Succesfull Fertility Preservation for Transfeminine Adolescents Via Semen Cryopreservation or Testicular Sperm Extraction
Conditions: Gender Dysphoria, Adolescent
Sponsor: Amsterdam UMC, location VUmc

Title  
Successful freezing of sperm cells via a testicular 
sperm extraction (TESE) procedure or semen 
cryopreservation in transfeminine adolescents 
with gender dysphoria 
Document Date 
15-4-2022 
Version number 
1 
NCT number 
Not yet assigned  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
Submitter 
Drs. T.H.R. Stolk, t.h.stolk@amsterdamumc.nl 
Coordinating researcher 
Dr. N.M. van Mello, n.vanmello@amsterdamumc.nl 
Principal Investigator(s) 
Prof. dr. J.A.F. Huirne, j.huirne@amsterdamumc.nl 
Co-researcher(s) 
Dr. N.M. van Mello 
Dr. E. van den Boogaard, e.vandenboogaard1@amsterdamumc.nl 
Dr. A. Meißner, a.meissner@amsterdamumc.nl 
Client (provider) 
Amsterdam UMC, location VUMC 
 
Research proposal 
Rationale 
Gender dysphoria is the experience of significant dissatisfaction with 
one's assigned sex at birth, which does not align with one's gender 
identity. Gender dysphoria is associated with mood swings, anxiety 
disorders, and suicidality. In transgender and gender diverse 
individuals, gender identity and/or expression does not align with their 
assigned sex at birth. Gender-affirming treatments have been shown 
to have a positive effect on the well-being of transgender individuals 
(1). 
 
Most individuals begin their transition at a young age. The medical 
transition for transgender adolescents (10-20 years old, based on 
WHO criteria) involves puberty suppression through the use of a GnRH 
agonist, which can be initiated early in puberty. Puberty blockers 
reduce behavioral and emotional problems and depressive symptoms, 
leading to improved daily functioning (2). Initiating puberty blockers is 
known as the "Dutch protocol" and is internationally applied in the 
transition of adolescents, proving to be successful in significantly 
reducing the severe distress caused by gender dysphoria (3).  

Subsequently, from the age of 15, patients become eligible for cross-
sex hormones, and from the age of 18, after at least 1 year of 
hormone therapy, they can undergo gender-affirming surgeries such 
as gonadectomy. 
 
Hormonal and surgical steps in the transition can have reversible as 
well as irreversible effects on the fertility of transgender individuals. 
The advice of the World Professional Association for Transgender 
Health (WPATH) guideline is to counsel everyone about this, even if 
there is currently no desire for fertility preservation, for example, in 
adolescents(1). In the Knowledge and Care Center for Gender 
Dysphoria (KZcG), since 2021, we have established a protocol where 
all adolescents with gender dysphoria who start puberty suppression 
or cross-sex hormone therapy are counseled about the effects of 
treatment on their fertility and the possibilities for fertility 
preservation. 
 
The number of adolescents with gender dysphoria seeking care at the 
Knowledge and Care Center for Gender Dysphoria (KZcG) has been 
increasing annually (Figure 1). As a result, we are seeing a larger group 
of adolescents in our clinic to discuss fertility preservation. Part of 
them assigned male at birth (i.e. transfeminine). The rise in number of 
adolescents is similar to other countries across the world, for example, 
in Australia(4). 
 
Figure 1: Number of registrations per year of children and adolescents 
at the KZcG 
 
For this group, it is particularly important to provide fertility 
counseling before starting puberty blockers. Because, puberty 

blockers prevent or stop the production of testosterone, thereby 
inhibiting the development of secondary sexual characteristics. As a 
consequence, this also prevents or greatly reduces spermatogenesis, 
(i.e. the maturation of sperm cells). 
 
From Tanner stage 2, adolescents in our center become eligible for 
puberty suppression. However, spermatogenesis may not have started 
in everyone by that stage. It is often not possible to obtain mature 
sperm cells from ejaculation due to both the physical and mental 
inability to induce ejaculation, caused by dysphoria and age. 
Therefore, the option of cryopreserving ejaculated spermatozoa 
(semen cryopreservation) is often not available. 
 
