ular Human Reproduction, Vol. 7, No. 11, 1007-1013, November 2001
© 2001 European Society of Human Reproduction and Embryology
Reproductive endocrinology
Prostate-specific antigen, testosterone, sex-hormone binding globulin and androgen receptor CAG repeat polymorphisms in subfertile and normal men
Amparo Mifsud1,4, Aw Tar Choon2, Dong Fang3 and E.L. Yong1
1 Departments of Obstetrics & Gynaecology, 2 Laboratory Medicine and 3 Biostatistics, National University of Singapore, Singapore 119074
Abstract
The aim of this study was to understand the androgen-related factors which may regulate concentrations of the tumour marker, prostate-specific antigen (PSA). We therefore measured the serum concentrations of total and free testosterone and of sex hormone-binding globulin (SHBG) and determined the androgen receptor (AR) gene CAG repeat length, then compared these values to total and free PSA concentrations in 91 subjects with proven fertility, and 112 subfertile men with defective spermatogenesis. Concentrations of free testosterone and total testosterone, adjusted for SHBG, were 17–20% lower in subfertile men compared with those in their fertile counterparts. This subtle, but highly significant (P < 0.001), difference in testosterone between fertile and subfertile men was accentuated by the positive correlation between testosterone and AR gene CAG repeat length in fertile, but not subfertile, subjects. In subfertile subjects, testosterone strongly correlated (r = 0.354, P < 0.001) with PSA concentrations, and independent of testosterone, total PSA negatively correlated (r = –0.229, P = 0.011) with AR CAG length. Overall our data suggest that, firstly, PSA correlates with testosterone only in an environment of relatively low androgenicity, such as in subfertile men. Secondly, in such a low androgenic environment, short CAG tracts (associated with high AR activity) correlate positively with PSA concentrations. These results suggest that interpretation of PSA is best made in conjunction with testosterone concentrations and AR CAG length.
androgen receptor/infertility/prostate-specific antigen/testosterone
Introduction
Androgen action is the sum effect of bioactive androgens and the intrinsic responsiveness of the androgen receptor (AR) in target cells. The major circulating androgen in males is testosterone and >98% of testosterone molecules are bound to proteins in the blood, principally to sex hormone-binding globulin (SHBG) and also to albumin and cortisol-binding globulin. It is assumed that bound hormones cannot exit blood capillaries and are therefore not bio-available, and so SHBG concentrations are commonly measured as a supplement to total testosterone determinations (Cunningham et al., 1985Go). The measurement of unbound free testosterone has been proposed as a better measure of bio-active testosterone (Hayes, 2000Go). Free testosterone, on diffusing through the cell membrane, binds specifically to the AR in the cytoplasm causing nuclear translocation of the receptor. In the nucleus, the AR–androgen complexes bind to the genomic DNA of promoters containing androgen response elements, thereby switching on androgen-regulated genes.
All androgens act on a single AR which is encoded by the X-linked AR gene. Within the transactivation domain of the AR gene is a polymorphic tract composed of 11 to 35 repeats of the codon, CAG. The length of this CAG tract, encoding a polyglutamine stretch, has been shown to be inversely related to the function of the AR gene in vitro. Thus, shorter CAG tracts are associated with increased AR activity in reporter gene studies, a phenomenon attributed either to higher intrinsic activity (Mhatre et al., 1993Go; Chamberlain et al., 1994Go; Tut et al., 1997Go) and/or to increased expression of the short form of AR protein (Choong et al., 1996Go). Short AR CAG tracts are associated with earlier age of onset (Hardy et al., 1996Go), and increased severity (Giovannucci et al., 1997Go), of the androgen-dependent tumour, prostate cancer. Conversely, recent studies have indicated that long CAG tracts are associated with reduced AR activity in vitro and in vivo. Thus, pathological expansion beyond the polymorphic range (>40 CAG) results in a fatal neuromuscular disease, spinal bulbal muscular atrophy (La Spada et al., 1991Go), a condition associated with reduced virilization and low sperm counts. Subsequent studies in Chinese (Tut et al., 1997Go), Australian (Downsing et al., 1999Go) and USA (Mifsud et al., 2001Go) populations indicated that long CAG tracts, while remaining in the polymorphic range, are associated with severely depressed spermatogenesis and male infertility. These findings validate the concept that AR function is inversely regulated by the length of its CAG repeat tract.
