Preservation of Ovarian Cortex Tissue in Girls With Turner Syndrome
Phase: NA
Conditions: Fertility Preservation, Ovarian Tissue Cryopreservation, Turner Syndrome, Live Birth, Premature Ovarian Failure
Sponsor: Radboud University Medical Center

Protocol ID NL57738.000.16 
 
  
 
 
 
 
 
RESEARCH PROTOCOL 
 
 
 
 
 
Preservation of ovarian 
cortex tissue in girls with 
Turner syndrome  
 
A national exploratory intervention study  
 
 
 
Version 7.7 – June 2020 
 
This study protocol was developed with input and feedback of: 
Patient representatives of the patient support group (Turner Contact Nederland (TCN) and 
the Disorders of Sexual Differentiation (DSD) sounding board group of the Radboud 
university medical center)  
Dr. J. in ’t Hout, statistician Radboud university medical center 
Dr. M. van Gelder, methodologist Radboud university medical center 
Prof. Dr. E. Van Leeuwen, medical ethicist Radboud university medical center 
Prof. dr. O. Hovatta, gynaecologist who performed cryopreservation of ovarian biopsies in 
Swedish patients with Turner Syndrome (Sweden) 
Prof. dr. C.H. Gravholt, endocrinologist (Denmark), expert in Turner Syndrome Guideline 
Development 
Prof. dr. A.H. Balen, gynaecologist (United Kingdom), expert in fertility care 
 
 

Protocol ID NL57738.000.16 
 
  
 
 
 
 
PROTOCOL TITLE:  Preservation of ovarian cortex tissue in girls with Turner 
syndrome: A multicenter exploratory intervention study 
 
Protocol ID 
NL57738.000.16 
Short title 
TurnerFertility 
Version 
7.7 
Date 
June 2020 
Coordinating 
investigators/project leaders 
K. Fleischer, MD, PhD, gynaecologist specialized 
in reproductive medicine, Radboudumc, Nijmegen 
(NL) 
A.A.E.M. van der Velden, MD, PhD, paediatric 
endocrinologist, Radboudumc, Nijmegen (NL)  
R. Peek, PhD, senior scientist, biologist 
specialized in fertility preservation, Radboudumc, 
Nijmegen (NL) 
M.J. Schleedoorn, MD, PhD student, physician 
reproductive medicine, Radboudumc, Nijmegen 
(NL) 
S. Nadesapillai, MD, PhD student, physician 
reproductive medicine, Radboudumc, Nijmegen 
(NL) 
Principal investigators 
K. Fleischer, MD, PhD, gynaecologist specialized 
in reproductive medicine, Radboudumc, Nijmegen 
(NL) 
R.J.T. van Golde, MD, PhD, gynaecologist 
specialized in reproductive medicine, MUMC, 
Maastricht (NL) 
Subsidizing party 
Radboud university medical center 
Independent expert  
H.J.L.M. Timmers, MD, PhD, endocrinologist 
 
 
 

Protocol ID NL57738.000.16 
 
  
 
 
 
 
PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Head of Department: 
D.D.M. Braat, MD, PhD, professor of 
reproduct
ive medicine, Radboudumc, 
N
ijmegen (NL) 
 
June 2020  
Coordinating Investigators / Project 
leaders / Principal Investigators: 
K. Fleischer, MD, PhD, gynaecologist 
special
ized in reproduct
ive medicine, 
Radboudumc, N
ijmegen (NL) 
 
June 2020 
M.J. Sch
leedoorn, MD, PhD student, 
physician reproduct
ive medicine, 
Radboudumc, N
ijmegen (NL) 
 
June 2020  
R.J.T. van Golde, MD, PhD, 
gynaecologist special
ized in 
reproduct
ive medicine, MUMC, 
Maastricht (NL) 
 
June 2020 
R. Peek, PhD, sen
ior scient
ist, 
biologist special
ized in fert
il
ity 
preservat
ion, Radboudumc, N
ijmegen 
(NL) 
 
June 2020  
A.A.E.M. van der Velden, MD, PhD, 
paediatric endocrinologist, 
Radboudumc, N
ijmegen (NL)  
 
June 2020 
S. Nadesapil
lai, MD, PhD student, 
physician reproduct
ive medicine, 
Radboudumc, N
ijmegen (NL) 
 
June 2020 
 
  

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TABLE OF CONTENTS 
 
 
SUMMARY ................................................................................................................................. 6 
1. INTRODUCTION AND RATIONALE ..................................................................................... 8 
2. OBJECTIVE ........................................................................................................................... 9 
3. STUDY DESIGN .................................................................................................................... 9 
4. STUDY POPULATION......................................................................................................... 10 
5. TREATMENT OF SUBJECTS ............................................................................................. 12 
6. METHODS ........................................................................................................................... 15 
7. SAFETY REPORTING......................................................................................................... 18 
8. STATISTICAL ANALYSIS.................................................................................................... 20 
9. ETHICAL CONSIDERATIONS ............................................................................................ 25 
10. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION............................... 31 
11. BIOBANK GOVERNANCE ................................................................................................ 32 
12. REFERENCES................................................................................................................... 35 
 
 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
ABR 
ABR form, General Assessment and Registration form, is the application 
form that is required for submission to the accredited Ethics Committee (In 
Dutch, ABR = Algemene Beoordeling en Registratie) 
AE 
AMH 
BMI 
Adverse Event 
Anti-Müllerian hormone 
Body Mass Index 
CA 
Competent Authority 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
CV 
DSD 
Curriculum Vitae 
Disorders of Sexual Differentiation 
DSMB 
E2 
e-CRF 
EDC 
FFPE 
FISH 
Data Safety Monitoring Board 
Estradiol  
Electronic case report forms 
Electronic Data Capture 
Formalin Fixated and Paraffin Embedded 
Fluorescence in situ hybridization 
FP 
FSH 
GCP 
HIV 
IBM SPSS 
Fertility Preservation 
Follicle-stimulating hormone  
Good Clinical Practice 
Human Immuno-deficiency Virus 
International Business Machines Corporation Statistical Package for the 
Social Sciences 
IC 
ICH 
 
IQR 
LBR 
Informed Consent 
international Conference on Harmonisation of technical requirements for 
registration of pharmaceuticals for human use 
Inter-quartile ranges 
Live birth rate 
LH 
METC  
 
OTC 
PhD 
POI 
POF 
Luteinizing hormone  
Medical research ethics committee (MREC); in Dutch: medisch ethische 
toetsing commissie (METC) 
Ovarian tissue cryopreservation 
Doctor (of Philosophy), Academic title 
Premature ovarian insufficiency 
Premature ovarian failure 
(S)AE 
SD 
(Serious) Adverse Event  
Standard deviation 
SPC 
Summary of Product Characteristics (in Dutch: officiële productinfomatie IB1-
tekst) 
Sponsor 
The sponsor is the party that commissions the organisation or performance 
of the research, for example a pharmaceutical 
company, academic hospital, scientific organisation or investigator. A party 
that provides funding for a study but does not commission it is not regarded 
as the sponsor, but referred to as a subsidising party. 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
TS 
Turner syndrome 
Wbp 
Personal Data Protection Act (in Dutch: Wet Bescherming Persoonsgevens) 
WMO 
Medical Research Involving Human Subjects Act (in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen 

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SUMMARY 
 
Rationale: Infertility due to premature ovarian insufficiency is a major concern for 
girls with Turner syndrome (TS) and their parents. Physicians are often asked about 
possible options to preserve their fertility. However, despite some experimental case 
reports, clear evidence for fertility preservation in these girls is lacking and many 
questions remain. Without evidence on the effectiveness of fertility preservation it 
cannot routinely be offered to girls with ovarian failure due to Turner syndrome. 
 
Objective: To investigate the occurrence of live birth in women with TS after ovarian 
tissue cryopreservation in childhood followed by auto transplantation in adulthood. 
 
Study design: A national multicentre exploratory intervention study  
 
Study population: Girls diagnosed with Turner Syndrome, aged 2-18 years. 
 
