Infertility is a significant social- and health problem in the Western World and at the moment in Denmark one in ten babies are born with the help of assisted reproduction. In 50% of infertile couples a male factor can be identified as a contributing cause (1).

Azoospermia is defined as the absence of spermatozoa in the ejaculate and it is a condition affecting 10-15% of infertile men (2, 3). Azoospermia is divided into obstructive azoospermia (OA) and nonobstructive azoospermia (NOA) of which the latter constitutes 60% (2, 3). In NOA the production of spermatozoa in the testis is either absent or markedly decreased.

Since 1999 microdissection testicular sperm extraction (mTESE) has become the preferred treatment option for NOA in many centers worldwide (4). The procedure is performed in general anesthesia using an operating microscope to carefully examine the entire testicular tissue for the presence of spermatozoa which can be used for assisted reproduction.

An alternative to mTESE is a percutaneous testicular sperm aspiration (TESA) or needle biopsy. This procedure is simple to perform using a biopsy needle to aspirate testicular tissue. The aspirated tissue is examined for the presence of spermatozoa that can be used in assisted reproduction.

Today there is no robust evidence on the optimal sperm retrieval protocol on men with NOA. This is in part due to the fact that no randomized trials have been performed to compare procedures. This study is the first to randomize procedures for surgical sperm retrieval.

Hypothesis In men with NOA, the investigators hypothesize that TESA is a viable first line approach compared to mTESE in regards to success rates of finding spermatozoa, complication rates and pregnancy outcomes.

A total of 110 men will be randomized to either mTESE or TESA and the rates of finding spermatozoa will be compared. However, for ethical reasons, because some believe mTESE have a greater chance of finding sperm cells, all men with a failed TESA will have a mTESE afterwards.

Background Infertility is a significant social- and health problem in the Western World and at the moment in Denmark one in ten babies are born with the help of assisted reproduction. In 50% of infertile couples a male factor can be identified as a contributing cause (1).

Azoospermia is defined as the absence of spermatozoa in the ejaculate and it is a condition affecting 10-15% of infertile men (2, 3). Azoospermia is divided into obstructive azoospermia (OA) and nonobstructive azoospermia (NOA) of which the latter constitutes 60% (2, 3). In NOA the production of spermatozoa in the testis is either absent or markedly decreased.

Since 1999 microdissection testicular sperm extraction (mTESE) has become the preferred treatment option for NOA in many centers worldwide (4). The procedure is performed in general anesthesia using an operating microscope to carefully examine the entire testicular tissue for the presence of spermatozoa which can be used for intracytoplasmic sperm injection (ICSI). The advantages of mTESE include high sperm retrieval rates (SRR), in recent systematic reviews reported around 52% (5-8), and low reported complication rates (9-11). However mTESE is a time-consuming invasive procedure requiring the right expertise, the right equipment and a proper setup of the fertility clinic.

An alternative to mTESE is a percutaneous testicular sperm aspiration (TESA). This procedure is simple to perform using a biopsy needle to aspirate testicular tissue. The aspirated tissue is examined for the presence of spermatozoa and SRR for this procedure is usually reported around 25% (5-8). The lower SRR compared to mTESE is the main limitation of this method, but the procedure is well tolerated by patients and due to its simplicity and short duration it can easily be performed in the out-patient clinic. In collaboration with University of Michigan the TESA technique has been improved by using an 18 gauge needle and performing multiple (50-100) passes throughout the entire testicular tissue while applying a vacuum. In the remainder of this document this multiple needle-pass TESA will be referred to as "TESA". Recently the investigators retrospectively evaluated this approach and found a SRR of 30% using TESA and interestingly the SRR of mTESE following a failed TESA was only 11% (12).

