Sperm-FISH analysis in a pericentric chromosome 1 inversion, 46,XY,inv(1)(p22q42), associated with infertility

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1 Molecular Human Reproduction Vol.13, No.1 pp , 2007 Advance Access publication October 31, 2006 doi: /molehr/gal094 Sperm-FISH analysis in a pericentric chromosome 1 inversion, 46,XY,inv(1)(p22q42), associated with infertility S.Chantot-Bastaraud 1, C.Ravel 1, I.Berthaut 1, K.McElreavey 2, P.Bouchard 3, J.Mandelbaum 1 and J.P.Siffroi 1,4 1 AP-HP, Hôpital Tenon, Service d Histologie, Biologie de la Reproduction et Cytogénétique, Université Pierre et Marie Curie-Paris6, EA 1533, 2 Unité de Reproduction, Fertilité et Développement, Département de Biologie du Développement, Institut Pasteur and 3 AP-HP, Hôpital Saint-Antoine, Service d Endocrinologie, Université Pierre et Marie Curie-Paris6, EA 1533, Paris, France 4 To whom correspondence should be addressed at: Hôpital Tenon, Service d Histologie, Biologie de la Reproduction et Cytogénétique, 4 rue de la Chine, Paris, France. jean-pierre.siffroi@tnn.aphp.fr No phenotypic effect is observed in most inversion heterozygotes. However, reproductive risks may occur in the form of infertility, spontaneous abortions or chromosomally unbalanced children as a consequence of meiotic recombination between inverted and non-inverted chromosomes. An odd number of crossovers within the inverted segment results in gametes bearing recombinant chromosomes with a duplication of the region outside of the inversion segment of one arm and a deletion of the terminal segment of the other arm [dup(p)/del(q) and del(p)/dup(q)]. Using fluorescence in-situ hybridization (FISH), the chromosome segregation of a pericentric inversion of chromosome 1 was studied in spermatozoa of a inv(1)(p22q42) heterozygous carrier. Three-colour FISH was performed on sperm samples using a probe mixture consisting of chromosome 1p telomere-specific probe, chromosome 1q telomere-specific probe and chromosome 18 centromere-specific alpha satellite DNA probe. The frequency of the non-recombinant product was 80.1%. The frequencies of the two types of recombinants carrying a duplication of the short arm and a deletion of the long arm, and vice versa, were respectively 7.6 and 7.2%, and these frequencies were not statistically significant from the expected ratio of 1:1. Sperm-FISH allows the further understanding of segregation patterns and their effect on reproductive failure and allows an accurate genetic counselling. Key words: chromosome 1/meiotic segregation/pericentric inversion/primary infertility/sperm-fish Introduction Pericentric inversions are structural intrachromosomal rearrangements resulting from two breaks on both sides of the centromere followed by the 180 rotation of the chromatin segment between these breaks. Most of them affect the chromosome 2 pericentric region, the heterochromatic regions of chromosomes 1, 9, 16 or the Y chromosome, all of them being considered as non-pathological polymorphisms with variable incidence values according to ethnical origin. As a consequence, the frequency of pericentric inversions in the general population vary from (Ravel et al., 2006) to 0.34% (Nielsen and Wohlert, 1991) or even from 1 to 2% (de la Chapelle et al., 1974; Kaiser, 1984) depending on the type of inversion reported. Most pericentric inversion carriers have a normal phenotype and usually a normal fertility. However, some of them can have some difficulties in conceiving a normal offspring because of the production of chromosomally unbalanced gametes following abnormal meiotic events. Indeed, when the pairing of an inverted chromosome with its normal homologue implies the formation of an inversion loop, the occurrence of an odd number of genetic recombinations within the loop leads to the formation of two abnormal chromosomes that are duplicated and deleted, respectively, for the regions outside the inversion (Figure 1). According to the size of the unbalanced chromosomal segment, such recombinant chromosomes lead to either spontaneous abortions or abnormal children. Contrary to chromosomal translocations, very few meiotic segregations of pericentric inversions have been described using either the in vitro penetration of hamster oocytes test (hamster test) or the fluorescence in-situ hybridization (FISH) (Anton et al., 2005, 2006; Malan