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Intratesticular Androgens and Spermatogenesis During Severe Gonadotropin Suppression Induced by Male Hormonal Contraceptive Treatment

THOMAS F. KALHORN

BRADLEY D. ANAWALT^*,

ALVIN M. MATSUMOTO^*,,

From the ^* Department of Medicine and Department of Medicinal Chemistry, University of Washington, Seattle, Washington; Department of Medicine, Veterans Affairs Puget Sound Health Care System; and Geriatric Research, Education and Clinical Center, Seattle, Washington.

Correspondence to: Dr Stephanie T. Page, Box 357138, 1959 NE Pacific, Seattle, WA 98195 (e-mail: page{at}u.washington.edu).

Received for publication March 8, 2007; accepted for publication May 3, 2007.

	Abstract

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Male hormonal contraceptive regimens function by suppressing gonadotropin secretion, resulting in a dramatic decrease in testicular androgen biosynthesis and spermatogenesis. Animal studies suggest that persistent intratesticular (iT)–androgen production has a stimulatory effect on spermatogenesis in the setting of gonadotropin suppression. We hypothesized that men with incompletely suppressed spermatogenesis (>1 000 000 sperm/mL) during male hormonal contraceptive treatment would have higher iT-androgen concentrations than men who achieved severe oligospermia (1 000 000 sperm/mL). Twenty healthy men ages 18–55 years enrolled in a 6-month male contraceptive study of transdermal testosterone (T) gel (100 mg/d) plus depomedroxyprogesterone acetate (300 mg intramuscularly every 12 weeks) with or without the gonadotropin releasing hormone (GnRH) antagonist acyline (300 µg/kg subcutaneously every 2 weeks for 12 weeks) were studied. During the 24th week of treatment, subjects underwent fine needle aspirations of the testes and iT-T and iT-dihydrotestosterone (iT-DHT) were measured in testicular fluid by liquid chromatography–tandem mass spectrometry. All men dramatically suppressed spermatogenesis; 15 of 20 men were severely oligospermic, and 5 of 20 suppressed to 1.5 million–3.2 million sperm per milliliter. In all subjects, mean iT-T and iT-DHT concentrations were 35 ± 8 and 5.1 ± 0.8 nmol/L. IT-androgen concentrations did not significantly differ in men who did and did not achieve severe oligospermia (P = .41 for iT-T; P = .18 for iT-DHT). Furthermore, there was no significant correlation between iT-T or iT-DHT and sperm concentration after 24 weeks of treatment. In this study of prolonged gonadotropin suppression induced by male hormonal contraceptive treatment, differences in iT-androgens did not explain differences in spermatogenesis. Additional studies to identify factors involved in persistent spermatogenesis despite gonadotropin suppression are warranted.

     Key words: Testosterone, dihydrotestosterone, INSL3, LH, FSH

Data from animal studies suggest that very low levels of residual iT-androgens may support spermatogenesis in the absence of gonadotropins. Reductions of iT-T in the rat to levels approximately 25% of control values do not affect spermatogenesis quantitatively; however, reductions in iT-T below this threshold (17 nmol/L) result in near complete abolition of sperm production (Zirkin et al, 1989). Moreover, administration of T to gonadotropin-suppressed rats has been shown to restore quantitatively normal spermatogenesis (Sharpe et al, 1988; Zirkin et al, 1989; Awoniyi et al, 1990, 1992). Similarly, in both the hpg mouse, which congenitally lacks both FSH and LH, and hypophysectomized cynomolgus monkeys, exogenous androgens are sufficient to initiate and support spermatogenesis (Marshall et al, 1986; Singh et al, 1995; Spaliviero et al, 2004). In men, higher doses of exogenous T were associated with persistent spermatogenesis when administered as part of a male hormonal contraceptive regimen (Meriggiola et al, 2002). Results from LH receptor–deficient mice also suggest that androgens are crucial for spermatogenesis. These animals continue to synthesize T at low levels despite the absence of an LH signal (Zhang et al, 2004) and continue to produce low numbers of sperm (Zhang et al, 2003). However, spermatogenesis in these animals is completely eliminated by treatment with the androgen receptor antagonist flutamide (Zhang et al, 2003). Together, these findings support the notion that minimal concentrations of residual iT-androgen signal can maintain low levels of spermatogenesis in a low-gonadotropin testicular environment.

