Presentation delivered at the Annual Meeting of the Nordic IVF Laboratory Society, 7th of September, 2010, Oslo, Norway

1. Sandro C. Esteves
ANDROFERT & University of Campinas (UNICAMP),
Campinas, BRAZIL
Aarhus University, DENMARK
Management of Infertile Men with
Non-obstructive Azoospermia:
clinical and IVF lab aspects

2. 1 out of 100 men have azoospermia; ~10% of infertile men
Esteves, 2
Modified from Esteves et al. Clinics 2011

3. Current scenario – Referral Center
% patients with
azoospermia
35%
Modified from Esteves et al. Clinics 2011; Source: ANDROFERT
3%
36%
61%
Hypo-hypo (NOA) Obstructive
Azoospermia
Spermatogenic
Failure (NOA)
Esteves, 3

4. Residual
spermatogenesis in
~50% cases, but
geographic location
unpredictable
Esteves & Agarwal, Clinics. 2013;68 Suppl 1:1-4.
Non-obstructive azoospermia does NOT necessarily imply
sterility
Esteves, 4

5. How to manage?
The key aspects
• Evaluate properly
• Give a fair estimate of outcome
• Develop a time-limited treatment plan
Esteves, 5

6. Esteves, 6

7. Differential diagnosis relatively simple; >90% accuracy to
determine type of azoospermia
Medical history
Cryptorchidism, testicular trauma, torsion,
infection, radio-/chemotherapy, congenital
abnormalities, systemic diseases
Physical examination
Small testes (<15 cc; long axis <4.6 cm)
Flat epididymis, palpable vas
Endocrine profile
Elevated FSH levels (>7.6 mIU/ml in 90% men)
Low testosterone levels (<300 ng/dl in up to
50%)
Modified from Esteves et al. Clinics 2011; 66:691-700
Esteves, 7

8. Esteves, 8
Esteves, Asian J Androl 2015; 17:1-12
Management of men with NOA - Step 2

9. Etiology equivocal to counsel about chances of
sperm retrieval success
Esteves, 9
Etiology Sperm retrieval
success
Cryptorchidism 50-70%
Post-infection ~60%
Torsion >50%
Post-chemotherapy/RT 25-75%
Genetic 0-60%
Idiopathic ~50%
Modified from Esteves et al Fertil Steril 2010; Raman & Schlegel J Urol 2003;
Hopps et al. Hum Reprod 2003; Damani et al JCO 2002

10. FSH levels
Testosterone
levels
Testicular
volume
electing candidates for SR
Can biomarkers predict SR success?
Diagnostic markers reflect global testicular function but
are not accurate to predict whether a site of active
spermatogenesis exists
Modified from Verza Jr & Esteves. Fertil Steril 2011; 96 (Suppl.): S53
Esteves, 10

11. Histopathology results helpful for counseling
but it does not provide definitive proof of whether sperm will
be found and might jeopardize future retrieval attempt
100%
40%
20%
Hypospermatogenesis Maturation Arrest Sertoli-cell only
Presence of sperm within the testicle
(N=357)
Modified from Esteves & Agarwal. Asian J Androl 2014; 16: 642
Testicular
histopathology
Esteves, 11

12. Modified from Esteves, Asian J Androl 2015 and Krausz et al. Andrologia 2014
Yq (AZF region) harbor key regulatory genes involved in
spermatogenesis
Esteves, 12

13. #4 Normal male
#5 AZFb microdeletion
#6 AZFc microdeletion
Multiplex PCR of Yq mandatory
Microdeletions of AZF region found in up to 15% of men with NOA
Esteves, 13

14. nil virtually nil ~50%
Presence of Yq microdeletion prognostic value
Sperm retrieval success
Esteves, 14

15. Esteves, 15
Modified from Esteves, Asian J Androl 2015; 17:1-12
Management of men with NOA - Step 3

16. High ITT levels essential for
spermatogenesis in combination
with FSH stimulation of Sertoli cells
Stimulation of Leydig/Sertoli cells
paradoxically weak due to high
baseline FSH/LH levels (relatively
low amplitudes)
Esteves, 16
Hypogonadism common feature in men with NOA
(Total testosterone <300 ng/dL or 10.4 nmol/L)
Shiraishi et al Hum Reprod 2012; 27: 331-9; Sussman et al Urol Clin N Am 2008; 35: 147-55

