Asian Journal of Andrology (2012) 14, 88–93

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REVIEW

Testicular biopsy: clinical practice and interpretation

Gert R Dohle1, Saad Elzanaty2 and Niels J van Casteren1

Testicular biopsy was considered the cornerstone of male infertility diagnosis for many years in men with unexplained infertility and
azoospermia. Recent guidelines for male infertility have limited the indications for a diagnostic testicular biopsy to the confirmation of
obstructive azoospermia in men with normal size testes and normal reproductive hormones. Nowadays, testicular biopsies are mainly
performed for sperm harvesting in men with non-obstructive azoospermia, to be used for intracytoplasmic sperm injection. Testicular
biopsy is also performed in men with risk factors for testicular malignancy. In a subgroup of infertile men, there is an increased risk for
carcinoma in situ of the testis, especially in men with a history of cryptorchidism and testicular malignancy and in men with testicular
atrophy. Ultrasonographic abnormalities, such as testicular microlithiasis, inhomogeneous parenchyma and lesions of the testes,
further increase the risk of carcinoma in situ (CIS) in these men. For an accurate histological classification, proper tissue handling,
fixation, preparation of the specimen and evaluation are needed. A standardized approach to testicular biopsy is recommended. In
addition, approaches to the detection of CIS of the testis testicular immunohistochemistry are mandatory. In this mini-review, we
describe the current indications for testicular biopsies in the diagnosis and management of male infertility.
Asian Journal of Andrology (2012) 14, 88–93; doi:10.1038/aja.2011.57; published online 12 December 2011

Keywords: carcinoma in situ of the testis; diagnosis; male infertility; testicular sperm extraction; testicular biopsy; testicular germ cell malignancies

INTRODUCTION TECHNIQUE
Unexplained male infertility is present in 30%–40% of men with Testicular biopsy can be performed under local and general anaesthe-
abnormal semen parameters.1 Potential explanations for unexplained sia. Usually, the procedure is performed as day-care surgery in an
male infertility are endocrine disruption of gonadal development dur- outpatient clinic setting. For a diagnostic testicular biopsy, a scrotal
ing early pregnancy, due to environmental pollution and genetic fac- incision of 2–3 cm could allow enough exposure of the tunica albu-
tors. Both genetic and environmental causes of infertility can result in ginea of the testis. A small incision in the capsule of the testicular of
testicular dysgenesis, and consequently in infertility, hypogonadism about 0.5 cm is made for obtaining a biopsy of about 33333 mm. For
and an increased risk of testicular malignancy.2 adequate classification of spermatogenesis, the removed tissue should
Testicular biopsy may be part of the diagnostic process of infertile contain at least 100 seminiferous tubules.5 The tissue should not be
men, but usually testicular histology does not explain the real cause of squeezed with a forceps since this may disrupt testicular tissue archi-
infertility. It merely confirms a disturbance of spermatogenesis in men tecture and hamper proper evaluation of the seminiferous tubules.
with low sperm concentrations and elevated follicle-stimulating hor- The wound is closed with absorbable sutures.
mone. Testicular biopsy plays a distinctive role in the diagnosis of An alternative to an open diagnostic testicular biopsy could be a
obstructive azoospermia: in these men, surgical repair of the genital transcutaneous puncture of the testis with either a true-cut needle or a
tract may be feasible and result in the presence of spermatozoa in the fine needle aspiration (FNA).6 The advantage of FNA of the testis is
semen and spontaneous pregnancies.3 In most cases, however, a rou- that is does not require surgical equipment and experience, and can
tine diagnostic testicular biopsy will not alter therapeutic options. be performed in an outpatient setting under local anaesthesia.7
For men with non-obstructive forms of severe oligozoospermia and Rammouu-Kinia et al.8 demonstrated a correlation of 88.5% between
azoospermia, intracytoplasmic sperm injection (ICSI) is the only fine needle biopsies and normal histology in different patient groups.
alternative if viable spermatozoa are present.4 In the era of ICSI, testicu- FNA may also be helpful in the diagnosis of small testicular lesions. It
lar biopsy is usually performed in men with non-obstructive azoosper- is, however, unclear if FNA can also accurately detect CIS of the testis.
mia (NOA) for spermatozoa extraction, the testicular sperm extraction FNA of the testis, using a small butterfly needle attached to a syringe,
(TESE) procedure. In about 50%–60% of men with NOA and in 100% may also be used to harvest spermatozoa for ICSI, especially in men
of men with OA, spermatozoa can be retrieved from the testes and used with OA.6,9 The technique is, however, associated with a lower sperm
for ICSI. In this review, we will discuss current indications for testicular retrieval rate in men with NOA compared to open biopsy techniques.
biopsy, the interpretation of testicular histology for infertility diagnosis, For TESE procedures, different techniques have been developed
the clinical (prognostic) value of the results of testicular biopsy and the and compared. The microsurgical TESE seems to have the highest
detection of carcinoma in situ (CIS) of the testis. sperm retrieval rate and may limit damage to the testicular tissue.
1
Department of Urology, Erasmus MC, 3000 CA Rotterdam, The Netherlands and 2Department of General Surgery, Urology Section, Kristianstad Central Hospital, Kristianstad SE
291 85, Sweden
Correspondence: Dr GR Dohle (g.r.dohle@erasmusmc.nl)
Received: 30 May 2011; Revised: 5 September 2011; Accepted: 5 September 2011; Published online: 12 December 2011
 Testis biopsy: clinical practice and interpretation
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89

