Conventional techniques to predict presence of sperm from reproductive tract for ICSI Barış Altay, MD Associate Professor of Urology Ege University School of Medicine Izmir, Turkey

Natural conception IUI Decreasing Risk and Cost Upgrading Fertility Status IVF/ICSI Ejaculated sperm IncreasedDesirability Surgical sperm retrieval for IVF/ICSI Donor sperm insemination Adoption

Etiology of Male Factor Infertility %

Infertility - Azoospermia: 5-20% %

History Physical examination Semen analysis (2x) Hormonal evaluation Genetic tests Radiologic imaging TREATMENT Evaluation of Azoospermic Men Biopsy/Cytology

Clinical characteristics of Obstructive Azoospermic Men Absolute diagnosis: Testicular biopsy/cytology → Normal spermatogenesis Relative findings:  Normal serum FSH  Ejaculate volume ↓  Testicular volume  16 ml.  Normal consistency  Dilated caput epididymis  Absence of vas deferens

Obstructive Infertility-Localization  Proximal  Intratesticular (15%) Congenital Congenital Acquired (Post-inflammatory, post-traumatic) Acquired (Post-inflammatory, post-traumatic)  Epididymal (60-75%) Congenital (CBAVD, Young’s syndrome) Acquired (infection, epididymitis) Acquired (infection, epididymitis)  Vasal Congenital (Agenesis of vas deferens) Acquired (Vasectomy, hernia repair, orchiopexy)  Distal (5-10%)  Ejaculatory duct obstruction

 Proximal Obstruction:  Microsurgical reconstruction  Vaso-vasostomy  Vasoepididymostomy  Assisted Reproductive Technology  Use of sperm for IVF/ICSI  Distal obstruction:  Endoscopic treatment (TUR-ED)  TRUS/Aspiration of Seminal fluid-sperm retrieval for ART Obstructive Azoospermia-Treatment

Pellet (-) Azoospermia Vas deferens (-) Absence of vas deferens (CBAVD) CFTR test Epididymal sperm-IVF/ICSI Vas deferens (+) Testis volume Low FSH Low Hypogonad.hypogonadism LH, prolactin Cranial imaging Normal Testis Bx Normal Abnormal Waiting for advanced technology Surgery Adoption High Primary failure Test. sperm- IVF/ICSI Adoption FSH Normal High Primary failure Test.sperm- IVF/ICSI Adoption GonadotropinsSurgery

Indications for Epididymal sperm aspiration Congenital bilateral absence of vas deferensCongenital bilateral absence of vas deferens Failed vasectomy reversal surgeryFailed vasectomy reversal surgery Presence of uncorrectable epididymal obstructionPresence of uncorrectable epididymal obstruction Impossible/failed vasal sperm aspirationImpossible/failed vasal sperm aspiration

MESA (Microsurgical Epididiymal 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: ~40.9x10 6 /ml Motility: 13-29% Motility: 13-29% Chen 1995 Collins 1996 Silber 1997 Belker 1994 Devroey 1995 Oates 1996 Holden 1997 Schroder-Printzen 2000

Outcomes of MESA Fertilization: 60-90%/cycle Pregnancy: 14-66%/cycle Delivery: 25-36% Belker 1994 Silber 1995 Ubaldi 1995 Zumbe 1996 Bispink 1997 Schroder-Printzen 1997

Sperm quality in Obstructive Azoospermia Increase in epididymal sperm qualityIncrease in epididymal sperm quality Normal Obstruction Normal ObstructionCaputCauda good good

Best Practice & Research Clinical Obstetrics & Gynaecology, 2003 FertilizationPregnancy Motile sperm 54% 26.4% Immotile sperm 29.8% 21.4% Sperm Motility and ICSI P=0.005

Clinical characteristics of Non- obstructive Azoospermia Absolute diagnosis: Testicular biopsy/Cytology Relative findings:  Normal ejaculate volume  Testicular volume < 15 ml.  Soft in consistency  Normal epididymis  Serum FSH   Testosterone/Estradiol  (<10, N: 14-16)  Serum inhibin B 

