Recent studies demonstrate that combinations of androgens and progestagens are highly effective in the suppression of spermatogenesis in normal volunteers. To test whether progestagen and androgen delivery systems designed to produce steady serum levels will be as effective as other androgen plus progestagen combinations, we compared Norplant II and testosterone (T) transdermal patch to T patch alone on the suppression of spermatogenesis in normal men. Thirty-nine healthy male volunteers (age, 20–45 yr) were randomly assigned to one of two groups. Group 1 (n = 19) received two transdermal T patches daily (Testoderm TTS, each patch designed to deliver about 5 mg/d T) alone, and group 2 (n = 20) received combined Norplant II Jadelle, four capsules delivering ∼160 μg/d levonorgestrel (LNG) plus T patch. Neither of these regimens were very effective, with suppression of spermatogenesis to severe oligozoospermia occurring in less than 60% of subjects.
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We then expanded the study to include two more groups to determine whether T patch or Norplant II was the main factor causing the inadequate suppression of spermatogenesis. Another 29 subjects were randomized to one of two groups. Group 3 (n = 15) received oral LNG (125 μg/d) plus T patch, and group 4 (n = 14) received Norplant II plus T enanthate (TE) injection (100 mg/wk im). After a pretreatment phase of 4 wk, all subjects received treatment for 24 wk, followed by a recovery period of 12–24 wk. Steady-state serum LNG levels (800–1200 pmol/liter) were achieved from wk 3–24 after Norplant II insertion and decreased rapidly after the removal of the implants at wk 24. Trough serum LNG levels after oral LNG administration were at a comparable range (940–1300 pmol/liter). Azoospermia was achieved in 24%, 35%, 33%, and 93%, and severe oligozoospermia (.
THE RESULTS OF multicenter clinical trials supported by the World Health Organization (WHO)/Contraceptive Research and Development (CONRAD) program have led to a renewed and energized interest in steroid base contraception for men (, ). In these studies, weekly im injections of testosterone enanthate (TE) in supraphysiological doses (200 mg/wk) resulting in serum T concentrations at or above the upper limit of the normal range induced azoospermia in 60–70% and oligozoospermia (20 × 10 6/ml; motility, 50%; and oval forms, 15%). The different ethnic groups were not represented equally in all treatment groups. Ethnic differences in responses were not tested statistically because the numbers in the nonwhite groups were too small to provide sufficient power for reliable analyses. Of the 68 subjects enrolled, 60 completed the study.
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Eight subjects did not complete the study (three from T patch group, one from Norplant II plus T patch group, and four from oral LNG plus T patch groups). Six subjects voluntarily withdrew due to inability to comply with the strict protocol schedule or moving away from the area; one discontinued because of severe depression due to preexisting disease not disclosed on screening; and another discontinued because of skin reactions to the T patch. Study design and medicationsAfter a pretreatment period of 4 wk, subjects were initially randomized into one of two groups. Group 1 (n = 19) applied T transdermal patches alone. Placebo Norplant II was not used or justified because this would require surgical procedures for insertion as well as removal. Group 2 (n = 20) received transdermal T patches plus Norplant II, two systems, i.e.
Four capsules for 24 wk. The subsequent 29 subjects were randomized into two additional groups. Group 3 (n = 15) received oral LNG (125 μg /d) plus T patches. Group 4 (n = 14) received Norplant II, two systems plus TE injections (100 mg/wk).The nonscrotal transdermal T patches (Testoderm TTS) were a gift from Alza Corp. (Mountain View, CA) obtained through Linda Atkinson, Ph.D. Each patch is 60 cm 2 and is designed to deliver 5 mg/d T. Two transdermal patches (delivering ∼10 mg/d T) were applied daily to the upper or lower back, shoulders, upper arms, or buttocks depending on the subjects’ preferences.
