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BRIEF REPORT |
Departments of Endocrinology and Andrology (T.S., P.R.M.), Gynecological Endocrinology (A.G.), and Pathology (R.T.), and Divisions of Infectious Diseases (L.B.) and Nephrology (F.S., G.G.), Spedali Civili, and Department of Medicine (E.A.R.), University of Brescia, 25123 Brescia, Italy
Address all correspondence and requests for reprints to: Dr. Alessandro Gambera, Via XXV Aprile, 10, 24058 Romano di L. (BG), Italy. E-mail: alessandro.gambera{at}tin.it.
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Abstract |
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Objective: The objective of the study was to examine the effects of this amyloidosis on testicular structure and function.
Design: This was an observational study in which patients with testicular amyloidosis were characterized.
Setting: The study was carried out at the Endocrinology Department of Brescia University.
Patients or Other Participants: Over a 13-yr period, 25 patients were found to be affected by leucine-75-proline apolipoprotein A-I testicular amyloidosis. Thirteen had the testicle as the first or only organ involved (group 1); in 12 testicular damage followed that of other organs (group 2).
Interventions: There were no interventions.
Main Outcome Measure: Hormone and lipidic profiles, semen analysis, echographic volume of testicles, testicular histology, and genetic analysis were carried out.
Results: Group 1 patients were younger than those of group 2. In group 1, eight had hypergonadotropic hypogonadism and five were normogonadic with high gonadotropins; in group 2 all subjects were hypogonadic. All men had low high-density lipoprotein values. Group 1 patients were macroorchid, whereas the testicular volume was at the highest limit in group 2 (group 1 vs. group 2, P < 0.05). All men in the first group and six in the second group were azoospermic; the remaining had oligoposia. Biopsies showed the germinal epithelium replaced by amyloid. Leydig cells were essentially preserved in normogonadic but not hypogonadic patients.
Conclusions: This amyloidosis may determine infertility, macroorchidism, and hypogonadism. Endocrine impairment follows spermatogenic impairment.
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Introduction |
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Twelve Apo A-I variants are known to be responsible for systemic amyloidosis, mainly involving the kidneys, heart, nerves, liver, and larynx (2).
Recently the new amyloidogenic variant Leucine-75-Proline (Leu75Pro), due to a thymine to cytosine transition in the Apo A-I gene, has been demonstrated to cause asymptomatic cholestatic hepatopathy and nephropathy (3, 4).
Testicular amyloidosis is rare. Recently isolated testicular amyloid deposits were found in two hypogonadic and infertile patients (5, 6) but also in a heterozygous man for a nondetermined type of familial amyloidosis, associated with an abnormal production of Apo A-I (7).
Few cases of testicular involvement in patients affected by primary systemic amyloidosis have been described, but in literature the cases of early testicular involvement, preceding cardiac or multiple organ disease are purely anecdotal (8, 9, 10, 11).
Secondary involvement of the testicles during chronic inflammatory diseases or neoplasias is much more frequent (6, 9, 12, 13).
The aim of this study is to examine the effects of systemic amyloidosis due to the Leu75Pro variant of ApoA-I on testicular structure and function.
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Subjects and Methods |
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Medical history was collected and all patients had a physical examination. Particular attention was paid to libido, erectile function, past testicular pathologies, difficulty in fathering, and age of conception.
All patients underwent an endocrine and lipidic profile, semen analysis, echographic evaluation of the testicles, testicular biopsies for histological diagnosis, and DNA analysis.
Total testosterone (T), SHBG, LH, and FSH, cholesterol, triglycerides, and HDL cholesterol were measured at 0900 h in a fasting condition. Free testosterone was calculated using the Vermeulen formula (14). Ultrasonographic evaluation was carried out by the same physician and testicular volume was calculated using the Lambert formula (15). A volume of 15–25 ml is considered normal (16). Semen analysis was read using the World Health Organization criteria.
