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ABSTRACT: There is ample documentation supporting the fact that androgens are required for normal spermatogenesis. A minority of infertile men have abnormal testosterone blood levels or mild androgen receptor mutations. We investigated the androgen receptor CAG and GGN repeat lengths in Chilean men with spermatogenic impairment. We studied 117 secretory azoospermic/oligozoospermic men (93 idiopathic and 24 excryptorchidic), without Y-chromosome microdeletions, and 121 controls with normal spermatogenesis (42 obstructive and 79 normozoospermic men). Peripheral blood was drawn to obtain genomic DNA for polymerase chain reaction and automated sequencing of CAG and GGN repeats. Testicular characterization included hormonal studies, physical evaluation, and seminal and biopsy analysis. The CAG and GGN polymorphism distributions were similar among idiopathic men, excryptorchidic men, and controls and among the different types of spermatogenic impairment. However, the proportion of the CAG 21 allele was significantly increased in idiopathic cases compared to controls (P = .012 by Bonferroni test, odds ratio = 2.99, 95% confidence interval, 1.27–7.0) and the CAG 32 allele only was observed in excryptorchidic patients (P < .0002, Bonferroni test). Idiopathic cases with Sertoli cell–only syndrome showed the highest proportion of the CAG 21 allele (P = .024, χ2 test). On the other hand, in idiopathic cases and controls the most common GGN allele was 23, followed by 24, but an inverse relation was found among excryptorchidic cases. The joint distribution of CAG and GGN in control, idiopathic, and excryptorchidic groups did not show an association between the 2 allele repeat polymorphisms (P > 0.05, χ2 test). Our results suggest that the CAG 21 allele seems to increase the risk of idiopathic Sertoli cell–only syndrome. Moreover, the GGN 24 allele could be contributing to deranged androgen receptor function, associated with cryptorchidism and spermatogenic failure.
Failure of spermatogenesis is largely responsible for male infertility, but its etiology remains unknown in nearly half of all cases (Bhasin, 2007; Krausz and Giachini, 2007). Until now, Y-chromosome microdeletions have constituted the most important known etiological factor for spermatogenic failure. Several studies indicate a prevalence of 5% to 20% in subjects with azoospermia or severe oligozoospermia (Vogt, 1998; Krausz et al, 1999), and only a few reports have found a higher prevalence in patients with severe testiculopathies, such as hypospermatogenesis, maturation arrest (MA), and Sertoli cell–only syndrome (SCOS; Foresta et al, 1998; Foresta, 2001; Ferlin et al, 2007).
Development of male phenotype and the initiation of spermatogenesis leading to production of male gametes are dependent on cellular events that respond to androgens. In fact, mutations in the androgen receptor (AR) gene cause a variety of defects, known collectively as the androgen insensitivity syndrome (AIS), which range from XY patients with female phenotype and high serum levels of testosterone and estradiol, known as complete insensitivity syndrome, to subjects with a mild AIS who have infertility as their primary or even sole symptom (Davis-Dao et al, 2007). Furthermore, a significant proportion of infertile males have a history of cryptorchidism, which may constitute an additional phenotypical expression of AIS. This is the most frequent congenital birth defect in males and represents the best-characterized risk factor for infertility and testicular cancer in adulthood, but its etiology remains mostly unknown (Ferlin et al, 2008; Foresta et al, 2008).
The AR contains 4 main functional domains: the amino-terminal transactivation domain (TAD), the centrally positioned DNA-binding domain, the hinge region, and the carboxyl-terminal ligand binding domain. Within TAD are 2 segments consisting of amino acid repeats, glutamine (encoded by CAG) and glycine (encoded by GGN). These repeat tracts are polymorphic, in that their size varies among individuals from a normal population (Lundin et al, 2003, 2007; Palazzolo et al, 2008). The CAG repeat lengths span from approximately 12 to 25 repeats, with a median number of 22, and in rare cases more than 35 contiguous CAGs (Palazzolo et al, 2008).
Longer CAG repeat lengths result in reduced AR transcriptional activity both in vivo and in vitro (Tut et al, 1997; Beilin et al, 2000; Crabbe et al, 2007). In fact, the CAG repeat tract has been the source of unprecedented interest in recent years because it was found that CAG expansion beyond 37 repeats leads to spinal bulbar muscular atrophy (also known as Kennedy disease), an adult-onset X-linked neurodegenerative disease that shows an inverse correlation between repeat length and the age of onset of gynecomastia, as well as clinical and hormonal evidence of androgen insensitivity (La Spada et al, 1991; Dejager et al, 2002; Palazzolo et al, 2008).
