Conventional techniques to predict Conventional techniques to predict

presence of sperm from presence of sperm from

reproductive tract for ICSIreproductive tract for ICSI

Barış Altay, MDBarış Altay, MDAssociate Professor of UrologyAssociate Professor of Urology

Ege University School of MedicineEge University School of MedicineIzmir, TurkeyIzmir, Turkey

Natural conception

IUI

DecreasingDecreasingRisk and CostRisk and Cost

Upgrading Fertility Upgrading Fertility StatusStatus

IVF/ICSI

Ejaculated spermEjaculated sperm

IncreasedIncreasedDesirabilityDesirability

Surgical sperm retrievalSurgical sperm retrievalfor IVF/ICSIfor IVF/ICSI

Donor sperm inseminationDonor sperm inseminationAdoptionAdoption

Etiology of Male Factor Infertility

0102030405060708090

100

Varicocele Obstruction Testisfailure

Idiopathic Hormonal Others

%

Infertility - Azoospermia: 5-20%

0

10

20

30

40

50

60

70

80

90

100

Obstruction Endocrine Genetic Testicular failure

%

HistoryPhysical examinationSemen analysis (2x)

Hormonal evaluationGenetic tests

Radiologic imaging

TREATMENT

Evaluation of Azoospermic MenEvaluation of Azoospermic Men

Biopsy/Cytology

10

20

Clinical characteristics of Obstructive Azoospermic Men

Absolute diagnosis:Absolute diagnosis: Testicular biopsy/cytology Testicular biopsy/cytology→ → Normal spermatogenesisNormal spermatogenesis

Relative findings:Relative findings: Normal serum FSH Normal serum FSH Ejaculate volume Ejaculate volume ↓↓ Testicular volume Testicular volume 16 ml. 16 ml.

Normal consistencyNormal consistency Dilated caput epididymisDilated caput epididymis Absence of vas deferensAbsence of vas deferens

Obstructive Infertility-LocalizationObstructive Infertility-Localization

ProximalProximal Intratesticular (15%)Intratesticular (15%)

CongenitalCongenital Acquired (Post-inflammatory, post-traumatic)Acquired (Post-inflammatory, post-traumatic)

Epididymal (60-75%) Epididymal (60-75%) Congenital (CBAVD, Young’s syndrome)Congenital (CBAVD, Young’s syndrome)

Acquired (infection, epididymitis)Acquired (infection, epididymitis) VasalVasal

Congenital (Agenesis of vas deferens)Congenital (Agenesis of vas deferens)Acquired (Vasectomy, hernia repair, orchiopexy)Acquired (Vasectomy, hernia repair, orchiopexy)

Distal (5-10%)Distal (5-10%)

Ejaculatory duct obstructionEjaculatory duct obstruction

Proximal Obstruction:Proximal Obstruction: Microsurgical reconstructionMicrosurgical reconstruction

Vaso-vasostomyVaso-vasostomy VasoepididymostomyVasoepididymostomy

Assisted Reproductive TechnologyAssisted Reproductive Technology Use of sperm for IVF/ICSIUse of sperm for IVF/ICSI

Distal obstruction:Distal obstruction: Endoscopic treatment (TUR-ED)Endoscopic treatment (TUR-ED) TRUS/Aspiration of Seminal fluid-sperm TRUS/Aspiration of Seminal fluid-sperm

retrieval for ARTretrieval for ART

Obstructive Azoospermia-TreatmentObstructive Azoospermia-Treatment

Pellet (-)Pellet (-) AzoAzooospermispermiaa

Vas deferens Vas deferens ((--))

Absence of vas deferens (CBAVD)Absence of vas deferens (CBAVD)

CFTR CFTR testtest

Epididymal sEpididymal spermperm-IVF/ICSI-IVF/ICSI

Vas deferens Vas deferens ((++))

Testis volumeTestis volume

LowLow

FSHFSH

LowLow

Hypogonad.Hypogonad.hypogonadismhypogonadism

LH, prolactinLH, prolactinCranial imagingCranial imaging

NormalNormal

Testis BxTestis Bx

NormalNormal

AbnormalAbnormal

Waiting for Waiting for advanced technologyadvanced technology

SurgerySurgery

AdoptionAdoption HighHigh

Primary failurePrimary failure

Test. sTest. spermperm--IVF/ICSIIVF/ICSI

AdoptionAdoption

FSHFSH

NormalNormal

HighHigh

Primary failurePrimary failure

Test.sTest.spermperm--IVF/ICSIIVF/ICSI

AdoptionAdoption

GonadotropinsGonadotropinsSurgerySurgery

Indications for Epididymal sperm Indications for Epididymal sperm aspirationaspiration

