% of unbalanced spermatozoa. These frequencies are significantly lower than those observed by Escudero et al. (Escudero et al., 2000). In particular, nullisomy rates were higher in this study. A nullisomy event can be due to either the absence of the studied chromosome or a decrease of hybridization efficiency. Only the use of normal control spermatozoa can allow one to distinguish between these two possibilities.
Sperm nuclei analysis of (14;21) translocation carriers have been reported using either heterospecific IVF (Balkan and Martin, 1983) or FISH (Rousseaux et al., 1995; Honda et al., 2000). In this study the frequencies of unbalanced spermatozoa varied from 7.0–8.7%. Again these frequencies are lower than those observed by Rousseaux et al. (18%) and Honda et al. (11.25%) (Rousseaux et al., 1995; Honda et al., 2000). For this reason, other FISH studies on spermatozoa from Robertsonian translocation carriers should be performed in order to give precise information to carriers about the genetic risks linked to these translocations.
All studies that have examined the outcome of pregnancies of Robertsonian translocation carriers have reported a relatively low (<2%) chromosomal imbalance rate at birth. For example, out of 230 amniocenteses performed on pregnancies of (13;14) translocation carriers, no unbalanced outcomes were described (Boué and Gallano, 1984). In addition, Robinson et al. showed that 21 out of 23 cases of trisomy 13 were consistent with a maternal origin of the extra chromosome (Robinson et al., 1996). In this series one case exhibited a paternal translocation (13;14); interestingly this trisomy 13 was due to a maternal meiotic non-disjunction. For male carriers of (14;21) translocations, Daniel et al. showed that they have a 4.3% risk of unbalanced chromosomal rearrangement at amniocentesis (Daniel et al., 1989). As expected, the frequency of unbalanced results after prenatal diagnosis (PND) is lower than the frequency of chromosomally unbalanced spermatozoa found in the present or previous studies. However, according to the present study, the frequency of unbalanced spermatozoa is lower than that reported at early embryo stage as proved by PGD. For example, Conn et al. reported the following results after three PGD cycles for a (13;14) paternal translocation: out of 27 embryos analysed, 4 (15%) were normal, 9 (33%) were aneuploid for chromosome 13 or 14 and 14 (52%) were chaotic (Conn et al., 1998). Escudero et al. also revealed a high rate of abnormal embryos after six PGD cycles for the same indication (Escudero et al., 2000). Thus, out of 30 embryos analysed, 15 (50%) were normal, 6 (20%) were aneuploid for chromosomes 13 or 14, 6 (20%) were haploid and 3 (10%) were complex abnormal. Presumably the (13;14) translocation is not causally related to all abnormalities found in the embryos at PGD. The difference between the results from PGD and PND suggests that there is a strong selection against unbalanced chromosomal constitution during early embryogenesis. In the present study, the total risk of unbalanced spermatozoa was estimated at around 10% in t(13;14) and t(14;21) carriers. This chromosomal risk may be considered as elevated, which would then justify the PGD practice. Knowing that no unbalanced result was published after PND for paternal (13;14) translocation, we recommend PGD only in cases of associated infertility in order to select balanced embryos. In cases of (14;21) translocation, PND results have shown that there is an existing risk which by itself justifies the PGD practice.
In conclusion, the present study shows that the proportion of unbalanced spermatozoa from Robertsonian translocation carriers is similar and in the order of 10%. We believe that this high frequency might justify PGD in these patients where the translocation is usually associated with infertility requiring intracytoplasmic sperm injections as it might improve the implantation rate and protect the couple from termination of pregnancy. All of these patients have been informed of their own genetic risk in relation to the sperm FISH study and have been included in our PGD programme. Further comparative analyses of chromosome complement in embryos and spermatozoa of translocation carriers are necessary to predict the rate of normal embryos expected in PGD cycles.
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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We wish to thank Dr T.Haaf (Max Planch Institut, Berlin) and Dr H.Loiseau (CHU Nantes) for providing the YAC and BAC probes.
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Notes |
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4 To whom correspondence should be addressed at: Service de Génétique et Reproduction, Hôpital Antoine Béclère 157, rue de la porte de Trivaux 92140 Clamart, France. E-mail: nelly.frydman{at}abc.ap-hop-paris.fr
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References |
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Submitted on April 4, 2001; accepted on August 10, 2001.
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