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ICSI patients
RBMOnline - Vol 16. No 4 2008 514-522 Reproductive BioMedicine Online; www.rbmonline.com/Article/3221 on web 22 February 2008
Symposium: Genetic and epigenetic aspects of assisted reproduction Underlying karyotype abnormalities in IVF/ICSI patients Jean Pierre Siffroi, MD PhD, has 25 years experience in the cytogenetics and genetics of infertility, first in Hôtel-Dieu hospital, Paris, then in Tenon hospital, and currently in the Armand Trousseau Hospital, a children’s hospital in Paris in which he manages the new Department of Medical Genetics and Embryology.
Dr Jean Pierre Siffroi S Chantot-Bastaraud, C Ravel, JP Siffroi1 Université Pierre et Marie Curie-Paris 6, EA 1533, Paris, F-75012 France and AP-HP, Hôpital Tenon, Service d’Histologie Biologie de la Reproduction et Cytogénétique, Paris, F-75020 France 1 Correspondence: e-mail: [email protected]
Abstract Cytogenetic investigations are performed in couples asking for IVF or intracytoplasmic sperm injection (ICSI) treatment. These serve a diagnostic purpose because male or female infertility might have a chromosomal origin. Chromosomal aberrations found in these patients include numerical abnormalities, such as Klinefelter syndrome, XYY karyotype or Turner syndrome and its variants; sex reversions, such as XX males or XY females; and also structural abnormalities, such as Robertsonian or reciprocal translocations and inversions. Finding the chromosomal origin of infertility in a patient also has a prognostic value because it aids the management of pregnancies obtained after IVF or ICSI and may lead to a proposal of prenatal or preimplantation genetic diagnosis. Keywords: chromosomal abnormalities, cytogenetics, ICSI, karyotype
Introduction
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Assisted reproduction techniques, and particularly intracytoplasmic sperm injection (ICSI), bypass natural barriers to fertilization such as negative selection against genetic factors leading to impairment of gametogenesis. As a consequence, they allow the transmission of these factors to the offspring, but also expose the latter to an increased risk of developing malformations or diseases of genetic origin.
to be present in a small, but significant percentage of couples consulting for infertility (Van Assche et al., 1996). Even if some of these chromosome abnormalities, such as Klinefelter or Turner syndromes, can be clinically suspected, neither particular familial history nor pathognomonic clinical findings allows the clinical diagnosis of most chromosomal anomalies in infertile patients who are otherwise phenotypically normal.
Because gametogenesis is a serial process, any defect in one of the hundreds or thousands of genes involved could theoretically lead to an arrest in the normal multiplication and/or maturation of gametes and to infertility. Research in transgenic animal models has described infertility associated with some phenotypes, mainly in males (Escalier, 2001). However, the identification of a particular gene defect responsible for failure of gamete production or maturation in humans is quite a rare event in clinical practice. On the other hand, chromosomal abnormalities have been shown
Therefore, karyotyping has become an obligatory biological examination in the management of these patients. In man, studies have shown a high frequency of chromosomal aberrations in blood cells from infertile patients (Chandley, 1979; Bourrouilou et al., 1985; DeBraekeleer and Dao, 1991; Pandyan and Jequier, 1996; Mau et al., 1997; Meschede et al., 1998; Peschka et al., 1999; Gekas et al., 2001; Vincent et al., 2002). The prevalence ranges from 2.2 to 13.1%, but most studies report values of about 4–5%, which represents an 8–10-fold increase when
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Symposium- Karyotype abnormalities in IVF/ICSI patients - S Chantot-Bastaraud et al.
compared with values observed in unselected newborns (Nielsen and Wohlert, 1991). Variations are due to different recruitment procedures (i.e. the severity of the sperm cell depletion in ejaculate), or to the interpretation of cytogenetic results (i.e. the question of whether variants in the karyotype are pathological or not). Whatever the frequency of chromosomal abnormalities in infertile men, the general consensus is that the probability of finding an abnormality in a patient’s karyotype is negatively correlated to sperm count. However, this assertion does not mean that men carrying an abnormal karyotype will necessarily develop infertility. Indeed, a recent study has shown that the frequency of abnormal karyotypes in normospermic sperm donors was the same as that at birth, which means that most newborns carrying an abnormal karyotype will never consult for fertility problems (Ravel et al., 2006). In infertile women or the female partner in an infertile couple, several studies have also found an increase in the frequency of chromosome abnormalities (Meschede et al., 1998; Sholtes et al., 1998; Peschka et al., 1999; Schreurs et al., 2000; Gekas et al., 2001; Morel et al., 2004; Clementini et al., 2005). Some authors have pointed out that chromosomal abnormalities were more frequent in women with secondary infertility than in those with primary infertility (Clementini et al., 2005; Papanikolaou et al., 2005). However, the relationship between abnormal karyotype and female infertility, apart from Turner syndrome, is not as clear as the situation in men. Most observations suggest that chromosome aberrations in women seldom interfere with the meiotic process and that the presence of a chromosomal abnormality is often compatible with oogenesis, leading to a higher risk of aneuploid gamete formation (Hunt and Hassold, 2002). The aim of this paper is to review the main chromosomal aberrations found in infertile couples seeking IVF treatment, with or without ICSI, to determine, as far as possible, how these abnormalities act on gametogenesis and to measure the genetic consequences on offspring.
Chromosomal abnormalities in infertile couples Male infertility may be the result of many causes such as cryptorchidism, structural abnormalities of the genital tract, genital infections, previous scrotal or inguinal surgery, varicoceles, hypogonadotrophic hypogonadism, chronic illness, medications and exposure to chemicals. However, in about 40% of men, no evident cause of the infertility is found and a genetic factor may be suspected. Determining the genetic cause of male infertility requires careful clinical and biological examination of the patient that can distinguish between obstructive and nonobstructive origins. In the former, the main genetic origin consists of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which are commonly associated with congenital vas deferens abnormalities (Georgiou et al., 2006; Ferlin et al., 2007). In non-obstructive infertility, a general consensus indicates that the study of the karyotype is justified only for oligozoospermia below 10 × 106 spermatozoa/ml and for non-obstructive azoospermia (Egozcue et al., 2000). Males with azoospermia are more likely to carry either a numerical or structural sex chromosome abnormality than those with oligospermia, whereas males with oligospermia are more likely to have an autosomal rearrangement in their karyotype
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(translocations, inversions, supernumerary markers) than those with azoospermia.
Numerical abnormalities 47,XXY males Klinefelter syndrome (KS) is the most frequent gonosomal anomaly in man. It occurs in 0.1–0.2% of newborn males, but the frequency rises from 0.5% in oligozoospermic individuals to 11% in azoospermic patients (Huynh et al., 2002). KS accounts for 67% of abnormal karyotypes in azoospermic males and for 19% in oligozoospermic patients (Mau-Holzmann, 2005). About 80% of KS cases carry a homogenous 47,XXY karyotype and azoospermia is almost inevitable (Lanfranco et al., 2004). The remaining 20% have a more complex karyotype that can include various mosaics, the most frequent of which is the 46,XY/47,XXY mosaicism. If it is easy to make the diagnosis of a mosaic karyotype when several cell lines are observed in blood cell metaphases, the reverse is not true, and in practice it is impossible to assert that an apparently non-mosaic 47,XXY man does not carry any tissular mosaicism, especially in the testis. This is an important point for the management of these patients because the question of whether a testicular biopsy should be proposed to 47,XXY azoospermic men often arises. Indeed, in these men, surgical sperm retrieval has revealed spermatozoa in up to half of cases (Schiff et al., 2005). Mosaic KS cases may be either oligozoospermic or azoospermic but the probability of finding testicular spermatozoa is much higher. Therefore, treating KS patients with ICSI raises some concerns that the offspring may inherit an XXY, XXX or another abnormal karyotype. A number of meiotic studies have revealed that XXY cells are unable to enter meiosis (Kjessler, 1966), but others have suggested that at least some of them can reach the primary spermatocyte stage (Skakkebaek et al., 1969; Vidal et al., 1984). Actually, most fluorescence in-situ hybridization (FISH) studies performed on mosaic and non-mosaic KS spermatozoa have shown a paradoxical low frequency of disomic XY or XX cells, only a small percentage (reviewed by Ferlin et al., 2005), which is quite different from the theoretical 50% monosomic:50% disomic cell ratio which could be expected if XXY germ cells were able to produce spermatozoa. These results suggest that abnormal XXY cells are unable to enter meiosis and that spermatozoa in KS patients originate from normal XY spermatogonia due to a testicular mosaicism (Guttenbach et al., 1997; Foresta et al., 1998; Blanco et al., 2001). The low, but significant, frequency of disomic sperm cells should be the result of an increase in segregation errors occurring in normal 46,XY germ cells within a deleterious testicular environment, as shown in XXY mouse models (Mroz et al., 1999). In testicular biopsies from KS patients, it has been observed that only XY primary spermatocytes are able to continue their differentiation (Bergère et al., 2002). However, the possibility that some XXY cells could achieve meiosis is not completely excluded. An increased frequency of chromosomal segregation errors in KS patients with spermatozoa raises the question whether chromosomes other than X and Y could also be involved, leading to potential autosomal abnormalities in offspring, such as trisomy 21 (Hennebicq et al., 2001). Although most children born after ICSI with sperm from KS patients have a normal karyotype
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(Lanfranco et al., 2004), the opportunity to propose either a prenatal or a preimplantation diagnosis in pregnancies obtained from KS fathers is still a matter of question.
47,XYY males This is the second most frequent gonosomal abnormality after KS. Most 47,XYY men are fertile but the frequency of this syndrome in infertile males is about four-fold higher than in the normal fertile population (0.2–0.3% versus 0.05% at birth) (Yoshida et al., 1997). Because most 47,XYY men are not usually diagnosed, the frequency observed in the population referred for karyotyping is about 0.1% overall. Such an estimation takes into account the fact that some 47,XYY children develop mental disorders, such as pervasive developmental disorders or autism (Nicolson et al., 1998; Geerts et al., 2003). Thus, spermatogenesis in XYY males ranges from normality to severe failure. No valuable explanation accounts for this variability other than different meiotic pairing configurations of the extra Y chromosome with the other sex chromosomes, which could explain a different impact on spermatogenesis (Blanco et al., 2001; Rives et al., 2003). The common property of some Y chromosomes to be prone to malsegregation errors in meiosis II and to carry a structural abnormality such as a microdeletion could be another explanation. However, this has never been demonstrated in XYY infertile men, although such deleted Y chromosomes have been shown to be mitotically unstable (Siffroi et al., 2000). Whatever it may be, most FISH studies performed on sperm cells from XYY men have shown that the majority of the spermatozoa produced by these patients contain a balanced karyotype, which suggests either the systematic elimination of the supernumerary Y chromosome during meiosis or the degeneration of the abnormal aneuploid cells (Shi and Martin, 2000).
45,X0/46,XY males This type of mosaicism is a common finding during the cytogenetic evaluation of infertile men and several studies have reported an increased frequency of low-level mosaics of this type in the male partner in couples examined prior to ICSI (Meschede et al., 1998; Peschka et al., 1999; Gekas et al., 2001). Morel et al. (2002) did not find any difference between fertile and ICSI-treated couples regarding the existence of sex chromosome mosaicism. However, the existence of a 45,X0 cell line in testis may be considered a pejorative element, which could accentuate the effect of other infertility factors, as shown in azoospermia factor c (AZFc)deleted patients (Jaruzelska et al., 2001).