• Congenital bilateral absence of vas Congenital bilateral absence of vas

deferens deferens

• Failed vasectomy reversal surgery Failed vasectomy reversal surgery

• Presence of uncorrectable epididymal Presence of uncorrectable epididymal

obstructionobstruction

• Impossible/failed vasal sperm aspirationImpossible/failed vasal sperm aspiration

MESA (Microsurgical Epididiymal MESA (Microsurgical Epididiymal Sperm Aspiration)Sperm Aspiration)

Advantageous:• Multiple sampling• More motile sperm• Cryopreservation

Disadvantageous:• Invasive procedure• Needed anesthesia• More cost

(Microscopic surgery)• More experience

• Ideal method in Obstructive azoospermia• Success rate: >90%• Sperm density: Sperm density: ~~40.9x1040.9x1066/ml/ml• Motility: 13-29%Motility: 13-29%

Chen 1995Collins 1996Silber 1997Belker 1994Devroey 1995Oates 1996Holden 1997Schroder-Printzen 2000

Outcomes of MESAOutcomes of MESA

• Fertilization: 60-90%/cycle

• Pregnancy: 14-66%/cycle

• Delivery: 25-36%Belker 1994Silber 1995Ubaldi 1995Zumbe 1996Bispink 1997

Schroder-Printzen 1997

Sperm quality in Obstructive Sperm quality in Obstructive AzoospermiaAzoospermia

• Increase in epididymal Increase in epididymal sperm qualitysperm quality

Normal Normal ObstructionObstruction

CaputCaput

CaudaCauda

goodgood

goodgood

Best Practice & Research Clinical Obstetrics & Gynaecology, 2003

FertilizationFertilizationPregnancyPregnancy

Motile sperm Motile sperm 54% 54% 26.4% 26.4%

Immotile sperm 29.8%Immotile sperm 29.8% 21.4% 21.4%

Sperm Motility and ICSISperm Motility and ICSI

P=0.005P=0.005

Clinical characteristics of Non-obstructive Azoospermia

Absolute diagnosis: Testicular biopsy/CytologyAbsolute diagnosis: Testicular biopsy/CytologyRelative findings:Relative findings: Normal ejaculate volumeNormal ejaculate volume Testicular volume < 15 ml.Testicular volume < 15 ml.

Soft in consistencySoft in consistency Normal epididymisNormal epididymis

Serum FSH Serum FSH Testosterone/Estradiol Testosterone/Estradiol (<10, N: 14-16) (<10, N: 14-16) Serum inhibin B Serum inhibin B

Non-obstructive Non-obstructive AzoAzooospermispermiaa

CorrectableCorrectable pat pathholoologygy (+) (+)

Treatment of vTreatment of variaricocelecoceleTreatment of hTreatment of hormonal ormonal failurefailureClear of gClear of gonadal toonadal toxinsxins

SSeemmen analysis/en analysis/ pellet test pellet test

Sperm (+)Sperm (+) Sperm (-)Sperm (-)

PregnancyPregnancySpontanSpontaneouseousIUIIUIIVFIVFIVF/ICSIIVF/ICSI

PatPathholoologygy (-) (-)

GenetiGeneticc test tests and counselings and counseling

TTesticular besticular biopsyiopsy

MatMatuurree sperm sperm

TESA-TESETESA-TESE(IVF/ICSI)(IVF/ICSI)

PregnancyPregnancy (+) (+) PregnancyPregnancy (-) (-)

TESA-TESE TESA-TESE (6 (6 months later)months later)

Use of freezed Use of freezed sperm sperm ororeembriyombriyoss

SpermatidSpermatid

ROSNIROSNI??

Sperm (-)Sperm (-)

AdoptionAdoption

Waiting-advanced Waiting-advanced tetechchnolonologygy

DonDonoor spermr spermiinseminansemination?tion?