17. Study Study design Study group Medication Findings
Pavlovich
et al. 2001
Case series
43 men with
T/E ratio <10
Testolactone No effect
Hussein et
al. 2005
Prospective
cohort
42 men with
favorable
hystology
Clomiphene
Sperm found in SA in 64.3%; All men
who remained azoospermic had
success at SR
Selman et
al. 2006
Prospective
cohort
49 men with
maturation
arrest
rec-hFSH and hCG
No return of sperm in ejaculate;
posttreatment SRR were 21.4%
Ramasamy
et al. 2009
Case series
56 men with
nonmosaic
Klinefelter
Testolactone or anastrozole,
alone or combined with hCG
SRR increased by 1.4-fold
Reifsnyder
et al. 2012
Retrospective
cohort
307 men with
hypogonadis
m
Aromatase inhibitors, hCG or
Clomiphene, alone or
combined
No effect
Shiraishi et
al. 2012
Prospective
cohort
28 men with
idiopathic SF
hCG alone or combined with
rec-hFSH
SR success in 21% of the treated men
vs. none in untreated men
Hussein et
al. 2013
Prospective
cohort
612
unselected
men
Clomiphene alone or
combined with hCG or hMG
Sperm found in SA in 10.9% of treated
males; SRR higher in men who
remained azoospermic and treated
(57.0 vs. 33.6%, p<0.001)
!
Low quality evidence indicates that hormonal therapy might
increase sperm production and sperm retrieval rates
Reviewed by Esteves, Asian J Androl 2015; 17:1-12

18. Increased intratesticular testosterone levels and spermatogonial
DNA synthesis after hCG therapy
Modified from Shinjo et al Andrology 2013;1:929-35 and Shiraishi et al Hum Reprod 2012;27:331-9.
273
1348
Before After
ITT (ng/dl)
ITT levels before and after
hCG-based therapy
Spermatogonial DNA
synthesis increased
Esteves, 18

19. Esteves, 19
Pre-sperm retrieval medical therapy to boost
intratesticular testosterone production
Modified from Esteves, Asian J Androl 2015; 17:1-12
Start with hCG (1,000-1,500 IU)
2x/week
Adjust dose to keep TT levels
~500-800 ng/dl (17-28 nmol/l)
Add aromatase inhibitor if T/E
levels <10
Add FSH 75-150 IU 2X/week if
levels drop to <1.5 IU/l
3 to 6-month treatment duration

20. MB, 46 yo., idiopathic NOA, history of previous negative SR by TESE
hCG dose
increased
FSH
added
Esteves, 20

21. Case study: idiopathic NOA treated with gonadotropins
before micro-TESE
hCG dose increased FSH added
Esteves, 21

22. VIALS
Case study: idiopathic NOA treated with gonadotropins before micro-TESE
Post-SR results
Esteves, 22

23. Spermatogonia B, pachytene
spermatocytes and early
spermatids highly vulnerable to
heat stress
Varicocele repair might improve
spermatogenesis and androgen
production
Esteves, 23
Varicocele found in ~5% men with NOA
Modified from Agarwal, Hamada & Esteves Nature Rev Urol 2012;9:678-90

24. 18 studies, 468 men with NOA subjected to varicocele
repair
44% had postoperative sperm return to ejaculate:
sperm count: 1.8 M/ml (95% CI: 0.98-2.77)
motility: 23% (95% CI: 12-33)
Histopathology the only prognostic factor for sperm
return to ejaculate:
hypospermatogenesis (56%)
maturation arrest (35%)
Sertoli cell only (10%)
Esteves, 24

25. Esteves et al. Outcome of varicocele repair in men with nonobstructive azoospermia: systematic
review and meta-analysis. Asian J Androl 2015;18:246-53
Although 2/3 remain azoospermic after varicocele repair,
SRR and LBR by ICSI higher in treated men
Sperm
retrieval
rate
Live birth
rate
OR=2.65
OR=2.19
Esteves, 25

