Molecular Human Reproduction, Vol. 8, No. 6, 589-595,
June 2002
© 2002 European Society of Human Reproduction and Embryology
Reproductive genetics |
Genetic follow-up of male offspring born by ICSI, using a multiplex fluorescent PCR-based test for Yq deletions
1 Monash Institute of Reproduction and Development, Monash University, Monash Medical Centre, 2 Prince Henry's Institute of Medical Research, Monash Medical Centre and 3 Monash IVF Pty Ltd, Clayton Rd, Clayton, Victoria 3168, Australia
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Abstract |
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De-novo deletions involving AZFa, b, c and d are one of the most common chromosomal aberrations in man resulting in defective spermatogenesis and male infertility. Currently, Yq deletion screening involves either single or multiplex PCR using Y-specific sequence tagged site markers and the subsequent analysis of the amplification products on ethidium bromide-stained agarose gels. To improve the practicality of routine and high throughput Yq testing, we have developed a more sensitive multiplex fluorescent (FL)–PCR screening system using genomic DNA extracted from cheek buccal cells as a readily available PCR template. For genetic follow-up studies of ICSI-conceived children, we also developed a DNA fingerprinting system based on the co-amplification of four highly polymorphic markers to validate family samples and detect any potential extraneous DNA contamination that could cause a misdiagnosis. Multiplex FL–PCR analysis of buccal cell DNA from two infertile men who conceived three sons by ICSI demonstrated that their Yq deletions were vertically transmitted. Fine mapping with additional Yq markers revealed identical deletion endpoints involving the loss of AZFdc sequences. This firstly indicates that the extent of the Yq deletion was unchanged on ICSI transmission and secondly supports the view that AZFdc deletions may arise by a common de-novo event. Analysis of paternal, maternal and sibling DNA fingerprints showed the co-inheritance of parental alleles by each male child and confirmed the expected relationship between each family member. The application of these new FL–PCR based screening tests in genetic follow-up studies will assist in confirming transmission of specific genetic defects to male offspring conceived by ICSI and provide a basis for genetic counselling and potential treatment options as these boys approach sexual maturity.
DNA fingerprinting/ICSI/male infertility/multiplex FL-PCR/Yq deletions
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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In one-third of couples with a history of infertility, the causative or underlying factors are associated with the male partner. In some infertile men, abnormalities in their semen have been related to a variety of chromosomal abnormalities, including aneuploidies, translocations, autosomal defects and microdeletions of the Y chromosome (Meschede and Horst, 1997
; Cooke et al., 1998; McLachlan et al., 1998). Yq microdeletions involving spermatogenic genes represent the most significant pathogenetic defect identified to date (Vogt, 1998). In studies of >5000 men with severe primary testicular failure and a sperm density of <5x106/ml the prevalence of Yq deletions is in the order of ~8% (range 1–35) (Foresta et al., 2001). Men with Yq deletions usually require assisted reproduction and ICSI to conceive children. Vertical transmission of Yq deletions to male offspring by ICSI has been reported (Kent-First et al., 1996a,b; Jiang et al., 1998; Chang et al., 1999; Kamischke et al., 1999; Page et al., 1999; Cram et al., 2000) and consequently, sons inheriting a Yq deletion from their father are likely to have a similar fertility phenotype (Bhasin et al., 1997).
Yq genes important for spermatogenesis appear to be concentrated within four azoospermia factor (AZF) regions known as AZFa, AZFb, AZFd and AZFc (Vogt et al., 1996; Kent-First et al., 1999). Microdeletion intervals have been defined by extensive PCR analysis of Y-specific sequence tagged sites (STSs) across these AZF regions (Ma et al., 1993; Reijo et al., 1995; Kent-First et al., 1996a,b; Najmabadi et al., 1996; Qureshi et al., 1996; Pryor et al., 1997; Simoni et al., 1997; Ferlin et al., 1999; Kleiman et al., 1999; Foresta et al., 2001). The majority (~80%) of all reported Yq deletions are associated with the loss of the AZFc region extending from distal AZFb sequences through AZFd to distal AZFc sequences in close proximity to the junction of the euchromatic and heterochromatic regions. The absence of the testes-specific DAZ (deleted in azoospermia) gene is tightly linked with this common AZFcd deletion and is usually associated with severe oligozoospermia or azoospermia (Najmabadi et al., 1996; Reijo et al., 1996; Pryor et al., 1997; Simoni et al., 1997; Oliva et al., 1998; Silber et al., 1998; Kleiman et al., 1999; Foresta et al., 2001). More recently, an infertile man with severe hypospermatogenesis has been shown to have a Yq deletion that maps specifically to the DAZ gene cluster (Moro et al., 2000) and this indicates for the first time that DAZ is a gene essential for normal spermatogenesis. Of the remaining Yq deletions, ~15% are restricted to AZFb and 5% to AZFa (Foresta et al., 2001). AZFb deletions are characterized by the loss of the testes-specific RBM (RNA binding motif) gene, RBM1 (Ma et al., 1993), whereas AZFa deletions involve either DFFRY (Drosophila Fat Facets Related Y, also termed USP9Y) or DBY (Dead Box Y) (Sun et al., 1999). Men with the most extensive Yq deletions involving AZFa, b, d and c sequences are invariably azoospermic and frequently exhibit testicular pathologies such as germ cell arrest and/or Sertoli cell-only syndrome (Vogt et al., 1996; Foresta et al., 2001).
