Hum. Reprod. Advance Access originally published online on December 22, 2005
Human Reproduction 2006 21(4):980-985; doi:10.1093/humrep/dei428
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Reduced recombination associated with the production of aneuploid sperm in an infertile man: a case report
1 Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, British Columbia and 2 Department of Biology, York University, Toronto, Ontario Canada
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Room 313, Willow Pavilion, 855 West 12th Avenue, Vancouver, British Columbia, Canada V5Z 1M9. E-mail: sai{at}interchange.ubc.ca
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Abstract |
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Studies using gene-linkage analysis have suggested that abnormal recombination during meiosis may lead to the production of aneuploid gametes; however, there is little direct evidence of a link between the two in human males. We analysed spermatocytes in the pachytene stage from a man with extremely high aneuploidy rates in his sperm. Testicular tissue specimens of the infertile man and two vasectomy reversals were processed with immuofluorescent techniques to visualize synaptonemal complex and recombination foci and fluorescent in situ hybridization on spermatocytes and sperm with probes for chromosomes 13, 21, 18, X and Y. We observed no recombination between sex chromosomes in the infertile man, while in two controls, we observed recombination rates of 79.3 and 81.0% between the sex chromosomes. This was associated with a total sex aneuploidy rate of 41.61% in testicular sperm of the infertile man (0.44 and 0.62% in two controls). Recombination on chromosome 21 was reduced in the infertile man, with 10.62% of spermatocytes showing no recombination (0 and 1.67% in two controls), as well as chromosome 13, with 53.98% having
1 recombination foci (22.05 and 21.67% in two controls). This was associated with increased aneuploidy for those chromosomes. Chromosome 18 aneuploidy was slightly increased, although there was no apparent decrease in recombination. These results provide the first evidence of both recombination and non-disjunction abnormalities in the same individual. This is also the only reported case of an infertile man who shows no recombination between the sex chromosomes, despite the formation of the sex body.
Key words: aneuploid sperm/male infertility/reduced recombination/spermatocytes/synaptonemal complex
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Introduction |
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Formation of a spermatozoon requires two successive cell divisions, meiosis I and meiosis II, to reduce the genetic content of a cell from 46 chromosomes (somatic) to 23 chromosomes (gametic). Pairing of homologous chromosomes (synapsis) and recombination occur during the first meiotic division and are critical events in the formation of haploid spermatozoa. Both of these events occur in a meiosis-specific protein structure called the synaptonemal complex (SC), which consists of SC proteins (SCP) 1, 2 and 3. If homologous chromosomes do not segregate properly, the resulting spermatozoon will be aneuploid and upon fertilization may result in spontaneous abortion or congenital malformation syndromes in the liveborn.
Some infertile men have been observed to have an increased frequency of chromosome-pairing abnormalities during meiosis, resulting in meiotic arrest (Judis et al., 2004
; Sun et al., 2004a). Furthermore, it is well established that some infertile men have increased levels of aneuploid sperm. It has been observed that aneuploidy can be transmitted from the sperm to the embryo during ICSI (Bonduelle et al., 2002; Tang et al., 2004). However, there is little direct evidence linking abnormal meiotic chromosome pairing/recombination, aneuploidy and male infertility in humans. Recently developed immunocytological techniques for visualizing recombination proteins related to the SC have opened up a new avenue for studying the early stages of meiosis during spermatogenesis (Hassold et al., 2000). Various important meiotic structures (SC in prophase, centromeres and sites of meiotic exchange) can be identified through the use of immuofluorescent antibodies. Antibodies against SCP1/SCP3 allow visualization of the calcinosis, Raynaud’s phenomenon, Esophageal dysfunction, Sclerodactyly, Telangiectasia (SC, CREST) allows visualization of the centromeres and antibodies against mut L homologue 1 (MLH1), a late-recombination protein that has been shown to co-localize to sites of chiasmata (Marcon and Moens, 2003), allow visualization of the recombination foci. These antibodies, in combination with centromere-specific muliticolour fluorescent in situ hybridization (FISH), have provided a method of quantifying the recombination foci on any of the 22 autosomal bivalents (Oliver-Bonet et al., 2003; Sun et al., 2004b). A study by Gonsalves and colleagues (2004) has also shown that some infertile men have decreased frequencies of recombination. However, in their study, rates of recombination on individual chromosomes were not measured, and there was no investigation of aneuploid spermatozoa of infertile men.
