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Pharmacogenetic Polymorphisms of the AR and Metabolism and Susceptibility to Hormone- Induced Azoospermia

Department of Medicine, University of Sydney (B.Y., D.J.H.); Department of Molecular and Clinical Genetics, Royal Prince Alfred Hospital (B.Y.); and ANZAC Research Institute, Concord Hospital (D.J.H.), Sydney 2139, Australia

Address all correspondence and requests for reprints to: Prof. D. J. Handelsman, ANZAC Research Institute, Sydney, New South Wales 2139, Australia. E-mail: djh{at}med.usyd.edu.au

Abstract

Clinical trials of hormonal male contraceptive regimens have identified a consistent population polymorphism in susceptibility to hormone-induced azoospermia. Using identical hormonal regimens, fewer men of European origin (approximately two thirds) become azoospermic compared with Asian men who virtually all become azoospermic. This variation within and between populations remains unexplained. To investigate pharmacogenetic differences in androgen metabolism or action that might explain variable susceptibility to hormonal-induced azoospermia, we studied single nucleotide polymorphism in the CYP3A4 gene, which encodes the major hepatic T-inactivating enzyme, and CAG and GGC triplet repeats in the AR gene in 75 Australian volunteers participating in a male hormonal contraceptive study and 106 population controls. These men were classified into groups according to whether 6 months of weekly T enanthate injections produced azoospermia (n = 54), near-azoospermia (n = 7), and nonazoospermia (n = 14). Mutagenically differentiated PCR was designed to identify A/G variants in the promoter region of the CYP3A4 gene. Fluorescent-labeled DNA fragments containing either CAG or GGC repeats were amplified from the genomic DNA, and their sizes were determined based on the capillary electrophoresis. The G allele of CYP3A4 gene was absent from the nonazoospermia and near-azoospermia groups, but overall this single nucleotide polymorphism distribution did not differ significantly between men in the azoospermia group or population controls. There were no significant differences in distribution of CAG and GGC triplet repeats among three groups or between them and the population controls based on the maximum likelihood estimate of the odds ratio and CLUMP II analyses. These results suggested that neither genetic polymorphisms in the AR gene (CAG and GGC repeats) nor that in hepatic androgen metabolism (CYP3A4 A/G variant) were the major contributors to the within-population variations in susceptibility to T-induced azoospermia.

ANDROGEN-BASED hormonal regimens have been widely evaluated in development of novel forms of male contraception (1). Such regimens are designed to deplete intratesticular T and eliminate FSH action through negative feedback suppression of pituitary LH and FSH secretion. This is achieved by administration of exogenous androgen with or without a second gonadotropin-suppressing agent. This approach exploits the fact that spermatogenesis is hormonally regulated by both FSH and T (the product of LH-stimulated Leydig cell steroidogenesis) acting upon Sertoli cells, which express their cognate receptors, whereas these receptors are lacking in germinal cells. As spermatogenesis is dependent on the maintenance of physiological pituitary gonadotropin secretion, pharmacological suppression of gonadotropins by withdrawing hormonal stimulation of Sertoli cells should lead consistently to reversible cessation of spermatogenesis.

Empirically, however, clinical trials have shown that androgen-based regimens fail to achieve uniform azoospermia in a consistent fashion, suggestive of a population polymorphism in hormone action. Men from Southeast Asian populations of differing genetic background (China, Indonesia) show near uniform rates of hormonally induced azoospermia. They are far more likely to suppress to azoospermia than men of European background, of whom only about two thirds become azoospermic with similar treatment. Previous studies have been unable to identify clinical, anthropometric, or hormonal determinants of this population polymorphism (2). It remains unclear whether resistance to hormonally induced azoospermia results from genetic and/or environmental factors. Genetic influence are suggested by ethnic difference in susceptibility to hormonally induced azoospermia (1); yet, the consistently high susceptibility of Asian men of different genetic backgrounds as well as the variable susceptibility of men within European populations require explanation.

This study was based on the hypothesis that population variation in functional aspects of androgen action may be responsible for these variations in apparent susceptibility to hormonal action on the testis. Therefore, in this study well described genetic population polymorphisms in the major enzyme involved in hepatic metabolic inactivation of T as well as in the AR were studied to determine whether they explained the apparent population polymorphism in susceptibility to androgen-induced azoospermia. Genotypes for the CYP3A4 gene encoding the major hepatic T-inactivating enzyme and the AR were determined for men participating in two WHO-supported male contraceptive efficacy studies who were classified according to their susceptibility to hormonally induced azoospermia.