In this study, we sought to determine the relationship between iT-androgens and human spermatogenesis during male hormonal contraceptive treatment. We hypothesized that men with persistent spermatogenesis after male hormonal contraceptive treatment would have significantly higher concentrations of testicular androgens than men with severely suppressed sperm production. Therefore, we measured iT-T and its potent metabolite dihydrotestosterone (DHT) from testicular fluid aspirates obtained in healthy men after 6 months of male hormonal contraceptive treatment.

	Methods

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Study Design

All procedures involving human subjects were approved by the Institutional Review Board at the University of Washington. Written informed consent was obtained prior to screening. This study was performed as a substudy of a larger male hormonal contraception trial that was published recently (Page et al, 2006). Following screening, 44 subjects were randomly assigned by the research pharmacist to 1 of 2 treatment groups: 1) T + depomedroxyprogesterone (DMPA) group: 1% T gel, 100 mg topically daily (Auxilium Pharmaceuticals, Norristown, Pa), + DMPA, 300 mg intramuscularly every 3 months (UpJohn Pharmaceuticals, Kalamazoo, Mich), or 2) acyline + T + DMPA group: acyline (NeoMDS, San Diego, Calif), 300 µg/kg subcutaneously every 2 weeks for 12 weeks, plus T gel + DMPA as in group 1. Throughout the drug exposure and recovery periods, subjects were asked to provide a semen sample obtained by masturbation every 2 weeks after at least 48 hours of ejaculatory abstinence. Blood was collected monthly. Subjects underwent a physical examination and blood draw for measurement of complete blood count, serum chemistries including liver function tests, LH, FSH, T, and sex hormone binding globulin (SHBG). All serum samples were centrifuged and stored at –70°C until the end of study analyses.

Testicular Fluid Aspiration

Thirty-eight men completed the protocol and, of these, 23 agreed to undergo percutaneous testicular fluid aspiration during the final week of drug exposure. Blood was collected on the day of the aspiration procedure for comparison of serum hormones and iT-androgen levels. Testicular fluid was sampled by fine needle aspiration using the technique of Jarow and colleagues (Jarow et al, 2001). With the subject lying on an examination table in the supine position, the skin over the spermatic cord at the external ring was cleansed with alcohol. Then, a bilateral spermatic cord block was performed with 10 mL of 1% buffered lidocaine. The skin over the anterosuperior aspect of the testes was then cleansed with alcohol, and a 19-gauge butterfly needle inserted into the testicular parenchyma. Negative pressure was generated with an attached syringe until testicular fluid was obtained. The testicular fluid sample was immediately placed on ice and centrifuged at 300 x g. The aspirate supernatant was stored at –70°C. The procedure was aborted in 2 men prior to the aspiration after failure to obtain sufficient local anesthesia; fluid could only be obtained from 1 of 2 testicles in 1 subject. There were no complications or serious adverse events.

Serum Hormone, SHBG, and INSL3 Measurements

In all cases, samples from a given individual were run in a single assay. Serum hormone assays for this study have been previously described (Page et al, 2006). FSH and LH levels were measured by immunofluorometric assay (Delfia, Wallac Oy, Finland). Serum T (Diagnostic Products Corp, Los Angeles, Calif) and SHBG (Delfia) were measured by radioimmunoassay (RIA). Free T was calculated as described by Södergard et al (1982) and validated by Vermeulen et al (1999). Insulin-like factor-3 (INSL3) is a circulating protein of testicular origin that is a marker of Leydig cell function (Foresta et al, 2004; Bay et al, 2005, 2006). INSL3 was measured by RIA (Phoenix Pharmaceuticals Inc, Belmont, Calif).

Seminal Fluid Analyses

Screening semen samples were examined within 60 minutes of collection and assessed for volume, sperm count, motility, and morphology according to World Health Organization criteria (1999). In subsequent seminal fluid analyses, sperm counts were measured manually after centrifugation and confirmed using the IVOS-Hamilton counter (Hamilton Thorne Research, Beverly, Mass). All individuals classified as azoospermic had no detectable sperm in the final 2 seminal fluid analyses of the study; the sperm were collected 2–4 weeks apart, with the last one the week of the aspiration procedure. Classification as severe oliogospermia or nonsevere oligospermia was based on the final seminal fluid analyses. For examination of the relationship between sperm concentration and iT-androgens and INSL3, the concentration of the final seminal fluid analyses collected within 1 week of the aspiration procedure was used.

