1 Contraceptive Development Network and 2 MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, University of Edinburgh, Edinburgh and 3 Drug Control Centre, King's College London, UK
4 To whom correspondence should be addressed at: MRC Human Reproductive Sciences Unit, The Chancellors Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK. Email: r.a.anderson{at}hrsu.mrc.ac.uk
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Abstract |
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Key words: etonogestrel/male contraception/progestogen/spermatogenesis/testosterone
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Introduction |
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Compared with oral administration, a long-acting drug delivery system has advantages, including dose-sparing and the avoidance of hepatic exposure to high doses, both of which may contribute to the reduction of unwanted adverse effects. Moreover, it may be preferred by some individuals because of ease of compliance (Martin et al., 2000a). Etonogestrel, the active metabolite of oral desogestrel, has been marketed recently in many countries as a long-acting implant (Implanon®, NV Organon, Oss, The Netherlands) providing 3 years of contraceptive efficacy in women. We have reported previously our experience with one or two etonogestrel implants in combination with depot testosterone pellets (Anderson et al., 2002
). Although profound suppression of spermatogenesis with minimal non-reproductive side effects was induced, azoospermia was achieved in only 64 and 75% of the one and two implant groups, respectively. Etonogestrel implants release
50 µg/day, thus even with two implants the daily dose is markedly lower than the optimally effective dose of 300 µg desogestrel, which has
80% oral bioavailability (Hasenack et al., 1986
). There was therefore evidence for significant dose-sparing with the implant preparation but, as spermatogenic suppression was not complete in all men, we hypothesized that the addition of a third etonogestrel implant may enhance this spermatogenic suppression. In this study, we additionally have extended the duration of treatment to 48 weeks to investigate whether the steady decline in etonogestrel release from the implants will maintain suppression of gonadotrophins and thus spermatogenesis for that length of time, using the same testosterone regimen we have used previously in the investigation of both oral desogestrel and etonogestrel implants.
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Methods |
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Study design and medication
This study was a single-group open investigation of the effects of etonogestrel implants with testosterone pellets. The duration of the treatment period was 48 weeks, with those subjects who were not azoospermic discontinuing treatment if they wished at 24 weeks. Following pre-treatment assessment, three implants each containing 68 mg etonogestrel (Implanon, NV Organon, Oss, The Netherlands) were inserted s.c. in the medial aspect of the non-dominant upper arm to all subjects. All subjects additionally received 400 mg testosterone pellets (2x200 mg, NV Organon) inserted s.c. under local anaesthetic into the anterior abdominal wall on the day of insertion of the etonogestrel implants, and 12 weekly thereafter for the duration of the treatment period, i.e. at 12, 24 and 36 weeks.
During treatment and recovery, subjects attended at 4 weekly intervals for medical review, and for semen analysis and venesection. Additional blood samples were drawn pre-treatment and at weeks 4 and 12 between 07.30 and 09.30 (a.m. samples) and between 16.30 and 18.30 (p.m. samples) for testosterone measurement. Subjects were examined at weeks 12, 24, 36, 48 and at final visits, and a morning first-void urine sample was obtained at the same time points for measurement of epitestosterone. Bio-electrical impedence was determined as described (Davies and Preece, 1988; Gregory et al., 1991
) using the Holtain Body Composition Analyser (Holtain Ltd, Dyfed, UK) and fat-free mass and percentage body fat determined for each subject at screening, 12 weekly thereafter and at 16 weeks of the recovery period. Throughout the study, any adverse events were noted at each visit. During the recovery phase, subjects attended at 4 weekly periods for a minimum of 16 weeks up to 24 weeks until semen analysis returned to normal by WHO criteria. Subjects with semen analysis below normal WHO criteria were followed-up beyond this period until normal values were attained.
