Mol. Hum. Reprod. Advance Access originally published online on October 3, 2007
Molecular Human Reproduction 2007 13(10):751-756; doi:10.1093/molehr/gam048
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Breakpoint characterization: a new approach for segregation analysis of paracentric inversion in human sperm
1 INSERM U847, Montpellier, France 2Department of Reproduction Biology, CHU Montpellier, Montpellier, France 3Department of Medical Genetics, CHU Montpellier, Montpellier, France 4 Institute of Human Genetics and Anthropology, Jena, Germany 5 Laboratory of Cytogenetics, CHR Chambery, France
6 Correspondence address. Department of Medicine and Biology of Reproduction, Arnaud de Villeneuve Hospital, CHRU Montpellier, 371, avenue du Doyen Gaston Giraud, 34295 Montpellier Cedex 5, France. Tel: +33 4 67 33 64 04; Fax: +33 4 67 33 62 90; E-mail: f-pellestor{at}chu-montpellier.fr
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Abstract |
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Paracentric inversions (PAI) are structural chromosomal rearrangements generally considered to be harmless. Nevertheless, cases of viable recombinants have been reported, indicating the interest of studying the meiotic behaviour of these chromosomal abnormalities. To date, the few studies reported have been performed using either the human–hamster fertilization system or fluorescence in situ hybridization with centromeric or telomeric DNA probes. In order to improve the assessment of meiotic segregation in PAI, we present a new strategy based on the use of bacterial artificial chromosome (BAC) probes which allow a precise localization of chromosome breakpoints and the identification of all meiotic products in human sperm. Sperm samples from carriers of an inv(5) and an inv(14) were used to test this new high-resolution procedure.
Key words: paracentric inversion/FISH/breakpoint characterization/segregation
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Introduction |
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Paracentric inversions (PAI) are chromosomal rearrangements that occur after two breaks on a chromosomal arm followed by 180° rotation of the chromosomal segment and reunion of the segment. In humans, the incidence of PAIs is estimated to be 0.1–0.5% (Fryns et al., 1986; Pettenati et al., 1995), and they are generally considered to be harmless chromosomal rearrangements without phenotypic consequences. However, during meiosis the occurrence of crossing over within the inverted segment may lead to the production of chromosomally abnormal gametes, through the formation of either acentric or dicentric recombinant chromatids, in addition to normal and inverted chromatids. The dicentric chromatid or dicentric bridge can break leading to duplication or deficiency (Madan, 1995). In addition to meiotic crossing over within the inversion loop, a U-loop recombination mechanism can also generate unbalanced gametes when a crossover at the entry points of the loop produces either a duplicated or deleted chromatid (Feldman et al., 1993; Mitchell et al., 1994). Phelan et al. (1993) proposed another mechanism of breakage and reunion of the sister chromatids which generates duplicated, deficient and normal chromatids. The mechanisms by which chromosome imbalances are generated from PAI are schematized in Fig. 1. The risk of liveborn recombinant offspring in paracentric inversion carriers is low, but such births have been documented (Worsham et al., 1989; Phelan et al., 1993; Mitchell et al., 1994; Lefort et al., 2002). Spontaneous abortion and infertility have also been reported among paracentric inversion carriers (Madan, 1995, Madan and Nieuwint, 2002), indicating that recombination in PAI could arise more frequently than suspected from liveborn data.
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To date, very few segregation analysis studies have been performed for PAI. Two cases have been studied using human sperm/hamster oocyte method (Martin, 1986, 1999). Although this method gave direct information about both numerical and structural chromosomal abnormalities, the procedure remained labour-intensive, time-consuming, and provided data on small numbers of sperm nuclei (94 and 120). The fluorescence in situ hybridization (FISH) technique has offered an alternative approach for investigating the meiotic segregation of chromosomal rearrangements in human sperm. Only four PAIs have been investigated using sperm FISH procedure (Devine et al., 2000; Anton et al., 2006; Vialard et al., 2007). In one case, an inv(2)(q14.2q24.3) was analyzed using a combination of centromeric
-satellite DNA probes (Devine et al., 2000), and in the three other cases (inv(4)(p14p15.3), inv(11)(q13.2q14.3) and inv(12)(q15q24.1) were studied with telomeric probes (Anton et al., 2006; Vialard et al., 2007). These multicolour FISH analyses resulted in data on large samples of human sperm (from 496 to 8158), but they did not allow one to distinguish all the meiotic segregants, especially those generated through U-loop formation or breakage/reunion mechanism of sister chromatids. The accurate identification of all the possible types of meiotic products is important not only for the genetic and reproductive counselling of the carriers, but also for our understanding of the mechanisms of chromosomal inversion formation that remain unclear due to lack of molecular studies concerning their breakpoints.
