1. 2012 Summer Internship Program
Cleveland Clinic Reproductive Research Center
Sandro Esteves, MD, PhD
Director, ANDROFERT
Center for Male Reproduction and Infertility
Campinas, BRAZIL

2. Learning Objectives
Understand azoospermia and the differences
between obstructive and nonobstructive subtypes
Learn the available methods for sperm acquisition
in azoospermia and their indications
Learn the success rates of sperm retrieval in
different azoospermia scenarios
Understand the reproductive potential of
azoospermic men undergoing assisted conception
Esteves, 2

3. Review this lecture at:
http://bit.ly/ccfsummerinterns2012
Pdf slides
Videos
Reference papers

4. Spermatogenesis
Where do we stand compared to our relatives?
Chimpanzee Human Gorilla
100 lbs 180 lbs 600 lbs
64 cc 20 cc 14 cc
>1 64 5
billion/mL million/mL million/mL

5. Sperm Count in Humans
General Population of Unscreened Men
Centiles
2.5% 50% 97.5%
Sperm count
per mL (x106) 4 64 237
Azoospermia
• Complete absence of sperm in the ejaculate
• 1-3% male population
• ~10% male infertility population
Cooper et l. Hum Reprod Update 2009; Esteves et al, CLINICS 2011

6. Semen Analysis and
Azoospermia
Centrifugation
at 3,000g for
15 minutes
The supernatant is
discharged and the
pellet is examined
Esteves, 6

7. Types of Azoospermia
Non-
Obstructive obstructive
• Normal sperm production • Absent or minimal sperm
• Mechanical blockage anywhere production within the
along the reproductive tract testicles
• Epididymis • Testicular failure
• Vas Deferens
• Ejaculatory Duct

8. Etiology of Azoospermia
Obstructive Nonobstructive
Congenital
Congenital bilateral absence of vas Testicular dysgenesis/cryptorchidism
deferens (CBAVD) Genetic abnormalities (Klinefelter’s
Ejaculatory duct and prostatic cysts syndrome, Yq microdeletions)
Germ cell aplasia (SCO syndrome)
Spermatogenic (maturation) arrest
Acquired
Post-infection (epididymitis, prostatitis, Testicular torsion; Trauma
seminal vesiculitis)
Post-inflammatory (eg. mump’s orchitis)
Post-surgical (vasectomy, epididymal cysts, Exogenous factors (steroid medications,
hernia, scrotal surgery, bladder neck surgery,
cytotoxic drugs, irradiation, heat)
prostatectomy)
Testicular Cancer; Systemic diseases
Iatrogenic (urological endoscopic
(liver cirrhosis, renal failure)
instrumentation)
Varicocele
Idiopathic (Unknown etiology)

9. Management of Azoospermia
OA NOA
non-
treatable
treatable
ductal sperm
reconstruc- retrieval
tion and ICSI
Esteves et al. An update on the initial assessment of the infertile male.
CLINICS 2011;66:1-10.

10. 1. Azoospermia is a descriptive
term of ejaculates that lack
spermatozoa without
implying a specific underlying
cause.
2. Azoospermia is not synonymous of sterility.
Treatment options are microsurgical ductal
reconstruction (selected cases of OA) and sperm
retrieval coupled with in vitro fertilization (ICSI).

11. Sperm Retrieval Goals
Immediate
use for
Obtain ICSI
sperm
for ICSI
Cryopreservation
Future
retrievals
Minimize
damage
Testicular
function

12. Sperm Retrieval Techniques
Technique Acronym Indications
Percutaneous Epididymal PESA OA cases only
Sperm Aspiration
Microsurgical Epididymal MESA OA cases only
Sperm Aspiration
Testicular Sperm Aspiration TESA; Failed PESA in OA
TEFNA1 Epididymal agenesis in CAVD
Favorable histopathology in NOA
Previous SR success in NOA
Testicular Sperm Extraction TESE Failed PESA or TESA in OA
(single or multiple biopsies) NOA cases
Microsurgical Testicular Micro-TESE NOA cases only
sperm Extraction
Esteves et al. Sperm Retrieval Techniques for Assisted Reproduction.
Esteves, 12
Int Braz J Urol 2011; 37(5):570-83