The Norplant II system (Jadelle, Leiras Pharmaceuticals, Turku, Finland) was provided by Harold Nash, Ph.D., from the Population Council (New York, NY). Each Norplant II system consists of two capsules. Each capsule is a 43-mm rod releasing 36–49 μg LNG/implant per day from a cured homogeneous mixture of LNG (about 75 mg per capsule) and a polydiniethyl siloxane elastomer covered by a thin-walled silicone rubber tubing. Four capsules (two systems) were implanted and removed by one of the investigators (A.N.) who has extensive experience with the Norplant systems. The capsules were implanted in the sc tissue of the inner upper third of the arm under local anesthesia at Harbor-University of California Los Angeles (UCLA) Medical Center General Clinical Research Center (GCRC). At the end of 24 wk of treatment, Norplant II capsules were removed under local anesthesia. LNG was obtained from Wyeth-Ayerst Laboratories, Inc.
(Philadelphia, PA), through Michael Gast, M.D. The oral LNG was formulated into capsules at the University of Washington (Seattle, WA), through Alvin Matsumoto, M.D. Each capsule contained 125 μg LNG. Subjects took one capsule of LNG orally daily. TE 200 mg/ml in 5-ml vials distributed by Bio-Technology General Corp. (Delatestryl, Iselin, NJ) was obtained through our hospital pharmacy. Subjects received TE 100 mg im on d 1 and continued at weekly intervals.
All TE injections were administered by medical personnel with the exception of a few subjects who lived far away from the study center. These subjects were given instructions on self-injections. All subjects underwent treatment for 24 wk and then completed a recovery period of 3–6 months until return of two consecutive sperm concentrations to the subject’s average pretreatment level or more than 20 × 10 6/ml. This study was approved by the Institutional Review Board of the Harbor-UCLA Research and Education Institute.
All study subjects gave informed written consent.All subjects had complete physical exams and interviews done by a physician at screening and wk 6, 12, 24, 36, and 48. Semen analysis was performed every 3 wk on samples obtained by masturbation after at least 48 h of abstinence during the 24-wk treatment period and monthly during the recovery phase until sperm count returned to baseline levels. Blood samples were obtained before administration of T patch, TE injection, or oral LNG every 3 wk for serum T, free T, SHBG, FSH, and LH measurements. Serum LNG levels were also measured every 3 wk until wk 28 in subjects with Norplant II implants. In group 2, eight subjects had delayed application of transdermal T patch until wk 3 to allow for the measurement of serum levels of LNG without the possible interference by the T administration.
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In these subjects, serum LNG levels were measured at 1, 2, 4, and 8 h, and on days 1, 2, 3, 4, 5, 7, 10, 14, and 21 after Norplant II insertion, and days 1, 2, 3, and 7 after Norplant II removal. In addition, serum hormone levels for LH, FSH, and SHBG were drawn on d 2, 3, 4, 5, 7, 10, and 14 after Norplant II insertion. In another seven subjects receiving oral LNG and T patches, additional serum LNG levels were measured before and at 0.5, 1, 2, 4, 8, and 24 h after the oral dose of LNG on wk 12. Fasting safety laboratory tests including serum lipid profile and prostate-specific antigen (PSA) levels were drawn at screening and at wk 12, 24, and 36.
Subjects completed a 7-d sexual diary before treatment, every 6 wk during the treatment phase, and at posttreatment wk 36 and 48. MethodsSerum LNG was measured by RIA after extraction with hexane and ethyl acetate using reagents obtained through Saulat Sufi, Ph.D., from the WHO Collaborating Center (London, UK). The lower limit of quantitation (LOQ) of LNG in serum measured by this assay is 125 pmol/liter. The intra-assay and interassay coefficients were 6.2% and 8.1%, respectively.
Serum T levels were measured after extraction with ethyl acetate and hexane by a specific RIA using reagents from ICN Biochemicals, Inc. (Costa Mesa, CA). The LOQ of serum T measured by this assay was 0.87 nmol/liter. All results below this value were reported as 0.87 nmol/liter.