In order to accurately localize inversion breakpoints and determine the segregation patterns of chromosomal inversions, we developed a high-resolution mapping strategy based on the use of human specific bacterial artificial chromosomes (BACs) spanning the chromosome breakpoint. For the first time, we present the application of this high-resolution mapping approach on human sperm for the segregation analysis of two PAI.
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Materials and methods |
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Preparation of metaphase chromosome spreads
Initial cytogenetic studies of the paracentric inversion carriers were performed on peripheral blood lymphocytes according to standard procedures for G- and R-banding. The chromosomal preparations were also used for testing probes and optimizing the in situ hybridization conditions. The slides were kept at –20°C until their use for FISH assays.
BAC clone selection and preparation
BAC clones were chosen within specific bands adjacent to the chromosomal breakpoints or spanning them, as determined by conventional cytogenetic analysis. The BAC clones were selected using NCBI database (Wellcome trust genome campus, Hinxton, Cambridge) and Ensembl database (http://www.ensembl.org/Homosapiens/index.html). Clones were requested from The Department of Genetics and Microbiology (University of Bari, Bari, Italy) and The Wellcome trust Sanger institute (Wellcome trust genome campus, Hinxton, Cambridge) or purchased from Invitrogen Ltd (Paisley, Scotland). Clones were chosen from an interval of 
500 Kb around the chromosomal breakpoints, and ordered proximal or distal to the breakpoints. The selected BACs were mapped until clones that spanned breakpoints were identified. The BAC clones in contiguity to either side of the BACs spanning the breakpoint were used to prepare the contig probes. The BAC DNA was isolated using Nucleobond DNA isolation kit (Macherey-Nagel Gmbh, Duren, Germany), labelled with digoxigenin-11-dUTP or biotin-16-dUTP, using commercially available labelling kit (Invitrogen, Carlsbad, CA) and diluted in a 10 µl hybridization mix (60% formamide).
FISH on metaphase preparations
Before FISH procedure, the slides were immersed for 5 min in a pepsin solution (50 ng/ml in 0.01 M HCl) pre-warmed to 37°C, and washed 5 min in x1 PBS. The slides were then treated with formaldehyde (1%), dehydrated through an ethanol series (70, 90 and 100%) and air-dried. The slides were denatured for 3 min at 73°C in 70% formamide/x2 SSC, pH 7.0, dehydrated in 70, 80 and 100% ethanol for 2 min each step, and allowed to air-dry.
The probes were denatured by incubating at 75°C for 7 min and applied to the denatured slides, covered with coverslips, and sealed with rubber cement. The hybridization was allowed to proceed overnight at 37°C in a humidified chamber. The slides were then washed twice in 50% formamide, x2 SSC, pH 7.0, at 46°C for 7 min, followed by two washes in x2 SSC at 46°C for 7 min. The detection was performed using either anti-dig FITC (2 µg/ml) or streptavidin Rhodamine (5 µg/ml), followed by two washes in x2 SSC/0.1% Tween 20 at 45°C for 5 min. Ultimately, slides were mounted with DAPI (100 ng/ml) in Vectashield antifade solution. The slide were observed under a Leica DMRA 2 fluorescence microscope equipped with adequate filter sets and coupled to a cooled CCD camera with the Isis software (Metasystems, Germany).