Extra structurally abnormal chromosomes
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Small supernumerary marker chromosomes (sSMC) are defined as additional centric chromosome fragments, which are too small to be identified or characterized unambiguously by banding cytogenetic techniques. Their identification by FISH has revealed that they are often made of two symmetrical short arms of acrocentric chromosomes separated by a centromere (i.e. disposed in mirror). Men carrying a supernumerary marker chromosome are usually phenotypically normal and fertile. However the frequency of sSMC is increased in the infertile population, they are found in 0.043% of newborns, in 0.035% of sperm donors and in 0.125% of infertile subjects (Tuerlings et al., 1998; Eggermann
et al., 2002; Ravel et al., 2006; Liehr and Weise, 2007). Their role in spermatogenesis failure is not clear but they could act by disrupting normal chromosome movements and/or sex body formation during meiosis.
Turner syndrome and variants Turner syndrome (TS) phenotype is characterized by short stature, gonadal dysgenesis and other somatic stigmata such as webbed neck and cubitus valgus. The prevalence is 1 in 1500 to 1 in 2500 females. Ovarian dysfunction in women with TS seems to be due to an abnormal and rapid degeneration of oocytes and follicles, resulting in primary amenorrhoea. Because of a total lack of germ cells, these patients cannot enter assisted reproduction programmes, except gamete donation, and therefore a non-mosaic 45,X0 karyotype is rarely reported in cytogenetic studies dealing with a couple’s infertility. However, about 30% of mosaic TS patients have partial ovarian function at puberty but only 2–5% of them become pregnant. In some TS patients, the karyotype shows both a normal X and a structurally rearranged X chromosome. The existence of an isochromosome for the long arm of the X chromosome [i(Xq)] accounts for up to 16% of TS cases and it is usually associated with a 45,X0 cell line (Kleczkowska et al., 1990). Most of these women also suffer from gonadal dysgenesis without any possibility of natural or medically assisted pregnancy. The phenotype associated with deletions of the short arm [del(Xp)] is often less severe and fertility may be preserved. In addition, structurally abnormal X chromosomes, like large ring X [r(X)], can be devoid of any consequence on ovarian function and be transmitted from fertile mothers to their daughters. The main problem encountered in cytogenetic surveys performed in infertile women is the high frequency of gonosomal mosaicism in the blood cells of patients. A basal level of low mosaicism probably exists in many fertile women, as seen in oocyte donors (Ravel et al., 2007) and the question arises whether or not such mosaicism may be responsible for female infertility when found in the course of IVF or ICSI procedures. It is likely that an abnormal 45,X/46,XX karyotype in association with clinical signs of TS may constitute a true constitutional mosaicism and that the risk for an abnormal pregnancy outcome may be substantial (Kaneko et al., 1990). On the other hand, Gardner and Sutherland (2004) suggest that sex chromosome mosaicism with less than 10% of abnormal cells probably does not indicate any specific reproductive risks in women without TS stigmata.
47,XXX syndrome Triple X women are usually fertile and give birth to children with a normal karyotype (i.e. without either a XXY or a XXX constitution), which means that the extra X chromosome is eliminated during female meiosis. Among a group of 4327 women karyotyped before ICSI, four were found to have a 47,XXX karyotype, which corresponds directly to the frequency found at birth (Mau-Holzmann, 2005). However, some studies have pointed out that XXX women could have an increased risk of developing premature ovarian failure (POF), probably through the occurrence of immunological disorders (Castillo et al., 1992; Guichet et al., 1996; Holland, 2001).
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Symposium- Karyotype abnormalities in IVF/ICSI patients - S Chantot-Bastaraud et al.
Sex reversion: XX males and XY females 46,XX males This rare anomaly is found in about 1 in 20,000 newborns but in approximately 0.9% of azoospermic patients (Mau-Holzmann, 2005). In most cases, normal male differentiation occurs during embryogenesis because of the presence of the SRY gene at the tip of the paternal X chromosome. Indeed, an abnormal recombination during paternal meiosis between the terminal short arms of both X and Y chromosomes, just proximal to the homologous pseudoautosomal region PAR1, leads to the translocation of the SRY gene onto the X chromosome. In some very rare cases, the SRY gene is absent and the male phenotype results either from the inactivation of a testis-determining repressor or from the activation of another gene implied in the sex determination cascade leading to testis formation (Ergun-Longmire et al., 2005). These patients present a testis hypotrophy and a lack of germinal cells resulting in total sterility. Treatment by ICSI is not possible and they must be referred to centres of gamete donation.
46,XY females Male to female sex reversion may be due to multiple causes affecting either the first step of sex determination (deletion of the short arm of the Y chromosome or a mutation in the SRY gene) or the subsequent steps of fetus masculinization (mutations in the androgen receptor gene or in the 5-alpha reductase gene). In all cases, females present with primary amenorrhoea and are sterile because they have non-functional streak gonads or intraabdominal testes. However, discovering a 46,XY female may also occur in the course of genetic investigation of infertility.
Structural abnormalities Chromosome behaviour at meiosis is a complex process, which is easily disturbed by the existence of an abnormality in the structure of chromosomes. The normal pairing of homologous chromosomes at the pachytene stage is an absolute prerequisite for meiosis and every disruption in chromosome pairing will lead to the death of the germ cells and their elimination by apoptosis. In the case of chromosomal exchanges or fusion, homologous chromosomes are not able to form normal bivalents and pair as tri or tetravalents, which are complex meiotic figures. Studies of affected meiosis, by silver staining or three-dimensional reconstruction in electron microscopy, have revealed many regions of asynapsis or heterosynapsis (Solari, 1999). As a consequence, in the male, but not in the female, abnormal chromosomes are often associated with the sex body, which is rich in proteins with a high affinity for the non-synapsed regions of the X and Y chromosomes. By their physical contact with the sex body, the autosomal segments would thus be inactivated (Guichaoua et al., 1991) and their transcription level would be decreased, if not abolished (Jaafar et al., 1993). The inactivation of autosomal chromosomal fragments associated with a structural abnormality of the karyotype may occur in a variable percentage of meiotic cells. This explains the moderate testis alteration seen in some patients and the differences observed between two individuals carrying the same chromosomal abnormality in a family. Therefore, infertility in translocation carriers appears to RBMOnline®
be the result of an impairment of the nuclear events in prophase I rather than the consequence of gene disruption at chromosomal breakpoints, although some testis-expressed transcripts have been shown to map near recurrent translocation sites found in infertile men (Olesen et al., 2001).
Robertsonian translocations Also called centric fusion, Robertsonian translocation consists of the fusion of two acrocentric chromosomes (i.e. chromosomes 13, 14, 15, 21 and 22) at their centromere (Figure 1). The loss of the short arms of the chromosomes involved is neutral for the cells, since the genes located in these regions are highly repeated in all acrocentric chromosome short arms. All combinations between acrocentric chromosomes are possible but the more frequent ones are the t(13;14) and the t(14;21). Robertsonian translocations occur in about 1 in 1000 newborns and the frequency rises to 0.2% in azoospermic males and to 1.5% in oligozoospermic men. Curiously, the prevalence of t(13;14) in normospermic gamete donors is the same as that in newborns, which means that most carriers do not develop fertility problems in adulthood (Ravel et al., 2006). The way in which Robertsonian translocations act on male germ cell development is probably through an association between the translocated chromosomes, which form a trivalent at meiosis, and the sex body. However, translocation carriers have very different fertility status, even in the same family, so the precise mechanism by which these translocations affect fertility is not clear. In women, Robertsonian translocations do not seem to affect gametogenesis. When receiving IVF or ICSI treatment, Robertsonian translocation carriers can transmit their translocation in an unbalanced state, thus leading to a trisomic fetus for one or the other chromosome involved. Except for trisomy 21, all other trisomies for acrocentric chromosomes are thought to terminate early in pregnancy or to give rise to severe fetal anomalies that are easily recognised at ultrasound examination. However, clinicians must be aware that such trisomies can be repaired at the first division of the zygote by a mechanism called trisomy rescue, which potentially leads to uniparental disomy. For chromosomes 14 and 15, both paternal and maternal uniparental disomies are pathological and give rise to severely affected children (i.e. bone malformations, growth retardation, Angelman or Prader-Willi syndromes) despite an apparently normal or balanced karyotype.
Reciprocal autosomal translocations Reciprocal translocations are defined as an exchange of chromosomal material between arms of two non-homologous chromosomes, usually without any apparent change in the global amount of genetic material (Figure 2). Like Robertsonian translocations, reciprocal translocations affect about 1 in 1000 newborns, which means that 1 in 500 individuals in the general population carry a chromosomal translocation. The frequency of balanced reciprocal translocation in infertile males is estimated to be 5–10 times higher than among newborns, according to size population and sperm criteria for patient inclusion, with a mean frequency at 0.7% (review by Mau-Holzmann., 2005). The cellular mechanism by which reciprocal translocations impair spermatogenesis is thought to be the same as that in Robertsonian translocations (i.e. an abnormal association with the sex body at
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13;14
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Figure 1. Robertsonian t(13;14) translocation in RHG banding.
the pachytene stage), except that homologous segments of the translocated chromosomes associate one to each other to form a quadrivalent figure. This complex meiotic type of synapsis adopts a more-or-less flat configuration, according to the size of both centric and non-centric segments, which is known to play a role in the modes of segregation at the end of pachytene: in other words, the more flat a quadrivalent is, the higher is the probability that the chromosomes will segregate in an unbalanced mode (Cans et al., 1993). Therefore, the diagnosis of a reciprocal translocation in an infertile patient has important consequences for both ICSI treatment and pregnancy management. In some rare cases, reciprocal translocations involve more than two chromosome pairs and the probability of obtaining a chromosomally balanced spermatozoon for ICSI becomes very low. Other assisted
a
b Figure 2. (a) GTG banded karyotype showing a t(1;22)(p13;q11.21) translocation (arrows) in an infertile man. (b) Fluorescence insitu hybridization on metaphase spreads with whole chromosome painting probes of chromosome 1 and chromosome 22. (Whole chromosome painting probes, Oncor, Gaithersburg, MD, USA.)
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reproduction procedures, such as gamete donation, might be proposed to couples in these cases (Siffroi et al., 1997). Although reciprocal translocations are not thought to interfere with the process of female meiosis, the frequency of these chromosomal rearrangements in women is particularly high in couples receiving ICSI treatment, at about 0.7%, which is very similar to that observed in infertile men (review by MauHolzmann, 2005). In the general population, female carriers of reciprocal translocations are diagnosed most often after recurrent spontaneous abortions or after the birth of a child carrying an unbalanced karyotype. Finding a high proportion of such carriers in ICSI couples means that these women probably develop a variable level of hypofertility, which has no consequence when their male partner is normally fertile, but leads to couple infertility and to ICSI when a contributing male factor is present. As a consequence, cytogenetic investigations in ICSI couples should be performed in both members.