3-12 months3-12 months

Predictive factors for testicular Predictive factors for testicular sperm retrieval in Non-obstructive sperm retrieval in Non-obstructive

azoospermiaazoospermia

Conventional Conventional Testicular sperm retrieval techniquesTesticular sperm retrieval techniques Histopathological examinationHistopathological examination Serum hormone levels (FSH, inhibin B)Serum hormone levels (FSH, inhibin B) Age Age Testicular volumeTesticular volume

Genetic markersGenetic markers

Radiologic methodsRadiologic methods

Testicular sperm retrieval Testicular sperm retrieval techniquestechniques

Testicular sperm aspiration (TESA)Testicular sperm aspiration (TESA)

Testicular sperm extraction (TESE)Testicular sperm extraction (TESE) MacroscopicMacroscopic Microscopic (Micro-TESE)Microscopic (Micro-TESE)

TESETESE (%)(%)

microTESEmicroTESE (%)(%)

Schlegel et alSchlegel et al. . (1)(1) 32 32 5858

Schlegel et al.Schlegel et al. (2)(2) 45 45 63 63

Tsujimura et alTsujimura et al.. 3535 4343

Raman et alRaman et al.. 5858 6161

Okada et alOkada et al.. 1717 4545

Amer et alAmer et al.. 3030 4747

AVERAGEAVERAGE 3636 5353(1) Schlegel PN 2005 (2) Schlegel PN 1999Tsujimura A et al. Human Reprod 2002Raman J et al. J Urol 2003Okada H et al., J Urol 2002Amer M et al,. Human Reprod 2000

Sperm Retrieval Ratios in NOASperm Retrieval Ratios in NOA

TESE TESE mimiccroTESE roTESE PP valuevalue

HHyypospermatogenepospermatogenesissis 50 50%% 8181%% 0.350.35

MaturaMaturatition Aon Arrrestrest 20 20%% 4444%% 0.290.29

SCOSCO 2929%% 4141%% 0.030.03

Sperm Retrieval Rates and HistopathologySperm Retrieval Rates and Histopathology

Schlegel Schlegel et al,et al, Urology 2005 Urology 2005

Microscopic TESEMicroscopic TESE

• Tubules containing spermatogenesis– Dilated– Opaque/white

No identification of these tubules with lower magnification

Schlegel PN, Human Reprod Schlegel PN, Human Reprod 19991999

MicroTESE-UpdateMicroTESE-Update

• N=684

• Sperm retrieval rate: 61%

• Fertilization: 55% (per oocyte)

• Clinical pregnancy: 47%

Schlegel P, AUA 2007Schlegel P, AUA 2007

MicroTESE after unsuccessful MicroTESE after unsuccessful conventional TESEconventional TESE

• NOA• N=50 primary • N=7 unsuccessful conventional TESE • N=18 unsuccessful microTESE• Salvage microTESE

Sperm retrieval (%)Sperm retrieval (%)

Unsuccessful conventional Unsuccessful conventional TESETESE

5757

Unsuccessful microTESEUnsuccessful microTESE 66

Primary NOAPrimary NOA 5656

Okada H, AUA 2007Okada H, AUA 2007

Histopathologic findings of the Testis

Obstructive AzoospermiaObstructive Azoospermia Normal spermatogenesis (>15 spermatid-sperm/ Normal spermatogenesis (>15 spermatid-sperm/

tubule)tubule)

Non-obstructive AzoospermiaNon-obstructive Azoospermia Complete sclerozisComplete sclerozis Complete germ cell aplazia Complete germ cell aplazia

Sertoli cell-only syndromeSertoli cell-only syndrome Focal spermatogenesis/germ cell aplazia Focal spermatogenesis/germ cell aplazia

Adult type SCOAdult type SCO Maturation arrestMaturation arrest

Spermatogonium, spermatocyte Spermatogonium, spermatocyte Spermatid arrestSpermatid arrest

Partial maturation arrestPartial maturation arrest HypospermatogenesisHypospermatogenesis

Testis volume

• Testicular volume is inversely correlated with the probability of the presence of sperm in the testis.

• However, spermatozoa can be successfully retrieved from a testis with a volume <5ml.

• Testicular volume• Serum FSH level• Age• Testosterone level• Serum Inhibin B level

• Only testicular histopathology is a predictor for sperm retrieval in NOA.