Prostate cancer is the most diagnosed malignant tumour and the second leading cause of cancer-related deaths in American men (Boring et al., 1994Go). Androgens are required for normal development of the prostate, as well as for its neoplastic transformation. The AR regulates transcription of prostate-specific antigen (PSA), a single chain glycoprotein enzyme (serine protease) that is present in urine, seminal plasma, and serum (Polascik et al., 1999Go). Serum PSA concentrations are a common oncogenic marker for prostate cancer since they parallel oncogenic growth in the initial androgen-dependent phase and in early stages of the androgen-independent phases of the tumour. Despite the widespread clinical use of PSA as a tumour marker, the relationship between androgen action and PSA concentrations in men without prostate hyperplasia or cancer remains unclear. It is therefore of public health interest to determine how variations in PSA concentrations in healthy men are affected by changes in SHBG, AR CAG polymorphisms and bioavailable testosterone.
These concerns prompted us to investigate the relationship of testosterone, SHBG and AR CAG polymorphisms with PSA concentrations in a large population of normal men with no known prostatic disease. Our subjects included subfertile men with depressed spermatogenesis because previous studies (Tut et al., 1997Go; Downsing et al., 1999Go; Mifsud et al., 2001Go) indicate that they are likely to have longer CAG tracts and therefore lower AR androgenicity. Our finding that these subfertile men also have significantly lower testosterone concentrations compared to their fertile counterparts, allowed us to examine the relationship of androgen action to PSA concentrations, in two steady-state environments of normal and below-normal concentrations of testosterone.
Materials and methods
Study population
Subfertile males were recruited from the infertility and andrology clinics of the National University Hospital, Singapore. A complete history and physical examination were performed, and the use of any medications or previous surgery was recorded. Patients who had prostatic disease, chromosomal abnormalities, hypopituitarism, hyperprolactinaemia, undescended testes, obstructive syndromes of the genital tract, and hypogonadism secondary to surgery, trauma, or chemotherapy were excluded from this study. Sperm parameters were assessed according to standard criteria (World Health Organization, 1999Go) and were the mean of at least two analyses carried out 3 months apart. Azoospermia was defined as the absence of any spermatozoa despite centrifugation of the semen specimen, severe oligozoospermia was sperm concentration <5x106/ml, and oligozoospermia defined as the mean sperm concentration being <20x106/ml. Control subjects were men of proven fertility with no previous infertility history or treatment, and without any genetic disease. Ethical committee approval was received and informed consent was obtained from all subjects and controls. Venous blood was collected from all subjects. DNA was extracted from peripheral leukocytes and serum stored at –20°C for subsequent hormonal analyses.
Hormonal analysis
Total and free testosterone, SHBG, total and free PSA were measured. Assays for each parameter were performed in one batch to reduce interassay variability.
Free testosterone was measured with the Coat-A-Count free testosterone solid phase 125I radioimmunoassay (Diagnostic Products Corp., Los Angeles, CA, USA). 125I-labelled testosterone analogue competed for a fixed time with free testosterone in the patient sample for binding to a testosterone-specific antibody immobilized to the wall of a polypropylene tube. The tube was then decanted and the antibody-bound fraction was isolated and counted in a gamma counter. The amount of free testosterone was interpolated from a standard curve calibrated in free testosterone concentrations, and was not calculated as a function of total testosterone and SHBG. In this respect, it differed from conventional equilibrium dialysis methods and from testosterone free index determinations.