In most IVF laboratories, genetic follow-up of male offspring born following ICSI is not routinely performed to confirm the vertical transmission of known Yq deletions or to detect the occurrence of de-novo Yq deletion events, which represent one of the most common structural chromosomal abnormalities in men (Edwards and Bishop, 1997). Furthermore, as other genetic causes of male infertility are identified, it will become increasingly important to assess the frequency of causative mutations and their incidence of transmission to offspring. Genetic screening of ICSI-conceived offspring can be reliably performed by PCR analysis of genomic DNA isolated from peripheral blood samples taken by venepuncture at 4–6 months of age. However, non-invasive sampling of cheek buccal cells has been reported as a more viable option to obtain genomic DNA from children (Rudbeck and Dissing, 1998). When genetic follow-up of ICSI children becomes a more widespread practice, new approaches will be necessary to accommodate high throughput analyses and confirm unequivocally that the test results are actually derived from the original family samples. Therefore, the aim of this work was to develop highly sensitive and specific multiplex fluorescent (FL)–PCR and DNA fingerprinting systems for the genetic analysis of small quantities of genomic DNA from patients' buccal cells.
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Materials and methods |
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Clinical patient details
In clinical studies of 650 infertile men with sperm counts <5x106/ml, we have identified 29 Yq deletions, with an overall frequency of 4.5%. Three of these men have since fathered sons by ICSI and two families have consented to genetic follow-up studies. Both cases were of severe idiopathic seminiferous tubule failure and long standing primary infertility.
Case study 1
This case involved a 26-year-old man with 1 year of primary infertility and idiopathic extreme oligospermia (only occasional sperm seen in fresh ejaculate). His testicular volumes were 12/10 ml. He had a normal testosterone level of 24.2 nmol/l (normal range 8–27) but a markedly elevated serum FSH of 22.9 IU/l (normal range 1.5–8). His karyotype was 46XY. The couple underwent two ICSI cycles with a total of 13 oocytes injected and six embryos were created. They achieved a singleton normal pregnancy resulting in the birth of a son.
Case study 2
The second case involved a 36-year-old man with 18 months of primary infertility and idiopathic severe oligospermia [sperm density 2x106/ml, 46% total motility and 90% abnormal forms. His testicular volumes were 15/10 ml with bilateral varicoceles. He had slightly elevated serum FSH (10.7 IU/l, normal range 1.5–8), normal serum LH (4.3 IU/l, normal range 1–6), low–normal serum testosterone (9.1 nmol/l, normal range 8–27) and a normal 46XY karyotype. Over a 3 year period the couple underwent four ICSI cycles, with the overall collection of 48 oocytes and the creation of 31 embryos. Two normal pregnancies resulted in the birth of two sons, 2 years apart.
Human buccal cell samples
Buccal cells were collected from each family member by twirling a cytology brush (EndoScanPlus; Medico, USA) on the inner cheek for 30 s. The head of the brush was immersed in a 1.5 ml microcentrifuge tube containing 750 µl of phosphate-buffered saline (PBS). DNA was prepared from the buccal cells immediately or after 24 h at room temperature.
