Dose-finding study of oral desogestrel with testosterone pellets for suppression of the pituitary–testicular axis in normal men*

Cameron W. Martin1, Simon C. Riley1, Dawn Everington1, Nigel P. Groome2, Rudolph A. Riemersma3, David T. Baird1 and Richard A. Anderson4,5

1 Department of Obstetrics and Gynaecology, Centre for Reproductive Biology, University of Edinburgh, 2 School of Biological and Molecular Sciences, Oxford Brookes University, Oxford, 3 Cardiovascular Research Unit, University of Edinburgh and 4 MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, University of Edinburgh, Edinburgh, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prototype hormonal male contraceptive regimens generally achieve only incomplete suppression to azoospermia with potentially adverse metabolic effects. We have carried out a short-term dose-finding study to investigate the potential of an oral gestogen, desogestrel, with testosterone pellets. Normal men received a single dose of 300 mg testosterone with 75 µg, 150 µg or 300 µg desogestrel daily for 8 weeks (n = 10 per group). LH and FSH were rapidly suppressed, with little difference between groups. Testosterone concentrations fell slightly during treatment with evidence of a linear dosage effect. Plasma inhibin B showed minor changes, but in seminal plasma it was suppressed, becoming undetectable in all men in the 300 µg desogestrel group. There were no significant changes in lipoproteins, fibrinogen or sexual behaviour during treatment, and minor falls in haematocrit and haemoglobin concentration. Sperm concentration fell in a dose-dependent manner, with three men, one man and seven men in the three groups respectively achieving severe oligozoospermia (<3x106/ml), and three men achieving azoospermia in the 300 µg group despite the short duration of the study. The combination of oral desogestrel with depot testosterone thus results in profound suppression of gonadotrophin secretion without adverse metabolic or behavioural effects. Desogestrel with a long-acting testosterone preparation is a promising approach to hormonal male contraception.

Key words: androgen/desogestrel/male contraception/testis/testosterone


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hormonal male contraceptives must suppress gonadotrophin secretion with consequent inhibition of spermatogenesis. This can be achieved using an androgenic steroid alone or in combination with a second, progestogenic steroid or gonadotrophin releasing-hormone (GnRH) agonist or antagonist (Patanelli, 1977Go; WHO, 1990 WHO, 1996; Pavlou et al., 1991Go; Behre et al., 1992Go; Meriggiola and Bremner, 1997Go; Swerdloff et al., 1998Go). The degree of spermatogenic suppression achieved when various steroid regimens are used is variable and often incomplete. While suppression to azoospermia provides reliable contraception (WHO, 1990), residual low rates of spermatogenesis are associated with significant pregnancy rates (WHO, 1996). Furthermore, `contraceptive' doses of testosterone alone or in combination with progestogens such as levonorgestrel have significant adverse metabolic effects, including a reduction of high density lipoprotein cholesterol (HDL-C) concentration, increases in haematocrit values and changes in body composition (Bagatell et al., 1994aGo; Anderson et al., 1995aGo,bGo; Bebb et al., 1996Go; Wu et al., 1996Go), which compromise their potential for further development. While administration of the progestogen/antiandrogen cyproterone acetate in combination with testosterone enanthate appears a very promising approach (Meriggiola et al., 1996Go), the goal of a hormonal male contraceptive to achieve universal azoospermia without significant metabolic effects remains elusive.