et al., 2006). Therefore, the individual evaluation of the recombination risk in each inversion carrier is important for providing an accurate genetic counselling and allowing an appropriate management of pregnancies. In this study, FISH analysis was used to estimate the proportion of recombinant chromosomes in sperm from a heterozygous inversion carrier of inv(1)(p22q42). Materials and methods Patient A 30-year-old African man was referred to the laboratory for primary infertility. Sperm analysis was performed according to WHO s criteria and revealed abnormal sperm parameters, including a low volume of ejaculate (0.8 ml), an oligozoospermia (5 9 million spermatozoa/ml) and a teratozoospermia with 96% of spermatozoa showing an abnormal shape. The subject gave informed consent for genetic investigations. Karyotype was performed using standard methods, and chromosomes were analysed after G and R banding. It revealed a large pericentric inversion of one chromosome 1, 46,XY,inv(1)(p22q42) (Figure 2). Y chromosome screening for AZF microdeletions was normal. No familial data regarding the inversion, like parental karyotypes, were available, but pedigree did not reveal any history of congenital malformations, miscarriages or mental retardation. The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org 55

2 S.Chantot-Bastaraud et al. Cambridge, UK), and the chromosome 1q telomere-specific probe, labelled with Texas Red (Aquarius, Cytocell Technologies), were hybridized simultaneously. A chromosome 18 centromere-specific alpha satellite DNA probe labelled with Spectrum Aqua (Abbott, Vysis-Abbott, Downer s Grove, IL, USA) was also used as an internal autosomal control to distinguish between disomy and diploidy. The localization of all probes was checked on blood cell metaphases from the patient and the control. Figure 1. (a) Diagram of a pericentric inversion. (b) Possible meiotic configuration of a pericentric inversion: inversion loop with a crossover. (c) Resultant recombinant chromosomes. Preparation of sperm and sperm-fish Patient and control semen samples were obtained by masturbation and incubated until liquefaction. The spermatozoa were isolated using the Puresperm technique (JCD, Lyon, France) and then diluted in phosphate-buffered saline (PBS) and washed three times by centrifugation for 10 min at 750 g. The pellet was resuspended in fresh fixative (3:1 methanol : acetic acid). The spermatozoa suspension was spread onto clean glass slides and air-dried. The sperm nuclei were decondensed and denatured by incubation in 1 M NaOH for 5 min at room temperature, dehydrated in an ethanol series (70, 85 and 100%) and air-dried. The probe mixture was denatured at 72 C for 10 min. After application of the three-probe mixture onto the slide, the hybridization was performed overnight at 37 C in a moist chamber. The slide was washed for 2 min at 72 C in 0.4 saline sodium citrate (SSC)/0.1% Tween 20 and 2 SSC/0.1% Tween 20 at room temperature and then stained with antifade medium containing 4,6-diamidino-2-phenyl-indole (DAPI). Scoring criteria The slides were examined with a DM400B Leica epifluorescence microscope (Wetzlar, Germany) equipped with filter sets optimized for DAPI, FITC, Texas Red and Aqua. Cells were captured with the Leica microscope and a cooled CCD camera using Genikon Imaging System (Alphelys, Plaisir, France). Same-colour signals were counted as two if they were separated by at least one signal diameter and had the same intensity, size and shape. Main informative signal types for the probe mixture consisted of the following: 1. A nucleus with one red, one green and one blue signal representing balanced sperm nuclei carrying either a normal or inverted chromosome A nucleus with one blue signal and two green signals representing abnormal sperm nuclei carrying a recombinant chromosome with two p-telomeres. 3. A nucleus with two red signals and one blue signal representing sperm nuclei carrying a recombinant chromosome with two q-telomeres. 