The mean accuracy (recovery) of the T assay, determined by spiking steroid free serum with varying amounts of T (0.9–52 nmol/liter), was 104% (range, 92–117%). The intra- and interassay coefficients of the T assay were 7.3% and 11.1%, respectively, at the normal adult male range, which in our laboratory were 10.33–36.17 nmol/liter (298–1043 ng/dl), respectively. Serum free T was measured by RIA in the dialysate after equilibrium dialysis using reagents provided by ICN Biochemicals, Inc./Nichols (San Juan Capistrano, CA). The LOQ for this assay is 0.03 nmol/liter. The intra- and interassay coefficients were 12.9% and 21.5%, respectively. The normal range was 0.12–0.62 nmol/liter. Serum FSH and LH were measured by the highly sensitive and specific fluoroimmunometric assays with reagents provided by Delfia (Wallace, Gaithersburg, MD).
The intra-assay coefficients of variation for LH and FSH were 4.3% and 5.2%, respectively; and the interassay variations for LH and FSH are 11.0% and 12.0%, respectively (adult normal male range, LH, 1.0–8.1 IU/liter; FSH, 1.0–6.9 IU/liter). For both LH and FSH assays, the LOQ is determined to be 0.2 IU/liter.
SHBGs were measured by fluoroimmunometric assay with reagents provided by Delfia. The LOQ is determined to be 0.5 nmol/liter.
The intra- and interassay coefficients were 2.3% and 6.1%, respectively. The normal range for this assay is 10.8–46.6 nmol/liter. All samples from a subject were measured in an assay to minimize interassay variability. When results were below the LOQ, the LOQ value was reported and used in data analysis.Semen samples were collected by masturbation after 48–72 h of abstinence into sterile plastic containers and were analyzed after liquefaction. Semen analyses were performed according to the recommended methods described in the WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. Sperm concentration was assessed using the hemocytometer method, and visual assessment was used for motility and morphology.Sexual function and mood were assessed by questionnaires that the subjects answered daily for 7 consecutive days before clinic visits. The subjects recorded whether they had sexual daydreams, anticipation of sex, flirting, sexual interaction (sexual motivation parameters) and orgasm, erection, masturbation, ejaculation, intercourse (sexual performance parameters) on each of 7 d.
The value was recorded as 0 (none) or 1 (any) for analyses, and the number of days the subjects noted a parameter was summed for the 7-d period. The average of the four sexual motivation parameters was taken as the sexual motivation score, and that of the five sexual performance parameters was taken as the sexual performance mean score (0–7). The subjects also assessed their level of sexual desire, sexual enjoyment, and satisfaction of erection using a seven-point Likert-type scale (0–7) and the percentage of full erection from 0–100%. The subjects rated their mood using a 0–7 score. The parameters assessed included the following positive mood responses: alert, friendly, full of energy, and well/good feelings; and negative mood responses: angry, irritable, sad, tired, and nervous. Weekly average scores were calculated.
The details of this questionnaire had been described previously (, ). T patch compliance was assessed by asking the subjects to bring back both used and unused patches for counting. LNG pills were also assessed by pill counting at each visit. Statistical analysisDescriptive statistics, either means and sd values or frequency distributions, were calculated for each variable at each time period. Outcome variables such as semen parameters, FSH, LH, T, free T, and SHBG levels were analyzed using ANOVA. One-way ANOVA was used for comparison of groups on baseline measures or change at 24 wk. Pairwise comparisons were made using the Student-Newman-Keuls test.
Repeated measures ANOVA was used to analyze the longitudinal course of the outcome variables. Time in treatment (including control values) was included as the repeated (within subject) factor in the ANOVA and treatment, as between the subject factors. Differential effects of different treatments were assessed by testing for significant interactions between time and treatment. Comparisons of baseline to final values within groups during treatment were made using paired t tests. Comparisons of rates of azoo- or oligozoospermia in two or four groups were made using Fisher Exact test. Type 1 error was set at 0.05, and actual P values were reported to provide further information on the strength of the finding. The results are represented as mean ± sem.