X autosome translocations in women The effects on fertility of balanced X-autosome translocations are variable and depend on several parameters such as breakpoint localization and X chromosome inactivation profile. Usually the normal X chromosome is inactivated to avoid the abnormal inactivation of the autosomal translocated segment and female carriers are phenotypically normal and fertile. However gonadal dysgenesis may occur in some of them if the breakpoint falls within the two critical Xq regions known to be involved in premature ovarian failure (POF) (POF1 at Xq21.3-q27 and POF2 at Xq13.3-q21.1) (Schlessinger et al., 2002; Portnoï et al., 2006).
Chromosomal inversions Chromosomal inversions are structural intrachromosomal rearrangements resulting from two breaks within a single chromosome followed by a 180° rotation of the chromatin between these breaks. According to the position of the centromere, within or outside the inverted segment, pericentric and paracentric inversions are observed. Autosomal inversions are the second most common type of chromosomal rearrangements after translocations. They are found in 0.022% of children at birth and 0.088% of sperm donors (Ravel et al., 2006). Most pericentric inversions affect chromosome 2 or the heterochromatic regions of chromosomes 1, 9, 16 and Y and they are considered as non-pathological variants. Other autosomal pericentric inversions are not usually associated with infertility but rather with the birth of an abnormal child carrying the inversion in an unbalanced state. Indeed, meiotic pairing of the inverted chromosomes with its own homologous partner sometimes needs the formation of an inversion loop, depending on the length of the inverted segment. The occurrence of an odd number of crossovers within the loop results in the production of chromosomes that are duplicated or deleted for the regions outside the inversion. Sperm FISH studies of male carrier spermatozoa is a good way to assess the ratio of balanced and unbalanced sperm cells before any conception, with or without assisted reproduction treatment (Chantot-Bastaraud et al., 2007).
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Most inversions are identified fortuitously but, in some cases, autosomal pericentric inversions have been associated with azoospermia or oligospermia (Gabriel-Robez et al., 1988; Navarro et al., 1993; Meschede et al., 1994; Chantot-Bastaraud et al., 2007) and some studies have pointed out that pericentric inversions could reduce male fertility (De Braekeleer and Dao, 1991; Guttenbach et al., 1997; Mau-Holzmann, 2005). In the same way as translocations, the existence of a synapsis failure at the base or in the inversion loop could explain spermatogenesis impairment. Moreover, in paracentric inversion, recombinations within the loop give both dicentric and acentric chromosomes, which are likely to block meiosis, and thus lead to germ cell death and apoptosis. In female partners of ICSI couples, an increased frequency of inversions has also been described, which could be due to fertilization and/or implantation failures because of the existence of a high proportion of recombinant unbalanced chromosomes in oocytes (review by Mau-Holzmann, 2005).
Heterochromatic polymorphism The specific case of the Y chromosome Tiepolo and Zuffardi (1976), by studying large final deletions of Yq among 6 azoospermic patients, described a region controlling spermatogenesis in the proximal euchromatic part of the Y long arm (Yq11), named AZF for azoospermia factor. The existence of this factor has subsequently been confirmed by many cytogenetic observations showing various abnormal Y chromosomes in infertile men (i.e. isochromosomes Yp, dicentric isochromosomes Yp, Y ring chromosomes or Y-autosome translocations) and by the description of molecular deletions of the AZF region (TuckMuller et al., 1995; Vogt et al., 1995). The latter are not detectable at classical cytogenetic examination and have been extensively reviewed elsewhere (McElreavey et al., 2000). The frequency of Y-autosome translocations reported is approximately 1 in 2000 (Alves et al., 2002). Breakpoints are generally localized in Yq12 (heterochromatic region) and Yq11 (AZF region). In the former, only Y heterochromatic material is transposed on another chromosome, most often on an acrocentric short arm, and the carrier is usually fertile. In contrast, when the breakpoint is localized in Yq11, azoospermia generally occurs. A particular mention should be made of isodicentric Y chromosomes, which consist of two short arms disposed in mirror, separated by two centromeres and a fragment of long arm. These chromosomes are probably the result of abnormal sister chromatid recombinations in the long arm. They have lost a great part of the AZF regions and thus are observed in azoospermic men. However, because the resulting chromosome has a size nearly similar to that of a normal Y chromosome, Y isodicentric are underestimated in routine cytogenetic studies and need particular FISH techniques to be diagnosed (Figure 3).
Genetic management of IVF/ICSI pregnancies Possible health problems in IVF/ICSI children are thought to be due to parental infertility itself or as a consequence of increased frequency of multiple pregnancies (Van Steirteghem et al., 2002; Cohen, 2007). However, pregnancies obtained after ICSI
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Figure 3. Cut-out of the sex chromosomes shows the abnormal idic(Yp) chromosome in RHG banding (a) and GTG banding (b). The abnormal Y chromosome is the correct size and may easily be mistaken for a normal one. Fluorescence in-situ hybridization (FISH) on metaphase spreads shows (c) SRY probe duplication (double arrows); (d) DYZ3 (Y centromere) probe duplication (double arrows); (e) SHOX (RP13–391G2 at Xp22.33) probe duplication in (double left arrows) and UTY (RP11–386L3 at Y q11.2 near AZFa region) (single right arrow). All FISH results indicate the existence of two short arms, two centromeres and a fragment of the long arm with a breakpoint between AZFa and AZFb. (Probes obtained from either Vysis, Abbott Laboratories or BACPAC Resources. BAC-probes selected from the University of California Santa Cruz Genome Browser.)
procedures are known to have a higher risk of de novo sex chromosomal aberrations and paternally inherited structural abnormalities (Van Steirteghem et al., 2002). However, the slightly increased risk generally observed does not justify any invasive prenatal diagnosis procedures, owing to the fact that many of these pregnancies have been achieved after several years of infertility.
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Most abnormal karyotypes in infertile couples are diagnosed at the initial genetic screening made in these cases. As a consequence, couples at risk of giving birth to a child carrying an unbalanced karyotype are aware of the situation before the pregnancy and can ask for a prenatal diagnosis after chorionic villus or amniotic fluid sampling. To date, many of them enter a preimplantation genetic diagnosis (PGD) procedure, which significantly increases their probability of having a chromosomally normal pregnancy and, at term, delivery of a healthy baby (Otani et al., 2006). Seeking PGD appears particularly easy, since most of these couples need IVF or ICSI to achieve a pregnancy, but the exact consequences of blastomere biopsy are still debated (Cohen and Grifo, 2007).
When performed for cytogenetic reasons, PGD allows the indirect karyotyping of a zygote using FISH techniques with appropriate fluorescent probes. Although FISH-PGD is unable to distinguish between zygotes carrying a normal karyotype and those with a translocated but balanced one, it allows the diagnosis of all the chromosomal imbalances generated by both Robertsonian and reciprocal translocations (Mackie Ogilvie and Scriven, 2004; Munné, 2005) and also those resulting from chromosomal inversions (Escudero et al., 2001).
Conclusion Couples engaged in IVF/ICSI procedures often present with male and/or female idiopathic infertility, which is known to have a frequent genetic origin. Among these, chromosomal abnormalities have been described as a major cause of gametogenesis failure. They can be easily diagnosed using conventional cytogenetic techniques and a large number of patients have been reported. RBMOnline®
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When engaged in one or other assisted reproduction technique, and particularly ICSI, couples carrying a chromosomal abnormality must be aware of the consequences for the offspring. Indeed, by selecting a single spermatozoon and an oocyte for injection, ICSI bypasses the natural process of selection, which is thought to occur during both natural conception and conventional IVF. As a consequence, it may allow the fertilization and the development of zygotes with potentially unbalanced karyotypes. Proposals of prenatal diagnosis or of PGD, when available, must be discussed with these couples.
Acknowledgements The authors thank Ken McElreavey for his helpful correction of the manuscript.
References Alves C, Carvalho F, Cremades N et al. 2002 Unique (Y;13) translocation in a male with oligozoospermia: cytogenetic and molecular studies. European Journal of Human Genetics 10, 467–474. Bergère M, Wainer R, Nataf V et al. 2002 Biopsied testis cells of four 47,XXY patients: fluorescence in-situ hybridization and ICSI results. Human Reproduction 17, 32–37. Blanco J, Egozcue J, Vidal F 2001 Meiotic behaviour of the sex chromosomes in three patients with sex chromosome anomalies (47,XXY, mosaic 46,XY/47,XXY and 47,XYY) assessed by fluorescence in-situ hybridization. Human Reproduction 16, 887–892. Bourrouillou G, Dastugue N, Colombies P 1985 Chromosome studies in 952 infertile males with a sperm count below 10 million/ml. Human Genetics 71, 366–367. Cans C, Cohen O, Mermet MA et al. 1993 Human reciprocal translocations: is the unbalanced mode at birth predictable? Human Genetics 91, 228–232. Castillo S, Lopez F, Tobella L et al. 1992 The cytogenetics of premature ovarian failure. Revista Chilena de Obstetricia y Ginecología 57, 341–345. Chandley AC 1979 The chromosomal basis of human infertility. British Medical Bulletin 35, 181–186. Chantot-Bastaraud S, Ravel C, Berthaut I et al. 2007 SpermFISH analysis in a pericentric chromosome 1 inversion, 46,XY,inv(1)(p22q42), associated with infertility. Molecular Human Reproduction 13, 55–59. Clementini E, Palka C, Iezzi I et al. 2005 Prevalence of chromosomal abnormalities in 2078 infertile couples referred for assisted reproductive techniques. Human Reproduction 20, 437–442. Cohen J 2007 Infertile couples, assisted reproduction and increased risks to the children. Reproductive BioMedicine Online 15, 245–246. Cohen J, Grifo JA 2007 Multicentre trial of preimplantation genetic screening reported in the New England Journal of Medicine: an in-depth look at the findings. Reproductive BioMedicine Online 15, 365–366. De Braekeleer M, Dao TN 1991 Cytogenetic studies in male infertility: a review. Human Reproduction 6, 245–250. Eggermann K, Mau UA, Bujdoso G et al. 2002 Supernumerary marker chromosomes derived from chromosome 15: analysis of 32 new cases. Clinical Genetics 62, 89–93. Egozcue S, Blanco J, Vendrell JM, et al. 2000 Human male infertility : chromosomal anomalies, meiotic disorders, abnormal spermatozoa and recurrent abortion. Human Reproduction Update 6, 93–105. Ergun-Longmire B, Vinci G, Alonso L et al. 2005 Clinical, hormonal and cytogenetic evaluation of 46,XX males and review of the literature. Journal of Pediatric Endocrinology and Metabolism 18, 739–748. Escalier D 2001 Impact of genetic engineering on the understanding