Intratesticular Steroid Measurements

Testicular fluid aspirate volumes ranged from 3 to 35 µL. Water was added to each sample to achieve a final volume of 100 uL; 50 pg of deuterated (D3)–DHT and D3-T were added to 60 µL of the diluted aspirate as internal standards; 1.0 M NaOH was then added and the samples left for 1 hour, after which they were by extracted with methylene chloride. The organic phase was evaporated to dryness under nitrogen, and the sample was reconstituted in 0.1 M hydroxyamine hydrochloride in 50% MeOH/water, vortexed, and heated at 60°C for 1 hour. Standards for DHT and T were prepared in parallel. The resulting oximes were analyzed by liquid chromatography-dual mass spectroscopy using a Waters Asquity High Performance Liquid Chromatography and Premier XE mass spectrometer (Milford, Mass). Ions monitored were at/m/z/309 and a306 for quantifying DAT-IS and DHT respectively and at/m/z/124 for T-IS and T after fragmentation for T-IS and T, respectively. This procedure resulted in a lower limit of quantitation of 100 and 500 attomoles per sample for T and DHT, respectively. The iT-androgen concentrations from the right and left testis were averaged for each individual. Intra-assay coefficients of variation generated using human serum for high-, mid-, and low-range samples were 3.5%, 3.1%, and 3.8% for T and 6.3%, 4.3%, and 15.8% for DHT, respectively.

Statistical Analyses

For baseline characteristics, means and standard deviations were computed. Due to non-normal distributions, hormone concentrations in individuals with severe oligospermia (sperm concentration 1 million per milliliter or below) and those with sperm concentrations above 1 million per milliliter were graphed as box and whiskers plots, and differences between these groups were compared with a Wilcoxon signed rank test. The relationship between serum hormone levels and sperm status (azoospermia and severe oligospermia) was calculated using the Mann-Whitney U test. Correlations between iT-androgens and hormone and sperm concentrations were performed using the Spearman technique. For all comparisons, a 2-sided of less than 0.05 was considered significant. Statistical analyses were performed using STATA version 8.0 (StataCorp LP, College Park, Tex).

	Results

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Study Population

Of 38 men enrolled in the trial (Page et al, 2006), 21 underwent successful percutaneous testicular fluid aspiration during the final week of drug exposure. Of the 21 subjects in whom sufficient iT fluid was collected, 1 subject was noted to have unsuppressed gonadotropins at the time of the aspiration (LH, 5.1 IU/L; FSH, 8.4 IU/L). Consistent with this, his iT-androgen concentrations were similar to those reported for untreated healthy men (Coviello et al, 2004; Coviello et al, 2005; Matthiesson et al, 2005) and approximately 100-fold greater than the other subjects, strongly suggesting that he did not adhere to the treatment regimen (ie, did not apply his T gel on a daily basis). He was therefore excluded from the reported analyses. However, analyses were performed both with and without this subject included, and the conclusions were unchanged.

Baseline characteristics of the study subjects are shown in Table 1. There were no significant differences at baseline between the subjects who underwent a testicular aspiration procedure and those who did not, except the men who consented to undergo testicular aspiration were older on average than the entire group in the initial study (35.5 ± 9.3 versus 29.1 ± 8.3, P = .04). There were no differences in gonadotropin suppression, sperm suppression, or iT-androgens between the 11 subjects randomized to acyline + T + DMPA and the 9 men randomized to T + DMPA (data not shown). Mean testicular aspiration volumes were 0.019 ± 0.023 mL for the right testes and 0.015 ± 0.010 mL for the left. There were no significant relationships between aspiration volume and iT-androgens, sperm concentration, or serum hormones (data not shown).

At the end of treatment, 15 of 20 subjects were severely oligospermic (sperm concentration 1 million or less per milliliter) when the testicular aspiration procedure was performed and had been severely oligospermic on 2 consecutive occasions (the week of the aspiration and 2 weeks prior). Of these, 9 were azoospermic (and had been so 2 weeks prior) and 6 had sperm counts of 10 000–100 000/mL. Five 20 subjects were not severely oligospermic at the time of the aspiration. Sperm concentrations in these men ranged between 1.5 million and 3.2 million per milliliter.

Serum Hormones and iT-Androgens

In this subset of 20 men, there was a significant decrease in gonadotropins compared with baseline (Table 2) with male hormonal contraceptive treatment; however, differences in end of treatment LH and T levels did not explain the degree of sperm suppression we observed (Table 2). Lower FSH levels were, however, significantly associated with azoospermia (P = .009) but not with severe oligospermia (1 000 000 sperm/mL).