Even though CAG tract lengths correlate inversely with sperm concentration in normal men (von Eckardstein et al, 2001), several studies involving infertile men have reported conflicting results, in part related to ethnicity, sample size, and inclusion criteria, with some showing no increase (Dadze et al, 2000; Sasagawa et al, 2001; von Eckardstein et al, 2001; Ferlin et al, 2004; Martinez-Garza et al, 2008; Westerveld et al, 2008), and others reporting an increased length with respect to controls (Tut et al, 1997; Dowsing et al, 1999; Mifsud et al, 2001; Patrizio et al, 2001; Wallerand et al, 2001). In 2007, Davis-Dao et al provided support for an association between the CAG length and idiopathic male infertility by a meta-analysis, but recommended measurement of additional AR length polymorphisms, such as GGN repeat length sequence. Moreover, a recent study investigated different CAG lengths in the normal range (16, 22, and 28) together with the GGN 23 allele and found that the highest AR activity was confined to CAG = 22 and not to CAG = 16, indicating some CAG alleles into the normal range may show no linearity between length and sensitivity of the AR (Nenonen et al, 2010).
The functional consequences of variations in the GGN repeat are even less clear, and epidemiological investigations of the association between the number of GGN repeats in male infertility have produced inconsistent results (Tut et al, 1997; Lundin et al, 2003; Ferlin et al, 2004). In general, the GGN repeats span from 10 to 27 and the predominant allele has 23 repeats (Lundin et al, 2003). In addition, in vitro data has indicated that ARs with glycine numbers other than 23 have low transactivating capacity in response to both testosterone and 5-α dihydrotestosterone (DHT; Lundin et al, 2007).
Recently, other studies have investigated the distribution of different CAG/GGN combinations in infertile men and controls (Ferlin et al, 2004, 2005; Ruhayel et al, 2004). In particular, the same 2 CAG/GGN haplotypes (CAG = 21/GGN = 24 and CAG ≥ 21/GGN ≥ 24) showed an increased susceptibility to idiopathic secretory infertility (Ferlin et al, 2004) and to cryptorchidism (Ferlin et al, 2005), associated with spermatogenic damage in an Italian population. Similar results were found in a Swedish population who showed evidence for a protective effect in <21 CAG and GGN = 23 length repeat carriers (Ruhayel et al, 2004).
Therefore, our aim was to study the CAG and GGN repeat lengths alone and in combination in Chilean men with primary spermatogenic failure, idiopathic or with a history of cryptorchidism, compared to controls with normal spermatogenesis.
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Normal levels of androgens and a functional receptor are essential for development and maintenance of the male phenotype and for spermatogenesis (Quigley et al, 1995; Hiort and Holterhus, 2000). A number of genetic factors that include chromosomal aberrations, Y-chromosome microdeletions, mutations in the CFTR gene, and several types of mutations in the AR gene may be responsible for about 15% of infertile men (Vogt, 1998; Foresta, 2001; Ferlin et al, 2006; Bhasin, 2007). To our knowledge, this is the first report of CAG and GGN polymorphisms in a South American group of patients with primary severe spermatogenic failure.
The distribution of CAG and GGN repeats in our cases and controls was within the normal range, and this was consistent with findings in Caucasian populations (Lumbroso et al, 1997; Sasaki et al, 2003; Ferlin et al, 2004; Ruhayel et al, 2004). Even though we did not observe significant differences in the distribution of all CAG or GGN alleles, we observed that CAG 21 was significantly more frequent in idiopathic cases than in controls. Although CAG 21 may be within the normal range, it can be associated with a 3-fold increased risk for idiopathic SCOS (OR = 2.99, 95% CI, 1.27–7.0). However, the mechanism by which this allele seems to increase susceptibility for this severe spermatogenic impairment is not clear.
The number of GGN repeats in idiopathic cases and controls showed that GGN 23 was the predominant allele and GGN 24 was the second most common allele. Conversely, an inverse relation was found in cases with a history of cryptorchidism, where GGN 24 was the prevalent allele compared to GGN 23. Our findings are similar to those of Aschim et al (2004), who found the same relationship in a similar group of Swedish excryptorchidic men compared to controls. In vitro characterization has showed a lower transactivating capacity for the GGN 24 allele and GGN 27 or GGN 10, compared to GGN 23, with a constant CAG repeat number of CAG 22, in response to testosterone analogs (R1881) and DHT (Lundin et al, 2007). Therefore, our results and those mentioned above suggest that the GGN 24 allele can increase susceptibility to cryptorchidism and infertility. In order to obtain more conclusive results, however, more patients with primary testiculopathies and a history of cryptorchidism should be studied. We were not able to assess the contribution of cryptorchidism to spermatogenic damage, because our subjects underwent orchidopexy at a relatively late age.
Recently, Foresta et al (2008) reviewed the role of genetic, hormonal, and environmental factors regarding human cryptorchidism. Evidence of possible genetic causes includes chromosomal alterations or mutations in insulinlike factor 3 (INSL3), INSL3 receptor (also known as RXFP2 or LGR8), and AR gene (Ferlin et al, 2008; Foresta et al, 2008). The first transabdominal phase of testicular descent is essentially INSL3-dependent. The role of AR in normal testis descent is related to the second phase of a 2-step process, the inguinoscrotal phase, in which testes move from the inguinal region to the scrotum. However, it has been suggested that the involvement of AR point mutations in isolated cryptorchidism is unclear (Ferlin et al, 2008; Foresta et al, 2008).
