Testicular biopsy

PRESENTER: Dr. Harmanpreet Kaur

MODERATOR: Dr. Rajender Kumar Thakral
 OVERVIEW
• TESTIS
• SPERMATOGENESIS

• BIOPSY
 Indications
 Methods
 Fixatives
 Interpretation in infertility
 Features in other pathological states

• FNAC OF TESTIS
• SUMMARY
• REFERENCES
 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
• 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.
 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.
• Undifferentiated germ cells in basal compartment of tubule –
Type A spermatogonia

• These multiply and form Type B spermatogonia

• Type B spermatogonia are committed to production of

spermatozoa
• 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.

• 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

SPERMATOGENESIS
Spermatogenesis

Figure 28.7
 BIOPSY INTRODUCTION
• First introduced by Charny and Hotchkiss in 1940.

• Testicular biopsy involves the process of obtaining a small

bit/core of testicular tissue sample, which is then sent for
microscopic examination.
 INDICATIONS
• DIAGNOSTIC
• Infertility- Incresed FSH in primary hypogonadism.
Obstructive azoospermia: To confirm the presence of
normal spermatogenesis before surgical correction of obstruction.
• Diagnosis of germ cell neoplasia in situ (GCNIS) of testis:
increased risk with
1) H/O cryptorchidism
2) H/O testicular germ cell tumour
3) Idiopathic testicular atrophy
4) Testicular microlithiasis
5) Inhomogeneous testicular parenchyma on USG
6) Radiologically apparent solid testicular lesions
THERAPEUTIC

 Non obstructive azoospermia (NOA) : Testicular sperm extraction

and microdissection to harvest spermatozoa.

Part of the specimen should be used for histological examination to

predict chance of future successful sperm harvesting and to diagnose
GCNIS.

Distinguish obstructive azoospermia from nonobstructive

azoospermia – most frequent reason.

Assisted reproductive techniques- to identify presence of

spermatozoa, spermatids and their subsequent extraction.
• Management of patients with nonobstructive azoospermia who are
candidates for sperm retrieval and IVF.

• Diagnose vasculitis

• Determine viability in cases of torsion

 METHODS
• Open incisional biopsies

• Wedge biopsies

• Percutaneous – core needle, fine needle

 Open incisional biopsy
 Atraumatically dropped in suitable fixative
 Optimal method
 Tunica vasculosa not obtained – vasculitis cases – wedge
biopsy satisfactory
 Touch preparation
 WEDGE BIOPSY
• Contain tunica albuginea and underlying parenchyma.

• It is possible to prepare touch preparations with the biopsy

specimen followed by immediate spraying with fixative.
PERCUTANEOUS- CORE NEEDLE , FINE
NEEDLE
•Percutaneous testis biopsy with a spring-loaded biopsy gun used for
prostatic needle biopsy diagnosis has been used successfully for the
diagnosis of male infertility.

•FNA more sensitive, equally specific as testis biopsy for sperm

detection, equally sensitive and specific as the biopsy touch imprint.
•Touch preparation at the time of testis biopsy and hematoxylin
and eosin (H&E) staining allow all cell types to be rapidly
identified.

•Distinction can be made between maturation arrest at the late

spermatid level and excurrent duct obstruction.

• Perfusion mapping with the use of color Doppler ultrasound

has been successful in identifying high-quality motile sperm.
 COMPLICATIONS
• Bleeding
• Infection
• Biopsies from small atrophic testes – increased risk of
hypogonadism
 FIXATION
• 10% formalin
 Nuclei shrink, appear denser
 Undulating tubular margins
 Tubules shrink
• Bouin, Hollande solutions preferred – superior nuclear detail
 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

• 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
• For electron microscopy - fixed with 4% commercial
paraformaldehyde and 1% glutaraldehyde in a neutral buffer.

• Histochemistry or immunofluorescence - frozen in liquid

nitrogen
 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
BIOPSY INTERPRETATION IN
INFERTILITY
 CAUSES OF MALE INFERTILITY
PRETESTICULAR
• Disorders of hypothalamus or pituitary
• Endocrine diseases(thyroid or adrenal disorder)
• Metabolic disorders (renal or liver diseases)
• Chronic infection
• Drugs

TESTICULAR
• Idiopathic hypospermatogenesis or aspermatogenesis
• Developmental and genetic disorder
• Circulatory- Varicocele or torsion
• Inflammatory lesions- Infectious or immune
• Iatrogenic- Chemical, radical or surgical
• Environmental
POST TESTICULAR

• Congenital anomalies of excretory ducts and accessory glands.

