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,
From the * Department of Internal Medicine, the
Center for Research in Reproduction and
Contraception, the Department of Obstetrics
and Gynecology, and the || Department of Medicinal
Chemistry, University of Washington, Seattle, Washington; and the
Geriatric Research, Education and Clinical
Center, Veterans Affairs Puget Sound Health Care System, Seattle,
Washington.
| Correspondence to: Dr Mara Y Roth, University of Washington, 1959 NE Pacific St, Box 357138, Seattle, WA 91895 (e-mail: mylang{at}u.washington.edu). |
| Received for publication May 28, 2009; accepted for publication September 21, 2009. |
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Abstract |
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Key words: Contraception, hormone, infertility, testis
Understanding the hormonal requirements necessary to support spermatogenesis in men has been difficult. Until recently, methods for measuring intratesticular hormone concentrations in men required testis tissue obtained by testicular biopsy or at the time of orchidectomy (Morse et al, 1973; Takahashi et al, 1982; Marie et al, 2001). These methods are considered to be too invasive for assessment of normal, healthy men; therefore, prior studies were in infertile men. In 2001, Jarow et al demonstrated that fine-needle tissue aspiration of the testes could be used to quantify intratesticular hormone concentrations. This procedure used to obtain intratesticular fluid for quantification of steroids by radioimmunoassay was further refined with measurement of testicular aspiration fluid steroid hormone levels by mass spectrometry (Zhao et al, 2004); however, comparison with contemporaneously measured serum hormone concentrations and between the testes in a given individual was performed only for testosterone. In this study, we performed testicular aspirations in a group of normal men to better understand the relationship between intratesticular concentrations of testosterone and its active metabolites, as well as the relationship among intratesticular sex steroids, circulating gonadotropins (LH and follicle-stimulating hormone [FSH]), and sex steroid levels in normal men.
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Materials and Methods |
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Prior to the aspiration procedure, each subject's vital signs were taken and a blood sample was drawn for assessment of serum hormone levels (15–20 minutes prior to testicular aspiration). After local anesthesia administration at the spermatic cord with 1% buffered lidocaine, a 19-gauge needle was used to perform bilateral testicular aspirations as previously described (Jarow et al, 2001; Coviello et al, 2005). Subjects were evaluated 1 week following the procedure, and 1 month later a follow-up semen analysis was performed.
The institutional review board of the University of Washington approved this study protocol prior to study initiation (National Clinical Trial 00756561). Informed consent was obtained from all subjects prior to screening evaluation.
Measurements
Testicular fluid samples were placed immediately on ice and centrifuged at
300 x g. Supernatant fluid was stored at –70°C. We
measured right and left testicular fluid samples for IT-T, intratesticular
dihydrotestosterone (IT-DHT), and intratesticular estradiol (IT-E2) by liquid
chromatography–tandem mass spectrometry (LC/MS/MS) on a Waters Aquity
UPLC coupled with a Micromass Premiere-XE tandem quadrupole mass spectrometer
(Waters Corp, Milford, Massachusetts) as described previously
(Kalhorn et al, 2007). For
IT-E2, human serum samples (100 µL) or intratesticular fluid samples were
diluted with water to a final volume of 0.5 mL. The samples were left at room
temperature for 1 hour, at which time 4.0 mL t-butyldimethyl ether (TBDME) was
added. The tubes were sealed, extracted on a horizontal shaker, centrifuged,
and flash frozen on dry ice. The top organic phase was decanted into a conical
screw-top tube and evaporated to dryness under nitrogen. Additional TBDME (150
µL) was added, the tubes vortexed, and the solvent again removed under
nitrogen. The residue was dissolved in 40 µL 100 mM pH 10.5 sodium
carbonate followed by the addition of 40 µL 1.0 mg/mL dansyl chloride in
acetonitrile. The tubes were sealed and heated at 60°C for 5 minutes. The
tubes were centrifuged and the supernatant removed for analysis. Samples were
analyzed in triplicate and injected twice.
The lower limit of quantification for all 3 sex steroid assays was 0.04 pg/mL. The intra-assay and interassay coefficients of variation for testosterone were 3.5% and 7.7% respectively, and for dihydrotestosterone (DHT) they were 3.5% and 6.3%. The intra-assay coefficient of variation for estradiol was 14% using human serum. Serum LH and FSH were quantified by immunofluorometric assay (Page et al, 2007). The intra-assay coefficient of variation for LH was 5.6%, and the interassay coefficient was 13.9%. For FSH, the intra-assay and interassay coefficients of variation were 3.0% and 5.0% respectively. All samples for all subjects were batched and measured in 1 assay.