Intervention: Ovarian tissue cryopreservation in childhood followed by auto 
transplantation in adulthood. In order to obtain the ovarian tissue for 
cryopreservation, all girls must undergo a laparoscopy under general anaesthesia 
which will be performed in academic/university clinics with paediatric surgery. During 
the laparoscopic intervention, a unilateral oophorectomy will be performed, thereby 
leaving the other ovary intact for hormone production, ovulation, spontaneous 
pregnancies and as an auto transplantation site for cryopreserved-thawed ovarian 
cortical tissue later on. Furthermore, a small sample of the ovarian cortex will be 
used to assess the oocyte quality and genetics (e.g. the presence of germ line 
mosaicism). Oocytes will be karyotyped by using Fluorescence in situ hybridization 
(FISH). Karyotypic and hormonal data will be collected once at the yearly clinical visit 
at the paediatric-endocrinologist. Therefore, a buccal swab, one portion of urine, and 
one extra blood sample of 3.5mL will be taken and evaluated during the routine 
laboratory evaluation. 
In the future, auto transplantation of frozen-thawed ovarian cortex strips will be 
performed. 
 
Main study parameters/endpoints: The main study endpoint is live birth after auto 
transplantation of cryopreserved-thawed ovarian cortical tissue in the future.   
 
Nature and extent of the burden and risks associated with participation, benefit 
and group relatedness:  
The primary objective remains to preserve the fertility of the respective (minor) 
patient, facing a very high risk of POI of 95-98%. Disadvantages of participating in 
this study are the potential risk of complications related to the laparoscopic unilateral 
oophorectomy and/or the unknown effect on future fertility of these girls. Moreover, 

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the procedure might raise false hope in patients (and/or parents) about the chance of 
getting pregnant after auto transplantation of cryopreserved-thawed ovarian tissue in 
the future. However, we attempt to overcome this by extensive and objective 
information provision by both written materials and face to face counseling.  
 
 
 
 
 

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1. INTRODUCTION AND RATIONALE 
 
Turner syndrome (TS) is a condition in which a female is partly or completely missing 
an X chromosome. The complete or partial loss of an X chromosome leads to 
dysmorphic signs and a constellation of physical findings that often includes short 
stature, premature ovarian insufficiency (POI) or failure (POF), and cardiac and renal 
abnormalities. Signs and symptoms vary among girls with TS, and intelligence is 
generally normal.  
TS is the most common chromosomal abnormality in females, affecting 1 on 2,500 
live born girls [1-3]. In some cases, TS is diagnosed prenatally by chorionic villus 
sampling or amniocentesis in case of cystic hygroma or intra-uterine growth 
retardation. The majority of girls receive the diagnosis TS before the age of 5 
because of growth retardation. Occasionally, the diagnosis TS is concluded in 
adolescence because of delayed puberty. 
Several interview studies [4, 5] show that, regardless of age, the possible or probable 
infertility due to POI is the main concern for girls/women with TS and also for their 
parents. Due to an accelerated loss of germ cells, most girls with TS undergo ovarian 
failure at a very early age, starting as early as 18 weeks fetal age [6, 7]. The timeline 
at which this occurs is less clear, and may be different for each patient with TS [7]. 
Spontaneous pregnancies are rare, and occur in approximately 2-5% of women with 
TS [8-10]. Up to 30% of females with TS have some pubertal development and up to 
10% experience spontaneous menarche [11]. Hence, 90% of girls with TS will suffer 
from POI during childhood, and thus do not enter puberty.   
A Swedish research group reported that primordial follicles can be found in the 
ovaries of both mosaic and non-mosaic girls with TS up to 17 years of age [12]. Due 
to this new insight, physicians are often asked by girls with TS and their parents 
about the options of fertility preservation [13]. Fertility preservation includes the 
cryopreservation of the patient’s own gametes, either by preserving mature oocytes 
or ovarian tissue containing primordial follicles. Cryopreservation of mature oocytes 
is a proven fertility preservation approach but can be performed only in post pubertal 
females, since oocyte maturation requires ovarian stimulation with exogenous FSH 
administration followed by transvaginal ultrasound-guided oocyte retrieval [14]. 
Ovarian tissue cryopreservation, however, appears to be a promising technique to 
preserve the fertility of younger girls with TS as it provides the possibility to store a 
larger number of primordial follicles before their disappearance [15]. 
Cryopreservation of ovarian tissue is accepted and established in girls and young 
women undergoing gonadotoxic cancer treatments with a risk of POI ≥ 50% [16-19]. 
Over the past decades several clinical guidelines [20-29] and decision tools [30-35] 
for ovarian tissue cryopreservation in cancer patients have been developed and 
evaluated. Auto transplantation of cryopreserved-thawed ovarian cortical tissue in 
cancer survivors, has resulted in restoration of ovarian function (63-67% of cases), 
pregnancies (18-35% of cases) and live births (10-25% of cases) [19, 36-42].  

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Despite the fact that fertility preservation in girls facing fertility threatening cancer 
treatment with a risk of POI ≥ 50% is accepted and established [16-18], there are 
currently no recommendations on fertility preservation in girls with TS facing a very 
risk of POI of 90%. Still, preservation procedures have been performed 
experimentally in girls and adolescents with TS [12, 15, 43, 44]. The promise of 
fertility preservation is at present hypothetical, given that no girl with TS who has 
undertaken these approaches thus far has returned for auto transplantation or IVF 
with embryo transfer. Further research is needed to provide supporting evidence for 
the efficiency of fertility preservation techniques in this specific group of patients. 
With regard to the occurrence of spontaneous pregnancies (2-5%), it is fundamental 
to identify which girls with TS might benefit from fertility preservation. Hence, there is 
a need for reliable markers to estimate the prognosis of the ovarian function for each 
individual patient as early in life as possible, to more precisely pinpoint the urgency of 
fertility preservation. Herewith, proper counseling is possible and if wished, treatment 
options can be chosen for each individual patient (i.e. cryopreservation of ovarian 
tissue, cryopreservation of mature oocytes or awaiting spontaneous pregnancy). 
Furthermore, a decision aid based on the information needs of girls with TS and their 
parents is needed to help them to make a deliberate decision. 
 
2. OBJECTIVE 
To investigate the occurrence of live birth in women with TS after ovarian tissue 
cryopreservation in childhood followed by auto transplantation in adulthood. 
 
3. STUDY DESIGN 
A multicenter exploratory intervention study. 
 
The participating centres are: 
• Radboud university medical centre, Nijmegen, The Netherlands (Principal 
Investigator): 75 patients 
• Maastricht University Medical Centre, Maastricht, The Netherlands: 25 
patients 
 
Patients will be included between 2017 – 2020.

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4. STUDY POPULATION 
 
4.1 Population (base) 
The study population includes girls with TS. 
 
4.2 Inclusion criteria 
In order to be eligible to participate in this study, a subject must meet all of the 
following criteria: 
 
• Girls and young females with classic Turner (i.e. 45X monosomy) or Turner 
variants (e.g. 45X/46XX mosaicism, ring X mosaicism, isochromosome X), 
• Aged 2 through 18 years, 
• who completed the diagnostic work up phase of TS including routine cardiac 
screening*, 
• whose agreement to participate in this study has been signed by the parents 
(girls aged 2-11 years old),  
• whose agreement to participate in this study has been signed by the patient 
and her parents (girls aged 12-15 years old), 
• whose agreement to participate in this study has been signed by the patient 
(girls aged 16-18 years old). 
 
4.3 Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from 
participation in this study: 
 
• Contra-indications for laparoscopic unilateral oophorectomy under general 
anaesthia (e.g. severe cardiovascular comorbidity and/or BMI >40 kg/m2)*, 
• Contra-indications for cryopreservation (i.e. active HIV, hepatitis-B or hepatitis-
C infection) 
 
*Based on the international Cincinatti Turner Guideline Concensus Meeting, July 
20161 and consultation of Dutch cardiologists, paediatric-cardiologists and 
anaesthesists between 2016-2017 there are no absolute cardiovascular contra-
indications for surgical intervention and/or pregnancy. Advice against surgical 
intervention and/or pregnancy should be based on the patient-specific cardiovascular 
risk profile. The 2% mortality risk due to acute aortic dissection is based on one 
survey and literature review study [45] that reported the outcomes of 101 
pregnancies in patients with TS after oocyte donation. Only 50% of the patients were 
screened by a cardiologist before entering the oocyte donation programme.  
Therefore, all girls who want to participate in this study should have completed the 
diagnostic work up phase of TS including routine cardiac screening and will be 
                                                 
1 Claus H. Gravholt, N.H.A., Gerard S. Conway, Olaf M. Dekkers, Mitchell E. Geffner, Karen O. Klein, Angela E. Lin, Nelly Mauras, Charmian 
A. Quigley, Karen Rubin, David E. Sandberg, Theo C.J. Sas, Michael Silberbach, Viveca Söderström-Anttila, Kirstine Stochholm, Janielle A. van 
Alfen-van derVelden, Joachim Woelfle, and Philippe F. Backeljauw. On behalf of the International Turner Syndrome Consensus Group., 
Clinical Practice Guidelines for the Care of Girls and Women with Turner Syndrome. Eur J Endocrinology, 2017. 

