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ICSI and the transmission of X-autosomal translocation: a three-generation evaluation of X;20 translocation: Case report
Sai Ma1,3,
Basil Ho Yuen1,
Maria Penaherrera2,
David Koehn2,
Larry Ness1 and
Wendy Robinson2
1 Department of Obstetrics and Gynecology and
2 Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada
3 To whom correspondence should be addressed. e-mail: sai@interchange.ubc.ca
Published reports show that male carriers of an X-autosome translocation, which is either inherited from their mother or is de novo, are generally sterile, regardless of the position of the breakpoint in the X chromosome. We report a three-generation propagation of such a translocation in a family with a case of male factor infertility. Due to the condition of severe oligozoospermia, the proband and his wife underwent ICSI, which resulted in the birth of a normal healthy female. Cytogenetic (chromosome) analyses and X-chromosome inactivation (XCI) assays were done on the family. The cytogenetic analysis of the proband, a man with severe oligozoospermia, revealed an X-autosomal translocation, 46,Y,t(X;20)(q10;q10), which was inherited from his mother. His brother had the same translocation. Amniocentesis and post-natal umbilical cord analyses revealed that the female infant carried the same translocation as her father. XCI studies showed highly skewed inactivation of the normal X chromosome in the female infant, her paternal grandmother, and her mother who had a normal karyotype. In contrast to the data from the literature, our study suggests that men with a certain type of X-autosomal translocation could conceive children through ICSI in conditions in which a few spermatogonia are able to complete meiosis II. The literature involving X-autosomal translocation in males is also reviewed and the importance of the study of X-chromosomal inactivation in female infants discussed.
Key words: intracytoplasmic sperm injection (ICSI)/male infertility/X-autosomal translocation/X chromosome inactivation
X-autosomal translocations are generally of maternal origin or arise de novo (Kalz-Füller et al., 1999). Fertility effects of a balanced X-autosome translocation vary depending on the sex of the carrier (Madan, 1983; Kalz-Füller et al., 1999), the position of the translocation breakpoints (Madan, 1983; Kalz-Füller et al., 1999) and the pattern of X-inactivation (Schmidt and Du Sart, 1992; Waters et al., 2001). Most carriers of an X-autosome translocation are phenotypically normal (Madan, 1983; Kalz-Füller et al., 1999; Waters et al., 2001). In female carriers, gonadal dysgenesis may occur, and ~9% may have multiple anomalies and/or mental retardation (Schmidt and Du Sart, 1992). When the breakpoint occurs within a gene, a ‘classic’ X-linked disorder can be phenotypically expressed in a female (van Bakel et al., 1995). In male carriers, azoospermia is the most common finding, although a few cases have been reported with severe oligozoospermia (Fraccaro et al., 1977).
The cause of the spermatogenetic failure in carriers of an X-autosome translocation is unknown, but spermatogenesis generally is much more sensitive to meiotic disruption than oogenesis due to a number of meiotic cell cycle checkpoints (Hunt and Hassold, 2002). Although metaphase I divisions with quadrivalent structures have been found in human carriers, some disturbance of pairing around the breakpoints was evident, as was non-homologous pairing in some cells (Quack et al., 1988). Unpaired regions of balanced chromosomal rearrangements tend to pair with any available unpaired region (like those of sex chromosomes) at pachytene, and this has been associated with meiotic arrest (Grao et al., 1989). Another theory involves the X chromosome undergoing extensive condensation in early spermatogenesis, with the timing of pairing and meiotic segregation of the sex chromosomes being precocious in comparison with that of the autosomes (Armstrong et al., 1994). This condensation is thought to reduce non-homologous pairing between the unpaired region of the X chromosome and may also prevent the unpaired X chromosome from triggering the cell cycle checkpoints that would occur in spermatogenesis in the presence of unpaired univalents (Handel et al., 1994; Turner et al., 2001). Thus, disrupted chromatin condensation may lead to failure of meiotic progress in spermatocytes by either the facilitation of non-homologous pairing or signalling the checkpoint of a cell in pachytene to undergo apoptotic arrest.