Genetic alterations, including mutations in the INSL3 receptor and Klinefelter syndrome, have been associated with bilateral persistent cryptorchidism and with progressive testicular damage, whereas early orchidopexy may reduce the risk for these sequelae (Ferlin et al, 2008). Likewise, studies regarding CAG polymorphisms and alterations in the AR gene are not associated with idiopathic azoospermia (Sasagawa et al, 2001) or cryptorchidism (Sasagawa et al, 2000), and the combined contribution of both polymorphisms has been poorly studied.
In this report, patients with a history of cryptorchidism showed a trend for a higher proportion of the combination GGN 24/CAG > 22. This may be explained because, besides a reduced transactivating capacity of the GGN 24 allele, CAG alleles above 22 have shown decreased in vivo and in vitro transactivation (La Spada et al, 1991; Tut et al, 1997).
One report by other authors (Ferlin et al, 2005) comparing cryptorchidic patients, with or without spermatogenic damage, and normal fertile men have found that 2 CAG/GGN haplotypes (CAG = 21/GGN = 24 and CAG ≥ 21/GGN ≥ 24) were more frequent in men with bilateral cryptorchidism (with and without spermatogenic impairment), who frequently had severe spermatogenic failure. In another report from the same authors (Ferlin et al, 2004), they studied men with idiopathic infertility and observed that the CAG = 21/GGN = 24 combination appeared to increase susceptibility to infertility. In those studies the combination CAG = 21/GGN = 24 was associated with a higher risk for both idiopathic spermatogenic impairment (21 of 163 cases vs 6 of 115 controls; OR = 2.7, 95% CI, 1.05–6.9) and cryptorchidism (8 of 50 cases vs 6 of 115 controls; OR = 3.4, 95% CI, 1.13–10.6). In contrast, our study showed that allele CAG = 21 per se was associated with a 3-fold greater risk for idiopathic SCOS (OR = 3.06, 95% 95% CI, 1.32–7.09) and not for spermatogenic impairment in excryptorchidic cases.
The possible implication of the CAG 21 allele on AR activity, which could be related to severe spermatogenic impairment and infertility in our patients, is not clear. In this regard, no reports had documented an increased frequency for this allele in patients with idiopathic SCOS. Several in vitro analyses to determine the effect of CAG length in AR transcriptional activity have been reported in the literature, with most of them showing that a progressive expansion of the CAG repeat in human AR caused a linear decrease of transactivation function. However, none of them determined the effect of CAG = 21. Tut et al (1997) compared the effect of CAG = 15, CAG = 20, and CAG = 31, determining that CAG = 20 had a mean activity between CAG = 15 (high activity) and CAG = 31 (lower activity), whereas Beilin et al (2000) compared the effect of CAG = 15, CAG = 24, and CAG = 31, and observed similar results, because CAG = 24 had a mean activity between CAG = 15 (high activity) and CAG = 31 (lower activity). These results would indicate that CAG = 21 probably does not have a transcriptional activity very different from that of other similar alleles. However, a recent study (Nenonen et al, 2010), investigated different CAG lengths in the normal range (16, 22, and 28) together with the GGN 23 allele observed that the highest AR activity was confined to CAG = 22 and not to CAG = 16, suggesting that subtle differences in the number of CAG repeats close to CAG = 21 can produce differences in transcriptional activity of the AR.
On the other hand, our results may be chance findings that may not allow firm conclusions regarding the biological importance of these combinations in men with spermatogenic defects. Therefore, we suggest that further studies of these polymorphisms should be performed, including in vitro transactivation studies using appropriated models for different tissues. In this regard it has been reported that some AR mutations observed in infertile patients showed a diminished transactivational response using extensive analysis with relevant in vitro systems, in particular with the PEM promoter (Zuccarello et al, 2008).
Even though we studied a relatively small number of patients with a history of cryptorchidism, our findings distinguished 2 different types of patients, excryptorchidic and those with idiopathic spermatogenic impairment. We observed a higher prevalence of CAG 21 in idiopathic cases and an inverse relation of the GGN 23 and GGN 24 in excryptorchidic cases. Moreover, we performed a detailed biopsy analysis in most of our patients that allowed us to select only subjects with severe spermatogenic impairment, finding a higher prevalence of the CAG 21 allele among idiopathic infertile patients with SCOS.
In summary, we suggest that the CAG 21 allele seems to increase the susceptibility for idiopathic SCOS, and the GGN 24 allele may contribute to deranged AR function, associated with cryptorchidism and spermatogenic failure.