4. A nucleus with two signals from each telomere and two centromeric signals was considered diploid. Figure 2. Partial karyotype and RHG banding for pericentric inversion of chromosome 1. (a) Normal chromosome 1. (b) Chromosome 1 with inversion. Control A healthy man with normal semen parameters and no familial history of infertility, radiotherapy, chemotherapy, chronic illness or medication was used as control. This man was a sperm donor; he gave informed consent for karyotyping and sperm-fish analysis. Choice of DNA probes As abnormal recombinant chromosomes carry either two short-arm or two long-arm telomeres after genetic recombination within an inversion loop, the frequency of abnormal chromosomes in gametes can be easily assessed by FISH using probes coding for the sub-telomeric sequences of the inverted chromosome. Three probes to assess the frequency of recombinant chromosome were used. The chromosome 1p telomere-specific probe, labelled with fluorescein isothiocyanate (FITC) (Aquarius, Cytocell Technologies, Results The localization of the different probes used was checked on metaphases obtained from peripheral blood lymphocytes of the carrier (Figure 3a) and the control (data not shown) and revealed a normal localization of the signals. A total of 2133 sperm nuclei from the patient and 2145 sperm nuclei from the healthy control were analysed. In the control, 97.1% of sperm nuclei exhibited one red, one green and one blue signal, corresponding to normal meiosis products, whereas 2.6% of sperm heads had other signal combinations that probably result from incomplete hybridization (Table I). The frequency of diploid cells in control was 0.3%. In the inversion carrier, the frequency of sperm nuclei with one signal of each colour (Figure 3b) was 81%, which is significantly different from the control (P < ) (Table I). These nuclei contain a non-recombinant chromosome 1, a chromosome 1 with an even number of recombinations within the loop or with recombination events outside of the loop. The frequencies of the two types of recombinants, rec(1)dup(1p) (Figure 3c) and rec(1)dup(1q) (Figure 3d), were 7.6 and 7.2%, respectively. These frequencies were not significantly different from the 1:1 ratio, which was expected to be found because these abnormal spermatozoa are the result of a unique meiotic event. The frequency of diploid sperm was 0.4%, which is not significantly different than in 56

3 Sperm-FISH analysis in a pericentric chromosome 1 inversion Figure 3. Fluorescence in-situ hybridization (FISH) results with chromosome 1p telomere-specific probe (green), chromosome 1q-specific probe (red) and centromere-18 probe (aqua). (a) Hybridization of partial lymphocyte metaphase from inversion 1 carrier. (b) Sperm from the inversion-1 carrier: one normal sperm (red, green and aqua). (c) One dup(p)/del(q) sperm (two green and aqua). (d) One dup(q)/del(p) sperm (two red and aqua). Table I. Frequency of recombinant and non-recombinant sperm in heterozygous carrier for pericentric inversion in chromosome 1 by fluorescence in-situ hybridization (FISH) analysis FISH segregation type Patient [nb spz (%)] Control [nb spz (%)] Chi-square Non recombinant Normal chromosome (80.1) 2083 (97.1) P < or inverted chromosome 1 Recombinant chromosome dup(p)/del(q) 162 (7.6) del(p)/dup(q) 153 (7.2) Subtotal 315 (14.8) P < Diploid 8 (0.4) 6 (0.3) Not significant Other a 101 (4.7) 56 (2.6) P < Total del, deletion; dup, duplication; FISH, fluorescence in-situ hybridization; nb spz, number of spermatozoa. a Other may represent non-classical recombinants or classical recombinant or disomic sperm with incomplete hybridization. control. Other probe combinations, such as a single q or p signal associated with the blue (18) signal or more complex signals, like two red, one green and one blue, were found in 4.7% of nuclei. These unclassifiable combinations may represent non-classical recombinants or classical recombinant or disomic sperm with incomplete hybridization. Discussion Unlike chromosomal translocations, pericentric inversions are rarely associated with infertility and are usually identified fortuitously. Indeed, autosomal inversions are found in, respectively, 0% and 0.3% of azoospermic and oligozoospermic men and between 0.3 and 0.4% of women asking for IVF with or without ICSI (Mau-Holzmann, 2005). However, some authors suggest that pericentric inversions may affect male fertility owing to the fact that 12% of carriers are ascertained through sterility (Guttenbach et al., 1997). Our patient had no evident factor of infertility raising the question whether or not the chromosomal inversion might be responsible for spermatogenesis impairment in his case. Once paired, the interaction of the rearranged chromosome with the XY body during meiosis, as observed in infertile translocation carriers, has never been investigated in men with a chromosomal inversion. In our patient, no data were available concerning the meiotic configuration of the bivalent chromosome 1, and therefore its possible association with the XY body was impossible to analyse. However, like in translocation carriers, meiotic failure could occur in inversion carriers 57