For individuals unable to ejaculate, testicular sperm extraction (TESE) 
is offered. In this procedure, tissue is taken from the testes through 
biopsies, with the aim of finding mature sperm cells in the laboratory 
and freezing them for future use. The care for transgender 
adolescents was established in 2021 at Amsterdam UMC, location 
AMC. 
 
Since 2014, in the Netherlands, the moratorium that prohibited the 
use of surgically obtained sperm for achieving pregnancy, which had 
been in effect since 1996, was lifted after the publication of the results 
of the study titled "ICSI with testicular sperm in men with 
azoospermia: an observational study with follow-up of children" 
conducted by Radboud UMC and Amsterdam UMC (5, 6). These 
studies demonstrated the safety of testicular sperm extraction (TESE) 
by monitoring pregnancies and children born through ICSI with 
testicular sperm. Since then, quality standards have been established, 
and the TESE procedure has been recognized and deemed safe. It has 
since become a standard clinical practice in specialized centers and 
can be used, among other cases, for fertility preservation in men with 
(testicular) cancer, Klinefelter patients without current desire for 
children, or trans women with azoospermia. 

 
Furthermore, there is a significant amount of expertise in taking 
testicular biopsies in adolescents, particularly in the context of pre- 
and peripubertal oncology patients undergoing infertility-inducing 
treatments like chemotherapy. Since 2011, small pieces of testicular 
tissue have been frozen before the start of the treatment in these 
patients, allowing for the potential isolation and multiplication of stem 
cells from the frozen tissue in the future, to be reintroduced into the 
testes to fulfill the desire for children through natural conception (7). 
The operative risks associated with this procedure, such as infection 
and bleeding, are low, ranging from 1.3% to 2.6% (8). Additionally, no 
long-term negative effects have been observed in this group (9, 10). 
However, for transfeminine adolescents, this technique cannot be 
offered at present due to the need for testosterone replacement, 
which is suppressed in trans women through testosterone blockers 
and gender-affirming female hormones, or due to prior gonadectomy. 
Therefore, for the prepubertal execution of the TESE procedure, 
successful in vitro spermatogenesis must first be developed. 
 
To maximize the chances of a successful TESE, spermatogenesis should 
already be underway, for which the production of testosterone is 
crucial. However, testosterone is also responsible for (irreversible) 
virilization, such as deepening of the voice and facial hair growth. 
Therefore, it is highly burdensome for this group of patients to 
postpone puberty suppression in order to secure their fertility for the 
future, especially considering that active fertility desire does not exist 
at the start of puberty. 
 
Currently, there is a lack of valid clinical factors to predict when 
spermatogenesis has sufficiently progressed in transfeminine 
adolescents. The intratesticular testosterone level is significantly 
higher than the peripheral blood testosterone level, and the blood 
testosterone value is not determinative of the intratesticular level. 
Ideally, a high intratesticular testosterone level would be beneficial for 

sperm production, while maintaining the lowest possible peripheral 
testosterone level to minimize virilization. 
 
In the current clinical practice, the child endocrinologist determines 
the Tanner stage before initiating puberty suppression, followed by 
fertility counseling with the gynecologist. If fertility preservation is 
desired afterward, a referral to the andrologist is made to assess the 
indication for TESE and determine the optimal timing for the 
procedure. In cases where ejaculation is not possible, the andrologist 
uses ultrasound measurements of testicular volume, serum 
testosterone, LH, and FSH levels to make this determination. Recent 
Australian research in transfeminine adolescents has shown that 
testicular volume appears to be the most predictive factor. This 
finding is based on a retrospective cohort study with a small sample 
size of N=31 (11). 
 
If the TESE procedure is performed too early, it is possible that no or 
insufficient suitable sperm cells can be found. TESE is an invasive 
procedure that can have complications such as infection and bleeding, 
although the chances of these complications are low. However, 
complications may occur even without freezing sperm cells. 
Additionally, it should be noted that the patient is constantly 
reminded of their genitalia during the postoperative period, which can 
cause dysphoria. The procedure should be performed in a way that 
avoids permanent damage to the testes, ensuring that the process is 
reversible in case the patients choose their assigned gender at birth or 
if there is sufficient tissue available to assess the presence of sperm 
cells at the time of gonadectomy. 
 
