1. Discuss normal vs. abnormal semen analysis 2. Evaluate different treatments of varicocele 3. Assess azoospermia and discuss micro dissection testicular sperm extraction 4. Diagnose Klinefelter syndrome and genetic abnormalities in men with infertility

1. Microsurgery for male fertility :Microsurgery for male fertility :
Case based discussionCase based discussion
Ranjith Ramasamy, M.D.Ranjith Ramasamy, M.D.
Director, Male Reproductive UrologyDirector, Male Reproductive Urology
Miller School of MedicineMiller School of Medicine
University of MiamiUniversity of Miami
ramasamy@miami.eduramasamy@miami.edu

2. Case # 1
 28 year old male with a history of infertility,
an unremarkable medical history, no prior
pregnancies
 Semen analysis revealing oligospermia
– Concentration - 14 million sperm per cc
– Motility - 38% motile
– Morphology - 5% normal forms by WHO criteria
– Volume 3cc

3. Next step?
 Physical examination
 Repeat the semen analysis

4. Case: 1 Oligospermia
• On exam -
Large left sided varicocele
• Repeat semen analysis
– Volume — 3cc
– Conc —16 million sperm per mL
– Morphology — 5%
– Motility — 42% sperm
with forward movement

5. Semen parameter 5th
percentile 50th
percentile 95th
percentile
Semen volume (mL) 1.5 3.7 6.8
Sperm density
(M/mL)
15 75 213
Sperm motility (%) 40% 60% 78%
WHO 2010 Semen ParametersWHO 2010 Semen Parameters

6. 5% 95%50%

7. Case # 1
 28 year old male with a history of subfertility,
an unremarkable medical history, no prior
pregnancies comes with 25 yo healthy wife
1st
Semen analysis
• Concentration - 14 million / cc
• Motility - 38% motile
• Morphology - 5% normal
• Volume 3cc
2nd
Semen analysis
• Concentration - 16 million / cc
• Motility - 41% motile
• Morphology - 5% normal
• Volume 3cc
BOTH SEMEN ANALYSES ARE ABNORMAL

8. VaricocelesVaricoceles
Varicoceles can increase intratesticular temperature
affecting spermatogenesis and testosterone production

9. Clinical Varicocele Classification
Classification Definition
Clinical (palpable):
Grade III ( Large)
Grade II (Medium)
Grade I ( Small)
Subclinical ( not palpable)
Easily visible
Palpable at rest (without
Valsalva maneuver), invisible.
Palpable with Valsalva
maneuver only
Vein larger than 3 mm on
ultrasound; Doppler reflux on
Valsalva maneuver

10. Indications for varicocelectomy
1. A varicocele is palpable.
2. The couple has documented infertility.
3. The female has normal fertility or potentially
correctable infertility.
4. The male partner has one or more abnormal
semen parameters .

11.  Varicocelectomy superior to observation in
infertile men with palpable varicoceles and
impaired semen quality
 Spontaneous pregnancy
 13.9% (control) versus 32.9% (treatment)
 odds ratio (OR) - 3.04
 Number needed to treat - 6 men

12.  Statistical postop improvementStatistical postop improvement
 Sperm count (14 → 35M)Sperm count (14 → 35M)
 Motile sperm (6.7→15.4M)Motile sperm (6.7→15.4M)
 ImprovedImproved clinical pregnancy rateclinical pregnancy rate
 48 vs 73%, p=0.0448 vs 73%, p=0.04
 ImproveImprove live birth ratelive birth rate
 37 vs 51%, p=0.0337 vs 51%, p=0.03
Esteves S, et al, J Urol, 184:1442-6, 2010. 5*

13. Varicocelectomy decreases DNA fragmentation

14. How to treat varicocele?
 Embolization
 Microsurgical varicocelectomy
 Laparoscopic varicocelectomy

15. Technique
Artery
Preserved
Hydrocele
(%)
Failure
(%)
Potential for
Serious
Morbidity
* Microscopic
inguinal or sub-
inguinal
Retroperitoneal
Conventional
inguinal
Laparoscopic
Balloon
Yes
No
No
Yes
Yes
0
7
3-30
12
0
1.4
15-25
5-15
5-15
15-25
No
No
No
Yes
Yes
Techniques of VaricocelectomyTechniques of Varicocelectomy
Goldstein, et al., CampbellGoldstein, et al., Campbell’s Urology 9th Edition, 2007’s Urology 9th Edition, 2007
* Microsurgical approach has lowest morbidity and failure rate.* Microsurgical approach has lowest morbidity and failure rate.