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of spermatogenesis. Human Reproduction Update 7, 191–210. Escudero T, Lee M, Stevens J et al. 2001 Preimplantation genetic diagnosis of pericentric inversions. Prenatal Diagnosis 21, 760–766. Ferlin A, Raicu F, Gatta V et al. 2007 Male infertility: role of genetic background. Reproductive BioMedicine Online 14, 734-745. Ferlin A, Garolla A, Foresta C 2005 Chromosome abnormalities in sperm of individuals with constitutional sex chromosomal abnormalities. Cytogenetics and Genome Research 111, 310-316. Foresta C, Galeazzi C, Bettella A et al. 1998 High incidence of sperm sex chromosomes aneuploidies in two patients with Klinefelter’s syndrome. Journal of Clinical Endocrinology and Metabolism 83, 203-205. Gabriel-Robez O, Ratomponirina C, Croquette M et al. 1988 Synaptonemal complexes in a subfertile man with a pericentric inversion in chromosome 21. Heterosynapsis without previous homosynapsis. Cytogenetics and Cell Genetics 48, 84–87. Gardner RJM, Sutherland GR 2004 Chromosome abnormalities and genetic counselling, 3rd edition. Oxford University Press, New York. Geerts M, Steyaert J, Fryns JP 2003 The XYY syndrome: a follow-up study on 38 boys. Genetic Counseling 14, 267–279. Gekas J, Thepot F, Turleau C et al. 2001 Chromosomal factors of infertility in candidate couples for ICSI: an equal risk of constitutional aberrations in women and men. Human Reproduction 16, 82–90. Georgiou I, Syrrou M, Pardalidis N et al. 2006. Genetic and epigenetic risks of intracytoplasmic sperm injection method. Asian Journal of Andrology 8, 643–673. Guichaoua MR, de Lanversin A, Cataldo C et al. 1991 Three dimensional reconstruction of human pachytene spermatocyte nuclei of a 17;21 reciprocal translocation carrier: study of XYautosome relationships. Human Genetics 87, 709–715. Guichet A, Briault S, Moraine C, Turleau C 1996 Trisomy X: ACLF (Association des Cytogeneticiens de Langue Francaise) retrospective study. Annales de Génétique 39, 117–122. Guttenbach M, Engel W, Schmid M 1997 Analysis of structural and numerical chromosome abnormalities in sperm of normal men and carriers of constitutional chromosome aberrations. A review. Human Genetics 100, 1–21. Hennebicq S, Pelletier R, Bergues U, Rousseaux S 2001 Risk of trisomy 21 in offspring of patients with Klinefelter’s syndrome. Lancet 357, 2104–2105. Holland CM 2001 47,XXX in an adolescent with premature ovarian failure and autoimmune disease. Journal of Pediatric and Adolescent Gynecology 14, 77–80. Hunt PA, Hassold TJ 2002 Sex matters in meiosis. Science 21, 2181–2183. Huynh T, Mollard R, Trounson A 2002, Selected genetic factors associated with male infertility. Human Reproduction Update 8, 183-198. Jaafar H, Gabriel-Robez O, Rumpler Y 1993 Chromosomal anomalies and disturbance of transcriptional activity at the pachytene stage of meiosis: relationship to male sterility. Cytogenetics and Cell Genetics 64, 273–280. Jaruzelska J, Korcz A, Wojda A et al. 2001 Mosaicism for 45,X cell line may accentuate the severity of spermatogenic defects in men with AZFc deletions. Journal of Medical Genetics 38, 798–802. Kaneko N, Kawagoe S, Hiroi M 1990 Turner’s syndrome-review of the literature with reference to a successful pregnancy outcome. Gynecologic and Obstetric Investigation 29, 81–87. Kjessler B 1966 Karyotype, meiosis and spermatogenesis in a sample of men attending an infertility clinic. Monographs in Human Genetics 2, 1–93. Kleczkowska A, Dmoch E, Kubien E et al. 1990 Cytogenetic findings in a consecutive series of 478 patients with Turner syndrome. The Leuven experience 1965-1989. Genetic Counseling 1, 227–233. Lanfranco F, Kamischke A, Zitzmann M, Nieschlag E 2004 Klinefelter’s syndrome. Lancet 364, 273–283. Liehr T, Weise A 2007 Frequency of small supernumerary marker chromosomes in prenatal, newborn, developmentally retarded
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and infertility diagnostics. International Journal of Molecular Medicine 19, 719–731. Mackie Ogilvie C, Scriven PN 2004 Preimplantation genetic diagnosis (PGD) for reciprocal translocations. Prenatal Diagnosis 24, 553–555. Mau UA, Backert IT, Kaiser P, Kiesel L 1997 Chromosomal findings in 150 couples referred for genetic counselling prior to intracytoplasmic sperm injection. Human Reproduction 12, 930–937. Mau-Holzmann UA 2005 Somatic chromosomal abnormalities in infertile men and women. Cytogenetic and Genome Research 111, 317–336. McElreavey K, Krausz C, Bishop CE 2000 The human Y chromosome and male infertility. Results and Problems in Cell Differentiation 28, 211–232. Meschede D, Froster UG, Bergmann M, Nieschlag E 1994 Familial pericentric inversion of chromosome 1(p34q23) and male infertility with stage specific spermatogenic arrest. Journal of Medical Genetics 31, 573–575. Meschede D, Lemcke B, Exeler JR et al. 1998 Chromosome abnormalities in 447 couples undergoing intracytoplasmic sperm injection-prevalence, types, sex distribution and reproductive relevance. Human Reproduction 13, 576–582. Morel F, Douet-Guilbert N, Le Bris MJ et al. 2004 Chromosomal abnormalities in couples undergoing intracytoplasmic sperm injection. A study of 370 couples and review of the literature. International Journal of Andrology 27, 178–182. Morel F, Gallon F, Amice V et al. 2002 Sex chromosome mosaicism in couples undergoing intracytoplasmic sperm injection. Human Reproduction 17, 2552–2555. Mroz K, Hassold TJ, Hunt PA 1999 Meiotic aneuploidy in the XXY mouse: evidence that a compromised testicular environment increases the incidence of meiotic errors. Human Reproduction 14, 1151–1156. Munné S 2005 Analysis of chromosomal segregation during preimplantation genetic diagnosis in both male and female translocation heterozygotes. Cytogenetic and Genome Research 111, 305–309. Navarro J, Benet J, Martorell MR et al. 1993 Segregation analysis in a man heterozygous for a pericentric inversion of chromosome 7(p13;q36) by sperm chromosome studies. American Journal of Human Genetics 53, 214–219. Nicolson R, Bhalerao S, Sloman L 1998 47,XYY karyotypes and pervasive developmental disorders. Canadian Journal of Psychiatry 43, 619–622. Nielsen J, Wohlert M 1991 Chromosome abnormalities found among 34910 newborn children: results from a 13-year incidence study in Århus, Denmark. Human Genetics 87, 81–83. Olesen C, Hansen C, Bendsen E et al. 2001 Identification of human candidate genes for male infertility by digital differential display. Molecular Human Reproduction 7, 11–20. Otani T, Roche M, Mizuike M et al. 2006 Preimplantation genetic diagnosis significantly improves the pregnancy outcome of translocation carriers with a history of recurrent miscarriage and unsuccessful pregnancies. Reproductive BioMedicine Online 13, 869–874. Pandiyan N, Jequier AM 1996 Mitotic chromosomal anomalies among 1210 infertile men. Human Reproduction 11, 2604–2608. Papanikolaou EG, Vernaeve G, Kolibianakis E et al. 2005 Is chromosome analysis mandatory in the initial investigation in normovulatory women seeking infertility treatment? Human Reproduction 20, 2899–2903. Peschka B, Leygraaf J, Van der Ven K et al. 1999 Type and frequency of chromosome aberrations in 781 couples undergoing intracytoplasmic sperm injection. Human Reproduction 14, 2257–2263. Portnoï MF, Aboura A, Tachdjian G et al. 2006 Molecular cytogenetic studies of Xq critical regions in premature ovarian failure patients. Human Reproduction 21, 2329–2334. Ravel C, Letur H, Le Lannou D et al. 2007 High incidence of chromosomal abnormalities in oocyte donors. Fertility Sterility 87,
439–441. Ravel C, Berthaut I, Bresson JL et al. 2006 Prevalence of chromosomal abnormalities in phenotypically normal and fertile adult males: large-scale survey of over 10,000 sperm donor karyotypes. Human Reproduction 21, 1484–1489. Rives N, Simeon N, Milazzo JP et al. 2003 Meiotic segregation of sex chromosomes in mosaic and non-mosaic XYY males : case report and review of the literature. International Journal of Andrology 26, 242–249. Schiff JD, Palermo GD, Veeck LL et al. 2005 Success of testicular sperm extraction and intracytoplasmic sperm injection in men with Klinefelter syndrome. Journal of Clinical Endocrinology and Metabolism 90, 6263–6267. Schlessinger D, Herrera L, Crisponi L et al. 2002 Genes and translocations involved in POF. American Journal of Medical Genetics 111, 328–333. Scholtes MC, Behrend C, Dietzel-Dahmen J et al. 1998 Chromosomal aberrations in couples undergoing intracytoplasmic sperm injection: influence on implantation and ongoing pregnancy rates. Fertility and Sterility 70, 933–937. Schreurs A, Legius E, Meuleman C et al. 2000 Increased frequency of chromosomal abnormalities in female partners of couples undergoing in vitro fertilization or intracytoplasmic sperm injection. Fertility and Sterility 74, 94–96. Shi Q, Martin RH 2000 Multicolor fluorescence in situ hybridization analysis of meiotic chromosome segregation in a 47,XYY male and a review of the literature. American Journal of Medical Genetics 93, 40–46. Siffroi JP, Le Bourhis C, Rouba H et al. 2000 Sex chromosome mosaicism in infertile males carrying Y chromosome long arm deletions. Human Reproduction 15, 2559–2562. Siffroi JP, Benzacken B, Straub B et al. 1997 Assisted reproductive technology and complex chromosomal rearrangements: the limits of ICSI. Molecular Human Reproduction 3, 847–851. Skakkebaek NE, Philip J, Hammen R 1969 Meiotic chromosomes in Klinefelter’s syndrome. Nature 221, 1075–1076. Solari AJ 1999 Synaptonemal complex analysis in human male infertility. European Journal of Histochemistry 43, 265–276. Tiepolo L, Zuffardi O 1976 Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Human Genetics 34, 119–124. Tuck-Muller CM, Chen H, Martinez JE et al. 1995 Isodicentric Y chromosome: cytogenetic, molecular and clinical studies and review of the literature. Human Genetics 96, 119–129. Tuerlings JH, de France HF, Hamers A et al. 1998 Chromosome studies in 1792 males prior to intra-cytoplasmic sperm injection: the Dutch experience. European Journal of Human Genetics 6, 194–200. Van Assche E, Bonduelle M, Tournaye H et al. 1996 Cytogenetics of infertile men. Human Reproduction 11 (Suppl. 4), 1–24. Van Steirteghem A, Bonduelle M, Devroey P, Liebaers I 2002 Followup of children born after ICSI. Human Reproduction Update 8, 111–116. Vidal F, Navarro J, Templado C et al. 1984 Synaptonemal complex studies in a mosaic 46,XY/47,XXY male. Human Genetics 66, 306–308. Vincent MC, Daudin M, De MP et al. 2002 Cytogenetic investigations of infertile men with low sperm counts: a 25-year experience. Journal of Andrology 23, 18–22. Vogt PH, Edelmann A, Hirschmann P, Kohler MR 1995 The azoospermia factor (AZF) of the human Y chromosome in Yq11: function and analysis in spermatogenesis. Reproduction, Fertility and Development 7, 685–693. Yoshida A, Miura K, Shirai M 1997 Cytogenetic survey of 1,007 infertile males. Urologia Internationalis 58, 166–176. Received 8 October; refereed 26 October 2007; accepted 28 November 2007.
