1 Laboratoire de Cytogénétique, Faculté de Médecine, Place Henri Dunant, 63000 Clermont-Ferrand and 2 Laboratoire de Biologie de la Reproduction et du Développement et CECOS, Hôtel-Dieu, Boulevard Léon Malfreyt, 63058 Clermont-Ferrand, France
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Abstract |
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Key words: balanced translocation/chromosome segregation/FISH/sperm
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Introduction |
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Until recently, the best method for sperm chromosomal analysis was to perform a zona-free hamster egg penetration test which is still now the only way to get a complete analysis of all chromosomes (Spriggs et al., 1992; Guttenbach et al., 1997
). However, although effective, this technique is difficult, time-consuming and expensive. In addition, only a few cells can be studied. This limited number of analysed sperm is not generally enough to provide a statistically meaningful analysis of the entire sperm population. Furthermore, sperm from certain subgroups of infertile men show an impaired capacity to penetrate eggs. The relative ability of genetically abnormal sperm to penetrate hamster eggs has not been extensively studied.
The fluorescence in-situ hybridization (FISH) technique has now been used in several studies for the detection of sperm aneuploidy (Robbins et al., 1993; Downie et al., 1997
; Colls et al., 1998
; Cifuentes et al., 1999
; Giltay et al., 1999
; Lim et al., 1999
; Rives et al., 2000
; Sloter et al., 2000
; Ushijima et al., 2000
). In contrast to the zona-free hamster egg penetration test, FISH allows the study of a high number of sperm and in a faster and more precise way. Thus, the statistical analysis is meaningful in these conditions.
We report here the study of a couple with a history of recurrent miscarriage and infertility. The patient bore a t(17, 22) (q11; q22) balanced translocation in his somatic cells and we studied the sperm cell chromosomes using FISH with specific probes to the particular loci. The main purpose was to evaluate the distribution of this translocation, after meiosis had occurred, in the entire sperm population, in order to give the couple an adapted genetic counselling.
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Materials and methods |
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Sperm were studied from a fresh ejaculate, after processing by migration on a two layer discontinuous Percoll gradient (47.595%) (Sigma-Aldrich, Saint Quentin Fallavier, France). First, the entire semen was washed 4 times by centrifugation in Earle's solution (3 min, 700 g, 20°C). The final pellet was resuspended in 510 µl of Earle's solution and dropped onto clean glass slides. The slides were air-dried and sperm were fixed by alcohol/ether (1/1, V/V, 15 min).
Sperm nuclear decondensation was obtained by plunging the slides into a solution of NaOH 1N for 1min at room temperature. The slides were rinsed in x2 sodium chloride/sodium citrate (x2 SSC, pH = 7). The sperm were then dehydrated in an ethanol dilution series from 70 to 100% and air dried.
DNA probes used for FISH
Three specific probes were used in this study and were purchased from Vysis (Vysis Inc., Voisins, Le Bretonneux, France). Her2/neu is located on 17q11.2-q12 and labelled with Spectrum OrangeTM. Two pericentromeric probes of the chromosome 17 (CEP 17) were used: one is labelled with Spectrum GreenTM and the other one with Spectrum OrangeTM. The combination of these two labelling gave a yellow colour. Bcr is located on 22q11.2 and is labelled with Spectrum GreenTM. Thus a three-colour FISH was performed. These probes can identify: (i) a locus from 17 pter to 17 q11: CEP 17 probe (yellow spot), on the centromere of the chromosome 17; (ii) a locus from 17 q11 to 17 qter on the long arm of the chromosome 17: Her2/neu probe (red spot), under the breakpoint of this chromosome; (iii) a locus from 22 pter to 22 q11 on the long arm of the chromosome 22: Bcr probe (green spot) and above the breakpoint of this chromosome.
FISH
The probes were denatured for 2 min at 73°C. The hybridization mixture (1 µl of each probe, 1 µl H2O, 7 µl of hybridization solution) was applied to each slide and covered with a coverslip 20x40 mm (Labonord, Templemars, France). This hybridization mixture is a solution of dextran sulphate, formamide in SSC (pH = 7). Each slide was then sealed with Rubber Cement (Kleertak; Mecanorama, France) before hybridization was carried out overnight in a moist chamber at 37°C. After hybridization, the slides were washed for 3 min in a solution of x0.4 SSC at 73°C and a second time for 30 s in a solution of x2 SSC/0.1% Nonidet P40. After the final wash, slides were air dried in the dark. The slides were counterstained with a solution of 4',6-diamidino-2-phenylindole.2HCl (DAPI II; Vysis Inc.) diluted in an antifade mounting medium (Vectashield; Valbiotech, France).
Scoring criteria and data collection
The slides were then examined with an epifluorescence microscope (DMRB®, Leica, Germany) at a magnification of x1000. The nuclear decondensation resulted in a sperm morphology similar to that already published. (Martini et al., 1995), i.e. each cell seen under the microscope could be clearly identified as a mature spermatozoon because of its structure and could be distinguished from other cells. Only clearly identifiable sperm were scored and only the spots appearing on the sperm head were counted. Retained spots were clearly seen and of equivalent size. Two spots were separated by at least the size of one of them, and if not, they were not taken into account.
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Results |
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Patient t(17; 22) (q11; q12)
Nineteen percent of the analysed sperm were balanced (alternate segregation): der(22), der(17) or 17, 22 bearing sperm. Due to the probes we used, the FISH technique did not allow the distinction between these two patterns.