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screened by a paediatric anaesthesist. Exclusion will be based on the patient specific 
risk profile. 
4.4 Sample size calculation 
As this is an exploratory study, the estimated LBR for girls with TS is currently 
unknown. Hence, the sample size calculation should be based on data obtained from 
performing OTC in other patient groups (e.g. girls undergoing gonadotoxic 
treatments).  
The LBR per transplantation of earlier cryopreserved ovarian tissue in cancer 
survivors is about 25%  [19].  
 
In order to describe the dichotomous outcome live birth rate (LBR), we used the 
sample size calculation of Hulley et al. 2013 [46] via http://www.sample-
size.net/sample-size-conf-interval-proportion: 
 
Confidence level (CI) =95% 
Expected proportion (P) = 25% (LBR) 
Total width of confidence interval (W) =20% (5% - 45%) 
α = (1-CL)/2 = 0.025 
Standard normal deviate for α = Zα = 1.960 
 
Sample size (N) = 4Zα2P(1-P)/(W2) = 72 
Expected positive results in sample (x) = 18 
 
One should consider that auto transplantation will not be performed in all girls who 
are participating in this study, due to several reasons (e.g. in the case that no follicles 
are found, in the case of de novo contra-indications for auto transplantation in the 
future and/or on patient’s preference). Furthermore, the number of miscarriages in 
patients with TS is known to be higher i.e. 30% versus 15% in healthy women [10].  
However, in our opinion, performing an exploratory intervention study in >200 minors, 
is unrealistic and inappropriate.  
Therefore, we aim to include primarily a total number of 100 girls with TS in this 
‘proof of concept’ study.   

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5. TREATMENT OF SUBJECTS 
 
Recruitment of participants and informed consent 
Girls with TS and their parents will be informed about this study by their paediatrician 
in the Dutch hospitals and/or by the Dutch TS patient support group (Turner Contact 
Nederland, or TCN). Girls with TS who meet the inclusion criteria and are potentially 
interested, will receive the study information for patients and/or parents during their 
yearly visit at their paediatric endocrinologist.  Needless to say, the girls and their 
parents are given time as individually requested to consider participation in this 
study. If the girl and the parents are interested in the study, they will be counseled by 
a gynaecologist. For the counseling, an additional document can be used where the 
existing options (i.e. awaiting spontaneous pregnancy, vitrification of oocytes, oocyte 
donation, high technological surrogacy, adoption and/or fostership) are compared 
with ovarian tissue cryopreservation.  Furthermore, we will further develop a decision 
aid based on appendix E4b and E4c and input of (parents of) patients and 
professionals, to help girls with TS and their parents to make a deliberate decision to 
participate in this study or not. The current decision aid includes a flowchart and 
background information on the existing options (i.e. awaiting spontaneous 
pregnancy, vitrification of oocytes, oocyte donation, high technological surrogacy, 
adoption and/or fostership) and ovarian tissue cryopreservation (see attachment E4b 
and E4c). Written consent must be given by the (parents of) patients before study 
participation. Study consent forms and the cryopreservation contract will be collected 
by one of the research nurses.  
Every year, the participant (and her parents) are asked for reconsent via the 
cryopreservation contract. When a paediatric participant becomes 16 years old, she will 
receive a new cryopreservation contract for reconsent. 
 
Data collection 
Castor Electronic Data Capture (EDC) will be used for Good Clinical Practice (GCP)-
compliant data collection. All participant data will be reported in electronic case report 
forms (e-CRF). Medical record data will be collected only by the members of the 
research group. Missing data will be supplemented by the paediatric endocrinologists.  
 
Procedures 
If not previously performed, a buccal swab will be taken during the yearly visit at the 
paediatric-endocrinologist to minimize the burden of travelling. Also, one portion of 
urine will be taken during one of the planned hospitals visits. Furthermore, one extra 
blood sample of 3.5mL will be taken for hormonal analysis and Hepatitis B/Hepatitis 
C/ HIV screening during the routine laboratory evaluation to minimize the number of 
vena punctures.  
In some cases, girls with a minimum age of 10 and 45,X karyotype will take an 
additional blood sample of 3,5mL during counseling (e.g. when certain hormones are 
missing or when the last hormonal analysis was carried out more than a year ago). In 
this way this specific group, will be informed properly about their chances of 
preserving fertility with cryopreservation of ovarian tissue. 
 
All participating girls with TS will undergo preoperative screening and risk 
assessment by a paediatric anaesthetist, and on indication by a paediatric-

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cardiologist. High-risk patients and their parents will be informed about their risks and 
excluded from this study in order to ensure patient safety. In low-risk participants the 
surgical procedure, followed by a hospital stay of one night, will be planned. Ovarian 
cortical tissue will be harvested by performing a unilateral oophorectomy via 
laparoscopic approach, thereby leaving the other ovary intact. A collaborating team of 
well-trained paediatric surgeons, gynaecologists specialized in reproductive 
medicine, and laboratory workers specialized in human ovarian cortical tissue 
cryopreservation, will ensure safe and efficient cryopreservation of the ovarian tissue. 
Cryopreservation of the ovarian tissue fragments will be performed according to the 
Dutch protocol ‘Cryopreservation and transplantation of ovarian tissue’ (Dutch 
Network Fertility preservation, September 2012). A safe storage of the cryopreserved 
ovarian tissue will be provided at the registered cryobank Radboud university medical 
centre. Our cryobank is ISO-accredited (accreditation number M101, ISO 15189) and 
registered by the Dutch Ministry of Health, Welfare and Sport (VWS) (registration 
number 5515 L/EO). A biopt of 5x5mm of the ovarian cortex will be used to assess 
the oocyte quality and genetics (e.g. the presence of germ line mosaicism). The 
DXZ1 probe will be used to detect the X chromosome in the isolated primordial 
follicles and denuded primordial follicles, the isolated primordial follicle derived 
granulosa cells and the ovarian stromal cells, by using fluorescence in situ 
hybridization (FISH). FISH analyses will be performed in close collaboration with the 
department of Genetics (Radboudumc).  
In the future, all women with TS who are interested in auto transplantation of earlier 
cryopreserved ovarian tissue, will be counseled by the gynaecologist. Afterwards, all 
interested participants will again undergo preoperative screening and risk 
assessment by a specialized cardiologist and anaesthetist. During a multidisciplinary 
meeting, the individual risk profile of each interested participant is discussed to see if 
there are any contra-indications for the laparoscopic procedure and/or future 
pregnancy. In low-risk participants the surgical procedure, followed by a hospital stay 
of one night, will be planned by the gynaecologist. Orthotopic transplantation of the 
autograft is performed via laparoscopic approach [19, 36, 38, 42]. High-risk patients 
will be informed about their risks by the gynaecologist, and excluded from this study 
in order to ensure patient safety. 
 
Hospital stay and follow-up 
Cryopreservation of ovarian tissue: 
Participants will be discharged after a hospital stay of 1 night. A follow-up visit with 
the gynaecologist is planned 6 weeks after surgery to inform patients about their 
results. When primordial follicles are found, an additional blood sample of 3,5ml 
blood will be taken three to six months after surgery to gain insight into the hormonal 
status (FSH, LH, AMH, E2, inhibin B ) and to start hormonal therapy when needed. The 
blood collection will take place during a routine follow-up visit with the paediatric-
endocrinologist or paediatrician. Thereafter the hormonal status will be monitored 
during the yearly visit at the paediatric-endocrinologist or paediatrician by collecting 
3,5ml extra blood (FSH, LH, AMH, E2, inhibin B ) during the yearly blood collection 
within their usual care. All blood samples will be sent to the Radboudumc in 
Nijmegen for hormonal analysis. 
In addition, girls with TS who are having a menstruation will use a period calendar to 
monitor their cycle. This is possible by using an App or they can write it down on 
paper.  
 