Suppression of gonadotrophin secretion by a progestogen or GnRH analogue results in a hypogonadal state which requires testosterone replacement to prevent the symptoms of androgen deficiency. Most available androgen preparations, such as testosterone enanthate, cause widely fluctuating and intermittently supraphysiological plasma testosterone concentrations (Behre et al., 1990Go; Anderson and Wu, 1996Go). This effect can be reduced or avoided using depot testosterone preparations such as fused crystalline testosterone pellets (Handelsman et al., 1992Go; Jockenhövel et al., 1996Go). When given alone, testosterone pellets suppress spermatogenesis to a similar degree as testosterone injections, but without the supraphysiological plasma testosterone concentrations and related side effects (Handelsman et al., 1992Go). Alternative approaches such as GnRH antagonists with low-dose androgen replacement indicate that androgen-dependent behaviour can be maintained without adverse changes in lipid profile (Pavlou et al., 1991Go). The pharmaceutical development of such agents has been slow and problematical, although preparations which can be given less frequently are becoming available (Behre et al., 1997Go). Progestogens have several advantages, including a long record of experimental and clinical usage and a wide variety of available preparations. While the more androgenic progestogens such as levonorgestrel may reduce the metabolic benefit of the lower dose of testosterone needed (Bebb et al., 1996Go), the `third-generation' gestogens, such as the orally active desogestrel and gestodene, are characterized by potent progestogenicity with low androgenicity (Fotherby and Caldwell, 1994Go; Darney, 1995Go; Kuhl, 1996Go). Reduced androgenicity may be of value even when administered to men, as it may reduce the androgenic effect of the hepatic first pass. Desogestrel is converted to the active metabolite 3-ketodesogestrel (etonogestrel) by the liver. When administered alone to men, desogestrel results in significant gonadotrophin and testosterone suppression, and although a preliminary report showed no changes in lipid metabolism at doses up to 450 µg (Bellis et al., 1996Go), a reduction in plasma total cholesterol, HDL-C and low density lipoprotein cholesterol (LDL-C) concentrations has been recently reported (Wu et al., 1999Go). Depot administration of the progestogen medroxyprogesterone acetate has also been demonstrated to enhance the suppression of spermatogenesis by testosterone pellets without suppression of HDL-C (Handelsman et al., 1996Go). An oral formulation is, however, the first choice for a hormonal male contraceptive among those men in Edinburgh in whom we have recently carried out a survey (Martin et al., 2000Go). We have therefore performed a dose-finding study of oral desogestrel with testosterone implants at a dose sufficient to provide androgen replacement at the lower limit of the physiological range. This allows for some potential androgenic effect of desogestrel, without overall androgen concentrations being elevated. The primary objectives were to investigate whether this combination would result in sufficient gonadotrophin suppression to merit further investigation as a potential male contraceptive, while avoiding adverse metabolic and behavioural effects.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects and treatment protocol
Thirty-one healthy Caucasian men aged 23 to 42 years (mean 33 years) were recruited from the general population. Inclusion criteria included a normal medical history and physical examination, biochemical (including total cholesterol, HDL-C and LDL-C) and haematological analyses within the normal range, and pre-treatment semen samples with normal values (WHO, 1992) on two occasions at least 2 weeks apart. Subjects were allocated at random into three treatment groups, taking either 75 µg, 150 µg or 300 µg desogestrel (NV Organon, Oss, The Netherlands) orally daily. In addition, all subjects received 300 mg fused crystalline testosterone implants (one 200 mg plus one 100 mg pellet; Organon) s.c. into the lower abdominal wall under local anaesthetic on day 1 of desogestrel treatment. After 8 weeks the subjects discontinued the desogestrel, thus entering the recovery phase which continued for 4 weeks. Subjects were reviewed and examined at 1, 2, 4, 6 and 8 weeks during the treatment phase, and at 2 and 4 weeks in the recovery phase. Each subject gave informed written consent, and this study received ethical approval from the Lothian Reproductive Medicine Ethical Review Committee. The study was performed to Good Clinical Practice standards with full drug accountability.

Blood sampling and hormone assays
Blood samples were obtained between 07:30 and 11:00 h on two occasions at least 2 weeks apart before treatment, and after 1, 2, 4, 6 and 8 weeks of treatment. Further samples were obtained 2 and 4 weeks after discontinuing desogestrel. Blood samples were centrifuged to separate the plasma, which was removed and stored at –20°C until hormone assay.

Total testosterone and oestradiol were measured by radioimmunoassay, as described previously (Corker and Davidson, 1978Go; Glasier et al., 1989Go) in single assays. Intra-assay coefficients of variation were 6.4 and 7.1% respectively. Plasma LH and FSH were measured by time-resolved immunofluorometric assay (Delfia, Wallac, Turku, Finland) and by highly sensitive immunoradiometric assay respectively (Netria, St Bartholomew's Hospital, London, UK) respectively: assay sensitivity was 0.12 IU/l in both cases. Sex hormone-binding globulin (SHBG) was measured by immunoradiometric assay (DPC, Los Angeles, CA, USA).

Inhibin B in blood and seminal plasma and inhibin forms containing pro and {alpha}C (pro-{alpha}C) immunoreactivity were measured as previously described (Groome et al., 1995Go, 1996Go; Anderson et al., 1998Go). Briefly, the inhibin B ELISA uses a monoclonal capture antibody (C5) raised against the human inhibin ßB subunit, partially immunopurified inhibin from human follicular fluid calibrated against recombinant 32 kDa human inhibin B (Genentech, San Francisco, CA, USA) as standard, and detection using a mouse monoclonal antibody (R1; produced in-house) raised against the inhibin {alpha}C-subunit conjugated to alkaline phosphatase, using an alkaline phosphatase amplification kit (Life Technologies, Paisley, UK). The detection limit of the assay was 15 pg/ml. The intra- and inter-plate coefficients of variation were 7% and 10% respectively. Recovery of inhibin B standard from seminal plasma has been demonstrated previously to be quantitative, and serial dilution of seminal plasma gave a dose–response parallel to that of the standard (Anderson et al., 1998Go). Inhibin isoforms containing pro-{alpha}C immunoreactivity were also detected by ELISA, using a highly purified preparation of pro-{alpha}C as standard with a monoclonal capture antibody (INPRO; produced in-house) directed against a sequence of the pro-{alpha}C {alpha}-subunit. The same detection antibody (R1) was used as in the dimeric inhibin B assay, and alkaline phosphatase activity was measured using p-nitrophenylphosphate (pNPP) as substrate. The detection limit was 3 pg/ml, and intra- and inter-plate coefficients of variation were 6% and 8%.