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Symposium: Genetic and epigenetic aspects of assisted reproduction Underlying karyotype abnormalities in IVF/ICSI patients Jean Pierre Siffroi, MD PhD, has 25 years experience in the cytogenetics and genetics of infertility, first in Hôtel-Dieu hospital, Paris, then in Tenon hospital, and currently in the Armand Trousseau Hospital, a children’s hospital in Paris in which he manages the new Department of Medical Genetics and Embryology.
Dr Jean Pierre Siffroi S Chantot-Bastaraud, C Ravel, JP Siffroi1 Université Pierre et Marie Curie-Paris 6, EA 1533, Paris, F-75012 France and AP-HP, Hôpital Tenon, Service d’Histologie Biologie de la Reproduction et Cytogénétique, Paris, F-75020 France 1 Correspondence: e-mail: [email protected]
Abstract Cytogenetic investigations are performed in couples asking for IVF or intracytoplasmic sperm injection (ICSI) treatment. These serve a diagnostic purpose because male or female infertility might have a chromosomal origin. Chromosomal aberrations found in these patients include numerical abnormalities, such as Klinefelter syndrome, XYY karyotype or Turner syndrome and its variants; sex reversions, such as XX males or XY females; and also structural abnormalities, such as Robertsonian or reciprocal translocations and inversions. Finding the chromosomal origin of infertility in a patient also has a prognostic value because it aids the management of pregnancies obtained after IVF or ICSI and may lead to a proposal of prenatal or preimplantation genetic diagnosis. Keywords: chromosomal abnormalities, cytogenetics, ICSI, karyotype
Introduction
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Assisted reproduction techniques, and particularly intracytoplasmic sperm injection (ICSI), bypass natural barriers to fertilization such as negative selection against genetic factors leading to impairment of gametogenesis. As a consequence, they allow the transmission of these factors to the offspring, but also expose the latter to an increased risk of developing malformations or diseases of genetic origin.
to be present in a small, but significant percentage of couples consulting for infertility (Van Assche et al., 1996). Even if some of these chromosome abnormalities, such as Klinefelter or Turner syndromes, can be clinically suspected, neither particular familial history nor pathognomonic clinical findings allows the clinical diagnosis of most chromosomal anomalies in infertile patients who are otherwise phenotypically normal.
Because gametogenesis is a serial process, any defect in one of the hundreds or thousands of genes involved could theoretically lead to an arrest in the normal multiplication and/or maturation of gametes and to infertility. Research in transgenic animal models has described infertility associated with some phenotypes, mainly in males (Escalier, 2001). However, the identification of a particular gene defect responsible for failure of gamete production or maturation in humans is quite a rare event in clinical practice. On the other hand, chromosomal abnormalities have been shown
Therefore, karyotyping has become an obligatory biological examination in the management of these patients. In man, studies have shown a high frequency of chromosomal aberrations in blood cells from infertile patients (Chandley, 1979; Bourrouilou et al., 1985; DeBraekeleer and Dao, 1991; Pandyan and Jequier, 1996; Mau et al., 1997; Meschede et al., 1998; Peschka et al., 1999; Gekas et al., 2001; Vincent et al., 2002). The prevalence ranges from 2.2 to 13.1%, but most studies report values of about 4–5%, which represents an 8–10-fold increase when
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Symposium- Karyotype abnormalities in IVF/ICSI patients - S Chantot-Bastaraud et al.
compared with values observed in unselected newborns (Nielsen and Wohlert, 1991). Variations are due to different recruitment procedures (i.e. the severity of the sperm cell depletion in ejaculate), or to the interpretation of cytogenetic results (i.e. the question of whether variants in the karyotype are pathological or not). Whatever the frequency of chromosomal abnormalities in infertile men, the general consensus is that the probability of finding an abnormality in a patient’s karyotype is negatively correlated to sperm count. However, this assertion does not mean that men carrying an abnormal karyotype will necessarily develop infertility. Indeed, a recent study has shown that the frequency of abnormal karyotypes in normospermic sperm donors was the same as that at birth, which means that most newborns carrying an abnormal karyotype will never consult for fertility problems (Ravel et al., 2006). In infertile women or the female partner in an infertile couple, several studies have also found an increase in the frequency of chromosome abnormalities (Meschede et al., 1998; Sholtes et al., 1998; Peschka et al., 1999; Schreurs et al., 2000; Gekas et al., 2001; Morel et al., 2004; Clementini et al., 2005). Some authors have pointed out that chromosomal abnormalities were more frequent in women with secondary infertility than in those with primary infertility (Clementini et al., 2005; Papanikolaou et al., 2005). However, the relationship between abnormal karyotype and female infertility, apart from Turner syndrome, is not as clear as the situation in men. Most observations suggest that chromosome aberrations in women seldom interfere with the meiotic process and that the presence of a chromosomal abnormality is often compatible with oogenesis, leading to a higher risk of aneuploid gamete formation (Hunt and Hassold, 2002). The aim of this paper is to review the main chromosomal aberrations found in infertile couples seeking IVF treatment, with or without ICSI, to determine, as far as possible, how these abnormalities act on gametogenesis and to measure the genetic consequences on offspring.
Chromosomal abnormalities in infertile couples Male infertility may be the result of many causes such as cryptorchidism, structural abnormalities of the genital tract, genital infections, previous scrotal or inguinal surgery, varicoceles, hypogonadotrophic hypogonadism, chronic illness, medications and exposure to chemicals. However, in about 40% of men, no evident cause of the infertility is found and a genetic factor may be suspected. Determining the genetic cause of male infertility requires careful clinical and biological examination of the patient that can distinguish between obstructive and nonobstructive origins. In the former, the main genetic origin consists of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which are commonly associated with congenital vas deferens abnormalities (Georgiou et al., 2006; Ferlin et al., 2007). In non-obstructive infertility, a general consensus indicates that the study of the karyotype is justified only for oligozoospermia below 10 × 106 spermatozoa/ml and for non-obstructive azoospermia (Egozcue et al., 2000). Males with azoospermia are more likely to carry either a numerical or structural sex chromosome abnormality than those with oligospermia, whereas males with oligospermia are more likely to have an autosomal rearrangement in their karyotype
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(translocations, inversions, supernumerary markers) than those with azoospermia.
Numerical abnormalities 47,XXY males Klinefelter syndrome (KS) is the most frequent gonosomal anomaly in man. It occurs in 0.1–0.2% of newborn males, but the frequency rises from 0.5% in oligozoospermic individuals to 11% in azoospermic patients (Huynh et al., 2002). KS accounts for 67% of abnormal karyotypes in azoospermic males and for 19% in oligozoospermic patients (Mau-Holzmann, 2005). About 80% of KS cases carry a homogenous 47,XXY karyotype and azoospermia is almost inevitable (Lanfranco et al., 2004). The remaining 20% have a more complex karyotype that can include various mosaics, the most frequent of which is the 46,XY/47,XXY mosaicism. If it is easy to make the diagnosis of a mosaic karyotype when several cell lines are observed in blood cell metaphases, the reverse is not true, and in practice it is impossible to assert that an apparently non-mosaic 47,XXY man does not carry any tissular mosaicism, especially in the testis. This is an important point for the management of these patients because the question of whether a testicular biopsy should be proposed to 47,XXY azoospermic men often arises. Indeed, in these men, surgical sperm retrieval has revealed spermatozoa in up to half of cases (Schiff et al., 2005). Mosaic KS cases may be either oligozoospermic or azoospermic but the probability of finding testicular spermatozoa is much higher. Therefore, treating KS patients with ICSI raises some concerns that the offspring may inherit an XXY, XXX or another abnormal karyotype. A number of meiotic studies have revealed that XXY cells are unable to enter meiosis (Kjessler, 1966), but others have suggested that at least some of them can reach the primary spermatocyte stage (Skakkebaek et al., 1969; Vidal et al., 1984). Actually, most fluorescence in-situ hybridization (FISH) studies performed on mosaic and non-mosaic KS spermatozoa have shown a paradoxical low frequency of disomic XY or XX cells, only a small percentage (reviewed by Ferlin et al., 2005), which is quite different from the theoretical 50% monosomic:50% disomic cell ratio which could be expected if XXY germ cells were able to produce spermatozoa. These results suggest that abnormal XXY cells are unable to enter meiosis and that spermatozoa in KS patients originate from normal XY spermatogonia due to a testicular mosaicism (Guttenbach et al., 1997; Foresta et al., 1998; Blanco et al., 2001). The low, but significant, frequency of disomic sperm cells should be the result of an increase in segregation errors occurring in normal 46,XY germ cells within a deleterious testicular environment, as shown in XXY mouse models (Mroz et al., 1999). In testicular biopsies from KS patients, it has been observed that only XY primary spermatocytes are able to continue their differentiation (Bergère et al., 2002). However, the possibility that some XXY cells could achieve meiosis is not completely excluded. An increased frequency of chromosomal segregation errors in KS patients with spermatozoa raises the question whether chromosomes other than X and Y could also be involved, leading to potential autosomal abnormalities in offspring, such as trisomy 21 (Hennebicq et al., 2001). Although most children born after ICSI with sperm from KS patients have a normal karyotype
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(Lanfranco et al., 2004), the opportunity to propose either a prenatal or a preimplantation diagnosis in pregnancies obtained from KS fathers is still a matter of question.
47,XYY males This is the second most frequent gonosomal abnormality after KS. Most 47,XYY men are fertile but the frequency of this syndrome in infertile males is about four-fold higher than in the normal fertile population (0.2–0.3% versus 0.05% at birth) (Yoshida et al., 1997). Because most 47,XYY men are not usually diagnosed, the frequency observed in the population referred for karyotyping is about 0.1% overall. Such an estimation takes into account the fact that some 47,XYY children develop mental disorders, such as pervasive developmental disorders or autism (Nicolson et al., 1998; Geerts et al., 2003). Thus, spermatogenesis in XYY males ranges from normality to severe failure. No valuable explanation accounts for this variability other than different meiotic pairing configurations of the extra Y chromosome with the other sex chromosomes, which could explain a different impact on spermatogenesis (Blanco et al., 2001; Rives et al., 2003). The common property of some Y chromosomes to be prone to malsegregation errors in meiosis II and to carry a structural abnormality such as a microdeletion could be another explanation. However, this has never been demonstrated in XYY infertile men, although such deleted Y chromosomes have been shown to be mitotically unstable (Siffroi et al., 2000). Whatever it may be, most FISH studies performed on sperm cells from XYY men have shown that the majority of the spermatozoa produced by these patients contain a balanced karyotype, which suggests either the systematic elimination of the supernumerary Y chromosome during meiosis or the degeneration of the abnormal aneuploid cells (Shi and Martin, 2000).
45,X0/46,XY males This type of mosaicism is a common finding during the cytogenetic evaluation of infertile men and several studies have reported an increased frequency of low-level mosaics of this type in the male partner in couples examined prior to ICSI (Meschede et al., 1998; Peschka et al., 1999; Gekas et al., 2001). Morel et al. (2002) did not find any difference between fertile and ICSI-treated couples regarding the existence of sex chromosome mosaicism. However, the existence of a 45,X0 cell line in testis may be considered a pejorative element, which could accentuate the effect of other infertility factors, as shown in azoospermia factor c (AZFc)deleted patients (Jaruzelska et al., 2001).