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Xenotransplantation 
Part of the ovarian cortex fragment that is available for research purposes is used to 
determine the density of the ovarian follicles and, when follicles are present, FISH 
analysis is performed on the follicular cells and the stromal cells (see procedures). 
Our recent results of these FISH analyses [70,71] has shown that in most patients 
with numerical X chromosomal aberrations the follicular granulosa cells are all 45,X. 
To study the effect of these 45,X granulosa cells on folliculogenesis we will perform 
xenotransplantation in mice with the remaining part of the ovarian cortex research 
fragment of selected patients (approximately 20 samples). This xenotransplantation 
model is used frequently in research directed at ovarian cortex cryopreservation and 
will provide us with important information on the functionality of follicles in TS girls. 
These experiments will be performed in collaboration with the group of Prof. 
M.M.Dolmans at the Université Catholique de Louvain (Brussels-Belgium). This 
group has extensive experience with xenotransplantations of human ovarian cortex 
tissue [72,73] and all the necessary local permissions.   
 
Auto transplantation of cryopreserved-thawed ovarian tissue: 
Participants will be discharged after a hospital stay of 1 night. A follow-up visit with 
the gynaecologist is planned 6 weeks after surgery. No other hospital visits are 
foreseen. 
 
Temporarily results 
Participants and their parents will be informed about their individual results (e.g. 
blood test results) during the foreseen hospitals visits. The number of follicles 
found and the results of the FISH-analysis will be reported to the participant and/or 
her parents during the follow-up visit with the gynecologist 6 weeks after surgery. 
Ovarian tissue cryopreservation will be performed in all participants, regardless the 
number of primordial follicles or oocyte genetics.  
 
Financial aspects 
Fertility preservation care in girls under 18 years old in the Netherlands is currently 
organized as following: the costs of cryopreservation and storage of ovarian tissue is 
funded by the Department of Obstetrics and Gynaecology of the hospital where the 
cryopreservation is performed. The costs for the laparoscopic removal of 1 ovary 
under general anaesthesia followed by a 1-night hospital stay are declared to the 
health insurance. Since there is no deductible excess for minors, no costs should be 
declared to the participating girls and/or their parents. 
 
 

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6. METHODS 
 
6.1 Study parameters/endpoints 
6.1.1 Main study parameter/endpoint 
• Live birth after auto transplantation of cryopreserved-thawed ovarian cortical 
tissue (i.e. live birth rate or LBR)   
6.1.2 Proximate  
• The number of primordial follicles found in the ovarian tissue 
6.1.3 Secondary study parameters/endpoints  
• The association between patient’s age at cryopreservation and LBR  
• The association between patient’s genotype and LBR 
• The association between patient’s AMH level at cryopreservation and LBR 
• The association between patient’s FSH level at cryopreservation and LBR 
6.1.4 Tertiary study parameters/endpoints  
• The willingness of girls with TS to perform a unilateral oophorectomy for 
fertility preservation (i.e. the study participation rate) 
• The number of eligible participants 
• The age of the participant 
• The incidence of somatic mosaicism in buccal cells, peripheral lymphocytes 
and urine cells 
• The incidence of germ cell mosaicism (i.e. oocytes versus somatic cells) 
• The developmental capacity of small follicles 
• Serum hormone levels (i.e. FSH, LH, AMH, E2, inhibin B) 
• The number of complications related to the laparoscopic procedure 
• The incidence of spontaneous puberty and/or spontaneous menarche after 
laparoscopic oophorectomy 
• The incidence of spontaneous pregnancies after laparoscopic oophorectomy 
• The incidence of menstruation cycle recovery after auto transplantation of 
cryopreserved-thawed ovarian tissue in the future 
• The incidence of pregnancies after auto transplantation of cryopreserved-
thawed ovarian tissue in the future 
• The number of ongoing pregnancies after auto transplantation of 
cryopreserved-thawed ovarian tissue in the future 
• The number of miscarriages after auto transplantation of cryopreserved-
thawed ovarian tissue in the future 
• Time to pregnancy after auto transplantation of cryopreserved-thawed ovarian 
tissue in the future 
• Time to live birth after auto transplantation of cryopreserved-thawed ovarian 
tissue in the future 
 

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6.2 Randomisation, blinding and treatment allocation 
Laparoscopic removal of one of both ovaries followed by cryopreservation of the 
ovarian cortical tissue and auto transplantation in the future will be performed without 
randomization, blinding or treatment allocation.  
 
6.3 Study procedures 
A non-invasive imaging technique will be used to assess the number of follicles in the 
ovarian tissue fragments. A small biopsy of 5x5mm of the ovarian cortex will be used 
to assess the oocyte quality and genetics (i.e. germ cell mosaicism). During 
embryogenesis the primordial follicle is assembled by the association of granulosa 
cells and a single oocyte. Both cell types are crucial for follicular development but 
have different embryonic origins. In case of mosaicism the karyotype of the oocytes 
might therefore be different from that of the granulosa cells and/or the stromal cells. 
To investigate the mosaicism at the level of the follicle and ovary, oocytes, granulosa 
cells and ovarian stromal cells will be karyotyped by using fluorescence in situ 
hybridization analysis (FISH analysis) using an X-chromosome specific probe. The 
remaining part of the ovarian tissue will be cryopreserved for fertility preservation 
purposes. The 3.5mL blood sample will be used for hormonal analysis (i.e. FSH, LH, 
AMH, E2 and inhibin B) and screening on Hepatitis B/ Hepatitis C / HIV. The cells 
isolated from the buccal swab and the urine sample will be karyotyped by using the 
FISH analysis described above. In all subjects, classic karyotyping on lymphocytes 
(white blood cells) has been performed before diagnosis of TS. 
 
6.4 Withdrawal of individual subjects 
Subjects may withdraw from the trial at any time. Subjects do not need to state a 
reason for withdrawal. The investigator may withdraw subjects who do not comply 
with the study requirements. 
 
6.5 Replacement of individual subjects after withdrawal 
Subjects who withdraw from the study during recruitment will be replaced.  
 
6.6 Follow-up of subjects withdrawn from treatment 
Subsequent treatment options for withdrawn subjects will be subject to the clinician’s 
judgment. Participation in this study will not negatively impact the subject’s standard 
of care.  
 
6.7 Premature termination of the study 
Safety reviews are planned primarily to guard against the unfavourable results in this 
study. Adverse events and complications related to the laparoscopic unilateral 
oophorectomy and/or general anaesthesia will be closely monitored in order to pick 
up any (unexpected) trends. Furthermore, the number of primordial follicles will be 
monitored as a proximate. A data and safety monitoring board (DSMB) at the 
initiating centre Radboudumc will be formed to control this trial (see 7.4 for more 
information about this DSMB). Only the DSMB may temporarily or permanently 

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discontinue the trial at a single site or at all sites for safety, ethical, compliance or 
other reasons. If this is necessary, the DSMB will endeavour to provide advance 
notification to the site. If the site or trial is suspended or discontinued, the principal 
investigator will be responsible for ensuring prompt notification to the CCMO. Where 
required by local regulations, the principal investigator will be responsible for 
informing the CCMO of trial or site discontinuation.  
A safely storage of the ovarian tissue will be provided till the participant has reached 
the maximum age for autotransplantation of ovarian tissue (cryocontract). All coded 
patient data will be safely stored  till the endpoint of this study has been reached, i.e. 
the last livebirth has been registered. In case of an early study termination due to 
safety reasons, each individual patient and/or her parents may freely decide about 
the continuation of the storage of the ovarian strips at the Radboud university 
medical center, transportation of the ovarian strips to another hospital or the 
elimination of tissue. 
 