Until now, our understanding of the link between abnormal meiotic recombination and aneuploid sperm has been limited to gene-linkage analysis, which is limited to specific chromosomal sequences and by hereditary/transmission constraints, and only provides an indirect relationship (Hassold et al., 1991; Sherman et al., 1991; Thomas et al., 2000; Shi et al., 2001). Establishment of a direct correlation between abnormal recombination and aneuploidy could improve risk assessment for ICSI.
When performing ICSI, there is a risk of transmission of aneuploidy from the sperm to the conception. This may be due to increased levels of aneuploidy in the sperm of some infertile men, the invasiveness of the procedure, or bypassing the natural selection of normal sperm. Since most chromosomal abnormalities are lethal, miscarriages are more likely to occur after ICSI. We report on such a case in an abortus, derived from ICSI, with a 45,X karyotype of paternal origin (Tang et al., 2004). FISH analysis on the sperm of this infertile man showed extremely high levels of aneuploidy, particularly for the sex chromosomes. We postulated that defects in recombination and pairing might be the direct cause of the man’s infertility and chromosomal abnormalities in the sperm. We used newly developed immunocytogenetic techniques to progress further in understanding the aetiology of non-disjunction in this man with severe oligoasthenoteratozoospermia (OAT). We also attempted to provide direct evidence in humans for a link between abnormal recombination/pairing and an increased incidence of aneuploidy.
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Patient report
The infertile father was 41 years old, had a normal somatic karyotype, a normal hormonal profile, no Y-chromosomal microdeletions and had severe OAT (<1 x 106 sperm/ml). Subsequent histological analysis of testicular tissue showed partial meiotic arrest. Due to the severity of the sperm parameters, the patient and his 36-year-old wife underwent ICSI treatment. A singleton pregnancy was established after transferring four eight-cell stage embryos. At 8 weeks of gestation, the patient had a spontaneous abortion. Cytogenetic analysis revealed that the abortus had a 45,X karyotype. Furthermore, microsatellite markers used to determine the origin of the 45,X karyotype showed that the missing X was of paternal origin (Tang et al., 2004).
FISH was performed on the ejaculate sperm using probes for chromosomes 18, X, Y (Tang et al., 2004), 13 and 21. Rates of aneuploidy in all studied chromosomes in the sperm were significantly increased when compared with the control. In the sperm of this infertile man, sex chromosome aneuploidy was markedly increased: sex chromosome nullisomy 19.58% (0.28% control), XY disomy 18.56% (0.10% control) (Tang et al., 2004). Thus, the 45,X karyotype in the abortus may have been the result of fertilization of the oocyte by a sperm lacking a sex chromosome. Approval was obtained from the University of British Columbia Ethics Committee before initiating this study.
Preparation of testicular tissue
The patient agreed to undergo a testicular biopsy for retrieval of a small amount of tissue for immunofluoresence analysis. Control tissue was obtained during vasectomy reversals from fertile men, aged 35 and 60 years. The tissue was processed using a modification of the method used by Barlow and Hulten (1998). In room temperature phosphate-buffered saline (PBS) (pH 7.4) the seminiferous tubules were separated and cut into 3–5 mm segments. The tissue was allowed to incubate for 45–60 min at room temperature in freshly prepared hypo-extraction buffer [30 mM Tris, 50 mM sucrose, 17 mM citric acid, 5 mM EDTA, 0.5 mM dithiothreitol (DTT) and 0.1 mM phenylmethylsulphonyl flouride]. The tissue was then deposited on a microscope slide with 20 µl of 100 mM sucrose (pH 8.2). Using fine forceps, the tubules were squeezed to release their contents, and 10 µl of the germ cell/sucrose slurry was transferred to a new slide overlain with 1% paraformaldehyde with 0.2% Triton X. Slides were incubated at room temperature for 24 h in a humid chamber.
