Lack of intraindividual variation of unbalanced spermatozoa frequencies from a 46,XY,t(9;22)(q21;q11.2) carrier: Case report

F. Morel1,2, N. Douet-Guilbert1,2, M.-J. Le Bris2, A. Herry1,2, C. Marchetti3, V. Lefebvre3, B. Delobel4, V. Amice2, J. Amice1,2 and M. De Braekeleer1,2,5

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


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
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The meiotic segregation pattern of 83 men carrying a balanced reciprocal translocation between two autosomes has already been published. Nevertheless, the question of intraindividual variations has not been addressed yet. A 32-year-old patient was found to be a carrier of a t(9;22)(q21;q11.2) during the investigations for a couple with infertility for 3 years. Two sperm samples were obtained at more than 3 months interval. Both sperm samples were analyzed in triple FISH with the D9Z1 and LSI BCR/ABL ES translocation probes. 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 statistically significant difference was found between both segregation profiles. Four studies have analyzed the meiotic segregation pattern of translocations within families; they found similar profiles of meiotic segregation in each family, but not between families. This suggests, along with our results, that meiotic segregation is not a random process. More studies on intraindividual variations are necessary to allow a better understanding of the meiotic behaviour of chromosomal rearrangements and the practical interest of studies of this kind.

Key words: FISH/male infertility/meiotic segregation/reciprocal translocation/spermatozoa


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several studies have reported an increased frequency of chromosomal abnormalities in infertile men and in male partners of couples referred for intracytoplasmic sperm injection (ICSI) (De Braekeleer and Dao, 1991Go; Gekas et al., 2001Go). Balanced reciprocal translocations are among the most frequent structural chromosomal rearrangements found in these men who have a normal phenotype but generally oligozoospermia or even azoospermia.

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., 2004aGo). 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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient's history and cytogenetic analysis
This 32-year-old patient was found to be a carrier of a t(9;22)(q21;q11.2) during the investigations for a couple with infertility for 3 years. His wife had had two miscarriages but no cytogenetic studies were performed on these two losses. A total of six intra-uterine inseminations (IUI) and two ICSI cycles were performed, all without success.

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, 1999Go). 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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Table I. Characteristics of both sperm samples from a 46,XY,t(9;22)(q21;q11.2) carrier

 
Analysis of chromosomal segregation in sperm cells
Both sperm samples of the patient were analyzed in triple FISH with the specific alphoid probe of chromosome 9 (probe D9Z1, spectrum aqua, Abbott, Rungis, France) and LSI BCR/ABL ES translocation probes which are a mixture of the LSI ABL probe (~650 kb, spectrum orange, Abbott) and the LSI BCR probe (~300 kb, spectrum green, Abbott).

The hybridization procedure and analysis have been previously described (Morel et al., 2004bGo). 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 {chi}2 test was used to compare the profiles of segregation between both samples. The level of statistical significance was set at P≤0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 2573 and 1551 spermatozoa, from samples 1 and 2 respectively, were analyzed after triple FISH with the D9Z1 and LSI ABL/BCR probes. In both ejaculates, the majority of the analyzed nuclei showed normal or balanced equipment resulting from alternate segregation during meiosis (Table II). Because of adjacent 1 segregation, 27.09% (sample 1) and 25.21% (sample 2) of the spermatozoa were unbalanced, with equal frequencies of them showing two red and one blue signal (RRB) or two green and one blue signal (GGB).


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Table II. Results of the meiotic segregation pattern in the two different sperm samples from a 46,XY,t(9;22)(q21;q11.2) carrier

 
Adjacent 2 segregation led to 11.39% (sample 1) and 9.34% (sample 2) of the nuclei having an unbalanced chromosomal equipment resulting from six different combinations. 6.02% and 6.58% of spermatozoa, from samples 1 and 2 respectively, segregated in the 3:1 mode whereas 0.58% and 0.64% of the spermatozoa were diploid or segregated in the 4:0 mode. No statistically significant difference was found between both segregation profiles.


    Discussion
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 Abstract
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 Materials and methods
 Results
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In this study, triple FISH using CEP9 and LSI BCR-ABL did not distinguish normal from balanced genotypes, as they displayed the same pattern of fluorescent signals (one red, one green and one blue). Moreover, this same pattern could also be produced by adjacent 1 segregation involving a crossing-over within one of the interstitial segments, as illustrated in Figure 1.



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Figure 1. The various segregation patterns of a t(9;22)(q21;q11.2) translocation.

