Single and multi-dose pharmacology of recombinant and urinary human chorionic gonadotrophin in men.

OBJECTIVE: Human choriogonadotrophin (hCG) treatment of gonadotrophin-deficient infertile men uses hCG of urinary (uhCG) or recombinant (rhCG) origin, but these treatments have not been compared nor are there studies defining rhCG dosing in men. DESIGN: hCG products were studied in randomized cross-over single-dose studies of standard (Study 1, 1500 IU and 62.5 µg, respectively) or high (Study 2, 5000 IU and 250 µg) dose and a multi-dose population pharmacology study of hCG use. PARTICIPANTS: Eight (Study 1) and seven (Study 2) volunteers in cross-over and 52 gonadotrophin-deficient men in the multi-dose study MEASUREMENTS: In cross-over studies, serum testosterone (T), dihydrotestosterone (DHT) and estradiol by liquid chromatography-mass spectrometry (LCMS) and serum hCG, LH, FSH, SHBG and T (observational study) by immunoassays. RESULTS: After standard and high-dose injection, serum hCG and testosterone responses had similar timing and peak concentrations except for a mildly lower early (<48 h) serum testosterone with uhCG. In the multi-dosing study, both hCGs had similar pharmacokinetics (pooled half-life 5.8 days, p < .001), while serum testosterone concentrations were stable after injection and did not differ between hCG products. Bench testing verified that 20% of pens from 4/10 individuals were used inappropriately. CONCLUSIONS: Although hCG pharmacokinetics are not formally bioequivalent, the similar pharmacodynamic effects on serum testosterone indicate that at the doses tested both hCGs provide comparable clinical effects. The starting dose of rhCG for treating gonadotrophin-deficient men should be 62.5 µg (6 clicks) of the rhCG pen.

Dried blood spot sampling of testosterone microdosing in healthy females.

Capillary dried blood spot (DBS) analysis coupled with multi-analyte steroid liquid chromatography mass spectrometry (LCMS) is attractive for field studies, home-based self-sampling as well as clinical trials by eliminating costly and laborious sample processing involving venipuncture and frozen storage/shipping while providing multiple steroid measurements from a single small sample. We investigated steroid measurements in DBS samples stored for four years at room temperature prior to analysis compared with the original venipuncture serum samples. Healthy women (n=12) provided paired DBS and blood samples over two weeks run-in before seven days treatment with daily transdermal T gel (12.5 mg) and after the end of treatment on days 0, 1, 2, 4, 7 and 14. Compliance with treatment and sampling was high and no adverse effects were reported. Testosterone (T), androstenedione (A4), 17 hydroxyprogesterone (17OHP) and progesterone (P4) were measured in extracted DBS samples as whole blood concentrations with and without adjustment for hematocrit. Using the same LCMS methods, DBS T and A4 measurements had high correlation with minimal bias from prior serum measurements with DBS T displaying the same pattern as serum, with or without hematocrit adjustment. However, serial whole blood measurements of T without hematocrit adjustment provided the best fitting model compared with serum, urine, or hematocrit-adjusted whole blood T measurements. These finding facilitate and simplify DBS methodology for wider field and home-based self-sampling studies of reproductive steroids indicating the need for hematocrit adjustment may be superfluous.

Sex steroids and androgen biomarkers in the healthy man study: within-person variability and impact of fasting.

OBJECTIVE: Serum testosterone measurements in clinical practice mostly utilize "direct" (non-extraction) immunoassays which have method-specific bias due to steroid cross-reactivity and nonspecific matrix artifacts. Although more accurate, sensitive, and specific liquid chromatography-mass spectrometry (LCMS) dominates in clinical research, the within-person variability of serum testosterone in healthy men using LCMS measurement is not reported. DESIGN: Longitudinal multi-sampling observational study of men in excellent health over 3 months. METHODS: Elite healthy men (n = 325) over 40 years of age in excellent, asymptomatic health provided 9 blood samples over 3 months with serum testosterone, dihydrotestosterone (DHT), estradiol (E2), and estrone (E1) measured by validated LCMS with conventional biochemical and anthropometric variables. RESULTS: Quantitative estimates of within-person variability within day and between day, week, month, and quarter were stable other than an increase due to fasting. The androgen biomarkers most sensitive to age and testosterone among widely used biochemical and anthropometric variables in middle-aged and older men were identified. CONCLUSIONS: This study provides estimates of variability in serum testosterone and the best androgen biomarkers that may prove useful for future studies of androgen action in male ageing.

Detection of testosterone microdosing in healthy females.

