Hum. Reprod. Advance Access originally published online on October 25, 2006
Human Reproduction 2007 22(3):702-707; doi:10.1093/humrep/del414
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Miglustat has no apparent effect on spermatogenesis in normal men
1 Department of Medicine 2 Department of Urology, University of Washington, Seattle, WA, USA 3 Schering AG, Berlin, Germany 4 Department of Ophthalmology, University of Washington, Seattle, WA and 5 Drug Metabolism, Berlex Biosciences, Richmond, CA, USA
6 To whom correspondence should be addressed at: Department of Medicine, University of Washington, Box 356429, 1959 NE Pacific Street, Seattle, WA 98195, USA. E-mail: jamory{at}u.washington.edu
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Abstract |
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BACKGROUND: In mice, administration of the glycosphingolipid biosynthesis inhibitor miglustat results in reversible infertilty, characterized by impaired sperm motility and markedly abnormal sperm morphology. This observation suggested that miglustat might have utility for fertility control in man. To ascertain the impact of miglustat on human spermatogenesis, we conducted a pilot study of miglustat administration in normal men. METHODS: After a 2-week baseline period, seven normal men were administered miglustat 100 mg, orally, twice daily for 6 weeks. During treatment, subjects had frequent seminal fluid analyses to assess the impact of treatment on sperm concentration, motility and morphology and the ability to undergo the acrosome reaction by in vitro assays. RESULTS: Five subjects completed all aspects of the study. In these subjects, there was no apparent effect of miglustat on sperm concentration, motility or sperm morphology after 6 weeks of therapy. In addition, no changes in acrosome structure or function were observed with treatment, despite therapeutic concentrations of miglustat in the serum and seminal plasma. All subjects experienced gastrointestinal upset, diarrhoea and mild weight loss during treatment. No other abnormalities in blood counts, serum chemistries, vision or overall health were observed. CONCLUSION: In contrast to the observations in mice, the oral administration of miglustat does not appear to affect human spermatogenesis. Further elucidation of the mechanism underlying the species specificity of miglustat may improve our understanding of the role of glycosphingolipids in spermatogenesis and result in alternative approaches to male fertility control.
Key words: miglustat/spermatogenesis/Gaucher disease/glycosphingolipid/male contraceptive
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Introduction |
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Currently available methods of male fertility control are limited to vasectomy and condoms, with only the latter being truly reversible. Therefore, there is great interest in developing additional methods. Most experimental male-based regimens use hormones to suppress spermatogenesis, leading to azoospermia in 50–80% of men (Amory et al., 2006
). Despite years of research into hormonal approaches to male fertility control, an acceptable regimen with universal efficacy has remained elusive. Non-hormonal experimental approaches such as gossypol (Gu et al., 2000) and 
-chlorohydrin (Jones, 1983) have shown promise in human or animal studies, but neither compound has proven safe and effective enough to be introduced clinically. Therefore, alternative targets in men are needed.
Enzymes involved in sperm glycosphingolipid biosynthesis may be good targets for fertility control as glycosphingolipids are abundant in sperm (Ritter et al., 1987; Honke et al., 2002) and thought to be necessary for normal spermatogenesis. Moreover, mice deficient in these enzymes have severely impaired fertility (Takamiya et al., 1998; Fujimoto et al., 2000). Recently, it was demonstrated that the oral administration of miglustat, an inhibitor of glycosphingolipid biosynthesis, to male mice rapidly resulted in reversible infertility without discernible adverse side effects (van der Spoel et al., 2002). Analysis of sperm from miglustat-treated mice revealed no change in sperm counts; however, sperm motility was decreased by more than 75%, and sperm and acrosome morphology were markedly abnormal. Mating studies during treatment revealed complete infertility, yet there were no changes in serum reproductive hormones or sexual behaviour. The authors speculated that an alteration in glycosphingolipid metabolism mediated by the oral administration of miglustat impaired spermiogenesis, resulting in normal numbers of non-functional and morphologically abnormal sperm, leading to treatment-induced infertility (van der Spoel et al., 2002; Suganuma et al., 2005). Importantly, after treatment was stopped, both sperm parameters and fertility rapidly returned to normal. Subsequent studies of miglustat in mice have demonstrated that reversibility is maintained even with the administration of miglustat for periods of up to 1 year (Walden et al., 2006). Lastly, treatment does not appear to affect the genetic integrity of the sperm, as oocytes microinjected with deformed spermatozoa from treated animals developed normally and gave rise to fertile, phenotypically normal offspring (Suganuma et al., 2005).
