European Journal of Endocrinology (2012) 166 291–294 ISSN 0804-4643

CLINICAL STUDY Reproductive function in men affected by X-linked /adrenomyeloneuropathy T J Stradomska, J Kubalska1, R Janas2 and A Tylki-Szyman´ska3 Department of Biochemistry and Experimental Medicine, The Children’s Memorial Health Institute, Al. Dzieci Polskich 20, 04-730 Warsaw, Poland, 1Department of Genetics, Institute of Psychiatry and Neurology, Warsaw, Poland, 2Department of Radioimmunology and 3Clinic of Metabolic Diseases, The Children’s Memorial Health Institute, Warsaw, Poland (Correspondence should be addressed to T J Stradomska; Email: [email protected])

Abstract Background: X-linked adrenoleukodystrophy (X-ALD) is the most frequent, severely neurodegenerative, clinically heterogeneous , the signs of which are a consequence of , adrenal cortex, and testes impairment. Objective: We studied testosterone, LH, and FSH levels in X-ALD/adrenomyeloneuropathy (AMN) patients. We evaluate the ability to procreate of these patients by analysis of pedigree and family screening by detection of very long-chain (VLCFA) levels. Subject and methods: Seventeen patients with X-ALD/AMN (16 with AMN and one asymptomatic) aged 24–48 (meanGS.D., 34.7G5.9) years, were identified based on the clinical picture, magnetic resonance imaging, and the presence of increased serum VLCFA levels. Nine X-ALD/AMN patients’ daughters, mean ages GS.D.Z7.7G3.8 years, were identified as heterozygote by elevated VLCFA levels. Serum VLCFA levels were determined as ester derivatives by a gas chromatography method. Serum testosterone, LH, and FSH levels in X-ALD/AMN patients were detected by IRMAs. Results: Serum testosterone levels were at the lowest levels of normal range but serum LH and FSH concentrations were increased in 57.1 and in 42.9% of X-ALD/AMN patients respectively. Among the 11 investigated of X-ALD/AMN married adult men, nine had produced offspring, a total of 13 children. All patients’ daughters showed elevated serum VLCFA at heterozygote levels. Conclusion: In this study, we report that in a group of X-ALD/AMN married adult men, we did not find a significant decrease in fertility compared with the Polish population (18.2 vs 15%).

European Journal of Endocrinology 166 291–294

Introduction progressive demyelination of the central and peripheral nervous systems, and adrenal insufficiency (3). Patients X-linked adrenoleukodystrophy (X-ALD; MIM 300100), with adrenomyeloneuropathy (AMN) also present is a neurodegenerative disorder characterized by hypogonadism, impairment of Leydig cells, and a progressive demyelination within the central and decrease in testosterone level (4, 5). The aim of this peripheral nervous systems. Biochemically, it is charac- study was to evaluate the procreative ability of adult terized by the accumulation of very long-chain fatty males affected by X-ALD/AMN. acids (VLCFAs), particularly lignoceric (C24:0) and cerotic (C26:0) acids in blood and tissues, a cause of disturbance in the peroxisomal b-oxidation process. Subjects and methods X-ALD results from mutations in the ABCD1 gene (300371) encoding an integral peroxisomal membrane Subjects protein (ALDP), belonging to the ATP-binding cassette- Seventeen patients with X-ALD/AMN (16 with AMN transporter superfamily (1). Accumulation of VLCFA and one asymptomatic) aged 24–48 (meanGS.D., contributes to X-ALD pathogenesis, although the 34.7G5.9) years, were identified based on the clinical mechanism is unclear (2). X-ALD is clinically hetero- picture, magnetic resonance imaging, and the presence geneous, and can present at a variety of ages. The of increased serum VLCFA levels or family screening (6). resulting symptoms include effects on the nervous The median duration of neurological symptoms was systems, adrenal gland, and testes. Hemizygotes present 6 years, range !1 to 18.

q 2012 European Society of Endocrinology DOI: 10.1530/EJE-11-0490 Online version via www.eje-online.org