Total testosterone, total PSA and free PSA were measured by the Architech assay systems (Abbott Laboratories, Diagnostic Division, Abbott Park, IL, USA) according to manufacturer's instructions. These immunoassays utilized specific antibody-coated paramagnetic microparticles and chemiluminescent technology. The concentrations of total PSA (both free PSA and PSA complexed to alpha-1-antichymotrypsin) and free PSA were measured in a two-step immunoassay.
Concentrations of SHBG were measured with the Immulite SHBG assay (Diagnostic Products Corp.). The assay tube was coated with an alkaline phosphatase-conjugated monoclonal antibody specific for SHBG. The chemiluminescent substrate, a phosphate ester of adamantyl phosphate, underwent hydrolysis in the presence of alkaline phosphatase enzyme. Emission of light was proportional to the concentration of SHBG in the sample. The sensitivity of the assay was of 0.2 nmol/l.
For all the tests mentioned above, the inter- and intra-assay coefficients of variation were <15%.
Determination of the number of CAG repeats
The number of CAG repeats in the AR gene was determined by automated GeneScan analyses following a previously described protocol (Mifsud et al., 2000Go). Briefly, the AR CAG repeat tract was amplified by PCR using specific primers. The sizes of the samples were analysed on a 377 DNA Sequencer running Genescan 672 software using internal size markers. Some samples were sequenced to gain further accuracy in size determination. These samples of known length were subsequently used in every gel as controls.
Y-chromosome microdeletion screening
Since Y-chromosome microdeletions are associated with impaired spermatogenesis, all samples were screened for genetic deletions with primer pairs specific for the AZFa, AZFb and AZFc regions according to a previously described protocol (Liow et al., 1997).
Statistical analyses
The mean values of the various parameters studied were calculated and compared between the infertile and fertile group, using a two-tailed independent sample t-test. The correlations among variables were analysed using Pearson's correlation coefficients (r) and, when appropriate, adjustments for potentially confounding variables were made by multiple regression analysis. Statistical analyses were performed using SPSS Version 9.01 (SPSS Corp., Chicago, IL, USA) computer software. Statistical significance was defined as a two-sided P < 0.05 and data were reported as mean ± SE.
Results
Endocrine parameters important for androgen action and metabolism were measured in 91 subjects with proven fertility, and 112 subfertile men with varying degrees of spermatogenic defects (azoospermic, 29.4%; severe oligospermic, 54.1%; oligospermic, 16.5%). The mean ages for the fertile and subfertile men were 35.57 ± 0.58 and 33.98 ± 0.50 years respectively. The racial composition of both groups were similar, being predominantly of chinese ethnic origin. No Y-chromosomal microdeletions were detected in the 112 subfertile males.
Testosterone is lower in subfertile males
The mean values of PSA (free and total), SHBG, AR CAG repeat lengths, and testosterone (free, total, and corrected for SHBG) between fertile and subfertile males were compared using the two sided t-test (Table IGo). Total testosterone, testosterone adjusted for SHBG, and free testosterone were ~17–20% lower in subfertile men compared to that in their fertile counterparts. Differences in free testosterone and testosterone/SHBG were highly significant (P < 0.001). The frequency distribution of testosterone concentrations in subfertile subjects was shifted to the left with respect to the fertile group, indicating that low testosterone concentrations were a general feature of the subfertile group, and that data were not skewed by a few severely hypogondal patients (Figure 1Go). This relative hypogonadism in subfertile men was not age-related since, on average, subfertile subjects were younger than fertile controls. There was a tendency, not reaching statistical significance, for the AR CAG length to be longer in the subfertile group. No differences in SHBG, or in free and total PSA concentrations were observed between subfertile patients and fertile controls. Free testosterone and total testosterone concentrations were very strongly correlated (P < 0.0001) in both fertile and subfertile populations, suggesting that use of the more laboratory-friendly total testosterone assay might be sufficient for most purposes.
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Table I. Descriptive statistics of the parameters regulating androgen action
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Figure 1. Frequency distribution of total testosterone concentrations in fertile and subfertile subjects. Serum total testosterone in each individual was measured and grouped to the closest even value. Fertile men, n = 91; subfertile men, n = 112.