Extra structurally abnormal chromosomes
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Small supernumerary marker chromosomes (sSMC) are defined as additional centric chromosome fragments, which are too small to be identified or characterized unambiguously by banding cytogenetic techniques. Their identification by FISH has revealed that they are often made of two symmetrical short arms of acrocentric chromosomes separated by a centromere (i.e. disposed in mirror). Men carrying a supernumerary marker chromosome are usually phenotypically normal and fertile. However the frequency of sSMC is increased in the infertile population, they are found in 0.043% of newborns, in 0.035% of sperm donors and in 0.125% of infertile subjects (Tuerlings et al., 1998; Eggermann
et al., 2002; Ravel et al., 2006; Liehr and Weise, 2007). Their role in spermatogenesis failure is not clear but they could act by disrupting normal chromosome movements and/or sex body formation during meiosis.
Turner syndrome and variants Turner syndrome (TS) phenotype is characterized by short stature, gonadal dysgenesis and other somatic stigmata such as webbed neck and cubitus valgus. The prevalence is 1 in 1500 to 1 in 2500 females. Ovarian dysfunction in women with TS seems to be due to an abnormal and rapid degeneration of oocytes and follicles, resulting in primary amenorrhoea. Because of a total lack of germ cells, these patients cannot enter assisted reproduction programmes, except gamete donation, and therefore a non-mosaic 45,X0 karyotype is rarely reported in cytogenetic studies dealing with a couple’s infertility. However, about 30% of mosaic TS patients have partial ovarian function at puberty but only 2–5% of them become pregnant. In some TS patients, the karyotype shows both a normal X and a structurally rearranged X chromosome. The existence of an isochromosome for the long arm of the X chromosome [i(Xq)] accounts for up to 16% of TS cases and it is usually associated with a 45,X0 cell line (Kleczkowska et al., 1990). Most of these women also suffer from gonadal dysgenesis without any possibility of natural or medically assisted pregnancy. The phenotype associated with deletions of the short arm [del(Xp)] is often less severe and fertility may be preserved. In addition, structurally abnormal X chromosomes, like large ring X [r(X)], can be devoid of any consequence on ovarian function and be transmitted from fertile mothers to their daughters. The main problem encountered in cytogenetic surveys performed in infertile women is the high frequency of gonosomal mosaicism in the blood cells of patients. A basal level of low mosaicism probably exists in many fertile women, as seen in oocyte donors (Ravel et al., 2007) and the question arises whether or not such mosaicism may be responsible for female infertility when found in the course of IVF or ICSI procedures. It is likely that an abnormal 45,X/46,XX karyotype in association with clinical signs of TS may constitute a true constitutional mosaicism and that the risk for an abnormal pregnancy outcome may be substantial (Kaneko et al., 1990). On the other hand, Gardner and Sutherland (2004) suggest that sex chromosome mosaicism with less than 10% of abnormal cells probably does not indicate any specific reproductive risks in women without TS stigmata.
47,XXX syndrome Triple X women are usually fertile and give birth to children with a normal karyotype (i.e. without either a XXY or a XXX constitution), which means that the extra X chromosome is eliminated during female meiosis. Among a group of 4327 women karyotyped before ICSI, four were found to have a 47,XXX karyotype, which corresponds directly to the frequency found at birth (Mau-Holzmann, 2005). However, some studies have pointed out that XXX women could have an increased risk of developing premature ovarian failure (POF), probably through the occurrence of immunological disorders (Castillo et al., 1992; Guichet et al., 1996; Holland, 2001).
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Symposium- Karyotype abnormalities in IVF/ICSI patients - S Chantot-Bastaraud et al.
Sex reversion: XX males and XY females 46,XX males This rare anomaly is found in about 1 in 20,000 newborns but in approximately 0.9% of azoospermic patients (Mau-Holzmann, 2005). In most cases, normal male differentiation occurs during embryogenesis because of the presence of the SRY gene at the tip of the paternal X chromosome. Indeed, an abnormal recombination during paternal meiosis between the terminal short arms of both X and Y chromosomes, just proximal to the homologous pseudoautosomal region PAR1, leads to the translocation of the SRY gene onto the X chromosome. In some very rare cases, the SRY gene is absent and the male phenotype results either from the inactivation of a testis-determining repressor or from the activation of another gene implied in the sex determination cascade leading to testis formation (Ergun-Longmire et al., 2005). These patients present a testis hypotrophy and a lack of germinal cells resulting in total sterility. Treatment by ICSI is not possible and they must be referred to centres of gamete donation.
46,XY females Male to female sex reversion may be due to multiple causes affecting either the first step of sex determination (deletion of the short arm of the Y chromosome or a mutation in the SRY gene) or the subsequent steps of fetus masculinization (mutations in the androgen receptor gene or in the 5-alpha reductase gene). In all cases, females present with primary amenorrhoea and are sterile because they have non-functional streak gonads or intraabdominal testes. However, discovering a 46,XY female may also occur in the course of genetic investigation of infertility.
Structural abnormalities Chromosome behaviour at meiosis is a complex process, which is easily disturbed by the existence of an abnormality in the structure of chromosomes. The normal pairing of homologous chromosomes at the pachytene stage is an absolute prerequisite for meiosis and every disruption in chromosome pairing will lead to the death of the germ cells and their elimination by apoptosis. In the case of chromosomal exchanges or fusion, homologous chromosomes are not able to form normal bivalents and pair as tri or tetravalents, which are complex meiotic figures. Studies of affected meiosis, by silver staining or three-dimensional reconstruction in electron microscopy, have revealed many regions of asynapsis or heterosynapsis (Solari, 1999). As a consequence, in the male, but not in the female, abnormal chromosomes are often associated with the sex body, which is rich in proteins with a high affinity for the non-synapsed regions of the X and Y chromosomes. By their physical contact with the sex body, the autosomal segments would thus be inactivated (Guichaoua et al., 1991) and their transcription level would be decreased, if not abolished (Jaafar et al., 1993). The inactivation of autosomal chromosomal fragments associated with a structural abnormality of the karyotype may occur in a variable percentage of meiotic cells. This explains the moderate testis alteration seen in some patients and the differences observed between two individuals carrying the same chromosomal abnormality in a family. Therefore, infertility in translocation carriers appears to RBMOnline®
be the result of an impairment of the nuclear events in prophase I rather than the consequence of gene disruption at chromosomal breakpoints, although some testis-expressed transcripts have been shown to map near recurrent translocation sites found in infertile men (Olesen et al., 2001).
Robertsonian translocations Also called centric fusion, Robertsonian translocation consists of the fusion of two acrocentric chromosomes (i.e. chromosomes 13, 14, 15, 21 and 22) at their centromere (Figure 1). The loss of the short arms of the chromosomes involved is neutral for the cells, since the genes located in these regions are highly repeated in all acrocentric chromosome short arms. All combinations between acrocentric chromosomes are possible but the more frequent ones are the t(13;14) and the t(14;21). Robertsonian translocations occur in about 1 in 1000 newborns and the frequency rises to 0.2% in azoospermic males and to 1.5% in oligozoospermic men. Curiously, the prevalence of t(13;14) in normospermic gamete donors is the same as that in newborns, which means that most carriers do not develop fertility problems in adulthood (Ravel et al., 2006). The way in which Robertsonian translocations act on male germ cell development is probably through an association between the translocated chromosomes, which form a trivalent at meiosis, and the sex body. However, translocation carriers have very different fertility status, even in the same family, so the precise mechanism by which these translocations affect fertility is not clear. In women, Robertsonian translocations do not seem to affect gametogenesis. When receiving IVF or ICSI treatment, Robertsonian translocation carriers can transmit their translocation in an unbalanced state, thus leading to a trisomic fetus for one or the other chromosome involved. Except for trisomy 21, all other trisomies for acrocentric chromosomes are thought to terminate early in pregnancy or to give rise to severe fetal anomalies that are easily recognised at ultrasound examination. However, clinicians must be aware that such trisomies can be repaired at the first division of the zygote by a mechanism called trisomy rescue, which potentially leads to uniparental disomy. For chromosomes 14 and 15, both paternal and maternal uniparental disomies are pathological and give rise to severely affected children (i.e. bone malformations, growth retardation, Angelman or Prader-Willi syndromes) despite an apparently normal or balanced karyotype.
Reciprocal autosomal translocations Reciprocal translocations are defined as an exchange of chromosomal material between arms of two non-homologous chromosomes, usually without any apparent change in the global amount of genetic material (Figure 2). Like Robertsonian translocations, reciprocal translocations affect about 1 in 1000 newborns, which means that 1 in 500 individuals in the general population carry a chromosomal translocation. The frequency of balanced reciprocal translocation in infertile males is estimated to be 5–10 times higher than among newborns, according to size population and sperm criteria for patient inclusion, with a mean frequency at 0.7% (review by Mau-Holzmann., 2005). The cellular mechanism by which reciprocal translocations impair spermatogenesis is thought to be the same as that in Robertsonian translocations (i.e. an abnormal association with the sex body at
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13;14
14
Figure 1. Robertsonian t(13;14) translocation in RHG banding.
the pachytene stage), except that homologous segments of the translocated chromosomes associate one to each other to form a quadrivalent figure. This complex meiotic type of synapsis adopts a more-or-less flat configuration, according to the size of both centric and non-centric segments, which is known to play a role in the modes of segregation at the end of pachytene: in other words, the more flat a quadrivalent is, the higher is the probability that the chromosomes will segregate in an unbalanced mode (Cans et al., 1993). Therefore, the diagnosis of a reciprocal translocation in an infertile patient has important consequences for both ICSI treatment and pregnancy management. In some rare cases, reciprocal translocations involve more than two chromosome pairs and the probability of obtaining a chromosomally balanced spermatozoon for ICSI becomes very low. Other assisted
a
b Figure 2. (a) GTG banded karyotype showing a t(1;22)(p13;q11.21) translocation (arrows) in an infertile man. (b) Fluorescence insitu hybridization on metaphase spreads with whole chromosome painting probes of chromosome 1 and chromosome 22. (Whole chromosome painting probes, Oncor, Gaithersburg, MD, USA.)
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reproduction procedures, such as gamete donation, might be proposed to couples in these cases (Siffroi et al., 1997). Although reciprocal translocations are not thought to interfere with the process of female meiosis, the frequency of these chromosomal rearrangements in women is particularly high in couples receiving ICSI treatment, at about 0.7%, which is very similar to that observed in infertile men (review by MauHolzmann, 2005). In the general population, female carriers of reciprocal translocations are diagnosed most often after recurrent spontaneous abortions or after the birth of a child carrying an unbalanced karyotype. Finding a high proportion of such carriers in ICSI couples means that these women probably develop a variable level of hypofertility, which has no consequence when their male partner is normally fertile, but leads to couple infertility and to ICSI when a contributing male factor is present. As a consequence, cytogenetic investigations in ICSI couples should be performed in both members.