Extraction of genomic DNA from human buccal cells
Microcentrifuge tubes containing the head of the cytology brush were centrifuged for 1 min at 4500 g and the brush was carefully removed with sterile tweezers to avoid dislodging the cell pellet at the bottom of the tube. The tube was centrifuged again for 1 min at 6000 g, the supernatant decanted and the cell pellet resuspended in 600 µl PBS buffer. Following centrifugation as before, the supernatant was decanted and the cell pellet resuspended in 10 µl lysis buffer (200 mmol/l KOH, 50 mmol/l dithiothreitol) (Cui et al., 1989), vortexed briefly and the tube heated at 65°C for 10 min. The solution containing the DNA was then neutralized by the addition of 10 µl neutralizing buffer (900 mmol/l Tris–HCl, pH 8.3, 200 mmol/l HCl, 300 mmol/l KCl) (Cui et al., 1989), mixed, and centrifuged for 10 min at 13 000 rpm. Very carefully, 20 µl of the supernatant containing the DNA was transferred to a sterile 0.5 ml microcentrifuge tube and stored at –20°C. By ethidium bromide staining of the genomic DNA resolved on agarose gels, this alkaline buccal cell extraction method yielded low amounts of DNA (2–8 ng/µl).
FL–PCR
From mapping studies using a range of Y-specific STSs, the analysis of DAZ and RBM1 gene markers is sufficient to identify the majority of all reported Yq deletions in infertile men (Foresta et al., 2001). On this basis, two multiplex FL–PCR formats were developed to include two independent primer sets for DAZ and RBM1, to reduce the possibility of a false negative result. In addition, single primer sets for the control genes SRY and GAPDH were included, to internally monitor PCR performance and the quality of the genomic DNA template.
All primer pairs were synthesized as dried oligonucleotides by Sigma, Australia. The forward primer of each pair was modified by 5' fluorescent labelling using 6-FAM. Each primer pair was diluted in molecular biology grade H2O (Sigma) to 200 pmol/µl stock solutions under sterile conditions and stored in aliquots at –20°C until use. Table I displays the primer sequences, gene location and the size of the respective FL–PCR products for the Y-specific genes DAZ, RBM1 and SRY and the control gene GAPDH.
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Two multiplex PCR formats were developed consisting of three primer pairs each; namely, multiplex 1 (DAZ
, DAZ 
and RBM1 F/R) and multiplex 2 (RBM1 F/V, SRY F/R and GAPDH F/R). The optimized multiplex FL–PCR reaction consisted of 3 µl of 10x Taq PCR buffer (500 mmol/l KCl, 100 mmol/l Tris–HCl and 15 mmol/l MgCl2), 0.5 µl of 10 mmol/l dNTPs, 0.2 µl of Taq polymerase (5 units/µl) (Amersham Pharmacia Biotech, Melbourne, Australia), 21.3 µl MilliQ–H2O, 2 µl of template buccal cell genomic DNA (2–8 ng/µl) and 3 µl of primer mix, making a final volume of 30 µl. The final amount of each primer pair in each PCR tube was 4 pmol of DAZ , 4 pmol of RBM1 F/R and 20 pmol of DAZ (multiplex 1), and 4 pmol of GAPDH F/R, 20 pmol of SRY F/R and 20 pmol of RBM1 F/V (multiplex 2). FL–PCR was performed using the 9700 Thermocycler PCR machine (PE Applied Biosystems, Melbourne, Australia). The thermal cycling conditions for the FL–PCR reaction included an initial denaturation step of 3 min at 94°C, followed by 40 cycles consisting of denaturation for 45 s at 94°C, annealing for 45 s at 58°C and extension for 45 s at 72°C. The final step was a 5 min hold at 72°C to completely extend all the PCR products. With each FL–PCR reaction, two positive controls consisting of genomic DNA extracted from the blood (100 ng/µl) of a fertile man and woman and a negative control, blank sample (no DNA) were run in parallel.
DNA fingerprinting
Details of the four tetranucleotide microsatellite markers and amelogenin selected for the DNA fingerprinting system are summarized in Table II. The optimized multiplex FL–PCR consisted of 2.5 µl of 10xTaq PCR buffer, 0.5 µl of 10 mmol/l dNTPs (200 µmol/l), 0.3 µl of Taq polymerase (5 units/µl), 12.6 µl MQ–H2O and 9.1 µl of primer mix, making a final volume of 25 µl. Multiplex FL–PCR was performed using the 9700 Thermocycler PCR machine. All tubes underwent manual `Hot Start' followed by thermal cycling conditions involving 30 cycles consisting of denaturation for 45 s at 94°C, annealing for 45 s at 60°C and extension for 1 min at 72°C. With each multiplex FL–PCR, positive controls using known male genomic DNA and a negative control, blank sample (no DNA) were always included, as described above.