SHBG is positively correlated with testosterone
Pearson's correlation coefficients were calculated for the factors in the androgen system and their relationships were examined with bivariate analyses. In both fertile and subfertile patients, SHBG concentrations directly correlated with that of testosterone (Tables II and IIIGoGo). The combined correlation between total testosterone and SHBG, for fertile and subfertile men grouped together, was highly significant (r = 0.546, P < 0.0019). This was true whether testosterone was expressed as free testosterone or total testosterone. This finding was surprising since androgens, unlike oestrogens, were considered to have an inhibitory effect and thus to be negatively correlated with SHBG (Plymate et al., 1983Go). AR CAG repeat length was positively correlated with free testosterone and SHBG in fertile men (P < 0.05), but not in subfertile patients.
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Table II. Results of the bivariate correlations established in the fertile group
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Table III. Results of the bivariate correlations established in the subfertile group
Testosterone, AR CAG repeats, and PSA
Testosterone strongly correlated with PSA concentrations in subfertile men (Table IIIGo). The relationship between testosterone and PSA in subfertile men remained highly significant (P < 0.001) when testosterone was measured as total or free testosterone, or when total or free PSA was assayed. Since PSA values in normal populations are highly scattered, we calculated the log total PSA and observed that log total PSA and testosterone was tightly correlated (r = 0.356, P < 0.001). However, in the fertile controls, no relationship between testosterone and PSA was observed (Table IIGo). There was a negative correlation between AR CAG repeat length and total PSA, or log total PSA, in subfertile patients (r = –0.229, P = 0.011) (Table IIIGo). The correlation between AR CAG length and PSA was still present after adjusting for testosterone (r = –0.216, P = 0.023), and was thus independent of the effect of testosterone on PSA.
Discussion
One of the most significant observations in this study was that serum testosterone concentrations in subfertile patients were significantly lower than that in fertile controls. It is generally not appreciated that, in terms of testosterone production, the subfertile male population is relatively hypogonadic. While still within normal limits, there was a shift to the left in the frequency distribution of testosterone concentrations in subfertile patients compared with fertile controls. Low testosterone concentrations in infertile men have been reported in a limited number of older studies. Thus, total testosterone was found to be below normal in 32% of 60 men from the USA with oligozoospermia (Check et al., 1995Go), and serum concentrations of testosterone, and its metabolite oestradiol, were found to be significantly decreased in infertile Japanese men (Yamamoto et al., 1995Go). The cause for low testosterone concentrations in our subfertile men is not clear, but is unlikely to be due to chromosomal abnormalities, hypopituitarism, hyperprolactinaemia, undescended testes, or hypogonadism secondary to surgery, trauma, or chemotherapy, as these cases were excluded from this study. Y-chromosome microdeletions are also not an aetiological factor. Nevertheless, our large study population, of more than 200 men in total, can be divided into two groups: fertile men with normal concentrations of testosterone, and subfertile patients whose testosterone concentrations were on average 17–20% lower. This finding gave us the opportunity to explore the steady-state effect of normal versus low–normal concentrations of testosterone on SHBG and PSA concentrations.
The place of SHBG in the androgen system is controversial. On one hand, it is generally accepted that androgens, unlike oestrogens, reduce SHBG concentrations. Thus, SHBG concentrations are lower in males than in females, and syndromes of hyperandrogenization in females [polycystic ovarian syndrome (PCOS), hirsutism and acne] are associated with decreased SHBG concentrations (Rosenfield, 1971Go). Administration of testosterone results in a 2-fold lowering of SHBG in normal and hypogonadal men (Plymate et al., 1983Go). On the other hand, concentrations of testosterone and SHBG in males appear to be positively correlated (Carlstrom et al., 1990Go; Gann et al., 1996Go). Furthermore, in-vitro experiments with hepatic cell lines indicate that androgens increase, rather than decrease, the synthesis and secretion of SHBG (Edmunds et al., 1990Go). In our study, SHBG concentrations in the serum of both fertile and subfertile men were directly proportionate with testosterone. This has important implications for androgen action since ~40% of testosterone is physiologically bound to SHBG, and is therefore not biologically active. The positive correlation of SHBG with testosterone will tend to minimize and moderate the androgenic effects of changing total testosterone in men.
