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Sandro Esteves, MD, PhD
Director, ANDROFERTCenter for Male Reproduction and Infertility
Campinas, BRAZIL
Sperm Retrieval Techniques:
Looking for a Needle in the Haystack
2012 Summer Internship Program Cleveland Clinic Reproductive Research Center
Esteves, 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
Review this lecture at:
http://bit.ly/ccfsummerinterns2012
Pdf slides
Videos
Reference papers
Sperm Retrieval Techniques:
Looking for a Needle in the Haystack
100 lbs
64 cc
>1 billion/mL
SpermatogenesisWhere do we stand compared to our relatives?
600 lbs
14 cc
5 million/mL
180 lbs
20 cc
64 million/mL
HumanChimpanzee Gorilla
Centiles
2.5% 50% 97.5%Sperm count per mL (x106) 4 64 237
Sperm Count in HumansGeneral Population of Unscreened Men
Cooper et l. Hum Reprod Update 2009; Esteves et al, CLINICS 2011
Azoospermia• Complete absence of sperm in the ejaculate• 1-3% male population• ~10% male infertility population
Esteves, 6
Semen Analysis and Azoospermia
Centrifugation at 3,000g for 15 minutes
The supernatant is discharged and the pellet is examined
Types of Azoospermia
• Normal sperm production• Mechanical blockage
anywhere along the reproductive tract• Epididymis• Vas Deferens• Ejaculatory Duct
Obstructive• Absent or minimal sperm production within the testicles• Testicular failure
Non-obstructive
Obstructive Nonobstructive
Etiology of Azoospermia
Post-infection (epididymitis, prostatitis, seminal vesiculitis)
Post-surgical (vasectomy, epididymal cysts, hernia, scrotal surgery, bladder neck surgery,
prostatectomy)
Iatrogenic (urological endoscopic instrumentation)
Testicular torsion; Trauma
Post-inflammatory (eg. mump’s orchitis)
Exogenous factors (steroid medications, cytotoxic drugs, irradiation, heat)
Testicular Cancer; Systemic diseases (liver cirrhosis, renal failure)
Varicocele
CongenitalCongenital bilateral absence of vas
deferens (CBAVD)
Ejaculatory duct and prostatic cysts
Testicular dysgenesis/cryptorchidism
Genetic abnormalities (Klinefelter’s syndrome, Yq microdeletions)
Germ cell aplasia (SCO syndrome)
Spermatogenic (maturation) arrest
Acquired
Idiopathic (Unknown etiology)
OA NOA
non-treatabletreatable
ductal reconstruc-
tion
sperm retrieval and ICSI
Management of Azoospermia
Esteves et al. An update on the initial assessment of the infertile male. CLINICS 2011;66:1-10.
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).
Sperm Retrieval GoalsImmediate use for ICSI
Cryopreservation
Future retrievals
Testicular function
Minimize damage
Obtain sperm
for ICSI
Technique Acronym Indications
Percutaneous Epididymal Sperm Aspiration
PESA OA cases only
Microsurgical Epididymal Sperm Aspiration
MESA OA cases only
Testicular Sperm Aspiration TESA; TEFNA1
Failed PESA in OAEpididymal agenesis in CAVD
Favorable histopathology in NOA Previous SR success in NOA
Testicular Sperm Extraction (single or multiple biopsies)
TESE Failed PESA or TESA in OANOA cases
Microsurgical Testicular sperm Extraction
Micro-TESE NOA cases only
Sperm Retrieval Techniques
Esteves, 12
Esteves et al. Sperm Retrieval Techniques for Assisted Reproduction. Int Braz J Urol 2011; 37(5):570-83
Sperm Retrieval in Obstructive Azoospermia
• Epididymis• Testicle• Simple and 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, 13
Please visit http://androfert.com.br/videos to watch this video
Percutaneous Sperm Retrieval in Obstructive Azoospermia
Success of Sperm Retrieval Using
Percutaneous Methods in OA
100% 96.6% 96.3%
CBAVD Vasectomy Post-infection
OBSTRUCTIVE AZOOSPERMIA
Esteves et al. Reproductive potential of men with OA undergoing percutaneous sperm retrieval and ICSI according to the cause of obstruction. J Urol 2012,
submitted.Esteves, 15
Successful Retrievals
78.1%
97.3%
PESA alonePESA + rescue TESA
%2PN Fertiliza-tion
%Top quality embryos
% Pregnancy % Miscarriage
70
48.543.2
12.1
73.6
46.351.3
20
Epididymal/Testicular sperm Ejaculated sperm
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.
P>0.05
Esteves, 16
Success of ICSI using surgically-retrieved sperm from men with OA
% Live birth Gestational age (wks)
Birth weight (gramsx10)
34.4 35.8
265
32.2 37.0
277
36.4 35.5
250
CBAVD Post-vasectomy Post-infection
P>0.05
Esteves, 17
Neonatal Outcome of Babies Born after ICSI in OA according to the
cause of obstruction
Esteves et al. Reproductive potential of men with OA undergoing percutaneous sperm retrieval and ICSI according to the cause of obstruction. J Urol 2012,
submitted.
Maformation rate: 1.5%Perinatal mortality: 1.5%
Sperm Retrieval In Obstructive Azoospermia
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
Non-obstructive Azoospermia Untreatable
condition
Esteves, 19
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
Predictive Value of Noninvasive Tests for Sperm Retrieval in NOA
Verza Jr. & Esteves. Fertil Steril 2011; 96: S53
FSH Testosterone
Testicular Volume
Esteves, 21
Prevalence of Yq microdeletions:1:2.000-3.000 newborns
Azoospermic men: 5-12%
Esteves, 22Esteves, Miyaoka & Agarwal. An update on the initial assessment of the
infertile male. CLINICS 2011; 66:1-10.