The ready detectability of synthetic androgens by mass spectrometry (MS)-based antidoping tests has reoriented androgen doping to using testosterone (T), which must be distinguished from its endogenous counterpart making detection of exogenous T harder. We investigated urine and serum steroid and hematological profiling individually and combined to determine the optimal detection model for T administration in women. Twelve healthy females provided six paired blood and urine samples over 2 weeks prior to treatment consisting of 12.5-mg T in a topical transdermal gel applied daily for 7 days. Paired blood and urine samples were then obtained at the end of treatment and Days 1, 2, 4, 7, and 14 days later. Compliance with treatment and sampling was high, and no adverse effects were reported. T treatment significantly increased serum and urine T, serum dihydrotestosterone (DHT), urine 5α-androstane-3α,17ß-diol (5α-diol) epitestosterone (E), and urine T/E ratio with a brief window of detection (2-4 days) as well as total and immature (medium and high fluorescence) reticulocytes that remained elevated over the full 14 posttreatment days. Carbon isotope ratio MS and the OFF score and Abnormal Blood Profile score (ABPS) were not discriminatory. The optimal multivariate model to identify T exposure combined serum T, urine T/E ratio with three hematological variables (% high fluorescence reticulocytes, mean corpuscular hemoglobin, and volume) with the five variables providing 93% correct classification (4% false positive, 10% false negatives). Hence, combining select serum and urine steroid MS variables with reticulocyte measures can achieve a high but imperfect detection of T administration to healthy females.

Depot Pure GnRH Antagonist for Long-term Treatment of Ovarian Hyperthecosis Monitored by Multisteroid LCMS Profiling.

Ovarian hyperthecosis (OHT), severe hyperandrogenism after menopause in the absence of ovarian or adrenal tumors, is usually treated by surgical excision. We report a 58-year-old woman presenting with severe hyperandrogenism (serum testosterone 15.7-31.0 nmol/L, normal female <1.8 nmol/L) with menopausal gonadotropins and virilization but no adrenal or ovarian lesions. Multisteroid profiling by liquid chromatography mass spectrometry (LCMS) of adrenal and ovarian vein samples identified strong gradients in the left ovarian vein (10- to 30-fold vs peripheral blood in 17OHP4, 17 hydroxyprogesterone, 17 hydroxypregnenolone, androstenedione, testosterone, dehydroepiandrosterone) but the right ovarian vein could not be cannulated with the same findings in a second ovarian vein cannulation. OHT diagnosis was confirmed by an injection of a depot pure gonadotropin-releasing hormone (GnRH) antagonist (80 mg Degarelix, Ferring) producing a rapid (<24 hour) and complete suppression of ovarian steroidogenesis as well as serum luteinizing hormone and follicle-stimulating hormone lasting at least 8 weeks, with reduction in virilization but injection site reaction and flushing and vaginal spotting ameliorated by an estradiol patch. Serum testosterone remained suppressed at 313 days after the first dose despite recovery of menopausal gonadotropins by day 278 days. This illustrates use of multisteroid LCMS profiling for confirmation of the OHT diagnosis by ovarian and adrenal vein sampling and monitoring of treatment by peripheral blood sampling. Injection of a depot pure GnRH antagonist produced rapid and long-term complete suppression of ovarian steroidogenesis maintained over 10 months. Hence a depot pure GnRH antagonist can not only rapidly confirm the OHT diagnosis but also induce long-term remission of severe hyperandrogenism without surgery.

Optimal injection interval for testosterone undecanoate treatment of hypogonadal and transgender men.

OBJECTIVE: To define the optimized inter-injection interval of injectable testosterone undecanoate (TU) treatment for hypogonadal and transmen based on individual dose titration in routine clinical practice. DESIGN AND METHODS: A prolective observational study of consecutive TU injections in men undergoing testosterone replacement therapy for pathological hypogonadism or masculinization of female-to-male transgender (transmen) subject to individual dosing titration to achieve a stable replacement regimen. RESULTS: From 2006 to 2019, 6899 injections were given to 325 consecutive patients. After excluding the 6-week loading dose, 6300 injections were given to 297 patients who had at least three and a median of 14 injections. The optimal injection interval (mean of last three injection intervals) had a median of 12.0 weeks (interquartile range 10.4-12.7 weeks). The interval was significantly influenced by age and body size (body surface area, BSA) but not by diagnosis or trough serum LH, FSH, and SHBG. Longer (≥14 weeks; 68/297, 23%), but not shorter (≤10 weeks; 22/297, 7.4%), intervals were weakly correlated with age but not diagnosis or other covariables. Low blood hemoglobin increased with trough serum testosterone to reach plateau once testosterone was about 10 nmol/L or higher. CONCLUSION: Optimal intervals between TU injection after individual titration resulted in the approved 12-week interval in 70% of patients with only minor influence for clinical application of BSA and not of trough serum LH, FSH, and SHBG. Individually optimized inter-injection interval did not differ between men with primary or secondary hypogonadism or transmen.

Rate and Extent of Recovery from Reproductive and Cardiac Dysfunction Due to Androgen Abuse in Men.