However, there is no robust evidence on the optimal sperm retrieval protocol on men with NOA. This is in part due to the fact that no randomized trials have been performed to compare the two procedures. Often patients from different countries are required to pay the infertility treatment themselves and the cost for a TESA is around $500 while a mTESE is around $8.000. This makes it difficult to perform a randomized trial in most countries but because of the public health systems of Denmark and Sweden there is a unique possibility to be the first in the world to do a randomized trial on this matter.

Hypothesis In men with NOA, the investigators hypothesize that TESA is a viable first line approach compared to mTESE in regards to SRR, complication rates and pregnancy outcomes.

Screening and randomization Potential study patients and their partners will initially be identified in their work-up during their infertility treatment. When a patient is identified as having NOA, the patient will be screened according to inclusion- and exclusion criteria. If these are met and the patient provides an informed consent for participation, the patient is included and randomized to undergo mTESE or TESA. Patients will be recruited from the fertility clinics at Herlev and Gentofte Hospital, Malmö University Hospital, Holbaek Hospital and Rigshospitalet in collaboration with Department of Growth and Reproduction, Rigshospitalet.

Planning the treatment and surgery Treatment of the infertile couples will follow regular treatment protocols at the respective fertility clinics. This includes freezing retrieved sperm cells and thawing them in relation to the oocyte retrieval. As an alternative to sperm cell freezing, retrieved oocytes will be frozen and thawed in relation to the surgical sperm retrieval. The aim is to have 6-8 oocytes stored before operation for couples following this route. All patients will undergo full anesthesia. If the patient is randomized to TESA and this fails, then a salvage mTESE can be done immediately while in the operating theatre.

Visits Before visit 1 all patients and their partners will have received written and oral information on the study and will have had time to provide an informed consent.

At visit 1 patients and possibly their partners (mandatory in cases of oocyte cryopreservation) meet with the primary investigator. It will be secured that an informed consent for participation is signed. The focus of visit 1 will be on accessing the men's evaluation leading to the NOA diagnosis and checking inclusion- and exclusion criteria. In addition, information regarding medical history will be obtained and a physical examination of the external genitalia will be carried out. Patients will fill out questionnaires on psychosexual health and pain before the visit and their answers will be reviewed together. A blood sample will be taken for the purpose of measuring hormone levels and for storage in the biobank. Likewise patients will bring a semen sample for storage in the biobank. If not previously done patients will be tested with regards to karyotype, AzF microdeletions and CFTR mutations if deemed necessary.

Visit 2 will be the actual procedure. Follow-up is comprised by patients filling out the mentioned questionnaires, reporting any complications and having a blood sample drawn at visit 3 and 4. Follow-up will primarily be done by phone/mail, but at visit 3 and 4 patients will have to go to the Department of Growth and Reproduction, Rigshospitalet (for Danish patients) or the fertility clinic at Malmö University Hospital (for Swedish patients) to have blood samples drawn. Samples collected in Sweden will be send for analysis at the Department of Growth and Reproduction, Rigshospitalet. Pregnancy outcome will be reported by phone/mail 9-15 months after the procedure is done.

Biobank A research biobank will be made containing blood samples, semen and testicular tissue. Blood samples will be used for genotyping and to measure relevant biomarkers including piRNAs, miRNAs, protein markers and biochemical markers. Semen will also be used to measure relevant biomarkers. Testicular tissue will be used for testing for molecular and biochemical markers of spermatogenesis and to measure intratesticular testosterone levels. Additionally testicular tissue showing maturation arrest will be used for attempts to maturate germ cells in vitro (not to be used for fertilization, only research). The purpose of the above is to find markers of successful sperm retrieval and to get a better understanding of the biology of NOA and potentially to develop pharmacological strategies in the future for treating certain forms of NOA as e.g. maturations arrest.

Safety and adverse events:

Hematoma As TESA/ mTESE is a surgery there is a risk of post-operative bleeding leading to scrotal or spermatic cord hematomas. Estimated risk <1,5%.