The same pathologist examined the biopsies after hematoxylin-eosin and Congo-red staining under polarized light. An antibody panel against 
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-chains, serum amyloid-A, fibrinogen, and apoA-I (Dako, Milan, Italy) was used on the paraffin-embedded sections to immunohistochemically characterize the amyloid deposits.
DNA analyses were carried out using standard procedures and direct sequencing was used to search for any Apo A-I mutation in the whole gene (3).
A written informed consent was given by all patients and the institutional review board approved the study.
Statistical analysis
All values were expressed as mean ± SD. Student’s unpaired t test was used to compare the hormone levels, lipid parameters, and echographic measures of the different groups of patients. P < 0.05 was considered statistically significant.
Hormone assays
Hormones were determined by an automated analyzer using chemiluminescent immunoassay: the Architect System (Abboth Diagnostic Division, Milan, Italy) for LH, FSH, and T (reagents by Ortho-Clinical Diagnostics, Amersham, UK); and Immulite Automated Analyzer 2000 (Diagnostic Product Corp. Medical Systems, Genoa, Italy) for SHBG (reagents by Diagnostic Products Corp., Los Angeles, CA).
Our laboratory participates in the External Quality Assessment Schemes (UK-NEQAS, Birmingham, UK) and Immunocheck (IFC-CNR-EQAS, Pisa, Italy).
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Results |
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shows the clinical characteristics of the two groups of patients at diagnosis. Men affected by testicular amyloidosis as early or single manifestation of the disease were included in group 1. Group 2 consisted of those with testicular disease after other organ involvement: six renal; one hepatic; four renal and hepatic; and one renal, hepatic, and adrenal.
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On physical examination, 11 men in the first group (84.6%) but only four in the second (33.3%) were macroorchid with testicles appearing smooth and tough.
Group 1 came to observation due to infertility, reduced libido, erectile dysfunction, and gynecomastia, isolated or variously associated, and only three by screening when a family member was affected.
All group 2 subjects were diagnosed by screening as being patients with other organ damage. They complained of reduced libido, erectile dysfunction, but only 8.3% of infertility (Table 1
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Only five men in group 1 (38.5%) had managed to father, from 3 to 27 yr before the diagnosis of testicular amyloidosis, whereas 11 (91.7%) men in group 2 had fathered, from 10 to 48 yr before the onset of testicular disease.
Table 2 shows the clinical, endocrine, echographic, and lipidic parameters of men with early and late testicular amyloidosis.
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Normogonadic patients had normal testosterone levels with high gonadotropins, whereas the hypogonadic subjects had significantly lower androgen levels and higher gonadotropins (P < 0.05).
All patients in the second group had clear hypergonadotropic hypogonadism, with androgen values significantly lower and LH significantly higher if compared with those of group 1. No statistical differences in androgen and gonadotropins levels were found between group 2 and the hypogonadic patients of group 1.
In all groups, the mean SHBG, triglycerides, and cholesterol appeared normal. HDL values were low in both groups, without statistical differences.
Ultrasonographic investigation confirmed the increased volume of the testicles in the first group (19–44 ml), without statistical differences between the two subgroups.
In the second group, ultrasonography revealed a normal volume for the left testicle with the right testicle at the top of the normal values. Group 2 testicles were smaller than those of group 1 (P < 0.05). Mean left volume of group 2 was also significantly lower than that of the hypogonadic men in group 1.
Semen analysis in group 1 showed complete azoospermia in 100% of men. Moreover, a 29-yr-old man had previous semen analyses showing an evolution from normozoospermia to azoospermia in only 23 months. Six men in group 2 had azoospermia, and in six the examination resulted not feasible due to oligoposia.
Biopsies showed abundant deposits of amorphous eosinophilic material positive to Congo Red staining. Amyloid was deposited in the epithelium of seminiferous tubules, causing their narrowing or obstruction with the disappearance of Sertoli cells in most cases. Only in two subjects of the normogonadic subgroup was the histological picture characterized by areas of complete replacement of the epithelium or with only Sertoli cells mixed with areas of partial damage of the germinal epithelium, with the evidence of all stages of spermatogenesis to spermatids.