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Polyacrylamide denaturing gel electrophoresis and genescan analysis
Following FL–PCR, the products were subjected to agarose gel electrophoresis in order to determine their intensity and molecular size. FL–PCR products were analysed by an ABI Prism 377 DNA Sequencer (PE Applied Biosystems) following electrophoresis on a 4.2% polyacrylamide gels (containing 8% urea). Depending on the intensity of the bands observed on the agarose gel, either 2 µl of product was appropriately diluted in MQ–H2O or directly added to 3 µl of a loading dye cocktail (0.5 µl formamide and 2.5 µl 2500 Genescan standard). Samples were denatured at 95°C for 3 min, placed on ice and 2.5 µl was loaded into the pre-formed wells. Samples were electrophoresed in 1xTBE buffer for 5 h at 3000 volts. All PCR products were identified and sized using the ABI Prism 377 DNA Sequencer associated Genescan 672 software (PE Applied Biosystems).
Probability of sibship
Based on the knowledge of all four parental alleles, the probability that any two siblings would have identical DNA fingerprints was calculated at 1 in 173 for this DNA fingerprinting system, based on the heterozygosity indices of the selected four microsatellite markers (Table II
) as follows: 4x(0.88) * 4x(0.88) * 4x(0.93) * 4x(0.94) = 173.30.
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Results |
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Multiplex 1 and 2 PCR analysis of control genomic DNA (100 ng) produced the expected DAZ, RBM1, SRY and GAPDH bands for a fertile man and the expected single GAPDH band for a fertile woman (Figure 1
). Genomic DNA from two infertile men with a known Yq deletion and their male offspring conceived by ICSI were similarly tested using this multiplex PCR format. In the first case, father F001 had one son, S001. In the second case, father F002 had two sons, S002a and S002b, in separate IVF cycles. Buccal cells were taken from all members of each family and genomic DNA was prepared as a PCR template. Multiplex 1 and 2 PCR analysis of the male DNA samples confirmed the absence of the DAZ and DAZ bands in father F001 and in his son S001 (Figure 1), indicating vertical transmission of the Yq deletion by ICSI. Likewise, sons S002a and S002b also inherited their father's Yq DAZ deletion (data not shown). In all cases, the band intensities on the agarose gels were variable with some bands quite faint in multiplex 1 and 2.
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To verify the complete absence of the DAZ bands, the 6-FAM-labelled PCR products were also subjected to electrophoresis on 4.2% denaturing polyacrylamide gels and analysed by Genescan software (Figures 2 and 3
). The control male DNA produced all five Y-specific peaks for DAZ, RBM1 and SRY and a peak for GAPDH, whereas the control female DNA produced the expected GAPDH peak. Analysis of DNA from F001 and F002 and their ICSI-conceived sons showed uniform peaks for RBM1F/R in multiplex 1 and peaks for RBM1F/V, SRY and GAPDH in multiplex 2. There was no evidence of any DAZ or DAZ peaks, confirming unequivocally that the DAZ gene was indeed absent in these samples.
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DNA fingerprints of the buccal cell DNA from each family member showed strong and clear amplification of all five markers with no evidence of non-specific background interference. Based on the heterozygosity of each microsatellite marker (Table II
) and the knowledge of all four parental alleles, the probability of any two siblings having identical DNA fingerprints was calculated at 1 in 173 (refer to Materials and methods for calculation). The allelic profiles of S001, S002a and S002b displayed a predictable inheritance pattern of the maternal and paternal alleles for each microsatellite marker, thus confirming that these ICSI-conceived sons were the progeny of their respective parents (Figure 4). As expected, DNA fingerprints for siblings S002a and S002b were unique.
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The extent of the Yq deletions in the father and son pairs were finely mapped by conventional PCR using an additional 14 Y-specific markers concentrated around the distal ends of AZFb and AZFc (Jones et al., 1994
). Both infertile men (F001 and F002) and their respective male offspring (S001 and S002a/b) had the same marker pattern, indicative of identical deletions involving the total loss of AZFdc sequences between GY149 and SY159 (Table III). These results of father and son pairs suggest that no further expansion of the deletion occurred on ICSI transmission. Interestingly, six further DNA samples of infertile men with the same RBM1+/DAZ- genotype also had the identical Yq deletion endpoints (data not shown in Table III).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Yq deletions are the most significant genetic cause of male infertility identified to date. The vast majority of Y chromosome deletions involve the testes-specific DAZ and RBM1 genes which are located in the AZFc and AZFb regions of Yq respectively. On this basis, we have developed a new multiplex FL–PCR system, based on the analysis of the Y-specific markers DAZ and RBM1, that has the capacity to reliably detect Yq deletions in low amounts of genomic DNA extracted from cheek buccal cell samples. In studies of two couples where the male partners had severe oligospermia and a known Yq deletion, multiplex FL–PCR analysis and readout of amplified products on both ethidium bromide-stained agarose and DNA sequencing Genescan gels showed the absence of the DAZ gene and confirmed that their ICSI-conceived sons had inherited their Yq deletion. This observation is in keeping with previous studies that have consistently shown the vertical transmission of Yq deletions to male offspring by ICSI (Kent-First et al., 1996a
,b; Jiang et al., 1998; Chang et al., 1999; Kamischke et al., 1999; Page et al., 1999; Cram et al., 2000). Parallel analysis of individual DNA fingerprints with four polymorphic markers also showed that each son inherited the expected alleles from his parents, thus validating the samples taken from each family member. Together, these findings suggest that FL–PCR-based protocols using buccal cell DNA as a template may provide an alternative approach for easy, reliable and high throughput Yq screening of infertile men and their ICSI-conceived sons.