HDL-C was measured after precipitation with dextran sulphate-magnesium, and total cholesterol and triglycerides (TG) were measured enzymatically (Wood et al., 1987Go). LDL-C was calculated indirectly (Friedewald et al., 1972Go). Haemoglobin, renal (urea and creatinine) and liver function tests (bilirubin, albumin, alkaline phosphatase and transaminases) were determined by routine autoanalyser. Fibrinogen was measured in citrated plasma using the Clauss technique.

Behavioural assessment
Subjects and their partners were both assessed for possible effects of treatment on behaviour. A structured interview was administered to each subject pre-treatment, after 4 and 8 weeks treatment, and 4 weeks after discontinuing desogestrel. This interview was modified from a previously described method (Cawood and Bancroft, 1996Go), and was used to derive frequency of sexual intercourse and masturbation over the preceding 2 weeks. At the same time points, subjects completed SES 2 (the psychosexual stimulation scale) and SES 3 (the sexual motivation scale) of the Frenken Sexual Experience Scales (Frenken and Vennix, 1981Go). This measuring instrument has a substantial body of normative data, and has been shown to have satisfactory psychometric properties. SES 2 refers to the extent that someone seeks or allows (rather than avoids or rejects) sexual stimuli of an auditory–visual or imaginary kind. A low score means high allowance of sexual stimuli or higher psychosexual arousability. SES 3 refers to sexual interaction with one's partner. A low score means a strong approach tendency or `sexual appetite'. Weighted scores were used; this gives a mean score of zero for normative data as obtained by Frenken and Vennix. We have previously used both the structured interview and SES scales in the assessment of hormonal effects on mood and sexuality in normal and hypogonadal men (Anderson et al., 1992Go, 1999Go).

Subjects' partners were given a questionnaire after 8 weeks treatment. The questionnaire requested the partner's opinion as to whether there had been any changes in eight mood states (`cheerful', `tense', `relaxed', `ready to fight', `unhappy', `irritable', `energetic', and `interest in sex') during the treatment phase. Data are presented as the number of responses of `increase', `decrease', or `no change' for each mood state.

Semen samples
Semen samples were submitted after 3–7 days abstinence on two occasions before treatment at a minimum of 2 weeks apart, and after 4 and 8 weeks treatment. A further sample was submitted 4 weeks after discontinuing desogestrel. Severe oligozoospermia was defined as sperm concentration <3x106/ml, and azoospermia was confirmed by examination of the pellet after centrifugation. Seminal plasma was separated by centrifugation at 3000 g for 5 min and stored at –20°C until assayed for inhibin B.

Statistical analyses
Results are presented as mean ± SEM. The primary hypothesis tested was that the degree of suppression of gonadotrophin concentration was related to the dose of desogestrel, i.e. a linear treatment effect. The concentrations of testosterone, plasma inhibin B, pro-{alpha}C, SHBG and lipids were analysed by analysis of variance (ANOVA) since these variables were normally distributed with equal error variances for the treatment groups. One-way ANOVA was carried out to compare baseline concentrations between the three treatment groups. There were no significant inter-group differences, except in the case of testosterone. Analysis of covariance (ANCOVA) was used to test for a linear effect of the dose of desogestrel after adjusting for baseline values. Paired t-tests were used to investigate at what time points a significant treatment effect was seen for each group. Non-parametric testing was used where the distribution was not normal (FSH, oestradiol, seminal plasma inhibin B and sperm density) or where there was significant non-homogeneity of variance (LH). The Kruskal–Wallis test was used to compare baseline values, and Spearman rank correlation to test for a linear dose effect on the differences from baseline at each time point. Wilcoxon signed rank tests were used to investigate the time points at which there were significant changes during treatment. Spearman's rank correlation was used to examine the strength of a linear relationship between pairs of variables. Statistical analyses were carried out using SPSS software.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects and pre-treatment values
Insertion of testosterone pellets was well tolerated, and the pellets were extruded in only one case (one of the two pellets extruded). This occurred during the recovery phase; therefore results from this individual are included in the analysis. One subject failed to complete the study, withdrawing for personal reasons (family bereavement) after 4 weeks desogestrel treatment. Data from this individual were excluded. No significant adverse effects were reported by any subject.

Compliance is clearly important in the investigation of an orally administered agent. This was assessed by direct questioning at each visit, and substantiated by pill counting in returned bottles. Two subjects reported missing two consecutive pills, but no missed pills were reported by other subjects. Data from these two individuals did not appear to be affected.

There were no differences in gonadotrophin, oestradiol, SHBG, inhibin B, pro-{alpha}C or lipid concentrations between groups before treatment. Analysis of variance demonstrated that pre-treatment plasma testosterone concentrations differed between groups (P = 0.039), being lower in the group receiving 300 µg desogestrel. Analysis of group differences in pre-treatment sperm concentrations (mean of two samples) approached significance (P = 0.05), with higher concentrations in the group receiving 300 µg desogestrel.