Assays
Blood samples were obtained in fasting subjects (for glucose and lipids) and plasma separated by centrifugation at 4000 g for 15 min and stored at 20°C until hormone assay. Testosterone was measured by radioimmunoassay (Corker and Davidson, 1978), and LH, FSH and sex hormone-binding globulin (SHBG) by a time-resolved immunofluorometric in-house assay. Assay sensitivity was 0.3 nmol/l for testosterone, 0.5 nmol/l for SHBG, 0.03 IU/l for FSH and 0.15 IU/l for LH. The intra-assay coefficients of variation (CVs) were <10% for testosterone, FSH and LH, and 4% for SHBG. The inter-assay CVs were 12.4% for testosterone, <10% for FSH and LH, and 8.8% for SHBG. Free testosterone was calculated as described (Vermeulen et al., 1999
). Urinary epitestosterone concentrations (aglycone plus free fraction) were determined by gas chromatographymass spectrometry as described and validated previously (Kicman et al., 1995
; Coutts et al., 1997
). Between-assay precision was <8% for epitestosterone concentrations between 27 and 133 nmol/l, and 13.4% at 5 nmol/l. The assay sensitivity was 0.87 nmol/l. Inhibin B was measured in both serum and seminal plasma by methods previously described (Groome et al., 1996
; Anderson et al., 1998
) with an assay sensitivity of 7.8 pg/ml. Etonogestrel was measured by in-house radioimmunoassay by Organon NV, assay sensitivity 30 pg/ml. Intra-assay CV was 9% and inter assay CV was 14%. Samples were analysed for general haematological and biochemical values (including total cholesterol and HDL-C) by routine autoanalyser at 12 weekly intervals.
Semen analysis
At all assessments, semen analysis was carried out using WHO methodology (World Health Organization, 1999). Local normal values for motility are >27% grade a+b, or >36% grade a+b + c and normal morphology >15%. Azoospermia was confirmed following centrifugation of the whole semen sample. Centrifugation was performed at 3660 g for 15 min, and a sample was classified as azoospermic only after a systematic examination of the re-suspended precipitate indicated the complete absence of spermatozoa.
Behavioural assessment
Sexual activity and interest were investigated by means of a structured questionnaire used to quantify sexual activity over the preceding 2 week period (Anderson et al., 1992). This was carried out before treatment and at 12 weekly intervals thereafter.
Statistical analysis
Results are presented as mean±SEM. Hormone data were log transformed and semen concentrations cube root transformed before analysis by ANOVA (analysis of variance) for repeated measures. Paired t-tests were used to investigate at what time points significant treatment effects were evident, with the exception of behavioural data which were analysed using the Wilcoxon matched pair test for non-parametric testing. For all comparisons, a P-value of <0.05 was considered significant.
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Results |
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Testosterone and epitestosterone concentrations
Serum testosterone concentrations remained within the normal physiological range throughout the treatment period, with fluctuations according to the timing of testosterone pellet re-administration (Figure 2a). A gradual decline was observed from pre-treatment values reaching statistical significance at week 4 (P=0.0006) with a nadir at week 12. Following re-administration of testosterone at week 12, concentrations rose to levels that were not significantly different from baseline at week 16, with a similar pattern of fluctuation throughout the remainder of the treatment period. During the recovery phase, testosterone concentrations rapidly returned to pre-treatment concentrations. Calculated free testosterone concentrations showed a similar pattern, with nadir concentrations significantly lower than pre-treatment (P<0.01, Table II) and returning to pre-treatment levels during the recovery phase. During the treatment phase, free testosterone concentrations showed a gradual rise from week 12 (0.30±0.03 nmol/l) to week 48 (0.39±0.03 nmol/l), which was not statistically significant.
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A diurnal variation in serum testosterone concentrations was observed pre-treatment (Figure 3), concentrations in the morning being an average of 35% higher than in the early evening (P=0.002). After 4 weeks of treatment, this was lost, with no significant differences between morning and evening concentrations. Concentrations at both times of day at 4 weeks, however, were not significantly different from pre-treatment early evening concentrations. At 12 weeks of treatment, mean testosterone concentrations were low, this being immediately prior to re-administration of the testosterone pellets, but were again similar in the morning and evening. Comparison of the diurnal variation in testosterone concentrations between pre-treatment and 12 weeks showed a significant difference (P<0.05).