16. Case # 2 Azoospermia
• 33-year-old healthy male with33-year-old healthy male with
31 year old healthy wife31 year old healthy wife
• Azoospermic with bilateral 4cc testesAzoospermic with bilateral 4cc testes

17. Hormonal evaluation
 FSH: 25.225.2 mIU/mL (normal 2-8)
 LH: 15.1 mIU/mL (normal 2-8)
 Testosterone: 344 ng/dL
 (normal 300 – 800)
 Prolactin: 8.5
 Estradiol: <20

18. Next step?
 Diagnosis of non-obstructive vs. obstructive
azoospermia

19. Schoor et al. J Urol 2002
90% of men with nonobstructive azoospermia had
FSH > 7.6 mIU/ml, or testicular long axis < 4.6 cm
High FSH with small testis
– azoospermia likely due to production defect
– no diagnostic biopsy needed

20. Case # 2 Azoospermia
• 33-year-old healthy male with33-year-old healthy male with
31 year old healthy wife31 year old healthy wife
• Azoospermic with bilateral 4cc testesAzoospermic with bilateral 4cc testes
• FSH 25.2FSH 25.2
• Dx: NOADx: NOA
• Next step?Next step?
• Medical Therapy?Medical Therapy?

21. Hormonal Therapy for Male InfertilityHormonal Therapy for Male Infertility
 Clomiphene citrateClomiphene citrate
 Selective Estrogen ReceptorSelective Estrogen Receptor
ModulatorModulator
 AnastrazoleAnastrazole
 Blocks peripheral conversion ofBlocks peripheral conversion of
TTEE
 AntioxidantsAntioxidants
 Evidence empiricEvidence empiric
 Controlled trials w clearlyControlled trials w clearly
defined outcomes neededdefined outcomes needed
 TestosteroneTestosterone
 Contraindicated in menContraindicated in men
attempting conceptionattempting conception
Higher CentersHigher Centers
GnRHGnRH
HypothalamusHypothalamus
Anterior PituitaryAnterior Pituitary
Sertoli CellsSertoli Cells Leydig CellLeydig Cell
FSH LH
+ +
-
-
Testosterone
Estradiol
SERMs:SERMs:
ClomipheneClomiphene
SERMs:SERMs:
ClomipheneClomiphene
AnastrazoleAnastrazoleAnastrazoleAnastrazole

22. Medical Therapy - IndicationsMedical Therapy - Indications
 Role of medical therapyRole of medical therapy
– Low serum total testosterone (<300 ng/dL)Low serum total testosterone (<300 ng/dL)
– Normal or subnormal LH, irrespective of FSHNormal or subnormal LH, irrespective of FSH

23. Who is the appropriate candidate for medical therapy?Who is the appropriate candidate for medical therapy?
 32 yo M with oligospermia or azoospermia32 yo M with oligospermia or azoospermia
 T – 270 ng/dL (normal 300 – 800 ng/dL)T – 270 ng/dL (normal 300 – 800 ng/dL)
 FSH – 11.3 mIU/mL (normal 2 -8)FSH – 11.3 mIU/mL (normal 2 -8)
 LH 5.2 mIU/mL (normal 2 -8)LH 5.2 mIU/mL (normal 2 -8)
 E – 30 pg/mL (normal < 40 pg/mL)E – 30 pg/mL (normal < 40 pg/mL)
 Treat with clomiphene citrate 25mg everyTreat with clomiphene citrate 25mg every
other dayother day

24. Who is the appropriate candidate for medical therapy?Who is the appropriate candidate for medical therapy?
 32 yo M with oligospermia or azoospermia32 yo M with oligospermia or azoospermia
 T – 270 ng/dL (normal 300 – 800 ng/dL)T – 270 ng/dL (normal 300 – 800 ng/dL)
 FSH – 11.3 mIU/mL (normal 2 -8)FSH – 11.3 mIU/mL (normal 2 -8)
 LH 5.2 mIU/mL (normal 2 -8)LH 5.2 mIU/mL (normal 2 -8)
 E –E – 90 pg/mL90 pg/mL (normal < 40 pg/mL)(normal < 40 pg/mL)
 T/E ratioT/E ratio is 2.7is 2.7 (normal 10)(normal 10)
 Treat with clomiphene citrate 25mg everyTreat with clomiphene citrate 25mg every
other day + anastrazole 1mg every other dayother day + anastrazole 1mg every other day

