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: aneuploidy in sperm/FISH/ICSI/parental origin of chromosomal abnormality
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Introduction |
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It has been recognized that abnormal chromosome constitutions occur more frequently in infertile men (Chandley et al., 1979). In addition, men with a normal somatic karyotype may have chromosomal abnormalities limited to only the germ cells (Egozcue et al., 1983
; Calogero et al., 2001
). Several fluorescent in-situ hybridization (FISH) studies have indicated an increased frequency of sex chromosome abnormalities in men with severe oligoasthenoteratozoospermia (OAT) (Moosani et al., 1995
; Bernardini et al., 1997
; Pang et al., 1999
) and in men with abnormal semen parameters (Colombero et al., 1999
). Despite the existence of much data on the aneuploidy rate in sperm from FISH studies and for aneuploidy rate in ICSI outcomes, the correlation between the two remains speculative.
FISH studies that looked at the sex aneuploidy rate in sperm from men who fathered (by natural conception) children with Turner syndrome (Martinez-Pasarell et al., 1999) and Klinefelter syndrome (Eskenazi et al., 2002
) have also shown a significant increase in XY disomy and sex-null sperm. Such a retrospective approach would prove more informative and provide a more direct connection between increased aneuploidy found in the sperm of the father seeking conception by ICSI and any abnormalities in an ICSI pregnancy. We report on one such example where ICSI produced a pregnancy with a 45,X karyotype, which was found to lack the paternal sex chromosome, and was associated with an extremely high rate of sex chromosomal aneuploidy found in the sperm of the father. The mechanisms of meiotic segregation of the sex chromosomes that could be involved in the origin of a sex aneuploid pregnancy, such as 45,X, will also be discussed.
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Case report |
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Cytogenetic and origin of abnormality analyses
Aborted material from the spontaneous miscarriage was submitted for cytogenetic analysis, involving standard cell culture and karyotype of metaphase spreads as well as by comparative genomic hybridization (CGH) to rule out maternal contamination.
If aneuploidy was detected, a molecular assay to test for origin of abnormality would be done. DNA was extracted from maternal and paternal blood samples, and chorion from the aborted material using standard protocols. All DNA samples were amplified using highly polymorphic X-linked microsatellite markers (androgen receptor: AR; fragile X mental retardation: FMR1) (described in Allen et al., 1992; Carrel and Willard, 1996
; Hecimovic et al., 1997
). Analyses of PCR products were done with an ABI Prism 310 Genetic Analyzer, with GeneScan Analysis Software version 3.1.2.
If paternal origin was concluded, sperm collected from the father would be analysed by multi-coloured FISH, with probes specific for chromosomes of interest.
Ascertainment and processing of sperm
Semen samples were obtained from the treatment couple and two other normal healthy males (aged 35 and 40 years) of assumed normal fertility. Semen samples were washed in 1xHanks buffer, and fixed in methanol:glacial acetic acid (3:1). Cell pellets were stored at 20°C until processed for FISH.
Fixed sperm were dropped onto pre-cleaned slides and washed twice in 2xSSC (saline sodium citrate). The slides were then incubated in 20 mmol/l dithiothreitol in 1 mol/l TrisHCl buffer (pH 8), and adequate decondensation of sperm was achieved when the diameter of sperm heads increased to a level that would allow for efficient hybridization and visualization of FISH probes while still being able to detect the sperm tails. The slides were then washed in 2xSSC and phosphate-buffered saline and dehydrated in ethanol series (70100%) and air-dried.
Fluorescence in-situ hybridization
All sperm samples (one test and two controls) were processed with directly labelled DNA probes specific to alpha-satellite repeat clusters in the centromeric region of chromosomes 18 [CEP 18 (D18Z1) SpectrumAqua; Vysis Inc., USA] and X and Y (CEP X SpectrumGreen/CEP Y SpectrumOrange; Vysis Inc.). Denaturation, hybridization and detection procedures were as recommended by Vysis. Each specimen slide was denatured in 70% formamide/2xSSC at 75°C for 5 min, placed in an ethanol series (70100%) and air-dried. The probe mixture was denatured at 73°C for 5 min and spread onto the denatured specimen slide. A coverglass was applied to the slide, sealed and allowed to hybridize overnight at 37°C. After hybridization, slides were washed in 0.4xSSC/0.3% NP-40 at 73°C for 2 min and 2xSSC/0.1% NP-40 at ambient temperature for 30 s and air-dried. Sperm were counterstained with DAPI II (Vysis Inc.) and assessed with an epifluorescent microscope (Nikon Elipse E600W) equipped with a triple bandpass filter (DAPI/FITC/Cy3), a dual band pass filter (FITC/Cy3), and single bandpass filters for Aqua, and fluorescein isothiocyanate (FITC) or cyanine (Cy3).
Scoring criteria
Scoring of nuclei was only done in an area of the slide where consistent hybridization was evident on initial screening of the slide. Only nuclei with intact morphology and long sperm tails were scored to select for mature sperm and to exclude any other cell type or artefact present on the slide. Any nuclei that have abnormal morphology would not be scored to avoid overlapping cells being scored as one. Two signals of the same colour were scored as two copies of the corresponding chromosome when they were comparable in brightness and size and were separated from each other by a distance longer than the diameter of each signal. Nullisomy of any individual chromosome was considered when the sperm clearly contained at least one of the other chromosomal signals, while complete nullisomic sperm, absence of any signal, was scored as such. Two-sample z-tests of two proportions were used for the statistical analysis.