Su LM et al J Urol 1999

No Predictive Value

Okada H, J Urol 2002

Friedler S ve ark. Human Reprod 2002

Ostad M ve ark. Urology 1998

Su LM ve ark J Urol 1999

Tournaye H, Hum Reprod 1996

Verneave V, Gynecol Obstet Fertil 2004

Kochinski I, Hum Reprod 2005

Ramasamy R, J Urol 2007

Testis Histopathology

• In contrast to the predominant spermatogenetic pattern, the most advanced pattern appears to affect the TESE results.

• Ramasamy-Schlegel, J Urol April 2007.

New Classification for testicular biopsies

1. Normal testicular biopsy

2. Hypospermatogenesis

3. Germ cell arrest

4. SCO appearence (syndrome)

5. Seminiferous hyalinization

6. Carcinoma in situ (CIS)

7. Immature testis (prepubertal)Mc Lachlan Hum Reprod 2007

Su Seo Amer Sousa Tsujimura

Okada Schlegel Koscinski Ramasamy Mean

Hypospermatogenesis 79 89 85.7 97.7 100 100 74 100 100 91.7

Maturation Arrest 47 62 53.3 75 75 40 45.5 83 60.1

Spermatid arrest 80 80

Primary spermatocyte arrest

33.3 33.3

SCO 24 16 33.3 29.8 22.5 33.9 40 23.5 51 30.4

Su LM et alJ Urol 1999

Seo TJ et al, Int J Androl 2001

Amer M. Et al,. Hum Reprod 2000

Sousa M., Hum Reprod 2002

Tsujimura A et al, Human Reprod 2002,

Okada H et al, J Urol 2002

Schlegel et al. Urology 2005

Koscinski I, Hum Reprod 2005

Ramasamy R, J Urol 2007

Sperm Retrieval Rates and Histopathology

Testis Biopsy

• Diagnostic biopsies were preferred to determine whether sperm + for ICSI.

• Unfortunately, diagnostic biopsy has limited prognostic value to predict microdissection TESE (extensive multiple biopsies may be needed)

Predictive factors of sperm Predictive factors of sperm recoveryrecovery

178 males with non-obstructive azoospermia 178 males with non-obstructive azoospermia

Undergoing TESE procedure for IVF/ICSIUndergoing TESE procedure for IVF/ICSI Spermatozoa recovery: 94/178 (52.8%)Spermatozoa recovery: 94/178 (52.8%)

Determination of predictive factorsDetermination of predictive factors Testicular volume, Histology, FSH Testicular volume, Histology, FSH

Spermatozoa recovery has no correlation with Spermatozoa recovery has no correlation with

testicular volume or serum FSH level testicular volume or serum FSH level

Only testicular histopathology can be used as a predictor of successful Only testicular histopathology can be used as a predictor of successful

sperm recoverysperm recovery

Seo and Ko, Int J Androl, 2001Seo and Ko, Int J Androl, 2001

Predictive factors of sperm Predictive factors of sperm recoveryrecovery

30 patients with non-obstructive azoospermia 30 patients with non-obstructive azoospermia Undergoing TESE procedure for IVF/ICSIUndergoing TESE procedure for IVF/ICSI Spermatozoa recovery: 21/30 (70%)Spermatozoa recovery: 21/30 (70%)

Determination of predictive factorsDetermination of predictive factors Age, Histology, FSH Age, Histology, FSH

Neither patient age nor FSH was predictive Neither patient age nor FSH was predictive Only testicular histopathology can be used as a predictor of Only testicular histopathology can be used as a predictor of

successful sperm recoverysuccessful sperm recovery

Mulhall JP et al, Urology, 1997Mulhall JP et al, Urology, 1997

SummarySummary Testicular volume, age and serum hormone levels have no Testicular volume, age and serum hormone levels have no

predictive value. predictive value.

Only testicular histopathology is a valid predictor for the Only testicular histopathology is a valid predictor for the

successful testicular sperm recovery by testicular successful testicular sperm recovery by testicular

biopsies.biopsies.

Based on conventional techniques, there are no standard Based on conventional techniques, there are no standard

preoperative criteria to predict prospectively presence or preoperative criteria to predict prospectively presence or

absence of sperm on TESE for an individual man. absence of sperm on TESE for an individual man.

Therefore, a new additional techniques and markers are Therefore, a new additional techniques and markers are

needed to improve sperm harvesting success from the needed to improve sperm harvesting success from the

reproductive tract. reproductive tract.