Subjects and Methods

Study populations

Samples from men of European background living in Sydney, Australia, who had participated in the two WHO male contraceptive efficacy studies were used in this study. The design, methods, and outcomes of those studies were described in detail previously (3, 4). Briefly, 86 eligible men entered the suppression phase of this study and were treated with weekly im injections of 200 mg T enanthate. For this study, men were classified as azoospermic (no sperm in the centrifuged deposit of 3 consecutive semen samples), near-azoospermic (lowest sperm concentration, <1 million/ml), or nonazoospermic (lowest sperm concentration, 1 million/ml) according to their lowest sperm output in semen analyses obtained at least monthly for 6 months; men who did not complete the 6-month suppression phase were considered unclassifiable for the present study and were excluded from analysis. Male blood donors (n = 106) were also studied as the population frequency controls. These studies received approval from the Central Sydney Area Health Service human ethics committee (RPAH zone) within National Health and Medical Research Council Guidelines for Human Experimentation.

DNA analysis

DNA for genotyping was obtained from the study participants using QIAamp Tissue Kit (QIAGEN, Clifton Hill, Australia) to extract DNA from 100 µl whole semen samples. For population controls, DNA was obtained from peripheral blood samples using the QIAamp Blood Kit (QIAGEN). The single nucleotide polymorphism (SNP) A/G in the CYP3A4 gene was studied using mutagenically separated PCR for genotyping (5). The differentiated primers are 5'-(Tet) aaa ACA aCC ATA GAG ACA AGG GCA A-3' and 5'-(Hex) ACA GCC ATA GAG ACA AGG GtA G-3'. Tet and Hex are the fluorescent labels, and lower case letters indicate the deliberately introduced mismatches for either differentiation or release of secondary structure. The common reverse primer is 5'-ACA CAC ACC ACT CAC TGA CCT C-3'. The total PCR volume was 25 µl, which contained 0.5 U Taq Gold (PE Applied Biosystems, Foster City, CA), 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 0.25 mM of each deoxy-NTPs and 0.28 µM of each primer. PCRs were performed in the GeneAmp PCR System 9600 (PE Applied Biosystems), which was programmed as follows: initial denaturing at 95 C for 12 min; then five cycles of 94 C for 30 sec, 64 C for 30 sec, and a decrease of 1 C per cycle; followed by 32 cycles of 94 C for 30 sec, 58 C for 30 sec, and finally 72 C for 7 min.

Two tandemly arranged trinucleotide repeats were studied in the exon 1 of the AR gene. They are AR-CAG and AR-GGC, and their primer sequences were derived from Macke et al. (6), except that the 5'-end was labeled by Tet and Hex, respectively. The total PCR volume was 25 µl, which contained 0.5 U AmpliTaq (PE Applied Biosystems), PCRx Enhancer system including 10 x buffer with 2.0 mM MgSO₄ (Life Technologies, Inc., Gaithersburg, MD), 0.25 mM of each deoxy-NTP, and 0.2–0.5 µM of each primer. PCRs were performed using GeneAmp PCR System 9600 (PE Applied Biosystems), which was programmed as follows: initial denaturing at 95 C for 4 min and then 36 cycles of 94 C for 30 sec, 71 C for 30 sec, and 72 C for 40 sec, followed by 72 C for 7 min. Genotypes were analyzed using the ABI Prism 310 Genetic Analyzer (PE Applied Biosystems), which had very high accuracy and reproducibility (7). The coefficient of variation is 0.73–1.83%, as estimated from repeated (n = 30) injections and electrophoresis of two DNA fragments (199 and 207 bp).

Statistical analysis

Intercooled Stata 6.0 for Windows NT (Stata Corp., College Station, TX) was used for different data analyses, which included ANOVA, ² test, and maximum likelihood estimate of the odds ratio analysis. P < 0.05 was considered statistically significant. The CLUMP II program (8) was used for the allele distribution analysis, and 10,000 simulations were performed to determine the significance levels.

Results

Subjects

Study participants were divided into 3 groups according to how their sperm output responded to the hormonal contraceptive regimen. Among 86 men entering the studies, 75 men were divided into those achieving azoospermia (n = 54), near-azoospermia (n = 7), and nonazoospermic (n = 14) groups; the remaining 11 men who could not be classified were excluded from further analysis.

CYP3A4 gene

The functional SNP is an A to G nucleotide change in the 5'-promoter region of human CYP3A4 gene, and its genotyping was based on the capillary electrophoresis (Fig. 1).

View larger version (36K): [in this window] [in a new window]   Figure 1. Genescan analyses of SNP in the CYP3A4 gene and of CAG and GGC repeats in the AR gene. The upper panel shows the G and A alleles with Hex and Tet fluorescent labels (represented by black and green, respectively). The CAG and GGC repeats are also shown in black and green, respectively in the lower panel. The length of the triplets was measured based on the internal Genscan-500 (TAMRA) size standard that was electrophoresed simultaneously with the triplet PCR products.