Gonadotrophins
Concentrations of both gonadotrophins fell in all three groups during desogestrel/testosterone treatment, with no linear relationship between the degree of suppression and the dose of desogestrel. A marked decline in both LH and FSH was detected after only 1 week of treatment in all three groups (P = 0.005, P = 0.008, P = 0.012 for FSH; P = 0.005, P = 0.015 and P = 0.012 for LH in the 75 µg, 150 µg and 300 µg desogestrel groups respectively). In all groups, both LH and FSH concentrations reached a nadir after 2–4 weeks with a partial recovery thereafter, particularly in the 75 µg desogestrel group. Thus LH concentrations rose from 1.31 ± 0.31 IU/l after 4 weeks treatment to 2.58 ± 0.68 IU/l after 8 weeks treatment (P = 0.028) in the 75 µg desogestrel group, but without significant change in the two higher dose groups. Analysis by the number of samples in which FSH was undetectable during treatment (seven, six and 14 in the three groups respectively) also suggested a dose-dependent effect. In all three groups, plasma gonadotrophin concentrations rapidly recovered on discontinuing desogestrel.

Testosterone and oestradiol
Testosterone concentrations fell during desogestrel/testosterone treatment in all three groups (Figure 1Go). The fall was evident after 1 week (P = 0.001 in each group), and plasma concentrations then remained constant for the duration of desogestrel treatment, recovering to pre-treatment levels within 4 weeks of discontinuing desogestrel. Despite differences in pre-treatment testosterone concentrations between groups, there were no differences during the recovery phase. Analysis of covariance demonstrated a significant linear dose effect of desogestrel on testosterone concentration after 2 and 4 weeks treatment (P = 0.031 and P = 0.043 respectively). Plasma oestradiol concentrations followed the same pattern as testosterone (Figure 1Go), with a rapid decline in all three groups after 1 week of treatment, followed by stable concentrations for the remainder of the treatment phase. There was a significant linear dose effect at 6 weeks treatment (P = 0.019).



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Figure 1. Plasma concentrations of (a) LH, (b) FSH, (c) testosterone, (d) oestradiol and (e) sex hormone-binding globulin (SHBG) at pre-treatment baseline (–2 and 0 weeks), during desogestrel/testosterone treatment (weeks 0–8), and following withdrawal of desogestrel treatment (weeks 10 and 12). Dose of desogestrel: 75 µg (•); 150 µg ({blacktriangleup}); 300 µg ({blacksquare}), p.o. daily. All men received 300 mg testosterone s.c. The duration of desogestrel treatment is indicated by the shaded bar. Values are mean ± SEM (n = 10 men per group).

 
Sex hormone-binding globulin
SHBG concentrations fell in the 150 µg and 300 µg desogestrel groups (P = 0.018 and P < 0.001 respectively after 8 weeks treatment (Figure 1eGo). There was no significant change in plasma SHBG concentration in the group receiving 75 µg desogestrel. Analysis of covariance showed evidence of a linear dose effect after both 4 and 8 weeks treatment (P = 0.028 and P = 0.008 respectively). SHBG recovered to pre-treatment concentrations after discontinuing desogestrel.

Inhibin forms
There were only minor changes in plasma inhibin B concentrations in any of the three groups during testosterone/desogestrel treatment (Figure 2aGo). There were, however, statistically significant decreases in the groups receiving 75 µg at 1 and 6 weeks (P = 0.018, P = 0.035) and 150 µg at 2 and 8 weeks (P = 0.018, P =0.049).



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Figure 2. (a) Plasma concentrations of inhibin B and pro-{alpha}C inhibin forms and (b) seminal plasma concentrations of inhibin B at pre-treatment baseline (–2 and 0 weeks), during desogestrel/testosterone treatment (weeks 0–8), and following withdrawal of desogestrel treatment (week 12). Seminal plasma inhibin B is plotted on a log scale. Inhibin B, filled symbols; pro-{alpha}C, open symbols. Dose of desogestrel: 75 µg ({circ},•); 150 µg ({triangleup},{blacktriangleup}); 300 µg ({square},{blacksquare}), p.o. daily. All men received 300 mg testosterone s.c. The duration of desogestrel treatment is indicated by the shaded bar. The broken line indicates the limit of detection. Values are mean ± SEM (n = 10 men per group).

 
Pro-{alpha}C inhibin forms showed a rapid fall during treatment (Figure 2aGo). The concentration of pro-{alpha}C was significantly lower than pre-treatment after 1 week in all three groups, and mean concentrations were lowest after 2 weeks treatment. Pro-{alpha}C remained suppressed during continuing treatment in the 150 µg and 300 µg desogestrel groups, and although there appeared to be a partial recovery in the 75 µg desogestrel group, analysis of covariance showed no significant linear dose effect at any time point. Following discontinuation of desogestrel treatment, pro-{alpha}C concentrations showed a prompt recovery to pre-treatment concentrations in all three groups by 2 weeks.

Inhibin B concentrations in seminal plasma showed a wide pre-treatment variation, from undetectable in two men to 6.16 ng/ml (median concentration 275 pg/ml). During testosterone/desogestrel treatment, seminal plasma inhibin B concentrations fell in all three groups (Figure 2bGo), but this reached statistical significance only for the 75 µg (P = 0.043) and 300 µg groups (P = 0.008). In the 300 µg desogestrel group, seminal plasma inhibin B became undetectable in all men after 8 weeks treatment, whereas it became undetectable in only two men in each of the other two groups. There was no significant correlation between seminal plasma inhibin B and sperm concentration before treatment, but these two variables did show a significant correlation after 8 weeks treatment (P = 0.047). Seminal plasma inhibin B concentrations also showed a significant correlation with FSH after 8 weeks treatment (P = 0.048) but not with LH, or with either gonadotrophin pre-treatment.