Statistical Analysis
Because of nonnormal distributions, all hormone concentrations were natural
log–transformed prior to analysis using parametric statistics.
Correlations between serum hormone levels and intratesticular hormones, and
between testosterone and other steroid hormones, were performed using the
Pearson technique. Statistical analyses were performed using STATA version
10.0 (Stata Corporation, College Park, Texas). For all analyses, a P
value of <.05 was considered significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Subject characteristics are shown in Table 1. Six subjects were Caucasian, 2 Asian, 1 African-American, and 1 native Hawaiian. The range of IT-T was 119–1251 ng/mL, with a median (interquartile [IQ] range) of 486 (429–897) ng/mL. The range of IT-DHT was 1.1–7.9 ng/mL, with a median (IQ range) of 3.7 (1.1–4.7) ng/mL. The range of IT-E2 was 0.4–3.9 ng/mL, with a median (IQ range) of 2.7 (1.3–2.4) ng/mL (Table 2). Serum testosterone measured at the time of the testicular aspiration ranged from 1.4 to 8.2 ng/mL, with a median (IQ range) of 3.0 (2.3–3.9) ng/mL. Serum DHT ranged from 82 to 525 pg/mL, with a median (IQ range) of 200 (120–240) pg/mL. Serum estradiol ranged from 14 to 33 pg/mL, with a median (IQ range) of 25 (19–29) pg/mL.
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To investigate the relationship between serum gonadotropins and intratesticular steroid concentrations that could contribute to the wide variability in intratesticular hormone levels observed, especially in testosterone, we looked for correlations between gonadotropins and intratesticular hormones. Serum LH, drawn approximately 15–20 minutes prior to the testicular aspiration, correlated strongly with IT-T (r = 0.87, P = .001; Figure 1A) and IT-E2 (r = 0.70, P = .025; Figure 1B), but not with IT-DHT (r = 0.25, P = .5; data not shown). Serum FSH also correlated with IT-T (r = 0.70, P = .024; Figure 1C), but not with IT-E2 (r = 0.50, P = .13; Figure 1D) or IT-DHT (r = 0.09, P = .82; data not shown).
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Discussion |
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Our results regarding IT-T concentrations are comparable to previous reports using this technique (Zhao et al, 2004) and add to the small body of data in this regard. We used the same minimally invasive percutaneous testicular aspiration technique under local anesthesia to obtain intratesticular fluid. However, we measured simultaneous serum steroid hormone concentrations in addition to intratesticular fluid. We also used a LC/MS/MS assay, which allows for a more sensitive and specific assay, in comparison to most previous data using radioimmunoassay (Jarow et al, 2001; McLachlan et al, 2002; Coviello et al, 2004; Matthiesson et al, 2005). Earlier reports of intratesticular concentrations used extracts from testis tissue obtained by biopsy or at orchidectomy, but evaluated primarily infertile men or men with prostate cancer, and used general anesthesia, which can alter hormone concentrations (Morse et al, 1973; Takahashi et al, 1982; Marie et al, 2001). Although our IT-T concentrations measured by LC/MS/MS are similar to those reported by Zhao et al (2004), our IT-E2 and IT-DHT values are somewhat lower. Increased sample size in future studies may narrow this apparent discrepancy, although we cannot rule out that differences in assay methodology might contribute to these differences.
Although the range of IT-T levels was quite broad, this variation seems to be explained by variation in LH and may reflect a pulsatile concentration of IT-T, similar to pulsatility in serum testosterone levels (Baker et al, 1975). The strong correlations between serum LH and IT-T and between serum LH and estradiol illustrate that IT-T and estradiol likely vary with LH pulses. Further evidence for this comes from the observation that such a relationship between IT-T and gonadotropins does not exist in men who have undergone prolonged gonadotropin suppression using exogenous testosterone in combination with a progestin (Page et al, 2007).
The significant correlations between IT-T and serum FSH, and between IT-E2 and serum FSH, were partially due to the presence of 1 subject with the highest IT-T and FSH. When this subject was omitted from these analyses, the correlations were no longer significant, implying that these correlations may be due to chance. In any case, it is clear that this relationship is not as strong as that between IT-T and LH and IT-E2 and LH. This is likely due to the known stimulatory role of LH on testosterone biosynthesis in Leydig cells, whereas FSH is not known to play a role in this process. It seems possible that the observed correlations are more likely due to cosecretion of FSH and LH from the pituitary rather than any direct effect of FSH on IT-T. Similarly, the significant correlation between serum T and IT-T is due to the presence of a signal outlier (not the same subject as omitted above) because removal of this subject reduced the P value from .03 to .07. This further illustrates the need for additional study of these relationships in a larger sample of men.