 
 
 

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7. SAFETY REPORTING 
 
7.1 Temporary halt for reasons of subject safety 
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the 
study if there is sufficient ground that continuation of the study will jeopardize subject 
health or safety.  The sponsor will notify the CCMO without undue delay of a 
temporary halt including the reason for such an action. The study will be suspended 
pending a further positive decision by the CCMO. The investigator will take care that 
all subjects are kept informed.  
7.2 AEs and SAEs 
7.2.1 Adverse events (AEs) 
Adverse events are defined as any undesirable experience occurring to a subject 
during the study period, whether or not considered related to the investigational 
product/ trial procedure/ the experimental intervention. All adverse events reported or 
observed by the investigator or his staff during the first study period (i.e. from 
informed consent till the follow-up visit at the gynaecologist 6 weeks post-surgery) 
and the second study period (i.e. from ART-request in the future till end of study)  will 
be recorded. 
7.2.2. Serious adverse events (SAEs) 
A serious adverse event is any untoward medical occurrence or effect that  
- 
results in death; 
- 
is life threatening (at the time of the event); 
- 
requires hospitalization or prolongation of existing in patients’ 
hospitalization; 
- 
results in persistent or significant disability or incapacity; 
- 
is a congenital anomaly or birth defect; or 
- 
any other important medical event that did not result in any of the outcomes 
listed above due to medical or surgical intervention but could have been 
based upon appropriate judgment by the investigator. 
Any other important medical event that did not result in any of the outcomes listed 
above due to medical or surgical intervention, but could have been based upon 
appropriate medical judgment. An elective hospital admission will not be considered 
as a serious adverse event. SAE´s should be reported to the Sponsor, registered by 
the Coordinating Investigator and reported to the CCMO when section 10, subsection 
4 of the WMO is applicable. The local investigator will report all SAEs to the main 
principal investigator without undue delay after obtaining knowledge of the events.  
The principal investigator will report the SAEs through the web portal ToetsingOnline 
(https://toetsingonline.nl) to the CCMO that approved the protocol, within 7 days of 
first knowledge for SAEs that result in death or are life threatening followed by a 
period of maximum of 8 days to complete the initial preliminary report. All other SAEs 
will be reported within a period of maximum 15 days after the principal investigator 
has first knowledge of the serious adverse events. 
 
 
 

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7.3. Annual safety report 
 
In addition to the expedited reporting of SAEs, the main principal investigator will 
submit, once a year throughout the clinical trial, a safety report to the CCMO and 
competent authority.  
This safety report consists of: 
• a list of all suspected (unexpected or expected) serious adverse 
reactions, along with an aggregated summary table of all reported 
serious adverse reactions, ordered by organ system 
• a report concerning the safety of the subjects, consisting of a complete 
safety analysis and an evaluation of the balance between the efficacy 
and the harmfulness of the intervention. 
 
Follow-up of adverse events 
All AEs will be followed until they have abated, or until a stable situation has been 
reached. Depending on the event, follow up may require additional tests or medical 
procedures as indicated, and/or referral to the general physician or a medical 
specialist. SAEs need to be reported till end of study within the Netherlands, as 
defined in the protocol.  
 
7.4. Data and Safety Monitoring Board (DSMB) / Safety Committee 
An Independent Data and Safety Monitoring Board (DSMB) is established comprising 
of independent experts who have no conflict of interest and agree with the outline of 
the protocol. Members of the DSMB are M.C. de Vries, M.D., PhD, paediatric 
endocrinologist and medical ethicist at the Leiden University Medical Center (NL) 
(Chair), T.C.J. Sas, M.D., PhD, paediatric endocrinologist at the Albert Schweitzer 
Hospital and Sophia Children’s Hospital (NL) (clinician), and M.M.H.J. van Gelder, 
PhD, assistant professor in Epidemiology at the Radboud university medical center 
(NL) (methodologist). The committee will meet at least once in 6 months during the 
inclusion period to evaluate the results of the interim analysis. During the study, the 
committee may decide to change the frequency of discussion. All data presented at 
this meeting will be considered confidential. Following this meeting, the DSMB will 
report to the principal investigator their advice on whether or not to continue the 
study. The committee may recommend changes in the conduct of the study. The 
advice(s) of the DSMB will be sent to the principal investigator of the study. Should 
the principal investigator decide not to fully implement the advice of the DSMB, the 
principal investigator will send the advice to the CCMO, including a note to 
substantiate why (part of) the advice of the DSMB will not be followed. 
 

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8. STATISTICAL ANALYSIS 
 
All data in this pilot study will be analyzed on both intention-to-treat and per-protocol 
analysis. Data of girls who are lost to follow-up will be included as far as possible. 
Missing data will be reported along with the reason. Baseline data will be described 
quantitatively. Continuous variables will be summarized as means with standard 
deviations (SDs) or as medians with inter-quartile ranges (IQRs), depending on their 
distribution. Dichotomous and ordinal data will be summarized as percentages. For 
all analyses, IBM SPSS will be used. 
 
8.1 Descriptive analysis 
Descriptive statistics will be used to analyze the number of primordial follicles in the 
ovarian tissue, and the number of (ongoing) pregnancies, miscarriages and live born 
children after auto transplantation of earlier cryopreserved ovarian tissue. 
The primary outcome (dichotomous) ‘live birth after auto transplantation of 
cryopreserved-thawed ovarian tissue’ will be assessed and reported as a 
percentage. The number of primordial follicles (continuous) will be reported as a 
mean with standard deviations (SDs). The number of pregnancies, ongoing 
pregnancies and miscarriages (dichotomous) will be reported as a percentage  
The time to pregnancy and the time to live birth after auto transplantation will be 
reported as a mean with standard deviation (SD).  
Furthermore, the relationship between the age of the participant when ovarian tissue 
cryopreservation (OTC) is performed (years), serum FSH level (IU/L), and serum 
AMH level (ng/ml), versus the number of primordial follicles, the incidence of 
(ongoing) pregnancy, and live birth will be described using Spearman’s correlation 
coefficient. In addition the number of primordial follicles, the incidence of (ongoing) 
pregnancy, and live birth will be described by the patient’s karyotype (i.e. 45,X 
monosomy or mosaicism).   
8.2 Interim analyses  
Interim analyses on safety and futility are planned every 6 months until the last 
participant (n=100) has undergone the laparoscopic unilateral oophorectomy 
followed by ovarian tissue cryopreservation. The first interim analysis will be 
performed after the inclusion of the first 10 patients.  Each interim analysis will be 
reported to the independent DSMB (Data and Safety Monitoring Board) by the 
coordinating investigator on a structured way by using the interim analysis framework 
(table 1 - Interim analysis framework). 
The percentage of participants with follicles in their ovary will be used as a futility 
indicator. Unfortunately, there is not much information on the expected success 
probability of ovarian tissue cryopreservation in girls with Turner syndrome. 
According to the findings of Hovatta et al. follicles could be found in 26% of girls with 
Turner syndrome. Primordial follicles were found in girls with a 45X, 46XX mosaicism 
genotype in 86% of cases, and in 11% of cases in girls with a 45X monosomy 
genotype. However, their findings are based on the examinations of one small 
formalin-fixated and paraffin-embedded ovarian tissue sample instead of evaluating 

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the whole ovary. The question remains whether this ovarian tissue sample correctly 
represents the presence and number of primordial follicles the whole ovary. It is 
known that primordial follicles are unevenly distributed throughout the cortex with a 
variation up to 20,000. 
Therefore, we will not implement a formal futility stopping rule but the DSMB will at 
each interim analysis evaluate the number of primordial follicles that has been 
retrieved per girl. 
The percentage of participants who had one or more complications related to the 
laparoscopic oophorectomy and/or anaesthesia will be used as a safety indicator. 
The DSMB will evaluate the number and type of complications at each interim 
analysis. 
Missing data will be reported along with the reason for missingness. Baseline data 
will be described quantitatively. The number of primordial follicles (continuous) will be 
reported as a median with a range. Descriptive statistics will be used to analyze the 
number of complications related to anaesthesia and/or the laparoscopic procedure.  
In addition, the percentage of participants with follicles in their ovary and the 
percentage of patients with one or more complications will be categorized by 
genotype and age-group. For all analyses, IBM SPSS will be used. 
The DSMB will discuss the results of each interim analysis and advice the principal 
investigator on the continuation of the trial based on the balance of futility and safety. 
The principal investigator will report each DSMB advice to the CCMO. Should the 
principal investigator decide not to fully implement the advice of the DSMB, the 
principal investigator will send the advice to the CCMO, including a note to 
substantiate why (part of) the advice of the DSMB will not be followed. 
  