X autosome translocations in women The effects on fertility of balanced X-autosome translocations are variable and depend on several parameters such as breakpoint localization and X chromosome inactivation profile. Usually the normal X chromosome is inactivated to avoid the abnormal inactivation of the autosomal translocated segment and female carriers are phenotypically normal and fertile. However gonadal dysgenesis may occur in some of them if the breakpoint falls within the two critical Xq regions known to be involved in premature ovarian failure (POF) (POF1 at Xq21.3-q27 and POF2 at Xq13.3-q21.1) (Schlessinger et al., 2002; Portnoï et al., 2006).
Chromosomal inversions Chromosomal inversions are structural intrachromosomal rearrangements resulting from two breaks within a single chromosome followed by a 180° rotation of the chromatin between these breaks. According to the position of the centromere, within or outside the inverted segment, pericentric and paracentric inversions are observed. Autosomal inversions are the second most common type of chromosomal rearrangements after translocations. They are found in 0.022% of children at birth and 0.088% of sperm donors (Ravel et al., 2006). Most pericentric inversions affect chromosome 2 or the heterochromatic regions of chromosomes 1, 9, 16 and Y and they are considered as non-pathological variants. Other autosomal pericentric inversions are not usually associated with infertility but rather with the birth of an abnormal child carrying the inversion in an unbalanced state. Indeed, meiotic pairing of the inverted chromosomes with its own homologous partner sometimes needs the formation of an inversion loop, depending on the length of the inverted segment. The occurrence of an odd number of crossovers within the loop results in the production of chromosomes that are duplicated or deleted for the regions outside the inversion. Sperm FISH studies of male carrier spermatozoa is a good way to assess the ratio of balanced and unbalanced sperm cells before any conception, with or without assisted reproduction treatment (Chantot-Bastaraud et al., 2007).
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Most inversions are identified fortuitously but, in some cases, autosomal pericentric inversions have been associated with azoospermia or oligospermia (Gabriel-Robez et al., 1988; Navarro et al., 1993; Meschede et al., 1994; Chantot-Bastaraud et al., 2007) and some studies have pointed out that pericentric inversions could reduce male fertility (De Braekeleer and Dao, 1991; Guttenbach et al., 1997; Mau-Holzmann, 2005). In the same way as translocations, the existence of a synapsis failure at the base or in the inversion loop could explain spermatogenesis impairment. Moreover, in paracentric inversion, recombinations within the loop give both dicentric and acentric chromosomes, which are likely to block meiosis, and thus lead to germ cell death and apoptosis. In female partners of ICSI couples, an increased frequency of inversions has also been described, which could be due to fertilization and/or implantation failures because of the existence of a high proportion of recombinant unbalanced chromosomes in oocytes (review by Mau-Holzmann, 2005).
Heterochromatic polymorphism The specific case of the Y chromosome Tiepolo and Zuffardi (1976), by studying large final deletions of Yq among 6 azoospermic patients, described a region controlling spermatogenesis in the proximal euchromatic part of the Y long arm (Yq11), named AZF for azoospermia factor. The existence of this factor has subsequently been confirmed by many cytogenetic observations showing various abnormal Y chromosomes in infertile men (i.e. isochromosomes Yp, dicentric isochromosomes Yp, Y ring chromosomes or Y-autosome translocations) and by the description of molecular deletions of the AZF region (TuckMuller et al., 1995; Vogt et al., 1995). The latter are not detectable at classical cytogenetic examination and have been extensively reviewed elsewhere (McElreavey et al., 2000). The frequency of Y-autosome translocations reported is approximately 1 in 2000 (Alves et al., 2002). Breakpoints are generally localized in Yq12 (heterochromatic region) and Yq11 (AZF region). In the former, only Y heterochromatic material is transposed on another chromosome, most often on an acrocentric short arm, and the carrier is usually fertile. In contrast, when the breakpoint is localized in Yq11, azoospermia generally occurs. A particular mention should be made of isodicentric Y chromosomes, which consist of two short arms disposed in mirror, separated by two centromeres and a fragment of long arm. These chromosomes are probably the result of abnormal sister chromatid recombinations in the long arm. They have lost a great part of the AZF regions and thus are observed in azoospermic men. However, because the resulting chromosome has a size nearly similar to that of a normal Y chromosome, Y isodicentric are underestimated in routine cytogenetic studies and need particular FISH techniques to be diagnosed (Figure 3).
Genetic management of IVF/ICSI pregnancies Possible health problems in IVF/ICSI children are thought to be due to parental infertility itself or as a consequence of increased frequency of multiple pregnancies (Van Steirteghem et al., 2002; Cohen, 2007). However, pregnancies obtained after ICSI
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Figure 3. Cut-out of the sex chromosomes shows the abnormal idic(Yp) chromosome in RHG banding (a) and GTG banding (b). The abnormal Y chromosome is the correct size and may easily be mistaken for a normal one. Fluorescence in-situ hybridization (FISH) on metaphase spreads shows (c) SRY probe duplication (double arrows); (d) DYZ3 (Y centromere) probe duplication (double arrows); (e) SHOX (RP13–391G2 at Xp22.33) probe duplication in (double left arrows) and UTY (RP11–386L3 at Y q11.2 near AZFa region) (single right arrow). All FISH results indicate the existence of two short arms, two centromeres and a fragment of the long arm with a breakpoint between AZFa and AZFb. (Probes obtained from either Vysis, Abbott Laboratories or BACPAC Resources. BAC-probes selected from the University of California Santa Cruz Genome Browser.)
procedures are known to have a higher risk of de novo sex chromosomal aberrations and paternally inherited structural abnormalities (Van Steirteghem et al., 2002). However, the slightly increased risk generally observed does not justify any invasive prenatal diagnosis procedures, owing to the fact that many of these pregnancies have been achieved after several years of infertility.
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Most abnormal karyotypes in infertile couples are diagnosed at the initial genetic screening made in these cases. As a consequence, couples at risk of giving birth to a child carrying an unbalanced karyotype are aware of the situation before the pregnancy and can ask for a prenatal diagnosis after chorionic villus or amniotic fluid sampling. To date, many of them enter a preimplantation genetic diagnosis (PGD) procedure, which significantly increases their probability of having a chromosomally normal pregnancy and, at term, delivery of a healthy baby (Otani et al., 2006). Seeking PGD appears particularly easy, since most of these couples need IVF or ICSI to achieve a pregnancy, but the exact consequences of blastomere biopsy are still debated (Cohen and Grifo, 2007).
When performed for cytogenetic reasons, PGD allows the indirect karyotyping of a zygote using FISH techniques with appropriate fluorescent probes. Although FISH-PGD is unable to distinguish between zygotes carrying a normal karyotype and those with a translocated but balanced one, it allows the diagnosis of all the chromosomal imbalances generated by both Robertsonian and reciprocal translocations (Mackie Ogilvie and Scriven, 2004; Munné, 2005) and also those resulting from chromosomal inversions (Escudero et al., 2001).
Conclusion Couples engaged in IVF/ICSI procedures often present with male and/or female idiopathic infertility, which is known to have a frequent genetic origin. Among these, chromosomal abnormalities have been described as a major cause of gametogenesis failure. They can be easily diagnosed using conventional cytogenetic techniques and a large number of patients have been reported. RBMOnline®
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When engaged in one or other assisted reproduction technique, and particularly ICSI, couples carrying a chromosomal abnormality must be aware of the consequences for the offspring. Indeed, by selecting a single spermatozoon and an oocyte for injection, ICSI bypasses the natural process of selection, which is thought to occur during both natural conception and conventional IVF. As a consequence, it may allow the fertilization and the development of zygotes with potentially unbalanced karyotypes. Proposals of prenatal diagnosis or of PGD, when available, must be discussed with these couples.
Acknowledgements The authors thank Ken McElreavey for his helpful correction of the manuscript.
References Alves C, Carvalho F, Cremades N et al. 2002 Unique (Y;13) translocation in a male with oligozoospermia: cytogenetic and molecular studies. European Journal of Human Genetics 10, 467–474. Bergère M, Wainer R, Nataf V et al. 2002 Biopsied testis cells of four 47,XXY patients: fluorescence in-situ hybridization and ICSI results. Human Reproduction 17, 32–37. Blanco J, Egozcue J, Vidal F 2001 Meiotic behaviour of the sex chromosomes in three patients with sex chromosome anomalies (47,XXY, mosaic 46,XY/47,XXY and 47,XYY) assessed by fluorescence in-situ hybridization. Human Reproduction 16, 887–892. Bourrouillou G, Dastugue N, Colombies P 1985 Chromosome studies in 952 infertile males with a sperm count below 10 million/ml. Human Genetics 71, 366–367. Cans C, Cohen O, Mermet MA et al. 1993 Human reciprocal translocations: is the unbalanced mode at birth predictable? Human Genetics 91, 228–232. Castillo S, Lopez F, Tobella L et al. 1992 The cytogenetics of premature ovarian failure. Revista Chilena de Obstetricia y Ginecología 57, 341–345. Chandley AC 1979 The chromosomal basis of human infertility. British Medical Bulletin 35, 181–186. Chantot-Bastaraud S, Ravel C, Berthaut I et al. 2007 SpermFISH analysis in a pericentric chromosome 1 inversion, 46,XY,inv(1)(p22q42), associated with infertility. Molecular Human Reproduction 13, 55–59. Clementini E, Palka C, Iezzi I et al. 2005 Prevalence of chromosomal abnormalities in 2078 infertile couples referred for assisted reproductive techniques. Human Reproduction 20, 437–442. Cohen J 2007 Infertile couples, assisted reproduction and increased risks to the children. Reproductive BioMedicine Online 15, 245–246. Cohen J, Grifo JA 2007 Multicentre trial of preimplantation genetic screening reported in the New England Journal of Medicine: an in-depth look at the findings. Reproductive BioMedicine Online 15, 365–366. De Braekeleer M, Dao TN 1991 Cytogenetic studies in male infertility: a review. Human Reproduction 6, 245–250. Eggermann K, Mau UA, Bujdoso G et al. 2002 Supernumerary marker chromosomes derived from chromosome 15: analysis of 32 new cases. Clinical Genetics 62, 89–93. Egozcue S, Blanco J, Vendrell JM, et al. 2000 Human male infertility : chromosomal anomalies, meiotic disorders, abnormal spermatozoa and recurrent abortion. Human Reproduction Update 6, 93–105. Ergun-Longmire B, Vinci G, Alonso L et al. 2005 Clinical, hormonal and cytogenetic evaluation of 46,XX males and review of the literature. Journal of Pediatric Endocrinology and Metabolism 18, 739–748. Escalier D 2001 Impact of genetic engineering on the understanding