Currently, there is no agreed set of Yq markers for clinical diagnostic PCR screening of infertile men with sperm counts of <5x106/ml in their semen. Because the absence of a PCR product defines a deletion event, single or multiplex PCR of one or more Yq markers targeted to each AZF region has been the most common practice to avoid a false negative result (Vogt, 1998). Whilst such PCR strategies are technically sound, DNA contamination caused by inadvertent introduction of extraneous DNA or cross-contamination of male samples is not routinely monitored by PCR and could potentially lead to a misdiagnosis of a Yq deletion. Conventional PCR using highly sensitive and specific primers and 30–45 cycles of PCR is sufficient to amplify DNA sequences from low amounts of DNA template (Findlay et al., 1998) and even single cells (Sherlock et al., 1998). The use of Southern blot hybridization as a secondary confirmation of the PCR results is one strategy that can avoid a misdiagnosis (Kent-First et al., 1996a; Cram et al., 2000), because low levels of DNA contamination in the primary samples would not be detectable. However, as a laboratory screening technique, Southern blotting has limited utility as it requires large amounts of high quality DNA and is very labour intensive and time consuming. Our new strategy for Yq screening of infertile men based on FL–PCR of the DAZ and RBM1 genes using two independent primer pairs combined with DNA fingerprinting provides increased sensitivity over conventional PCR, as well as the capacity to detect low levels of male to male DNA contamination in genomic DNA samples. Furthermore, the compatibility of FL–PCR with low amounts of genomic DNA extracted from buccal cells will reduce DNA preparation time and provide a more acceptable and accessible sampling method for genetic follow-up studies of male offspring conceived by ICSI. Moreover, this multiplex FL–PCR has the capacity to incorporate additional Y-specific STS markers and thus provide a more comprehensive coverage of Yq deletion events across all four AZF regions.
De-novo deletions of Yq occur at a frequency of ~1 in 5000 in men and are believed to arise by recombination events between highly repetitive DNA sequences (Edwards and Bishop, 1997). Recent physical mapping and DNA sequencing of the AZFa region (Sun et al., 2000) has identified the presence of conserved HERV15 class endogenous retroviral sequences proximal and distal to deletion breakpoints. Sequence analysis of two azoospermic men with AZFa deletions demonstrated that homologous recombination had occurred between specific domains in the proviral sequences. This finding supports the notion that AZFb and AZFc deletion events could occur by a similar mechanism. In fine mapping studies using highly targeted markers, we have demonstrated an apparently identical deletion interval for the frequently occurring AZFdc Yq deletion (RBM1+/DAZ- ). This also points to a common deletion event on Yq involving sequences in the distal region of AZFb and AZFc. DNA sequencing both proximal and distal to the deletions' endpoints will ultimately determine if all AZFdc deletions are indeed identical and provide clues as to the possible mechanism(s) of their formation.
In light of the fact that the Yq region does not undergo any significant homologous recombination with the X chromosome, it is conceivable that the Yq region has accumulated mutations along its length and, potentially, in genes important for normal spermatogenesis. Sequencing of the human genome (International Human Genome Sequencing Consortium, 2001 ; Venter et al., 2001) has revealed that the Y chromosome encodes only 20–30 functional genes. These include TTY1, PRY, TTY2, BPY1, XKRY and BPY2 that are expressed exclusively in the testis and DBY, USP9Y, TB4Y, EIFAY and UTY that are ubiquitously expressed (Lahn and Page, 1997). Any of these Yq genes could therefore harbour mutations that would account for unexplained defects in the semen analysis of men with idiopathic infertility. Indeed, mutation screening by single strand conformational polymorphism has recently revealed a functionally significant mutation in the AZFa USP9Y gene, associated with non-obstructive azoospermia (Sun et al., 1999). With the increased use of ICSI to treat severe male infertility, genetic defects of the Y chromosome including Yq deletions and point mutations in spermatogenic genes will undoubtedly be transmitted to first generation male offspring by ICSI and cause infertility in later years. For Yq deletions, a similar infertility phenotype exhibited by the father would be expected, although phenotype genetic correlations of DAZ- men with apparently identical AZFdc deletions (Reijo et al., 1996) suggest that genetic background could either exacerbate or alleviate the severity of the infertility. In the case of defects on the X chromosome causing male infertility, 50% of sons conceived by first generation ICSI-conceived female offspring would be expected to exhibit a similar infertility phenotype as their grandfather.
