2. OVERVIEW
• TESTIS
• SPERMATOGENESIS
• BIOPSY
Indications
Methods
Fixatives
Interpretation in infertility
Features in other pathological states
• FNAC OF TESTIS
• SUMMARY
• REFERENCES
3. Introduction
• Growth and development of testis
Static: from birth to 4 yrs
Growth: 4-10 yrs
Maturation: 10yrs-puberty
• At birth tubules are compactly filled with small
undifferentiated cuboidal cells
• Leydig cells are seen in newborn but then
disappear to reappear later
4. • At age 10 yrs, a growth spurt in tubules and
cell size, Leydig cells in interstitium
• 11 yrs – primary, secondary spermatocytes
appear
• 12 yrs – numerous spermatids
• Finally spermatozoa appear
• Maturing tubules with active spermatogenesis
increases gradually until adult stage is
reached.
5.
6.
7.
8.
9.
10. Spermatogenesis
• Production of male gametes is known as
SPERMATOGENESIS
• Development of male gamete into a motile
spermatozoon- SPERMIOGENESIS
• Takes approximately 70 days
• Occurs in testis ; final maturation to spermatozoa
occurs in epididymis
11. • Undifferentiated germ cells in basal
compartment of tubule – type A
spermatogonia
• These multiply and form spermatogonia type
B
• Type B spermatogonia are committed to
production of spermatozoa
12. • Spermatogonia type A- large round or oval
nucleus , condensed chromatin , peripheral
nucleoli and prominent nuclear vacuole
• Spermatogonia type B – dispersed chromatin ,
central nucleoli and no nuclear vacuole
• Both have sparse poorly stained cytoplasm
13. • Primary spermatocyte – copious cytoplasm ,
large nuclei ;coarse clumps or thin thread of
chromatin
• Secondary spermatocyte – rapidly undergo
division and are seldom seen
• Spermatids – small pointed nuclei
18. Indications
• Evaluate infertility
Increased FSH to three times normal is
sufficient evidence of primary hypogonadism
to obviate need for biopsy
If clinical findings are pathognomonic for
obstruction/testicular failure – biopsy is not
required to establish cause of azoospermia
19. Distinguish obstructive azoospermia from
nonobstructive azoospermia – most frequent
reason
Identify presence of spermatozoa, spermatids
as well as a source for ART
Management of patients with nonobstructive
azoospermia who are candidates for sperm
retrieval and IVF
20. Most perform bilateral biopsies but in patients
with discrepant testicular volume, some
perform biopsy on larger testis only
For adequate classification of
spermatogenesis tissue should contain at least
100 seminiferous tubules
21. • Diagnose vasculitis
• Determine viability in cases of torsion
• Identify presence of tumor cells after
chemotherapy – in ALL
bilateral biopsies on completion of
chemotherapy
22. • Discover malignant germ cells in patients with
increased risk of malignancy –
cryptorchid testes
contralateral germ cell tumor or history of one
Infertility
USG evidence of microlithiasis
24. Open incisional biopsy
Atraumatically dropped in suitable fixative
Optimal method
Tunica vasculosa not obtained – vasculitis
cases – wedge biopsy satisfactory
Touch preparation
25. • Percutaneous biopsy with spring loaded
biopsy gun was used successfully for male
infertility diagnosis
26. • FNA more sensitive, equally specific as testis
biopsy for sperm detection
• Information as to architectural
malorganisation, matrix components and
interstitial compartment is lost
29. Fixatives for testicular biopsy
• Stieve’s fixative
solution A mercury chloride
distilled water
solution B Formaldehyde ( 38 % )
glacial acetic acid
Mix 38ml of solution A + 12ml of solution B
30. • Bouin’s solution
saturated picric acid
formaldehyde (38%)
glacial acetic acid
fix for 24 hrs
washed several times with 50% percent alcohol
solution in order to eliminate excess picric acid
31. • For electron microscopy - fixed with 4%
glutaraldehyde solution
• histochemistry or immunofluorescence -
frozen in liquid nitrogen
32. Special stains
• Masson trichrome – increased tubular and
interstitial collagen
• PAS stain – cytoplasmic glycogen
• Elastic tissue stains – elastic fibers in walls of
postpubertal tubules, evaluation of blood
vessels in cases of suspected vasculitis
