Preserving ovARian function through cryoprEservation and informing girLs with cancer about infertility due to gonadotoxic treatment

Childhood cancer treatment has resulted in excellent survival rates over the
past decades. Hence, awareness for serious early and long term toxicity is
increasing. Impaired fertility potential is one of the most relevant long term
morbidities as rated by survivors and their families. Currently, at
presentation of a new child with cancer, the focus is on an optimal and rapid
diagnostic process, in order to start treatment as soon as possible. In that
process, informing the girl with cancer and her family about toxicity of the
treatment, and in particular about the risk of potential gonadal damage, does
not always have the highest priority in pediatric oncology care.

This needs to be urgently changed, as patients and their families benefit from
knowing when the risk is low, as that gives them, at least some, relief. On the
other hand, subsets of patients with high risk of gonadal damage may benefit
from referral to fertility experts, who may advise them to preserve gonadal
material, for future purpose, preferably before childhood cancer treatment is
started. Of course this requires careful consideration in terms of potential
delays in anti-cancer treatment, balanced against the time required for
fertility counselling and preservation. However, in principal, a standard
infrastructure is needed to identify, assess on risk, inform all girls with
cancer and their families, counsel and offer preservation if applicable. Thus a
fertility care plan has been developed in the Princess Máxima Center.

Preservation options such as oophoropexy and oocyte vitrification are widely
accepted as standard of care, but for many patients not an appropriate option
for preservation. Recently, preservation methods, such as ovarian tissue
cryopreservation (OTC), have become available for children potentially at risk
for gonadal damage. Currently, for prepubertal girls with cancer, OTC is the
only possible way to potentially guarantee future biological offspring, by
utilizing the preserved ovarian tissue for auto-transplantation in the future.
Worldwide more than a 1000 children have undergone OTC without any major
complications and 298 (29%) were under the age of 13 years at time of harvest.
However, there is limited experience with autotransplantation of ovarian tissue
in girls and adolescents, thus outcome, efficacy and safety data in large
cohorts in this age group have not been published. In addition, 200 births
after auto-transplantation of OTC material, harvested in postpubertal women,
have been reported. Sixteen girls in which OTC was performed prepubertally or
peripubertally already underwent ovarian tissue transplantation (OTT) with the
aim to restore fertility, which led to 11 pregnancies and 9 live births.
specifically. OTC in adult women and girls is considered safe and the Princess
Máxima Centre offers OTC as standard of care. However, remaining challenges
regarding autotransplantation of ovarian tissue include the infiltration of
tumor cells in the harvested ovary, loss of primordial follicles in the
neovascularization period after OTT and the options regarding oocytes in the
medulla. Generally, medullar tissue is not cryopreserved for future use,
however, it can be used to detect minimal residual disease (MRD) in the ovary
and to develop maturation techniques. We aim to biobank all rest material after
preparation of the tissue for cryopreservation (medulla tissue) and biobank 15%
of the harvested ovarian tissue. The research on the 15% and rest material is
focused on the safety and efficacy of future use of the tissue and may include,
but is not limited to, identification of the presence of minimal residual
disease (MRD) in the ovarian tissue (Supplemental Table S1), the presence of
follicles in various stages in the medullar tissue, the viability of the germ
cells and explore the techniques of ex vivo maturation of follicles in the
cortex and medulla.
A gap of knowledge exists in the field of fertility care and OTC as a fertilty
preservation method and this study aims to fill these gaps.

Patients will be included in 3 prospective cohorts, cohort A, B, C and 1
retrospective cohort, cohort 0.
Cohort A:
The primary objective of cohort A is to show that by implementing a standard
infrastructure all newly diagnosed girls with cancer (or relapse) and their
families can be informed on fertility in a structured manner by the navigator
(nurse practitioner), prior to the start of gonadotoxic cancer treatment about
their particular risk of fertility impairment due to their gonadotoxic
treatment.
The secondary objectives of cohort A are:
- to describe the impact of receiving information regarding fertility at the
time of cancer diagnosis (at the end of treatment in the Kwaliteit van Leven In
Kaart (KLIK) portal),
- to determine the influence of diagnosis and treatment on the remaining
gonadal reserve, reflected by anti-müllerian hormone (AMH),
follicle-stimulating hormone (FSH), estradiol and luteinizing hormone (LH)
levels and menstrual cycles (secondary amenorrhea) at diagnosis and end of
treatment.

