This document summarizes a training program in assisted reproductive technology focusing on azoospermia. It discusses the differences between obstructive and non-obstructive azoospermia and various sperm retrieval techniques such as PESA, MESA, TESA, and TESE. Success rates of sperm retrieval are provided for different causes of azoospermia. Predictive markers for sperm retrieval success are also examined, such as hormone levels, testicular volume, histopathology, and microdeletions. Micro-TESE is highlighted as a promising surgical method for non-obstructive azoospermia.

1. Training Program in Assisted Reproductive Technology 2011
Cleveland Clinic Reproductive Research Center
Sandro Esteves, MD, PhD
Director, ANDROFERT
Center for Male Reproduction and Infertility
Campinas, BRAZIL

2. Learning Objectives
Understand the difference between obstructive and
non-obstructive azoospermia
Overview the sperm retrieval techniques and
laboratory processing methods for retrieved sperm
Learn the success rates of sperm retrieval in
obstructive and non-obstructive azoospermia
Understand what is the reproductive potential of
azoospermic men undergoing assisted conception
Esteves, 2

3. Azoospermia
• It is not a synonymous of sterility
Non-
Obstructive obstructive
• Normal sperm production • Sperm production deficient
or absent
• Mechanical blockage
• Cryptorchidism, Orchitis, Ra
• Vasectomy, Post- diation, Chemotherapy, Trau
infectious, Congenital ma, Genetic, Varicocele, Go
nadotoxins, Unexplained

4. 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; TEFNA1 Failed PESA in OA
Epididymal agenesis in CAVD cases
Favorable testicular histopathology2 in NOA
Previous successful TESA attempt in NOA
Testicular sperm extraction TESE Failed PESA or TESA in OA
(single or multiple NOA cases
biopsies)
Microsurgical testicular Micro-TESE NOA cases only
sperm extraction
Esteves SC et al. Sperm Retrieval Techniques for Assisted Reproduction.
Int Braz J Urol 2011, in press.
Esteves, 4

5. Obstructive Azoospermia
Sperm • Epididymis
• Testicle
Retrieval • Simple and
for ART effective
Esteves SC & Agarwal A. Sperm Retrieval Techniques; In: Gardner D et al (Eds.), Human
Assisted Reproductive Technology. Cambridge University Press, pp. 41-53, 2011.
Esteves, 5

6. Please visit http://androfert.com.br/videos to see the video

7. Laboratory Sperm Processing
Verza Jr & Esteves, Atlas of Human Reproduction, in press

8. PERCUTANEOUS RETRIEVAL
Esteves SC, Verza S, Prudencio C, Seol B. Success of percutaneous sperm
retrieval and intracytoplasmic sperm injection (ICSI) in obstructive azoospermic (OA)
men according to the cause of obstruction. Fertil Steril. 2010;94 (Suppl):S233.

9. Non-obstructive Azoospermia
 10-20% of infertile men attending ART Clinics
 60-70% of azoospermic men
 Causes are: Male Infertility
• Pre-testicular: HH Diagnosis
• Testicular causes
7.7
• Genetic 19.5
 Y chromosome microdeletion
 Klinefelter syndrome 72.8
 Varicocele
 Cryptorchidism
Other
 Chemotherapy/Radiation
Non-obstructive azoospermia
 Infection Obstructive azoospermia
 Idiopathic
Source: ANDROFERT, Brazil

10. Non-obstructive Azoospermia
Sperm • Sperm production
Untreatable reduced or absent
Retrieval • Geographic location
condition
for ART unpredictable
TESA
TESE

11. Non-obstructive 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

12. 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, 12

13. Non-obstructive 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.

14. Please visit http://androfert.com.br/videos to see the video

15. TESE/TESA Sperm Processing
Verza Jr & Esteves, Atlas of Human Reproduction, in press
Esteves, 15

16. Micro-TESE
RESULTS

17. Micro-TESE vs TESE
Success Rates in Controlled Series
Study N Micro-TESE TESE
Schlegel 1999 27 63% 41%
Amer et al. 2000 100 45% 30%
Okada et al. 2002 98 45% 17%
Okubu et al. 2002 17 48% 24%
Tsujimura et al. 2002 93 43% 35%
Ramon et al. 2003 321 62% 58%
Esteves et al. 2011 60 45% 25%
Total 716 53% 41%
Microdissection provides sperm retrieval
for 1/5 of men who fail standard TESE

18. 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, 18

19. Predictive Values of Noninvasive Tests for
Sperm Retrieval in NOA
Marker Sensitivity % Specificity % Overall
Predictive Value
%
Testicular Volume 7.6-50 6.7-71
FSH 9-71 40-90
Inhibin B 44.6 63.4
FSH, Testosteron 71 71.4
e, Inhibin B
Testicular Volume 80.8
+ Hormones
Doppler US 47.3 89
Carpi. Controversies in nonobstructive azoospermia. Fertil Steril 2009.