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Serum inhibin B concentrations showed a gradual decline over the course of treatment, continuing to week 48 (P<0.001; Figure 4). This reached statistical significance from week 4 of treatment onwards (P=0.047). By week 16 of the recovery phase, serum inhibin B levels showed only limited evidence of recovery, remaining significantly lower than pre-treatment (P<0.001).
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SHBG showed a gradual decline over the treatment period (Table II). This reached statistical significance by week 4 (P=0.0002) and continued to week 48. During recovery, SHBG returned to pre-treatment concentrations.
Etonogestrel
Serum etonogestrel concentrations were highest 4 weeks after implant insertion, with a mean concentration of 765±57 pg/ml. Etonogestrel concentrations showed a gradual decline thereafter (Figure 5), being 63% of peak levels at 24 weeks and 43% at week 48. Etonogestrel was undetectable in all subjects 4 weeks after implant removal. The individual who showed partial spermatogenic recovery had serum etonogestrel concentrations close to the group mean.
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A small rise in haemoglobin concentrations was evident at week 48 (P=0.003), which remained elevated during the recovery period. Haematocrit remained unchanged.
Body composition
There were no significant changes in body weight during the treatment or recovery periods. Likewise, body composition analysis showed no changes in fat free mass or percentage body fat (Table III).
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Discussion |
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The present data demonstrate profound suppression of spermatogenesis with the combination of three etonogestrel implants and depot testosterone pellets, with all subjects achieving azoospermia. This compares favourably with our previous data using one (64% azoospermia) and two implants (75% azoospermia) over a 24 week period (Anderson et al., 2002) and is similar to that achieved with an oral dose of 300 µg desogestrel with the same regimen of testosterone administration (Kinniburgh et al., 2002
). Although sample sizes do not allow demonstration of statistically greater spermatogenic suppression with three than two etonogestrel implants, increased efficacy is supported by the more consistent suppression of gonadotrophins and of both serum and seminal inhibin B with three implants. The onset of suppression was rapid, with all subjects having sperm concentrations of <1x106/ml by week 16 of treatment. However, the time taken to reach azoospermia was considerably more variable, with three men maintaining very low but detectable numbers of sperm in the ejaculate to up to 28 weeks. Similar data are evident from the recent Australian efficacy study (Turner et al., 2003
) despite the very rapid suppression achieved by that combination of testosterone pellets and depot medroxyprogesterone acetate (DMPA), whereby 94% of men achieved a sperm concentration of <1x106/ml within 3 months. This may have significant implications for the practicality of the method, depending on the threshold required for acceptable contraceptive efficacy (Nieschlag, 2002
).
Serum etonogestrel concentrations of 1200 and 500800 pg/ml were reported for 300 and 150 µg oral desogestrel, respectively (Wu et al., 1999
; Anawalt et al., 2000
). In the present study, the serum etonogestrel concentration at 12 weeks was
600 pg/ml. Thus the suppressive effect of this preparation is similar to that of 300 µg desogestrel per day, whereas the dose is similar to 150 µg/day. Dose-sparing is also evident with this preparation of testosterone (Handelsman et al., 1992
), which maintains relatively stable serum concentrations and particularly avoids the supraphysiological peaks observed with esters such as testosterone enanthate (World Health Organization Task Force on Methods for the Regulation of Male Fertility, 1990
). The dose of testosterone administered here has no significant suppressive effect on spermatogenesis when given alone (Handelsman et al., 2000
), and in combination with a progestogen may be the minimum effective dose. The advantageous features of this testosterone preparation will contribute to minimizing the intratesticular testosterone concentration which is recognized to be of importance in maximizing spermatogenic suppression (Meriggiola et al., 2002
; Zhang et al., 2003
).