25. Who is NOT the appropriate candidate for medicalWho is NOT the appropriate candidate for medical
therapy?therapy?
 32 yo M with oligospermia or azoospermia32 yo M with oligospermia or azoospermia
 T –T – 480480 ng/dL (normal 300 – 800 ng/dL)ng/dL (normal 300 – 800 ng/dL)
 FSH –FSH – 18.318.3 mIU/mL (normal 2 -8)mIU/mL (normal 2 -8)
 LH -LH - 14.514.5 mIU/mL (normal 2 -8)mIU/mL (normal 2 -8)
 E – 30 pg/mL (normal < 40 pg/mL)E – 30 pg/mL (normal < 40 pg/mL)
Men with normal testosterone and elevated LH
should NOT be started on
clomiphene citrate

26. Hormonal evaluation in our patient
 FSH: 25.225.2 mIU/mL (normal 2-8)
 LH: 15.1 mIU/mL (normal 2-8)
 Testosterone: 344 ng/dL
 (normal 300 – 800)
 Prolactin: 8.5
 Estradiol: <20
Clomiphene citrate or anastrazole is NOT indicated due to
normal testosterone, elevated FSH/LH and normal
estradiol

27. Next step?
 What is the role of testis biopsy in the
management of men with NOA?

28. Testis Biopsy
Testicular biopsy : the seminiferous tubules are found to be
completely devoid of sperm cells or gonocytes.

29. Indications for testis biopsy
 A testicular biopsy is NOT NECESSARY for diagnosis or
prognosis of nonobstructive azoospermia (i.e. testicular
atrophy or markedly elevated FSH).
 Patients who have a normal serum FSH should undergo
a diagnostic testicular biopsy, as a normal serum FSH
level does not assure the presence of normal
spermatogenesis

31. Prior Biopsy and Microdissection TESEPrior Biopsy and Microdissection TESE
Ramasamy & Schlegel, J Urol 177:1447, 2007
2/32/3
(66%)(66%)
26/2726/27
(96%)(96%)
10/1010/10
(100%)(100%)
HypospermatogenesisHypospermatogenesis
1/51/5
(20%)(20%)
15/2115/21
(71%)(71%)
10/1210/12
(83%)(83%)
Maturation arrestMaturation arrest
1/91/9
(11%)(11%)
33/9033/90
(37%)(37%)
47/9247/92
(51%)(51%)
Sertoli cell onlySertoli cell only
3-4 (%)3-4 (%)1-2 (%)1-2 (%)0 (%)0 (%)Pathologic diagnosisPathologic diagnosis
Number of prior biopsies per testis:Number of prior biopsies per testis:
There was no threshold of prior biopsies over which
• microTESE was unsuccessful,
• and sperm was retrieved in many patients with SCO!
Pathologic diagnosisPathologic diagnosis

32. Success of microTESE after prior unsuccessfulSuccess of microTESE after prior unsuccessful
biopsiesbiopsies
Ramasamy & Schlegel, J Urol 177:1447, 2007
Unindicated diagnostic biopsies can DECREASE sperm retrieval
success with micro-TESE

33. Case # 2 Azoospermia
• 33-year-old healthy male with33-year-old healthy male with
31 year old healthy wife31 year old healthy wife
• Azoospermic with bilateral 4cc testesAzoospermic with bilateral 4cc testes
• FSH 25.2FSH 25.2
• Dx: NOADx: NOA
• Testis biopsy – SCOTestis biopsy – SCO
• Genetic testingGenetic testing

34. Who Should be Offered Genetic Testing?Who Should be Offered Genetic Testing?
• Men with sperm concentration < 5-10 million sperm/ml
- Y Chromosome Testing
- Karyotype Testing
• Men with absent vas deferens on examination, idiopathic
genital ductal obstruction
- CFTR Testing offered to partner, patient (assume male is
affected)
The male infertility best practice policy committee: report on optimal evaluation of the infertile male. Fertil
Ster 2006: 86: S202