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Among the patients, 11 men, aged 29–48 years Results (meanGS.D., 35.5G5.4), were married. Nine of these (eight with AMN and one asymptomatic) had produced In patients with AMN the VLCFA ratios (meanGS.D.) offspring, a total of 13 children (nine girls and four were: C24:0/C22:0Z1.634G0.089 and C26:0/C22:0Z boys). The date of conception of the children was 1–12 0.056G0.013. In the patients’ daughters, the presence years before the primary onset of the disease. In spite of of increased VLCFA was proven (C24:0/C22:0Z the fact that the other two marriages were open to 1.098G0.110 and C26:0/C22:0Z0.019G0.017 mg/ml; having children, they did not have them, the reasons Table 1). Mean VLCFA levels in patients’ daughters were for this being unknown. elevated at heterozygote levels. Nine X-ALD/AMN patients’ daughters, mean ages The serum concentration of testosterone was from GS.D.Z7.7G3.8 years, were obligatory heterozygotes. 10.5 to 26.5 nmol/l for all X-ALD/AMN patients and from 10.5 to 23.1 nmol/l for the group of married men, and was within the normal range. MeanGS.D. testoster- Blood sampling onevalueswere18.5G5.5 and 16.1G4.9 nmol/l respectively. Three patients from those who were fathers Blood (0.5 ml) was drawn after an overnight (12 h) fast. had the lowest testosterone levels (P!0.0012 vs all The serum was separated and stored frozen at below K patients). The mean testosterone level of this group 20 8C before analysis. constituted only about 60% of the mean testosterone for all patients. Analysis of VLCFA by gas chromatography The patients’ serum concentration of LH and method FSH was (meanGS.D.) 7.2G3.2 (range 2.9–11.8) IU/l and 7.5G7.9 (range 1.7–14.8) IU/l respectively. LH Serum VLCFA levels were determined as ester deriva- concentration was increased in 57.1% and FSH in tives by a gas chromatography technique (GC), 42.9% of patients compared with reference values. according to a previously described method (7). An analysis of family history and screening by detection of VLCFA as a biomarker for X-ALD/AMN Analysis of testosterone shows that nine out of 11 married men had produced offspring (nine girls and four boys), being Serum testosterone level was assayed using a CIS Bio 81.8% (9/11). International (Gif sur Yvette Cedex, France) TESTO-CT2 RIA kit. The intra-assay coefficient of variation (CV%) was 7.5% and inter-assay 7.0%. Reference values for men ranging from 8.2 to 34.6 nmol/l were used. Discussion The first published data in 1986, by Libber et al. (8), Analysis of LH and FSH found normal plasma testosterone and minor testicular deficiency in X-ALD/AMN patients. Moser et al. (9) in IRMA kits for the in vitro quantitative determination of 1991 found that serum testosterone levels were low in human LH and human FSH in serum or plasma were 11% and at the lower levels of normal in an additional purchased from DIAsource ImmunoAssays S.A., 11% of 69 AMN patients. Brennemann et al. (4) (49 Nivelles, Belgium. Detection limit for the LH-IRMA patients, mean age 36 years) showed mean testosterone was 0.2 mIU/ml, and intra- and inter-assay CV were 3.9 did not differ significantly between patients and healthy and 8.0%. The respective values for the FSH-IRMA controls, with two results at upper normal values, but were 0.1 mIU/ml, and 2.0 and 4.4% respectively. the mean free testosterone concentration was signi- Reference values for men ranging from 1.0 to 5.3 IU/l ficantly lower in comparison with controls. In retro- for LH and 1.3 to 8.1 IU/l for FSH were used. spective studies, Assies et al. (10) (26 patients, with

Table 1 Serum very long-chain fatty acid ratios in hemi- and heterozygotes X-linked adrenoleukodystrophy/adrenomyeloneuropathy (X-ALD/AMN) (meanGS.D.).

Group Number Age at diagnosis (years) C24:0/C22:0 C26:0/C22:0

Hemizygotes X-ALD/AMN 17 34.7G5.9 1.634G0.089 0.056G0.013 Daughters of X-ALD/AMN 9 7.7G3.8 1.098G0.110 0.019G0.017 patients Heterozygotesa X-ALD/AMN 30 (K) 1.150G0.120 0.028G0.012 Controlb 35 (K) 0.782G0.054 0.008G0.003 aHeterozygotes X-ALD/AMN – 30 mothers X-ALD/AMN patients, aged 22–50 years (21). bControl – 35 healthy subjects (17 women and 18 men, aged 14–50 years (21).