All biopsies showed amyloid deposits also in the interstitium, in the walls of the arteries, veins, and capillaries, leading to an increase in the vessel walls.
Biopsies belonging to hypogonadic patients showed no Leydig cells or few cells without a well-structured organization. On the contrary, few normal aggregates of Leydig cells were present in normogonadic men. Immunohystochemical analysis showed considerable immunoreactive amyloid deposits with the anti-Apo A-I antibody but not with the other antibodies.
Direct DNA sequencing showed that all 25 patients were heterozygous due to the same thymine to cytosine mutation at position 1772 of the Apo A-I gene, corresponding to a leucine to proline replacement at position 75 of the mature polypeptidic chain. No patient had other Apo A-I variants.
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Discussion |
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The progressive spermatogenic impairment, shown by the two biopsies with mixed tissue morphology, proceeds to the gradual endocrine damage. In fact, in group 1 five azoospermic patients showed Leydig cells only focally involved and the high gonadotropin levels being able to maintain a normal T secretion (subclinical hypogonadism). On the contrary, complete damage to the Leydig cells was seen in hypogonadic patients.
Spermatogenetic damage may be caused by the obliteration of intratesticular canaliculi, the direct effect of amyloid on the seminiferous tubules (12), or hypoxia due to vessel lumen obstruction (9).
Usually pathologies causing impairment of spermatogenesis lead to microorchidism (congenital and chromosomal abnormalities, Klinefelter, cryptorchidism, myotonic dystrophy), whereas the testicular volume in this kind of amyloidosis is normal or high. In particular, the testicular volume of group 1 is increased, whereas that of group 2 is normal or high.
The testicular volume of elderly men (group 2) was significantly lower than that of the hypogonadic men in the first group, who had similar degree of hypogonadism.
In literature age-related reduction in testicular volume is demonstrated only from the eighth decade of life (17), a clearly higher age than that of group 2 patients (range 50–79 yr).
We can suppose that the mechanism of testicular damage may be different between the younger patients (group 1) and the older ones (group 2). Studies on transthyretin amyloidosis show that the oligomeric intermediates of the fibril assembly are directly cytotoxic, causing oxidative stress and activation of apoptosis (18). Testicular damage in group 1 may be mainly due to a rapid replacement of parenchyma by amyloid, leading to macroorchidism, whereas in the second group, the cytotoxic mechanism may be predominant, causing failure without significant increase in volume.
The gene penetrance, the proportion of testicular damage in carriers or in patients already affected by other organ disease, is still unknown.
The high number of patients described in this study is likely to be due to a founder effect, i.e. to a dominant mutation in previous generations.
Azoospermic men, normogonadic or not, with or without macroorchidism and low HDL levels are to be considered highly suspicious, even if there is no family history. Because this disease, also shown in a different ethnic background (19), may involve only the testicle, a small part of idiopathic infertilities may be so explained.
Why the testicle is a target organ is still unknown. Apo A-I in the testicle removes cholesterol from Sertoli cells and carries it to Leydig cells (20). Conformational changes of the mutated protein during cholesterol trafficking may start proteolitic remodeling, producing fibrillogenic oligomers.
In conclusion, infertility, subclinical or overt hypogonadism, and macroorchidism are clinical features of Leu75Pro Apo A-I amyloidosis. Testicular involvement may be the first or only manifestation of disease, with deterioration of spermatogenesis and steroidogenesis from the third decade of life. Semen analysis is recommended annually in young asymptomatic carriers of the mutation, and sperm retrieval is suggested. Repeated evaluation of endocrine parameters is advisable annually to start androgen replacement therapy, when necessary.
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Footnotes |
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First Published Online February 19, 2008
Abbreviations: Apo, Apolipoprotein; HDL, high-density lipoprotein; Leu75Pro, leucine-75-proline; T, testosterone.
Received July 25, 2007.
Accepted February 12, 2008.
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References |
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