IVF Unit, Shaare-Zedek Medical Center, Ben Gurion University of the Negev, Jerusalem 91031, Israel
The first two authors contributed equally to the paper.
1 Current address: Bioinformatic Department, Jerusalem Center of Technology, Jerusalem, Israel
2 To whom correspondence should be addressed at: IVF Unit, Shaare-Zedek Medical Center, P.O.Box 3235, Jerusalem 91031, Israel. e-mail: gevat{at}szmc.org.il
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Abstract |
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Key words: androgen receptor gene/CAG repeat length/male subfertility/sperm morphology/teratozoospermia
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Introduction |
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An eight exon single copy gene in chromosome Xq11-12 encodes the AR (Tsai and OMalley, 1994). The first exon, which encodes the transactivation domain, contains a segment of CAG repeats, translated to polyglutamine. This glutamine-repeat tract is polymorphic and its size varies from nine to 36 in normal individuals (Andrew et al., 1997
).
The phenomenon of polyglutamine tracts, often of extensive length, is widespread. Expansion of CAG repeats in genes has been implicated in the pathogenesis of certain progressive neurodegenerative diseases (Lieberman and Fischbeck, 2000). CAG repeat tract expansion in the AR is associated with spinal bulbar muscular atrophy (SBMA, Kennedy disease) (La Spada et al., 1991
). This fatal, X-linked, adult onset neuromuscular disease is significantly linked with expansion of CAG tract of >40 repeats and characterized by hypovirilization and testicular atrophy resulting in marked oligozoospermia or azoospermia.
In vitro studies have demonstrated a negative correlation between CAG repeat size and AR function (Chamberlain et al., 1994). Moreover, while short alleles were found to be associated with prostate cancer (Giovannucci et al., 1997
; Stanford et al., 1997
; Hsing et al., 2000
) and polycystic ovary syndrome (Mifsud et al., 2000
; Ibanez et al., 2003
), longer AR CAG repeats were associated with moderate-to-severe undermasculinization (Lim et al., 2000
) and with breast cancer (Levine and Boyd, 2001
; Haiman et al., 2002
).
The data regarding male subfertility is less consistent. Groups from China, Singapore, Japan, Australia, Greece, Germany and North America have shown that longer CAG repeats are associated with defective spermatogenesis (Tut et al., 1997; Dowsing et al., 1999
; Yoshida et al., 1999
; Mifsud et al., 2001
; Patrizio et al., 2001
; Kukuvitis et al., 2002
; Pan et al., 2002
; Asatiani et al., 2003
; Casella 2003
). However, other studies conducted in Swedish, Finnish, German, Indian and Japanese populations have failed to show a significant relationship between idiopathic defective spermatogenesis and the length of CAG repeats (Giwercman et al., 1998
; Hiort et al., 1999
; Dadze et al., 2000
; Sasagawa et al., 2001
; Van Golde et al., 2002
; Rajpert-De Meyts et al., 2002
; Thangaraj et al., 2002
; Lund et al., 2003
). One European study showed inverse correlation between sperm concentration and CAG repeat length in fertile men but no association with infertility (von Eckardstein et al., 2001
). Two additional European studies have demonstrated an association between CAG repeat length and male subfertility, but found no correlation with sperm analysis variables (Legius et al., 1999
; Wallerand et al., 2001
). Recently some authors have suggested that differences in population distribution of CAG repeats and local environmental conditions are responsible for these disparities (Dadze et al., 2000
; Patrizio et al., 2001
).
Concern about the possible transmission of a premutation for neuromuscular disease and male subfertility makes interest in the clinical significance of CAG repeat expansion more than academic. The aim of this study was to assess the distribution of CAG repeat expansion in Israeli men and to determine its association with sperm parameters.
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Materials and methods |
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Semen analysis
Values for semen parameters were calculated as means of at least two analyses taken at least 1 month apart. Normal semen analysis was defined according to World Health Organization (WHO, 1999) recommendations: sperm concentration >20x106/ml, motility >50% and >14% normal forms (strict criteria, Kruger et al., 1987
). Patients with at least one abnormal semen parameter were included in the study group. Only those patients who had all three semen parameters within the normal range were considered as controls. Additional data were collected on specific morphological abnormalities, including abnormality location (head or non-head) and type of head abnormality (round/small or amorphous).