Fluorescence immunostaining
The slides were air dried for 30 min and washed twice in 0.4% PhotoFlo (Kodak 200 solution). The slides were then soaked in x1 ADB (1% donkey serum, 0.3% bovine serum albumin, 0.005% Triton X, PBS; pH 7.2) at room temperature for 30 min. The primary antibody cocktail [rabbit antihuman MLH1 (Oncogene, San Diego, CA, USA), 1 : 37.5; SCP3 antimouse immunoglobulin (Ig)G1 (produced by P.Moens, York University), 1 : 300, SCP1 antimouse (produced by P.Moens), 1 : 300, CREST antisera, 1 : 25; 1x ADB was applied to drained but not dry slides. A cover slip was applied, and the slide was incubated in a humid chamber at 37°C for 24 h. The slides were then soaked for 20 min in x1 ADB, followed by a subsequent wash in x1 ADB for 48 h at 4°C. The secondary antibody cocktail [Flourescein (FITC) Donkey antirabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) 1 : 50, Rhodamine (TRITC) Goat antimouse IgG (Jackson ImmunoResearch) 1 : 100, (AMCA) Donkey antihuman IgG (Jackson ImmunoResearch) 1 : 50, 1 x A
B] was applied and slides were incubated in a 37°C humid chamber. The slides were washed three times in PBS (10, 20 and 30 min) with agitation every 5 min. Slides were drained, antifade was added and a coverslip was applied. Slides were scanned with a Zeiss Axioplan epifluorescent microscope equipped with appropriate filters. Images of the SCP3/SCP1 fragments, MLH1 and CREST sites were captured using Cytovision V2.81 Image Analysis software (Applied Imaging International, San Jose, CA, USA). Pachytene cells were captured if MLH1 labelling was clear and the sex chromosomes were identifiable. Cell co-ordinates were recorded and prints were analysed for the numbers of MLH1 and abnormalities in the SC.
FISH on spermatocytes and sperm
After analysis of SCP3/MLH1 on the pachytene spermatocytes, FISH was performed to identify chromosomes 13, 18 and 21. Coverslips were removed and slides were soaked twice for 5 min in x4 saline sodium citrate (SSC)/0.05% Tween-20 solution, followed by drying in an ethanol series (70, 80, 90 and 100%). After air drying, slides were washed for 5 min in PBS, fixed in 10% formalin phosphate for 5 min, washed 5 min in PBS, rinsed in deionized water and dried in an ethanol series (70, 85 and 100%). Slides were air dried in darkness. Probe CEP 18 (Spectrum Blue) and probe mixture LSI 13 (Green)/LSI 21 (Red) (Vysis Inc., Downers Grove, IL, USA) were added to slides, coverslip was added and sealed with rubber cement. Slides were co-denatured on hotplate for 5 min at 75°C, then placed in a 37°C humid chamber overnight. Coverslips were removed and slides were washed at 75°C in x0.4 SSC/0.3% NP-40 solution for 2 min, followed by a 30 s rinse in room temperature x 2 SSC/0.1% NP-40 solution. After air drying in the dark, antifade and coverslip were added. The cells captured beforehand were relocated and chromosomes 13, 18 and 21 were identified.
Sperm were pre-treated with a 5 min wash in x2 SSC, followed by 15–30 min in 10 mM DTT/100 mM Tris (pH 8.0). When sufficient decondensation was achieved, slides were washed for 5 min in x2 SSC followed by dehydration in an ethanol series (70, 80, 90 and 100%). Procedures for sperm denaturation, hybridization of probes and post-hybridization wash were the same as those for spermatocytes. A probe mixture of CEP 18 (aqua)/CEP X (green)/CEP Y (red) was hybridized to sperm (Vysis Inc., Downers Grove, IL, USA). A second slide of denatured sperm was hybridized with a probe mixture of LSI 13 (green)/LSI 21 (red).
Data analysis
Fisher’s exact test was used to compare the percentage of spermatocytes in different stages of prophase, the percentage of XY bodies with MLH1 foci, the percentage of cells with one or more autosomal bivalent with no MLH1 foci and the percentage of cells with synaptic anomalies between the OAT patient and controls. The mean number of MLH1 foci in pachytene cells and on bivalents 13, 18 and 21 in the OAT patient was compared with controls using the Mann–Whitney U-test because the frequencies of MLH1 foci were not normally distributed. FISH results of the OAT patient and controls were compared using Fisher’s exact test and the Chi-squared test.