CONTEXT: Androgen abuse impairs male reproductive and cardiac function, but the rate, extent, and determinants of recovery are not understood. OBJECTIVE: To investigate recovery of male reproductive and cardiac function after ceasing androgen intake in current and past androgen abusers compared with healthy non-users. METHODS: Cross-sectional, observational study recruited via social media 41 current and 31 past users (≥3 months since last use, median 300 days since last use) with 21 healthy, eugonadal non-users. Each provided a history, examination, and serum and semen sample and underwent testicular ultrasound, body composition analysis, and cardiac function evaluation. RESULTS: Current abusers had suppressed reproductive function and impaired cardiac systolic function and lipoprotein parameters compared with non- or past users. Past users did not differ from non-users, suggesting full recovery of suppressed reproductive and cardiac functions after ceasing androgen abuse, other than residual reduced testicular volume. Mean time to recovery was faster for reproductive hormones (anti-Mullerian hormone [AMH], 7.3 months; luteinizing hormone [LH], 10.7 months) than for sperm variables (output, 14.1 months) whereas spermatogenesis (serum follicle-stimulating hormone [FSH], inhibin B, inhibin) took longer. The duration of androgen abuse was the only other variable associated with slower recovery of sperm output (but not hormones). CONCLUSION: Suppressed testicular and cardiac function due to androgen abuse is effectively fully reversible (apart from testis volume and serum sex hormone binding globulin) with recovery taking between 6 to 18 months after ceasing androgen intake with possible cumulative effects on spermatogenesis. Suppressed serum AMH, LH, and FSH represent convenient, useful, and underutilized markers of recovery from androgen abuse.

Testosterone for Androgen Deficiency-Like Symptoms in Men Without Pathologic Hypogonadism: A Randomized, Placebo-Controlled Cross-over With Masked Choice Extension Clinical Trial.

BACKGROUND: Off-label testosterone prescribing for androgen deficiency (AD)-like sexual and energy symptoms of older men without pathologic hypogonadism has increased dramatically without convincing evidence of efficacy. METHODS: In a randomized, double-blind, placebo-controlled study with three phases, we entered 45 men aged at least 40 years without pathologic hypogonadism but with AD-like energy and/or sexual symptoms to either daily testosterone or placebo gel treatment for 6 weeks in a cross-over study design with a third, mandatory extension phase in which participants chose which previous treatment they preferred to repeat while remaining masked to their original treatment. Primary endpoints were energy and sexual symptoms as assessed by a visual analog scale (Lead Symptom Score [LSS]). RESULTS: Increasing serum testosterone to the healthy young male range produced no significant benefit more than placebo for energy or sexual LSS. Covariate effects of age, body mass index, and pretreatment baseline serum testosterone on quality-of-life scales were detected. Only 1 out of 22 indices from seven quality-of-life scales was significantly improved by testosterone treatment over placebo. Participants did not choose testosterone significantly more than placebo as their preferred treatment in the third phase. CONCLUSIONS: Six-week testosterone treatment does not improve energy or sexual symptoms more than placebo in symptomatic men without pathologic hypogonadism.

Pharmacokinetics and Acceptability of Subcutaneous Injection of Testosterone Undecanoate.

CONTEXT: Can injectable testosterone undecanoate (TU) be administered effectively and acceptably by the subcutaneous (SC) route? OBJECTIVE: To investigate the acceptability and pharmacokinetics (PK) of SC injection of TU. DESIGN: Randomized sequence, crossover clinical study of SC vs IM TU injections. SETTING: Ambulatory clinic of an academic andrology center. PARTICIPANTS: Twenty men (11 hypogonadal, 9 transgender men) who were long-term users of TU. injections. Intervention: Injection of 1000 mg TU (in 4 mL castor oil vehicle) by SC or IM route. Main Outcome Measures: Patient-reported pain, acceptability, and preference scales. PK by measurement of serum testosterone, dihydrotestosterone (DHT), and estradiol (E2) concentrations with application of population PK methods and dried blood spot (DBS) sampling. RESULTS: Pain was greater after SC compared with IM injection 24 hours (but not immediately) after injection but both routes were equally acceptable. Ultimately 11 preferred IM, 6 preferred SC, and 3 had no preference. The DBS-based PK analysis of serum testosterone revealed a later time of peak testosterone concentration after SC vs IM injection (8.0 vs 3.3 days) but no significant route differences in model-predicted peak testosterone concentration (8.4 vs 9.6 ng/mL) or mean resident time (183 vs 110 days). The PK of venous serum testosterone, DHT, and E2 did not differ according to route of injection. CONCLUSIONS: We conclude that SC TU injection is acceptable but produces greater pain 24 hours after injection that may contribute to the overall majority preference for the IM injection. The PK of testosterone, DHT, or E2 did not differ substantially between SC and IM routes. Hence whereas further studies are required, the SC route represents an alternative to IM injections without a need to change dose for men for whom IM injection is not desired or recommended.