With the use of ICSI, it is now possible for men with oligozoospermia to father a child (Palermo et al., 1992; Ma and Ho Yuen, 2001). Since low sperm count or azoospermia in men has been shown to be associated with a 10-fold increase in chromosomal anomalies compared with the general male population (Retief et al., 1984), infertility treatment with ICSI concerns a population of men with a particular risk of carrying chromosomal aberrations or structural anomalies. In cases of males with balanced X-autosomal translocation, it is unknown whether the few rare sperm produced would carry a balanced chromosome complement, given the extreme disruption to meiosis. If a few sperm in men with a balanced X-autosomal translocation carry the translocation, then ICSI might pass it on to the offspring and allow for a pattern of transmission of an X-autosome translocation different from that normally seen (i.e. maternal inheritance).
X-chromosome inactivation (XCI) is a mechanism of dosage compensation, which results in silencing of the majority of genes on one of the two X chromosomes in somatic cells of females. In cases of balanced X-autosomal translocation in female carriers, the normal X chromosome is usually inactivated, leaving the derivative X chromosome in the active state. Female carriers of a balanced X-autosome translocation generally are phenotypically normal. However, infertility because of gonadal dysgenesis is common among those women in whom the breakpoint in the derivative X chromosome involves the critical region Xq13–q26.
We report a three-generation family that represents the first case of X-autosomal translocation transmitted from father to daughter through ICSI. We also discuss the differential effects on reproductive fitness of X-autosomal translocation in female and male carriers, as well as the implications of XCI in female carriers.
Clinical details
The family was ascertained via a primary infertile but otherwise normal 37-year-old man (Figure 1) with normal sized testes. His height was 183 cm and weight was 84 kg. The presence of severe oligozoospermia was established by means of several semen analyses. Serum gonadotrophins and testosterone were normal. He has had an ongoing history of infertility for 11 years. His only brother also has a long history of infertility, but managed to father two phenotypically normal daughters after 7 years of infertility. Neither semen analysis from his brother, nor paternity testing and karyotypes of his two daughters were available. The age of the proband’s mother at the birth of her sons was 35 and 42 years old respectively, and she experienced menopause at the age of 48. The female partner had no evidence of tubal, ovulatory or pelvic infertility factors. Apart from the infertility, no obvious abnormalities were present in his wife, himself or any other family members.
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Figure 1. Pedigree of a familial translocation. Dotted circles represent balanced carriers of the translocation. The arrow indicates the proband patient.
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ICSI
The couple underwent IVF combined with ICSI for the treatment of male factor infertility. A standard luteal phase ‘long protocol’, of controlled ovarian stimulation using a GnRH agonist and recombinant FSH with intravaginal progesterone as luteal support, was undertaken in the female partner. Of the 20 oocytes retrieved, 16 metaphase II oocytes were identified by the presence of a single polar body. Only a few sperm (~50) were found after concentration of the semen sample into small pellets. Motile sperm were selected from the concentrated pellets and then transferred to the polyvinylpyrrolidone (PVP) solution. The detailed ICSI procedures have been described previously (Ma and Ho Yuen, 2000).
Somatic chromosome studies
Chromosome analysis of peripheral blood was performed for the proband, his wife, his brother and his parents. Umbilical cord blood was used for the chromosomal analysis of the newborn baby. Primary cultures were established and metaphase chromosomes were harvested using standard methods. In addition, genetic counselling and prenatal diagnosis for chromosomal analysis were offered to the proband couple. Chromosomal banding was performed by the trypsin–Giemsa method. A detailed protocol is provided in Lam et al. (2001).
X-Chromosome inactivation (XCI) studies
DNA was extracted from cord blood using standard techniques. An assay to examine XCI status requires a means of distinguishing the active from inactive X chromosome as well as a polymorphism to distinguish between the two chromosomes. This is usually accomplished by analysis of an expressed polymorphism or by determining the methylation status of CpG dinucleotides near the polymorphism. In the present study, XCI was tested predominantly by methylation analysis at the androgen receptor gene (AR), the fragile X mental retardation gene (FMR1) and DXS6673E. DNA was digested with HpaII, a methylation-sensitive restriction enzyme, and both digested DNA and control undigested DNA were amplified by PCR with the respective primers. Methylated DNA was not cut by HpaII and was therefore amplified, whereas unmethylated alleles were digested and were not amplified. Non-random XCI was revealed by a difference in quantative intensity of the two alleles. The details of the method and results on placental XCI of this case have been described previously (Peñaherrera et al., 2003). This study was conducted with the approval of the Clinical Ethics Board at the University of British Columbia and included informed consent from all participants in the study.