26. Esteves, 26
Modified from Esteves, Asian J Androl 2015; 17:1-12
Management of men with NOA - Step 4

27. Sperm retrieval methods for men with NOA include
percutaneous and open surgical procedures
Technique Acronym Success
Testicular Sperm
Aspiration TESA 15-50%
Testicular Sperm
Extraction TESE 20-60%
Microdissection
Testicular Sperm
Extraction
Micro-TESE 40-67%
Modified from Esteves et al. Nature Rev Urol. 2018 and Esteves et al. Int Braz J Urol 2013
Esteves, 27

28. Micro-TESE 1.5 times more likely to result in successful sperm
retrieval than conventional TESE
Esteves, 28
Modified from Verza Jr & Esteves. Fertil Steril 2011
Controlled
series (N=60)
P<.01
Micro&TESE*was*1.5*1mes*more*likely*(95%*
CI:*1.4–1.6)*to*result*in*successful*SR*than*
conven1onal*TESE.**
*
Micro&TESE*vs*cTESE*
Modified from Bernie et al. Fertil Steril 2015; 104:1099-1103

29. http://androfert.com.br/videos
From Esteves Int Braz J Urol 2013; 39(3):440 (video)
Esteves, 29

30. Esteves, 30
Modified from Esteves, Asian J Androl 2015; 17:1-12
Management of men with NOA - Step 5

31. Esteves, 31
Process Procedure Techniques Main Goals
Testicular tissue
handling
Extraction of
minimum amount of
tissue
Micro-TESE
Optimal tissue processing and
searching efficiency
Mechanical mincing
Disruption of seminiferous tubules using
needles/microscissors, and forced passing through
small lumen catheters
Tubular break down and cellular
content loss
Enzymatic mincing
Incubation of testicular suspensions with
collagenase type IV (1,000 IU ml-1) and/or DNAse
(25 µg ml-1)
Tubular break down and cellular
content loss
Erythrocyte lysing
Incubation of testicular suspensions with
erythrocyte lysing buffer solution
Elimination of excessive blood cells
Motility enhancement
Incubation of testicular suspensions with
pentoxifylline
Selection of viable sperm for ICSI
Laboratory
environment and
"good laboratory
practices"
Air quality control HEPA & VOC air filtration
Secure optimal safety conditions for
gamete handling, sperm injection and
embryo culture
Temperature and pH
stability
Quality control and quality assurance of
instruments, equipment and reagents
Avoid iatrogenic cellular damage
Centrifugation
Simple washing or gradient centrifugation using low
centrifugation forces (200-300g)
Avoid iatrogenic cellular damage
Sterile techniques
Manipulation of gametes and embryos in laminar
flow cabinets or cleanrooms
Secure optimal safety conditions for
gamete handling, sperm injection and
embryo culture
Intracytoplasmic
sperm injection
Sperm selection
Hyposmotic swelling test
Mechanical touch technique
Laser-assisted sperm selection
Selection of viable immotile sperm for
ICSI
Testicular sperm
storage
Cryopreservation Sperm freezing using small volume carriers
Enhancement of post-thaw sperm
recovery and survival

32. Unpublished data; Source: ANDROFERT; 2007-2017; Mean age: 36.9 years (range: 23-64)
Results – Micro-TESE in NOA
% Sperm retrieval success
56.4%
Esteves, 32
91%
52%
21%
Hypospermatogenesis Maturation arrest SCO
SRR according to predominant testis
histopathology
N=864

33. Results – Micro-TESE in NOA
746 cycles (2007-2017)
Male age: 36.9 yo. (23-64)
Female age: 35.8 (21-44)
2PN: 60.4%
Blastulation: 45.3%
CPR per transfer: 36.4% (244/670)
Miscarriage: 15.6% (38)
LBR: 30.7% (226/680)
Esteves, 33

34. Modified from Esteves SC. JARG 2016; 33(10):1319-35
Sperm freezing in low volume carriers (*Cell Sleeper®)
*Trademark of NIPRO Co., Japan
Esteves, 34