Infection As TESA/ mTESE is a surgery there is a risk of infection. Estimated risk <1% Pain Pain is expected to occur after both procedures but especially after mTESE Drop in testosterone level In the litterature this is reported as a transient event following mTESE however there is a risk of more lasting androgen deficiency. Careful surgical techniques will be used to limit the amount of tissue damage.

Possible disadvantages:

Lower SRR following TESA SRR are reported lower for TESA compared to mTESE. For this reason patients with an unsuccessful TESA will be offered a salvage mTESE Oocyte cryopreservation In the past fertilization outcomes after cryopreservation have been lower compared to fresh oocytes. However with the evolution of cryopreservation techniques (vitrification) IVF pregnancy rates with the use of cryopreserved oocytes are now similar to those of fresh oocytes. (15) Grading of complications Complications will be graded according to the Clavien-Dindo classification of surgical complications (14).

Ethical evaluation This study will follow the newest edition of the Helsinki Declaration and at all times follow applicable laws. It will be reported to "Datatilsynet", "Videnskabsetisk komite" and the "Ethical Committee of Lund University".

Some of the ethical aspects related to this project have been briefly described in the safety and adverse events section. Both mTESE and TESA are safe procedures and although the investigators previously found a higher complication rate with mTESE compared to TESA (12), the general literature describe both procedures with comparable risks related to infection and hematomas.

To make the study possible to complete, either the sperm cells or the oocytes retrieved from the women will have to undergo cryopreservation. Sperm cell cryopreservation is standard practice at the fertility clinics today. Previously only fresh oocytes were used for in-vitro fertilization but recently it has been shown that treatment using cryopreserved oocytes give similar pregnancy rates to those of fresh oocytes. Thus freezing the oocytes should not be a disadvantage to the couples.

When looking at sperm retrieval rates, mTESE has until now been superior to TESA. However with the improved TESA results are expected to be more equal. In addition patients randomized to TESA will have the benefit of a more simple and less invasive treatment compared to mTESE. Moreover patients randomized to TESA will be provided with an option to receive a salvage mTESE that can be carried out the same day because all patients will undergo full anesthesia. In this way all patients included in the study will receive equal treatment options for NOA.

Attempts of maturation of germ cells in vitro will be made using tissue with partial or total maturation arrest. If successful, the germ cells maturated in vitro will not be used for any attempt of fertilization but only as "proof of concept" regarding treatment of azoospermia due to maturation arrest.

In a broader picture this project will be the first randomized study on this matter and therefore it will bring a clearer picture on which is the optimal sperm retrieval technique for men with NOA. This will benefit many patients with NOA in the future.

Publication of results:

Results from the study will be published in international peer-reviewed scientific journals, national journals and hospital websites. This will happen no matter the outcome of the study meaning that both positive, negative and inconclusive results will be made public. Additionally results will be presented at conferences and meetings. All published results will be made anonymous.

Inclusion Criteria:

• Azoospermia verified in at least two semen samples within the past six months, including assessment of the centrifuged pellet as per the WHO 5th edition (13)
• Testis volume (Prader's orchidometer) ≤ 15ml on both sides
• No indication of obstructive causes of azoospermia in medical history or physical examination (ex. absent vas deferens, vasectomy, scrotal trauma/injury, hernia repair or other operations potentially damaging the vas deferens)
• Capable and legally competent individual

Exclusion Criteria:

• Previous attempts of surgical sperm retrieval
• Previous testicular biopsy
• Anejaculation
• Retrograde ejaculation
• Bleeding disorders rendering surgery too high a risk
• Klinefelters Syndrome
• XX male
• AZFa/b microdeletion
• CFTR mutation
• Inability to understand and/or stick to the written information
• Patients not deemed suitable for general anesthesia

Exclusion during follow-up

• A patient can at any time during the study withdraw their consent of participation
• Normal histology on testis biopsy following TESA or mTESE

46XY

The randomization will be performed when the patient is asleep.