Assisted reproductive technology and complex chromosomal rearrangements: the limits of ICSI

Molecular Human Reproduction vol.3 no.10 pp. 847 851, 1997 Assisted reproductive technology and complex chromosomal rearrangements: the limits of ICSI J.P.Siffroi 1,5, B.Benzacken 2, B.Straub 1, C.Le Bourhis 1, M.O.North 1, G.Curotti 3, V.Bellec 3, S.Alvarez 4 and J.P.Dadoune 1 1 Laboratoire d Histologie, Biologie de la Reproduction et Cytogénétique, Hôpital Tenon, 4 Rue de la Chine, 75020 Paris, 2 Laboratoire d Histologie, Embryologie, Cytogénétique et Biologie de la Reproduction. Hôpital Jean Verdier, Bondy, 3 Laboratoire Marcel Merieux, 94 96 rue Chevreul, 69007 Lyon, and 4 Service de Gynécologie Obstétrique, Hôpital Tenon, 4 Rue de la Chine, 75020 Paris, France 5 To whom correspondence should be addressed Complex chromosomal rearrangements are very rare events in the human population. According to our knowledge on the consequences of simple reciprocal translocations for male fertility, translocations involving three or more chromosomes are thought to lead to severe reproductive impairments in terms of meiotic disturbance or chromosomal imbalance of gametes. We report the case of a 48 year old man whose sperm count revealed either oligozoospermia ( 10 3 spermatozoa/ml) or azoospermia. He was referred to the laboratory for in-vitro fertilization after intracytoplasmic sperm injection. Cytogenetic investigations showed a complex chromosomal rearrangement involving firstly a translocation between the short arm of chromosome 7 and the long arm of chromosome 13 and secondly a translocation between the short arm of the same chromosome 13 and the short arm of chromosome 9. Diagnosis was ascertained by fluorescence in-situ hybridization and staining of the nucleolar organizer regions. Theoretical study of the translocated chromosomes predicted a chain configuration of the hexavalent at the pachytene stage of meiosis. In all, 32 modes of segregation were considered and only one resulted either in a normal or a balanced gamete karyotype. Genetic counselling and choice of appropriate artificial reproduction technique are discussed. Key words: complex chromosomal rearrangement/genetic counselling/icsi/meiosis Introduction Complex chromosomal rearrangements (CCRs) are defined as reciprocal exchanges between three or more chromosomes. They are rare events in human pathology and only ~100 CCRs have been defined as constitutional findings (Batista et al., 1994). According to our knowledge of the consequences of simple reciprocal translocations on male fertility, CCRs are thought to lead to severe reproductive impairments in terms of meiotic disturbance or chromosomal imbalance in gametes. Spermatogenesis dysfunction in translocation carriers can now be bypassed by intracytoplasmic sperm injection (ICSI) but the question still remains as to whether or not a technique with such a very high genetic risk may be reasonably proposed for pregnancy. We report the case of a complex translocation, with four chromosomal breakpoints, in a man asking for assisted reproductive technology (ART) and we propose guidelines for genetic counselling. Case report The patient was a 48 year old man, referred in the laboratory for semen analysis. There was no history of infertility, miscarriages, hereditary diseases or malformations in his family. His wife was a 35 year old healthy woman and they were unrelated. Spermograms revealed either severe oligospermia ( 1000 spermatozoa/ml) or azoospermia. Biochemical semen parameters, plasma follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone were normal. Cytogenetic investigations were performed on white blood cells using a high resolution technique after cell culture synchronization and BrdU incorporation; it revealed a CCR involving, firstly, a translocation between the short arm of chromosome 7 and the long arm of chromosome 13 and, secondly, a translocation involving the short arm of the same chromosome 13 and the short arm of chromosome 9 (Figure 1). Cytogenetic diagnosis was ascertained by fluorescence insitu hybridization (FISH) using painting probes of chromosomes 7, 9 and 13 (Cambio Biosys) (Figure 1). Staining of the nucleolar organizer regions (NORs), which are localized on the short arms of acrocentric chromosomes in humans (13, 14, 15, 21 and 22), confirmed the translocation of the 13 NORs on the short arm of chromosome 9 (Figure 1). Therefore, the patient s karyotype was apparently balanced: 46,XY, der (7) t(7;13) (p14;q21) der (9) t(9;13) (p11.2;p21) der (13) t(7; 13) (q14;q21) t (9;13) 9p11.2;p21). No familial cytogenetic investigation was performed. Since the patient declined testicular biopsy, a theoretical meiotic study of the translocated chromosomes was carried out which it predicted a chain configuration of the hexavalent at the pachytene stage of meiosis (Figure 2). From this diagram, 64 different gamete karyotypes were considered, corresponding European Society for Human Reproduction and Embryology 847

