Testicular biopsies in young boys diagnosed with cancer to preserve future fertility- towards a safe and feasible autologous cell therapy in future.

More than 600 children per year are diagnosed with cancer in the Netherlands
(www.kwf.nl). Due to effective cancer treatments for these children, more than
80% of them will now survive. Currently, one in 250 of the adults is expected
to be a long-term survivor of childhood cancer (Brougham & Wallace, 2005).
Given this apparent success in paediatric oncology, long-term adverse effects
of cancer treatment are becoming increasingly important.
One of the major concerns with current treatment regimens are adverse effects
on fertility. Our follow up data indicate that infertility is common in
childhood survivors in general (~25%) and almost certain in young boys treated
with high dose chemotherapy, high dose alkylating agents or radiotherapy to the
testis region (Green 2014 lancet oncology p1215, Kenny 2018 JCO p2160). Having
survived their cancer, many of these boys/men and their -future- partners wish
to start a family just like other couples of reproductive age (van den Berg et
al, 2007).
In contrast to adolescent and adult men, for whom cryopreservation of semen
prior to start of chemotherapy is available and widely used, there are
currently no means to preserve reproductive potential in young boys diagnosed
with cancer. A high percentage of male childhood cancer survivors is therefore
doomed to remain childless, unless new technology is developed that allows them
to fulfil their child wish. Such technology is now appearing at the horizon:
cryopreservation of testicular tissue and autotransplantation of spermatogonial
stem cells (SSCs).
In 1994, successful transplantation of SSCs from one mouse to another was
reported (Brinster & Zimmermann, 1994). This landmark publication immediately
drew attention to the fact that this method could potentially be used to
preserve fertility in male cancer survivors. Since then, many groups, including
centers in the Netherlands (Amsterdam UMC, location AMC and UMC Utrecht) have
successfully performed SSC transplantation in various animal model systems
including mouse, rat, and bovine (Creemers et al., 2002; van Pelt et al., 2002;
Izadyar et al., 2003, Zhang et al., 2006, Mulder et al., 2018, Kadam et al.,
2019).
In recent years, preclinical work by the group of van Pelt (Amsterdam UMC,
location AMC, Amsterdam) has shown that cryopreservation and propagation of
human SSCs from testicular biopsies of adult human males is possible
(Sadri-Ardekani et al., 2009). Following this study, the same group has also
shown proof of principle, to a certain extent, of human pre-pubertal SSCs
propagation in vitro (Sadri-Ardekani et al., 2011). Many other research groups
are also focussed on creating efficient SSC propagation techniques with an
attempt at getting closer to the possibility of clinical application within the
next few years (Hou et al., 2015, Guo et al., 2015, Dong et al., 2019). One of
the main limitations of these studies is the lack of consensus on informative
human SSC markers. Therefore, more recent studies are aimed towards generating
robust candidate markers for identifying and characterising human SSCs against
other cell types within the testes (Guo et al., 2018, Sohni et al., 2019). This
will be significantly beneficial in isolating and propagating human
pre-pubertal SSCs with increased efficacy in comparison to the current
available method (Sadri-Ardekani et al., 2011).
Therefore, in the group of children who are treated with chemotherapy and/or
radiotherapy with high risk of infertility, affecting spermatogenesis
permanently, it is important to start cryopreservation of testicular tissue now
to be able to transplant SCCs later in life, which may well be more than 15
years after the cancer treatment.
Our request is in line with international activities. Ethical approval for
collection of testicular material was already approved in Amsterdam UMC,
location AMC ( Project NL 27690.000.09 which was active from September 2009
till September 2018) the Netherlands has already been approved for children.
Also in other countries such as Belgium (Vrije Universiteit Brussels, Centrum
voor Reproductieve Geneeskunde (www.brusselsivf.be / prof.dr. Herman Torunaye)
and the Katholieke Universiteit Leuven (Wyns et al, 2008;Wyns et al, 2007 /
prof.dr. Christine Wyns) and Sweden (Karolinska Institute / Keros et al., 2007
/ prof.dr. Outi Hovatta) the procedure is in place for the group of young boys
of who we expect high risk of infertility. We feel it is important to now also
offer young boys diagnosed with cancer in our centralized paediatric oncology
center, the Princess Maxima Center in Utrecht in the Netherlands the
possibility to store testicular tissue for possible future SSC
autotransplantation.

1. To preserve testicular tissue of young boys with cancer with high risk of
infertility for possible autologous transplantation in the future if
infertility has become apparent, and to identify germcells in the testicular
tissue.
2. To gain insight in the molecular profile of isolated testicular cell
fractions, including SSCs and supportive cells, before and after propagation in
vitro to develop the most optimal and safe standard operation protocol for SSC
isolation and in vitro propagation.
3. To follow up the unique cohort of testicular biopsied prepubertal boys (AMC,
Amsterdam) diagnosed with cancer on testicular damage by determining size
(ultrasound), hormonal profiles and pubertal progression and if possible semen
analysis during their yearly visit to the outpatient clinic. This procedure
will also be applied to the prospective cohort.

Young boys defined as Tanner stage lower or equal to P3G3 testes volume < 16 cc
(diagnosed with cancer at the Princess Maxima Center Utrecht who are scheduled
to undergo therapy that is predicted to have a high risk for infertility are
eligible for the study if unable to produce semen containing spermatozoa of
adequate quality by masturbation or electrostimulation. At our hospital, the
boys and parents are given an individual estimate on the probability of
infertility in relation to their scheduled treatment and estimated risk for
relapse. In addition, they will be informed about the procedure necessary to
obtain testicular tissue and the possible side-effects. It is then up to the
parents and patient to decide whether or not they want to proceed with storing
testicular tissue. Based on our previous investigations, the majority of
parents are willing for their son to undergo a testis biopsy for possible SSC
autotransplantation in the future (van den Berg et al, 2007, Gajjar et al.,
2015).
Follow-up cohort: The young boys from the above prospective cohort and the boys
of the previous cohort (CCMO NL 27690.000.09) who are still alive will receive
an informed consent if they would like to participate in the follow up project,
to identify possible damage as result of the biopsy. At visit to the clinic,
pubertal stage will be assessed, together with measurements of height, weight,
sitting height, and parental heights. The growth chart will be reconstructed
and bone age will be assessed. Laboratory evaluation will include measurements
of LH, FSH, testosterone, AMH and Inhibin B concentrations ultrasound to look
at defects of the testes and the volume, and if possible semen analysis.

1)To preserve testicular tissue of young boys with cancer and high risk for
infertility

.2) Long term follow up of the total cohort prepuberal boys who have undergone
a testicular biopsy. (AUMC NL 27690,000.09 + new cohort )

Under general anaesthesia a testicular biopsy will be taken; provided that a
signed informed consent is obtained from both parents. Scheduling the
testicular biopsy at the moment of performing pre-treatment procedures (such as
placement of a long term tunneled catheter) minimizes the burden for the
patient and will not postpone start of therapy. To minimize the risk of the
biopsy, the procedure will only be done on one testis under the condition that
there is no history of testicular torsion or cryptorchidism. The biopsy will be
approximately 0.6-0.8 ml in size and will never exceed 30% of one testis. The
technique will ensure that substantial loss of testicular tissue does not
occur. Complication rate is extremely low. a hematoma or infection can occur
but is mild and in most cases does not lead to postponement of treatment.
Follow-up will be done at the moment of follow-up for the malignancy. No
additional hospital visits are foreseen in conjunction with this proposal.