Testicular Failure in Humans: Current management and future perspectives

Sandro C. Esteves, MD., PhD. Medical Director, ANDROFERT

Campinas, Brazil

Testicular Failure in Humans Current Management and

Future Perspectives

V ISABR 2014

Learning objectives At the completion of this talk participants should

be able to: •  Overview the technical aspects of ICSI as

carried out in humans •  Understand why spermatogenic failure (SF) is

one of the most challenging condition in human reproduction

•  Learn how we manage men with SF seeking fertility at Androfert

ANDROFERT androfert.com.br

ANDROLOGY AND HUMAN REPRODUCTION CLINIC - REFERRAL CENTER FOR MALE REPRODUCTION S ESTEVES, 2 2014 OCTOBER

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http://www.androfert.com.br/review

Testicular Failure in Humans Current Management and Future

Perspectives



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Esteves & Agarwal. Sperm Retrieval Techniques; In: Gardner et al. Human Assisted Reproductive Technology. Cambridge, pp. 41-53, 2011

Spermatogenic failure is the worst prognostic condition in male infertility

§  Up to 1% of all men; 10-15% of infertile

males §  Irreversible condition; often termed as

“sterile” §  Semen analysis reveals “Azoospermia”

Should be confirmed in multiple specimens after examination of pelleted semen



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Spermatogenic failure is an irreversible condition

Testicular torsion; Trauma Post-inflammatory (eg. Mumps orchitis) Exogenous factors (eg. Cytotoxic drugs, irradiation) Testicular cancer Systemic diseases (eg. Liver cirrhosis, renal failure)

Congenital Testicular dysgenesis/cryptorchidism Genetic abnormalities (Klinefelter syndrome, Yq microdeletions, etc.)

Acquired

Idiopathic (Unknown etiology) Esteves et al. Clinics 2011; 66: 691-700



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Esteves et al Int Braz J Urol 2014; 40: 443-53

Goals of semen analysis are to reduce analytical error and enhance precision

Examination of pelleted

semen Differentiation between ‘true’ azoospermia (lack of any sperm) and cryptozoospermia (very few sperm)

Minimum 2-3 analyses Transient azoospermia due to medical conditions and biological variability



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Supernatant is discharged

Pellet is meticulously examined

Centrifugation at 3,000g for 15 minutes

Prognosis and management differentially affected by type of azoospermia

Azoospermia

Obstructive

Non-obstructive

Types

Hypo-hypo

Spermatogenic failure

Clinical Picture

FSH/LH: ñ or nl TT: low or nL

Testes: small or nl

NL testes NL FSH, LH, TT

Mechanical blockage

FSH/LH <1.2 mUI/mL, Low TT, small testis,

poor virilization



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Disrupted

Normal

Spermatogenesis

Diagnostic parameters provide >90% prediction of whether azoospermia is due

to spermatogenic failure 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%)



ANDROFERT Esteves et al Clinics 2011

Verza Jr & Esteves, Atlas of Human Reproduction SBRH 2013

Isolated diagnostic biopsy rarely indicated provide no definitive proof of whether sperm will be

found; may jeopardize future retrieval attempts

Differential diagnosis with obstructive azoospermia Work-up in SF associated to maturation arrest is unrevealing

Wet examination and cryopreservation if sperm found

Hypospermatogenesis

Maturation arrest

Sertoli cell-only



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Sperm retrieval highly successful regardless of cause of obstruction and method of retrieval



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Obstructive azoospermia is a favorable prognostic condition in male infertility

100% 96.6% 96.3%

CBAVD Vasectomy Post-‐infection

OBSTRUCTIVE AZOOSPERMIA

Management options include reconstructive surgery and ART

OA (N=146)

Esteves et al. J Urol. 2013;189: 232-7

ICSI outcome in obstructive azoospermia comparable with fertile donors

64 61 47

34 61 66

50 38

2PN Fertilization

(%)

High quality embryos (%)

Clinical pregnancy (%)

Live birth (%)

Obstructive azoospermia (N=146) Donor sperm (N=40)



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p=NS

Esteves et al. Asian J Androl 2014; 16: 602-6

Baseline Posttreatment Esteves & Papanikolaou Fertil Steril 2011; 96 (Suppl.): S230

Series of men with adult-onset hypo-hypo Recombinant hCG (Ovitrelle 250 mcg); SC, qw

Hypogonadotropic hypogonadism favorable prognosis and simple management



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Frequency of azoospermia among 2,383 patients attending an Infertility Clinic

Esteves et al. Clinics 2011; 66: 691-700.