J.P.Siffroi et al. Figure 3. Trisomic and/or monosomic chromosomal imbalances in the 64 expected gamete karyotypes. The 15 karyotypes inside the step shape area lead to potentially viable fetuses. Figure 1. Partial R banded karyotype showing the complex chromosomal rearrangement between chromosomes 7, 9 and 13. Confirmation by fluorescent in-situ hybridization (FISH) using whole chromosome painting and by nucleolar organizer regions (NORs) staining. communication) (Figure 3). Only one 3:3 segregation mode was thought to give either a normal (chromosomes 7, 9, 13) or a balanced (der7, der9, der13) gamete karyotype. Among the 62 other expected unbalanced gamete karyotypes, 13 were considered to lead to potentially viable children according to the viability thresholds developed by Cohen et al. (1994) (Table I). Considering these predictions, the patient received genetic counselling and the choice of artificial insemination with donor spermatozoa instead of ICSI was made. Figure 2. Schematic representation of the theoretical configuration of the translocated chromosomes at the pachytene stage of meiosis. Values indicate the percentages of haploid autosomal length (HAL) represented by normal chromosomes (surrounded) and by centric and translocated segments of the derivative (der) chromosomes. to 32 theoretical segregation modes (one 6:0 mode, six 5:1 modes, 15 4:2 modes and 10 3:3 modes). The lengths of trisomic or monosomic chromosomal segments, except the short arm of chromosome 13 whose imbalance has no phenotypic effect, were calculated in percentages of haploid autosomal length (HAL) according to data furnished by the Reci-Conseil Data Bank (Grenoble, France; O.Cohen, personal Discussion Precise identification of all the chromosomes involved in a CCR is the prerequisite to every appropriate genetic counselling. FISH is now widely used as an important adjunct to classical cytogenetics for the diagnosis of cryptic or CCR (Rosenberg et al., 1992; Batista et al., 1994). In combination with high resolution techniques of chromosome banding, it represents an essential tool to determine if a complex abnormal karyotype is apparently balanced or not, especially in prenatal diagnosis (Mercier et al., 1996). Using a combination of six different fluors for chromosome labelling, Speicher et al. (1996) have developed epifluorescence filter sets and computer software for the unequivocal discrimination of each human chromosome pair. Such a technique, named multiplex-fish (M-FISH), could be used for the characterization of complex karyotypes, especially in tumour cells. The introduction of ART, and particularly ICSI, represents a new departure in genetic counselling; indeed, men with severe oligozoospermia and even azoospermia, who were considered previously as definitely sterile, can now reproduce using ICSI. Because of the high frequency of chromosomal 848

ART and complex chromosomal rearrangements Table I. Details of the chromosomal characteristics of the 15 gamete karyotypes leading to potentially viable fetuses Segregation Expected gamete Expected chromosome Percentage Viability mode chromosomes imbalance HAL 3:3 7, 13, 9 0 Normal 3:3 der7, der13, der9 0 Balanced 3:3 7, 9, der13 Partial trisomy 7 Partial trisomy 9 1,97 trisomy Partial monosomy 13 1,73 monosomy 3:3 der7, der9, 13 Partial trisomy 13 Partial monosomy 7 1,73 trisomy Partial monosomy 9 1,97 monosomy 3:3 7, der9, 13 Partial monosomy 9 0.75 monosomy 3:3 7, der9, der13 Partial trisomy 7 1,22 trisomy Partial monosomy 13 1,73 monosomy 3:3 der7, 9, 13 Partial trisomy 13 1,73 trisomy Partial monosomy 7 1,22 monosomy 3:3 7, der13, 9 Partial trisomy 9 0,75 4:2 der 7, der13, 9, der 9 Trisomy 9 4,68 trisomy 4:2 7, 13, 9, der 9 Partial trisomy 9 3,93 trisomy 4:2 der 7, der13, 13, der 9 Trisomy 13 3,84 trisomy 4:2 der 7, 13, der13, 9 Trisomy 13 Partial trisomy 9 4,59 trisomy 4:2 7, der13, 13, der 9 Partial trisomy 7 Partial trisomy 13 2,84 trisomy 4:2 der 7, 9 Partial monosomy 7 Partial monosomy 13 2,84 monosomy 4:2 7, 13, der13, 9 Partial trisomy 7 Partial trisomy 9 Partial trisomy 13 3,59 trisomy HAL haploid autosomal length. abnormalities found in this population, a number of questions arise concerning the genetic risk for their progeniture. The case that we report illustrates the well-known relationships between male infertility and chromosomal aberrations. The incidence of karyotype abnormalities in infertile men depends on several factors and particularly sperm numeration taken together with chromosome analysis. Indeed, oligozoospermia is defined as a sperm count of 20 10 6 spermatozoa/ ml, but most clinicians and laboratory teams are in agreement with the fact that karyotyping must be proposed to men with 10 10 6 spermatozoa/ml (Retief et al., 1984), especially those