13. Sperm Retrieval in Obstructive
Azoospermia
• Epididymis
• Testicle
• Simple and Effective
Esteves SC & Agarwal A. Sperm Retrieval Techniques; In: Gardner D et al (Eds.), Human
Esteves, 13 Assisted Reproductive Technology. Cambridge University Press, pp. 41-53, 2011.

14. Percutaneous Sperm Retrieval in
Obstructive Azoospermia
Please visit http://androfert.com.br/videos to watch this video

15. PESA alone
PESA + rescue TESA
97.3
%
OBSTRUCTIVE AZOOSPERMIA
100% 96.6% 96.3%
78.1
%
Successful Retrievals CBAVD Vasectomy Post-infection
Esteves et al. Reproductive potential of men with OA undergoing percutaneous
sperm retrieval and ICSI according to the cause of obstruction. J Urol
Esteves, 15
2012, submitted.

16. Epididymal/Testicular sperm Ejaculated sperm
70 73.6
P>0.05
48.5 46.3 51.3
43.2
20
12.1
%2PN %Top quality % Pregnancy % Miscarriage
Fertilization embryos
Verza Jr S & Esteves SC. Sperm defect severity rather than sperm source is associated
with lower fertilization rates after intracytoplasmic sperm injection.
Int Braz J Urol 2008; 34:49-56.
Esteves, 16

17. CBAVD Post-vasectomy Post-infection
P>0.05 265 277 250
Maformation rate: 1.5%
Perinatal mortality: 1.5%
34.4 32.2 36.4 35.8 37.0 35.5
% Live birth Gestational age (wks) Birth weight
(gramsx10)
Esteves et al. Reproductive potential of men with OA undergoing percutaneous
sperm retrieval and ICSI according to the cause of obstruction. J Urol
Esteves, 17
2012, submitted.

18. Sperm Retrieval
In Obstructive Azoospermia

19. Non-obstructive
Untreatable Azoospermia
condition
 Small testes/elevated FSH/”sterile”
 Absent or minimal production for
sperm to appear in ejaculate
 Heterogeneity of sperm production:
600-800 seminiferous tubules/testis;
Single focus of production adequate to
retrieve spermatozoa for ICSI
 Goal: To identify and retrieve
sperm for ICSI, but…
Geographic location unpredictable
Esteves, 19

20. Can We Predict Sperm Retrieval
Success in NOA?
Important because:
1. Can minimize emotional and financial cost of IVF
cycles.
2. Can minimize trauma/damage to testis during sperm
harvesting.
Esteves, 20

21. Predictive Value of Noninvasive Tests
for Sperm Retrieval in NOA
FSH Testosterone
Testicular Volume
Verza Jr. & Esteves. Fertil Steril 2011; 96: S53
Esteves, 21

22. Predictive Value of Noninvasive
Tests for Sperm Retrieval in NOA
Y Chromosome Microdeletion Screening
Prevalence of Yq microdeletions:
1:2.000-3.000 newborns
Azoospermic men: 5-12%
Esteves, 22
Esteves, Miyaoka & Agarwal. An update on the initial assessment of the
infertile male. CLINICS 2011; 66:1-10.

23. Predictive Value of Noninvasive
Tests for Sperm Retrieval in NOA
Y Chromosome Microdeletion Screening
AZFb
deletion
Absence of
retrievable sperm
Esteves SC & Agarwal A. Novel concepts in male infertility.
Esteves, 23
Int Braz J Urol 2011; 37:5-15.