4 S.Chantot-Bastaraud et al. by the same mechanism of association with the XY body and spreading of the gonosomal inactivation process towards autosomal segments. Infertility in inversion carriers can also occur after disruption or deletion of a specific gene or gene family implied in sperm cell production. Unless the exact determination of the chromosomal breakpoint is not performed, such an assertion cannot be checked. However, an excess of chromosome 1 breakpoints has been described in infertile males leading to the hypothesis that infertility in our patient could be due to this mechanism (Bache et al., 2004). Finally, a meiotic failure could also be due either to a reduction of recombination events within the loop (Brown et al., 1998) or to a synapsis impairment within the bivalent (Anton et al., 2005). When compared with the time-consuming hamster test, FISH is now a reliable method for evaluating sperm chromosomal content and allows the analysis of either accidental aneuploidy events or the segregation of a paternal structural rearrangement like a translocation or an inversion (Guttenbach et al., 1997). However, unlike the observation of a whole human chromosome set in a hamster s oocyte, FISH can only distinguish balanced and unbalanced gametes and, therefore, cannot discriminate between normal and translocated or inverted chromosomes. Despite the fact that sperm-fish studies are useful for predicting the risk of transmission of chromosomally abnormal gametes, thus allowing a more accurate genetic counselling, only seven studies have been reported to date in chromosomal pericentric inversion carriers (Jaarola et al., 1998; Anton et al., 2002, 2006; Yakut et al., 2003; Mikhaail-Philips et al., 2004; Mikhaail-Philips et al., 2005; Malan et al., 2006), two of which imply the chromosome 1 (Jaarola et al., 1998; Yakut et al., 2003). In chromosomal inversions, recombinant gametes are produced by a single or an odd number of genetic recombinations occurring within the inversion loop. However, the meiotic behaviour of two homologues chromosomes, one of which carries an inversion, depends on the chromosomes involved in rearrangement, the morphology and the length of the inverted chromosome fragments and the localization of chromosomal breakpoints (Ashley, 1988). All these factors are crucial for the meiotic configuration adopted by the bivalent. Effective homologous pairing is a prerequisite for recombination events. In short inversions, asynapsis is likely to take place, and inverted segments often remain as asynaptic balloon where no or rare recombination events occur. On the contrary, in very large inversions, the normal synapsis of the inverted segment with its noninverted homologue prevails over pairing of the terminal segments, and the regions outside of the inversion may remain unpaired or aligned through heterosynapsis. If the inverted and non-inverted chromosomal segments pair through the formation of an inversion loop, then a homologous synapsis is effective all the bivalent long, but the occurrence of a single recombination within the loop will determine the possibility of producing unbalanced chromosomes, i.e. duplicated/ deleted for either one or the other regions outside the inversion. Empirical reports have demonstrated that a minimum of 30% of the total length of the chromosome must be involved in the inversion to produce recombinant chromosome (Winsor et al., 1978). Furthermore, the location of the breakpoint in the rearrangement seems to affect the likelihood of forming an inversion loop. Ashley (1988) has suggested that a lack of homology would be recognized if G-light bands were aligned with G-light bands and an inversion loop would be formed. If, however, one or the two breaks lie in a G-dark band, the lack of homology could not be recognized and heterosynapsis would occur leading to the suppression of recombination. Because heterochromatin