with very low sperm counts who wish for ICSI (Baschat et al., 1996). From a review of 10 different studies (Guichaoua et al., 1993), the mean frequency of abnormal karyotypes in infertile men has been established as 5.3%, of which 1.5% are structural chromosomal aberrations. In a number of cases, Klinefelter syndrome can be suspected from physical examination but, except for sperm count and familial history of patients, neither seminal nor clinical characteristics may be of predictive value for structural chromosomal abnormalities (Pandiyan and Jequier, 1996). The close association between CCRs and male infertility is less well documented (Joseph and Thomas, 1982; Rodriguez et al., 1985; Saadallah and Hulten, 1985), but a high prevalence of maternal origin has been observed in familial CCRs which is consistent with spermatogenetic failure in male CCR carriers (Kleczkowska et al., 1982); in a recent study of 30 cases of familial CCRs, Batista et al. (1994) reported only four families in which a complex translocation was transmitted through spermatogenesis. In our patient, parental karyotypes were not performed but, on account of the lack of miscarriage, abnormal children or other infertile men in the proband s family, it can be assumed that this CCR is de novo. The genetic risk to the progeniture of infertile men treated by ICSI has been the subject of recent debate (Persson et al., 1996). An increased frequency of sex chromosome aneuploidy in children born after ICSI has been reported (Bonduelle et al., 1995); such an excess may represent a significant incidence of 46,XY/47,XXY mosaicism in fathers, even when a normal karyotype has been found in blood cells. Indeed, comparison by FISH of spermatozoa of nine oligoasthenoteratozoospermic patients with nine fertile donors has revealed a 10-fold increase in the extra sex chromosome rate in infertile group (Pang et al., 1995). ICSI could also be responsible for an abnormal segregation of maternal chromosomes during second polar body extrusion leading to an aneuploid embryo (Rosenbusch and Sterzik, 1996). In males carrying structural chromosomal aberrations, the genetic risk for offspring concerns unbalanced transmission of the rearrangement resulting in partial trisomy/ monosomy for the translocated chromosomal segments. In reciprocal chromosomal translocations, study of meiotic segregation can be achieved using the technique of in-vitro human hamster fertilization. For each patient, several hundred sperm karyotypes can be analysed by this method (Martin, 1988; Pellestor et al., 1989; Jenderny, 1992). Results show that, whatever the translocation may be, all modes of segregation are found in gametes, even for translocations which give only one type of imbalance at term such as the well-known t(11;22) (q23;q21). The proportion of balanced (alternate segregation) and unbalanced (adjacent 1,2 and 3:1 segregations) sperm 849

J.P.Siffroi et al. karyotypes is highly variable but, among unbalanced segregations, the prevalence of adjacent 1 mode is emphasized by many authors (Pellestor et al., 1989). Using human sperm injection in mouse oocytes, Lee et al. (1996) found a positive correlation between abnormal amorphous, round and elongated sperm heads and frequency of sperm structural chromosomal aberrations such as chromosome or chromatid breaks or gaps. Nevertheless, these anomalies might reflect technical artefacts in the sperm preparation or the inability of mouse oocytes to repair sperm DNA damage and none of them were found in somatic cells of patients. Therefore, even though not all spermatozoa with abnormal heads are chromosomally abnormal, the use of such spermatozoa in ICSI increases the risk of abnormal fertilization. As zona-free hamster egg fertilization is a labour- and timeconsuming method, FISH has been proposed as an alternative technique for analysing meiotic segregation in male translocation carriers (Goldman and Hulten, 1993; Lu et al., 1994). Using whole chromosome painting in combination with paracentromeric probes, FISH allows the study of frequency and distribution of chiasma at metaphase I and detailed analysis of the first meiotic segregation (Goldman and Hulten, 1993). It has been emphasized that interstitial chiasmata, which are chromatid exchanges between the centromere and the translocation breakpoint, render alternate and adjacent 1 segregation products cytologically indistinguishable and could be an explanation to the prevalence of the adjacent 1 segregation mode in gametes. In our case, azoospermia, at the time of the patient s examination, did not allow sperm cytogenetic analysis. Therefore, genetic counselling was given according to theoretical data concerning the risk of fetal unbalanced karyotypes. A number of methods have been developed for predicting segregation modes in reciprocal translocations: the pachytene diagram predictive method (PDP