Puberty progresses differently in each individual. In some cases, 
secondary changes occur faster than sperm maturation, leading to 
high levels of mental distress due to dysphoria, which may cause 
patients to refrain from undergoing TESE. This period can also be 
stressful and uncertain for both the child and the parents. Therefore, it 

is important to provide comprehensive counseling and 
multidisciplinary support throughout the process. 
 
Currently, there are no ideal clinical non-invasive parameters to 
determine the optimal timing for TESE. To determine the appropriate 
moment for a successful TESE there is need to set up a cohort study of 
all transfeminine adolescents who are referred for TESE. This will 
enable us to make better predictions in the future and optimize our 
fertility counseling and care. 
 
Furthermore, we intend to improve the counseling and support 
provided to both parents and the child by examining the experiences 
of the referred group throughout the entire process and reflecting on 
the experiences of the group that has undergone TESE. 
Objective 
When is the TESE process most successful by: 
 
1. Identifying clinical non-invasive parameters that indicate a high 
likelihood of finding mature sperm cells. 
2. Evaluating the experience of the process for patients and parents to 
determine the potential physical and emotional burden of the TESE 
process and identifying factors that can reduce this burden to improve 
feasibility. 
Study design  
For part 1 of the research question "when is the sperm yield from TESE 
most successful," the study design is observational, including both 
retrospective and prospective cohort research, utilizing data from 
medical records. 
 
To address the second part of the research question, we intend to 
employ a descriptive research method. This will involve qualitative 
research through interviews and quantitative research through 
questionnaires administered to both the children and their parents 
during or after the treatment. 
Study population 
Healthy adolescents assigned male at birth with gender dysphoria and 
a desire for surgical fertility preservation prior to or after the 

temporary discontinuation of puberty suppression, who have been 
referred to the andrologist 
Inclusion criteria 
Patients will be included if they meet the following criteria: 
 
(Parents of) Individuals assigned male at birth who identify as 
transfeminine (trans girl, non-binary person, etc.). 
 
Under the care of a specialized gender center or clinic 
 
Have a diagnosis of gender dysphoria according to the 
Diagnostic and Statistical Manual of Mental Disorders, Fifth 
Edition (DSM-5) or a diagnosis of gender incongruence 
according to the International Classification of Diseases, 11th 
Revision (ICD-11). 
 
Have not yet started or have discontinued puberty 
suppression before the start of gender affirming hormone 
treatment. 
 
Referred or scheduled for referral to the andrologist following 
fertility counseling. 
Exclusion criteria  
Patients will be excluded if they do not wish to participate in this 
research. 
Sample size 
We aim to conduct the study over a period of 2 years to include a 
sufficient number of 30-40 adolescents, allowing us to generate valid 
data. 
Recruitment of 
participants 
The participants who have already undergone TESE will be approached 
by the primary treating physician during their clinic visit or via 
telephone. The participant information will then be sent to them via 
email or mail. During the next physical appointment that the 
participant has at gender clinic, the consent form will be signed in the 
presence of the researcher. 
 
If the referral has not yet been made, the primary treating physician 
will inform the participant about the study during the fertility 
counseling session and provide them with the participant information 
after the consultation. If a referral for TESE is desired, the primary 
treating physician will contact the participants and their parents by 
phone to confirm their interest in participation. A one-week 