Androgen action is further modulated by our finding that AR CAG length correlates directly with free testosterone in fertile men. There is increasing evidence from cellular (Mhatre et al., 1993Go; Chamberlain et al., 1994Go; Tut et al., 1997Go) and human (Irvine et al., 1994; Stanford et al., 1997Go; Rebbeck et al., 1999Go) studies that activity of the AR is inversely regulated by the length of its CAG tract. Thus long AR CAG tracts are associated with moderate to severe undermasculinized genitalia in XY males (Lim et al., 2000Go). Although not observed in all populations (Dadze et al., 2000Go), depressed spermatogenesis has been associated with long CAG tracts in men from Singapore (Tut et al., 1997Go), Australia (Downsing et al., 1999Go), Japan (Yoshida et al., 1999Go) and USA (Mifsud et al., 2001Go; Patrizio et al., 2001Go), sperm production being an exquisitely androgen-dependent process. On the other hand, short CAG tracts are associated with androgen-dependent prostate cancer in Caucasoid (Irvine et al., 1994; Hardy et al., 1996Go; Giovannucci et al., 1997Go; Stanford et al., 1997Go) and Chinese (Hsing et al., 2000Go) subjects, with PCOS (Mifsud et al., 2000Go) and with male pattern baldness (Sawaya and Shallta, 1998). Accordingly, the effect of low testosterone in fertile men would be balanced by the increased activity of the AR associated with shorter CAG tracts and vice versa. Strikingly, no association between testosterone and AR CAG tract was observed in subfertile patients. On the contrary, the AR CAG tracts tended to be longer in our subfertile men, thereby accentuating the effects of low testosterone, contributing to the resultant low androgenicity in these patients.
Although the mechanisms regulating PSA concentrations remain ambiguous, it is acknowledged that androgens have an important role. When men in the early stages of prostate growth are given anti-androgen therapy, PSA concentrations and prostate volume decrease. The promoter region of the PSA gene has two androgen-response elements located 170 and 400 bp upstream of the transcription start site (Cleutjens et al., 1996Go). In-vitro studies have demonstrated expression of the PSA gene by the action of androgens and AR (Perez-Stable et al., 2000Go). Despite the importance of PSA as a screen for prostate cancer, the mechanisms regulating PSA concentrations remain largely unknown. Clinical interpretation is confounded by the wide range of PSA concentrations encountered in normal men. For example, PSA ranged from 0.09 to 5.96 ng/ml in our subjects, all of whom have no history of prostatic disease. In our study, testosterone strongly correlated with PSA concentrations only in subfertile men, suggesting that testosterone drives PSA concentrations only in conditions of relatively low testosterone. Thus, testosterone (free testosterone or total testosterone) was highly correlated (P < 0.0001) with both free PSA and total PSA in these subfertile men, with low to normal testosterone concentrations. This observation is consistent with previous studies which state that PSA and testosterone are related in conditions where testosterone concentrations are low, that is in pubertal boys (Kim et al., 1999Go), in women (Escobar-Morreale et al., 1998Go) and in transsexual women where testosterone was administered externally (Goh, 1999Go). However, no correlation between testosterone and PSA was observed in our fertile men with normal testosterone concentrations. Interestingly, administration of testosterone to healthy young males does not increase the concentrations of serum PSA (Monath et al., 1995Go; Cooper et al., 1998Go). One possible explanation is that in a high androgenic environment, the androgen response mechanism is saturated and further increases in testosterone cannot induce additional AR activity or an increase in PSA concentrations. It is noteworthy that while the androgen dependence of the prostate gland has long been accepted, the participation of oestrogens and the effects of oestradiol/testosterone ratio on prostatic growth has recently been recognized (Farnsworth, 1999Go). Nevertheless the effects of oestrogen on prostatic volume and PSA production are likely to be minor compared to androgen, since prostate volumes are reduced after long-term oestrogen therapy in male to female transsexuals (Jin et al., 1996Go; van Kesteren et al., 1996Go).