36. Qualitative analysis
• After review of all available tubules assign
predominant pattern of pathologic change
• One biopsy may have one or more patterns
and one pattern often predominates
37. • Rapid identification of those who are unlikely
to benefit from therapy
• Severe hypoplasia, sertoli cell only tubules or
tubular hyalinization unlikely to regain fertility
from surgical therapy
38. • Evaluate size, number, thickness of tubules
• Relative number and type of germ cells
• Degree of interstitial fibrosis
• Presence and condition of leydig cells
39. • Average number of late spermatids in tubules
closely correlates with sperm count in non
obstructed males
• Sperm count lower than expected from biopsy
is evidence of partial obstruction
40. Patterns of damage
• Normal histology
• Immature testes
• Sloughing of immature cells
• Hypospermatogenesis
• Maturation arrest
• Sertoli cell only pattern
• Peritubular fibrosis and tubular hyalinisation
41. • Damage may not be uniform across all tubules
• More than one pattern may be found within
one biopsy specimen
• Damage may differ between two gonads
42. • Biopsy is rarely pathognomonic of single
etiology
• Results of biopsy narrows the differential
diagnosis
• When coupled with quantitative analysis,
provides prognosis for fertility
43. Infertility with normal histology
• Germ cells in all stages are seen in tubules
• Not all tubules contain all stages
• All tubules actively undergoing
spermatogenesis
• Number of late spermatids correlates with
sperm counts
47. • Normal postpubertal testis
• Often seen with obstructive azoospermia
• Most common congenital lesion – atresia of
tail of epididymis and proximal portions of vas
deferens
• Absence/atresia of the vasa – dominant cause
of azoospermia in patients with cystic fibrosis
48. • 40-50% cases of obstuctive azoospermia –
infectious – acute epididymitis
>35 yrs – E Coli
<35 yrs – N Gonorrhoeae, C Trachomatis
49. • Vasectomy
Some authors – in absence of infection,
ischemia – no adverse effects on the germinal
epithelium or leydig cells
50. • Others –
maturation arrest at spermatocyte level
thickening of tubular basement membranes
reduced spermatogenesis
germinal cell vacuolization
51. Infertility associated with immature
testis in an adult
• Testes are identical to prepubertal testes
• Tubules are small, lumenless, lined by
immature sertoli cells and germ cells not
beyond the stage of spermatogonia or primary
spermatocyte
• Sertoli cell junctional complexes absent
52. • Peritubular elastic fibers absent
• Mature leydig cells absent
• Immature leydig cell precursors may be seen
54. • Tumors, cysts or trauma in sella or suprasellar
areas will cause panhypopituitarism
• Craniopharyngioma – most common cause for
organic GnRH deficiency related gonadal
failure –
<15 yrs
suprasellar calcification
anterior pituitary failure
diabetes insipidus
55. • Hypogonadotropic eunuchoidism – congenital
deficiencies of LH and/or FSH in adults who
gave a history of never having undergone
normal puberty
Kallman syndrome – secondary to congenital
defect in GnRH secretion by hypothalamus
Laurence-Moon-Biedel syndrome
Prader-Willi syndrome
57. Infertility associated with sloughing of
immature cells
• Orderly pattern of maturation is lost
• Jumbled germinative epithelium
• Immature germ cells including primary
spermatocytes are found in tubular lumina
58. • Mild peritubular, interstitial fibrosis
• Normal leydig cells
• Cases with >50% sloughing should be placed
in this category
60. Infertility associated with
hypospermatogenesis
• Normal/ slightly decreased diameter of
tubules
• All germinative elements in normal
proportions
• Numbers of germ cells reduced, thinning of
germinative epithelium
• Tunica propria, leydig cells are normal
63. Infertility associated with maturation
arrest
• Spermatogenesis stops abruptly at early stage
usually primary spermatocyte level
• Arrested cells increased, sloughed into lumina
• Sertoli cells, leydig cells, tunica propria normal
64. • Complete – germ cells mature only to a
certain point
• Incomplete – similar except few late
spermatids along luminal border
65.
66.