As new candidate male infertility genes are revealed by gene targeting and N-ethyl-N-nitrourea mutagenesis experiments in mice (Nolan et al., 2000) and mutations are identified in the human homologues by techniques such as denaturing high pressure liquid chromatography (Liu et al., 1998), it will be obligatory for IVF units to offer comprehensive pre-IVF screening for infertile men as well as genetic follow-up of all ICSI-conceived children. The application of FL–PCR-based techniques combined with DNA fingerprinting will guarantee a high degree of reliability and accuracy that will be essential for high throughput genetic testing. Such testing will also provide important information to couples about the potential risks of assisted reproduction. Furthermore, although the natural history of spermatogenesis is not known for men with Yq deletions, it is conceivable that during adolescence the testes may have some capacity to produce sperm of reasonable quality and number. If this is the case, a prior knowledge of a Yq deletion or other genetic cause in male offspring conceived by ICSI may provide the opportunity for them to produce a semen sample for freezing before sperm production declines and thus increase their fertility prospects by assisted reproduction later in life.
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We thank Pam Audrins for organizing the collection of buccal cell samples from the families who conceived sons by ICSI. This research was funded by a grant from Monash IVF Pty Ltd.
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4 To whom correspondence should be addressed at: MIRD, level 3, 27–31 Wright St, Clayton, Victoria 3168, Australia.E-mail: mandy.katz{at}med.monash.edu.au
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Bhasin, S., Ma, K. and deKretser, D.M. (1997) Y chromosome micro-deletions and male infertility. Ann. Med., 29, 261–263.[Web of Science][Medline]
Chang, P.L., Sauer, M.V. and Brown, S. (1999) Y chromosome microdeletion in a father and his four infertile sons. Hum. Reprod., 14, 2689–2694.
Cooke, H.J., Hargreave, T. and Elliott, D.J. (1998) Understanding the genes involved in spermatogenesis: a progress report. Fertil. Steril., 69, 989–995.[Web of Science][Medline]
Cram, D.S., Ma, K., Bhasin, S., Arias, J., Pandjaitan, M., Chu, B., Audrins, P., Saunders, D., Quinn, F., deKretser, D. et al. (2000) Y chromosome analysis of infetile men and their sons conceived through intracytoplasmic sperm injection: vertical transmission of deletions and rarity of de novo deletions. Fertil. Steril., 74, 909–915.[Web of Science][Medline]
Cui, X., Li, H., Goradia, T.M., Lange, K., Kazazian, H.H., Galas, D. and Arnheim, N. (1989) Single sperm typing: Determination of genetic distance between the G
-globin and parathyroid hormone loci by using the polymerase chain reaction and allele specific oligomers. Proc. Natl Acad. Sci. USA, 86, 9389–9393.
Edwards, R.G. and Bishop, C.E. (1997) On the origin and frequency of Y chromsome deletions responsible for male infertility. Mol. Hum. Reprod., 3, 549–554.
Ferlin, A., Moro, E., Garolla, A. and Foresta, C. (1999) Human male infertility and Y chromosome deletions: role of the AZF-candidate genes DAZ, RBM and DFFRY. Hum. Reprod., 14, 1710–1716.
Findlay, I., Toth, T., Matthews, P., Marton, T., Quirke, P. and Papp, Z. (1998) Rapid trisomy diagnosis (21, 18 &13) using fluorescent PCR and short tandem repeats: applications for prenatal diagnosis and preimplantation genetic diagnosis. J. Assist. Reprod. Genet., 15, 266–275.[Web of Science][Medline]
Foresta, C., Moro, E. and Ferlin, A. (2001) Y chromosome microdeletions and alterations of spermatogenesis. Endocr. Rev., 22, 226–39.
International Human Genome Sequencing Consortium (2001) Initial sequencing and analysis of the human genome. Nature, 409, 860–920.[Medline]
Jiang, M.C., Lien, Y.R., Chen, S.U., Ko, T.M., Ho, H.N. and Yang, Y.S. (1998) Transmission of de novo mutations of the deleted in azoospermia genes from a severely oligozoospermic male to a son via intracytoplasmic sperm injection. Fertil. Steril., 71, 1029–1032.