All other sperm (81%) were unbalanced and their distribution was unequal among each of all the theoretical possibilities (see Table I).
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All the results are presented in Table I. Fewer than 2% of all the spots scored were unexplained.
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Discussion |
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No other cases with similar breakpoints were found in other databases, such as HC FORUM (Dr O.Cohen, Grenoble, France).
In our study, 20% of the sperm bore a normal or balanced genetic complement. This is in agreement with the patient's history considering that he naturally conceived a healthy boy. In addition, his father, bearing the same balanced translocation, also had two naturally conceived healthy children. His sister had no history of miscarriages and also naturally conceived two healthy children. Unfortunately, her karyotype was unknown. The t(17; 22) (q11; q12) patient's severe oligozoospermia could possibly be a consequence of his karyotype abnormality. However, his cryptorchidism was the most likely cause of this infertility. His weak testicular function can be related to either or both abnormalities.
The history of four miscarriages could clearly be explained by unbalanced translocation bearing sperm: 80% of the sperm scored had an unbalanced karyotype. However, it seems surprising that so many abnormal sperm can reach complete maturation. One could expect that the major part of the sperm population would be represented by normal sperm regarding their karyotypes, but in our study, they only represented 20% of the entire population. As a hypothesis, we propose that all mis-segregations were much more common than the normal segregations; thus, even if some did not reach complete maturation because of their abnormal karyotype, a large part of them could finally pass through the spermatogenesis control check points.
The unequal rates between unbalanced translocation and its complement (for example: 17, der(22): 3.2% and der(17), 22: 9.7%) could possibly be explained by the quality and the quantity of the genes involved in this translocation: in some cases missing genes could be more important to reach complete maturation than others.
The rate of the 3:1 segregation was another astonishing point. Sperm bearing one chromosome were much more frequent than sperm bearing three chromosomes: 20% bore der(17) only versus 0.1% bore 17, der(22), 22 for example. A hybridization failure could explain these results. However only 3.3% of the control sperm showed abnormal results, which represent any abnormality, including hybridization failure. Nearly 45% of the patient's sperm showed only one hybridization symbol; thus this high percentage is not only related to hybridization failure. In addition, this particular point seemed in opposition with what is known about the conceptus products: autosomal monosomia, even partial, are very lethal. However, this fact could correspond to a recruitment bias: these kind of monosomia might be so lethal that they stopped their development very early and resulted in very early abortions which were not noticed.
According to Durak et al. (Durak et al., 1999), who studied sperm from two translocation carriers, t(4; 8) (p15; p12) and t(15; 22) (q23; q13.2), the 3:1 segregation was rare and they only considered 2:2 segregation in their study. Therefore what they suggested cannot be compared with our results. A study of a balanced translocation t(2;3) (p24;q26) (Martin, 1994
) where, in these sperm, the segregation 3:1 was only present at the rate of 1.2 versus 50% in ours and even 58% if we also consider the 3:1 segregation followed by a second meiosis abnormality. The alternate segregation, adjacent 1 segregation and adjacent 2 segregation were at the rate of 55.4, 3.6 and 7.2% versus 20, 13 and 6% in ours respectively. Other results (Estop et al., 1997
; Hummelen et al., 1997
; Martini et al., 1998
; Mercier et al., 1998
; Honda et al., 1999
) were in agreement with those of Martin (Martin, 1994
). In contrast, in a case of another reciprocal translocation t(11; 22) described by Estop et al. (Estop et al., 1999
), the 3:1 segregation was found at a higher rate (40.1%) than the adjacent 1 (17.6%) and adjacent 2 (12.5%) segregation. Furthermore, in a case of (5; 7) translocation, the rate of 3:1 segregation was very close to the adjacent 2 segregation rate: 17 versus 16.6% (Estop et al., 1995
). These authors considered these results as unusual.
Finally, according to Honda et al. (Honda et al., 2000), meiotic segregation analysis of a Robertsonian translocation carrier revealed a rate of unbalanced sperm of 88.4%, significantly different from the theoretical frequency of 33.3%.
Hence, we believe that in these cases of balanced translocation, no general features can be drawn. Each one must be considered as a particular case. The chromosomes and the genes implied that their breakpoints are likely to be one of the important points, although it is currently not possible to elucidate this fully. Thus, genetic counselling remains an important step before assisted reproductive technologies, if needed, are performed for these couples. In addition, our results show that genetic counselling must be done with care, in view of the different conclusions that have been reached by various investigators.
To date, the history of recurrent miscarriages with a clear cytogenetic origin is relevant for pre-implantation genetic diagnosis. This kind of assisted reproductive technology could help the couple to have the desired pregnancy with fewer risks than for a naturally conceived pregnancy. FISH techniques can successfully be used, giving an individual result on very few embryonic cells and allowing the medical team to select an embryo or embryos devoid of the investigated chromosomal abnormalities.
In conclusion, our study shows that a higher incidence of the nullisomia compared with the disomia is found in the sperm of a t(17; 22) (q11; q12) balanced translocation carrier. This is not in agreement with the well-known higher incidence of trisomia than of monosomia (except for the gonosomes) in ongoingpregnancies.
Hence, as no general features can be drawn for the distribution of the sperm karyotypes of balanced translocation bearing men, the FISH technique is an important way to estimate their distribution and to help the medical team to adapt genetic counselling.
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Acknowledgements |
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Notes |
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Submitted on February 22, 2001; resubmitted on July 2, 2001
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References |
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accepted on October 1, 2001.