Sperm FISH procedure
Sperm samples were collected in a sterile container after 3 days of sexual abstinence. After liquefaction at room temperature, the samples were washed three times in x1 PBS by centrifugation (5 min at 2000 rpm). The final pellets were fixed for 1 h in fresh fixative (methanol: glacial acetic acid 3:1) at –20°C. The sperm suspensions were then dropped onto clean microscopic slides and air-dried. Slides were aged 2 days at room temperature before use for FISH reactions.
Before in situ labelling assays, the slides were treated with pepsin (50 ng/ml in 0.01 M HCl) pre-warmed at 37°C for 10 min, followed by a treatment with x1 PBS for 5 min. The slides were then dehydrated through an ethanol series (70, 90 and 100%, 2 min each step) and air-dried. The sperm nucleus decondensation and DNA denaturation was performed by slide incubation in 0.5 N NaOH solution at room temperature for 8–12 min, followed by a wash in x2 SSC, a dehydration through an ethanol series and an immersion in 70% formamide/x2 SSC solution 3 min at 73°C. Finally, the slides were washed in x2 SSC, dehydrated through an ethanol series and air-dried.
The probes were denatured separately for 7 min at 75°C in a water bath. Each probe mix was applied to the denatured slides and slides were covered with coverslips, sealed with rubber cement and hybridized overnight in a dark, moist chamber at 37°C. After hybridization, coverslips were gently removed and the slides were washed twice in 50% formamide/50% x2 SSC solution at 46°C for 7 min, then twice in x2 SSC at 46°C for 7 min. The detection was performed using either anti-dig FITC (2 µg/ml) or streptavidin–Rhodamine (5 µg/ml), followed by two washes in x2 SSC/0.1% Tween 20 at 45°C for 5 min. The slides were mounted with DAPI (100 ng/ml) in Vectashield antifade solution, and observed under a Leica DMRA 2 fluorescence microscope equipped with adequate filter sets. Only individual and well-delineated sperm nuclei were taken into consideration for the screening. Overlapping sperm nuclei, disrupted nuclei or large nuclei with diffuse signals were not considered.
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Results |
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Characterization of breakpoints
The selected BAC probes were hybridized to the inversion carrier metaphase spreads in order to classify them as either proximal or distal to the breakpoints, in order to narrow down the critical chromosomal location of the inversion breakpoints, and finally identify the clones spanning the breakpoints.
Case 1: inv(5)(q13.3q33.1)
The breakpoint at 5q13.3 was characterized using eight BAC clones (Table 1) of the 5q13.3 region (Fig. 2). The breakpoint was mapped to chromosome position 74998605–75170876 bp at 5q13.3. The other breakpoint of the 5q33.1 region was characterized using seven BAC clones (Table 1) of the region 5q33.1 (Fig. 2). The breakpoint was mapped to chromosome position 150265542–150418860 bp. The BACs spanning the breakpoints of 5q13.3 and 5q33.1 regions were RP11-179H5 and RP11-12A04, respectively. As shown in Fig. 3A, the BACs, RP11-179H5 labelled with FITC and RP11-12A04 labelled with Rhodamine, gave clear split signals on the inverted chromosome 5. These BACs were used in sperm for identifying meiotic segregation patterns. In the sperm harbouring the inverted chromosome, BACs gave specific patterns with either two yellow signals or two red and two green signals, corresponding, respectively, to the perfect or the incomplete co-localization of BAC clones (Fig. 3B). In contrast, the sperm with a normal chromosome 5 gave one red and one green signal pattern (Fig. 3C).
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The analysis of 128 sperm nuclei gave the following segregation pattern: 60 nuclei were normal, 59 had the inversion and 9 had a duplicated or deficient for part of chromosome 5.