is a site where crossing over is suppressed, the presence of heterochromatin region within an inverted segment may account also for a lower frequency of recombinant product. In a compilation of inversion segregation studies reported in previous publications (eight by FISH technique and five by the analysis of sperm chromosome complement), for which sperm segregations were available, Anton et al. (2005) have calculated the size of the inverted segments, their proportion within the chromosome and their pairing ability and have confirmed that these parameters were closely related to the production of recombinant chromosomes and thus to unbalanced gametes. According to their estimations, the production of significantly increased levels of unbalanced recombinant gametes would require a minimum of inverted segment size close to 100 Mbp and a ratio of around 50% of the chromosome length. The existence of a recombination threshold, requiring the inversion of at least half of a chromosome length to produce a significant percentage of recombinant chromosomes, is illustrated by the study of two other pericentric inversion of chromosome 1 carrying different chromosomal breakpoints (Jaarola et al., 1998; Yakut et al., 2003). In the case described by Jaarola et al. (1998), <0.4% of recombinant chromosomes were observed, in relation to an inversion comprising only one-third of the chromosome 1. On the contrary, Yakut et al. (2003) described an inversion corresponding to almost half the chromosome 1 length and observed that 16% of 1636 sperm nuclei were recombinant. In view of the large size of this inversion (p36q32 44), the percentage of recombinant chromosomes should have been even greater than 16%, but the existence of the heterochromatic region at the proximal part of the chromosome 1 long arm, in which recombination is suppressed, may be an explanation for this moderate rate of recombinant chromosomes. A direct relationship between the size of an inversion and the length of a chromosome may be the cause of the high percentage of recombinant gametes observed by Malan et al. (2006) in a patient carrying a chromosome 21 inversion. Indeed, despite the small length of this chromosome (about 47 Mbp), this patient exhibited more than 32% of recombinant gametes which could be explained by the fact that the inversion spanned over nearly the total length of the chromosome. In our case, the size of the inversion was estimated to be 130 Mbp, which represents 52.2% of the chromosome 1 total length. Again, a 14.8% recombinant chromosome rate could be explained by the lack of recombination within the chromosome 1 heterochromatic region. In 0.4% of sperm cells, FISH signal revealed a diploid content of nuclei, which is in agreement with the frequency found in healthy and chromosomally normal men (Rubes et al., 2002). We also found 4.7% of sperm nuclei that exhibited other atypical probe combinations. Data obtained from controls demonstrated that at least half of these could have occurred because of incomplete hybridization of telomeric probes, the other half representing unexplained recombination processes. Very recently, Morel et al. (2006) analysed the meiotic segregation in spermatozoa of six pericentric inversion carriers by multicolour FISH. This study includes a new pericentric inversion of chromosome 1 with the inverted segment size corresponding to 95% of the length of chromosome 1. The authors found a high rate of recombinant gametes (30.4%). Unfortunately, the relationship with infertility is not discussed although four of these patients were referred for infertility. In conclusion, application of multicolour sperm-fish analysis is of special value for investigating the rate of recombinant and nonrecombinant products of pericentric inversion in males. In carrier women, such a large-scale evaluation is impossible because of the very few number of available gametes but can be approached by using preconceptional or preimplantation genetic diagnosis (PGD). Whatever the results of FISH in sperm cells of carrier men may be, this approach provides useful indications for appreciating the opportunity of a prenatal diagnosis or a PGD. Moreover, it allows a better understanding of the segregation patterns in chromosomal inversions and their effect on reproductive failure.

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