The impact of miglustat on human spermatogenesis in normal men has not been ascertained, but miglustat is approved for use in humans for the treatment of Gaucher disease. Gaucher disease is a rare autosomal recessive inherited disease of glycosphingolipid excess, which is characterized by enlargement of the spleen and liver, impaired haematopoiesis, osteoporosis and, in some cases, neurological symptoms (Cox et al., 2003; Elstein et al., 2004; Pastores et al., 2005). If the effects on sperm morphology and motility in man are similar to those observed in mice, miglustat or other alkylated imino sugars might have potential utility as non-hormonal male contraceptives. Therefore, to determine the contraceptive potential of this compound, we conducted a 6-week pilot study to ascertain whether miglustat administration would impair sperm motility and sperm morphology in normal men.
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Methods |
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Subjects
Seven healthy men, ages 18–50, were recruited by newspaper advertisements and posted flyers. Inclusion criteria were the following: general good health, normal medical history, absence of routine medication use, normal physical examination, normal serum laboratory tests including complete blood count, liver function tests, total testosterone and two normal seminal fluid analyses as defined by World Health Organization criteria (1999)
(sperm count >20 million/ml, motility >50% and morphology >15% normal forms). Exclusion criteria were significant chronic or acute psychiatric illness, history of alcohol or anabolic steroid abuse and participation in another research study within the preceding 3 months. The Institutional Review Board at the University of Washington approved all procedures. Written, informed consent was obtained before screening in all cases. In addition, this protocol was reviewed by the US Food and Drug Administration and granted an Investigational New Drug number.
Study procedures
This was an open-label, single-arm study consisting of a 2-week control period, a 6-week treatment period and a 6-week recovery period. Following screening, subjects were administered miglustat (ZavescaTM, Actelion Pharmaceuticals, Basel, Switzerland), 100 mg orally, twice daily for 6 weeks. Subject compliance was assessed via a self-completed drug log. Subjects were asked to provide semen samples obtained by masturbation after at least 48 h of ejaculatory abstinence every week throughout drug exposure. In addition, before the morning dose of miglustat, blood was collected weekly for complete blood counts, serum chemistries including liver function tests, serum FSH, LH and testosterone. In addition, pre-dose serum and semen miglustat levels were measured at baseline, after 3 and 6 weeks of treatment and 4 weeks into follow-up. After treatment was complete, subjects provided semen samples and had blood drawn every 2 weeks for 6 weeks. Sperm acrosome reaction was assessed at baseline, after 3 and 6 weeks of treatment and at the end the follow-up period. Subjects also underwent a slit lamp ophthalmologic examination before taking the drug, and after completing the study, as some concern existed about cataract formation in humans, because some laboratory animals developed cataracts on high doses of miglustat (although this has not been reported in humans). Lastly, subjects completed a validated questionnaire regarding sexual function (O’Leary et al., 1995), at baseline, after 3 and 6 weeks of treatment and after 6 weeks of follow-up.
Measurements
Seminal fluid samples were analysed in the Male Fertility Laboratory in the Department of Urology at the University of Washington for sperm concentration and motility by World Health Organization (WHO, 1999) methods and for morphology by Tygerberg strict criteria (Menkveld et al., 1990) using computer-assisted sperm analysis. Motile sperm were separated from the semen using a 40/80% Percoll gradient. Purified sperm from the 80% Percoll fraction were prepared for capacitation by washing with 0.5% human serum albumin (HSA) in Ham’s F10 media and incubating at 37°C in 5% CO2. Acrosome reaction tests were modified from our previous methods (Cross et al., 1986; Ravnik et al., 1995) and used a calcium ionophore (A23187
[GenBank]
) and progesterone (Sigma, St. Louis, MO, USA). Treatments were applied for 15 min in the presence of 3.5% HSA after 4 (calcium ionophore) and 24 h (progesterone) of capacitation. Untreated control sperm were incubated with 3.5% HSA for 15 min. To quantify the acrosome reaction, we stained the prepared slides with fluorescein-conjugated lectin Pisum sativum agglutinin (Vector Labs, Burlingame, CA, USA). For each data point, at least 200 sperm were analysed, with replicate counts, using fluorescence microscopy. Results were expressed as a percentage of spermatozoa observed to have undergone the acrosome reaction.