Genotyping of AR-CAG and AR-GGC repeats is illustrated in Fig. 1. There were 19 distinct CAG and 13 GGC alleles in our study populations (Fig. 2). The CAG and GGC repeat sizes of different groups were within normal ranges (Table 2), and no pathological triplet expansion was observed. Whether considered as discrete or continuous, the sizes of CAG repeats were not significantly different among the azoospermia, near-azoospermia, and nonazoospermia groups based on the ANOVA analysis (F = 1.14; P = 0.35). Combining the nonazoospermia and near-azoospermia groups gave the same results, with no difference from the azoospermia groups (F = 1.05; P = 0.42).

View larger version (31K): [in this window] [in a new window]   Figure 2. Frequency distributions of the CAG and GGC repeats in the AR gene in different groups.

There was no significant difference in GGC repeat number among the three groups or compared with the population controls (P > 0.05). Allele distribution analysis (CLUMP II) was also consistent with the results from the maximum likelihood estimate of the odds ratio.

Discussion

Spermatogenesis is pituitary gonadotropin and androgen dependent. Initiation during puberty and maintenance throughout manhood requires pituitary gonadotropin (FSH and LH) secretion, leading to adequate intratesticular T via LH-stimulated Leydig cell steroidogenesis. Both T and FSH act primarily on the Sertoli cell, which has cognate hormonal receptors, whereas germ cells do not. Hence, withdrawal of pituitary gonadotropin secretion combined with depletion of intratesticular T should result in reversible inhibition of spermatogenesis. This mechanism, which reverses the normal pubertal initiation of spermatogenesis and replicates the reversibility of spermatogenesis in seasonal animals, is the conceptual basis of male hormonal contraceptive regimens whether employing androgens alone or combined with a second gonadotropin-suppressing agent (progestins and GnRH antagonist). Many different regimens of these types are highly effective at suppressing spermatogenesis; however, several WHO studies have shown a consistent variation within and between populations in susceptibility to hormonally induced azoospermia. Among populations of European origin, azoospermia is produced in about two thirds of men (9, 10, 11), whereas men living in China (3, 4) and Indonesia (12, 13) exhibit almost universal azoospermia in response to the same hormonal regimens. Retrospective analysis of participants in WHO studies identified corollary hormonal changes, but failed to disclose any plausible mechanisms to account for these within and between population differences (2). Whether this population variability in hormone pharmacology represents genetic (e.g. polymorphisms in hormone metabolism or action) and/or environmental (e.g. nutritional effects) factors remains unclear. The similarly high susceptibility to hormonally induced azoospermia in two populations of Asian men with differing genetic backgrounds are more consistent with epigenetic factors being important, such as nutritional influences on hormonal pharmacology. Conversely, within-population differences among men of European background are more consistent with underlying genetic differences. Mechanistically, resistance to hormonal-induced azoospermia may represent effects of reduced androgen action, such as via incomplete suppression of FSH secretion (as FSH is harder to suppress than LH), or direct androgen effects, such as stimulatory effects of exogenous T on the Sertoli cells. In the current pharmacogenetic study we investigated for the first time the potential association of known functional polymorphisms in the AR and in hepatic androgen metabolism as possible explanations for variable susceptibility to hormonally induced azoospermia.

CYP3A4 is a member of the cytochrome P450 family involved in the oxidation of T to 2ß-, 6ß, or 15ß-hydroxytestosterone (14). It is the most abundant hepatic cytochrome P-450, has great significance for drug metabolism, and exhibits both very broad substrate specificity and significant between-individual variability in enzyme activity. Substantial interindividual variability has been observed in the metabolism of specific compounds by CYP3A4 (15). Recently, an A to G transition change that alters the 10-bp nifedipine-specific element was identified in the 5'-regulatory region of the CYP3A4 gene (5). Such an alteration might modulate genetic expression of CYP3A4 in the liver and thereby influence T inactivation. For example, this polymorphism has been suggested to influence the pathogenesis of prostate cancer (5). Nevertheless, the functional significance of this polymorphism has also been questioned (16, 17). In the present study we examined this SNP in the CYP3A4 using a highly sensitive and specific purpose-developed, mutagenically separated PCR. Using fluorescent labels incorporated into the distinct PCR products, different alleles can be automatically identified by capillary electrophoresis (7). In this study the G allele frequency was similar in the hormone-induced azoospermia group and the population controls. Interestingly, the G allele was not found in either the nonazoospermia or near-azoospermia group. If the absence of G allele was associated with more efficient inactivation of exogenous T, this could contribute to the insufficient suppression of spermatogenesis in those two groups. Nevertheless, this difference was not statistically significant, and the power of the study was sufficient to conclude that this hypothesis is unlikely to be true.