We also examined the relationship between intratesticular and
contemporaneous serum hormone concentrations. Intratesticular hormone
concentrations are significantly higher than serum concentrations, as
previously shown in both mice and humans
(Turner et al, 1984;
Jarow et al, 2001).
Interestingly, the intratesticular to serum ratios of testosterone and
estradiol are nearly 200 and 100 respectively, but serum DHT is only about 15
times higher than IT-DHT. This suggests that DHT is formed primarily at
peripheral sites rather than within the testes. This is compatible with
studies of 5
- reductase expression, which demonstrate high levels of
expression in the skin, gut, kidney, and prostate and only modest expression
in the male reproductive tract (Thigpen et
al, 1993). Indeed, DHT may not be critical for spermatogenesis
because chronic inhibition of 5 - reductase has a minor impact on sperm
concentrations in most men (Amory et al,
2007) and does not appear to augment sperm suppression when added
to other male hormonal contraceptive agents
(Kinniburgh et al, 2001).
Intratesticular concentrations of testosterone are highly correlated
between the right and left testes. The correlation for estradiol between the 2
testes did not quite reach statistical significance, likely because of our
small sample size. Interestingly, there was no correlation between DHT levels
in the right and left testes in this small sample of normal men. Many factors
could contribute to this finding, including possible differential geographic
expression of the enzyme 5-reductase that metabolizes testosterone into
DHT (Mahony et al, 1998).
Blood flow in the right and left testes is also known to be unequal because of
variations in the anatomy of the blood supply to each testis
(Fritjofsson et al, 1969),
which might theoretically impact nontesticular-derived sources of DHT within
each testicle.
There are several weaknesses with our study. In particular, the small sample size prevents us from having adequate statistical significance to define normative ranges for intratesticular hormone concentrations. However, the strengths of this study include our ability to use a highly sensitive LC/MS/MS assay in normal men and the use of a minimally invasive technique that allows the subjects to avoid general anesthesia, which can alter serum steroid concentrations. Future studies using the same assay will allow us to develop a larger cohort of normal men from whom to calculate a normal range for intratesticular hormones. Serum hormone concentrations also fluctuate with circadian rhythms (Plymate et al, 1989), yet we did not attempt to time our aspirations with identified LH pulses, which may also alter intratesticular hormone concentrations.
Measurement of intratesticular steroid hormone concentrations will provide essential information for future studies of the hormonal regulation of spermatogenesis. Our findings have implications for the treatment of male infertility and male contraceptive development. For example, the relationship between intratesticular hormones and infertility has not been explored systematically in large numbers of infertile compared with normal men, despite the known central contribution of testosterone to spermatogenesis. The use of the technique presented here, which allows for repeat assessment of intratesticular hormones in an individual, combined with sensitive assay techniques using LC/MS/MS, will allow us to explore the relationship between intratesticular steroid hormone concentrations and spermatogenesis in future cross-sectional and interventional studies. These findings also have implications for the development of male hormonal contraception. Male hormonal contraception uses exogenously administered androgens and progestins to suppress hypothalamic release of gonadotropin-releasing hormone and pituitary release of gonadotropins (LH and FSH). This suppresses endogenous production of testosterone, and subsequently spermatogenesis, while providing systemic androgens to maintain activity at peripheral sites and prevent symptomatic hypogonadism. Although rates of azoospermia with use of both androgens and progestin reach 90%–95% (Page et al, 2008), we have little understanding of why the remaining 5%–10% of men fail to achieve azoospermia. Animal models suggest that gonadotropin-independent androgen production may support residual spermatogenesis in the setting of gonadotropin ablation (Zhang et al, 2004). Alternatively, high-dose exogenous androgens used in such regimens could diffuse into the testes and support spermatogenesis. These hypotheses have been difficult to test in humans, but could be examined using our technique of testicular aspiration in men during trials of experimental male hormonal contraceptives.
In summary, our study has confirmed and extended earlier work demonstrating that testosterone, DHT, and estradiol can be measured by a minimally invasive, percutaneous, fine-needle aspiration technique of the testes, and that these concentrations are much higher than those in the serum. In addition, we have demonstrated that serum LH correlates very strongly with IT-T and estradiol, accounting for some of the wide range of normal values and providing evidence for the importance of gonadotropins in the regulation of intratesticular steroid concentration. A strong correlation also exists between testosterone concentrations in the right and left testes, indicating that a unilateral aspiration technique is likely sufficient to determine IT-T and estradiol concentrations. Future studies will include larger populations to establish a normal hormone range for intratesticular hormone concentrations. This paradigm, coupled with hormone manipulation, will allow us to identify the critical relationships and thresholds for intratesticular hormones to support spermatogenesis in men.
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Acknowledgments |
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Footnotes |
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