All routine laboratory tests (blood counts, serum kidney and liver tests) were performed in the clinical laboratory at the Department of Laboratory Medicine, University of Washington. Serum testosterone was measured by radioimmunoassay (Diagnostic Products Corporation, Los Angeles, CA, USA). The assay sensitivity for testosterone was 0.5 nmol/l. For low-, mid- and high-pooled values of 3.8, 10.6 and 24.4 nmol/l of testosterone, the intra-assay coefficients of variation were 4.4, 5.1, and 6.0%, and the inter-assay coefficients of variation were 17.5, 11.8, and 12.9%, respectively. FSH and LH levels were measured by immunofluorometric assay (Delfia, Wallac Oy, Turku, Finland). The sensitivity of the assay for FSH and LH was 0.016 IU/l and 0.019 IU/l, respectively. For low-, mid- and high-pooled values of 0.05, 1.0 and 21 IU/l of FSH, the intra-assay coefficients of variation were 5.9, 3.0 and 3.0%, respectively, and the inter-assay coefficients of variation were 20.7, 5.0 and 6.2%, respectively. For low-, mid- and high-pooled values of 0.06, 1 and 16 IU/l of LH, the intra-assay coefficients of variation were 12.6, 5.6 and 4.1%, respectively, and the inter-assay coefficients of variation were 16.5, 13.9 and 10.5%, respectively.
Serum and seminal plasma miglustat levels were measured by high-performance liquid chromatography-mass spectroscopy (HPLC-MS). Frozen samples were thawed at room temperature. The calibration standards were generated by serial dilution of miglustat (from 1 mM methanol stock solutions) into control serum/seminal plasma. Fifty microlitre aliquots of each of the serum/seminal plasma standards and experimental samples was precipitated by the addition of four volumes of cold acetonitrile (200 µl) containing 1 µM of a suitable internal standard. The precipitated serum/seminal plasma proteins were removed by centrifugation at 30000 g for 20 min at room temperature. The supernatant was evaporated to dryness (GeneVac, EC-2) at 40°C and reconstituted with 200 µl of 20% acetonitrile solution. Approximately 80 µl of the resultant supernatant was transferred to HPLC vial inserts for analysis. The quantification range of the calibration curve was from 0.041 to 30 µM, where 0.041 µM (
8 ng/ml) represents the lower limit of quantification in serum/seminal plasma. Liquid chromatographic separation was carried out utilizing a Shimadzu 10AD binary pump system connected to a Leap autosampler (Leap HTS PAL, Switzerland). The elution solvent system consisted of water with 0.025% NH4OH (v/v) and acetonitrile. A Polaris C18-A column (2.1 mm x 50 mm., Varian Corporation, Walnut Creek, CA, USA) was used for the gradient HPLC separation. The organic modifier gradient was 5–95% acetonitrile in 3 min, followed by 2-min wash with 95% acetonitrile, then 5% acetonitrile for 2 min (resulting in a total of 7 min per analytical run). The flow rate was set at 0.3 ml/min, and 5 µl of sample was injected in each case.
An Applied Biosystem API 4000 QTrap triple-quadruple mass spectrometer (Foster City, CA, USA) equipped with a TurboIonSpray source operated in a positive ion mode was utilized in this study. The most intense product analyte ion (220.2
158.2 atomic mass unit) was selected as the ion for quantification. Probe source temperature was held at 400°C in this study, and heating gas (nitrogen) was maintained at 8 l/min. The transient ion current (precursor ion to product ion) was measured for analyte and internal standard. The ratio of the integrated peak areas of the analyte to the internal standard was used to generate a calibration curve for the quantification of miglustat in the serum and seminal plasma samples (Analyst version 1.4).
Statistical analysis
Pre- and on-treatment means and SDs were calculated for all study time points. Sperm count, motility, morphology, acrosome reaction and seminal and serum miglustat levels between the control and treatment phases were compared using a repeated measures analysis of variance with subjects serving as their own controls. Sperm concentrations were log-transformed before analysis (Berman et al., 1996), but all other values were analysed without transformation. Sexual function parameters and mean serum and seminal plasma miglustat levels were compared to baseline using a paired t-test with unequal variance. Statistical analyses were performed using STATA (College Park, TX). For all comparisons a P-value of <0.05 (two-tailed) was considered significant.
