1 Service de Cytogénétique, Cytologie et Biologie de la Reproduction, CHU Morvan, Brest, 2 Laboratoire d'Histologie, Embryologie et Cytogénétique, Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, Brest, 3 Service de Biologie de la Reproduction, CHRU Lille and 4 Centre de Génétique Chromosomique, Hôpital Saint Antoine, Lille, France
5 To whom correspondence should be addressed at: Laboratoire de Cytogénétique, Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, 22 avenue Camille Desmoulins, CS 93837, F-29238 Brest cedex 3, France. Email: marc.debraekeleer{at}univ-brest.fr
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Abstract |
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Key words: FISH/male infertility/meiotic segregation/reciprocal translocation/spermatozoa
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Introduction |
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The meiotic segregation pattern of 39 men, studied by heterospecific fecundation, and 44 men, using fluorescence in situ hybridization (FISH) or PRINS, carrying a balanced reciprocal translocation between two autosomes has already been published. Whatever the technique used, the frequencies of unbalanced spermatozoa varied from 19% to more than 80% (Morel et al., 2004a). Thus, the risk of producing unbalanced offspring in these patients is important. Nevertheless, the question of intraindividual variations has not been addressed yet.
In the present study, we analyzed and compared the meiotic segregation pattern in spermatozoa of two different samples from a 46,XY,t(9;22)(q21;q11.2) carrier by multicolor FISH. To the best of our knowledge, this is the first study on intraindividual variations of unbalanced spermatozoa from a man with a chromosomal aberration.
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Materials and methods |
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Two sperm samples were obtained at more than 3 months interval (Table I). Semen analysis was performed according to WHO guidelines and morphology was judged using the strict criteria (World Health Organization, 1999). The analysis showed a severe oligozoospermia (5 millions of spermatozoa/ml) and asthenoteratozoospermia (global motility of 40% and teratozoospermia of 84%) in the first sample and asthenoteratozoospermia (global motility of 30% and teratozoospermia of 94%) combined with a very severe oligozoospermia (0.5 million of spermatozoa/ml) in the second sample. Prior to this study, the patient was informed of the investigations and gave his consent.
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The hybridization procedure and analysis have been previously described (Morel et al., 2004b). Briefly, before hybridization, DNA slides were immersed in a jar of 2 x SSC/0.4% NP40 solution for 30 min at 37°C and then immediately passed through an ethanol series (70, 90 and 100%). The denaturation was performed simultaneously on spermatozoa and probes for 1 min at 75°C. The slides were incubated overnight in a dark humidified chamber at 37°C. They were washed for 45 s in 0.4 x SSC/0.3% NP40 at 72°C and 20 s in 2 x SSC/0.1% NP40 at room temperature. Finally, they were counterstained with 4',6-diamidino-2-phenyl-indole (DAPI).
The slides were analyzed using a Zeiss AxioPlan Microscope (Zeiss, Le Pecq, France). Subsequent image acquisition was performed using a CCD camera with Isis (in situ imaging system) (MetaSystems, Altlussheim, Germany).
Statistical analysis
An independent 2 test was used to compare the profiles of segregation between both samples. The level of statistical significance was set at P
0.05.
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Results |
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Discussion |
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Adjacent 2 segregation generated six different combinations and provided data on the frequency of recombination events within the interstitial segments on the derivative chromosomes 9 and 22 (Figure 1). In the absence of recombination, equal frequencies of 23,9, + der(22) and 23, + der(9),22 spermatozoa were obtained. If a recombination event occurred in the interstitial segment of chromosome 9, two other types of gametes were produced: 23,+9,22 and 23,der(9), + der(9),22. If it occurred in the interstitial segment of chromosome 22, gametes with a 23,9,+22 and 23,9,der(22), + der(22) constitution were produced.
An excess of hypohaploid spermatozoa compared to hyperhaploid spermatozoa was seen following the 3:1 segregation. Such an observation was already made in other studies (Rousseaux et al., 1995; Van Hummelen et al., 1997
; Blanco et al., 1998
; Estop et al., 1999
; Oliver-Bonet et al., 2001
; Geneix et al., 2002
). This excess of spermatozoa with 22 chromosomes compared to those with 24 chromosomes could be due to an over-estimate of hypohaploidies. Possible explanations include technical problems involved in the effectiveness of hybridization of the probes, superposition of the signals, size and intensity of the spots which can be different according to the type of probes used (centromeric, telomeric, specific locus). For Blanco et al. (1998)
, the unequal frequencies of the complementary products could be related to differences in viability of the spermatocytes and spermatids according to their chromosomal equipment.
The frequency of gametes exhibiting a chromosomal imbalance was 45.32% and 42.1% in samples 1 and 2 respectively, with the unbalanced spermatozoa resulting from adjacent 1, adjacent 2, and 3:1 segregation in decreasing frequencies. No intraindividual variations in the chromosomally unbalanced spermatozoa frequencies were found between the two sperm samples. This suggests that meiotic segregation is not a random process (Morel et al., 2004b). However, no other study on intraindividual variations in male carriers of a reciprocal translocation has been published. However, four studies have analyzed the meiotic segregation pattern of translocations within families (Estop et al., 1992
; Rousseaux et al., 1995
; Cora et al., 2002
; Morel et al., 2004b
). Similar profiles of meiotic segregation were found in each family, but not between families, confirming that the risks of meiotic imbalances vary primarily according to the nature of the chromosomes involved in the rearrangement (size of the arms, centromere position) and the breakpoints position.
Our results showed that >40% of the spermatozoa were unbalanced. The risk of zygotic chromosomal imbalance related to the presence of the translocation is thus >40% if we accept, as generally done by most teams, that the unbalanced spermatozoa are as fertilizing as the normal or balanced spermatozoa. This evaluation is very important because, recently, Escudero et al. (2003) found a correlation between the percentage of abnormal spermatozoa and that of abnormal embryos from couples in whom the male was a carrier of a translocation. Therefore, meiotic segregation studies should be integrated in the genetic exploration of the infertile man with a translocation to give a personalized risk assessment of unbalanced spermatozoa.
In our study, we found no variations of unbalanced spermatozoa frequencies in two different sperm samples from a 46XY,t(9;22)(q21;q11.2) carrier. Nevertheless, intraindividual variations cannot be excluded in other chromosomal rearrangements. Should our results be confirmed by other studies on different chromosomal translocations, it could imply that a sole semen sample could be sufficient to counsel a patient on the risk of unbalanced offspring.
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References |
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Submitted on January 16, 2004; resubmitted on May 17, 2004; accepted on July 6, 2004.
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