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Azoospermia 35%

61%

36%

3% Hypo-hypo

OA

SF

Esteves et al. Sperm Retrieval Techniques. Int Braz J Urol 2011; 37: 570-83

About 40-50% of men with SF have residual spermatogenesis within the testis



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§  Not enough for sperm to appear in ejaculate

§  600-800 seminiferous tubules §  Goal is identify site of

production and retrieve sperm for ICSI

§  Geographic location unpredictable

Challenges faced by health professionals providing care for men with SF are manifold

§  Counseling about the chances of finding testicular sperm

§  Usefulness of any medical intervention before sperm retrieval

§  Which sperm retrieval method to apply §  Reproductive potential of retrieved

gametes in ICSI treatment §  Health of offspring



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1. Counsel about the

chances of finding

sperm for ICSI

2. Select who could benefit

from interventions before sperm

retrieval

3. Select the best

SR method

4. Use of state-of-art lab

techniques to handle the retrieved gametes



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Clinical management of men with spermatogenic failure at Androfert



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chances of finding

sperm for ICSI



retrieval

3. Select the best

SR method



FSH levels Testosterone levels

Testicular volume

elec%ng candidates for SR Can biomarkers predict SR success?

Diagnostic markers reflect global testicular function but not the presence of a site of

active spermatogenesis



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Verza Jr & Esteves. Fertil Steril 2011; 96 (Suppl.): S53

Biopsy helpful for counseling but does not provide definitive proof of whether sperm will be found; may jeopardize future retrieval attempts

100%

40.3% 19.5%

Hypospermatogenesis Maturation Arrest Seroli-cell only

Presence of sperm within the testicle (N=357)

Esteves & Agarwal. Asian J Androl 2014; 16: 642

Testicular histopathology



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Esteves et al Fertil Steril 2010; Raman & Schlegel J Urol 2003; Hopps et al. Hum Reprod 2003; Damani et al JCO 2002

Etiology category Success in finding sperm

Cryptorchidism 52-74% Post-infection 67% Torsion >50% Post-chemotherapy/RT 25-75% Genetic 25-70% Idiopathic 50-60%

Etiology cannot determine whether or not sperm will be found within the testis



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Genetic assessment of AZF region within the Y chromosome helpful to determine the

chances of finding testicular sperm

Microdeletions within AZF region found in up to 15% of men with SF

Hamada et al. Clinics 2013; 68 (Suppl 1): 39-60; Krausz et al. Andrologia 2014; 2: 5-19



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Multiplex-PCR molecular diagnosis of YCMD

Krausz et al. Andrologia 2014; 2: 5-19

#4 Normal male #5 AZFb microdeletion #6 AZFc microdeletion



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✕✕✕nil nil 50-70%

Hamada et al. 2013; Krausz et al. 2014; Esteves et al. 2013

Complete AZFa, AZFb or AZFa+b microdeletions unfavorable prognosis;

SR not recommended



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Chances of finding sperm

within the testis

1.  Counseling about chances of finding sperm and who are candidates for SR

Key messages



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§  Diagnostic parameters not accurate to determine if residual spermatogenesis exists

§  YCMD screening mandatory to “deselect” men with AZFa-b deletions from undergoing SR

§  Male offspring of fathers with AZFc microdeletions will inherit the Yq deletion (infertility)

2.  Identify who may benefit from interventions before sperm retrieval



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Rationale for medication Paradoxically weak stimulation of Leydig and

Sertoli cells by endogenous gonadotropins - Due to high baseline FSH and LH levels the

relative amplitudes are low Hypogonadism (TT<300 ng/dl) in up to 50%

men with SF - High ITT levels essential for regulating

spermatogenesis in combination with Sertoli cell stimulation with FSH



ANDROFERT Shiraishi et al Hum Reprod 2012; 27: 331-9; Sussman et al Urol Clin N Am 2008; 35: 147-55



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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)

!

There may be a role for medical treatment but more studies are needed

Mechanism of action unclear but may be related to high ITT that stimulate residual

spermatogenic areas

Shinjo E et al Andrology 2013; 1: 929-35; Shiraishi et al Hum Reprod 2012; 27: 331-9.

273

1348

Before After

ITT (ng/dl)

ITT levels increased after hCG-based therapy

Spermatogonial DNA synthesis increased



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PCNA expression

Testosterone and estradiol levels

<300 ng/dL

(10.4 nmol/L)

Hypogonadism category

Pure

Medication algorithm at Androfert Treatment aimed at

boosting endogenous T production

Aromatase inhibitor (anastrozole 1mg

orally qid)

Rec-hCG (250 mcg SC qw); rec-FSH added

(75 IU SC biw) if FSH levels <1.5 mIU/ml



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T/E ratio <10

T/E ratio >10 (nl)

Aromatase hyperactivity

Varicocele associated with impaired spermatogenesis

Proposed mechanisms

include heat-mediated stress and ischemia

Spermatogonia B, pachytene spermatocytes and early spermatids highly vulnerable to heat stress