Predictive Value of Noninvasive Tests for Sperm Retrieval in NOAY Chromosome Microdeletion Screening
Esteves SC & Agarwal A. Novel concepts in male infertility. Int Braz J Urol 2011; 37:5-15.
AZFa deletion
Absence of retrievable
sperm
AZFc deletion
70% success to retrieve testicular
sperm
AZFb deletion
Absence of retrievable sperm
Esteves, 23
Predictive Value of Noninvasive Tests for Sperm Retrieval in NOAY Chromosome Microdeletion Screening
Testicular Histopathology
Predictive Value of Invasive Tests for Sperm Retrieval
in NOA
Esteves, Miyaoka & Agarwal. Surgical Treatment of Male Infertility in the ICSI Era. CLINICS 2011; 66:1463-77. Esteves, 24
Sensitivity (95% CI)
Specificity (95% CI)
Accuracy (%)
HYPO 93 (66-100) 70 (54-82)81.9MA 64 (31-89) 59 (44-73)
SCO 20 (08-37) 20 (07-41)
Predictive Value of Testing for Sperm Retrieval in NOA
Sperm Retrieval in Nonobstructive Azoospermia
OPEN BIOPSY
Controlled studies for NOA men
Needle Aspiration
Open Biopsy
Friedler et al., Human Reprod 12:1488, 1997
4/37 (11%) 16/37 (43%)
Ezeh et al. Human Reprod 13:3075, 1998
5/35 (14%) 22/35 (63%)
Nonobstructive Azoospermia
TESA vs. TESE
Conventional TESE (open biopsy) in NOA
1 2 3 4 7 8 9 10 140
5
10
15
20
25
Number of testicular fragments excised
Nu
mb
er o
f p
atie
nts
Ostad et al., Urology 52:692, 1998.
Esteves, 28
Nonobstructive AzoospermiaTesticular 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.
Please visit http://androfert.com.br/videos to watch this video
Schlegel 1999
Amer et al. 2000
Okada et al. 2002
Okubu et al. 2002
Tsujimura et al. 2002
Ramon et al. 2003
Esteves et al. 2011
Micro-TESE
43%-53%
TESE
25%-41%
Success of Sperm Retrieval by Method in NOA
– controlled series –
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.
Microsurgical vs Single-Biopsy TESE in Nonobstructive Azoospermia
Overall Hypospermatogenesis Maturation Arrest Sertoli-cell Only
45%
93%
64%
20%25%
64%
9% 6%
Sperm Retrieval Success Rates
Micro-TESE single-biopsy TESE
Controlled series of 60 patients
Microsurgical versus conventional single-biopsy testicular sperm extraction in nonobstructive azoospermia: a prospective controlled study
Verza Jr S, Esteves SC. Fertil Steril 2011; 96 (3): S53
Histology categoriespairwise
comparisons P<0.0001
MethodP=0.0005
Esteves, 33
80 µM
320 µM
90 µM
140 µM
x100
Sertoli cell-only
Hypospermatogenesis
80 µM
230 µM
Verza Jr & Esteves, Atlas of Human Reproduction, SBRH 2012
Tissue Removal
Open Large Single-Biopsy
TESE
Micro-TESE P-value
Tissue Removed (mg) 65 ± 25 8.9 ± 2.5 <0.01
Verza Jr & Esteves. Fertil Steril 2011; 96 (3): S53
Microsurgical vs Single-Biopsy TESE in Nonobstructive
Azoospermia
Esteves, 36
Conventional TESE Micro-TESE
Fragment weight Fragment weight
Success of Sperm Retrieval by Cause of NOA
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
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%
No. of Patients 255
% Retrieval Rate 51.1
No. ICSI cycles 328
Mean ± SD Patient AgeMale
Female37.0 ± 7.632.4 ± 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
Success of ICSI using testicular sperm retrieved by micro-TESE in NOA
Androfert (2010)
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
Neonatal Outcome of Babies Born after Micro-TESE and ICSI in NOA - Androfert (2010)
Successful Sperm Retrieval Live Birth rate
97.9%
38.2%55.2%
25.0%
Obstructive (N=142) Nonobstructive (N=172)
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.
Sperm Retrieval Success Rates and Reproductive Potential of Azoospermic
Men undergoing ICSI
Sperm Retrieval In Obstructive Azoospermia
Sperm Retrieval in
NonobstructiveAzoospermia
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.
Esteves, 44
Understand azoospermia and the differences between obstructive and nonobstructive subtypes
Learning Objectives
Azoospermia is the lack of sperm in the ejaculateShould be confirmed by semen analysis with
centrifugation and pellet examination
Found in ~10% of the male infertility population
Types are Obstructive and NonobstructiveCongenital, acquired and unknown etiologies
AO: sperm production is normalNOA: sperm production severely abnormal or absent
Esteves, 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 NOAMicrodissection TESE for the most difficult cases of NOA
Esteves, 46
Learning ObjectivesLearn the success rates of sperm retrieval in
different azoospermia scenarios
Sperm retrieved in 40-60% of NOA casesCurrent 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
Sperm retrieved in virtually all cases of OANot related to collection method or cause of obstruction
Esteves, 47
Learning ObjectivesUnderstand the reproductive potential of
azoospermic men undergoing assisted conception
Success of ICSINot related to collection method
Related to the type of azoospermiaFollow-up of children born similar outcomes (few data)
Obstructive AzoospermiaSimilar (or better) results than ejaculated sperm ~40% live birth rates
Nonobstructive AzoospermiaLower results than other infertility causes
~25% live birth rates
Thank [email protected]