Non-obstructive Azoospermia Correctable pathology (+) Treatment of varicocele Treatment of hormonal failure Clear of gonadal toxins Semen analysis/ pellet test Sperm (+) Sperm (-) Pregnancy Spontaneous IUIIVFIVF/ICSI Pathology (-) Genetic tests and counseling Testicular biopsy Mature sperm TESA-TESE(IVF/ICSI) Pregnancy (+) Pregnancy (-) TESA-TESE (6 months later) Use of freezed sperm or embriyos Spermatid ROSNI? Sperm (-) Adoption Waiting-advanced technology Donor sperm insemination? 3-12 months

Predictive factors for testicular sperm retrieval in Non-obstructive azoospermia  Conventional  Testicular sperm retrieval techniques  Histopathological examination  Serum hormone levels (FSH, inhibin B)  Age  Testicular volume  Genetic markers  Radiologic methods

Testicular sperm retrieval techniques  Testicular sperm aspiration (TESA)  Testicular sperm extraction (TESE)  Macroscopic  Microscopic (Micro-TESE)

TESE (%) microTESE (%) Schlegel et al. (1) 3258 Schlegel et al. (2) 4563 Tsujimura et al Raman et al Okada et al Amer et al AVERAGE3653 (1) Schlegel PN 2005 (2) Schlegel PN 1999 Tsujimura A et al. Human Reprod 2002 Raman J et al. J Urol 2003 Okada H et al., J Urol 2002 Amer M et al,. Human Reprod 2000 Sperm Retrieval Ratios in NOA

TESE microTESE P value Hypospermatogenesis 50% 81% 0.35 Maturation Arrest 20%44% 0.29 SCO 29%41% 0.03 Sperm Retrieval Rates and Histopathology Schlegel et al, Urology 2005

Microscopic TESE Tubules containing spermatogenesis –Dilated –Opaque/white No identification of these tubules with lower magnification Schlegel PN, Human Reprod 1999

MicroTESE-Update N=684 Sperm retrieval rate: 61% Fertilization: 55% (per oocyte) Clinical pregnancy: 47% Schlegel P, AUA 2007

MicroTESE after unsuccessful conventional TESE NOA N=50 primary N=7 unsuccessful conventional TESE N=18 unsuccessful microTESE Salvage microTESE Sperm retrieval (%) Unsuccessful conventional TESE 57 Unsuccessful microTESE 6 Primary NOA 56 Okada H, AUA 2007

Histopathologic findings of the Testis Obstructive Azoospermia  Normal spermatogenesis (>15 spermatid-sperm/ tubule) Non-obstructive Azoospermia  Complete sclerozis  Complete germ cell aplazia  Sertoli cell-only syndrome  Focal spermatogenesis/germ cell aplazia  Adult type SCO  Maturation arrest  Spermatogonium, spermatocyte  Spermatid arrest  Partial maturation arrest  Hypospermatogenesis

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

SuSeoAmerSousaTsujim ura OkadaSchlegelKoscinskiRamasamyMean Hypospermatogenesis Maturation Arrest Spermatid arrest 80 Primary spermatocyte arrest 33.3 SCO 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 recovery 178 males with non-obstructive azoospermia  178 males with non-obstructive azoospermia  Undergoing TESE procedure for IVF/ICSI  Spermatozoa recovery: 94/178 (52.8%)  Determination of predictive factors  Testicular volume, Histology, FSH  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 sperm recovery Seo and Ko, Int J Androl, 2001

Predictive factors of sperm recovery  30 patients with non-obstructive azoospermia  Undergoing TESE procedure for IVF/ICSI  Spermatozoa recovery: 21/30 (70%)  Determination of predictive factors  Age, Histology, FSH  Neither patient age nor FSH was predictive  Only testicular histopathology can be used as a predictor of successful sperm recovery Mulhall JP et al, Urology, 1997

Summary  Testicular volume, age and serum hormone levels have no predictive value.  Only testicular histopathology is a valid predictor for the successful testicular sperm recovery by testicular biopsies.  Based on conventional techniques, there are no standard preoperative criteria to predict prospectively presence or absence of sperm on TESE for an individual man.  Therefore, a new additional techniques and markers are needed to improve sperm harvesting success from the reproductive tract.