Intracytoplasmic sperm injection
Of the 16 oocytes injected, 15 survived the procedure (94.1%) and 11 of them fertilized normally (73%), as shown by the presence of two pronuclei 18 h after injection. Four good-quality embryos, two at the 7-cell stage, one at the 6-cell stage and one at the 5-cell stage, were transferred on day 3 after oocyte retrieval. Six embryos at the pronuclear stage were cryopreserved on day 1 of fertilization. A positive pregnancy test was obtained 14 days after embryo transfer. At 6 weeks of pregnancy, the first ultrasound showed a single gestational sac with heartbeat. The pregnancy was carried on to term and no complications were noted. A healthy female baby was born at 40+ weeks. Apgar scores were 8 at birth and 10 after 5 min. The newborn baby weighed 3.46 kg, and no malformation was found. In follow-up after 1 year, the female infant did not exhibit any minor or major malformation. Cognitive and mental development appeared to be normal at that time.
Cytogenetic analyses
Conventional G-banding of phytohaemaglutinin-stimulated peripheral blood lymphocytes of the proband revealed a balanced whole arm translocation involving the X chromosome and chromosome 20. The breakpoints appear to be at the centromeres of both chromosomes followed by fusion of the two long arms and the two short arms. This translocation with 46,Y,t(X;20) (q10;q10) karyotype was evidenced in all cells examined, (Figures 2 and 3). The proband’s mother and brother were also found to carry the same translocation. The proband’s wife had a normal karyotype. The results of the amniocentesis showed that the fetus had the same translocation as the father. The post-natal umbilical cord study also showed the same result.
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Figure 2. Proband’s karyotype 46,Y,t(X;20)(q10;q10). The arrows indicate the chromosomes with the translocation.
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Figure 3. Chromosomes 20 and X of the child, her father (proband) and paternal grandmother.
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Determination of X inactivation ratios
Methylation analysis at the AR and FMR1 locus demonstrated a completely skewed X inactivation pattern in post-natal cord blood from the female infant. A highly skewed X inactivation ratio was also observed in her mother, who did not carry the balanced translocation (Table I). Based on the AR assay, the female infant’s mother showed preferential inactivation of the same allele that was inactivated preferentially in her daughter. For FMR1, however, the mother was also skewed, but towards the inactivation of a different allele from that transmitted to her daughter. The mother was uninformative for marker DXS6673E (Table I). For the paternal grandmother, who was also a carrier of the translocation, the AR assay showed highly skewed XCI with preferential inactivation of the normal X chromosome (represented by allele b), the same allele that was inactivated in the child, but was uninformative for both FMR1 and DSX6673E.
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Table I. Percentage of XCI in cord blood, and in maternal and paternal grandmother’s blood
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The clinical phenotype in patients who have a translocation involving the X chromosome and an autosomal chromosome frequently differ from those cases where the translocation involves two autosomes (Madan, 1983; Kalz-Füller et al., 1999). The phenotypic consequence, in terms of reproductive fitness, of an X-autosome translocation in our study family is consistent with known consequences of balanced X-autosomal translocation in carriers, i.e. all three carriers in this family had a normal phenotype, with proven fertility in the proband’s mother, and infertility in the male proband (Madan, 1983>; Kalz-Füller et al., 1999). This case is unique as it is, to our knowledge, the first reported instance where an X-autosome translocation was transmitted from a male to his offspring through ICSI. It seems likely that meiosis proceeded normally in at least some cells during spermatogenesis, in order to generate balanced haploid gametes.