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Results |
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FISH analysis of the testicular sperm using probes for chromosomes 13, 18, 21, X and Y showed increased levels of aneuploidy for all chromosomes when compared to FISH of testicular biopsies of fertile men, retrieved during vasectomy reversals (Table I). Surprisingly, there were significant differences between the rates of aneuploidy in the ejaculate sperm and the sperm in the testes. XY nullisomy was significantly lower in the testicular sperm than in the ejaculate sperm; however, disomy of the sex chromosomes and chromosome 21, as well as nullisomy of chromosomes 13, 18 and 21, was significantly greater in the testes than in the ejaculate sperm (P
0.05, Chi-square test).
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After immunostaining, spermatocytes in prophase were classified as being in one of four stages: (i) leptotene, if only short fragments of SCP3 were present (Figure 1A); (ii) zygotene, if complete SCP3 fragments were found with no association between homologous chromosomes, and therefore 46 CREST signals (Figure 1B); (iii) zygotene/pachytene, if some homologous chromosomes were fully synapsed, forming SCP3/SCP1 fragments, with little or no MLH1 foci (Figure 1C); and, finally, (iv) pachytene, if all homologous chromosomes were synapsed, resulting in 24 CREST signals and complete SCP3/SCP1 fragments with MLH1 foci on most bivalents (Figure 1D). In all individuals, the majority of cells in prophase I were in the pachytene stage; however, the OAT patient had significantly less cells in pachytene (56.5%) than the controls (mean = 80.8%) (P < 0.0001, Table II). In the OAT patient, meiotic cells appeared to be partially blocked in leptotene, with 32.3% of cells in this stage compared with 11.0 and 12.4% in the controls (P < 0.0001).
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The total number of MLH1 foci on autosomal chromosomes was counted for each pachytene spermatocyte (Figure 2), and the infertile man showed a significantly lower frequency of recombination when compared to both controls. The mean number of recombination sites per cell in the infertile man was 38.3 ± 5.6, which was significantly lower than the controls (42.9 ± 4.7 and 48.0 ± 4.7) in this study (P < 0.0001, Table III). The number of MLH1 foci in this infertile man was also significantly less than that seen for fertile men in other studies, which have ranged from 42.5 to 55.0 foci (Lynn et al., 2002; Gonsalves et al., 2004; Hassold et al., 2004; Codina-Pascual et al., 2005; Sun et al., 2005).
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Of the 133 pachytene cells analysed in the OAT patient, no MLH1 foci were seen in the XY body; the controls, however, showed rates of recombination in the XY bivalent at 79.3 and 81.0% (Table III). Neither of the controls displayed more than one MLH1 focus per XY bivalent. Recombination rates on autosomal chromosomes also appeared to be reduced in the patient, as 35.3% of cells had at least one autosomal chromosome without an MLH1 focus compared to 3.3 and 3.0% seen in the controls (P < 0.0001, Table III). There was no difference in the rates of gaps in the SC between the patient and controls; however, the patient did display unpaired regions in the SC in 14.3% of cells, which was significantly greater than the 3.0% in one of the controls (P = 0.0031).
We subsequently performed multicolour FISH on the immunolabelled testicular spreads using probes for chromosomes 13, 18 and 21 (Figure 3B). The same cells that were previously captured were relocated, and the number of recombination foci on chromosomes 13, 18 and 21 was determined (Figure 3A). There was no difference in the rate of achiasmate (no MLH1 focus) chromosome 13 bivalents between the patient and control. However, the chromosome 13 bivalent of the infertile man had only one MLH1 focus in 50.4% of cells compared with 20.6 and 21.7% of chromosome 13 bivalents in the fertile controls (P < 0.0003, Table IV). The majority of chromosome 13 bivalents in the controls had two or more MLH1 foci. The chromosome 21 bivalent in the patient was achiasmate in 10.6% of cells, which was significantly greater than the 0 and 1.7% observed in the controls (P = 0.004, P = 0.04, Table IV). We did not find a clear reduction in recombination on the chromosome 18 bivalent in the infertile man: 61.1% of the chromosome 18 bivalents in the infertile man showed only one MLH1 focus, compared with 64.7 and 41.7% in the control (Table IV). Aneuploidy rates in testicular sperm for chromosome 18 (3.36%), although significantly greater than the controls, were not as high as those seen for chromosomes 13 (6.05%) and 21 (7.64%) (Table I).