Jones, M.H., Khwaja, O.S.A., Briggs, H., Lambson, B., Davey, P.M., Chalmers, J., Zhou, C.Y., Walker, E.M., Zhang, Y., Todd, C. et al. (1994) A set of ninety-seven overlapping yeast artificial chromosome clones spanning the human Y chromosome euchromatin. Genomics, 24, 266–275.[Web of Science][Medline]
Kamischke, A., Gromoll, J., Simoni, M., Behre, H.M. and Nieschlag, E. (1999) Transmission of a Y chromosomal deletion involving the deleted in azoospermia (DAZ) and chromodomain (CDY1) genes from father to son through intracytoplasmic sperm injection: case report. Hum. Reprod., 14, 2320–2322.
Kent-First, M.G., Kol, S., Muallem, A., Ofir, R., Manor, D., Blazer, S., First, N. and Itskovitz-Eldor, J. (1996a) The incidence and possible relevance of Y-linked microdeletions in babies born after intracytoplasmic sperm injection and their infertile fethers. Mol. Hum. Reprod., 2, 943–950.
Kent-First, M.G., Kol, S., Muallem, A., Blazer, S. and Itskovitz-Eldor, J. (1996b) Infertility in intracytoplasmic-sperm-injection-derived sons. Lancet, 348, 332.[Web of Science][Medline]
Kent-First, M.G., Muallem, A. and Shultz, J. (1999) Defining regions of the Y-chromosome responsible for male infertility and identification of a fourth AZF region (AZFd) by Y-chromosome microdeletion detection. Mol. Reprod. Dev., 53, 27–41.[Web of Science][Medline]
Kleiman, S.E., Yogev, L. and Hauser, R. (1999) Genetic evaluation of infertile men. Hum. Reprod., 14, 33–38.
Lahn, B.T. and Page, D.C. (1997) Functional coherence of the human Y chromosome. Science, 278, 675–680.
Liu, W., Smith, D.I., Rechtzigel, K.J., Thibodeau, S.N. and James, C.D. (1998) Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations. Nucleic Acids Res., 26, 1396–1400.
Ma, K., Inglis, J.D., Sharkey, A., Bickmore, W.A., Hill, R.E., Prosser, E.J., Speed, R.M., Thomson, E.J., Jobling, M., Taylor, K., et al. (1993) A Y chromosome gene family with RNA-binding protein homology: candidates for the azoospermic factor AZF controlling human spermatogenesis. Cell, 75, 1287–1295.[Web of Science][Medline]
McLachlan, R.I., Mallidis, C., Ma, K., Bhasin, S. and de Kretser, D.M. (1998) Genetic disorders and spermatogenesis. Reprod. Fertil. Dev., 10, 97–104.[Medline]
Meschede, D. and Horst, J. (1997) The molecular genetics of male infertility. Mol. Hum. Reprod., 3, 419–430.
Moro, E., Ferlin, A., Hsiao Yen, P., Guanciali Franchi, P., Palka, G. and Foresta, C. (2000) Male infertility caused by a de novo partial deletion of the DAZ cluster on the Y chromosome. J. Clin. Endocrinol. Metab., 85, 4069–4073.
Najmabadi, H., Huang, V., Yen, P., Subbarao, M.N., Bhasin, D., Banaag, L., Naseeruddin, S., de Kretser, D.M., Baker, H.W., McLachlan, R.I. et al. (1996) Substantial prevalence of microdeletions of theY-chromsome in infertile men with idiopathic azoospermia and oligospermia detected using a sequence-tagged site-based mapping strategy. J. Clin. Endocrinol. Metab., 81, 1347–1352.[Abstract]
Nolan, P.M., Peters, J., Strivens, M., Rogers, D., Hagan, J., Spurr, N., Gray, I.C., Vizor, L., Brooker, D., Whitehill, E. et al. (2000) A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse. Nature Genet., 25, 440–443.[Web of Science][Medline]
Oliva, R., Margarit, E., Ballesca, J.-L., Carrio, A., Sanchez, A., Mila, M., Jimenez, L., Alvarez-Vijande, J.R. and Ballesta, F. (1998) Prevalence of Y chromosome microdeletions in oligospermic and azoospermic candidates for intracytoplasmic sperm injection. Fertil. Steril., 70, 506–510.[Web of Science][Medline]
Page, D.C., Silber, S. and Brown, L.G. (1999) Men with infertility caused by AZFc deletion can produce sons by intracytoplasmic sperm injection, but are likely to transmit the deletion and infertility. Hum. Reprod., 14, 1722–1726.