35. When to do Genetic Testing?When to do Genetic Testing?
Sperm <5 million/mLSperm <5 million/mL
No sperm/testis failureNo sperm/testis failure
Idiopathic Obstruction,Idiopathic Obstruction,
absent vas deferensabsent vas deferens
ScenarioScenario Y Delet.Y Delet. KaryotypeKaryotype Cyst. FibrCyst. Fibr
XX XX
XX XX
XX

36. Klinefelter syndromeKlinefelter syndrome
 Reduced testicular volume (usuallyReduced testicular volume (usually
<6cc)<6cc)
 IncreasedIncreased gonadotropingonadotropin levels (LHlevels (LH
and FSH)and FSH)
 Low serumLow serum testosteronetestosterone (<12nmol/l)(<12nmol/l)
and varying symptoms ofand varying symptoms of
hypogonadismhypogonadism
 AzoospermiaAzoospermia (<8% of Klinefelter(<8% of Klinefelter
patients have few sperm in theirpatients have few sperm in their
ejaculate)ejaculate)

37. Y Chromosome MicrodeletionY Chromosome Microdeletion
AZFa deletedAZFa deleted
Germ cell AplasiaGerm cell Aplasia
No retrievable spermNo retrievable sperm
AZFa deletedAZFa deleted
Germ cell AplasiaGerm cell Aplasia
No retrievable spermNo retrievable sperm
AZFb deletedAZFb deleted
Maturation ArrestMaturation Arrest
No retrievable spermNo retrievable sperm
AZFb deletedAZFb deleted
Maturation ArrestMaturation Arrest
No retrievable spermNo retrievable sperm
AZFc deleted
Hypospermatogenesis
70% chance of retrieving
testicular sperm for ICSI

38. Case # 2 Azoospermia
• 33-year-old healthy male with33-year-old healthy male with
31 year old healthy wife31 year old healthy wife
• Azoospermic with bilateral 4cc testesAzoospermic with bilateral 4cc testes
• FSH 25.2FSH 25.2
• Dx: NOADx: NOA
• Testis biopsy – SCOTestis biopsy – SCO
• Karyotype: 46 XYKaryotype: 46 XY
• YCMD: No deletionsYCMD: No deletions

39. Case # 2 Azoospermia
• 33-year-old healthy male with33-year-old healthy male with
31 year old healthy wife31 year old healthy wife
• Azoospermic with bilateral 4cc testesAzoospermic with bilateral 4cc testes
• FSH 25.2FSH 25.2
• Dx: NOADx: NOA
• Testis biopsy – SCOTestis biopsy – SCO
• Karyotype: 46 XYKaryotype: 46 XY
• YCMD: No deletionsYCMD: No deletions

40.  Fine needle aspirationFine needle aspiration
 Percutaneous biopsyPercutaneous biopsy
 Open biopsyOpen biopsy
 Multiple open biopsiesMultiple open biopsies
Next step – Surgical therapy?

41. Testis sperm
aspiration
(TESA)
Testis sperm
extraction
(TESE)
Testis
microdissection
(Micro-TESE)
Least Invasive Most invasive
Not all sperm retrieval procedures are the sameNot all sperm retrieval procedures are the same

42. Sperm retrieval inSperm retrieval in
non-obstructive azoospermianon-obstructive azoospermia
 Use of a microscope can identify sites of spermUse of a microscope can identify sites of sperm
production without removal of large areas of theproduction without removal of large areas of the
testistestis
 Microdissection TESE (testicular sperm extraction)Microdissection TESE (testicular sperm extraction)

43. MicroTESE identifies theMicroTESE identifies the
most advanced stages ofmost advanced stages of
spermatogenesisspermatogenesis
Microdissection Testicular Sperm ExtractionMicrodissection Testicular Sperm Extraction
Ramasamy and Schlegel 2006Ramasamy and Schlegel 2006

44. Microdissection TESEMicrodissection TESE

45. Management of testicular tissue specimensManagement of testicular tissue specimens
 Spermatozoa are within tubulesSpermatozoa are within tubules
 Effective dispersion of tubulesEffective dispersion of tubules
critical using mechanical dispersioncritical using mechanical dispersion
 Enzymatic dispersion (0.1%Enzymatic dispersion (0.1%
collagenase + RBC lysis buffer)collagenase + RBC lysis buffer)
 Incubate sample overnight withIncubate sample overnight with
PentoxifyllinePentoxifylline
Ramasamy R et al. F&S 2011