Case 2: inv(14)(q23.2q32.1)
The breakpoint at 14q23.2 was characterized using six BAC clones (Table 1) of the region 14q23.1–q23.3 (Fig. 2), a region of 8 Mb. The breakpoint was mapped to chromosome position 63768477–63912834 bp at 14q23.2. The other breakpoint of the 14q32.13 region was characterized using eight BAC clones (Table 1) of the 6 Mb region 14q32.11–14q32.13 (Fig. 2). The breakpoint was mapped to chromosome position 94090988–94303514. The BACs spanning the breakpoints of 14q23.2 and 14q32.13 regions were RP11-712C19 and RP11-986E7, respectively. As shown in Fig. 3D, both of the BACs, RP11-712C19 labelled with FITC and RP11-986E7 labelled with Rhodamine, gave clear split signals on the inverted chromosome 14. When tested upon sperm nuclei, the BACs gave either two yellow or two red and two green signals in the sperm harbouring the inverted chromosome 14 (Fig. 3E), and one red and one green signal pattern in sperm with normal chromosome 14 (Fig. 3F).
We analysed 250 sperm nuclei and found 121 normal,119 inverted and 10 with duplicated or deficient for part of chromosome 14.
Sperm FISH assays
The hybridization of the selected clones on sperm gave efficient results with a satisfactory visualization of labelling signals, as illustrated in Fig. 3B–E. The fluorescent signals were always uniform and bright, and thus easy to distinguish and score. The identification of the signals were never ambiguous because of the high signal/background ratio, indicating that this procedure could allow the identification of all meiotic products in sperm samples of inversion carriers. Thus, in the present study, sperm nuclei displaying inverted segments were clearly identified, unlike in previous sperm FISH studies using centromeric or telomeric probes.
In each case, the hybridization efficiency was estimated by scoring 100 sperm nuclei per slide. For the inv(5), 91% of scored sperm nuclei were labelled. For the inv(14), this increased to 97%.
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Discussion |
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The fluorescence in situ hybridization (FISH) technique has become the method of choice for the in situ detection of structural chromosomal rearrangements in isolated cells and the study of their meiotic segregation, as illustrated by its intensive use for preimplantation genetic diagnosis (PGD) of translocations (Harper et al., 2006) and the increased number of reports on sperm FISH analysis of Robertsonian or reciprocal translocations (Benet et al., 2005; Roux et al., 2005). On the other hand, the meiotic behaviour of chromosomal inversions has barely been explored. To date, only 16 pericentric inversions and 4 PAI have been investigated by sperm FISH (Anton et al. 2005; Morel et al., 2007; Vialard et al., 2007). This could be linked to the low risk of imbalances generally attributed to the inversions, but also to the technical difficulties inherent to the precise analysis of their meiotic segregation. All the published sperm FISH studies of inversions were performed using either centromeric or telomeric DNA probes which do not allow the in situ identification of all possible meiotic products. Thus, in the case of PAI, three meiotic mechanisms have been described to explain the formation of recombinant chromosomes. However, only normal, dicentric and acentric fragments can be visualized in situ by centromeric or telomeric probes whereas inverted chromosomes and other abnormal meiotic outcomes are also generated through meiosis as illustrated in Fig. 4.
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The objective of this study was to design a new approach allowing one to accurately determine the meiotic behaviour of specific inversions. Owing to the chromosomal nature of inversions, exact breakpoint localizations are required for the identification of all meiotic segregants. In this study, we demonstrated that the BAC clones spanning the breakpoint can be efficiently used as probes to directly determine the segregation of chromosomal inversions in human sperm. Of course, the molecular cytogenetic mapping of breakpoints is a labour-intensive procedure which takes longer than the generation of an in situ signal using commercially available probes. However, as a result of this effort, the precise position of breakpoints can be determined and a complete segregation analysis can be performed in sperm. Such data can significantly improve the genetic counselling provided to inversion carriers, especially because of the wide variability observed in the production of recombinant gametes (from 0 to 38%) and the documented occurrence of viable recombinant offsprings (Pettenati et al., 1995; Lefort et al., 2002). Thus, both the chromosomes 5 and 14 analysed in this study are among the most frequently implicated ones in paracentric inversion (Madan, 1995). By extension, this approach could be used in preimplantation genetic diagnosis after polar body or blastomere biopsy. Patients would greatly benefit from efficient procedures for identification of all possible segregants since the possibility of unpredictable unbalanced chromosome products exists. For unique cell analysis, it is crucial to get probes with the highest hybridization efficiency. Consequently, probe optimization is an important step, and in this way, the successful testing of BAC probes on difficult materials such as human sperm nuclei could be considered as a good indicator of efficiency and reliability of the procedure. Breakpoint mapping of chromosomal inversions could also be done by the multicolour banding (MCB) technique introduced by Liehr et al. (2002). The accuracy of this method based on the use of sets of region-specific micro-dissection derived libraries has been well documented on somatic cells. As pointed out by Weise et al. (2003), MCB can be efficiently used for roughly mapping breakpoints or narrowing critical regions, but precise localization of breakpoints required subsequent BAC mapping. In addition, a major limitation for the use of MCB on human sperm and unique blastomeres is the risk of overlapping of the fluorescent signals and subsequent inaccurate interpretation. In the case of using MCB on human sperm, the limitations can be overcome by combination of MCB and suspension (S)-FISH, a new procedure for performing FISH in suspension with 3D analysis of interphase nuclei (Steinhaeuser et al., 2002).