The AR is a ligand-activated transcription factor belonging to the steroid-thyroid hormone nuclear receptor superfamily (18). In exon 1 of the transcription activation domain, there are two triplet repeat polymorphisms, the polyglutamine and polyglycine tracts encoded by CAG and GGC repeats, respectively (19). It is well established that expansion of the CAG triplet repeats into the pathological range (>40 triplet repeats) cause a genetic form of motor neuron disease, known as spinal and bulbar muscular atrophy or Kennedy disease (20, 21), one of a variety of triplet repeat neurodegenerative disorders. Although subtle defects of in vitro function of the AR with expanded CAG triplet repeats have been reported (22, 23, 24), the normal sexual differentiation and pubertal development of affected men as well as the late-onset, progressive neurodegeneration and its absence in heterozygous female carriers of the defective AR gene are all more consistent with a toxic gain of function rather than loss of function defects in the AR. Within the nonpathological range of CAG triplet repeat lengths, additional functional consequences of polymorphisms in CAG triplet repeat length have been reported, but remain unresolved. A tendency to reduced spermatogenesis with longer CAG repeat lengths was reported in some (24, 25), but not all (26, 27), studies. As these men previously experienced normal urogenital sinus differentiation, a highly androgen-dependent process that is universally defective among men with dysfunctional ARs (19), the biological significance of such variations in AR structure remain to be clarified. Similarly, shorter CAG triplet repeat lengths have been associated with earlier onset and more aggressive prostate cancer (28) and benign prostate hyperplasia (29, 30). Some evidence linking the GGC triplet repeat to risk of prostate cancer has been reported (31). If triplet repeat variation does influence AR function, then their relationship with prostate carcinogenesis may be related to their influence on androgen sensitivity, although other studies do not support this postulate (32). In the present study the CAG and GGC repeats were genotyped using fluorescent-labeled primers and capillary electrophoresis. The lack of difference among the three groups in the WHO study and their similarity to the population controls suggest that variations in either AR triplet repeat length polymorphism cannot explain the populations difference among European men in resistance to hormone-induced azoospermia.

Limitations of the present study are its relatively small size and the few genes evaluated. The size of the study is limited by the difficulty in determining the phenotype of resistance to hormone-induced azoospermia as classification requires prolonged (6-month) administration of weekly T injections and monthly sperm counts. Such studies are arduous, invasive, and expensive to undertake and no surrogate short term measures are available. As a result, even the reproducibility of the phenotype in any one individual has not been established, although the proportions demonstrating the resistant phenotype have been consistent across all clinical studies. The present study was undertaken in the largest of the 16 centers participating in the WHO male contraceptive efficacy studies (3, 4). Nevertheless, the power of this study was sufficient to exclude major influences of naturally occurring functional polymorphisms in the major hepatic T-metabolizing enzyme or AR. This finding is consistent with the prior observation that there were no consistent differences in plasma T or LH and FSH either before or during treatment in the WHO studies that differentiated the men who did from those who did not achieve azoospermia (2, 3, 4). The present study does not exclude the possibility that functional genetic polymorphisms in other hormonal response genes or the AR may be involved in this population variation. Further studies of genes regulating gonadotropin secretion and action, local androgen metabolism (e.g. 5-reductase) (33) as well as environmental effects on androgen production (34) would be of interest. Indirect evidence that differences in 5-reductase activity determine susceptibility to hormone-induced azoospermia (35, 36, 37) remain to be verified. It is also possible that the differences in susceptibility to hormone-induced may be environmental rather than genetic (2, 34). Finally, polymorphisms may occur in synergistic or antagonistic combinations that may be difficult to identify individually.

The population polymorphisms in susceptibility to hormone-induced azoospermia are significant in connection with the requirement that hormonal male contraceptive regimens aim to provide uniform azoospermia (1) and the minority of European men with resistance to hormone-induced azoospermia would constitute a limitation on the feasibility of developing effective regimens. Hence, further understanding of the biological basis of this source of individual variability is considered of significance. Nevertheless, as modern combination hormonal regimens demonstrate that virtually universal azoospermia is achievable (38, 39, 40), the practical importance of elucidating such sources of variation has diminished. More importantly, these sources of within- and between-population variation in hormone responsiveness may have greater importance in elucidating the biological basis of individual susceptibility or resistance to hormone-dependent disease, such as naturally occurring as well as xenobiotic-induced disorders of testis, prostate, and the cardiovascular system. For this reason, further studies to identify the biological basis of variation in susceptibility to hormone-induced azoospermia may be warranted.

Acknowledgments

We are grateful to the staff of the Department of Andrology, Concord Hospital (formerly Andrology Unit, Royal Prince Alfred Hospital), for their skilful assistance in conducting the original WHO male contraceptive studies, and to Dr. I. T. Huhtaniemi for helpful discussion before the study.

Footnotes

Abbreviations: SNP, Single nucleotide polymorphism.

Received January 4, 2001.

Accepted May 14, 2001.

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