method), established by Jalbert et al. (1980), is based on the analysis of meiotic figures whereas the factorial discriminant analysis method (D method) takes into account specific information concerning the translocation and the carrier patient s characteristics (Cans et al., 1993a). These methods are successful in predicting segregation modes in 66% of the data for the PDP method and in 80% of the data for the D method. Using a logistic regression model, Cans et al. (1993b), proposed an accurate estimation of unbalanced offspring risk in reciprocal translocations: within the group of risk 5%, 97% of the individuals were correctly classified by this method. Unfortunately, all these methods have been validated in translocations involving two chromosomes and informative data concerning frequency of segregation modes and unbalanced offspring risk in CCRs are not available. Therefore, in our case, only a theoretical prediction of chromosomal segregation in gametes was possible, which gave 64 different karyotypes. Viability thresholds of chromosomal imbalances have been estimated as 5% of haploid autosomal length for pure trisomies, 3% for pure monosomies though, in combination imbalances, no value exceeds both 3.6% trisomy and 0.6% monosomy (Cohen et al., 1994). The resulting viability area has a step shape out of which every chromosomal imbalance 850 is considered as lethal. These values vary slightly with the segregation mode, the sex of the carrier parent and the genomic content of the unbalanced chromosomal segments. According to these estimations, beside the unique 3:3 segregation mode resulting in a chromosomal normal or balanced karyotype in offspring, 13 different gamete karyotypes might result in the birth of an abnormal child or in medical abortion after prenatal diagnosis. The other segregation processes were thought to lead either to fertilization failure or to early zygote development arrest and miscarriage. Therefore, disregarding the ratio of the 64 different possible karyotypes in mature gametes which cannot be estimated in CCRs, there was very little chance for our patient to have normal progeny. Such an evaluation is quite different from the appreciation of genetic risk in couples where both parents carry a separate translocation: in these rare cases, a model has been developed which calculates the risk for live-born unbalanced offspring by adding individual risks for each translocation minus the product of these separate risks, leading to a mean value of 6% (Campbell et al., 1995). ICSI has been successfully used by Testart et al. (1996) in couples carrying either paternal or maternal simple chromosomal rearrangements; among seven fetuses obtained by this technique, five were carriers of a balanced karyotype and two of a normal karyotype. Because ART in azoospermic men sometimes requires several testicular biopsies to retrieve a few spermatozoa suitable for ICSI, such a procedure may be proposed to couples with low genetic offspring risk or to men carrying simple karyotype rearrangements where chromosomal segregation can be accurately estimated. In CCR carrier patients, a successful result of ICSI is unlikely and it seems unreasonable to consider this technique as a valuable way for treating their infertility. References Baschat, A.A., Küpker, W., Al Hasani, S. et al. (1996) Results of cytogenetic analysis in men with severe subfertility prior to intracytoplasmic sperm injection. Hum. Reprod., 11, 330 333. Batista, D.A.S., Pai, G.S. and Stetten, G. (1994) Molecular analysis of a complex chromosomal rerrangement and a review of familial cases. Am. J. Med. Genet., 53, 255 263. Bonduelle, M., Legein, J. and Willikens, A. (1995) Follow-up study of children born after intracytoplasmic sperm injection. Hum. Reprod., 10 (Abstr. book 2), 54. Campbell, S.A., Uhlmann, W.R., Duquette, D. et al. (1995) Pregnancy outcome when both members of a couple have balanced translocations. Obstet. Gynecol., 85, 844 846. Cans, C., Cohen, O., Mermet, M.A. et al. (1993a) Human reciprocal translocation: is the unbalanced mode at birth predictable? Hum. Genet., 91, 228 232. Cans, C., Cohen, O., Lavergne, C. et al. (1993b) logistic regression model to estimate the risk of unbalanced offspring in reciprocal translocations. Hum. Genet., 92, 598 604. Cohen, O., Cans, C., Mermet, M.A. et al. (1994) Viability thresholds for partial trisomies and monosomies. A study of 1,159 viable unbalanced reciprocal translocation. Hum. Genet., 93, 188 194. Goldman, A.S.H. and Hulten, M.A. (1993) Analysis of chiasma frequency and first meiotic segregation in a human male reciprocal translocation heterozygote, t(1;11) (p36.3;q13.1), using fluorescence in situ hybridization. Cytogenet. Cell Genet., 63, 16 23. Guichaoua, M.R., Delafontaine, D., Noël, B. and Luciani, J.M. (1993) L infertilité masculine d origine chromosomique. Contracept. Fertil. Sex., 21, 113 121.