• Inflammatory lesions of excretory ducts and accessory glands.

• Iatrogenic or posttraumatic lesions of the excretory ducts,

accessory glands, or ejaculatory nerve plexus.
 LOW POWER SCREENING, X40

• Confirm proper tissue type

• Semiquantitative assessment for adequacy (>75 tubular cross

sections)

• Initial impression of overall heterogeneity, assess for intratubular

germ cell neoplasia, granulomas, interstitial fibrosis, or
inflammation.
• Qualitative analysis
• Semi quantitative analysis
• Quantitative analysis
• Medical history, previous paternity
• Semen analysis
• Physical findings
• Serum gonadotropin measurements
 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.

• 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
• 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.
• Sperm count lower than expected from biopsy is evidence of
partial obstruction.
 PATTERNS OF DAMAGE
• Normal histology
• Immature testis
• Sloughing of immature cells
• Hypospermatogenesis
• Maturation arrest
• Sertoli cell only pattern
• Peritubular fibrosis and tubular hyalinisation
 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.
 CAUSES
• Ductal obstruction – congenital, acquired
• Impaired sperm motility – immotile cilia syndrome
• Tubule hypercurvature and branching
• Hyperabsorption of sperm by epididymis
• Varicocele
• Inadequate sampling
• Idiopathic
• 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.

• 40-50% cases of obstuctive azoospermia – infectious – acute

epididymitis
 >35 yrs – E Coli
 <35 yrs – N Gonorrhoeae, C Trachomatis
•
 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.
• Peritubular elastic fibers absent
• Mature leydig cells absent
• Immature leydig cell precursors may be seen
 CAUSES

• Common denominator – prepubertal diminished/ absent

gonadotropin secretion
• 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
• 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
 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.
• Mild peritubular, interstitial fibrosis
• Normal leydig cells
• Cases with >50% sloughing should be placed in this category
 CAUSES
• Varicoceles commonly associated
• Prior vasectomy
• Mumps orchitis
• Idiopathic
 Infertility associated with
hypospermatogenesis

• Normal/ slightly decreased diameter of tubules.

• All stages of spermatogenesis are present but reduced.
• Numbers of germ cells reduced, thinning of germinative
epithelium.
• Tunica propria, leydig cells are normal.
• Mild hypospermatogenesis- changes in occasional tubules.
• Moderate hypospermatogenesis- changes are in between.
• Severe hypospermatogenesis- significant reduction in all
tubules.
 CAUSES
• Environmental – malnutrition, toxic chemicals, chemotherapy
• Genetic – advancing age, down syndrome
• Endocrine – hypo/hyper thyroidism, glucocorticoid excess,
hyperprolactinemia, adrenogenital syndrome
• Ductal obstruction
• Idiopathic
Interstitial fibrosis

Germ cell
disorganisation
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.
• Complete – germ cells maturity ceases at a specific point
frequently at primary spermatocyte level.
• Incomplete – similar except few late spermatids are present in
few tubules, patients usually oligospermic.
Numerous spermatogonia, few
spermatocytes,no mature spermatozoa.

Sertoli cell prominent since reduced

germ cells.

Tubules often contain degenerated

cells with irregular
Dense nuclei
• Complete maturation arrest – sperm counts are zero
• Incomplete maturation arrest - oligospermic
 CAUSES
• Environmental – chemicals, chemotherapy
• Genetic – XYY, cystic fibrosis, adrenogenital syndrome
• Uremia
• Mumps orchitis
• Endocrine – glucocorticoid excess, postpubertal gonadotropin
deficiency
• Spinal cord injury, varicocele, vasectomy
 Infertility associated with sertoli cell only
syndrome

• Germinal aplasia/ del Castillo syndrome

• Absent germinal epithelium.
• Two populations of tubules are seen-One population exhibiting
germ cell aplasia and other population has reduced
spermatogenesis.
• Tunica propria, basement membrane normal
• Variable leydig cell number
Tubules are reduced in
diameter and lined by
sertoli cells.