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Downloaded from Bioscientifica.com at 10/05/2021 06:52:47AM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (2012) 166 The production of offspring by X-ALD/AMN males 293 median age 34 years) found basal testosterone below absence of neurological defects and/or adrenocortical the normal value in 12% (3/26) of subjects. insufficiency. Our studies showed similar results, i.e. low serum In patients with AMN impairment of Leydig cells, testosterone levels but in the normal range. Interest- adrenocortical and testicular interstitial cells, a decrease ingly, the lowest testosterone levels (P!0.0012) were in testosterone level resulting in infertility and impo- noted in three patients, who were in the advanced stage tence (5) has been found. Powers & Schaumburg (14) of disease, and descended further 1 to 1.5 years later. described damaged sexual function as the first symptom This observation confirmed that disease progression is of AMN in five out of six men. connected with relapsed testicular hypofunction. Results of recent investigations agree with Powers’ The above data shows that testosterone levels in previous observations (5, 11). By detection, peroxisomal X-ALD/AMN hemizygotes are in the lower part of the marker proteins showed the presence of normal range. However, the testosterone increase in most cell types in the testes (except for mature after human chorionic gonadotropin stimulation was spermatozoa), and during the course of spermato- estimated to be insufficient in 88%, indicating deficient genesis. The highest levels of peroxisomal protein–lipid testicular steroidogenesis (10). transporters, i.e. ABCD1, were found in Sertoli cells It was claimed earlier that VLCFA cholesterol ester (15). The above results claimed that peroxisomal accumulates intercellularly in X-ALD/AMN, probably b-oxidation is essential for lipid homeostasis in the leading to a shortage in the cholesterol pool (11) as a testes and for male fertility. Inactivation of this process basal substrate of steroid hormone synthesis and in a study done on mice, by knocking out the difficulties in their production. On the other hand, multifunctional protein-2, and ACOX1 (which take incorporation of VLCFAs in cell membrane lipids part in three of the four stages of the b-oxidation influences membrane rigidity and permeability, thus process), causes full degeneration of the testes and interfering with receptor binding of ACTH (12). infertility (16, 17). However, it is known that the Biochemical studies by measurement of LH levels (for X-ALD/AMN defect concerns another, preliminary a description of the endocrine function of the Leydig stage of the b-oxidation process (transport substrate). cell) and FSH levels (the sensitive endocrine parameter Actually, our results were based on the determination of the quality of spermatogenesis (13)) confirm to of testosterone, LH, and FSH levels in AMN patients at various degrees lesions of the Leydig and Sertoli cells in the time of diagnosis, which was about 6 years after the X-ALD/AMN patients. presumed onset of the disease. A comparison of LH and FSH results in X-ALD/AMN However, an analysis of family history and screening patients presented in different studies is summarized by detection of VLCFA as a biomarker for X-ALD/AMN in Table 2. The highest values, increased to 63.3% for showed that nine out of 11 married men had produced LH levels and 53.1% for FSH, were detected by offspring at a time before the occurrence of clinical Brennemann et al. (4). According to Moser et al. (9), manifestations. the respective values were lower: 42 and 30%. Assies This evidence could cause some surprise compared et al. (10) referred to 16 and 32% increased results for with earlier morphological and biochemical results these parameters. Our results for LH were similar to showing testicular dysfunctions (4, 5). After all, the those of Brennemann et al. (4), and for FSH was located evaluation of fertility on the basis of clinical and in the area referred to earlier (9, 10, and 4). Dysfunction biochemical parameters is only approximate. The final of Leydig cells and/or of the Sertoli cells was reported to efficient positive proof is one or more offspring. The be as much as 81.6% of 49 AMN patients (4). In our mechanism leading to demyelination, spinal cord finding it concerned 71% of males affected by axonal loss and adrenal as well as steroid hormone X-ALD/AMN. These results are consistent with defective insufficiency in X-ALD/AMN is still unknown. Increased testicular hormonogenesis and compatible with obser- VLCFA levels have been detected in infants (18, 19),at vation of pathological changes in the and least a few years before any onset occurs. However, in seminiferous tubules (5, 11). Assies et al. (10) found our present state of knowledge, it is impossible to predict that 77% of 26 patients with X-ALD/AMN had clinical the form and course of the disease. Among different or subclinical testicular insufficiency, even in the phenotypes of X-ALD we could only examine the reproductive function in those with a late onset. It Table 2 Increased LH and FSH levels in X-linked adrenoleuko- seems that the amount of reduction in fertility due to dystrophy/adrenomyeloneuropathy patients in different studies. impairment of spermatogenesis is connected with the degree of progressive disorders (20). Certainly, it is a LH FSH Number of complex problem and requires detailed investigation. (%) (%) Age (years) patients Source It must be noted that the date of conception of 42 30 (21–53) 69 (9) children was 1–12 years before the primary onset of the 63.3 53.1 36.2G1.5 (18.5–59) 49 (4) disease. Fathers did not know about the risk of the 16 32 34 (18–78) 26 (10) 57.1 42.9 34.7G5.9 (24–48) 17 Our results disease and transmission of the mutant gene to all their daughters. Obviously, it is necessary to highlight that

