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 You!s.esteves@androfert.com.br