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Table 1 - Interim analysis framework: 
 
Interim analysis number: 
Study period: 
Date: 
 
1. Results of the current study period 
1.1 Inclusion Radboudumc 
Number of patients included: 
Number of patients excluded: 
Number of patients who have undergone the laparoscopic oophorectomy: 
 
 
1.2 Inclusion MUMC 
Number of patients included: 
Number of patients excluded: 
Number of patients who have undergone the laparoscopic oophorectomy: 
 
1.3 Participants in the current study period (n=): 
Hospital (Radboudumc=1, 
MUMC=2) 
 
 
 
 
Research number 
 
 
 
 
Age (years) 
 
 
 
Mean age (years) and 
standard deviation 
Genotype (45,X 
monosomy or mosaicism)  
 
 
 
% 45, X monosomy and 
% 45,X mosaicism 
FSH level (IU/l) 
 
 
 
Median FSH level (IU/l) 
and range 
AMH level (ug/l) 
 
 
 
Median AMH level (ug/l) 
and range 
Primordial follicles found in 
the ovarian tissue (yes/no) 
 
 
 
% of participants with 
primordial follicles in the 
ovarian tissue 

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The number of primordial 
follicles found in the 
ovarian tissue 
 
 
 
Median number of 
primordial follicles found 
in the ovarian tissue with 
range 
Complication related to the 
laparoscopic 
oophorectomy (yes/no and 
a description of 
complication and 
management) 
 
 
 
% participants with one or 
more complications 
related to the 
laparoscopic 
oophorectomy  
 
1.4 Futility indicators: 
The percentage of participants in the current study period with primordial follicles in 
the ovarian tissue: % 
 
Relationship with age:  
Age 2-11 years old: % 
Age 12-18 years old: % 
Relationship with genotype: 
45x monosomy genotype: % 
45x/46xx mosaicism genotype: % 
other genotypes: % 
 
1.5 Safety indicators: 
The percentage of participants in the current study period who had one or more 
complications related to the laparoscopic oophorectomy:  % 
 
Relationship with age:  
Age 2-11 years old: % 
Age 12-18 years old: % 
Relationship with genotype: 
45x monosomy genotype: % 
45x/46xx mosaicism genotype: % 
other genotypes: % 
 
 
 
 
 

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2. Overall study results (all periods) 
 
2.1 Inclusion Radboudumc 
Overall number of patients included: 
Overall number of patients excluded: 
Overall number of patients who have undergone the laparoscopic oophorectomy: 
 
2.2 Inclusion MUMC 
Overall number of patients included: 
Overall number of patients excluded: 
Overall number of patients who have undergone the laparoscopic oophorectomy: 
 
2.3 Futility indicators: 
The overall percentage of participants with primordial follicles in the ovarian 
tissue: % 
 
Relationship with age:  
Age 2-11 years old: % 
Age 12-18 years old: % 
Relationship with genotype: 
45x monosomy genotype: % 
45x/46xx mosaicism genotype: % 
other genotypes: % 
 
2.4 Safety indicators: 
The overall percentage of participants who had one or more complications 
related to the laparoscopic oophorectomy:  %  
 
Relationship with age:  
Age 2-11 years old: % 
Age 12-18 years old: % 
Relationship with genotype: 
45x monosomy genotype: % 
45x/46xx mosaicism genotype: % 
other genotypes: % 
 

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9. ETHICAL CONSIDERATIONS  
 
9.1 Equality  
The WHO states that all individuals have the right to decide freely the number, 
spacing and timing of their children and that patients should have access to all 
medical treatments [47]. The right to access fertility preservation services, however, 
is not legally stated.  
Over the last years, ovarian tissue cryopreservation (OTC) has been experimentally 
performed in hundreds of girls that already underwent or should undergo gonadotoxic 
cancer treatments. Auto transplantation of cryopreserved-thawed ovarian tissue in 
cancer survivors has resulted in restoration of ovarian function, multiple pregnancies 
and >90 live births [19, 36-42].  
Some people believe that fertility preservation services should be available for 
everybody, without discrimination. This has led to a new debate to expand OTC as a 
fertility preservation options to other patient groups than girls undergoing gonadotoxic 
cancer treatments (e.g. transgenders and girls with TS).  
The promise of fertility preservation for these patient groups is at present 
hypothetical, as it is for each individual oncofertility patient. However, the Oncofertility 
Consortium2 states that OTC is a promising approach to provide fertility options to 
girls with TS [48]. In some countries, OTC is already routinely offered to girls with TS 
[12, 15, 44, 48, 49]. We believe that such an intervention should not be offered to 
these girls as common clinical practice but in a safe and controlled research setting. 
Recently, ethical approval was given to perform OTC in Dutch transgenders in a 
research setting. We believe it is time to expand these options to girls with TS too. 
Qualitative research shows a strong need for new options like fertility preservation 
services (citation of a girl with TS: “I wish I could have kids. I wish I had the choice”).  
In daily practice, physicians are often asked by girls with TS and/or their parents 
about fertility preservation services. This is also stated by Grynberg et al. in a 
recently published review on fertility preservation in girls with TS [13], in the patient 
support letter that is attached to this research proposal (see attachment A3), and in 
the following citations from our  patient meeting: 
 
“Should we then accept what nature has decided?” (adolescent with TS)  
 
“At the moment you have no choice” (adolescent with TS) 
 
"One thing I know, her wish is very strong" (parent of a girl with TS, aged 12 years 
old) 
 
                                                 
2 The Oncofertility Consortium is a multi-institutional group that assessed the impact of cancer and its 
treatment on reproductive health. The term oncofertility refers to an interdisciplinary field that bridges 
oncology, reproduction, and women’s health research for the purpose of exploring and expanding 
options for the reproductive future of cancer patients. 

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9.2 Regulation statement  
This study will be performed in accordance with this study protocol, the Declaration of 
Helsinki, the Medical Research Involving Human Subjects Act (WMO), the ICH 
Harmonized Tripartite Guideline for Good Clinical Practices, and all other applicable 
regulatory requirements. 
 
9.3 Recruitment and consent  
As stated in The Medical Research Involving Human Subjects Act (WMO) states, all 
participating girls (and their parents) will be fully informed about the nature of this 
study, its purpose, procedures, expected duration and the potential risks and benefits 
involved in study participation along with any discomfort it may entail, in order to 
make a deliberated decision and to prevent false hope. Information will be age- 
specific, easy-to-read and complete. Furthermore, each subject will be informed that 
participation in the study is voluntary and that withdrawal of consent will not affect the 
right to the most appropriate treatment, neither the relationship with their doctor 
relationship. This informed consent will be given by means of a standard written 
statement. Each subject and/or her parents will be given enough time to read and 
understate the statement before signing consent and dating the document. Each girl 
and/or her parents will receive a copy of the written statement once signed (see 
appendix).  
 A safely storage of the ovarian tissue will be provided till the participant has reached 
the maximum age for autotransplantation of ovarian tissue (cryocontract). All coded 
patient data will be safely stored  till the endpoint of this study has been reached, i.e. 
the last livebirth has been registeredIn case of a study drop out or an early study 
termination due to safety reasons, each individual subject and/or her parents may 
freely decide about the continuation of the storage of the ovarian strips at the 
Radboud university medical center, transportation of the ovarian strips to another 
hospital or the elimination of tissue. 
 
9.4 Objection by minors  
The WMO states that minors aged 12 or older are able to understand the possibilities 
and limitations of medical investigations. In minors <12 years old, parents are 
deciding whether their daughter will undergo laparoscopy for future fertility insurance. 
In minors aged 12 - 15 years old, both parents and the participant have to give 
consent. This means that the procedure can only be done if they agree and their 
parents consent, but not if they object.  
Participants <12 years old may be legally not competent to make their own decision, 
requiring an approved consent from both parents. However, interviews with parents 
of girls with TS [4], showed that girls aged <12 years old already have major 
concerns regarding their future fertility and are distressed upon learning of their 
possible inability to become biological mothers. Tailored information can help to 
inform minors on this study in order to promote the autonomy of the minor and her 
parents. Therefore, we developed an illustrated brochure. 