20. Predictive Values of Noninvasive Tests for
Sperm Retrieval in NOA
 Chance of finding sperm is dependent on the
most advanced site of spermatogenesis within
the testis.
 Markers reflect global spermatogenic function
but not the most advanced site of sperm
production in a dysfunctional testis.
Esteves, Miyaoka & Agarwal: An update on the initial assessment of the infertile male.
CLINICS 2011; 66:1-10.

21. Chance of Sperm Retrieval by NOA
Diagnosis
 Cryptorchidism 52-74%
 Varicocele 63-68%
 Post-infection (mumps, etc.) 67%
 Torsion >50%
 Post-radiation/chemotherapy 55-75%
 Genetic (Klinefelter, Y microdeletion) 0-75%
 Idiopathic 50-60%
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

22. Predictive Values of Noninvasive Tests for
Sperm Retrieval in NOA
Y Chromosome Microdeletion
AZFb
deletion
Absence of
retrievable sperm
Esteves SC & Agarwal A. Novel concepts in male infertility.
Esteves, 22
Int Braz J Urol 2011; 37:5-15.

23. Predictive Values of Invasive Tests for
Sperm Retrieval in NOA
 Testicular Histopathology
Esteves, Miyaoka & Agarwal. Surgical Treatment of Male Infertility in the ICSI Era.
CLINICS 2011; 66:1463-77.
Esteves, 23

24. Microsurgical vs Single-Biopsy TESE in NOA: a
prospective controlled study
Verza Jr S & Esteves SC; ASRM 2011 (O-178)
Single Large Second Biopsy Micro-TESE
Open-Biopsy Fragment • Sperm Search
• Sperm Search • Histology
Esteves, 24

25. Microsurgical vs Single-Biopsy TESE in
Non-obstructive Azoospermia
• Controlled series of 60 patients
Sperm Retrieval Success Rates
Micro-TESE single-biopsy TESE
93%
P=0.02
64% 64%
45%
25% 20%
9% 6%
Overall Hypospermatogenesis Maturation Arrest Sertoli-cell Only
Verza Jr & Esteves, O-178, ASRM 2011

26. Sperm Retrieval Techniques
Advantages Disadvantages
PESA Fast and low cost; No surgery Few sperm retrieved; Cryopreservation limited
Minimal morbidity, repeatable Fibrosis and obstruction at aspiration site
Risk of hematoma/spermatocele
MESA Large number of sperm retrieved Increased cost and time-demanding
Sperm cryopreservation Microsurgical instruments and expertise
Reduced risk of hematoma Postoperative discomfort
TESA Fast and low cost; No surgery Low success rate/few sperm retrieved in NOA
Repeatable Cryopreservation limited
Minimal/mild postop discomfort Risk of hematoma/testicular atrophy
TESE No microsurgical expertise Low success rate/few sperm retrieved in NOA
Fast and repeatable Risk of testicular atrophy (multiple biopsies)
Postoperative discomfort
Micro-TESE Higher success rates in NOA Increased cost and time-demanding
Larger number of sperm Microsurgical instruments and expertise
retrieved Postoperative discomfort
Esteves, Miyaoka & Agarwal. Sperm Retrieval Techniques for Assisted Conception.
Int Braz J Urol in press

27. Reproductive
Potential of
Azoospermic Men
undergoing ART
Esteves, 27

28. Intracytoplasmic Sperm Injection Outcomes Using
Surgically-retrieved Sperm from Obstructive
Azoospermic Men
Epididymis Testicle p
Female Age (years) 31.5 7.7 36.3 5.1
Mature Oocytes Injected (n) 9.4 5.8 9.4 4.9
Embryo Transfer (n) 3.3 1.3 3.7 1.5
2PN Fertilization (%) 74.7% 21.2% 69.1% ± 19.6%
NS
TQE day 3 (%) 44.6% 30.5% 52.7% ± 29.6%
Clinical Pregnancy (%) 51.6% 50.0%
Miscarriage (%) 18.8% 25.0%
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, 28

29. Intracytoplasmic Sperm Injection Outcomes Using
Ejaculates vs. Surgically-retrieved Sperm from
Obstructive Azoospermic Men
Ejaculate Epididymis/Testicle
70 73
48 46 51 NS
43
20
12
Fertilization rate %TQE Pregnancy (%) Miscarriage (%)
(%)
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.