24. Predictive Value of Invasive
Tests for Sperm Retrieval
in NOA
 Testicular Histopathology
Sensitivity Specificity (95% Accuracy
(95% CI) CI) (%)
HYPO 93 (66-100) 70 (54-82)
MA 64 (31-89) 59 (44-73) 81.9
SCO 20 (08-37) 20 (07-41)
Esteves, Miyaoka & Agarwal. Surgical Treatment of Male Infertility in the ICSI Era.
Esteves, 24 CLINICS 2011; 66:1463-77.

25. Predictive Value of Testing
for Sperm Retrieval in NOA

26. Sperm Retrieval in
Nonobstructive Azoospermia
OPEN BIOPSY

27. Nonobstructive Azoospermia
TESA vs. TESE
Controlled studies Needle Open Biopsy
for NOA men Aspiration
Friedler et al., 4/37 (11%) 16/37 (43%)
Human Reprod 12:1488, 1997
Ezeh et al. 5/35 (14%) 22/35 (63%)
Human Reprod 13:3075, 1998

28. Conventional TESE (open biopsy)
in NOA
Number of patients 25
20
15
10
5
0
1 2 3 4 7 8 9 10 14
Number of testicular fragments excised
Ostad et al., Urology 52:692, 1998.
Esteves, 28

29. Nonobstructive Azoospermia
Testicular microdissection - micro-TESE
• Method to identify site(s) of
production
– Based on the diameter of
seminiferous tubules
• Microsurgical approach
– Identify site of production
– Preserve vasculature of testis
– Small quantity of tissue excised
Schlegel PN. Testicular sperm extraction: microdissection improves sperm yield with
minimal tissue excision. Hum Reprod. 1999;14:131-135.

30. Please visit http://androfert.com.br/videos to watch this video

31. Schlegel 1999
Amer et al. 2000
Micro-
Okada et al. 2002
TESE
43%-53%
Okubu et al. 2002
Tsujimura et al. 2002 TESE
25%-41%
Ramon et al. 2003
Esteves et al. 2011

32. 1. Sperm retrieval
techniques are surgical
methods to collect
spermatozoa from the
epididimys or the testis of
azoospermic men seeking
fertility.
2. The method of choice is based primarily upon
the type of azoospermia being obstructive or
nonobstructive.
3. Retrieved sperm should be used for ICSI or
cryopreserved for a future ICSI attempt.

33. Microsurgical vs Single-Biopsy TESE
in Nonobstructive Azoospermia
Controlled series of 60 patients
Sperm Retrieval Success Rates
Micro-TESE single-biopsy TESE
93%
Method Histology categories
P=0.0005 64% 64% pairwise
45%
comparisons
P<0.0001
25% 20%
9% 6%
Overall Hypospermatogenesis Maturation Arrest Sertoli-cell Only
Microsurgical versus conventional single-biopsy testicular sperm extraction in
nonobstructive azoospermia: a prospective controlled study
Esteves, 33 Verza Jr S, Esteves SC. Fertil Steril 2011; 96 (3): S53

35. Hypospermatogenesis
Sertoli cell-only
Verza Jr & Esteves, Atlas of Human Reproduction, SBRH 2012

36. Conventional TESE Micro-TESE
Microsurgical vs Single-
Biopsy TESE in
Nonobstructive
Azoospermia
Fragment weight Fragment weight
Tissue Removal
Open Large Micro- P-
Single-Biopsy TESE value
TESE
Tissue Removed (mg) 65 ± 25 8.9 ± 2.5 <0.01
Esteves, 36
Verza Jr & Esteves. Fertil Steril 2011; 96 (3): S53

37. Success of Sperm Retrieval
by Cause of NOA
Cryptorchidism 52-74%
Varicocele 63-68%
Post-infection 67%
Torsion >50%
Post-chemotherapy/RT 25-75%
Genetic (Klinefelter, AZFc Yq microdeletions) 25-70%
Idiopathic 50-60%
OVERALL 40-60%
Esteves et al., Fertil Steril 94; 2010; Raman and Schlegel. J Urol.170; 2003;
Hopps et al. Hum Reprod. 180, 2003; Damani et al. JCO. 15; 2002