35. Sperm freezing using Cell-sleepers® in NOA
25 patients (age: 35.7 yo.; range: 31-45)
• No. Sperm/’sleeper’: 21 (range: 6-60)
• No. ’Sleepers’/patient: 3 (range: 1-4)
• Post-thaw motility: 70% (range: 10%-90%)
• Sperm recovery rate: 67% (32%-100%)
28 cycles
• %2PN: 53.1% (range: 36%-63%)
• Blastulation rate: 48.2% (35%-61%)
• No. transfers: 6 (mean No. embryos: 1.5)
• Implantation rate: 44%
• OPR per transfer: 3/6 (50%)
Esteves, 35

36. Live birth rates in severe male factor infertility according to the
number of retrieved oocytes
Esteves, 36

37. Authors Region N Outcome Main findings
Palermo et al.
1999
USA 22 Congenital
abnormalities
No difference with obstructive azoospermia
4.5% vs 1.3%
Vernaeve et al.
2005
Belgium 61
Perinatal data;
Congenital
abnormalities
Lower gestational age (singletons); Increased
frequency of premature twins;
No difference with OA (4% vs 3%)
Fedder et al
2007
Denmark 76 Congenital
abnormalities
No difference with other infertility categories
(0% vs 4.0%)
Belva et al.;
2011
Belgium 193 Perinatal data;
Congenital
abnormalities
Similar perinatal outcomes;
No difference 4.2% NOA vs 5.2% OA
Esteves et al.;
2014
Brazil 137 Perinatal data;
Congenital
abnormalities
Similar perinatal outcomes;
No difference 2.1% NOA vs 1.5% OA
Modified from Esteves et al, Nature Rev Urol 2018; Esteves et al. Asian J Androl 2014 & Esteves & Agarwal. Clinics 2013
Neonatal Outcome of Babies
In general, health of offspring from azoospermic fathers reassuring but
data is limited as well as long-term follow-up
Esteves, 37

38. Lack of any sperm in about 50% men with NOA
Esteves, 38
Tanaka et al. Proceedings of the National Academy of Sciences of the
United States of America. 2015;112(47):14629-14634.

39. Lack of any sperm in about 50% men with NOA
Esteves, 39
Stem cell = An undifferentiated cell of a multicellular organism that is
capable of giving rise (by mitosis) to indefinitely more cells of the same
type, and from which certain other kinds of cell arise by differentiation

40. Stem cell therapy for aspermatogenic men
Modified from Aponte et al. Clinics 2013
Esteves, 40

41. Stem cell therapy for aspermatogenic men
Modified from Aponte et al. Clinics 2013
Esteves, 41

42. Take-home messages
NOA the most severe male infertility condition.
Despite lacking sperm in the ejaculate, ~50% of men with NOA have minimal
intratesticular sperm production. Sperm harvested from the seminiferous tubules can be
used for ICSI and result in viable offspring.
Optimal management of men with NOA seeking fertility includes (i) differential
azoospermia diagnosis, (ii) genetic testing and counseling, (iii) identification of men
eligible for medical and/or surgical interventions prior to SR, (iv) use of micro-TESE to
retrieve testicular spermatozoa, and (v) application of state-of-art IVF techniques.
A coordinated multidisciplinary effort involving urologists/andrologists, geneticists,
reproductive endocrinologists, and embryologists is essential to increase the chances of
achieving a biological offspring in men with NOA.
Innovative stem cell research aiming at creating artificial gametes might give
hope to those men with complete aspermatogenesis.
Esteves, 42

43. THANK YOU Dr. Sandro C. Esteves
Dr. Marcelo Scandiucci
Dr. José Eduardo Orosz
Dr. Renan Andreollo
Fabiola Bento
Cristiane Medina
Sidney Verza Jr.
Camila Pompeu
Luciana Oliveira
Vanessa Moreno
Ellen Silva
Roseane Oliveira
Thais Paiva
Sarah Queiroz
Katia Pereira
Sandra Souza
Leila Simplicio
Shirley Machado
Jonathan Santos
Dr. Silval Zabaglia
Dra. Fabiana Nakano
Dr. Julio Voget
Dr. Ricardo Miyaoka
Dr. Ricardo Barini
Dr. Wail Margeotto
Dra. Cristiane Moreira
Dr. Arnaldo Gomes
Marisa Russo
Ivanete Santos
Sandra Santana
Ana Paula Barbosa
Ana Pastorelli
Slides available at: www.slideshare.net/sandroesteves