testicular biopsy

Testicular biopsy

PRESENTER: Dr BAPTISTGUIDE: Dr ARUNA S

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 testisStatic: from birth to 4 yrsGrowth: 4-10 yrsMaturation: 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 spermatogonia type B

• 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

Indications

• Evaluate infertilityIncreased 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

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

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

• Diagnose vasculitis• Determine viability in cases of torsion• Identify presence of tumor cells after

chemotherapy – in ALLbilateral biopsies on completion of

chemotherapy

• Discover malignant germ cells in patients with increased risk of malignancy –

cryptorchid testescontralateral germ cell tumor or history of one InfertilityUSG evidence of microlithiasis

Methods

• Open incisional biopsies• Wedge biopsies• Percutaneous – core needle, fine needle

Open incisional biopsy

Atraumatically dropped in suitable fixativeOptimal methodTunica vasculosa not obtained – vasculitis

cases – wedge biopsy satisfactoryTouch preparation

• Percutaneous biopsy with spring loaded biopsy gun was used successfully for male infertility diagnosis

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

• Information as to architectural malorganisation, matrix components and interstitial compartment is lost

Complications

• Bleeding• Infection• Biopsies from small atrophic testes –

increased risk of hypogonadism

Fixation

• 10% formalinNuclei shrink, appear denserUndulating tubular marginsTubules 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% glutaraldehyde solution

• 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

• 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 testes• Sloughing of immature cells• Hypospermatogenesis• Maturation arrest• Sertoli cell only pattern• Peritubular fibrosis and tubular hyalinisation

• 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

• Biopsy is rarely pathognomonic of single etiology

• Results of biopsy narrows the differential diagnosis

• When coupled with quantitative analysis, provides prognosis for fertility

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

• VasectomySome authors – in absence of infection,

ischemia – no adverse effects on the germinal epithelium or leydig cells

• Others – maturation arrest at spermatocyte levelthickening of tubular basement membranesreduced spermatogenesisgerminal cell vacuolization

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 yrssuprasellar calcificationanterior pituitary failurediabetes 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 syndromePrader-Willi syndrome

• Prepubertal androgen excessAndrogen producing tumorAdrenogenital syndromeExogenous androgen administration

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 germinative elements in normal proportions

• Numbers of germ cells reduced, thinning of germinative epithelium

• Tunica propria, leydig cells are normal

causes

• Environmental – malnutrition, toxic chemicals, chemotherapy

• Genetic – advancing age, down syndrome, fertile eunuch syndrome

• Endocrine – hypo/hyper thyroidism, glucocorticoid excess, hyperprolactinemia, adrenogenital syndrome

• Ductal obstruction• Idiopathic

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 mature only to a certain point

• Incomplete – similar except few late spermatids along luminal border

• In a given patient block is at a consistent stage• 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• Small tubules lined exclusively by sertoli cells

perpendicular to basement membrane• Tunica propria, basement membrane normal• Variable leydig cell number

causes

• Chemotherapy, irradiation• Klinefelter, XYY, Down syndrome• Adrenogenital syndrome• Hyperprolactinemia• Uremia• Mumps orchitis• Varicocele• idiopathic

• GCA with scattered spermatogonia – germinal cell hypoplasia

• GCA/ germinal cell hypoplasia – look for malignant intratubular germ cells

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

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

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 system1) No cells in tubular sections2) No germ cells. Only sertoli cells present3) Spermatogonia are the only germ cells

present4) Only few spermatocytes (<5) and no

spermatids or spermatozoa present5) No spermatozoa, no spermatids but several

or many spermatocytes present

6) No spermatozoa and only few spermatids present (<5-10)

7) No spermatozoa but many spermatids present8) Only few spermatozoa present (<5-10)9) Many spermatozoa present but germinal

epithelium disorganised with marked sloughing

10) Complete spermatogenesis with many spermatozoa

• In a normal adult testismean score count should be at least 8.9060% 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 methodSilber and Rodriguez-RigauOval spermatids with dark densely stained

chromatin are countedAt least 20 tubular profiles evaluatedExpressed 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 spermatidselongated spermatidsSpermatozoa• 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 addressedFrequency of global sclerosisamount and type of matrix in tunica propria

and interstitiumintegrity of interstitial vesselsidentification 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 tumorsnon palpable USG detected lesionsretroperitoneal GCTfollow 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

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

Methods

• Open incisional biopsies• Wedge biopsies• Percutaneous – core needle, fine needle

Fixation

• 10% formalinNuclei shrink, appear denserUndulating tubular marginsTubules shrink• Bouin, Hollande solutions preferred – superior

nuclear detail

Biopsy interpretation in infertility

• Qualitative analysis• Semi quantitative analysis• Quantitative analysis

• 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

Patterns of damage

• Normal histology• Immature testes• Sloughing of immature cells• Hypospermatogenesis• Maturation arrest• Sertoli cell only pattern• Peritubular fibrosis and tubular hyalinisation

• 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

• Biopsy is rarely pathognomonic of single etiology

• Results of biopsy narrows the differential diagnosis

• When coupled with quantitative analysis, provides prognosis for fertility

REFERENCES1) Levin HS. Nonneoplastic Diseases of the Testis. In: Mills,

Carter, Greenson, Oberman, Reuter, Stoler (eds.) Sternberg’s Diagnostic Surgical Pathology. Vol.3. 4th ed. USA. Lippincott Williams and Wilkins; 2004. p2133-66.

2) Rosai J. Testis. In: Rosai J (ed.) Rosai and Ackerman’s 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 genitalia. In: Silverberg, Delellis, Frable, Livolsi, Wick (eds.) Silverberg’s Principles and Practice of Surgical Pathology and Cytopathology. Vol.2. 4th ed. China. Churchill Livingstone; 2006. p1731-90.

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: Weidner, Cote, Suster, Weiss (eds.) Modern Surgical Pathology. Vol.2. 1st ed. China. Saunders; 2003. p1215-56.

6) Guillermo MP, Orell SR. Male and female genital tract. In: Orell, Sterrett (eds.) Orell & Sterrett’s Fine Needle Aspiration Cytology. 5th ed. India. Elsevier; 2012. p339-69.

7) Cerilli LA, Kuang W, Rogers D. A practical approach to testicular biopsy interpretation for male infertility. Arch Pathol Lab Med. 2010;134(8):1197-204.

8) Dohle GR, Elzanaty S, van Casteren NJ. Testicular biopsy: clinical practice and interpretation. Asian J Androl. 2012;14(1):88-93.

9) McLachlan RI, Rajpert-De Meyts E, Hoei-Hansen CE, de Kretser DM, Skakkebaek NE. Histological evaluation of the human testis--approaches to optimizing the clinical value of the assessment: mini review. Hum Reprod. 2007;22(1):2-16.

Thank you