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of spermatogenesis. Human Reproduction Update 7, 191–210. Escudero T, Lee M, Stevens J et al. 2001 Preimplantation genetic diagnosis of pericentric inversions. Prenatal Diagnosis 21, 760–766. Ferlin A, Raicu F, Gatta V et al. 2007 Male infertility: role of genetic background. Reproductive BioMedicine Online 14, 734-745. Ferlin A, Garolla A, Foresta C 2005 Chromosome abnormalities in sperm of individuals with constitutional sex chromosomal abnormalities. Cytogenetics and Genome Research 111, 310-316. Foresta C, Galeazzi C, Bettella A et al. 1998 High incidence of sperm sex chromosomes aneuploidies in two patients with Klinefelter’s syndrome. Journal of Clinical Endocrinology and Metabolism 83, 203-205. Gabriel-Robez O, Ratomponirina C, Croquette M et al. 1988 Synaptonemal complexes in a subfertile man with a pericentric inversion in chromosome 21. Heterosynapsis without previous homosynapsis. Cytogenetics and Cell Genetics 48, 84–87. Gardner RJM, Sutherland GR 2004 Chromosome abnormalities and genetic counselling, 3rd edition. Oxford University Press, New York. Geerts M, Steyaert J, Fryns JP 2003 The XYY syndrome: a follow-up study on 38 boys. Genetic Counseling 14, 267–279. Gekas J, Thepot F, Turleau C et al. 2001 Chromosomal factors of infertility in candidate couples for ICSI: an equal risk of constitutional aberrations in women and men. Human Reproduction 16, 82–90. Georgiou I, Syrrou M, Pardalidis N et al. 2006. Genetic and epigenetic risks of intracytoplasmic sperm injection method. Asian Journal of Andrology 8, 643–673. Guichaoua MR, de Lanversin A, Cataldo C et al. 1991 Three dimensional reconstruction of human pachytene spermatocyte nuclei of a 17;21 reciprocal translocation carrier: study of XYautosome relationships. Human Genetics 87, 709–715. Guichet A, Briault S, Moraine C, Turleau C 1996 Trisomy X: ACLF (Association des Cytogeneticiens de Langue Francaise) retrospective study. Annales de Génétique 39, 117–122. Guttenbach M, Engel W, Schmid M 1997 Analysis of structural and numerical chromosome abnormalities in sperm of normal men and carriers of constitutional chromosome aberrations. A review. Human Genetics 100, 1–21. Hennebicq S, Pelletier R, Bergues U, Rousseaux S 2001 Risk of trisomy 21 in offspring of patients with Klinefelter’s syndrome. Lancet 357, 2104–2105. Holland CM 2001 47,XXX in an adolescent with premature ovarian failure and autoimmune disease. Journal of Pediatric and Adolescent Gynecology 14, 77–80. Hunt PA, Hassold TJ 2002 Sex matters in meiosis. Science 21, 2181–2183. Huynh T, Mollard R, Trounson A 2002, Selected genetic factors associated with male infertility. Human Reproduction Update 8, 183-198. Jaafar H, Gabriel-Robez O, Rumpler Y 1993 Chromosomal anomalies and disturbance of transcriptional activity at the pachytene stage of meiosis: relationship to male sterility. Cytogenetics and Cell Genetics 64, 273–280. Jaruzelska J, Korcz A, Wojda A et al. 2001 Mosaicism for 45,X cell line may accentuate the severity of spermatogenic defects in men with AZFc deletions. Journal of Medical Genetics 38, 798–802. Kaneko N, Kawagoe S, Hiroi M 1990 Turner’s syndrome-review of the literature with reference to a successful pregnancy outcome. Gynecologic and Obstetric Investigation 29, 81–87. Kjessler B 1966 Karyotype, meiosis and spermatogenesis in a sample of men attending an infertility clinic. Monographs in Human Genetics 2, 1–93. Kleczkowska A, Dmoch E, Kubien E et al. 1990 Cytogenetic findings in a consecutive series of 478 patients with Turner syndrome. The Leuven experience 1965-1989. Genetic Counseling 1, 227–233. Lanfranco F, Kamischke A, Zitzmann M, Nieschlag E 2004 Klinefelter’s syndrome. Lancet 364, 273–283. Liehr T, Weise A 2007 Frequency of small supernumerary marker chromosomes in prenatal, newborn, developmentally retarded
521
Symposium- Karyotype abnormalities in IVF/ICSI patients - S Chantot-Bastaraud et al.
522
and infertility diagnostics. International Journal of Molecular Medicine 19, 719–731. Mackie Ogilvie C, Scriven PN 2004 Preimplantation genetic diagnosis (PGD) for reciprocal translocations. Prenatal Diagnosis 24, 553–555. Mau UA, Backert IT, Kaiser P, Kiesel L 1997 Chromosomal findings in 150 couples referred for genetic counselling prior to intracytoplasmic sperm injection. Human Reproduction 12, 930–937. Mau-Holzmann UA 2005 Somatic chromosomal abnormalities in infertile men and women. Cytogenetic and Genome Research 111, 317–336. McElreavey K, Krausz C, Bishop CE 2000 The human Y chromosome and male infertility. Results and Problems in Cell Differentiation 28, 211–232. Meschede D, Froster UG, Bergmann M, Nieschlag E 1994 Familial pericentric inversion of chromosome 1(p34q23) and male infertility with stage specific spermatogenic arrest. Journal of Medical Genetics 31, 573–575. Meschede D, Lemcke B, Exeler JR et al. 1998 Chromosome abnormalities in 447 couples undergoing intracytoplasmic sperm injection-prevalence, types, sex distribution and reproductive relevance. Human Reproduction 13, 576–582. Morel F, Douet-Guilbert N, Le Bris MJ et al. 2004 Chromosomal abnormalities in couples undergoing intracytoplasmic sperm injection. A study of 370 couples and review of the literature. International Journal of Andrology 27, 178–182. Morel F, Gallon F, Amice V et al. 2002 Sex chromosome mosaicism in couples undergoing intracytoplasmic sperm injection. Human Reproduction 17, 2552–2555. Mroz K, Hassold TJ, Hunt PA 1999 Meiotic aneuploidy in the XXY mouse: evidence that a compromised testicular environment increases the incidence of meiotic errors. Human Reproduction 14, 1151–1156. Munné S 2005 Analysis of chromosomal segregation during preimplantation genetic diagnosis in both male and female translocation heterozygotes. Cytogenetic and Genome Research 111, 305–309. Navarro J, Benet J, Martorell MR et al. 1993 Segregation analysis in a man heterozygous for a pericentric inversion of chromosome 7(p13;q36) by sperm chromosome studies. American Journal of Human Genetics 53, 214–219. Nicolson R, Bhalerao S, Sloman L 1998 47,XYY karyotypes and pervasive developmental disorders. Canadian Journal of Psychiatry 43, 619–622. Nielsen J, Wohlert M 1991 Chromosome abnormalities found among 34910 newborn children: results from a 13-year incidence study in Århus, Denmark. Human Genetics 87, 81–83. Olesen C, Hansen C, Bendsen E et al. 2001 Identification of human candidate genes for male infertility by digital differential display. Molecular Human Reproduction 7, 11–20. Otani T, Roche M, Mizuike M et al. 2006 Preimplantation genetic diagnosis significantly improves the pregnancy outcome of translocation carriers with a history of recurrent miscarriage and unsuccessful pregnancies. Reproductive BioMedicine Online 13, 869–874. Pandiyan N, Jequier AM 1996 Mitotic chromosomal anomalies among 1210 infertile men. Human Reproduction 11, 2604–2608. Papanikolaou EG, Vernaeve G, Kolibianakis E et al. 2005 Is chromosome analysis mandatory in the initial investigation in normovulatory women seeking infertility treatment? Human Reproduction 20, 2899–2903. Peschka B, Leygraaf J, Van der Ven K et al. 1999 Type and frequency of chromosome aberrations in 781 couples undergoing intracytoplasmic sperm injection. Human Reproduction 14, 2257–2263. Portnoï MF, Aboura A, Tachdjian G et al. 2006 Molecular cytogenetic studies of Xq critical regions in premature ovarian failure patients. Human Reproduction 21, 2329–2334. Ravel C, Letur H, Le Lannou D et al. 2007 High incidence of chromosomal abnormalities in oocyte donors. Fertility Sterility 87,
439–441. Ravel C, Berthaut I, Bresson JL et al. 2006 Prevalence of chromosomal abnormalities in phenotypically normal and fertile adult males: large-scale survey of over 10,000 sperm donor karyotypes. Human Reproduction 21, 1484–1489. Rives N, Simeon N, Milazzo JP et al. 2003 Meiotic segregation of sex chromosomes in mosaic and non-mosaic XYY males : case report and review of the literature. International Journal of Andrology 26, 242–249. Schiff JD, Palermo GD, Veeck LL et al. 2005 Success of testicular sperm extraction and intracytoplasmic sperm injection in men with Klinefelter syndrome. Journal of Clinical Endocrinology and Metabolism 90, 6263–6267. Schlessinger D, Herrera L, Crisponi L et al. 2002 Genes and translocations involved in POF. American Journal of Medical Genetics 111, 328–333. Scholtes MC, Behrend C, Dietzel-Dahmen J et al. 1998 Chromosomal aberrations in couples undergoing intracytoplasmic sperm injection: influence on implantation and ongoing pregnancy rates. Fertility and Sterility 70, 933–937. Schreurs A, Legius E, Meuleman C et al. 2000 Increased frequency of chromosomal abnormalities in female partners of couples undergoing in vitro fertilization or intracytoplasmic sperm injection. Fertility and Sterility 74, 94–96. Shi Q, Martin RH 2000 Multicolor fluorescence in situ hybridization analysis of meiotic chromosome segregation in a 47,XYY male and a review of the literature. American Journal of Medical Genetics 93, 40–46. Siffroi JP, Le Bourhis C, Rouba H et al. 2000 Sex chromosome mosaicism in infertile males carrying Y chromosome long arm deletions. Human Reproduction 15, 2559–2562. Siffroi JP, Benzacken B, Straub B et al. 1997 Assisted reproductive technology and complex chromosomal rearrangements: the limits of ICSI. Molecular Human Reproduction 3, 847–851. Skakkebaek NE, Philip J, Hammen R 1969 Meiotic chromosomes in Klinefelter’s syndrome. Nature 221, 1075–1076. Solari AJ 1999 Synaptonemal complex analysis in human male infertility. European Journal of Histochemistry 43, 265–276. Tiepolo L, Zuffardi O 1976 Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Human Genetics 34, 119–124. Tuck-Muller CM, Chen H, Martinez JE et al. 1995 Isodicentric Y chromosome: cytogenetic, molecular and clinical studies and review of the literature. Human Genetics 96, 119–129. Tuerlings JH, de France HF, Hamers A et al. 1998 Chromosome studies in 1792 males prior to intra-cytoplasmic sperm injection: the Dutch experience. European Journal of Human Genetics 6, 194–200. Van Assche E, Bonduelle M, Tournaye H et al. 1996 Cytogenetics of infertile men. Human Reproduction 11 (Suppl. 4), 1–24. Van Steirteghem A, Bonduelle M, Devroey P, Liebaers I 2002 Followup of children born after ICSI. Human Reproduction Update 8, 111–116. Vidal F, Navarro J, Templado C et al. 1984 Synaptonemal complex studies in a mosaic 46,XY/47,XXY male. Human Genetics 66, 306–308. Vincent MC, Daudin M, De MP et al. 2002 Cytogenetic investigations of infertile men with low sperm counts: a 25-year experience. Journal of Andrology 23, 18–22. Vogt PH, Edelmann A, Hirschmann P, Kohler MR 1995 The azoospermia factor (AZF) of the human Y chromosome in Yq11: function and analysis in spermatogenesis. Reproduction, Fertility and Development 7, 685–693. Yoshida A, Miura K, Shirai M 1997 Cytogenetic survey of 1,007 infertile males. Urologia Internationalis 58, 166–176. Received 8 October; refereed 26 October 2007; accepted 28 November 2007.
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