ResultsBaseline clinical and biochemical characteristics of the 68 subjects at the time of randomization are shown in. The parameters were all within the normal range and not significantly different among the four groups. Sperm concentrationAnalysis of the primary outcomes (semen parameters) comparing subjects with delayed T patch application (wk 3) to those who began T patch at the start of treatment phase (d 1) in the Norplant II plus T patch group showed no significant differences; therefore, they were included as a single group in the subsequent analyses of the semen parameters.shows the sperm concentration of the subjects in logarithmic scale. Spermatogenesis was least suppressed in the T patch alone group (50% of baseline concentration) and most marked in the Norplant II plus TE group (7–11% of baseline), whereas the T patch plus Norplant II (21–29% of baseline) and T patch plus oral LNG (28–38% of baseline) were in between. Sperm motility decreased in all groups and followed the same pattern as that of sperm concentration (data not shown). Mean sperm motility (in the subjects with spermatozoa in the ejaculate) in the Norplant II plus TE group were suppressed to less than 10% from wk 15 onward.
Recovery of sperm concentration and motility was complete by 16 wk after withdrawal of treatment. Mean (± sem) sperm concentrations in the four groups of subjects. In this and subsequent figures, the groups are represented by ⋄, T patch only; □, Norplant II plus T patch; ▴, oral LNG plus T patch; and., Norplant II plus TE injections. Pre TX, Pretreatment.shows that the Norplant II plus TE group achieved significantly greater suppression of spermatogenesis in more subjects compared with the other three groups (four group comparison, P = 0.0001). Severe oligozoospermia (.
Percentage of subjects achieving azoospermia ( black bars) and sperm count 3 × 10 6m/liter ( open bars) during treatment and recovery. Serum LNG levelSerum LNG levels reached a peak (1900 ± 97 pmol/liter) 4 d after Norplant capsule insertion (, right top inset).
Thereafter, the levels fell slightly, and steady-state serum LNG levels ranging between 800 and 1200 pmol/liter were attained throughout the treatment period. Serum LNG levels returned to baseline levels 3 d after the Norplant capsules were removed (data not shown). In the oral LNG plus T patch group, throughout the treatment period, mean trough serum LNG levels were maintained between 940 and 1300 pmol/liter. At wk 12, the serum LNG level was 1080 ± 201 pmol/liter before oral LNG administration. Serum LNG levels reached a peak of 1907 ± 372 pmol/liter 30 min after oral administration, and levels were then maintained between 1000 and 1300 pmol/liter until the next dose at 24 h (, right bottom inset). Serum LNG levels during treatment and recovery phase.
The right top inset shows LNG levels during the 21 d after Norplant II insertion in the eight subjects with delayed T patch application. The right bottom inset shows serum LNG levels in four subjects at wk 12 of treatment with oral LNG 125 μg/d and T patch (0 h represents the trough level before the next dose). Serum LH and FSH levelsBoth serum LH and FSH levels were significantly suppressed in all groups during the treatment period compared with baseline ( P = 0.0001; ). In the eight subjects with delayed T patch application, mean serum LH and FSH levels were suppressed by Norplant II alone (wk 3, serum LH, 2.92 ± 0.78 IU/liter; serum FSH, 1.69 ± 0.35 IU/liter) but were further suppressed when T patch was applied (wk 6, serum LH, 1.65 ± 0.58 IU/liter; serum FSH, 0.75 ± 0.25 IU/liter;, insets).