 
Equal frequencies of both possible 23,der(9) and 23,der(22) combinations were obtained, as expected, by adjacent 1 segregation. However, these combinations could also result from alternate segregation with a recombination within one of the interstitial segments (Figure 1).

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., 1995Go; Van Hummelen et al., 1997Go; Blanco et al., 1998Go; Estop et al., 1999Go; Oliver-Bonet et al., 2001Go; Geneix et al., 2002Go). 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)Go, 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., 2004bGo). 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., 1992Go; Rousseaux et al., 1995Go; Cora et al., 2002Go; Morel et al., 2004bGo). 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)Go 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.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Blanco J, Egozcue J, Clusellas N and Vidal F (1998) FISH on sperm heads allows the analysis of chromosome segregation and interchromosomal effects in carriers of structural rearrangements: results in a translocation carrier, t(5;8)(q33;q13). Cytogenet Cell Genet 83, 275–280.[ISI][Medline]

Cora T, Acar H and Kaynak M (2002) Molecular cytogenetic detection of meiotic segregation patterns in sperm nuclei of carriers of 46,XY,t(15;17)(q21;q25). J Andrology 23, 793–798.[Abstract/Free Full Text]

De Braekeleer M and Dao TN (1991) Cytogenetic studies in male infertility: a review. Hum Reprod 6, 245–250.[ISI][Medline]

Escudero T, Abdelhadi I, Sandalinas M and Munne S (2003) Predictive value of sperm fluorescence in situ hybridization analysis on the outcome of preimplantation genetic diagnosis for translocations. Fertil Steril 79, 1528–1534.[CrossRef][ISI][Medline]

Estop AM, Cieply KM, Munne S and Feingold E (1999) Multicolor fluorescence in situ hybridization analysis of the spermatozoa of a male heterozygous for a reciprocal translocation t(11;22)(q23;q11). Hum Genet 104, 412–417.[CrossRef][ISI][Medline]

Estop AM, Levinson F, Cieply KM and Van Kirk V (1992) The segregation of a translocation t(1;4) in two male carriers heterozygous for the translocation. Hum Genet 89, 425–429.[ISI][Medline]

Gekas J, Thépot F, Turleau C, Siffroi JP, Dadoune JP, Wasels R, Benzacken B and Association des Cytogénéticiens de Langue Française (2001) Chromosomal factors of infertility in candidate couples for ICSI: an equal risk of constitutional aberrations in women and men. Hum Reprod 16, 82–90.[Abstract/Free Full Text]

Geneix A, Schubert B, Force A, Rodet K, Briançon G and Boucher D (2002) Sperm analysis by FISH in a case of t(17;22)(q11;q12) balanced translocation. Hum Reprod 17, 325–331.[Abstract/Free Full Text]

Morel F, Douet-Guilbert N, Le Bris MJ, Herry A, Amice V, Amice J and De Braekeleer M (2004a) Meiotic segregation of translocations during male gametogenesis. Int J Andrology (in press).

Morel F, Douet-Guilbert N, Roux C, Tripogney C, Le Bris MJ, De Braekeleer M and Bresson JL (2004b) Meiotic segregation of a t(7;8)(q11.21;cen) translocation in two carrier brothers. Fertil Steril 81, 682–685.[CrossRef][ISI][Medline]

Oliver-Bonet M, Navarro J, Codina-Pascual M, Carrera M, Egozcue J and Benet I (2001) Meiotic segregation analysis in a t(4;8) carrier: comparison of FISH methods on sperm chromosome metaphases and interphase sperm nuclei. Eur J Hum Genet 9, 395–403.[CrossRef][ISI][Medline]

Rousseaux S, Chevret E, Monteil M, Cozzi J, Pelletier R, Devillard F, Lespinasse J and Sele B (1995) Meiotic segregation in males heterozygote for reciprocal translocations: analysis of sperm nuclei by two and three colour fluorescence in situ hybridization. Cytogenet Cell Genet 71, 240–246.[ISI][Medline]

Van Hummelen P, Manchester D, Lowe X and Wyrobek AJ (1997) Meiotic segregation, recombination, and gamete aneuploidy assessed in a t(1;10)(p22.1;q22.3) reciprocal translocation carrier by three- and four-probe multicolor FISH in sperm. Am J Hum Genet 61, 651–659.[ISI][Medline]

World Health Organization (1999) Laboratory manual for the examination of human semen and semen-cervical mucus interaction. Cambridge University Press, New York, NY.

Submitted on January 16, 2004; resubmitted on May 17, 2004; accepted on July 6, 2004.





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