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Results |
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Study subjects
Of 17 men screened for the study, 7 met all the inclusion criteria and enrolled in the study. Of the 10 men who were not enrolled in the study, 4 subjects were excluded for subnormal semen parameters, and 6 were excluded for abnormal screening laboratory values. Seven men enrolled in the study, and five completed all study procedures: one subject discontinued study medication after 3 days because of depression, fatigue and dizziness, another subject was non-compliant with the study medication. These subjects were not included in the analyses. The baseline characteristics of the seven study subjects are summarized in Table I.
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Sperm and semen parameters
There were no significant changes compared with baseline in sperm concentration, sperm motility or sperm morphology during treatment with miglustat (Figure 1). In addition, no significant change due to treatment in in vitro assessments of acrosome reaction pre-capacitation, after capacitation, after treatment with calcium ionophore or after incubation with progesterone was observed (Table II). However, increased rates of acrosome reaction were observed with both calcium ionophore and progesterone treatments at all time points, demonstrating the effectiveness of these techniques in increasing acrosome reaction. The percentage of spermatozoa with an abnormal acrosome on microscopic analysis did not change with treatment [11.8 ± 5.8% at baseline versus 10.6 ± 5.6% at end of treatment; P = 0.66]. Similarly, the percentage of spermatozoa with a small acrosome did not change with treatment [7.4 ± 3.2% at baseline versus 7.3 ± 2.5% at end of treatment; P = 0.94]. Lastly, as compared with baseline, there were no changes in semen volume, semen pH or seminal white blood cell count during treatment (data not shown).
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Serum and seminal miglustat concentrations
Miglustat was not detectable in either serum or seminal plasma either at baseline or at the end of study visit. In the five compliant subjects, there was a significant increase in serum miglustat concentration during treatment (Figure 2; P = 0.02 compared with baseline), with all subjects experiencing a serum miglustat level above 4µM both 3 and 6 weeks into treatment. In one subject, serum miglustat level exceeded 9µM after 6 weeks of treatment. Miglustat was also significantly increased in the seminal plasma of subjects during treatment (Figure 2; P = 0.005). The seminal concentrations of miglustat were slightly, but non-significantly, higher than the serum concentrations.
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Serum hormones and sexual function
There was no significant impact of therapy on serum testosterone during treatment (Baseline: 11.3 ± 3.2 nmol/l versus 10.2 ± 2.3 nmol/l (treatment week 6); P = 0.48). Moreover, no change in serum LH or FSH concentration was observed [LH: baseline 2.8 ± 0.8 IU/l versus 2.8 ± 0.7IU/l, (treatment week 6); P = 0.98; FSH: baseline: 2.6 ± 0.5 IU/l versus 3.4 ± 1.1 IU/l, (treatment week 6); P = 0.10). In addition, sexual function was not significantly altered during treatment, and no subject complained of changes in sex drive, erectile or ejaculatory function or overall satisfaction with sexual activity (Table III).
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Adverse effects
There were no serious adverse events associated with miglustat therapy during treatment. All subjects experienced mild-to-moderate gastrointestinal side effects, including diarrhoea, flatulence and abdominal distention, which improved with time. In addition, subjects experienced a mean weight loss of 2.0 kg during treatment. Additional side effects included mild insomnia and ‘strange dreams’ in one subject. Importantly, there were no significant changes in the blood cell counts or serum measures of liver or kidney function. Lastly, there were no complaints of neurological or visual abnormalities with treatment, and all subjects had normal ophthalmologic and neurological examinations at the conclusion of the study.
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Discussion |
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Our results demonstrate that the oral administration of miglustat has no obvious short-term effect on spermatogenesis in normal men and do not support the notion that miglustat can be used as a ‘male pill’ (Cooper, 2003
; Robaire, 2003). Despite the use of a dose and serum levels consistent with a therapeutic effect in men with Gaucher disease (Cox et al., 2003; Elstein et al., 2004; Pastores et al., 2005), we saw no significant effect on sperm motility, sperm morphology or the ability of the sperm to undergo acrosome reaction in vitro. These results are consistent with a lack of an observed effect on the spermatogenesis of men with Gaucher disease who are receiving treatment with miglustat (A. Zimran, personal communication).
