Andrew Leung

Single Exposure to Heat Induces Stage-Specific Germ Cell Apoptosis in Rats: Role of Intratesticular Testosterone on Stage Specificity

Short term exposure of the testis to heat causes degeneration of germ cells. However, the mechanisms underlying this process are poorly understood. The major objectives of this study were to determine whether the heat-induced loss of germ cells in the adult rat occurs via apoptosis, to document its stage-specific and cell-specific distribution, and to examine whether intratesticular testosterone (T) plays any role in the stage specificity of heat-induced germ cell death. Testes of adult male Sprague-Dawley rats were exposed to 22 C (control) or 43 C for 15 min. Animals were killed on days 1, 2, 9, and 56 after heat exposure. Germ cell apoptosis was characterized by DNA gel electrophoresis and in situ terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling assay. The incidence of germ cell apoptosis [apoptotic index (AI)] was quite low in control rats (AI = 0.04–0.1). Mild hyperthermia within 1 or 2 days resulted in a marked activation (AI = 4.7–5.6) of germ cell apoptosis predominantly ...

Testicular Heat Exposure Enhances the Suppression of Spermatogenesis by Testosterone in Rats: The “Two-Hit” Approach to Male Contraceptive Development

The objectives of the study were to determine stage-specific changes in the kinetics of germ cell apoptosis induced by administration of exogenous testosterone (T) alone and to examine whether addition of a single testicular heat exposure would enhance the induction of germ cell apoptosis and the suppression of spermatogenesis by T. Adult male rats were implanted with 3-cm SILASTIC brand capsules (Dow Corning Corp.) containing T for up to 6 weeks. Intratesticular T levels declined to 2.9% of control values by 1 week and remained suppressed at 2, 3, and 6 weeks after T administration. The incidence of germ cell apoptosis (expressed as numbers per 100 Sertoli cells) was low in control rats (0–9.52). After T treatment, the mean incidence of apoptosis at stages VII–VIII increased significantly by 1 week (21.43 ± 3.33) and showed further increases by 6 weeks (56.30 ± 7.47); apoptotic rates remained low at early (I–VI) and later (XII–XIV) stages. To test whether the combination of T with a single testicular hea...

Validation of a Testosterone and Dihydrotestosterone Liquid Chromatography Tandem Mass Spectrometry Assay: Interference and Comparison with Established Methods

Testosterone (T) and its metabolite dihydrotestosterone (DHT) are androgens with different biologic profiles. T and DHT measurements are required for assessment of patients with ambiguous genitalia, hirsutism, during 5 alpha reductase treatment of prostate disorders, and new androgen formulations. Our laboratory has developed and validated a method to simultaneously measure serum T and DHT with liquid chromatography tandem mass spectrometry (LC-MS/MS) for use in a clinical chemistry laboratory. Analysis of sera from blood collected in tubes containing clot activator gave results of T that were fourfold higher than blood collected in plain tubes. Changing the ion pair selected for monitoring eliminated this interference by clot activators. Blood collected in fluoride-coated tubes gave serum T and DHT levels that were 20 and 15% lower, respectively than levels measured in blood collected in plain tubes (no additives). Addition of T enanthate to blood collected in plain tubes caused a dose related increase serum T levels due to the action of non-specific esterases in the red cells. This esterase activity could be avoided by using fluoride tubes for blood collection. Serum DHT levels were consistently lower when measured by LC-MS/MS versus radioimmunoassay. The differences were concentration dependent and the variance for the difference was large when serum DHT concentration was low. Celite chromatograph prior to radioimmunoassay reduced the differences between the two methods, thus confirming that higher levels of DHT obtained by immunoassays were probably due to interfering substances which were partially removed by Celite chromatography.

Effect of increased scrotal temperature on sperm production in normal men

Abstract Objective: To determine whether application of polyester-lined athletic supports to bring the testes closer to the abdomen increases scrotal temperature and decreases sperm production. Design: Prospective clinical study. Setting: University academic medical center. Patient(s): Twenty-one healthy male volunteers. Intervention(s): The study consisted of a pretreatment period of 6 weeks, a treatment phase of 52 weeks, and a recovery phase until return to normal sperm production. During the treatment phase, the men wore polyester-lined athletic supports (single layer, double layer, or double layer impregnated with aluminum) throughout the day. Main Outcome Measure(s): Semen parameters and sperm function tests. Result(s): In all three groups of subjects, scrotal temperature was consistently increased by 0.8 to 1°C while the subjects were wearing the athletic supports. Mean sperm concentration; sperm motility, morphology, and viability; sperm hyperactivation; and ability of spermatozoa to penetrate zone-free hamster oocytes were not affected by the increase in scrotal temperature. Conclusion(s): The increase in scrotal temperature induced by polyester-lined athletic supports was insufficient to cause significant suppression of spermatogenesis or alteration of sperm function.