Overall our data suggest that, firstly, PSA correlates with testosterone only in an environment of relatively low androgenicity, such as in subfertile patients; secondly, in a low androgenic environment, short CAG tracts (associated with high AR activity; see above) correlate positively with PSA concentrations. Our finding that PSA can vary with testosterone and AR CAG tract, in subfertile patients with relative hypogonadism, has implications for the screening of such men for prostatic disease. Our data would predict that a borderline high PSA concentration is more likely to indicate prostate disease in a subfertile man with low testosterone and long AR CAG tracts, compared with that in a fertile man with normal testosterone and AR CAG tracts. The utility of this paradigm has to be validated by prospective clinical trials.
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Figure 2. Differences in androgen system between fertile and subfertile subjects. Although testosterone concentrations in fertile and subfertile subjects overlap to some extent, differences in androgen receptor (AR) CAG tract would tend to accentuate the effects of low testosterone (T) in the subfertile group. In this environment of low androgenicity, PSA correlates with testosterone and correlates inversely AR CAG length in subfertile subjects. In contrast, uniformly high androgenicity can be maintained in fertile subjects because low testosterone is associated with increased AR activity, and vice versa. SHBG = sex hormone-binding globulin; PSA = prostate-specific antigen.
Acknowledgements
We wish to acknowledge the help of Dr Victor Goh and Mr Bahar in hormonal assays. This study was supported by the National Medical Research Council of Singapore.
Notes
4 To whom correspondence should be addressed at: Department of Obstetrics & Gynaecology, National University Hospital, Level 2, Lower Kent Ridge Road, Singapore 119074. E-mail:
[email protected] Back
References
Boring, C.C., Squires, T.S., Tong, T. et al. (1994) Cancer statistics. CA Cancer J. Clin., 44, 7–26.[ISI][Medline]
Carlstrom, K., Eriksson, A., Stege, R. et al. (1990) Relationship between serum testosterone and sex hormone-binding globulin in adult men with intact or absent gonadal function. Int. J. Androl., 13, 67–73.[ISI][Medline]
Chamberlain, N.L., Driver, E.D. and Miesfeld, R.L. (1994) The length and location of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect transactivation function. Nucleic Acids Res., 22, 3181–3186.[Abstract/Free Full Text]
Check, J.H., Lurie, D. and Vetter, B.H. (1995) Sera gonadotropins, testosterone, and prolactin levels in men with oligozoospermia or asthenozoospermia. Arch. Androl., 35, 57–61.[ISI][Medline]
Choong, C.S., Kemppainen, J.A., Zhou, Z.X. et al. (1996) Reduced androgen receptor gene expression with first exon CAG repeat expansion. Mol. Endocrinol., 10, 1527–1535.[Abstract]
Cleutjens, K.B., van Eekelen, C.C., van der Korput, H.A. et al. (1996) Two androgen response regions cooperate in steroid hormone regulated activity of the prostate-specific antigen promoter. J. Biol. Chem., 271, 6379–6388.[Abstract/Free Full Text]
Cooper, C.S., Perry, P.J., Sparks, A.E. et al. (1998) Effect of exogenous testosterone on prostate volume, serum and semen prostate specific antigen levels in healthy young men. J. Urol., 159, 441–443.[ISI][Medline]
Cunningham, S.K., Loughlin, T., Culliton, M. et al. (1985) The relationship between sex steroids and sex-hormone-binding globulin in plasma in physiological and pathological conditions. Ann. Clin. Biochem., 22, 489–497.