Serum FSH levels were significantly more suppressed in the Norplant plus T patch group compared with T patch only group ( P = 0.0139;, top panel). In the Norplant II plus TE group, mean serum FSH was suppressed to less than 0.5 IU/liter from wk 3–24. In many subjects, the serum FSH levels were below the LOQ. Mean serum LH levels were less than 0.5 1U/liter throughout the treatment period and were also suppressed to below the LOQ in the Norplant II plus TE group in most subjects. Suppression of LH was less marked and not to the LOQ in the groups treated with T patch alone, T patch plus Norplant II, or oral LNG. Both serum LH and FSH levels returned to baseline levels during the recovery period. Serum FSH ( top panel) and LH ( bottom panel) levels in four groups including all subjects.
In this and subsequent figures, the insets represent the levels measured in the eight subjects in the Norplant II plus T patch group in which application of the T patch was delayed until wk 3. Serum SHBGMean serum SHBG was not decreased in the T patch alone group but was suppressed to 58%, 69%, and 53% of baseline values in T patch plus Norplant II, T patch plus oral LNG, and Norplant II plus TE groups, respectively. To show that Norplant II suppressed SHBG levels even in the absence of androgens, in the eight subjects whose T patch application was delayed for 3 wk, mean serum SHBG was suppressed from 38.2 ± 2.9 nmol/liter at baseline to 29.3 ± 1.4, 23.4 ± 2.8, and 22.2 ± 2.1 nmol/liter at wk 1, 2, and 3, respectively, when the subjects were exposed to Norplant alone ( P = 0.0001) and not further suppressed (20.7 ± 2.3 nmol/liter at wk 6) after T patch was applied (, inset). Serum SHBG levels during pretreatment (Pre Tx), treatment, and recovery phases in the four groups of subjects. Serum total T and free T levelsSerum total and free T concentrations are shown in, top and bottom panels.
Serum total T levels remained within the adult normal range in all groups throughout the treatment period. In the T patch alone group, mean serum T levels were higher than baseline at wk 3, 6, and 9 ( P = 0.0070, 0.0223, and 0.0424, respectively). Mean serum total T levels were lower in all three groups with a progestagen component (, top panel).
The lower serum total T concentrations in the T patch plus Norplant II, T patch plus oral LNG, and Norplant II plus TE groups might be related to the decrease in SHBG levels resulting in lower SHBG-bound T fractions. Serum free T levels showed no significant change from baseline, remained within normal range, and were not different between the four groups throughout the treatment period (, bottom panel). Insets show the mean serum total T and free T levels in the eight subjects in the Norplant II plus T patch group who applied T patches at wk 3 during the treatment phase.
In these subjects with delayed T patch application, mean serum T and free T concentrations were significantly decreased at wk 3 ( P = 0.001 and 0.010, respectively) when compared with baseline, remained within the low normal range, and returned to their baseline values by wk 6 after T patch application. Serum total T ( top panel) and serum free T ( bottom panel) during pretreatment (Pre Tx), treatment, and recovery phases in the four groups of subjects. Sexual functionAnalyses of the psychosexual diaries collected for 7 d before clinic visits showed that the sexual motivation and performance scores as well as the positive (alert, friendly, full of energy, well/good feelings) and negative (angry, irritable, sad, tired, nervous) mood summary scores were not significantly changed with all treatment groups (data not shown). Body weight, testis volume, and safety parametersThere was no significant change in body weight in all four groups during and after treatment. The mean left or right testis weight decreased by 2–8 ml from baseline in the four groups. The mean decrease in testis volume was most marked in the Norplant II plus TE group at wk 24 (left testis volume decreased by 7.2 ± 1.1, and right by 8.9 ± 1.0 ml from baseline volumes).
None of the subjects appreciated the change in testis size. Total cholesterol and low-density lipoprotein (LDL) levels did not change significantly from baseline during treatment and recovery periods in all groups.