Improvement of the mapping of inversion breakpoints is also of great interest for a better understanding of the molecular mechanisms of inversion formation. There is some new evidence emerging from molecular studies that certain particular genomic regions might be prone to breakage and recombination. The sequences around the inversion breakpoints are frequently enriched for interspersed repetitive elements and/or co-localized with fragiles sites which might not only promote instability and double strand breaks (Stankiewicz and Lupski, 2002; Gilling et al., 2006), but also display large sequence homology (Schmidt et al., 2005). In addition, PAI are not always distinguishable from insertion, which is a three-break rearrangement. Whereas the risk for a paracentric inversion carrier of having a viable recombinant is expected to be low, the general risk for an insertion carrier is 15% (Madan and Nieuwint, 2002). In this case, only the use of region-specific probes like BAC probes can distinguish between inversion and insertion.
In conclusion, the precise analysis of inversion breakpoints is important both for genetic analysis and counselling of inversion carriers, as well as for a better knowledge of the meiotic mechanisms of formation and recombination of inversion. This study thus illustrates the usefulness of a BAC clone based strategy.
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Acknowledgements |
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We gratefully thank Professor Mariano Rocchi (University of Bari, Italy) and Wellcome Trust Sanger Institute for providing the BAC clones used in this study. This work was partially supported by Organon France and Ferring Pharmaceuticals. The authors also thank Deutsche Forschungs Gemeinschaft (436 ARM 17/11/06, LI 820/15-1) for its support.
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References |
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Anton E, Vidal F, Egozcue J, Blanco J. Genetic reproductive risk in inversion carriers. Fertil Steril (2006) 85:661–666.[CrossRef][ISI][Medline]
Benet J, Oliver-Bonet M, Cifuentes P, Templado C, Navarro J. Segregation of chromosomes in sperm of reciprocal translocation carriers: a review. Cytogenet Genome Res (2005) 111:281–290.[CrossRef][ISI][Medline]
Devine DH, Whitman-Elia G, Best RG, Edwards JG. Paternal paracentric inversion of chromosome 2: a possible association with recurrent pregnancy loss and infertility. J Assist Reprod Genet (2000) 17:293–296.[CrossRef][ISI][Medline]
Feldman GL, Weiss L, Phelan M, Shroer RJ, Van Dyke DL. Inverted duplication of 8p: ten new patients and review of the literature. Am J Med Genet (1993) 47:482–486.[CrossRef][ISI][Medline]
Fryns JP, Kleczkowska A, Berghe H van den. Paracentric inversions in man. Hum Genet (1986) 73:205–213.[CrossRef][ISI][Medline]
Gilling M, Dullienger JS, Gesk S, Metze-Heidemann S, Siebert R, Meyer T, Brondum-Nielsen K, Tommerup N, Ropers HH, Tummer Z, et al. Breakpoint cloning and haplotype analysis indicate a single origin of the common Inv (10) (p11.2q21.2) mutation among northern Europeans. Am J Hum Genet (2006) 78:878–883.[CrossRef][ISI][Medline]
Harper JC, Boelaert K, Geraedts J, Harton G, Kearns WG, Moutou C, Muntjewerff N, Repping S, SenGupta S, Scriven PN, et al. ESHRE PGD Consortium data collection V: cycles from January to December 2002 with pregnancy follow-up to October 2003. Hum Reprod (2006) 21:3–21.