The diurnal variation of testosterone concentrations in adult men has been well characterized (Faiman and Winter, 1971; Bremner et al., 1983
) if not understood. The dose of testosterone which is physiological is usually considered to be that which reproduces the peak concentration observed in men during the morning (Nieschlag et al., 1992
). This may result in the administration of a higher dose than that required for physiological replacement. In this study, we carried out a preliminary investigation of diurnal variation in serum testosterone before and during testosterone/progestogen administration, which we hypothesized would not be detectable during exogenous steroid administration if it was primarily due to variation in testosterone production rather than metabolism (Southren et al., 1967
). The data confirmed that the diurnal variation of testosterone was lost during treatment, at both 4 and 12 weeks. Testosterone concentrations at 4 weeks were similar to pre-treatment evening samples; however, they are probably lower than average over the duration of treatment. While the regimen used here provides the standard replacement dose for hypogonadal men (800 mg every 6 months; Behre et al., 2004
), administration of half the total dose every 12 weeks will result in slight under-replacement over the initial 12 weeks, with steady state reached after the second administration. The average testosterone concentration following second administration was 15.5 nmol/l, which matches accurately the average 24 h concentration determined by frequent sampling in a group of young healthy men (Plymate et al., 1989
). This regimen may therefore closely replace testosterone production based on physiological diurnal production rather than morning peaks. The lack of changes in non-reproductive functions such as lipoproteins, haematocrit and body composition observed in this study is strong evidence that the dose administered here (
5 mg/day at steady state) provides close to physiological replacement, but this will need confirmation in longer studies assessing a wide range of androgen-dependent functions.
Gonadotrophin secretion was profoundly suppressed during treatment. This was particularly marked with LH. Suppression of FSH was more variable, but greater than with one or two implants (Anderson et al., 2002). The 12 week testosterone administration regimen also appears more effective at preventing FSH escape than the same total dose administered at 24 week intervals (Turner et al., 2003
). Desogestrel and other progestogens may result in greater spermatogenic suppression than achieved by comparable gonadotrophin suppression using testosterone alone (McLachlan et al., 2002
), consistent with direct testicular effects on steroidogenesis (Satyaswaroop and Gurpide, 1978
; El-Hefwany and Huhtaniemi, 1998
; El-Hefwany et al., 2000
) or androgen metabolism (Mauvais-Jarvis et al., 1974
). In the present study, FSH was incompletely suppressed during weeks 2448 in three subjects, only one of whom showed spermatogenic recovery. While adequate suppression of FSH is clearly necessary for achievement of azoospermia (Narula et al., 2001
; Weinbauer et al., 2001
), it appears that there is no clear threshold below which azoospermia can be confidently predicted, and that FSH suppression is only one of a number of potential determining factors for incomplete suppression or escape of spermatogenesis. Consistent with the reproducible suppression of LH, urinary excretion of epitestosterone fell to
10% of pre-treatment values and remained at that level for the duration of treatment. Epitestosterone (17
-hydroxyandrost-4-en-3-one) is a natural epimer of testosterone secreted predominantly by the testis (Kicman et al., 1999
) which therefore provides a measure of endogenous testicular secretion. Epitestosterone excretion during the present treatment regimen was similar to that previously reported during oral desogestrel/testosterone treatment of normal men (Kinniburgh et al., 2002
), and is significantly higher than in hypogonadal men (Kicman et al., 1999
). Direct measurement of intratesticular testosterone also indicates low ongoing testosterone production despite near complete LH suppression (McLachlan et al., 2002
).