consideration period will then begin. If participation is desired, the 
consent form will be signed during the next physical appointment in 
the presence of the researcher. If the participant has a long distance 
to travel to the gender clinic and does not have any upcoming physical 
appointments in the near future, the consent form will be sent by mail 
with a prepaid return envelope. 
Intervention 
The participant information will include a request for permission to 
use data from the medical records for scientific research purposes.  
For the second part of the research question, we will also inquire 
whether the participant and their parents are willing to complete a 
questionnaire at least once and up to three times, with each 
questionnaire taking a maximum of 10 to 15 minutes. Additionally, a 
minimum of 5 study participants who have undergone TESE will be 
asked to participate in an interview, as well as their parents. Similarly, 
a minimum of 5 study participants who are referred to the andrologist 
will also be invited to participate in an interview, along with their 
parents. The interviews will take place once at location or via video 
call if there is a long travel distance, and they will last a maximum of 1 
hour. 
Standard care / 
Standard treatment 
All adolescents are referred for fertility counseling before starting 
puberty suppressants. In this consultation, a counseling session takes 
place regarding fertility preservation. If the patient expresses a desire 
for preservation and is eligible for TESE (due to age/early 
puberty/incapacity to produce ejaculation), they are referred to the 
andrologist. An intake appointment is scheduled, after which the 
indication for TESE is confirmed. Currently, the indication is based on 
blood values (testosterone, LH, FSH) and an ultrasound to determine 
testicular volume. If the testicular volume or laboratory values are still 
insufficiently indicative of a successful TESE, the examination is 
repeated after 3 to 6 months or upon the patient's request if puberty 
progresses more rapidly and intensely than expected, thus tailored to 
the patient. The TESE procedure is performed in an outpatient setting 
under general anesthesia with local anesthesia, using microsurgery 

techniques to obtain small pieces of testicular tissue, ensuring 
reversibility of the procedure. 
Study parameters 
Primary outcome measures: 
Part 1: 
Ultrasound-determined testicular volumes before TESE procedure, 
when this is not available, we will use the measurements of the 
Tanner stage at the time of the procedure, by the pediatric 
endocrinologist. 
Laboratory values (testosterone, LH, FSH) shortest before the TESE 
procedure, including subsequent measurements, collected as part of 
routine clinical care for non-study purposes 
Age at the time of TESE 
Presence of spermatozoa  
Number of cryopreserved straws 
Quality of obtained sperm cells (concentration/motility) 
Part 2: 
Experience of the TESE procedure for the patient and parents 
Experience of counseling for the patient and parents 
Experience of any waiting period for the patient and parents 
Feasibility for adolescents: Outcome of TESE after referral (whether 
the TESE procedure was carried out or not) 
Study endpoints 
 
Secondary outcome measures: 
 
Progression of secondary sexual characteristics/virilization from the 
time of referral to the TESE procedure 
Subgroup of patients who temporarily discontinued puberty 
suppressants 
Statistical analysis 
The statistical program SPSS will be used for analyzing the 
observational cohort and questionnaires. 
 
Descriptive analyses will be conducted to display the central tendency 
(mean, median, and mode) and variation (range and standard 
deviation) of the included sample. 

 
To demonstrate a statistically significant relationship between 
different variables and the observed outcomes, linear and multivariate 
regression analyses will be employed. If necessary, adjustments will be 
made for confounding factors. 
 
The questionnaires will be send via Castor and analyzed using SPSS.   
 
The interviews will be transcribed verbatim. Transcripts will be coded 
using the MaxQDA software program, from which thematic analysis 
themes will be derived. 
Burden for the participant 
The interviews are conducted face-to-face or, in the case of long travel 
distances, through video consultations. The interviews last for a single 
session of 45-60 minutes. The questionnaires are sent online and take 
approximately 15 to 20 minutes to complete. The topics of fertility and 
the physical changes related to the TESE procedure discussed in the 
interviews can be considered intimate information. However, it is 
important to note that these topics are also routinely discussed by 
physicians and psychologists during clinical visits. Therefore, 
participants may find it relatively normal to discuss these topics with 
their healthcare providers. Nevertheless, participants have the option 
to select "I do not want to complete this questionnaire" for each 
questionnaire, in which case the questions will be removed from the 
digital survey. Apart from the time invested in completing the 
questionnaires and/or participating in interviews, there is no 
additional burden on the participants. Blood tests are always part of 
the treatment process and there are no additional blood sampling 
moments. 
Risk for the participant 
The participant faces no risks. 
Benefits of participating in 
the research 
The participant does not have direct benefits from participating in the 
research. Indirect benefits may include the potential improvement of 
care and information provision within the Knowledge and Care Center 
for Gender Dysphoria, both nationally and internationally, based on 
the research outcomes. This can be perceived as highly meaningful. 