Sertoli cells are

perpendicular to basement
membrane.

Cells have nuclear

indentations and prominent
nucleoli.
 CAUSES
• Chemotherapy, irradiation
• Klinefelter, XYY, Down syndrome
• Adrenogenital syndrome
• Hyperprolactinemia
• Uremia
• Orchitis
• Varicocele
• Idiopathic
Infertility associated with peritubular fibrosis
and tubular hyalinization

• Germinal epithelium damaged by fibrosis interposed between

it and blood supply.

• Peritubular fibrosis, tubular fibrosis and tubular hyalinization.

• Germinal epithelium lost first, followed by sertoli cells and at

end stage – entire tubule is filled with collagen.
Germinal epithelium
is damaged by
fibrosis.

Peritubular fibrosis.

Sertoli cells are lost

and at end stage
entire tubule is filled
with collagen.
 CAUSES
• Chemotherapy, irradiation, alcoholism
• Klinefelter, XYY, adrenogenital syndrome
• Postpubertal androgen/estrogen excess
• Hypoprolactinemia
• Mumps orchitis
• Varicocele
• Vascular sclerosis
 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
 Johnsen scoring system
1) No cells in tubular sections
2) No germ cells. Only sertoli cells present.
3) Arrest at the spermatogonia: A type Spermatogonia multiplicate
but do not develop to maturing cells of spermatogenesis.
4) Arrest of spermatogenesis at the stage of primary spermatocytes:
a few primary spermatocytes are present.
5) Arrest of spermatogenesis at the stage of primary spermatocytes:
many spermatocytes border the lumen of the seminiferous
tubules.
6) Severely reduced spermatogenesis: Only few immature
spermatids, reduced height of germinal epithelium.
7) Considerably reduced spermatogenesis:No mature spermatids,
only immature spermatids, no spermiation.
8) Distinct reduced spermatogenesis: Few mature spermatids, no
spermiation.
9) Modest reduced spermatogenesis: reduced number of mature
spermatids, a few zones of spermiation.
10) Intact spermatogenesis: many mature spermatids and zone of
spermiation.
• In a normal adult testis
 mean score count should be at least 8.90
 60% or more of tubules should score 10.
Mean score +/- SD Diagnosis

9.38 +/- 0.24 Normal

3.80 +/- 1.80 Hypogonadotropic eunuchoidism

6.09 +/- 2.25 Acquired adult hypopituitarism

1.25 +/- 0.28 Klinefelter syndrome

4.43 +/- 2.30 Cryptorchid testes

2.0 +/- 0.03 Sertoli cell only

5.32 +/- 2.13 Severe hypospermatogenesis

7.80 +/- 1.26 Moderate hypospermatogenesis

 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
• 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
 Allows to compare sperm count with amount of
spermatogenesis predicted by biopsy
 Discrepancies suggest obstructive component
 Flow cytometry
• Quantitative, reproducible
• Biopsy material disaggregated by mechanical shearing and
protease digestion
• Cell suspension stained with propridium iodide or acridine
orange
• 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
• 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
• 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
 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

• Sertoli cells are displaced towards the lumen.

• Spermatogenesis in affected segment is always absent.

• Thickened peritubular basement membranes.

• PAS positive, diastase sensitive but also seen in nonneoplastic
spermatogonia and sertoli cells
• Antibodies against placental alkaline phosphatase – more
specific
 Cryptorchidism
• Arrest in development of germ cells
• Hyalinisation, thickening of basement membrane of tubules
• Increased interstitial stroma, prominent leydig cells
 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
• 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
 Varicocele
• Decreased spermatogenesis
• Sloughing of immature germinal cells
• Degeneration of germ cells
• Increased numbers of leydig cells
 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
 Vasculitis
• Biopsy from painful, enlarging or shrinking testis and should
consists of wedge containing capsule, tunica vasculosa and
testicular parenchyma
 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
 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.
 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
 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
 COMPLICATIONS
• Painful
• Only contraindication – acute orchitis with cellulitis of
scrotum
 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
 Distinguish obstructive azoospermia from nonobstructive
azoospermia – most frequent reason.
 Results of biopsy narrows the differential diagnosis
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Thank you