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Downloaded from Bioscientifica.com at 10/05/2021 06:52:47AM via free access 294 T J Stradomska and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2012) 166 in all daughters, obligatory heterozygotes showed an 8 Libber SM, Migeon CJ, Brown FR & Moser HW. Adrenal and increase in VLCFA parameters at heterozygote levels testicular function in 14 patients with adrenolekodystrophy or adrenomyeloneuropathy. Hormone Research 1986 24 1–8. (doi:10. (Table 1). 1159/000180533) The above results allow us to conclude that nine out 9 Moser HW, Bergin A, Naidu S & Ladenson PW. Adrenoleukodys- of 11 AMN-hemizygotes had a normal ability to trophy. Endocrinology and Metabolism Clinics of North America 1991 procreate in the pre-symptomatic period. Currently, 20 297–318. infertility of couples in Poland is estimated at about 10– 10 Assies J, Gooren LJ, van Geel B & Bart PG. Signs of testicular insufficiency in adrenomyeloneuropathy and neurologically 15% (22). Dysfunction in the ability to procreate in our asymptomatic X-linked adrenoleukodystrophy; a retrospective investigated group of AMN patients was evaluated at study. International Journal of Andrology 1997 20 315–321. 18.2% (2/11). The level is slightly higher than the index (doi:10.1046/j.1365-2605.1997.00066.x) for the whole Polish population. 11 Powers JM. Adreno- (adreno-testiculo- In X-ALD/AMN patients with late onset of the leuko-myelo-neuropathic complex). Clinical Neuropathology 1985 4 181–199. disease, the possibility of procreation and the resulting 12 Whitcomb RW, Linehan WM & Knazek RA. Effect of long-chain, inheritance by their daughters should be taken into saturated fatty acids on membrane microviscosity and adreno- consideration. All families should have genetic counsel- corticotropin responsiveness of human adrenocortical cells in vivo. ing concerning the inheritance of X-ALD. Journal of Clinical Investigation 1988 81 185–188. (doi:10.1172/ In our investigated group of X-ALD/AMN patients, JCI113292) significantly decreased fertility was not observed. 13 Bergmann M, Behr HM & Nieschlag E. Serum FSH and testicular morphology in male infertility. Clinical Endocrinology 1994 40 133–136. (doi:10.1111/j.1365-2265.1994.tb02455.x) Declaration of interest 14 Powers JM & Schaumburg HH. A fatal cause of sexual inadequacy in men: adrenoleukodystrophy. Journal of Urology 1980 124 The authors declare that there is no conflict of interest that could be 583–585. perceived as prejudicing the impartiality of the review reported. 15 Nenicu A, Lu¨ers GH, Kovacs W, Bergmann M & Baumgart-Vogt E. Peroxisomes in human and mouse testis: differential expression of peroxisomal proteins in germ cells and distinct somatic cell types of Funding the testis. Biology of Reproduction 2007 77 1060–1072. (doi:10. 