Lefort G, Blanchet P, Belgrade N, Rivier F, Chaze AM, Sarda P, Demaille J, Pellestor F. Stable dicentric duplication-deficiency chromosome 14 resulting from crossing-over within a maternal paracentric inversion. Am J Hum Genet (2002) 113:333–338.
Liehr T, Heller A, Starke H, Rubustov N, Trifonov V, Mrasek K, Weise A, Kuechler A, Claussen U. Microdissection based high resolution multicolour banding for all 24 human chromosomes. Int J Mol Med (2002) 9:335–339.[ISI][Medline]
Madan K. Paracentric inversions: a reivew. Hum Genet (1995) 96:503–515.[ISI][Medline]
Madan K, Nieuwint AW. Reproductive risks for paracentric inversion heterozygotes: inversion or insertion? That is the question. Am J Med Genet (2002) 107:340–343.[CrossRef][ISI][Medline]
Martin RH. Sperm chromosome analysis in a man heterozygous for a paracentric inversion of chromosome 7 (q11q22). Hum Genet (1986) 73:97–100.[CrossRef][ISI][Medline]
Martin RH. Sperm chromosome analysis in a man heterozygous for a paracentric inversion of chromosome 14 (q24.1q32.1). Am J Hum Genet (1999) 64:1480–1484.[CrossRef][ISI][Medline]
Mitchell JJ, Vekemans M, Luscombe S. U-type exchange in a paracentric inversion as a possible mechanism of origin of an inverted tandem duplication of chromosome 8. Am J Med Genet (1994) 49:384–387.[CrossRef][ISI][Medline]
Morel F, Laudier B, Guerif F, Couet ML, Royere D, Roux C, Bersson JL, Amice V, De-Braekleer M, Douet-Guilbert N. Meiotic segregation analysis in spermatozoa of pericentric inversion carrriers using fluorescence in situ hybridizatin. Hum Reprod (2007) 22:136–141.
Pettenati MJ, Rao PN, Phelan MC. Paracentric inversions in humans: a review of 446 paracentric inversions with presentation of 120 new cases. Am J Med Genet (1995) 55:171–187.[CrossRef][ISI][Medline]
Phelan MC, Stevenson RE, Anderson EV Jr. Recombinant chromosome 9 possibly derived from breakage and reunion of sister chromatids within a paracentric inversion loop. Am J Med Genet 15 (1993) 46:304–308.[CrossRef][ISI][Medline]
Roux C, Tripogney C, Morel F, Joanne C, Fellmann F, Claveguin MC, Bresson JL. Segregation of chromosomes in sperm of Robertsonian translocation carriers. Cytogenet Genome Res (2005) 111:291–296. Review.[CrossRef][ISI][Medline]
Schmidt S, Claussen U, Liehr T, Weise A. Evolution versus constitution: differences in chromosomal inversion. Hum Genet (2005) 117:213–219.[CrossRef][ISI][Medline]
Stankiewicz P, Lupski JR. Molecular-evolutionary mechanisms for genomic disorders. Curr Opin Genet Dev (2002) 12:312–319.[CrossRef][ISI][Medline]
Steinhaueuser U, Starke H, Nietzel A, Lindenau J, Ulmann P, Claussen U, Liehr T. Suspension (S)-FISH, a new technique for interphase nuclei. J Histochem Cytochem (2002) 50:1697–1698.
Vialard F, Delanete A, Clement P, Simon-Bouy B, Aubriot FX, Selva J. Sperm chromosome analysis in two cases of paracentric inversion. Fertil Steril (2007) 87:418.e1–415.
Weise A, Heller A, Mrasek K, Kuechler A, Pool-Zobel BL, Clausen U, Liehr T. Multitude multicolour chromosome banding (mMCB) – a comprehensive one-step multicolour FISH banding technique. Cytogenet Genome Res (2003) 103:34–39.[CrossRef][ISI][Medline]
Submitted on June 26, 2007; accepted on July 11, 2007.
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