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
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Abstract |
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Key words: intracytoplasmic sperm injection (ICSI)/male infertility/X-autosomal translocation/X chromosome inactivation
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Introduction |
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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 Xq13q26.
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.
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Case report |
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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 trypsinGiemsa 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.
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Results |
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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 probands mother and brother were also found to carry the same translocation. The probands 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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Discussion |
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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 probands brother.
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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.
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Acknowledgements |
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References |
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Submitted on December 13, 2002; accepted on February 24, 2003.