ORIGINAL ARTICLE: Accuracy of calculated free testosterone formulae in men

Background  As reference laboratory methods for measuring free testosterone (FT) by equilibrium dialysis (ED) are laborious, costly and nonautomatable, FT levels are often calculated (cFT) rather than measured. However, the predictive accuracy of such estimates in routine use relative to laboratory measurements is not well defined. We provide a large‐scale evaluation of the predictive accuracy for different FT formulae compared with laboratory ED measurement and an analysis of clinical factors that may influence accuracy.

Reexamination of pharmacokinetics of oral testosterone undecanoate in hypogonadal men with a new self-emulsifying formulation.

Many hypogonadal men prefer oral testosterone (T) treatment. Oral T undecanoate (TU) is available in many countries, but not in the United States. We aimed to assess the pharmacokinetics of oral TU in a new self-emulsifying drug delivery system formulation. Pharmacokinetics studies were conducted in 3 parts: 12 hypogonadal men were enrolled in 2 centers for a 1-day dosing study; 29 participants were enrolled from 3 centers for a 7-day dosing study; and 15 participants were enrolled from 1 center for a 28-day dosing study. Serial blood samples for serum sex hormone measurements by liquid chromatography-tandem mass spectrometry were drawn for up to 36 hours after oral TU administration. Mean serum T levels (C(avg)) after oral dosing of T 200 mg as TU twice daily with food were within the adult male range in most participants in the 1-, 7-, and 28-day dosing studies but were much lower in the fasting state. The dose-proportional increase in C(avg) of serum T after oral T 300 mg twice daily resulted in more participants with supraphysiologic serum T levels. In the 28-day study, trough serum T reached a steady state at day 7. Serum dihydrotestosterone and estradiol levels tracked serum T concentration. Dihydrotestosterone-testosterone ratios increased 3-fold after oral TU administration. Oral T 200 mg twice daily as TU in a new SEDDS formulation may be a viable therapy for hypogonadal men.

Single, escalating dose pharmacokinetics, safety and food effects of a new oral androgen dimethandrolone undecanoate in man: a prototype oral male hormonal contraceptive

The novel androgen, dimethandrolone (DMA) has both androgenic and progestational activities, properties that may maximize gonadotropin suppression. We assessed the pharmacokinetics of dimethandrolone undecanoate (DMAU), an orally bioavailable, longer acting ester of DMA, for male contraceptive development. Our objective was to examine the safety and pharmacokinetics of single, escalating doses of DMAU (powder in capsule formulation) administered orally with or without food in healthy men. We conducted a randomized, double‐blind Phase 1 study. For each dose of DMAU (25–800 mg), 10 male volunteers received DMAU and two received placebo at two academic medical centres. DMAU was administered both fasting and after a high‐fat meal (200–800 mg doses). Serial serum samples were collected over 24 h following each dose. DMAU was well tolerated without significant effects on vital signs, safety laboratory tests or electrocardiograms. When administered while fasting, serum DMA (active compound) was detectable in only 4/10 participants after the 800 mg dose. When administered with a 50% fat meal, serum DMA was detectable in all participants given 200 mg DMAU and showed a dose‐incremental increase up to 800 mg, with peak levels 4–8 h after taking the dose. Serum gonadotropins and sex hormone concentrations were significantly suppressed 12 h after DMAU administration with food at doses above 200 mg. This first‐in‐man study demonstrated that a single, oral dose of DMAU up to 800 mg is safe. A high‐fat meal markedly improved DMAU/DMA pharmacokinetics.