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Moreover, all girls involved in this study are free to decide whether they want to make 
use of auto transplantation of cryopreserved ovarian tissue in the future, or not. In 
case of a minor’s resistance regarding participation or continuation in this study, none 
of the study procedures will be performed. Not only capacity-based but also 
distressed-based child’s objections [50] regarding participation or continuation in this 
study will be respected.  
 
9.5 Benefits 
The Medical Research Involving Human Subjects Act (WMO) states that 
experimental medical research in children or minors is forbidden, unless the research 
is deemed of a net benefit (i.e. therapeutic research). Since this intervention study 
aims to preserve the fertility of the respective (minor) participant, facing a very high 
risk of POI of 95-98%, it relates to therapeutic research, and therefore, in principal 
permissible scientific research.  
 
9.5.1 Success rates of fertility preservation in other patient groups 
Recently, the success rate of cryopreservation of ovarian tissue in advance of 
cytotoxic therapies and later transplantation(s) of the tissue was reported [19]. The 
FertiPROTEKT study group found a restoration of ovarian function in 67% per 
transplantation. Pregnancy and live birth rates were 33 and 25% per transplantation, 
respectively [19]. The number of auto transplantation sessions is depending on the 
number of ovarian strips. However, we cannot automatically project this data on 
women with TS, neither is it possible to estimate the adjusted pregnancy and live 
birth rates for this patient group.  
 
9.5.2 The number of primordial follicles 
The occurrence of pregnancy and live birth after auto transplantation of frozen 
cortex strips is correlated with the number of primordial follicles found in the 
ovarian tissue [51]. Borgström et al.[15] reported the number of follicles found in 
the ovarian biopsies of both 45,X mosaic patients as patients with 45,X monosomy 
aged 8.0 - 19.8 years old. However, the number of follicles in the frozen ovarian 
strips could not be determined since there was no non-invasive imaging technique 
available. Instead, the study group performed a standard histological analysis of 
an adjacent formalin fixated and paraffin embedded (FFPE) ovarian tissue 
fragment. The number of follicles found in the FFPE-fragment varied between 0 
and 1200. The question remains if their results are representative for the frozen 
cortex strips, since it is known that primordial follicles are unevenly distributed 
throughout the ovarian cortex with a variation up to 20,000 [52, 53]. 
 
9.5.3 The functionality of the primordial follicles 
Furthermore, the functionality of the primordial follicles should be taken into 
account. Women with TS are known to have a higher risk of having a miscarriage 
(30.8% compared to 15% for healthy women [10]) and a slightly higher risk of 
having a child with a congenital disorder (the probability of a child with Down 
Syndrome is estimated to be 4% compared to 0.4% for healthy women, the 

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probability of a child with TS is estimated to be 11 % compared to 0.5% for healthy 
women)[10, 54]. However, we do not know if their increased risks are related to 
functionality and/or the chromosome profile of their follicles. 
In order to fully inform the participant and her parents about her chances, a small 
sample of the ovarian tissue will be karyotyped. The number of follicles found and 
the results of the FISH-analysis will be reported to the participant and/or her 
parents. 
 
9.5.4 Reduction of the psychosocial burden  
This study aims to reduce the burden related to the uncertainty about fertility in 
girls with TS hereby to improve their psychosocial wellbeing. Qualitative research 
[4] showed the impact of TS on the life of patients with TS aged 7-59 years. 
Regardless of age, infertility was the most frequently cited concern in this interview 
study. 
From girls undergoing gonadotoxic treatments, we know that uncertainty about 
fertility leads to a reduction of quality of life and can even lead to serious 
psychosocial disorders [55]. Girls who were not offered/counseled about fertility 
preservation services, experienced a worse quality of life [56]. About 70% of the girls 
who survived their cancer preferred to have genetically concordant children above 
any other form of parenting [57]. 
Decisions surrounding fertility preservation (FP) in children, adolescents, and adults 
can be difficult due to the distress of a cancer diagnosis and the time constraints for 
decision-making [58-61]. The decision-making process is promoted when counseling 
has been done by a specialized gynaecologist, when there was enough opportunity 
for patients to ask questions, and when a decision-aid was available [58-61].  
In girls with TS, we will be able to provide tailored counseling by the time that the 
child has completed the diagnostic work-up phase. In this patient group, we do not 
experience the time pressure as in oncological children. Proper counseling will be 
done by a specialized gynaecologist. Therefore, our concept decision aid will be 
further developed.  
 
9.6 Risks 
The laparoscopic removal of ovarian tissue may be in <1% complicated by injuries to 
the bladder, ureters or bowel, and the risk of infection and bleeding [62]. To minimize 
the risk of the laparoscopic procedure and to ensure safe and efficient 
cryopreservation of the ovarian tissue, the procedure will be performed by a 
collaborating team of well-trained paediatric surgeons, gynaecologists specialized in 
reproductive medicine, and laboratory workers specialized in human ovarian cortical 
tissue preservation. All children will be preoperatively screened for co morbidities.  A 
follow-up visit will be done 6 weeks after surgery. Moreover, participants and their 
parents will be well-informed about all aspects of this procedure including the 
benefits and risks. 
Like with any other new intervention or treatment, there might be a chance of raising 
false hope in the participating girls (and/or parents). However, we attempt to 
overcome this by extensive and objective information provision by both written 

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materials and face to face counselling. Therefore, all girls with TS and their parents 
will be fully-informed and properly counseled about all aspects of this procedure 
including the benefits and risks, and their other options (i.e. awaiting spontaneous 
pregnancy, vitrification of oocytes, oocyte donation, high technological surrogacy, 
adoption and/or fostership) [63].  
The possible effects of the removal of one ovary on future fertility of girls with TS are 
unknown. Recent studies [64-67] show that the surgical removal of one ovary in a 
healthy child does not affect the menstrual cycle and / or the chances of getting 
pregnant in the future. The surgical removal of one ovary in a healthy adult can lead 
to an earlier menopause up to 3 years in comparison to a woman who still has both 
ovaries [68]. It has been suggested that one intact ovary may compensate for the 
loss of the other [69].  
 
9.7 Benefits and risks assessment 
Due to a lack of evidence, it is not possible to predict the outcome for each individual 
participant. However, there are 3 possible scenarios: 
 
1) The participant belongs to the largest group of girls (95-98%) who are infertile at 
the end of adolescence. OTC could be successfully performed (e.g. follicles where 
found in the cortex strips). 
Due to early menopause, this girl does not have any spontaneous chances of getting 
pregnant. The removal of one ovary will not reduce her spontaneous pregnancy 
chances (0% → 0%). Her fertility might be saved for later. 
 
2) The participant belongs to the largest group of girls (95-98%) who are infertile at 
the end of adolescence. OTC was not successful (e.g. no follicles where found in the 
cortex strips). 
Due to early menopause, this girl does not have any spontaneous chances of getting 
pregnant. The removal of one ovary will not reduce her spontaneous pregnancy 
chances (0% → 0%). Unfortunately, her fertility could not be saved.  
The intervention does not have a personal benefit for this participant. 
 
3) The participant belongs to the smallest group of girls (2-5%) who may become 
spontaneously pregnant in the future. OTC could be successfully performed (e.g. 
follicles where found in the cortex strips). 
Due to a solid ovarian reserve in her remaining ovary, this participant still has a 
chance of spontaneously becoming pregnant. Her fertility might be saved for later if 
needed. 
All girls and their parents will be counseled about the 3 possible scenario’s before 
participating in this study.  
 
9.8 Are there other options? 
Needless to say, it would be preferable to offer fertility preservation services to girls 

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that are competent to make their own decision. However, the majority of girls is 
infertile by the end of adolescence due to the accelerated loss of germ cells. 
Cryopreservation of mature oocytes is a proven fertility preservation approach but 
can be performed only in a small group of girls with TS. Since oocyte maturation 
requires ovarian stimulation with exogenous FSH administration followed by 
transvaginal ultrasound-guided oocyte retrieval [17], this procedure can only be 
performed in post pubertal girls. Unfortunately, 90% of girls with TS will not have a 
spontaneous menstruation onset [11]. Furthermore, transvaginal ultrasound, the 
ovum pick up and the administration of exogenous FSH over a longer period are not 
appropriate in young (virgo) girls. 
 