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Discussion |
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FISH studies on human sperm have shown that infertile men are at an increased risk of sperm aneuploidy, particularly involving the sex chromosomes (Moosani et al., 1995
; Bernardini et al., 2000; Ushijima et al., 2000). The infertile man reported on in this study showed obvious increases in aneuploidies of chromosomes 13, 18, 21 and the sex chromosomes (Table I). We hypothesized that the extremely high rates of aneuploidy observed in this man may have been due to errors in recombination, leading to meiosis I non-disjunction (Tang et al., 2004). There were significant differences between aneuploidy rates in the testicular sperm and sperm in the ejaculate, with disomy of the sex chromosomes and chromosome 21, as well as nullisomy of chromosomes 13, 18 and 21, increased in the testes. Increased aneuploidy has been observed in sperm retrieved from the testes compared to the ejaculate, and it has been suggested that this is due to the ejaculate sperm reaching a higher degree of maturation (Gianaroli et al., 2005). Nevertheless, further studies comparing chromosomal complements in sperm from the ejaculate and testes in the same men will be needed to determine whether a true difference exists.
However, recent studies have reported on abnormal synapsis leading to complete (Gonsalves et al., 2004; Judis et al., 2004) and partial (Sun et al., 2004a) meiotic arrest in men with idiopathic infertility. In those cases, the proportion of cells in zygotene was increased, suggesting problems in the pairing of homologous chromosomes and synapsis. In our case, an increased proportion of cells in leptotene could suggest a partial blockage in the formation of the SC along sister chromatids, leading to partial meiotic arrest.
We observed significant interindividual variation in the rates of recombination between the controls in this study. The mean number of recombination sites per cell in the two controls (42.9 and 48.0) differed significantly, which was not unexpected, as significant interindividual variation in the number of meiotic exchanges has been observed among fertile men in almost all immunofluoresent studies of recombination (Lynn et al., 2002; Gonsalves et al., 2004; Hassold et al., 2004; Codina-Pascual et al., 2005; Sun et al., 2005). Reduced recombination has been associated with infertility in two previous studies (Gonsalves et al., 2004; Sun et al., 2004a). In both of these reports, as in our case, reduced recombination was associated with partial meiotic arrest.
We observed no recombination (0%) between the sex chromosomes in this infertile man, compared with 79.3 and 81.0% of the cells from the controls. Thus, it appears that the extremely high level of sex chromosome aneuploidy in the sperm of this man was caused by a lack of recombination, leading to non-disjunction at meiosis I. These results support previous findings that a reduction in meiotic exchange between the sex chromosomes is linked to aneuploid sperm (Hassold et al., 1991; Thomas et al., 2000; Shi et al., 2001). Previous investigations, however, relied on genetic linkage analysis of paternally derived 47,XXY offspring (Hassold et al., 1991; Thomas et al., 2000) or the analysis of single spermatozoa (Shi et al., 2001) to display a relationship between a lack of recombination in the pseudoautosomal region and the production of aneuploid sperm. Thus, those studies were unable to distinguish between a failure of pairing of the sex chromosomes or normal pairing without recombination. In this study, we have shown that lack of recombination, despite the formation of the XY body, in an infertile man can be an indicator for extremely high levels of sex chromosome aneuploidy.
