Departments of Pathology and Laboratory Medicine, and Obstetrics and Gynecology, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia
1 To whom correspondence should be addressed at: King Faisal Specialist Hospital and Research Center, P.O.Box 3354, MBC 10, Riyadh, 11211, Saudi Arabia. E-mail: serdar{at}kfshrc.edu.sa
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Abstract |
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Key words: germinal mosaic/microdeletion/multiple displacement amplification/teratozoospermia/Y chromosome
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Introduction |
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Teratozoospermia is defined as less than the reference value of normally shaped sperm in an ejaculate (World Health Organization, 1999). According to the strict criteria of Kruger, <16% normal sperm present in semen is the threshold for teratozoospermia (Kruger et al., 1986
). A role for Y chromosome on sperm morphological abnormalities in mice was suggested long ago (Krzanowska, 1969
) and has been extensively studied since then (reviewed by Burgoyne, 1998
). Several candidate genes including Rbm and Ssty have been suggested to play a role in sperm morphology (Mahadevaiah et al., 1998
; Toure et al., 2004
). In humans, studies on the genetics of teratozoospermia have mostly concentrated on sperm chromosomal aneuploidies (Lewis-Jones et al., 2003
; Vicari et al., 2003
; Burrello et al., 2004
). Moreover, Zeyneloglu et al. (2002)
reported Y chromosome microdeletions in a patient with globozoospermia.
Y chromosome microdeletions could be inherited, or of de novo origin that might arise during spermatogenesis or early embryonic development (Edwards and Bishop, 1997; Kleiman et al., 1999
; Calogero et al., 2002
; Komori et al., 2002
; Kuhnert et al., 2004
). The exact mechanism of de novo transmission is not known. Germinal mosaicism or abnormalities of meiosis have been suggested to play a role in de novo transmissions (Vendrell et al., 1999
; Le Bourhis et al., 2000
).
The aim of this study was to investigate Y chromosome microdeletions in men with teratozoospermia.
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Materials and methods |
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Semen specimens were evaluated for count and motility according to World Health Organization (1999) criteria and for morphology according to the Krugers strict criteria within 1 h of collections (Kruger et al., 1986
). Sperm were isolated by using Percoll gradient centrifugation before DNA extraction (Coskun et al., 2000
).
DNA extraction and PCR
Genomic DNA was prepared from peripheral blood or sperm samples using DNAzol standard technique according to the manufacturers instructions (Invitrogen, Carlsbad, CA, USA) with some modification. Briefly, the sperm pellet, after Percoll separation, was washed with phosphate-buffered saline (PBS) (Irvine Scientific, Santa Ana, CA, USA) and centrifuged at 2000 g five times in order to obtain a clean pellet of cells. Afterward, the same protocol was followed using DNAzol. Twenty Specific Targeted Sequences (STS-3 markers in AZFa, seven markers in AZFb, nine markers in AZFc and one marker between AZFa and AZFb) were assessed using PCR (Table I). PCR protocol consists of amplifying each locus separately without using multiplex PCR. A PCR run was set as 20 markers for the patient. Female DNA and water were used as negative controls. The PCR was carried out in a total volume of 20 µl. The reaction mixture included 40 ng of each DNA sample, 1 x PCR buffer, 1.5 mmol/l MgCl2, 200 µmol/l dNTP, 1 µmol/l of each primer pair and 1 IU Taq DNA polymerase (Invitrogene). The reactions were carried out in a thermal cycler (MJ Research, Waltham, MA, USA). After an initial denaturation step at 94°C for 5 min, PCR amplifications of 35 cycles were performed as follows: 95°C for 1 min for denaturation, 5562°C ramping for 80 s for primer annealing and 72°C for 60 s for the extension. The programs were followed by the final extension step at 72°C for 7 min. The reaction products were then analysed by electrophoresis at 100 V on 2% agarose gels (Sigma, St Louis, MO, USA). In the case of a deletion being observed, a second run was performed in order to confirm the deletion in the presence of a positive control for that deletion.
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Whole genome amplification for single sperm
Whole genome amplification of a single sperm DNA was performed by using REPLI-g kit (Qiagen, Valencia, CA, USA). Briefly, single sperm were collected under an inverted microscope using an ICSI needle in 0.5 ml PCR tubes containing 3 µl of PBS. Aliquots from the last wash droplets were taken to serve as blanks (two tubes). Samples and blanks were incubated 10 min on ice in the lysing buffer (0.1 mol/l dithiothreitol, 0.4 mol/l KOH, 10 mmol/l EDTA), followed by addition of 3.5 µl of stop buffer (solution B). Cell lysates (10 µl) were used directly for multiple displacement amplifications (MDA) by adding 40 µl of the master mix in a total volume of 50 µl. The mix was then incubated at 30°C for 6 h followed by heat inactivation at 65°C for 3 min. MDA products were then tested for the presence of SRY locus (Table I) and Y chromosome-carrying sperm were tested for Y chromosome microdeletions as described above. MDA products from a single sperm with no deletions were used as positive control.
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Results |
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Two patients out of four with deletions were available to provide peripheral blood and fresh semen for single sperm Y chromosome microdeletion analysis. Both patients had none of the STS deleted from the peripheral blood DNA. Y chromosome microdeletion analysis was performed in the MDA-amplified SRY-positive single sperm DNA (Figure 1). MDA yields 10 µg DNA from a single spermatozoon. To obtain 10 SRY-positive sperm, 19 and 22 sperm had to be analysed in each patient. Y chromosome microdeletion analysis in MDA-amplified single sperm DNA confirmed the presence of the same deletion in all 10 sperm for one patient and eight sperm out of 10 in the second patient.