ART and complex chromosomal rearrangements Jalbert, P., Sele, B. and Jalbert, H. (1980) Reciprocal translocations: a way to predict the mode of imbalanced segregation by pachytene-diagram drawing. A study of 151 human translocations. Hum. Genet., 55, 209 222. Jenderny, J. (1992) Sperm chromosome analysis of two males heterozygous for a t(2;17) (q35;p13) and t(3;8) (p13;p21) reciprocal translocations. Hum. Genet., 90, 171 173. Kleckowska, A., Fryns, J.P. and Van Den Berghe, H. (1982) Complex chromosomal rearrangements (CCR) and their genetic consequences. J. Genet. Hum., 30, 199 214. Lee, J.D., Kamiguchi, Y. and Yanagimachi, R. (1996) Analysis of chromosome constitution of human spermatozoa with normal and aberrant head morphologies after injection into mouse oocytes. Hum. Reprod., 11, 1942 1946. Lu, P.Y., Hammitt, D.G., Zinsmeister, A.R. and Dewald, G.W. (1994) Dual color fluorescence in situ hybridization to investigate aneuploidy in sperm from 33 normal males and a man with a t(2;4, 8) (q23;q27;p21). Fertil. Steril., 62, 394 399. Martin, R.H. (1988) Meiotic segregation of human sperm chromosomes in translocation heterozygotes: report of a t(9;10) (q34;q11) and a review of the literature. Cytogenet. Cell Genet., 47, 48 51. Mercier, S., Fellmann, F., Cattin, J. and Bresson, J.L. (1996) Molecular analysis by fluorescence in situ hybridization of a prenatally detected de novo complex chromosomal rearrangement t(2q;3p;4q;13q). Prenat. Diagn., 16, 1046 1050. Pandiyan, N. and Jequier, A.M. (1996) Mitotic chromosomal anomalies among 1210 infertile men. Hum. Reprod., 11, 2604 2608. Pang, M.G., Zackowski, J.L. and Hoegerman, S.F. (1995) Detection by fluorescence in situ hybridization of chromosome 7, 11, 12, 18, X and Y abnormalities in sperm from oligoasthenospermic patients of an in vitro fertilization program. J. Assist Reprod. Genet., 12, 53S. Pellestor, F., Sele, B., Jalbert, H. and Jalbert, P. (1989) Direct segregation analysis of reciprocal translocations: a study of 283 sperm karyotypes from four carriers. Am. J. Hum. Genet., 44, 66 67. Persson, J.W., Peters, G.B. and Saunders, D.M. (1996) Genetic consequences of ICSI. Is ICSI associated with risks of genetic disease? Implications for counselling, practice and research. Hum. Reprod., 11, 921 932. Rodriguez, M.T., Martin, M.J. and Abrisqueta, J.A. (1985) A complex chromosomal rearrangement involving four chromosomes in an azoospermic man. J. Med. Genet., 22, 66 67. Rosenberg, C., Blakemore, K.J., Kearns, W.G. et al. (1992) Detection of chromosomal rearrangements by fluorescence in situ hybridization. Am. J. Hum. Genet., 50, 700 705. Rosenbusch, B. and Sterzik, K. (1996) Irregular chromosome segregation following ICSI? Hum. Reprod., 11, 2337 2338. Saadallah, N. and Hulten, M. (1985) A complex three breakpoint translocation involving chromosomes 2,4 and 9 identified by meiotic investigations of a human male ascertained for subfertility. Hum. Genet., 71, 312 320. Speicher M.R., Gwyn Ballard S. and Ward D.C. (1996) Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat. Genet., 12, 368 375. Testart, J., Gautier, E., Brami, C. et al. (1996) Intracytoplasmic sperm injection in infertile patients with structural chromosome abnormalities. Hum. Reprod., 11, 2609 2612. Received on April 24, 1997; accepted on July 18, 1997 851