The concentration of inhibin B provides an overall measure of Sertoli cell number and function including spermatogenesis (Anderson and Sharpe, 2000). While it would be expected that effective hormonal contraceptive regimens would result in significant falls in inhibin B concentrations, this has not always proved to be the case (Anawalt et al., 1996
; Anderson et al., 1997
; Zhengwei et al., 1998
; Büchter et al., 1999
; Martin et al., 2000b
; Kinniburgh et al., 2002
). We have not found significant changes in our previous studies with both oral desogestrel and etonogestrel implants despite the high prevalence of azoospermia. It is likely that changes in circulating inhibin B require profound regression of spermatogenesis more consistently throughout the testis than is achieved with some regimens (Anderson and Sharpe, 2000
). This is supported by testis biopsy data showing variable degrees of spermatogenic regression between nearby seminiferous tubules despite induction of azoospermia (Zhengwei et al., 1998
; McLachlan et al., 2002
). It is possible that the fall in serum inhibin B contributed to the less consistent suppression of FSH than LH during the latter months of this study. The fall in inhibin B observed in the present study may reflect a greater consistency of suppression than is reflected purely by the prevalence of azoospermia. This is supported by the striking fall in the concentration of inhibin B in the ejaculate. A more variable fall was found in our previous study with one or two etonogestrel implants (Anderson et al., 2002
). In the present study, we confirm and enlarge on this finding that changes in seminal inhibin B are a sensitive window into the seminiferous epithelium, as seminal inhibin B was profoundly suppressed in all men to a median of <10 pg/ml at 24 weeks treatment. This is supported by observations in the individual who demonstrated recovery of spermatogenesis during treatment, as the appearance of sperm in the ejaculate intriguingly was preceded by a partial recovery of seminal plasma inhibin B. Interestingly, both serum and seminal inhibin B showed only limited recovery over 16 weeks, while sperm concentrations had largely returned to normal, indicating complex relationships between these various markers of testicular function. Further investigation is required to establish the time scale for recovery of the endocrine function of the seminiferous epithelium following gonadotrophin suppression.
Other approaches using long-acting preparations have involved implants and depot injections. Levonorgestrel has also been administered in implant formulation (Norplant II®), with azoospermia achieved in 35% of subjects when given with transdermal testosterone patches and 93% of subjects in combination with weekly testosterone enanthate (Gao et al., 1999; Gaw Gonzalo et al., 2002
). The combination with testosterone implants or long-acting injectable preparations has yet to be investigated. 7
-Methyl-19-nortestosterone (MENT), a synthetic androgen more potent than testosterone and resistant to 5
-reduction (Sundaram et al., 1993
), has also been developed recently as an implant and a potential long-acting male contraceptive. However, even when up to four implants were used (a dose which resulted in significant effects related to excess androgenicity), 30% of men still had significant numbers of sperm in the ejaculate (von Eckardstein et al., 2003
) consistent with the limitations of an androgen-only approach in Caucasian men. Thus, of implant approaches to date, the combination presented in this study exhibits higher levels of spermatogenic suppression with a more favourable side effect profile than any of the others. This beneficial therapeutic ratio is likely to reflect the pharmacokinetics of both the testosterone and progestogen preparations. Other promising long-term approaches include long-acting injectable testosterone undecanoate alone, achieving high levels of oligozoospermia and azoospermia among Chinese men (Gu et al., 2003
), the depot injectable combination of norethisterone enanthate and testosterone undecanoate (Kamischke et al. 2002
), and DMPA with testosterone pellets (Turner et al., 2003
).
In conclusion, the results in this study demonstrate that administration of etonogestrel implants at an appropriate dose together with a long-acting testosterone preparation induces profound and consistent suppression of spermatogenesis that can be maintained for a period of 1 year. Whether this time period could be extended remains to be investigated. The maintenance of testosterone concentrations within the eugonadal range and the dose-sparing effects of the delivery methods involving constant release may contribute to the lack of non-reproductive effects. This approach may be a template as the basis for an acceptable, long-acting, and reversible male hormonal contraceptive.
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Acknowledgements |
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Submitted on May 12, 2004; accepted on August 2, 2004.