67. • In a given patient block is at a consistent stage
• Complete maturation arrest – sperm counts
are zero
• Incomplete maturation arrest - oligospermic
76. Germinal cell aplasia and focal
spermatogenesis
• 2 populations of tubules
• Smaller exhibit GCA
• Tubules of increased diameter show reduced
spermatogenesis
• One tubule may harbor both changes
• Profoundly reduced sperm count
77.
78. Infertility associated with peritubular
fibrosis and tubular hyalinization
• Germinal epithelium damaged by fibrosis
interposed between it and blood supply
• Peritubular fibrosis
• Germinal epithelium lost first, followed by
sertoli cells and at end stage – entire tubule is
filled with collagen
81. Semiquantitative analysis
• Johnsen
• Each tubule assigned value corresponding to
pattern of damage and extent of loss of
germinative epithelium
• All tubular profiles evaluated
• score from 1 to 10
• Compiled values displayed as modal number ,
mean, standard deviation or histogram
82. Johnsen scoring system
1) No cells in tubular sections
2) No germ cells. Only sertoli cells present
3) Spermatogonia are the only germ cells
present
4) Only few spermatocytes (<5) and no
spermatids or spermatozoa present
5) No spermatozoa, no spermatids but several
or many spermatocytes present
83. 6) No spermatozoa and only few spermatids
present (<5-10)
7) No spermatozoa but many spermatids present
8) Only few spermatozoa present (<5-10)
9) Many spermatozoa present but germinal
epithelium disorganised with marked sloughing
10) Complete spermatogenesis with many
spermatozoa
84. • In a normal adult testis
mean score count should be at least 8.90
60% or more of tubules should score 10.
86. Quantitative analysis
1) Enumerating germ cells and determining
tubular cross sectional areas
2) Total germ cell-sertoli cell ratio by counting at
least 30 tubular cross sections.
Ratio is constant at about 13:1 in young
healthy men.
average of 12 sertoli cells per tubular cross
section is normal
87. • One efficient method
Silber and Rodriguez-Rigau
Oval spermatids with dark densely stained
chromatin are counted
At least 20 tubular profiles evaluated
Expressed as spermatids per tubular profile
and correlates with postoperative sperm
counts
88. Allows to compare sperm count with amount
of spermatogenesis predicted by biopsy
Discrepancies suggest obstructive component
89. Flow cytometry
• Quantitative, reproducible
• Biopsy material disaggregated by mechanical
shearing and protease digestion
• Cell suspension stained with propridium
iodide or acridine orange
90. • Percentage of haploid, diploid cells analysed
• Spermatozoa/spermatids are haploid/near
haploid
• Sertoli cells, leydig cells, secondary
spermatocytes, spermatogonia are diploid
• Primary spermatocytes are diploid
91. • Haploid cells can be separated into
round spermatids
elongated spermatids
Spermatozoa
• using AO staining or differential DNA staining
based on progressive condensation of
chromatin among these cell types
92. • FCM concentrates on cellular content of
testicular biopsies
• Issues not addressed
Frequency of global sclerosis
amount and type of matrix in tunica propria
and interstitium
integrity of interstitial vessels
identification of unexpected cell types
93. Intratubular germ cell neoplasia
• In postpubertal patients, most commonly
appears as germ cells with
enlarged,hyperchromatic nuclei and clear
cytoplasm along basal portion of the tubules
• Conspicuous nucleoli, frequent mitotic figures
94. • Sertoli cells are displaced towards the lumen
• Spermatogenesis in affected segment is
always absent
• Thickened peritubular basement membranes
95.
96.
97. • PAS positive, diastase sensitive but also seen
in nonneoplastic spermatogonia and sertoli
cells
• Antibodies against placental alkaline
phosphatase – more specific
98. Cryptorchidism
• Arrest in development of germ cells
• Hyalinisation, thickening of basement
membrane of tubules
• Increased interstitial stroma, prominent leydig
cells
99.
100.
101. Torsion and infarction
• If torsion lasts longer than 24 hrs, the testis
almost certainly will infarct
• Torsion that lasts less than 6 hrs will not cause
a testicular infarct
• Upto 6 hrs after torsion – venous congestion,
intestitial hemorrhage
• Biopsy at 5and ½ hrs – no nuclear pyknosis
102. • At 9 and ½ hrs – neutrophils in walls of
capillaries, severe interstitial hemorrhage but
no infarction
• At 4days – hemorrhagic infarction, coagulative
necrosis with neutrophils
• After 1-2 months – infarct, granulation tissue
105. Microlithiasis
• Microcalcification in tubules
• Discovered on ultrasound examination
• Consists of more than 5 foci of calcification,
less than 2mm, randomly distributed
• large majority of cases are not associated with
germ cell malignancy
106.