Cohort B:
The primary objective of cohort B is to offer counseling about fertility
preservation by the gynecologist to all families with a child with an
intermediate to high risk of infertility, which is clinically evaluated
according to the Edinburgh criteria and CED due to gonadotoxic treatment.
The secondary objectives of cohort B are:
- to explore the reasons for the decision to preserve or not,
- to qualitatively describe the impact of receiving information regarding
fertility at the time of cancer diagnosis combined with the counseling (1 - 2
months (range 1-6 months) after counseling).
- to determine the influence of diagnosis and treatment on the remaining
gonadal reserve, reflected by anti-müllerian hormone (AMH),
follicle-stimulating hormone (FSH), estradiol and luteinizing hormone (LH)
levels and menstrual cycles (secondary amenorrhea) at diagnosis, end of
treatment and 1 year after the end of treatment.

Cohort C:
The primary objective of cohort C is to provide safety data on the OTC
procedure in a large pediatric oncology population with respect to surgical
complications of ovarian tissue (OT) harvest for cryopreservation of ovarian
tissue within one month of the surgery and relatable to the surgery, including
the delay in initiation of cancer treatment.
The secondary objectives of cohort C are:
- to describe the number of OTC*s performed and the characteristics of patients
undergoing OTC,
- to determine the influence of OT harvest on the remaining gonadal reserve,
reflected by anti-müllerian hormone (AMH), follicle-stimulating hormone (FSH),
estradiol and luteinizing hormone (LH) levels and menstrual cycles (secondary
amenorrhea)
- to biobank all of rest material after preparation of the tissue for
clinical-use-cryopreservation (medulla tissue and tuba tissue).
- to biobank for research up to 15% of the harvested ovarian tissue.
The research on the 15% and rest material is focused on the safety and efficacy
of future use of the tissue and may include, but is not limited to,
identification of the presence of minimal residual disease (MRD) in the ovarian
tissue (Supplemental Table S1), the presence of follicles in various stages in
the medullar tissue, the viability of the germ cells and explore the techniques
of ex vivo maturation of follicles in the cortex and medulla.
- Check for MRD. Supplemental Table S1 describes the currently available
targets, and investigate whether the MRD presence in the ovarian cortex is also
present in the medullar tissue.
- The number of follicles in various stages in the harvested ovarian tissue in
the cortex and medulla
- To develop the optimal tools to identify and propagate oocytes in and from
this tissue to allow and enhance the probability of autologous transplantation
in the future if infertility has become apparent.
- To gain insight in the molecular profile of oocytes and supportive cells,
before and after propagation in vitro to develop the most optimal and safe
standard operation protocol for oocyte isolation and in vitro propagation, to
prepare for optimal circumstances of oocytes to mature.

Cohort 0:
The primary objective of the retrospective cohort 0 is to evaluate the
fertility care since the opening of the Princess Máxima Center in 2015 by
documenting number of informed patients
The secondary objectives of cohort 0 are:
- to describe the characteristics of informed and non-informed patients,
- to explore the reasons for the decision to preserve or not in counseled
patients since the opening of the Princess Máxima Center in 2015,
- to qualitatively describe the impact of receiving information regarding
fertility at the time of cancer diagnosis combined with the counseling in
counseled patients since the opening of the Princess Máxima Center in 2015.

Prospective longitudinal observational registry study design (cohort A,B,C).
Retrospective chart review in the first year of fertility care after opening of
the Maxima (cohort 0).

For patients in cohort A, B, C and the counseled subset of 0 extent and burden
are low. Cohort A receives standard of care only and no informed consent is
obtained. A blood sample (LH, FSH, estradiol and AMH levels) will be taken from
patients in cohort B and C as part of the research protocol one year after
cessation of treatment, if possible using existing vascular access ports, at
times when blood is collected for diagnostic/therapeutic purposes. In cases
that OTC is performed, one additional blood sample is collected the next day
preferably 27 hours after preservation (the timing may be altered for the
individual patient). Approximately at the end of the treatment patients and
parents in cohort A will answer a few questions in KLIK as SOC. 1-2 months
(range 1-6 months) after counseling patients and parents in cohort B are asked
to fill out a questionnaire on the impact of fertility information and
fertility counselling and the reason for the decision for or against fertility
preservation. Information concerning menses, weight and general health will be
collected at these time points during a routine visit at the outpatient clinic.

Benefits for the participants receiving the standard of care fertility care may
include a better knowledge on whether they carry a lower or higher risk of
gonadal impairment which may positively influence quality of life during and
after treatment, because patients and parents are informed on the effect of
cancer treatment on fertility. Another benefit is that patients and parents
have the possibility to improve fertility care and contribute to better
fertility care and preservation options for future patients. Preservation
options such as oophoropexy and oocyte vitrification are often not feasible for
children. Ovarian tissue cryopreservation (OTC) is therefore the only option to
preserve fertility and offered as SOC in the Princess Máxima Center.

To ensure standardized measurements and a complete follow up, all patients will
be invited to the outpatient clinic at the Princess Máxima Centre for follow-up
at these time points. (i.e. they will not go to *shared care hospitals* for
follow up at these time points.)