Dadze, S., Wieland, C., Jakubiczka, S. et al. (2000) The size of the CAG repeat in exon 1 of the androgen receptor gene shows no significant relationship to impaired spermatogenesis in an infertile Caucasoid sample of German origin. Mol. Hum. Reprod., 6, 207–214.[Abstract/Free Full Text]
Downsing, A.T., Yong, E.L., Clark, M. et al. (1999) Linkage between male infertility and trinucleotide repeat expansion in the androgen-receptor gene. Lancet, 354, 640–643.[ISI][Medline]
Edmunds, S.E., Stubbs, A.P., Santos, A.A. et al. (1990) Estrogen and androgen regulation of sex hormone binding globulin secretion by a human liver cell line. J. Steroid Biochem. Mol. Biol., 37, 733–739.[ISI][Medline]
Escobar-Morreale, H.F., Serrano-Gotarredona, J., Avila, S. et al. (1998) The increased circulating prostate-specific antigen concentrations in women with hirsutism do not respond to acute changes in adrenal or ovarian function. J. Clin. Endocrinol. Metab., 83, 2580–2584.[Abstract/Free Full Text]
Farnsworth, W.E. (1999) Estrogen in the etiopathogenesis of BPH. Prostate, 41, 263–274.[ISI][Medline]
Gann, P.H., Hennekens, C.H., Ma, J. et al. (1996) Prospective study of sex hormone levels and risk of prostate cancer. J. Natl. Cancer Inst., 88, 1118–1126.[Abstract/Free Full Text]
Giovannucci, E., Stampfer, M.J., Krithivas, K. et al. (1997) The CAG repeat within the androgen receptor gene and its relationship to prostate cancer. Proc. Natl. Acad. Sci. USA, 94, 3320–3323.[Abstract/Free Full Text]
Goh, V.H. (1999) Breast tissues in transsexual women|a nonprostatic source of androgen up-regulated production of prostate-specific antigen. J. Clin. Endocrinol. Metab., 84, 3313–3315.[Abstract/Free Full Text]
Hardy, D.O., Scher, H.I., Bogenreider, T. et al. (1996) Androgen receptor CAG repeat lengths in prostate cancer: correlation with age of onset. J. Clin. Endocrinol. Metab., 81, 166–170.
Hayes, F.J. (2000) Testosterone—Fountain of youth or drug of abuse? J. Clin. Endocrinol. Metab., 85, 3020–3023.[Free Full Text]
Hsing, A.W., Gao, Y.T., Wu, G. et al. (2000) Polymorphic CAG and GGN repeat lengths in the androgen receptor gene and prostate cancer risk: a population-based case-control study in China. Cancer Res., 60, 5111–5116.[Abstract/Free Full Text]
Irvine, R.A., Yu, M.C., Ross, R.K. et al. (1995) The CAG and GGC microsatellites of the androgen receptor gene are in linkage disequilibrium in men with prostate cancer. Cancer Res., 55, 1937–1940.[Abstract/Free Full Text]
Jin, B., Turner, L., Walters, W.A. et al. (1996) The effects of chronic high dose androgen or estrogen treatment on the human prostate. J. Clin. Endocrinol. Metab., 81, 4290–4295.[Abstract]
Kim, M.R., Gupta, M.K., Travers, S.H. et al. (1999) Serum prostate specific antigen, sex hormone binding globulin and free androgen index as markers of pubertal development in boys. Clin. Endocrinol. (Oxf.), 50, 203–210.[Medline]
La Spada, A.R., Wilson, E.M., Lubahn, D.B. et al. (1991) Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature, 352, 77–79.[Medline]
Lim, H.N., Chen, H., McBride, S. et al. (2000) Longer polyglutamine tracts in the androgen receptor are associated with moderate to severe undermasculinized genitalia in XY males. Hum. Mol. Genet., 9, 829–834.[Abstract/Free Full Text]
Liow, S.L., Ghadessy, F.J., Ng, S.C. et al. (1998) Y chromosome microdeletions, in azoospermic or near-azoospermic subjects, are located in the AZFc (DAZ) subregion. Mol. Hum. Reprod., 4, 763–768.[Abstract/Free Full Text]