There are several possible explanations for the clear differences between the results observed in mice (van der Spoel et al., 2002; Walden et al., 2006) and those observed in this study. The dose of miglustat used in this study, 100 mg twice daily, is two-thirds of the usual dose used for the treatment of individuals with Gaucher disease; however, this dose is also effective and is thought to significantly inhibit glycosphingolipid biosynthesis in man, as many subjects take lower doses with improvement in organomegaly and other signs and symptoms of active Gaucher’s disease (Cox et al., 2003; Elstein et al., 2004). In addition, the serum levels of miglustat achieved in this study are consistent with a therapeutic effect on glycosphingolipid metabolism seen in both animal (Platt et al., 1997; Jeyakumar et al., 1999) and human studies of glycosphingolipid metabolism (Cox et al., 2003). Furthermore, we have demonstrated that the drug is present in the male reproductive tract, at concentrations similar to those present in the serum immediately before dosing. Although we cannot exclude the possibility that the miglustat was excluded from the developing germ cells during a crucial window of time in spermiogenesis by the ‘blood–testis’ barrier, this seems unlikely given the small molecular weight of this compound and its ability to access the central nervous system (Platt et al., 1994). Lastly, the serum levels achieved in our study were well above the serum levels necessary for the contraceptive effect in mice, in whom effective contraception is achieved with serum miglustat levels under 1 µM (Platt F and van der Spoel A, personal communication).
Another possible explanation for the difference observed in this study and the results obtained in mice is that the treatment duration in this study may have been insufficient. In the mouse studies, effects on sperm motility and morphology were observed after 15 days, roughly one-half the duration of the murine spermatogenic cycle (Clermont, 1972). The human spermatogenic cycle is roughly 65 days (Missell et al., 2006). Therefore, a treatment duration of 6 weeks encompasses roughly two-thirds of a human spermatogenic cycle, making it extremely unlikely that a marked effect of miglustat on spermatogenesis could have been missed. By way of contrast, after 6 weeks of treatment with a hormonal contraceptive regimen, a greater than 50% reduction in sperm concentration is observed in a majority of men (Ly et al., 2005). Given the small sample size in this pilot study, we cannot exclude the possibility that some men might be more susceptible to the modulation of glycosphingolipid metabolism and demonstrate impairment of spermatogenesis with miglustat administration; however, we feel that the results from this study can exclude its utility as a fertility control method for all men at the dose and duration studied.
What then accounts for the difference in response to miglustat administration between mice and men? It is tempting to speculate that mice may be more susceptible to the alteration of glycosphingolipid metabolism by miglustat, or humans may have an additional mechanism for the synthesis of sperm glycosphingolipids that is absent in the mouse. In either case, further research into the species specificity of the response to miglustat administration will be required to fully understand these results. In addition, the precise biochemical basis for the effect in mice needs to be elucidated if the species specificity is to be better understood.
The adverse effects observed in this study, mainly diarrhoea, bloating and flatulence, are consistent with the known inhibition of intestinal disaccharides by miglustat (Lachmann, 2003). This enzyme inhibition leads to the retention of undigested carbohydrate within the intestinal lumen and results in an osmotic diarrhoea. This side effect is common in studies of miglustat administration to individuals with Gaucher’s disease and would likely pose a significant barrier to acceptance of a male fertility control method based on this mechanism of action.
In conclusion, we have shown that short-term administration of the oral glycosphingolipid biosynthesis inhibitor miglustat has no discernible effect on spermatogenesis in normal men. However, given the intriguing findings in mice, this approach to male contraception warrants additional study. Further elucidation of the mechanism behind the species specificity of miglustat may serve to both improve our understanding of the role of glycosphingolipids in spermatogenesis and result in alternative, novel approaches to male fertility control in humans.
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Acknowledgements |
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The authors thank Frances Platt and Aarnoud van der Spoel (University of Oxford, Department of Pharmacology) for many interesting insights in the inhibition of spermatogenesis in mice by miglustat. In addition, the authors also thank Ms. Kymberly Anable and Ms. Kathy Winter (University of Washington) for study coordination. This work was supported by a pilot grant from the National Institute of Child Health and Human Development, a division of the National Institute of Health through cooperative agreement U54 HD42454 as part of the Cooperative Contraceptive Research Centers Program and by a grant from Schering AG. Dr. Amory is supported, in part, by the National Institute of Child Health and Human Development, a division of the National Institute of Health by grant number K23 HD45386.
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Submitted on August 28, 2006; resubmitted on September 19, 2006; accepted on September 26, 2006.
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