We observed achiasmate chromosome 21 bivalents in 10.6% of cells, which was greater than that observed in controls (0 and 1.67%). Thus, it appears that reduced recombination on chromosome 21 was associated with aneuploid sperm. While failed recombination has been correlated to the production of maternally derived trisomy 21 (Sherman et al., 1991), little is known about the role of aberrant recombination in the paternal non-disjunction of chromosome 21. Nevertheless, these results, along with previous observations (Savage et al., 1998) suggest that chromosome 21 bivalents without recombination may be an indicator for paternally derived trisomy 21. We also observed a reduction in recombination on chromosome 13, with the majority of bivalents having one or no MLH1 focus, compared with two foci observed in the controls. It is believed that proper recombination and subsequent chiasmata formation is necessary for sufficient microtubule attachment and alignment of homologous chromosomes during metaphase I (Roeder, 1997). Thus, an absence or altered location of recombination may result in increased aneuploidy (Hassold and Hunt, 2001) or failed spermatogenesis and infertility (Hale, 1994).
We did not observe a clear reduction in recombination on the chromosome 18 bivalent. While aneuploidy of chromosome 18 in the testicular sperm was elevated (3.36%), it was not as high as aneuploidy rates for chromosomes 13 (6.05%) and 21 (7.64%). Thus, it appears that reduced recombination may only be an indicator for extremely high levels of aneuploidy. It has been shown that there is an excess of early recombination protein foci (such as RAD51, DMC1 and RPA), which ensure sufficient interactions between the homologous chromosomes (Moens et al., 2002). Thus, it appears that, in this infertile man, these early recombination proteins may be underexpressed, or the mechanism whereby these proteins are removed is overactive.
The results from this study provide the first evidence of both recombination and non-disjunction abnormalities in the same individual. Furthermore, we believe that this is the only reported case of an infertile man who shows no recombination between the sex chromosomes, despite the formation of the sex body. Abnormal patterns of recombination may be the underlying cause of both the partial meiotic arrest, leading to infertility, and the abnormally high rates of non-disjunction seen in this man. Future mutation detection analysis for genes encoding meiotic proteins will shed further light on the molecular basis of this man’s infertility and high levels of abnormal sperm.
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We gratefully thank the patients for donating samples, as well as the clinical and laboratory staff of the University of British Columbia IVF program in the Division of Reproductive Endocrinology and Infertility for their assistance in this study. The Canadian Institute of Health Research (MOP53067 to S.M) and The Hospital for Sick Children Foundation (XG 02–086 to S. M) provided the financial support.
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Submitted on September 17, 2005; resubmitted on November 1, 2005; accepted on November 14, 2005.
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 F. Sun, M. Mikhaail-Philips, M. Oliver-Bonet, E. Ko, A. Rademaker, P. Turek, and R.H. Martin Reduced meiotic recombination on the XY bivalent is correlated with an increased incidence of sex chromosome aneuploidy in men with non-obstructive azoospermia Mol. Hum. Reprod., July 1, 2008; 14(7): 399 - 404. [Abstract] [Full Text] [PDF]
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G. Kirkpatrick, K. A. Ferguson, H. Gao, S. Tang, V. Chow, B. H. Yuen, and S. Ma A comparison of sperm aneuploidy rates between infertile men with normal and abnormal karyotypes Hum. Reprod., July 1, 2008; 23(7): 1679 - 1683. [Abstract] [Full Text] [PDF]
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L. Uroz, O. Rajmil, and C. Templado Premature separation of sister chromatids in human male meiosis Hum. Reprod., April 1, 2008; 23(4): 982 - 987. [Abstract] [Full Text] [PDF]
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K. A. Ferguson, V. Chow, and S. Ma Silencing of unpaired meiotic chromosomes and altered recombination patterns in an azoospermic carrier of a t(8;13) reciprocal translocation Hum. Reprod., April 1, 2008; 23(4): 988 - 995. [Abstract] [Full Text] [PDF]
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E. C. Wong, K. A Ferguson, V. Chow, and S. Ma Sperm aneuploidy and meiotic sex chromosome configurations in an infertile XYY male Hum. Reprod., February 1, 2008; 23(2): 374 - 378. [Abstract] [Full Text] [PDF]
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 K. A. Ferguson, E. C. Wong, V. Chow, M. Nigro, and S. Ma Abnormal meiotic recombination in infertile men and its association with sperm aneuploidy Hum. Mol. Genet., December 1, 2007; 16(23): 2870 - 2879. [Abstract] [Full Text] [PDF]
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