30. Intracytoplasmic Sperm Injection Outcomes Using
Surgically-retrieved Sperm
Obstructive Non-obstructive
Azoospermia Azoospermia
2PN Fertilization Rate 73.6% 52.2%*
TQE transfer day 46.3% 35.7%*
Clinical Pregnancy Rate 51.3% 25.9%*
Miscarriage Rate 20.0% 14.3%
*
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, 30

31. Sperm Defect Severity Rather Than Sperm Source Is
Associated With Lower Fertilization Rates After
Intracytoplasmic Sperm Injection
Verza Jr S & Esteves SC; Int Braz J Urol 2008; 34
ICSI Ejaculated Sperm (n=220) Testicular/
Epididymal Sperm
Sperm Defect (n=93)
Normal Single Double Triple OA NOA
2PN Fertilization (%) 71.3 73.2 72.1 63.4* 73.6 52.2*
TQE on Day 3 (%) 48.4 50.5 46.9 48.3 46.3 35.7*
Clinical Pregnancy (%) 40.9 36.6 44.4 51.0 51.3 25.9*
Miscarriage (%) 14.9 9.1 12.5 12.0 20.0 14.3
* P<0.05 Esteves, Androfert

32. Sperm Retrieval Rates and Reproductive
Potential of Azoospermic Men undergoing ICSI
Obstructive (N=142) Non-obstructive (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 <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.

33. Sperm Retrieval Techniques
Obstructive Azoospermia
• Sperm retrieval and lab processing simple
• Sperm obtained in virtually all cases
• Chance of Retrieval and ICSI Outcomes:
• Independent on obstruction etiology
• Independent on retrieval technique
• Independent on sperm source
• Results similar or better than ejaculated sperm

34. Sperm Retrieval Techniques
Non-obstructive Azoospermia
• Sperm production deficient or absent
• Overall, retrieval rates ~50%
• Labor-intensive lab sperm processing
• Retrieval rates dependent on technique
• Micro-TESE yields better SRR
• Predictive factors: testis histology & Y-chromosome
• Reproductive potential by ICSI lower than OA
and non-azoospermic men

35. MCQ 1
 Azoospermic males presenting with:
a) obstructive azoospermia (OA) have normal spermatogenesis and a
mechanical block somewhere between the epididymis and the
ejaculatory duct. Common causes of OA include vasectomy, post-
infectious diseases and congenital conditions.
b) nonobstructive azoospermia (NOA) have extremely deficient or
absent sperm production within the testicles. Common causes of NOA
include cryptorchidism, orquitis, radio/chemotherapy, use of
gonadotoxic medication and steroids, and genetic origin.
c) nonobstructive azoospermia have retrieval rates dependent on the
method of collection. Testicular histopathology results and Y-
chromosome microdeletion testing are useful tools to predict the
likelihood of sperm retrieval.
d) obstructive azoospermia have virtually 100% successful retrievals.
Retrieval rates and ICSI outcomes are neither dependent on the method
of collection nor on the origin of sperm for ICSI (epididymal or
testicular).

36. MCQ 2
 The following techniques can be used to retrieve sperm in men with
nonobstructive azoospermia:
a) PESA (percutaneous epididymal sperm aspiration).
b) Micro-TESE (microdissection testicular sperm extraction).
c) TESA (testicular sperm aspiration).
d) Conventional TESE (testicular sperm extraction) using single or
multiple biopsies.

37. MCQ 3
 The following statements apply to sperm retrieval techniques:
a) Micro-TESE yields higher sperm retrieval success rates than
conventional TESE or TESA.
b) PESA is a fast, effective and safe method to retrieve sperm in
obstructive azoospermia. Expertise in microsurgery is required for
PESA.
c) TESA is safe and effective in cases of failed PESA. No expertise in
microsurgery is required for TESA.
d) MESA is indicated in obstructive azoospermia. Sperm retrieval rates
are comparable to PESA although higher sperm number is obtained.

38. MCQ 4
 Overall, sperm retrieval success and pregnancy rates by ICSI (using
retrieved sperm) in men with obstructive (OA) and nonobstructive
(NOA) azoospermia are:
a) 50% and 30%, 70% and 25%, respectively.
b) >90% and 50%, 40% and 25%, respectively.
c) 50% and 30%, respectively, and rates are not dependent on the type
of azoospermia being obstructive or nonobstructive.
d) 100% and 50% in OA men with vasectomy, and 0% in NOA men with
testicular histology showing germ cell aplasia (Sertoli cell-only).