Disadvantages of 
participating in the 
research 
The participant has no disadvantages of participating in the research. 
Compensation for the 
participant 
The participant will not receive any compensation for participating in 
the research. 
Administrative aspects 
The patient characteristics and treatment details (success, outcomes, 
etc.) will be collected from the medical records and stored in a 
pseudonymized SPSS file, which is password-protected. The database 
is stored on a secured departmental drive and is accessible only to the 
authorized researchers. Access to the database is monitored and 
recorded. The questionnaires will be administered using Castor EDC 
Surveys. This ensures that all data is stored in an encrypted format. 
The research data and the resulting database will be retained for up to 
15 years after the completion of the study and will then be destroyed. 
 
1. Coleman E, Radix AE, Bouman WP, Brown GR, de Vries ALC, Deutsch MB, et al. 
Standards of Care for the Health of Transgender and Gender Diverse People, Version 8 (vol 
23, pg S1, 2022). Int J Transgender Health 2022;23:S259-S61. 
2. de Vries AL, Steensma TD, Doreleijers TA, Cohen-Kettenis PT. Puberty suppression in 
adolescents with gender identity disorder: a prospective follow-up study. J Sex Med. 
2011;8(8):2276-83. 
3. de Vries AL, McGuire JK, Steensma TD, Wagenaar EC, Doreleijers TA, Cohen-Kettenis 
PT. Young adult psychological outcome after puberty suppression and gender reassignment. 
Pediatrics. 2014;134(4):696-704. 
4. Pang KC, de Graaf NM, Chew D, Hoq M, Keith DR, Carmichael P, Steensma TD. 
Association of Media Coverage of Transgender and Gender Diverse Issues With Rates of 
Referral of Transgender Children and Adolescents to Specialist Gender Clinics in the UK and 
Australia. Jama Network Open. 2020;3(7). 
5. Meijerink AM, Cissen M, Mochtar MH, Fleischer K, Thoonen I, de Melker AA, et al. 
Prediction model for live birth in ICSI using testicular extracted sperm. Human Reproduction. 
2016;31(9):1942-51. 
6. Meijerink AM, Ramos L, Janssen AJWM, Maas-van Schaaijk NM, Meissner A, Repping S, 
et al. Behavioral, cognitive, and motor performance and physical development of five-year-old 
children who were born after intracytoplasmic sperm injection with the use of testicular sperm. 
Fertility and Sterility. 2016;106(7):1673-+. 
7. Mulder CL, Zheng Y, Jan SZ, Struijk RB, Repping S, Hamer G, van Pelt AMM. 
Spermatogonial stem cell autotransplantation and germline genomic editing: a future cure for 
spermatogenic failure and prevention of transmission of genomic diseases. Human 
Reproduction Update. 2016;22(5):561-73. 
8. Uijldert M, Meissner A, de Melker AA, van Pelt AMM, van de Wetering MD, van Rijn RR, et 
al. Development of the testis in pre-pubertal boys with cancer after biopsy for fertility 
preservation. Human Reproduction. 2017;32(12):2366-72. 
9. Borgstrom B, Fridstrom M, Gustafsson B, Ljungman P, Rodriguez-Wallberg KA. A 
prospective study on the long-term outcome of prepubertal and pubertal boys undergoing 
testicular biopsy for fertility preservation prior to hematologic stem cell transplantation. Pediatr 
Blood Cancer. 2020;67(9):e28507. 

10. 
Kanbar M, de Michele F, Giudice MG, Desmet L, Poels J, Wyns C. Long-
term follow-up of boys who have undergone a testicular biopsy for fertility preservation. Hum 
Reprod. 2021;36(1):26-39. 
11. 
Peri A, Ahler A, Gook D, O'Connell MA, Bourne H, Nightingale M, et al. 
Predicting successful sperm retrieval in transfeminine adolescents after testicular biopsy. J 
Assist Reprod Genet. 2021;38(10):2735-43.