Sperm cryopreservation prior to gonadotoxic treatment: experience of a single academic centre over 4 decades.

STUDY QUESTION: What is the natural history of outcomes of sperm cryostorage at an Australian tertiary academic centre? SUMMARY ANSWER: Cryostorage is feasible in virtually all men facing gonadotoxic therapy but the timing of sperm disposal varies according to the reason for it. WHAT IS KNOWN ALREADY: Gonadotoxic treatment for cancer or non-cancer diseases damages spermatogenesis and impairs male fertility. Sperm cryopreservation is an established technique to preserve male fertility prior to gonadotoxic treatment. STUDY DESIGN, SIZE, DURATION: A retrospective review of clinical, anthropometric, semen analysis and hormonal data from 1978 to 2017 involving 2717 men comprising 2085 men with cancer, 234 non-cancer disease and 398 healthy controls, in a single tertiary academic centre with the same clinic and laboratory staff. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Sperm output was analysed according to diseases, the feasibility of sperm cryostorage notably for adolescents, regional access to an urban cryostorage facility, the determinants of sperm output and time-dependent disposal of cryostored sperm. Semen samples were assessed by contemporaneous WHO methods. MAIN RESULTS AND THE ROLE OF CHANCE: Of 2085 men with cancer, 904 (43%) had haematological malignancies, 680 (33%) testicular cancers and 136 (6.5%) were adolescents. Most men (89%) and adolescents (80%) could collect sperm. Sperm output for all cancers and non-cancer diseases was lower than controls. Sperm output correlated positively with total testicular volume (r = 0.44, P < 0.0001) and negatively with serum FSH and LH (r = -0.24, -0.12, respectively, both P < 0.0001) but not testosterone. For all stored samples, the median time in cryostorage was 8.5 years, 7% were transferred for use to induce pregnancy (median time 2.5 years) and 62.2% were discarded as no longer needed (return of fertility, 35.9% median 3.5 years; death, 26.3%, median 6.5 years), the high disposal rate reflecting regular annual follow-up to establish ongoing need for continued cryostorage. Cryostorage facilities are not available in remote and rural areas of the State and the proportion of outer regional and remote area residents cryostoring sperm was only about half that compared with urban residents. LIMITATIONS, REASONS FOR CAUTION: This study does not report the pregnancy outcomes of the patients who used the cryostored sperm, due to recent limitations on health data privacy. WIDER IMPLICATIONS OF THE FINDINGS: Sperm cryostorage is feasible for virtually all men, including sufficiently mature adolescents, who can collect semen to insure future paternity as well as making positive psychological preparation for the patient's survival. Disposal of cryostored material when no longer required is efficient with regular follow-up. Sperm cryopreservation should be an integral part of comprehensive treatment plan in men receiving gonadotoxic treatment but remains underutilized. STUDY FUNDING/COMPETING INTEREST(S): There was no external funding for this study and there were no relevant conflicts of interest.

Urine and Serum Sex Steroid Profile in Testosterone-Treated Transgender and Hypogonadal and Healthy Control Men.

Background: The impact of testosterone (T) treatment on antidoping detection tests in female-to-male (F2M) transgender men is unknown. We investigated urine and serum sex steroid and luteinizing hormone (LH) profiles in T-treated F2M men to determine whether and, if so, how they differed from hypogonadal and healthy control men. Method: Healthy transgender (n = 23) and hypogonadal (n = 24) men aged 18 to 50 years treated with 1000 mg injectable T undecanoate provided trough urine and blood samples and an additional earlier postinjection sample (n = 21). Healthy control men (n = 20) provided a single blood and urine sample. Steroids were measured by mass spectrometry-based methods in urine and serum, LH by immunoassay, and uridine 5'-diphospho-glucuronosyltransferase 2B17 genotype by polymerase chain reaction. Results: Urine LH, human chorionic gonadotropin, T, epitestosterone (EpiT), androsterone (A), etiocholanolone (Etio), A/Etio ratio, dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT), and 5α,3α- and 5ß,3α-androstanediols did not differ between groups or by time since last T injection. Urine T/EpiT ratio was <4 in all controls and 12/68 (18%) samples from T-treated men, but there was no difference between T-treated groups. Serum estradiol, estrone, and DHEA were higher in transgender men, and serum T and DHT were higher in earlier compared with trough blood samples, but serum LH, follicle-stimulating hormone, and 3α- and 3ß,5α-diols did not differ between groups. Conclusion: Urine antidoping detection tests in T-treated transgender men can be interpreted like those of T-treated hypogonadal men and are unaffected by time since last T dose. Serum steroids are more sensitive to detect exogenous T administration early but not later after the last T dose.