1095/biolreprod.107.061242) This research did not receive any specific grant from any funding 16 Huyghe S, Schmalbruch HK, De Gendt K, Verhoeven G, Guillou F, agency in the public, commercial, or non-profit sectors. van Veldhoven PP & Baes M. Peroxisomal multifunctional protein 2 is essential for lipid homeostasis in Sertoli cells and male fertility in mice. Endocrinology 2006 147 2228–2236. (doi:10.1210/en. References 2005-1571) 17 Baes M & Van Veldhoven PP. Generalised and conditional 1 Kemp S & Wander RJA. X-linked adrenoleukodystrophy: very- inactivation of Pex genes in mice. Biochimica et Biophysica Acta long-chain fatty acid metabolism. ABC half-transporters and the 2006 1763 1785–1793. (doi:10.1016/j.bbamcr.2006.08.018) complicated route to treatment. Molecular Genetics and Metabolism 18 Moser AB, Kreiter N, Bezman L, Shou-en BS, Raymond GV, 2006 90 268–276. (doi:10.1016/j.ymgme.2006.10.001) Sakkubai N & Moser HW. Plasma very long chain fatty acids 2 Hudspeth MP & Raymond GV. Immunopathogenesis of adreno- in 3,000 disease patients and 29,000 control. Annals leukodystrophy: current understanding. Journal of Neuroimmunology of Neurology 1999 45 100–110. (doi:10.1002/1531-8249 2007 182 5–12. (doi:10.1016/j.jneuroim.2006.10.009) (199901)45:1!100::AID-ART16O3.0.CO;2-U) 3 van Geel BM, Assies J, Haverkort EB, Koelman JH, Vereeten B Jr, 19 Stradomska TJ & Tylki-Szyman´ska A. VLCFA levels determined by Wanders RJA & Barth PG. Progression of abnormalities in gas chromatography in the diagnosis of peroxisomal disorders. adrenomyeloneuropathy and neurologically asymptomatic Folia Neuropathologica 2009 47 306–313. X-linked adrenoleukodystrophy despite treatment with “Lorenzo’s 20 Aversa A, Palleschi S, Cruccu G, Silvestroni L, Isidori A & Fabbri A. oil”. Journal of Neurology, Neurosurgery and Psychiatry 1999 67 Rapid decline of fertility in a case of adrenoleukodystrophy. Human 290–299. (doi:10.1136/jnnp.67.3.290) Reproduction 1998 13 2474–2479. (doi:10.1093/humrep/13.9. 4 Brennemann W, Kohler W, Zierz S & Klingmulle D. Testicular 2474) dysfunction in adrenomyeloneuropathy. European Journal of 21 Stradomska TJ & Tylki-Szyman´ska A. Decreasing serum Endocrinology 1997 137 34–39. (doi:10.1530/eje.0.1370034) VLCFA levels in ageing X-ALD female carriers. Journal of 5 Powers JM. The pathology of peroxisomal disorders with Inherited Metabolic Diseases 2001 24 851–857. (doi:10.1023/ pathogenic considerations. Journal of Neuropathology and Experi- A:1013992224811) mental Neurology 1995 54 710–719. (doi:10.1097/00005072- 22 Drosdzol A & Skrzypulec V. Evaluation of marital and sexual 199509000-00014) interactions of Polish infertile couples. Journal of Sexual Medicine 6 Moser HW, Raymond GV & Dubey P. Adrenoleukodystrophy: new 2009 6 3335–3346. (doi:10.1111/j.1743-6109.2009.01355.x) approaches to a neurodegenerative disease. Journal of the American Medical Association 2005 294 3131–3134. (doi:10.1001/jama. 294.24.3131) 7 Stradomska TJ, Tylki-Szyman´ska A & Bentkowski Z. Very-long- Received 3 June 2011 chain fatty acids in . European Journal of Pediatrics Revised version received 29 October 2011 1999 158 226–229. (doi:10.1007/s004310051055) Accepted 1 November 2011

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