Sperm concentration
Sperm concentration fell in all groups during desogestrel/testosterone treatment (Figure 3Go). This decline was statistically significant after 4 weeks in the 150 µg and 300 µg desogestrel groups, and after 8 weeks in all groups, with a highly significant linear dose effect (P = 0.014 and P < 0.001 at 4 and 8 weeks treatment). Median sperm concentrations after 8 weeks treatment were 24x106, 10x106 and 0.1x106/ml in the 75 µg, 150 µg and 300 µg desogestrel groups respectively. There were differences in the numbers of men achieving both azoospermia (n = 0, 0 and 3; P = 0.019) and severe oligozoospermia (<3x106/ml: n = 3, 1 and 7; P = 0.038) in the 75 µg, 150 µg and 300 µg desogestrel groups respectively. There were highly significant correlations between sperm concentration and both gonadotrophins (FSH, P = 0.001; LH, P < 0.001) after 8 weeks treatment. The start of recovery was observed 4 weeks after discontinuing desogestrel (Figure 3Go).



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Figure 3. Sperm concentration at pre-treatment baseline (–2 and 0 weeks), during desogestrel/testosterone treatment (weeks 0–8), and following withdrawal of desogestrel treatment (week 12), plotted on a log scale. Dose of desogestrel: 75 µg (•); 150 µg ({blacktriangleup}); 300 µg ({blacksquare}), p.o. daily. All men received 300 mg testosterone s.c. The duration of desogestrel treatment is indicated by the shaded bar. Values are mean ± SEM (n = 10 men per group).

 
Metabolic effects
There were no differences in plasma lipid fractions (total cholesterol, TG, HDL-C or LDL-C) between the three groups before treatment, or any changes during desogestrel/testosterone treatment (Figure 4Go) when the groups were analysed separately or combined. However, during the recovery phase there were significant increases in total cholesterol in the 75 µg and 300 µg groups (P = 0.007 and P = 0.009 respectively). There were no changes in routine biochemical variables (renal and liver function tests) during desogestrel treatment. There was a fall in haematocrit value after 8 weeks treatment in all three groups (P = 0.002, P = 0.026 and P = 0.049 in the 75 µg, 150 µg and 300 µg groups respectively) and in haemoglobin concentration in the 75 µg and 150 µg groups (P = 0.020 and P = 0.031 respectively), but not in the 300 µg desogestrel group (Table IGo). Post-treatment fibrinogen concentrations were not significantly different those measured pre-treatment in any group (Table IGo).



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Figure 4. Plasma concentrations of (upper panel) total cholesterol and high density lipoprotein cholesterol (HDL-C), and (lower panel) low density lipoprotein cholesterol (LDL-C) and triglyceride at pre-treatment baseline (–2 and 0 weeks), during desogestrel/testosterone treatment (weeks 0–8), and following withdrawal of desogestrel treatment (weeks 10 and 12). Filled symbols, total cholesterol and LDL-C; open symbols, HDL-C and triglyceride. Dose of desogestrel: 75 µg ({circ},•); 150 µg ({triangleup},{blacktriangleup}); 300 µg ({square},{blacksquare}), p.o. daily. All men received 300 mg testosterone s.c. The duration of desogestrel treatment is indicated by the shaded bar. Values are mean ± SEM (n = 10 men per group).

 

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Table I. Haemoglobin concentrations, haematocrit values and fibrinogen concentrations in men receiving desogestrel/testosterone treatment for 8 weeks
 
Behavioural effects
As not all men in the study had sexual partners, sexual activity was scored as the sum of sexual intercourse and masturbation in the 2 weeks preceding interview. One man in each of the 150 µg and 300 µg groups declined to participate in this aspect of the study. Frequency of sexual activity did not differ between the three groups pre-treatment, and was unchanged in all groups during treatment and in the recovery phase (Figure 5Go). The results of the SES2 and SES 3 questionnaires are also shown in Figure 5Go. There were no differences between the three groups pre-treatment, and there were no changes in any group either during treatment or in the recovery phase.



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Figure 5. (a) Frequency of sexual activity (sum of sexual intercourse and masturbation over 2-week period), (b) Sexual Experience Scales (SES) 2 score and (c) SES 3 score at pre-treatment baseline (open columns), after 4 weeks desogestrel/testosterone treatment (stippled columns), after 8 weeks treatment (hatched columns), and 4 weeks following discontinuation of desogestrel treatment (filled columns) in the three groups receiving the indicated dose of desogestrel. All men also received 300 mg testosterone s.c. Values are mean ± SEM (n = 10 in the 75 µg desogestrel group; n = 9 in the 150 µg and 300 µg groups as one man in each of these two groups declined to participate in this aspect of this study).