9.9 Compensation for injury  
The principal investigator has a liability insurance which is in accordance with article 
7 of the WMO. The principal investigator (also) has an insurance which is in 
accordance with the legal requirements in the Netherlands (Article 7 WMO). This 
insurance provides cover for damage to research subjects through injury or death 
caused by the study. The insurance applies to the damage that becomes apparent 
during the study or within 4 years after the end of the study. 
 

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10. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
 
10.1 Handling and storage of data and documents 
Data will be collected using Castor. Data monitoring will be done by the principal 
investigator and local investigators in each of the participating centres. Data handling 
will be done confidentially and coded, with the participant code only available to the 
investigator working in the local centre.  
 
10.2 Annual progress report 
The main principal investigator will submit a summary of the progress of the trial to 
the CCMO once a year. Information will be provided on the date of inclusion of the 
first subject, numbers of subjects included and numbers of subjects that have 
completed the trial, serious adverse events/ serious adverse reactions, other 
problems, and amendments.  
 
10.3 Temporary halt and (prematurely) end of study report 
The main principal investigator will notify the CCMO of the end of the study within a 
period of 8 weeks. The end of the study is defined as the last participant’s last visit. 
The main principal investigator will notify the CCMO immediately of a temporary halt 
of the study, including the reason of such an action. In case the study is ended 
prematurely, the main principal investigator will notify the CCMO within 15 days, 
including the reasons for the premature termination. Within one year after the end of 
the study, the main principal investigator will submit a final study report with the 
results of the study to the CCMO. 
 
10.4 Public disclosure and publication policy 
The trial will be registered at clinicaltrials.gov. The principal investigator will publish 
the results of the study as soon as appropriate.  
  

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11. BIOBANK GOVERNANCE 
 
In this study, all ovarian tissue will be handled and stored according to the World 
Medical Association (WMA) Declaration of Taipei on ethical considerations regarding 
health databases and biobanks. 67th WMA General Assembly. Taipei, Taiwan. 
October 2016, with consideration of the following governance arrangements. 
 
1. The purpose of the biobank 
Cryopreservation and storage of ovarian tissue for both clinical use, i.e. fertility 
preservation, and to expand scientific knowledge. 
 
2. The nature of health data and biological material that will be contained in the 
biobank 
This cryobank will contain cryopreserved ovarian tissue and patient data, including 
consent forms. All clinical data will be recorded in EPIC an electronic medical record 
system. 
Castor Electronic Data capture (EDC) will be used for Good Clinical Practice (GCP)-
compliant research data collection. Patient data, with exclusion of the cryotubes and 
the consent forms, is coded and will be reported in electronic case report forms (e-
CRF). Medical record data will be collected only by the members of the research 
group. 
 
3. Arrangements for the length of time for which the data or material will be stored 
A safely storage of the ovarian tissue will be provided till the participant has reached 
the maximum age for autotransplantation of ovarian tissue (cryocontract). All coded 
patient data will be safely stored  till the endpoint of this study has been reached, i.e. 
the last livebirth has been registered 
 
4. Arrangements for regulations of the disposal and destruction of data and material 
Once a year all subjects will be asked if they want to continue the storage of their 
ovarian tissue. Each individual patient and/or her parents may freely decide about the 
continuation of the storage of the ovarian tissue at our cryobank, transportation of the 
ovarian tissue to another cryobank, donation for research, or the elimination of their 
cryopreserved ovarian tissue. Storage, transportation, destruction or donation of 
ovarian tissue can be performed only with the signed consent of the person (s) 
legally entitled to decide what should happen with the tissue. Destruction of ovarian 
tissue is performed according to the guidelines of the Radboud university medical 
center in an anonymous and respectful manner. In case of disposal or destruction of 
ovarian tissue upon patient’s consent, the contract for the storage of ovarian tissue 
will be closed and discarded. In case of transportation, a new contract should be 
signed by the patient and the person(s) who are responsible for the governance of 
the recieving cryobank. 
Patients who are interested in auto transplantation of the cryopreserved ovarian 
tissue, will be counselled by the gynaecologist. Afterwards, all patients will again 
undergo preoperative screening and risk assessment by an anaesthesist and 
cardiologist. During a multidisciplinary meeting, the individual risk profile of each 
participant is discussed to see if there are any contra-indications for the laparoscopic 
procedure and/or future pregnancy, In low-risk patients the surgical procedure, 
followed by a hospital stay of one night, will be planned by the gynaecologist. 

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Orthopic transplantation of the autograft is performed via a laparoscopic procedure. 
All patients will be informed about the number of ovarian strips used for auto 
transplantation and the number of strips which are still remaining.  
High-risk patients will be informed about their risks by the gynaecologist. in such 
cases, auto transplantation is delayed or will be discouraged. However, patients can 
decide freely to transport their tissue to another hospital and ask for a second 
opinion. 
 
5. Arrangement for how the data and material will be documented and traceable in 
accordance with the consent of the concerned persons 
All research data and material will be documented and traceable in accordance with 
the consent of the patients involved.   
 
6. Arrangement for how the data and material will be dealt with in the event of 
change of ownership or closure 
In case of an early study termination or change of cryobank governance, all patients 
and/or their parents will be informed. Each individual patient and/or her parents may 
freely decide about the continuation of the storage of the ovarian tissue at our 
cryobank, transportation of the ovarian tissue to another cryobank, donation for 
research, or the elimination of their cryopreserved ovarian tissue. 
 
7. Arrangement for obtaining appropriate consent or other legal basis for data or 
material collection 
The collection, storage and use of data and ovarian tissue from the participating 
patients must be voluntary. Therefore, specific informed consent of the participants 
and/or their parents is obtained in accordance with the Declaration of Helsinki. In this 
study the following consent forms are used: 
1) Informed consent form for participation in this study for patients  
2) Informed consent form for participation in this study for parents of patients  
3) Informed consent form for the storage of ovarian tissue  
 
Research on ovarian tissue is carried out upon patient’s consent only, and in 
accordance with this research protocol on which a competent medical ethical 
committee has given a positive judgment.  
When a participant is interested in donating her tissue for research, she will be 
informed about the research purposes, the chance of unintended findings and how 
she will be informed about these findings. 
 
8. Arrangements for protecting dignity, autonomy, privacy and preventing 
discrimination 
The dignity, autonomy and privacy of the patients will be respected by the duty of 
confidentiality of all who are involved in handling data and biological material. There 
will be no discrimination. 
 
9. Criteria and procedures concerning the access to and the sharing of the health 
data or biological material 
A safe storage of all biological material and data, including the consent forms, is 
provided at our cryobank. Our cryobank is located at a restricted area in the Radboud 
university medical center. Access is permitted by electronic authorization only. Our 
cryobank is ISO-accredited (accreditation number M101, ISO 15189), and registered 

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by the Dutch Ministry of Health, Welfare and Sport (VWS) (registration number 5515 
L/EO). All data will be coded with exception of the cryotubes and the consent form for 
storage of ovarian tissue. Members of the research team, the Data and Safety 
Monitoring Board, and the Health Care Inspectorate are the only persons who have 
access to the key to the code. This key of code is stored in Castor Electronic Data 
capture (EDC). 
There will be no sharing of health data and/or biological material. 
 
10. The person or persons who are responsible for the governance 
The persons who are responsible for the governance of the Radboudumc ovarian 
tissue cryobank are L. Ramos (embryologist, head of the IVF laboratory) and C.C.M. 
Beerendonk (gynaecologist). 
 
11. The security measures to prevent unauthorized access or inappropriate sharing 
As mentioned under 9. 
 
12. The procedures for re-contacting participants where relevant 
All subjects will be asked for the continuation of the storage of the ovarian tissue 
yearly. When the participant turns 16 years old, she will be asked for re-consent to 
participate in this study and the continuation of storage of her ovarian tissue by mail. 
 
13. The procedures for receiving and addressing enquiries and complaints 
Enquiries and complaints can be addressed anonymously via the complaints 
committee of the Radboud university medical center. In case of any questions, 
patients and their parents can contact the independent physician dr. Timmers who 
has no further involvement in the investigation. 

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