After 24 weeks of male hormonal contraceptive treatment, mean iT-T and iT-DHT concentrations were 35 ± 8 and 5.1 ± 0.8 nmol/L, respectively. In contrast to results reported for healthy untreated men, where iT-T is approximately 100-fold the concentration in serum (Jarow et al, 2001; Coviello et al, 2004), in our subjects iT-T concentrations were similar to serum T levels. The iT-T and iT-DHT levels were highly correlated with one another (P < .01, r = 0.63). End-of-treatment iT-androgen concentrations were not significantly associated with end-of-treatment serum hormone levels (Table 3), including gonadotropins. Moreover, serum INSL3, a circulating protein of testicular origin that is a marker of Leydig cell function (Foresta et al, 2004) whose production, like androgens, is regulated at least in part by LH (Foresta et al, 2004; Bay et al 2005, 2006), did not correlate with iT-androgen concentrations after 6 month of male hormonal contraceptive treatment.

iT-Androgens and Spermatogenesis

In contrast to our initial hypothesis, we did not find a correlation between end-of-treatment iT-androgens and sperm concentration (Figure 1). We went on to determine whether iT-androgens were associated with clinically significant persistent spermatogenesis after male hormonal contraceptive treatment. Neither iT-T (Figure 2A) nor iT-DHT (Figure 2B) were significantly higher in men who failed to achieve severe oligospermia (P = .41 for iT-T, P = .18 for iT-DHT) or failed to achieve azoospermia versus those who did after 6 months of male hormonal contraceptive treatment.

View larger version (14K): [in this window] [in a new window]   Figure 1. Scatter plots of sperm concentration and (A) intratesticular testosterone (iT-T) or (B) iT-dihydrotestosterone during week 24 in men treated with T gel and depomedroxyprogesterone for male contraception (n = 20).

View larger version (19K): [in this window] [in a new window]   Figure 2. Box and whiskers plots of (A) intratesticular (iT) testosterone or (B) iT dihydrotestosterone by end-of-treatment sperm concentration status (n = 20).

	Discussion

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Our data are consistent with prior analyses of the relationship between iT-androgens and spermatogenesis in humans. In a pilot study of 7 individuals who underwent testicular aspirations following male hormonal contraceptive treatment, the lone subject studied who failed to achieve severe oligospermia after 6 months of treatment had nearly the highest iT-T (29 nmol/L); however, a second subject who became azoospermic had a similar iT-T level (30 nmol/L) (Coviello et al, 2004). Moreover, 2 studies measuring tissue hormone levels in testicular biopsy specimens obtained after 2–12 weeks of male hormonal contraceptive treatment have failed to demonstrate a relationship between sperm concentration or germ cell number and iT-T concentrations (McLachlan et al, 2002; Matthiesson et al, 2005). The results we report here are the largest series to date of iT-androgen concentrations after long-term male hormonal contraceptive treatment and the first to include a substantial portion of nonazoospermic men evaluated after a period considered adequate to assess a full clinical response. Together, these data suggest that there is not a threshold iT-T level below which azoospermia becomes universal.

It has been suggested that the active metabolite of T, DHT, a more potent androgen, may play a pivotal role in maintaining spermatogenesis during male hormonal contraceptive treatment (Anderson et al, 1996). Despite observing a large, significant decline in iT-T with 2–12 weeks of T + DMPA treatment, McLachlan et al noted only nonsignificant decreases in iT-DHT levels compared with controls (McLachlan et al, 2002). These authors hypothesized that increased expression of testicular 5-reductase triggered by the low-gonadotropin environment might be responsible for the relative preservation of iT-DHT that might support spermatogenesis in some men. In addition, Walton et al (2006) noted the addition of the progestin desogestrel (which improves suppression of spermatogenesis) to a 4-week regimen of a gonadotropin releasing hormone (GnRH) antagonist plus T resulted in decreased expression of 5-reductase in the testes and proposed that this might be a second mechanism by which progestins improve spermatogenic suppression. In contrast to McLachlan et al, iT-DHT was profoundly reduced in a short-term study of various male hormonal contraceptive regimens compared with controls (Matthiesson et al, 2005). Similarly, as a consequence of more prolonged gonadotropin suppression, iT-DHT levels in our study were 80%–90% lower than those reported for untreated healthy men (Zhao et al, 2004; Matthiesson et al, 2005). Although we observed a trend toward a correlation between iT-DHT and end-of-treatment sperm concentration (P = .09) in this study, we found no difference between iT-DHT in men who achieved azoospermia or severe oligospermia and those who did not (P = .19 and P = .18, respectively). Similarly, after 8 weeks of various combinations of male hormonal contraceptive treatment including the progestin, levonorgestrel, or the a 5-reductase inhibitor dutasteride, no association between germ cell number and iT-DHT was found (Matthiesson et al, 2005). Moreover, the addition of a 5-reductase inhibitor to a male hormonal contraceptive regimen was not shown to increase the effectiveness of spermatogenic suppression (McLachlan et al, 2000; Kinniburgh et al, 2001). Our data suggest that iT-DHT does not play a pivotal role in maintaining low-level spermatogenesis during male hormonal contraceptive treatment. However, we cannot rule out a small effect, because a much larger study would be required to address this issue. In the present study, we had an 80% power to detect a twofold difference in iT-androgens between men who achieved azoospermia and those who did not. To find a 30% difference in iT-DHT, or iT-T between groups with 90% power, we would need end-of-treatment testicular fluid from 60 or 120 subjects, respectively.