Mhatre, A.N., Trifiro, M.A., Kaufman, M. et al. (1993) Reduced transcriptional regulatory competence of the androgen receptor in X-linked spinal and bulbar muscular atrophy. Nat. Genet., 5, 184–188.[ISI][Medline]
Mifsud, A., Ramirez, S. and Yong, E.L. (2000) Androgen receptor gene CAG trinucleotide repeats in anovulatory infertility and polycystic ovaries. J. Clin. Endocrinol. Metab., 85, 3484–3488.[Abstract/Free Full Text]
Mifsud, A., Sim, C.K.S., Boettger-Tong, H. et al. (2001) Trinucleotide (CAG) repeat polymorphisms in the androgen-receptor gene: molecular markers of male infertility risk. Fertil. Steril., 75, 275–281[ISI][Medline]
Monath, J.R., McCullough, D.L., Hart, L.J. et al. (1995) Physiologic variations of serum testosterone within the normal range do not affect serum prostate-specific antigen. Urology, 46, 58–61.[ISI][Medline]
Patrizio, P., Leonard, D.G., Chen, K.L. et al. (2001) Larger trinucleotide repeat size in the androgen receptor gene of infertile men with extremely severe oligozoospermia. J. Androl., 22, 444–448.[Abstract]
Perez-Stable, C.M., Pozas, A. and Roos, B.A. (2000) A role for GATA transcription factors in the androgen regulation of the prostate-specific antigen gene enhancer. Mol. Cell. Endocrinol., 167, 43–53.[ISI][Medline]
Plymate, S.R., Leonard, J.M., Paulsen, C.A. et al. (1983) Sex hormone-binding globulin changes with androgen replacement. J. Clin. Endocrinol. Metab., 57, 645–648.[Abstract]
Polascik, T.J., Oesterling, J.E. and Partin, A.W. (1999) Prostate specific antigen: a decade of discovery—what we have learned and where we are going. J. Urol., 162, 293–306.[ISI][Medline]
Rebbeck, T.R., Kantoff, P.W., Krithivas, K. et al. (1999) Modification of BRCA1-Associated Breast Cancer Risk by the Polymorphic Androgen Receptor CAG Repeat. Am. J. Hum. Genet., 64, 1371–1377.[ISI][Medline]
Rosenfield R.L. (1971) Plasma testosterone binding globulin and indexes of the concentration of unbound plasma androgens in normal and hirsute subjects. J. Clin. Endocrinol. Metab., 32, 717–728.[ISI][Medline]
Sawaya, M.E. and Shalita, A.R. (1998) Androgen receptor polymorphisms (CAG repeat lengths) in androgenetic alopecia, hirsutism, and acne. J. Cutan. Med. Surg., 3, 9–15.[Medline]
Stanford, J.L., Just, J.J., Gibbs, M. et al. (1997) Polymorphic repeats in the androgen receptor gene: molecular markers of prostate cancer risk. Cancer Res., 57, 1194–1198.[Abstract/Free Full Text]
Tut, T.G., Ghadessy, F.J., Trifiro, M.A. et al. (1997) Long polyglutamine tracts in the androgen receptor are associated with reduced trans-activation, impared sperm production, and male infertility. J. Clin. Endocrinol. Metab., 82, 3777–3782.[Abstract/Free Full Text]
Van Kesteren, P., Meinhardt, W., van der Valk, P. et al. (1996) Effects of estrogens only on the prostates of aging men. J. Urol., 156, 1349–1353.[ISI][Medline]
World Health Organization (1999) Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction, 4th edn. Cambridge University Press, Cambridge.
Yamamoto, M., Hibi, H., Katsuno, S. et al. (1995) Serum estradiol levels in normal men and men with idiopathic infertility. Int. J. Urol., 2, 44–46.[Medline]
Yoshida, K.I., Yano, M., Chiba, K. et al. (1999) CAG repeat length in the androgen receptor gene is enhanced in patients with idiopathic azoospermia. Urology, 54, 1078–1081.[ISI][Medline]
Submitted on March 22, 2001; accepted on August 21, 2001.
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BUt the question is what about those who use multi compounds and not just test would this be the same or what