 
Questionnaires were returned by seven, seven and five partners in the 75 µg, 150 µg and 300 µg desogestrel groups respectively (Table IIGo). Increased interest in sex was reported by no partners in the 75 µg group, by three in the 150 µg group, and by three in the 300 µg group. Only one partner, of a man in the 150 µg desogestrel group, reported a fall in interest in sex. The majority of partners reported no change in mood states, with the exception being a tendency towards reporting increases in tenseness and irritability. These two mood states are highly correlated, with 6/8 of those reporting an increase in irritability also recording an increase in tenseness. An increase in tenseness was reported by three, three and one partner, and an increase in irritability by three, three and two partners in the 75 µg, 150 µg and 300 µg groups respectively; these data were not amenable to reliable statistical analysis.


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Table II. Summary of questionnaires completed by subject's partners after 8 weeks desogestrel/testosterone treatment
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The combination of oral administration of desogestrel with single administration of a depot preparation of testosterone resulted in rapid and profound suppression of gonadotrophin secretion at all three doses of desogestrel tested, without any effect on plasma lipid concentrations. This combination of potent hormonal effect without metabolic side effect is desirable for further development of any potential hormonal male contraceptive, indicating that desogestrel with testosterone pellets is a promising approach. A group receiving testosterone alone was not included, as higher doses of testosterone pellets given alone have been demonstrated to result in incomplete suppression of spermatogenesis (Handelsman et al., 1996Go).

Administration of testosterone with a progestogen has been investigated as a potential hormonal male contraceptive for many years (Alvarez-Sanchez et al., 1977Go; Brenner et al., 1977Go; Melo and Coutinho, 1977Go; Foegh et al., 1980Go). The combination of levonorgestrel with testosterone enanthate has been suggested to result in more rapid suppression of spermatogenesis than testosterone alone (Bebb et al., 1996Go), although the dose of testosterone used for comparison was submaximal. Depot preparations of both progestogen and androgen result in more complete, but not more rapid, suppression of spermatogenesis when administered in combination than depot testosterone given alone (Handelsman et al., 1996Go). As the adverse metabolic effects of many injectable testosterone/progestogen combinations may reflect a combination of the relatively high concentrations of testosterone and the androgenicity of the progestogen, we have investigated the use of an oral progestogen with very low androgenicity (Fotherby and Caldwell, 1994Go; Darney, 1995Go). The dose of testosterone used was chosen to provide replacement at the lower limit of the physiological range assuming near-complete suppression of endogenous production, based on the release characteristics of the pellets (Handelsman et al., 1990aGo) and the normal production rate for testosterone (Vierhapper et al., 1997Go).

Gonadotrophin secretion was markedly suppressed in all three groups. While there was little difference between treatment groups, there was some evidence of a dose-dependent effect as LH showed a partial recovery in the 75 µg group. Plasma testosterone however demonstrated dose-dependent suppression, as did spermatogenesis and seminal plasma inhibin B, illustrating the sensitivity of the testis as an in-vivo bioassay of gonadotrophin secretion. A greater suppression of LH than FSH has been reported following administration of desogestrel alone (Wu et al., 1999Go). The rate and degree of suppression of gonadotrophins is comparable with that obtained using either higher doses of testosterone alone (Anderson and Wu, 1996Go; Wu et al., 1996Go), or in combination with other progestogens (Bebb et al., 1996Go; Handelsman et al., 1996Go) or a GnRH antagonist (Behre et al., 1997Go). The partial recovery in LH concentration in the 75 µg group after 8 weeks treatment may reflect the slight reduction in release of testosterone from the pellets which occurs after this time (Handelsman et al., 1990aGo; Jockenhövel et al., 1996Go). This recovery was less evident in the FSH results, and in the groups receiving higher doses of desogestrel, probably as a result of greater suppression of the hypothalamus/pituitary by the desogestrel. The highly significant relationship between plasma gonadotrophins and sperm concentration after 8 weeks treatment suggests that part of the incomplete suppression of spermatogenesis in the men receiving 75 µg desogestrel reflects this reduction in exogenous testosterone, with consequent escape of gonadotrophin suppression. There was no detectable increase in the plasma concentration of testosterone during treatment in this group, reflecting inability to distinguish between endogenous and exogenous testosterone in this context, and illustrating the potential value of measurement of other specific steroidal products of the testis such as epitestosterone (Handelsman et al., 1996Go; Anderson et al., 1997aGo). These data therefore demonstrate the importance of the development of a testosterone preparation with truly zero-order release kinetics before the lowest effective doses of co-administered steroids can be determined.