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Discussion |
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Sperm DNA isolated from the cell pellet of semen has been previously used in the screening of Y chromosome microdeletions (Katagiri et al., 2004). In our study, sperm DNA has been preferred over blood DNA to detect any germinal mosaicism. Furthermore, the application of MDA into single cells (Hellani et al., 2004
, 2005
) made it possible to amplify enough DNA from a single sperm for Y chromosome microdeletion analysis. MDA yields a consistent amplification of STS in the control sperm DNA. Jiang et al. (2005)
have also successfully applied MDA as an effective method of global genome amplification in single sperm. Studying single sperm might answer many questions on de novo transmission of Y chromosome microdeletions and germinal mosaicisms.
Studies in mice have shown an important relationship between certain Y chromosome deletions and sperm head abnormalities (Suh et al., 1989; Styrna et al., 1991
, 2003
; Toure et al., 2004
). Toure et al. (2004
) showed that Yq deletion with abolished Ssty expression resulted in severe sperm morphological abnormalities. Similarly, reduced Rbm expression in mice has been suggested to be responsible for, or to contribute to, the abnormal sperm development (Mahadevaiah et al., 1998
). It is difficult from our study to pinpoint any region that could be responsible for teratozoospermia since all four patients have a scattered pattern of microdeletions. In humans, studies on the relationship between Y chromosome microdeletions and sperm morphological abnormalities have yet to be performed. There has been only a case report of a patient with globozoospermia and Y chromosome microdeletions (Zeyneloglu et al., 2002
). However, the causal effect of Y chromosome microdeletions to globozoospermia is not proven. The coexistence of Y chromosome microdeletions and sperm morphology has been reported previously (van Landuyt et al., 2000
; Rao et al., 2004
). However, in both reports, sperm count and motility abnormalities were also present. The first study described one patient who had microdeletion in AZFc region with severe oligozoospermia and abnormal morphology in sperm (van Landuyt et al., 2000
). The second described Y chromosome microdeletions in patients suffering from oligoasthenoteratozoospermia with varicocele (Rao et al., 2004
). To the best of our knowledge, our study is the first to report mosaic Y chromosome microdeletions in pure teratozoospermic patients.
Of 80 patients examined, four were found to be carriers of deletions on the Y chromosome long arm from sperm DNA. Fifty-three samples of sperm DNA extracted from anonymous donors with normal sperm count, motility and morphology showed the absence of any deletions using the same protocol. Deletions were further assessed in two patients who agreed to give blood and fresh semen samples. Leukocyte screening showed the presence of the entire AZF regions suggesting a mosaicism between sperm and peripheral blood as suggested earlier (Le Bourhis et al., 2000). A total of 10 SRY-positive sperm were screened for each patient after whole genome amplification of single sperm DNA. Interestingly, the result showed the deletion in all 10 sperm in one patient and eight out of 10 in the other. The presence of the loci in two out of 10 sperm might be due to a mosaicism in the germ cell line itself in this patient. Unfortunately, we were unable to test the other two patients because they were not available to give samples. The presence of almost complete mosaicism between somatic and germ cells could happen by the combination of two events. First is the observation of early germ cell differentiation during embryonic development (Edwards and Beard, 1999
). Pronuclear and embryonic origins of Y deletions have been proposed (Edwards and Bishop, 1997
) and deletions in the blastomere which is destined to be germ cell precursor could lead to almost complete mosaicism. Second is the generation of palindromic deletions occurring in these germ cell precursors which might result in non-contiguous deletions (Akgun et al., 1997
; Kuroda-Kawaguchi et al., 2001
). Similar non-contiguous deletions were previously reported in genomic DNA (Foresta et al., 1999
).
A correlation between teratozoospermia and Y chromosomal aneuploidy in sperm has been reported (Lewis-Jones et al., 2003; Vicari et al., 2003
). Though few data are available in humans, it is plausible to speculate roles of Y chromosome genes in the formation of a normal spermatozoon. The establishment of a relationship between teratozoospermia and Y chromosome deletions could not be done in patients carrying homogeneous deletions in all their tissue, since such deletions in AZF usually lead to a complete impairment of spermatogenesis. Those observations suggest that azoospermia or very severe oligozoospermia are the most commonly observed phenotypes in patients with homogeneous Y chromosome microdeletions. However, germinal mosaic Y chromosome microdeletions could lead to teratozoospermia probably because of missing genes responsible for spermiogenesis. This suggestion requires further research.
De novo transmission of Y chromosome microdeletions has been suggested and demonstrated in babies born after ICSI (Reijo et al., 1995, 1996
; Kent-First and Muallem, 1996
; Vogt et al., 1996
, Pryor et al., 1997
; Jiang et al., 1999
). Our two cases presented here nicely explain one of the mechanisms for de novo transmission of Y chromosome microdeletions. Germinal mosaic patients could have normal spermatogenesis, however. sperm morphology might be affected and the resulting sperm could lead to fertilization and development of Y chromosome-microdeleted boys.
This report is the first to show a possible relationship between teratozoospermia and Y chromosome microdeletion. Our observations suggest that some of the teratozoospermia might be related to gonadal mosaic Y chromosome microdeletion. Gonadal mosaicism can be a source of de novo transmissions of Y chromosome microdeletions. The application of MDA can yield enough DNA from a single sperm for genetic analysis. Patients with a high number of morphologically abnormal sperm might be advised to undergo sperm DNA Y chromosome microdeletion analysis. Further studies on the implication of Y chromosome microdeletions in germ cells are needed to elucidate the role of Y chromosome genes on spermiogenesis.
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Acknowledgements |
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References |
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Submitted on May 2, 2005; resubmitted on July 3, 2005; accepted on July 14, 2005.
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