DNA analysis
Peripheral blood from each patient was collected in EDTA-containing tubes. Leukocytes were digested by proteinase K and purified by phenol chloroform. The DNA was precipitated by ethanol. The primers were labelled with [-33P]ATP using T4-polynucleotide kinase from MPI Company. Ten nanograms of genomic DNA were subjected to 35 cycles of PCR amplification. The primers were 5'-TCCAGAATCTGTTCCAGAGCGTGC-3' and 5'-GCTGTG AAGGTTGCTGTTCCTCAT-3'. The PCR amplification was performed under the following conditions: 95°C for 30 s, 58°C for 30 s and 72°C for 1 min. PCR products were separated by electrophoresis in denaturing 6% ureapolyacrylamide formamide gel, followed by autoradiography. PCR products, for which their CAG repeat lengths were identified by sequencing, were used as reference size.
Statistical analysis
Comparisons of mean CAG repeat lengths across dichotomized semen variables (normal versus abnormal) were carried out using Students t-test and MannWhitney U-test. Comparisons of mean CAG repeat lengths across quartile groups and of frequency distributions were conducted using the KruskalWallis test for mean ranks. Binary logistic regression models were fitted to analyse the effects of CAG repeat length (independent variable) on sperm quality variables (dependent variables). Finally, analyses of correlations between CAG repeat length and severity of morphological abnormalities was carried out using Kendalls tau-b correlation coefficients. All statistical analyses were performed using SPSS software (version 10.0.5, SPSS Inc., USA). Results are expressed as mean ± 95% CI.
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Results |
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Discussion |
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In accordance with the findings of some authors (Giwercman et al., 1998; Dadze et al., 2000
; Sasagawa et al., 2001
; Rajpert-De Meyts et al., 2002
; Van Golde et al., 2002
; Asatiani et al., 2003
) and in contrast to those of others (Tut et al., 1997
; Dowsing et al., 1999
; Yoshida et al., 1999
; Mifsud et al., 2001
; Patrizio et al., 2001
), we found no association between CAG repeat elongation and semen concentration or motility. A possible explanation for these conflicting data is ethnic differences in the study populations, which may have led to different results in the European and Asian research. The CAG repeat length is highly polymorphic between ethnic groups (Sartor et al., 1999
). African Americans generally have shorter CAG repeats (La Spada et al., 1991
; Giovannucci et al., 1997
), which are less prevalent in Asian populations (Irvine et al., 1995
). This explanation is supported by the relative homogeneity of the European research results (Rajpert-De Meyts et al., 2002
), while studies in Asian populations are less consistent. Especially confusing are the results of studies in the Japanese population (Komori et al., 1999
; Yoshida et al., 1999
; Sasagawa et al., 2001
). Most participants in our study are of Semitic origin. Interestingly, there was no difference in CAG length distribution between men of different ethnic subgroups in our study (Arabs, Ashkenazi and Sepharadi Jews, Table I).
An attempt to categorize the semen analysis variables according to WHO and Krugers criteria and compare the groups for CAG repeat prevalence failed to yield significance. Recently, in a large multi-centre study, it was found that the accepted WHO criteria for normal semen analysis were poor predictors for fertility (Guzick et al., 2001). Even though sperm morphology appeared to be the most discriminating predictor of infertility. The sensitivity of sperm morphology as a predictor of infertility was corroborated by numerous studies, particularly since Kruger championed it as the main predictor of fertility (Kruger et al., 1987
; Vawda et al., 1996
; Menkveld et al., 2001
). In our study the only variable significantly associated with CAG repeat length was morphology, expressed as percentage of normal forms.
Our findings validate the concept that polymorphism of AR CAG length may contribute to spermatogenesis efficiency through a subtle modulatory effect on AR function. CAG repeats most likely do not have a major independent effect on reproduction, but rather modify or fine-tune endocrine feedback systems and hormone action. If this were not so, the specific polymorphism would be expected to disappear from the reproductive pool. The overall fertility status of an individual depends not only on AR sequence alterations but rather on interactions between other genomic and environmental parameters. Probably comparison of groups according to any categorised sperm analysis variables is a crude instrument for such fine AR activity modifier as CAG repeat.
In conclusion, we found a positive correlation between AR CAG repeat length and teratozoospermia. This finding validates the concept that the length of AR CAG repeats tract is negatively associated with spermatogenesis. However, we do not conclude that mild expansion of the CAG repeat tract directly cause teratospermia, but assume that men with longer CAG repeats might be more prone to spermatogenesis defects in response to any pathogen/epigenetic factors. Genetic screening for AR CAG length maybe offered to couples with male factor infertility, at least in those populations where an association with reproductive variables could be found.
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Acknowledgement |
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FOOTNOTES |
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References |
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Submitted on September 8, 2003; accepted on February 18, 2004.