SHBG concentrations also fell with increasing desogestrel dose, despite the fall in testosterone concentrations, which would be expected to have the opposite effect (Plymate et al., 1983Go). This decline in SHBG will result in a lesser fall in free testosterone than in total testosterone. As free testosterone is the biologically active moiety (Mendel, 1989Go; Rosner, 1990Go), these results confirm the difficulty of extrapolating the consequences of changes in total testosterone when changes in SHBG do not follow the same pattern. Any adverse effect of lowered total testosterone concentration may be reduced or prevented under these conditions, although this should be demonstrated prospectively in a long-term trial of this regimen. A decline in SHBG concentration with desogestrel treatment is well recognized in women (Darney, 1995Go; Kuhl, 1996Go), and has also been recently reported in men (Wu et al., 1999Go). The present demonstration of a dose-dependent effect using a progestogen with high selectivity (Fotherby and Caldwell, 1994Go; Kuhl, 1996Go) does not provide evidence as to whether this result is mediated by progestogen or androgen receptors, but the observation that the decline in SHBG occurred despite a decline in testosterone concentration in all groups suggests that it is indeed a direct effect of desogestrel. This hepatic effect of desogestrel may be a result of the high dose received by the liver during first-pass following absorption, and contrasts with the lack of effect of depot medroxyprogesterone acetate when given with testosterone pellets (Handelsman et al., 1996Go).

Inhibin B concentrations in blood showed little change during desogestrel/testosterone treatment, despite the profound suppression of gonadotrophin secretion. Inhibin B is derived from the testis, largely from the Sertoli cells, although the Leydig cells may also produce dimeric inhibin (Risbridger et al., 1989Go; Anawalt et al., 1996Go; Illingworth et al., 1996Go; Anderson et al., 1998Go). Secretion of both inhibin B and pro-{alpha}C is increased by FSH administration (Anawalt et al., 1996Go), and both are suppressed by administration of testosterone alone or with levonorgestrel (Anawalt et al., 1996Go; Anderson et al., 1997bGo). Dissociation between dimeric inhibin B and monomeric pro-{alpha}C secretion occurs during chemotherapy-induced testicular damage: inhibin B falls while pro-{alpha}C rises, associated with a sustained rise in FSH with little change in LH or testosterone (Wallace et al., 1997Go). The fall in plasma pro-{alpha}C and seminal plasma inhibin B reported here is consistent with this model of FSH-dependent secretion from the Sertoli cell, but the basis for the lack of decline in inhibin B is at present unclear. It suggests that the control of inhibin B secretion by Sertoli (and possibly also Leydig) cells may be less directly linked to the concentration of FSH than the secretion of pro-{alpha}C. A similar lack of change in serum inhibin B has been recently reported in a study of the effects of transdermal testosterone with oral levonorgestrel in men (Büchter et al., 1999Go).

Inhibin bioactivity has long been recognized in seminal plasma (Franchimont, 1972Go). The presence of dimeric inhibin B has recently been described (Anderson et al., 1998Go), with a much greater range of concentration than found in blood, and a significant correlation with sperm concentration suggesting that it may reflect the activity of the seminiferous epithelium. The present results demonstrate that administration of testosterone/desogestrel resulted in a rapid decline in seminal plasma inhibin B concentrations. The decline in seminal plasma inhibin B was related to the dose of desogestrel, and indeed seminal plasma inhibin B became undetectable after 8 weeks treatment in all men receiving 300 µg desogestrel. There was also a significant correlation between seminal plasma inhibin B and sperm concentration after 8 weeks treatment independent of treatment group, suggesting a continuing relationship between inhibin B secretion and spermatogenesis. These results provide further evidence for the differential regulation of the two components of the bi-directional secretion of inhibin by Sertoli cells, demonstrated in primates as well as in rodents (Handelsman et al., 1990bGo). A shift in the pattern of inhibin secretion with a reduction in secretion into the seminiferous tubular lumen and an increase in basal secretion has been demonstrated in rats depleted of pachytene spermatocytes (Maddocks et al., 1992Go). Suppression of spermatogenesis to azoospermia requires prolonged drug administration in some men: changes in seminal plasma inhibin B may rapidly and reliably predict the slower changes in sperm concentration. This would be of value in the development of hormonal male contraception, allowing more efficient screening of treatment regimens, and will be tested prospectively in a longer study.