46. Effects of microTESEEffects of microTESE
Ramasamy et al., Urology 65:1190, 2005

47. Effect of TESE on TestosteroneEffect of TESE on Testosterone
0%
20%
40%
60%
80%
100%
Preop 1-3
months
12
months
18
months
Testosterone
Ramasamy et al., Urology 65:1190, 2005

48. Effect of FSH on sperm retrievalEffect of FSH on sperm retrieval
Ramasamy et al., F&S, 92:590, 2009
High FSH should NOT be a contraindication for micro-TESE

49. Case 3: AzoospermiaCase 3: Azoospermia
• 33-year-old33-year-old with uwith unremarkable past medical
history with low libido and primary infertilitywith low libido and primary infertility
• Slight gynecomastia (2cm)
• Firm testicles approximately 3 ml in volume
• Semen analysis: azoospermia

50. Diagnosis
• Based on history and physical features, what is
the most likely diagnosis?

51. Klinefelter syndrome
Taller than average
height
Reduced libido
Reduced facial and
body hair
Gynaecomastia
Small testes
Fatigue
Depression
Osteoporosis
Fat accumulation
(abdomen, hips)
Poor erections
Infertility
Handelsman DJ, Zajac JD. Med J Aust 2004; 180: 529–35.

52. Diagnostic tests for Klinefelter syndrome
What tests, if any, would you request?

53. Hormonal evaluation + Karyotype
• Testosterone - 225 ng/dL (normal: 300 – 800ng/dL)
• FSH - 26.7 mIU/ml (normal: 1.0–8.5 mIU/ml)
• LH - 10.3 mIU/ml (normal: 1.0–10 mIU/ml)
Chromosomal analysis
– 47,XXY pattern

54. Genetic Testing
• Karyotype: abnormalities range from 10-15% in
azoospermic men; Klinefelter syndrome most
common
Y-chromosome microdeletions: 13% of men with
azoo or severe oligospermia
– AZFa/b – adoption
– AZFc – rare sperm in testis
Oates, RD. Genetic considerations in the treatment of male infertility.
Infert Reprod Med Clin N America. Vol 13 (2002) 551-585.

55. Next steps?
• Donor spermDonor sperm
• AdoptionAdoption
• Medical TxMedical Tx
• TESE with microdissectionTESE with microdissection

56. Micro-TESE and Klinefelter syndromeMicro-TESE and Klinefelter syndrome
114 attempts at sperm retrieval (in 88 men)114 attempts at sperm retrieval (in 88 men)
Sperm retrieved: 78/114 (68%) attemptsSperm retrieved: 78/114 (68%) attempts
– Fertilization & transfer: 66 cyclesFertilization & transfer: 66 cycles
Clinical pregnancies: 33/78 (42%)Clinical pregnancies: 33/78 (42%)
– 52% pregnancy rate/ET52% pregnancy rate/ET
Forty-four children born (46,XX or 46,XY)Forty-four children born (46,XX or 46,XY)
Higher sperm retrieval rates than previouslyHigher sperm retrieval rates than previously
reportedreported
Low risk of genetic anomalies in offspringLow risk of genetic anomalies in offspring
Ramasamy et al. J Urol 182:1108, 2009

57. • Men with T >
300ng/dL had the
best sperm
retrieval rate of
86%.
• Medical Rx 
Preop T >
250ng/dL 
higher SRR (77%
vs 55%)
Medical Tx before Micro-TESE in men with KSMedical Tx before Micro-TESE in men with KS
Ramasamy et al. J Urol 182:1108, 2009

58. SummarySummary
 Varicocelectomy can improve natural conceptionVaricocelectomy can improve natural conception
and ART outcomesand ART outcomes
 Semen analyses need to be carefully interpretedSemen analyses need to be carefully interpreted
along with clinical context – refrain from usingalong with clinical context – refrain from using
“ABNORMAL” and “NORMAL”“ABNORMAL” and “NORMAL”
 Medical therapy for male fertility remains empiricMedical therapy for male fertility remains empiric
– use along with proper indication– use along with proper indication
 Men with NOA have heterogeneous pattern ofMen with NOA have heterogeneous pattern of
sperm productionsperm production
 Microdissection TESE more effective for selectMicrodissection TESE more effective for select
patientspatients

59. ThankThank
@ranjithramamd
ramasamy@miami.edu