A surrogate serum marker for iT-androgens or for persistent spermatogenesis would allow for further analyses of these questions without requiring invasive procedures. Recently, in an analysis of 108 subjects from 3 hormonal contraceptive trials including the 38 men in our original study, we reported that higher circulating levels of the Leydig cell product, INSL3, were associated with persistent spermatogenesis (Amory et al, 2007). In the present study we failed to find a relationship between serum INSL3 and either iT-T, iT-DHT, or sperm concentration after prolonged gonadotropin suppression. This discrepancy is likely due to the relatively small differences in INSL3 levels between subjects.

This study has some limitations. Due to the invasiveness of the procedure, testicular aspirations were performed at only a single time point at the end of treatment. This single measurement of iT-androgens may not reflect androgen synthesis in an individual over time. In addition, sperm concentrations fluctuate over time. To address this, individuals were classified as azoospermic or severely oligospermic in our study based on consecutive seminal fluid analyses 2 weeks apart. Lastly, this study was underpowered to detect small differences in iT-androgens; however, it is the largest study to date examining iT-androgens and their relationship to spermatogenesis after 6 months of contraceptive treatment. Moreover, it is the first study with sufficient numbers of subjects to examine the relationship between iT-androgens in "responders" (ie, those achieving severe oligospermia, the acceptable clinical benchmark for entrance into the efficacy phase of a male contraceptive trial [Nieschlag, 2007]) compared with "treatment failures" associated with male hormonal contraceptive administration.

In summary, in this study we did not observe a relationship between persistent spermatogenesis and iT-androgen concentrations after male hormonal contraceptive treatment for 6 months. iT-T and iT-DHT were not significantly different in men who achieved azoospermia or severe oligospermia and those who did not. Moreover, circulating INSL3, a marker of Leydig cell function, did not correlate with iT-androgen concentrations or spermatogenesis when circulating gonadotropins were at the lower limit of detectability. Additional studies to identify factors involved in human spermatogenesis in the low-androgen testicular environment are necessary to better understand persistent spermatogenesis despite profound gonadotropin suppression induced by male hormonal contraceptive regimens and, ultimately, allow for clinical use of this method of contraception.

	Acknowledgments

 
We thank Ms Kathy Winter, Ms Marilyn Busher, Ms Janet Gilchrest, and Ms Kymberly Anable for assistance with the clinical aspects of the study, Ms Consuelo Pete for seminal fluid analyses, and Ms Dorothy McGuinness for performance of hormone assays. We are grateful to Auxilium Pharmaceuticals for the gift of 1% Testim gel and the National Institute of Child Health and Human Development for providing acyline.

	Footnotes

 
S.T.P. and T.F.K. have nothing to disclose. B.D.A. has received honoraria for speaking from Indevus Pharmaceuticals. A.M.M. has consulted for GlaxoSmithKline, Quatrx, Solvay, Amgen, Threshold, and GTx Pharmaceuticals and received grant support from GlaxoSmithKline, Ascend Therapeutics, and Solvay Pharmaceuticals. J.K.A. has received grant support from Schering AG, GlaxoSmithKline, and Merron Pharmaceutics. W.J.B. has received honoraria from Indevus Pharmaceuticals and has consulted for Quatrx; he also is an inventor on US patent 7138389.

S.T.P. is supported by the National Institute of Aging, a division of the National Institutes of Health (NIH), (grant K23 AG027238) and by the Endocrine Society Solvay Clinical Research Award. J.K.A. is supported in part by the NIH National Institute of Child Health and Human Development (grant K23 HD45386). The National Institute of Child Health and Human Development also supported this work through cooperative agreements U54-HD-12629 and U54 HD-42454 as part of the specialized Cooperative Centers Program in Reproductive Research and the Cooperative Contraceptive Research Centers Program (W.J.B.).

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