Pryor, J.L., Kent-First, M., Muallem, A., Van Bergen, A.H., Nolten, W.E., Meisner, L. and Roberts, K.P. (1997) Microdeletions in the Y chromosome of infertile men. N. Eng. J. Med., 336, 534–539.
Qureshi, S.J., Ross, A.R., Ma, K., Cooke, H.J., Intyre, M.A., Chandley, A.C. and Hargreave, T.B. (1996) Polymerase chain reaction screening for Y chromosome microdeletions: a first step towards the diagnosis of genetically-determined spermatogenic failure in men. Mol. Hum. Reprod., 2, 775–779.
Reijo, R., Lee, T.-Y., Salo, P., Alagappan, R., Brown, L.G., Rosenberg, M., Rozen, S., Jaffe, T., Straus, D., Hovatta, O. et al. (1995) Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein. Nature Genet., 10, 383–393.[Web of Science][Medline]
Reijo, R., Alagappan, R.K., Patrizio, P. and Page, D.C. (1996) Severe oligospermia resulting from deletions of the azoospermia factor gene on Y chromosome. Lancet, 347, 1290–1293.[Web of Science][Medline]
Rudbeck, L. and Dissing, J. (1998) Rapid, simple alkaline extraction of human genomic DNA from whole blood, buccal epithelial cells, semen and forensic stains for PCR. Biotechniques, 25, 588–592.[Web of Science][Medline]
Sherlock, J., Cirigliano,V., Petrou, M., Tutschek, B. and Adinolfi, M. (1998) Assessment of diagnostic quantitative fluorescent multiplex polymerase chain reaction assays performed on single cells. Ann. Hum. Genet., 62, 9–23.[Web of Science][Medline]
Silber, S.J., Alagappan, R., Brown, L.G. and Page, D.C. (1998) Y chromsome deletions in azoospermic and severly oligospermic men undergoing intracytoplasmic sperm injection after testicular sperm extraction. Hum. Reprod., 13, 3332–3337.
Simoni, M., Gromoll, J., Dworniczak, B., Rolf, C., Abshagen, K., Kamischke, A., Carani, C., Meschede, D., Behre, H.M., Horst, J. et al. (1997) Screening for deletions of the Y chromosome involving the DAZ (deletion in azoospermia) gene in azoospermia and severe oligospermia. Fertil. Steril., 67, 542–547.[Web of Science][Medline]
Straub, R.E., Speer, M.C., Luo, Y., Rojas, K., Overhauser, J., Ott, J. and Gilliam, T.C. (1993) A microsatellite genetic linkage map of human chromosome 18. Genomics, 15, 48–56.[Web of Science][Medline]
Sun, C., Skaletsky, H., Birren, B., Devon, K., Tang, Z., Silber, S., Oates, R. and Page, D.C. (1999) An azoospermic man with a de novo point mutation in the Y-chromosomal gene USP9Y. Nature Genet., 23, 429–432.[Web of Science][Medline]
Sun, C., Skaletsky, H., Rozen, S., Gromoll, J., Nieschlag, E., Oates, R., and Page, D.C. (2000) Deletion of azoospermia factor a (AZFa) region of human Y chromosome caused by recombination between HERV15 proviruses. Hum. Mol. Genet., 9, 2291–2296.
Toth, T., Findlay, I., Papp, C., Toth-Pal, E., Marton, T., Nagy, B., Quirke, P. and Papp, Z. (1998) Prenatal detection of trisomy 13 from amniotic fluid by quantitative fluorescent polymerase chain reaction. Prenat. Diagn., 18, 669–674.[Web of Science][Medline]
Venter, J.C., Adams, M.D., Myers, E.W., Li, P.W., Mural, R.J., Sutton, G.G., Smith, H.O., Yandell, M., Evans, C.A., Holt, R.A. et al. (2001) The sequence of the human genome. Science, 291, 1304–1352.
Vogt, P.H. (1998) Human chromosome deletions in Yq11, AZF candidate genes and male infertility: history and update. Mol. Hum. Reprod., 4, 739–744.
Vogt, P.H., Edelmann, A., Kirsch, S., Henegariu, O., Hirschmann, P., Kiesewetter, F., Kohn, F.M., Schill, W.B., Farah, S., Ramos, C. et al. (1996) Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum. Mol. Genet., 5, 933–943.
Submitted on February 7, 2001; resubmitted on November 8, 2001; accepted on March 5, 2002.
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