Functional role of progestin and the progesterone receptor in the suppression of spermatogenesis in rodents

Synthetic progestins such as levonorgestrel (LNG) are used in combination with testosterone (T) in male contraceptive clinical trials to suppress gonadotropins secretion, but whether progestins have additional direct effects on the testis are not known. This study aimed to examine the effect of a potent progestin, (LNG), alone or in combination with testosterone (T) on spermatogenesis in adult rats, and to evaluate the functional role of the progesterone receptors (PRs) in the testis. In comparison with a low dose of LNG treatment in adult rats for 4 weeks, T and T + LNG treatment decreased testicular sperm count to 64.1 and 40.2% of control levels respectively. LNG induced germ cell apoptosis at stages I–IV and XII–XIV; T increased apoptosis at stages VII–VIII; LNG + T treatment induced greater germ cell apoptosis at a wider range of seminiferous epithelial stages. RT‐PCR and Western Blots showed that PR was present in testes and up‐regulated during suppression of spermatogenesis induced by testicular hormonal deprivation. PR knockout (PRKO) mice had larger testes, greater sperm production, increased numbers of Sertoli and Leydig cells. Suppression of gonadotropin and intratesticular T by GnRH‐antagonist treatment induced PR promoter driven LacZ expression in Leydig cells of PRKO mice. This suggests that GnRH‐antagonist treatment while inducing germ cell apoptosis also up‐regulates PR. We conclude that (i) LNG + T induced greater suppression of spermatogenesis through increase in germ cell apoptosis involving a wider range of seminiferous epithelial stages than either treatment alone, (ii) up‐regulation of PR was associated with inhibition of spermatogenesis, (iii) PR knockout mice showed increased sperm production suggesting that testicular PR activated events play a physiological and pharmacological inhibitory role in the testis. These data support the hypothesis that in addition to its known suppressive effects on gonadotropins, progestins may have direct inhibitory actions on the testis.

Steady-state pharmacokinetics of oral testosterone undecanoate with concomitant inhibition of 5α-reductase by finasteride

Oral testosterone undecanoate (TU) is used to treat testosterone deficiency; however, oral TU treatment elevates dihydrotestosterone (DHT), which may be associated with an increased risk of acne, male pattern baldness and prostate hyperplasia. Co-administration of 5α-reductase inhibitors with other formulations of oral testosterone suppresses DHT production and increases serum testosterone. We hypothesized that finasteride would increase serum testosterone and lower DHT during treatment with oral TU. Therefore, we studied the steady-state pharmacokinetics of oral TU, 200 mg equivalents of testosterone twice daily for 7 days, alone and with finasteride 0.5 and 1.0 mg po twice daily in an open-label, three-way crossover study in 11 young men with experimentally induced hypogonadism. On the seventh day of each dosing period, serum testosterone, DHT and oestradiol were measured at baseline and 1, 2, 4, 8, 12, 13, 14, 16, 20 and 24 h after the morning dose. Serum testosterone and DHT were significantly increased into and above their normal ranges similarly by all three treatments. Co-administration of finasteride at 0.5 and 1.0 mg po twice daily had no significant effect on either serum testosterone or DHT. Oral TU differs from other formulations of oral testosterone in its response to concomitant inhibition of 5α-reductase, perhaps because of its unique lymphatic route of absorption.

Oligozoospermia induced by exogenous testosterone is associated with normal functioning residual spermatozoa

OBJECTIVE To determine the functional capacity of residual spermatozoa in semen samples from normal men with T enanthate-induced oligozoospermia. DESIGN Prospective clinical study. SETTING Academic research center. PATIENT(S) Twelve healthy men were studied while participating in a multicenter T enanthate contraceptive efficacy study. Data were analyzed from only eight subjects, whose sperm concentrations were between 1.3 and 10 x 10(6)/mL at the suppression phase. INTERVENTION(S) Testosterone enanthate (200 mg) was administered IM weekly during the suppression and treatment (efficacy) phases (total 15 months). MAIN OUTCOME MEASURE(S) Sperm function tests (stimulated acrosome reaction, sperm hyperactivation [HA], and zona-free hamster oocyte penetration tests) were performed during the pretreatment, suppression (usually after 6 to 10 weeks of treatment, when sperm concentration was anticipated to decrease to < 10 x 10(6)/mL), and recovery phases. Studies were not done during the contraceptive efficacy phase because only one of the subjects was not azoospermic. RESULT(S) Mean sperm concentration was reduced but sperm motility, motility characteristics, and morphology were not affected by T enanthate treatment. The residual spermatozoa in the ejaculate could acrosome react, exhibited normal HA, and maintained the capacity to penetrate and fuse with the oocyte. CONCLUSION(S) Suppression of spermatogenesis to moderate oligozoospermia (< 10 x 10(6)/mL) with exogenous T enanthate administration was not associated with impaired sperm function of the residual spermatozoa. The study did not exclude the possibility that disorders of sperm function might occur when spermatogenesis is suppressed further to very severe oligozoospermia (< 1 x 10(6)/mL), commonly observed in hormonal male contraceptive clinical trials.