Translocations involving a portion of the X chromosome have a profound impact on spermatogenesis, as indicated by the failure of most spermatocytes to enter into meiosis (Jamieson et al., 1996). In certain cases, spermatogenesis can proceed to the formation of elongated spermatids, but the process is remarkably inefficient, as indicated by the presence of a few sperm. Up to now, only two cases (excluding ours) produced two children, while most of the other reported cases involving X-autosomal translocation presented with azoospermia (Table II). Our case not only presented with severe oligozoospermia in the proband, but also included three other familial carriers, including the proband’s brother.
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Table II. Human reciprocal X-autosome translocations associated with infertility in males
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Review of 26 males (Table II) with X-autosomal translocation did not show marked preference for involvement of a specific autosome, but it appears that the breakpoint occurs more often in the critical region of Xq13–q26 (17 out of 26 cases). However, the fact that the breakpoints in nine cases were distributed outside the critical region suggests that, unlike female carriers who have reduced fertility predominantly associated with the breakpoint in Xq13–q26, the male carriers are invariably sterile, regardless of the position of the breakpoint on the X chromosome (Kalz-Füller et al., 1999). The breakpoint in our case involves the centromeric region in both the X chromosome and chromosome 20, followed by fusion of the two long arms and the two short arms. This balanced translocation may have fewer detrimental effects on spermatogenesis than other X chromosomal breakpoints, allowing a few spermatogonia to undergo meiotic events and consequently producing a few sperm with balanced translocation.
Because ICSI needs only one spermatozoon to fertilize an oocyte, most subfertile and infertile men, i.e. men with either very few sperm (extreme oligozoospermia) or no sperm (retrieval from testis) in the ejaculate, can now father a child (Ma et al., 2000; Silber, 2000). Our case illustrates this possibility of producing offspring from a patient with severe oligozoospermia by ICSI, and also demonstrates that germ cell maturation can exist in males with an X-autosome translocation with some sperm containing the balanced translocation. However, the risk of producing progeny with an unbalanced translocation also exists through ICSI. With the advent of ICSI, more cases with X-autosomal translocations are likely to be discovered and may produce offspring with balanced translocation in conditions where a few spermatogonia can complete meiosis II. Furthermore, with the development of microdissection testicular sperm extraction (TESE) (Silber, 2000), sperm or mature spermatids from some men with azoospermia and with X-autosomal translocation may be likely to be retrieved from the testis (Quack et al., 1988), and used for ICSI.
The risk of an aneuploid offspring for X-autosomal translocation carriers seems to be similar to that for reciprocal autosomal exchanges (i.e. it would depend on the autosome involved, the length of the translocated segments, the configuration at pachytene and the expected segregation) (Jalbert et al., 1980; Stene and Stengel-Rutkowsky, 1982). Moreover, the viability of the resulting imbalance could be favoured by a ‘selective’ inactivation. In this family, the infant and the paternal grandmother, who are both carriers of the balanced X;20 translocation, had skewed X-inactivation of the normal X chromosome, thus conforming to the normal X chromosome behaviour in X-autosomal translocations, i.e. the normal X in most female carriers is inactivated in order to keep a balanced dosage of expressed genes (Mattei et al., 1982). The normal phenotype in the females and fertility in the paternal grandmother of this family may be due to the translocation involving the entire short and long arm of the X chromosome, allowing the critical region for maintaining gonadal function to be uninterrupted.
In conclusion, our study confirms the transmission of an X-autosome translocation from mother to son. In contrast to the published data, the son transmitted the inherited translocation to his daughter, producing a three-generation X-autosome translocation, which can only result from ICSI under conditions in which a few spermatogonia can complete meiosis II. Since severely infertile males may have reciprocal translocations involving either the X and/or an autosomal chromosome, it is of clinical importance to determine to what degree skewed X-chromosomal inactivation is present in the resulting female newborns with X-autosomal translocation. This information can be used to predict the risk of an abnormal phenotype and presence of an X-linked disease in these newborns from ICSI so that the parents can be counselled accordingly.
We gratefully thank the family for donating the samples for this study, the clinical and laboratory staff of the University of British Columbia IVF Program in the Division of Reproductive Endocrinology and Infertility for their clinical work, and Mr Steven Tang for the help with preparation of the manuscript. This study was supported by The Hospital for Sick Children Foundation (grant no. XG 02-086).
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