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Results |
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The test of origin of the abnormality by PCR amplification with both the primers for AR and FMR1 gene loci of the DNA samples from the peripheral blood of both parents and chorion from the placenta indicated absence of the paternal X chromosome from the embryonic cell line (Table I). Thus, the 45,X karyotype was the result of the fertilization of the oocyte with a sperm lacking a sex chromosome.
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Discussion |
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FISH studies that investigate aneuploidy in human sperm have indicated that oligozoospermic men are especially at risk for sperm chromosomal abnormalities, particularly involving the sex chromosomes (Moosani et al., 1995; Bernardini et al., 2002
; Ushijima et al., 2002
). Their results indicate sex chromosome aneuploidy as the major contributor to aneuploidy in sperm of infertile men. A significantly higher rate of gonosomal aneuploidy, particularly XY disomy, has been seen in men with severe OAT (1.35% in Bernardini et al., 1997
; 2.357.41% in Pang et al., 1999)
, although increases in rates of non-disjunction have also been demonstrated in sperm cells with strictly normal morphology (1.89% in Ryu et al., 2001
).
In the current case, the increase in sex chromosomal abnormality is obvious and the marked pattern of aneuploidy in haploid sperm, namely the near equal increase in disomy XY and sex-nullisomy sperm, is consistent with an abnormality specifically occurring during meiosis I non-disjunction. Although the X and Y chromosomes have short homologous pairing segments that form a synaptonemal complex in which crossing-over takes place, they remain as univalents, ultimately leading to non-disjunction, much more often than the smallest autosomes. The occurrence of 47,XXY is attributable to an error in paternal meiosis I in nearly half of all cases (Hassold et al., 1991). Recently, Thomas and Hassold (2003
) discussed how the paternal meiosis I non-disjunction of XXY is related to recombination that is either reduced or absent, particularly in the pseudoautosomal region of Xp/Yp region (PAR1). Their suggestion of direct genetic linkage analysis of a single sperm (Shi et al., 2001
) to look at the level of recombination in 24, XY sperm would prove useful in determining if our case is indeed related to reduced recombination. Further study on this matter is needed. Although evidence for meiosis I non-disjunction in spermatogenesis has long been documented, its role in male factor infertility is still unclear.
While increased incidence of XY disomy and sex nullisomy might directly be explained by a meiosis I non-disjunction event, it does not account for the presence of the other abnormal constitutions found in our analyses. One possibility could be the existence of a mosaic population of primary spermatocytes with differential ability to undergo normal meiosis, which would explain the presence still of a large proportion of normal haploid sperm (59.2%). Also there are probably multiple non-disjunction events occurring at different stages of meiosis. In addition, there may be an underlying condition that predisposes the germ cells to proceed with error-prone cell division. The existence of stringent meiotic checkpoints in spermatogenesis has been proposed in studies of mice (reviewed by Hunt and Hassold, 2002) and corroborated by studies of infertile men (Chandley et al., 1979
) that prohibit the progression from metaphase I when univalent chromosomes are present. In the current case, the existence of the high rate of aneuploidy in mature sperm could be the result of diminished or absence of these controls.
Another explanation for the resulting variety of sex chromosomally abnormal constitutions might relate to the effects of an altered testicular environment. Mroz et al. (1998) studied XXY male mice and concluded that an abnormal testicular environment and not the existence of an XXY population in the germ cells caused an increase in multiple categories of numerical chromosomal abnormalities. Deficiencies in such an environment may predispose meiotic errors in germ cells and such a mechanism of abnormality could quite possibly be applied to other forms of male infertility, including those with normal somatic karyotypes, as in the current case where the father had a peripheral blood karyotype of 46,XY.
The high level of sex nullisomy (19%) in the sperm was most likely the reason for the 45,X conception, which would make this case a very good example of the transmission of chromosomal abnormality from sperm to the conceptus through ICSI. We have suggested some ideas about the mechanism for the abnormalities encountered in the spermatogenesis of our case, but clearly more conclusive studies need to be done. Confirming previous studies of ICSI males, we have encountered significantly increased sex chromosomal aneuploidy attributable to malsegregation during spermatogenesis. The mechanism of meiotic non-disjunction and the role it plays in spermatogenesis is yet to be understood completely, but with the use of novel molecular approaches applied to the level of the single sperm, we may be able to answer some of these questions. Although it is not possible to assess the genetic constitution of a particular sperm before it is used for ICSI, leaving preimplantation genetic diagnosis (PGD) as the only means of obtaining any genetic information about the embryo before transfer, information provided by FISH may be helpful in counselling couples interested in pursuing assisted reproductive technology about their risk of transmitting chromosomal abnormalities of paternal origin. According to the findings from FISH analysis of the sperm in this couple, the risk of conceiving a child with chromosomal aneuploidy, particularly involving the sex chromosomes, in any future attempt by ICSI is significant, despite best efforts to minimize this by selecting motile sperm (sperm with normal morphology was not available in this patient). PGD should be considered if this couple was to pursue future attempts at conception by ICSI. To estimate the true risk of abnormal conceptions through ICSI, large prospective studies are clearly needed to generate accurate rates of chromosomal aneuploidy. This can only be achieved if patient selection criteria and methodologies are standardized, correlating cytogenetic information in the progeny and parental origin of chromosomal abnormalities to information from molecular and cytogenetic analyses of sperm.
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Acknowledgements |
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Submitted on July 28, 2003; accepted on September 18, 2003.