The combination of desogestrel with testosterone pellets, in the doses used here, did not result in any change in lipoprotein concentrations. HDL-C concentrations are hormone-dependent within the physiological range of testosterone and oestradiol in normal men (Bagatell et al., 1994bGo), and are suppressed by some steroidal contraceptive regimens (Wallace and Wu, 1990Go; Bagatell et al., 1994aGo; Anderson et al., 1995bGo). The present results suggest that this can be avoided using a progestogen with low androgenicity in combination with a low dose of testosterone administered in a preparation with near zero-order pharmacokinetics. While a preliminary report of the effects of desogestrel alone in doses up to 450 µg suggested no effect on HDL-C (Bellis et al., 1996Go), a more recent report from the same group has demonstrated that administration of 150 µg and 300 µg desogestrel alone resulted in falls in total cholesterol, HDL-C and LDL-C; during subsequent addition of testosterone the fall in HDL-C was increased (Wu et al., 1999Go). It is unclear why those results differ from these authors' earlier study and those presented here. The present results do not allow distinction to be made between the relative importance of either the low androgenicity of desogestrel or this formulation of testosterone. The combination of depot medroxyprogesterone acetate (300 mg) with 800 mg testosterone pellets has also been reported to result in no change in HDL-C concentrations, while resulting in azoospermia in 9/10 subjects after 2–3 months (Handelsman et al., 1996Go), although administration of medroxyprogesterone acetate with testosterone enanthate resulted in a fall in HDL-C (Wallace and Wu, 1990Go). Administration of the progestogen/antiandrogen cyproterone acetate in combination with testosterone enanthate also resulted in no change in lipoprotein concentrations (Meriggiola et al., 1996Go), but a fall in haemoglobin concentration and haematocrit value. A small decline in haematocrit value, but no significant fall in haemoglobin concentration, was found with the largest dose of desogestrel in the present study, although falls in both were found with the two lower doses of desogestrel. These changes probably reflect the decline in testosterone concentration. An effect of venesection is unlikely as, after 4 weeks treatment when a fall in haematocrit value was detected, only 200 ml blood had been removed over a period of 6 weeks. No changes in either haematocrit or haemoglobin values were found when similar doses of desogestrel were combined with testosterone enanthate (Wu et al., 1999Go); this difference may result from the larger dose of testosterone used in that study. SHBG has been proposed to be a marker of excessive hepatic steroid effect (Handelsman et al., 1996Go); the present results, which show changes in SHBG but not HDL-C, support the use of SHBG as a sensitive index of hepatic steroid effect. Overall, these data illustrate the relative lack of effect of depot testosterone preparations on metabolic outcomes, although the effects of progestogens in combination with testosterone—particularly on lipoprotein metabolism—appear unclear. More detailed mechanistic studies involving ApoA1 synthetic rates and lipoprotein lipase activity are required to resolve these issues.

The reduction in circulating testosterone concentrations was well tolerated by all subjects. In particular, there was no evidence of loss of interest in sex using four outcome measures: self-report of frequency of sexual activity; SES 2 and 3 questionnaires; and partner's report. Indeed, partner's reports suggested a trend towards an increase in interest in sex dependent on the dose of desogestrel. There was also a tendency towards an increase in tenseness and irritability recorded by subjects' partners, but no evidence of a dose-dependent relationship with desogestrel. These symptoms may reflect the fall in circulating testosterone concentrations. As self-reported male sexual behaviour and mood are maintained at normal levels by mildly subphysiological testosterone concentrations (Kwan et al., 1983Go; O'Carroll et al., 1985Go; Wang et al., 1996Go), these results may point to the value of partners' reports in detecting small changes. We have used here a combination of approaches to maximize the chance of detecting small effects, but the limitations of currently available methods in this field are widely recognized.

While the results obtained here do not therefore provide evidence that androgen substitution was inadequate, longer-term studies are clearly required to assess the safety of such a regimen, especially in view of the suggestion based on epidemiological data that lower testosterone concentrations are associated with increased risk of cardiovascular disease (Barrett-Connor, 1996Go). Few relevant experimental data exist on the non-reproductive effects of androgens and progestogens in normal men, and such combinations may have different effects to testosterone alone. Desogestrel-containing oral contraceptive pills have been associated with increased risk of venous thrombosis in women (Spitzer et al., 1996Go); the relevance of this to men is uncertain, and is not addressed by these data. Nevertheless, the present results demonstrate the feasibility of reductions in the dose of exogenous testosterone required for adequate gonadotrophin suppression while avoiding the well-recognized and significant adverse effects of other testosterone preparations which continue to be used in the search for a hormonal male contraceptive.

Suppression of spermatogenesis was not a primary outcome measure of this short study. A minimum of 4 months is required for reliable data, and sperm concentration continues to fall in some men over a still longer period (WHO, 1995). The preliminary data obtained however—and in particular the high incidence of severe oligozoospermia and azoospermia in the 300 µg desogestrel group—provide support for this prototype. Direct effects of progestogens on Leydig cell steroidogenesis and androgen metabolism have been suggested to contribute to their effect on spermatogenesis (Meriggiola and Bremner, 1997Go; Wu, 1997Go), potentiating the effect of the withdrawal of LH and FSH. Reductions in the dose of testosterone may also contribute to an increased prevalence of azoospermia (Handelsman et al., 1996Go; Meriggiola and Bremner, 1997Go; Wu et al., 1999Go).

In summary, this study demonstrates that the combination of an oral progestogen with low androgenicity with depot administration of testosterone results in profound suppression of gonadotrophin secretion with no adverse effect on HDL-C concentration. While the degree of suppression of spermatogenesis and other possible metabolic effects (e.g. on bone metabolism) should be determined in longer studies, these results support the value of this approach.


    Acknowledgments
 
This work was supported by the Medical Research Council and the UK Department for International Development (Grant no. G9523250). The technical assistance of Margaret Millar, Nancy Evans and Claire Tierney is gratefully acknowledged. We are grateful to Organon for the provision of desogestrel tablets and testosterone pellets.


    Notes
 
5 To whom correspondence should be addressed at: MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, University of Edinburgh, 37 Chalmers St, Edinburgh EH3 9ET, UK. E-mail: r.a.anderson{at}ed-rbu.mrc.ac.uk Back

* Presented in part at the 13th European Society of Human Reproduction and Embryology meeting, Edinburgh, June 1997 Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on November 22, 1999; accepted on March 22, 2000.