Serum HDL-C levels decreased by 4.9, 6.1, 4.8, and 12.3% from baseline in the T patch only, Norplant II plus T patch, oral LNG plus T patch, and Norplant II plus TE groups, respectively. The change was different among the four groups ( P = 0.0128), with Norplant II plus TE showing more change than the other groups. The decrease was also significantly different from baseline in the Norplant II plus T patch group ( P = 0.0048) and Norplant II plus TE group ( P = 0.0437). Hematologic profiles, blood chemistries, and PSA levels did not change significantly from baseline throughout the study period.
There was no clinically significant abnormality found on digital rectal prostate exam performed at baseline, end of the treatment period, and recovery. Adverse effectsMild asymptomatic gynecomastia (increased breast tissue within the areola region) was found in nine patients (seven in the Norplant plus T patch, two in the T patch group). Mild acne on the face or trunk occurred in 10 subjects during the treatment period (3 in the Norplant plus T patch, 4 in the T patch, 3 in the Norplant plus TE group), with spontaneous resolution occurring during treatment period or after treatment withdrawal.
Mild dermal irritation described as mild erythema on T patch application sites (upper buttocks and upper arms) occurred in six subjects and was reduced by changing or rotating T patch application sites. One subject developed generalized hives to the T patches after first application and was immediately terminated from the study. Two subjects reported mild increase in emotional irritability and anger (one in the Norplant plus TE, one in the oral LNG plus T patch) in the first few weeks of the study, which resolved requiring no intervention. Two subjects developed transient decrease in libido. There was one serious adverse event unrelated to the treatment. This subject, in the T patch only group, did not disclose his past psychiatric history of multiple depressive episodes at enrollment. He developed depression 12 wk into the treatment phase, attempted suicide, and was admitted to a psychiatric ward for observation.
He was discontinued from the study. DiscussionThis study has explored the effects of different T and progestagen (LNG) delivery systems on suppression of spermatogenesis in experimental male contraceptive regimens. We chose Norplant II as a depot to provide a relatively steady delivery of progestagen.
Placebo implants were not considered justified because of the requirement of minor surgical procedures for insertion and removal. Despite their ability to achieve steady serum T levels in this study, T pellets were not used as a T delivery system because of reports of significant extrusion rate and the requirement of another surgical procedure to insert the pellets at another site. As a less invasive alternative, we elected to use transdermal T patches at twice the dose recommended for androgen replacement in hypogonadal men. This dose was selected because a previous report found that a single transdermal T patch delivering 5 mg/d was not efficient in suppressing spermatogenesis even in combination with oral LNG.
The T patch alone group was necessary for comparison with other groups because the effect of T patches designed to deliver 10 mg/d transdermally on suppression of spermatogenesis had not been previously studied in normal volunteers. When the preliminary data in the initial two groups (T patch alone and T patch plus Norplant II) showed that these treatment regimens were unable to achieve marked suppression of spermatogenesis to the same degree as previous studies, we added two additional groups to address the question of whether the route or dose of the androgen (T patch) or progestagen (Norplant II) was the main cause for this failure.We showed in this study that Norplant II, two systems (four capsules), provided steady-state serum levels of LNG within 2 wk after insertion.
This concentration was maintained throughout the treatment period of 6 months. Serum LNG concentration then returned to LOQ levels 3 d after implant removal, indicating that significant accumulation is unlikely. The steady-state serum LNG levels achieved with Norplant II are equivalent to the trough serum LNG concentrations achieved after oral administration of 125 μg LNG and comparable to those from prior studies in women , suggesting similar bioavailability of Norplant II in both sexes. The four LNG implants (Norplant II) were well- tolerated in men. There were no problems with implant insertion or removal. Norplant II implants alone suppressed serum LH and FSH levels. Addition of T patches to Norplant II further suppressed serum gonadotropins, although not to the lowest levels possible.
We showed that Norplant II plus T patch achieved azoo- or oligozoospermia in 60% of men and was more effective than T patch alone (24% of men became azoo- or oligozoospermic), thus confirming the additive effect of progestagens to androgens in the suppression of spermatogenesis.
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