107. Vasculitis
• Biopsy from painful, enlarging or shrinking
testis and should consists of wedge containing
capsule, tunica vasculosa and testicular
parenchyma
108.
109. Amyloidosis
• Systemic amyloidosis commonly involves testis
• Amyloid deposition occurs in relation to blood
vessles of interstitium and in walls of tubules
• Testis biopsy is more sensitive than rectal
biopsy for diagnosis of primary and secondary
amyloidosis
110. FNAC of testis
• Aim is to provide triage of cases of testicular
swelling into those who require surgery as the
first choice treatment and those who do not
111. Technical considerations
• 25 gauge needles
• No LA
• USG guidance in
partly cystic tumors
non palpable USG detected lesions
retroperitoneal GCT
follow up of lymphoma, leukemia patients
112. limitations
• Inadequate sample
• Sample not fully representative eg in large
mixed TGCT
• Pleomorphism in smears of normal testis
mistaken for TGCT or lymphoma
• Intrascrotal fluid may conceal tumor. Testis
reexamined after evacuation of fluid from
hydrocele/hematocele
114. SUMMARY
Main indication of testicular biopsy is
evaluation of infertility
Increased FSH to three times normal is
sufficient evidence of primary hypogonadism
to obviate need for biopsy
If clinical findings are pathognomonic for
obstruction/testicular failure – biopsy is not
required to establish cause of azoospermia
115. Distinguish obstructive azoospermia from
nonobstructive azoospermia – most frequent
reason
Identify presence of spermatozoa, spermatids
as well as a source for ART
Management of patients with nonobstructive
azoospermia who are candidates for sperm
retrieval and IVF
119. • Evaluate size, number, thickness of tubules
• Relative number and type of germ cells
• Degree of interstitial fibrosis
• Presence and condition of leydig cells
• Average number of late spermatids in tubules
closely correlates with sperm count in non
obstructed males
120. Patterns of damage
• Normal histology
• Immature testes
• Sloughing of immature cells
• Hypospermatogenesis
• Maturation arrest
• Sertoli cell only pattern
• Peritubular fibrosis and tubular hyalinisation
121. • Damage may not be uniform across all tubules
• More than one pattern may be found within
one biopsy specimen
• Damage may differ between two gonads
122. • Biopsy is rarely pathognomonic of single
etiology
• Results of biopsy narrows the differential
diagnosis
• When coupled with quantitative analysis,
provides prognosis for fertility
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Sternberg’s Diagnostic Surgical Pathology. Vol.3. 4th ed.
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Surgical Pathology. Vol.1. 9th ed. India. Elsevier; 2009.
p1412-56.
3) Tickoo SK, Amin MB, Cramer HM,Harik LR, Ulbright TM.
The testis, paratesticular structures, and male external
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(eds.) Silverberg’s Principles and Practice of Surgical
Pathology and Cytopathology. Vol.2. 4th ed. China.
Churchill Livingstone; 2006. p1731-90.
124. 4) Damjanov I, Bostwick DJ, Amin MB. Male
Reproductive System. In: Damjanov, Linder (eds.)
Anderson’s Pathology. Vol.2. 10th ed. USA. Mosby-
Year Book, Inc.; 1996. p2166-230.
5) Tickoo SK, Tamboli P, Warner NE, Amin MB. Testis
and Paratestis including spermatic cord. In:
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Surgical Pathology. Vol.2. 1st ed. China. Saunders;
2003. p1215-56.
6) Guillermo MP, Orell SR. Male and female genital
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Fine Needle Aspiration Cytology. 5th ed. India.
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125. 7) Cerilli LA, Kuang W, Rogers D. A practical
approach to testicular biopsy interpretation for
male infertility. Arch Pathol Lab Med.
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biopsy: clinical practice and interpretation. Asian
J Androl. 2012;14(1):88-93.
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