38. No. of Patients 255
% Retrieval Rate 51.1
No. ICSI cycles 328
Mean ± SD Patient Age
Male 37.0 ± 7.6
Female 32.4 ± 4.7
Mean ± No. Injected Oocytes 9.8 ± 7.2
Mean ± %2PN Fertilization 43.7 ± 27.9
No. Transfer 298
Mean ± No. Embryos Transferred 2.4
No. Clinical Pregnancy (%) 86 (28.9)
No. Live birth (%) 64 (21.5)
Esteves, 38

39. No. of Babies Born 102
No. Multiple Deliveries (%) 29 (28.4)
Mean ± SD Gestational Age 35.5 ± 2.7
Mean ± Birth Weight 2532 ± 601
Gender ratio; boy/girl 1.0/1.3
No. Perinatal Deaths 6 (5.9)
No. Birth Defects (%) 3 (2.9)
Esteves, 39

40. Sperm Retrieval Success Rates and
Reproductive Potential of Azoospermic
Men undergoing ICSI
Obstructive (N=142) Nonobstructive (N=172)
97.9%
55.2%
38.2%
25.0%
Successful Sperm Retrieval Live Birth rate
Odds-ratio 43.0 1.86
95% CI 10.3 – 179.5 1.03 – 2.89
P-value <0.01 0.03
Prudencio C, Seoul B, Esteves SC. Reproductive potential of azoospermic men
undergoing intracytoplasmic sperm injection is dependent on the type of azoospermia.
Fertil Steril 2010; 94(4):S232-3.

41. Sperm Retrieval
In Obstructive Azoospermia

42. Sperm Retrieval
in
Nonobstructive
Azoospermia

43. 1. Nowadays, the use of
surgically-retrieved
sperm and ICSI has
become an established
procedure for couples
wishing to obtain a
biological offspring in
whom the male partner
have azoospermia.
2. So far, the post-natal outcomes of babies born
from such fathers are reassuring.

44. Learning Objectives
Understand azoospermia and the differences
between obstructive and nonobstructive subtypes
Azoospermia is the lack of sperm in the ejaculate
Should be confirmed by semen analysis with
centrifugation and pellet examination
Found in ~10% of the male infertility population
Types are Obstructive and Nonobstructive
Congenital, acquired and unknown etiologies
AO: sperm production is normal
NOA: sperm production severely abnormal or absent
Esteves, 44

45. Learning Objectives
Learn the available methods for sperm acquisition
in azoospermia and their indications
Percutaneous (PESA, TESA) and open
(MESA, TESE, micro-TESE) techniques are
available
Epididymides and testicles are the target organs
Epididymal retrievals: Obstructive azoospermia
Testicular retrievals: AO and NOA
Microdissection TESE for the most difficult cases of NOA
Esteves, 45

46. Learning Objectives
Learn the success rates of sperm retrieval in
different azoospermia scenarios
Sperm retrieved in virtually all cases of OA
Not related to collection method or cause of obstruction
Sperm retrieved in 40-60% of NOA cases
Current testing not reliable to predict SR success
Success not related to the cause of NOA
Men with AZF a or b microdeletions not candidates
Higher SRR with micro-TESE
Esteves, 46

47. Learning Objectives
Understand the reproductive potential of
azoospermic men undergoing assisted conception
Success of ICSI
Not related to collection method
Related to the type of azoospermia
Follow-up of children born similar outcomes (few data)
Obstructive Azoospermia
Similar (or better) results than ejaculated sperm
~40% live birth rates
Nonobstructive Azoospermia
Lower results than other infertility causes
~25% live birth rates
Esteves, 47