ANDROFERT Agarwal, Hamada & Esteves Nature Rev Urol 2012; 9: 678-90

Rationale for varicocele repair Catch-up testicular growth among

adolescents following varicocele repair

Improvement in sperm parameters after varicocele repair

Abnormally-low T restored to normal levels in some men after varicocele repair



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Wang et al Fertil Steril 1991; 55: 152-5; Su et al J Urol 1995; 154: 1752-5; Çayan et al J Urol 2002; 168: 929731-4; Hamada et al Nat Rev Urol 2013; 10: 26-37



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Among 233 men with SF and clinical varicocele, about 1/3 had motile sperm in

postoperative ejaculate

Weedin et al J Urol 2010; 183: 2309-15

Varicocele success depend on the presence of spermatogenesis in histopathology Maturation arrest and hypospermatogenesis

favorable prognosis



ANDROFERT Weedin et al J Urol 2010; 183: 2309-15

Inci et al J Urol 2009; 182: 1500-5; Haydardedeoglu et al Urology 2010; 75:83-6

§  Inci 2009 OR: 2.63; 95% CI: 1.05-6.60; p=0.03)

Although 2/3 remain azoospermic after varicocele repair, sperm retrieval success

increased in treated individuals



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§ Haydardedeoglu 2010

53 30

Treated (N=66)

Untreated (N=30)

SR success (%)

61 38

Treated (N=31)

Untreated (N=65)

p<0.01

2.  Identify who may benefit from interventions before sperm retrieval

Key messages



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§  Medication to boost endogenous T production seems beneficial to improve spermatogenesis, particularly in men with low T levels and low T/E ratio

§  Repair of clinical varicocele should be considered in ICSI candidates before SR


chances of finding

sperm for ICSI



retrieval

3. Select the best sperm

retrieval method





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Methods for sperm retrieval in SF 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%

Esteves et al Int Braz J Urol 2013; 37: 570-83; Deruyver et al Andrology 2014; 2: 20-4



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Microsurgical method yields higher success regardless of histopathology findings

45%

93%

64%

20% 25%

64%

9% 6%

Overall Hypospermatogenesis Maturation Arrest Sertoli-cell Only

Sperm Retrieval Success Rates

Micro-TESE single-biopsy TESE

Controlled series (N=60)

Histology categories pairwise comparisons:

p<0.0001

Method P=0.0005



ANDROFERT Verza Jr & Esteves. Fertil Steril 2011; 96 (Suppl.): S53

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

Microsurgical testicular sperm extraction is key in SF



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3. Selecting the best sperm retrieval method

Key message


chances of finding

sperm for ICSI



retrieval

3. Select the best

SR method





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• Optimize sperm retrieval • Mechanical mincing • Enzymatic tissue digestion • Avoid iatrogenic damage



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Tissue removed (mg) Large Single-Biopsy TESE

Micro-TESE

P-value

65 ± 25 8.9 ± 2.5 <0.01

Optimizing sperm retrieval

Conven6onal TESE Micro-‐TESE

Fragment weight Fragment weight

Verza Jr & Esteves Fertil Steril 2011; Esteves & Varghese J Reprod Sci 2013



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Morphometric Evaluation of Seminiferous Tubules



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Median 25%-75% 5%-95% Raw Data

yes No

Presence of Sperm

160

180

200

220

240

260

280

300

320

340

360

380

400

420

Max

. Tub

ule

Dia

met

er

Verza Jr S, Esteves SC. Fertil Steril 2012; 98: S242

N=54; Tubule Diameter: KW-H (1;54) = 25.2; P<0.001



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On average, one top-quality additional embryo for transfer or cryopreservation

Clean Room Technology & ICSI Results2,315 patients; 14,660 embryos



ANDROFERT Esteves & Bento. Reprod Biomed Online 2013; 26: 9-21

4. Using state-of-art lab techniques to handle gametes of men with SF

Key messages



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§  Increase the chances of finding sperm from extracted testicular tissue

§  Avoid iatrogenic damage to the already compromised gametes

§  Optimize ICSI outcome

What we have achieved after doing this herculeous effort



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41.4 47.0 43.3

20.0 Sperm

retrieval success (%)

2PN fertilization

after ICSI (%)

High quality embryo

generated by ICSI (%)

Live birth per treatment cycle (%)

Spermatogenic failure (N=365)

Esteves et al Asian J Androl 2014; 16: 602-6

Why we do all that



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What about the health of resulting offspring

Esteves et al Asian J Androl 2014; 16: 602-6

Region N Outcome analyzed 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% SF vs 5.2% OA (ns)

Esteves & Agarwal. Clinics 2013; 68 (Suppl.1): 141-50

Neonatal Outcome of Babies Born Health of offspring reassuring

but a call for continuous monitoring needed due to limited data and lack of long-term follow-up



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What the future holds for men with spermatogenic failure…

Health & Medicine

Testicular Failure in Humans: Current management and future perspectives