Unravelling the mechanism of azoospermia in nephropathic cystinosis

General background
Cystinosis is an autosomal recessive lysosomal storage disorder characterized
by widespread lysosomal cystine accumulation and crystal formation in all body
tissues. Various endocrine organs are affected.
In a first organized report on reproductive function in male cystinosis
patients, primary hypogonadism has been documented in a substantial subset of
patients (approximately 70%) (Chik et al., 1993).
Recently, in a small male cystinosis patient cohort study performed by our
group, azoospermia was remarkably observed in all of these patients, despite a
normal pituitary-testicular axis, early cysteamine treatment and a normal renal
function (eGFR) (Besouw et al., 2010). However, testicular biopsy in one of
these patients showed a normal spermatogenesis (Johnson score 8 - 9) (Besouw et
al., 2010). As primary hypogonadism has shown to be present in a substantial
proportion of the adult patients, we can assume that fertility could gradually
decrease from the post-pubertal stage onwards, with increasing age.

1. Fertility effects of cysteamine.
Based on in vivo and in vitro data of animal studies as well as studies
performed on human subjects, it may be hypothesized that cysteamine treatment
could contribute to sub/infertility through (1) endocrine effects, (2) direct
spermicide effects, and (3) effects on posttranslational modifications during
epididymal sperm maturation.

1.1 Endocrine effects
Animal studies have shown a suppressive effect of cysteamine on gastric
somatostatin production (Szabo et al., 1981). Fukuhara et al documented an
inverse correlation between gastric density of somatostatin producing cells and
plasma somatostatin levels on one hand, and plasma ghrelin levels on the other
hand, after cysteamine administration (Fukuhara et al., 2005). These data
suggest an indirect stimulation of ghrelin secretion by cysteamine through
suppression of somatostatin.
In vitro and in vivo data have suggested a local gonadal effect of ghrelin on
Leydig cell proliferation, testosterone secretion and regulation of apoptosis
in spermatogenesis (Camninos et al., 2014). In vivo data in human male and
female subjects have also identified a systemic effect of ghrelin on the
hypothalamic pituitary axis. Suppression of LH and FSH production and increased
prolactinemia after ghrelin bolus injection has been clearly documented
(Camninos et al., 2014; Kluge et al, 2007; Lanfranco et al., 2008; Messini et
al., 2009).

1.2 Direct spermicide effects
The direct effect of cysteamine on sperm function has been demonstrated by a
potent dose- dependent reversible inhibition of sheep testicular hyaluronidase
(IC50 150 µg/ml), and a moderate reversible inhibition of human acrosin by
cysteamine (IC50 370 µg/ml) (Anderson et al., 1997). A weak sperm-immobilizing
capacity of cysteamine has been documented in a modified Sander-Cramer test
with an IC50 of 16 ± 3.5 mg/ml. A significant spermicidal effect of cysteamine
has been observed in a modified Sander-Cramer test when motility is evaluated
after 10 minutes, with 80% inhibition of rabbit sperm motility at 10µg/ml
cysteamine, and complete inhibition at cysteamine concentrations ranging from
50µg/ml to 500µg/ml (Anderson et al., 1997). Whether cysteamine penetrates the
blood-testes barrier and which concentrations are reached in human sperm is
unknown.
1.3 Effects on posttranslational modifications
Disulfide bond formation constitutes an important part of the posttranslational
modifications taking place during the epididymal sperm maturation process
(Sutovsky, 2014). Several organelles in the tail of spermatozoa (e.g. the outer
dense fibres, outer mitochondrial membrane, fibrous sheath, and connecting
piece) contain sulfhydryl rich groups which undergo oxidation during epididymal
transit. The disulphide bond formation in these organelles is important in
stabilizing the sperm tail structure and acquiring the normal wave pattern of
sperm motility. Since cysteamine is a disulphide bond breaking agent, it can be
hypothesized that it might exert a negative effect on these necessary
posttranslational modifications.

2. Animal models and cell lines in fertility of cystinosis
Unfortunately, in our previous study we have demonstrated that the male Ctns-/-
C57BL/6 murine model is not suitable for unravelling the pathogenesis of
infertility in cystinosis since it does not exert an infertility phenotype
(Besouw et al., 2012). Also, no other cystinosis animal models with an
infertility phenotype are currently available.

To clarify the mechanism of azoospermia in male cystinosis patients

To determine the cause of azoospermia in male cystinosis patients as
obstructive or non-obstructive by non-invasive means (clinical examination,
reproductive hormonal profile, ejaculated semen analysis, analysis of seminal
biomarkers, ultrasonographic biomarkers, urine sampling) in comparison to
patients with confirmed obstructive azoospermia (group B) and age-matched
healthy control subjects (group C).

This trial is a prospective, interventional, case-control study.

The burden and risks for the subjects involved in this study, are both minimal.
The burden exists in having a visit with an Andrology specialist, consisting of
a history and clinical examination, a venapuncture, a testicular ultrasound,
and making a sperm- and urine sample. Depending on the duration of abstinence
prior to making this sperm sample, it can be necessary to make a second
appointment for making this sperm sample.
Risks are limited to the (minimal) invasive procedure of a venipuncture;
although these risks are not different in nature or severity then in case of a
classical venipuncture.