Table 1 Studies comparing conventional testicular sperm extraction Klinefelter patients. In most patients, testosterone levels recover
(cTESE) to microsurgical testicular sperm extraction (mTESE) in to prebiopsy levels within 1 year due to an elevation of luteinizing
regard to sperm retrieval rate (SRR) hormone LH. With age, these men may be at risk for late-onset
Study cTESE mTESE hypogonadism.12 In patients with NOA, an assessment should be
made between the risk of injury due to multiple-sited biopsies and
n SRR (%) n SRR (%) the diagnostic and therapeutic benefits for the patient.
Okada et al.33 24 17 74 45
Ramasamy et al.40 83 32 460 58 TISSUE HANDLING
Tsujimura et al.41 37 35 56 43 All too frequently in clinical practice, the value of testicular biopsy is
Average SRR (%) 40 (16–63) 48 (42–58) diminished as a result of poor biopsy specimen handling and incorrect
interpretation of samples by pathologists, not thoroughly familiar
with the reproductive tract histology.13 A standardized approach of
the fixation and staining procedure of the biopsy and the classification
of the specimen are needed.13,14
For a reliable evaluation, the biopsies taken should be uniformly
provided to the pathologist. The specimen fixation is important as it
can influence staining patterns. A standardized approach for fixation
and staining of the specimen is desirable.15–17 The European Germ
Cell Cancer Consensus Group advocates the use of Stieve’s or Bouin’s
solution for fixation.18 The use of Stieve’s solution might aid in pre-
serving morphology, but hampers intranuclear staining methods if the
fixation process is prolonged. Bouin’s solution allows good fixation of
the tissue for histological evaluation of spermatogenesis, but is infre-
quently used because of its reduced shelf-life and toxicity.19 Formalin
is commonly used for fixation, but may cause shrinkage artefacts. This
Figure 1 OCT 3/4 staining of carcinoma in situ cells in seminiferous tubules of can be reduced by using neutral buffered formaldehyde.20 Also,
the testis.
immunohistochemistry with OCT 3/4 for the detection of CIS could
be false-negative if Stieve’s or Bouin’s solution is used.15
Spermatozoa can be retrieved from tubules that are dilated and For the diagnosis of CIS, Dieckmann et al.21 proposed to take two
this can be visualized with an operating microscope. This technique biopsies in each testis to increases the chance of detecting CIS by 18%.
needs microsurgical skills and general anaesthesia is usually required. This, however, applies to normal-size testis and not to atrophic testis.
Table 1 summarizes the different sperm retrieval rates obtained by Performing a two-sided biopsy in an atrophic testis might lead to
conventional TESE procedures and by microsurgical TESE. Especially testicular dysfunction and a single biopsy is recommended for routine
small-size testes may benefit from microsurgical TESE procedures. diagnostic purposes. Routinely, haematoxylin–eosin staining is usu-
Performing a testicular biopsy does not have a high complication ally applied for evaluation of spermatogenesis. For the detection of CIS
rate: sometimes bleeding and infection occur. Biopsies taken in of the testis, additional staining procedures are recommended.22 CIS is
small atrophic testicles might increase the risk of hypogonadism, difficult to identify in testicular biopsies. Several markers for CIS of the
which then results in life-long testosterone substitution.10 This con- testis have been developed, including placental alkaline phosphatase,
cern should be considered in advance and discussed with the AP2-gamma and stem cell factor receptor c-KIT.23 In a recent study
patient. The risk for hypogonadism after testicular biopsy specif- performed by our department, we found an extra diagnostic yield of
ically applies to TESE procedures performed in small-size testis. 20% in identifying CIS in biopsies from infertile men using the OCT 3/
Ishikawa et al.11 demonstrated that the risk of hypogonadism is 4 marker. OCT 3/4 is highly specific for CIS and currently, the best
increased in patients with Sertoli cell-only syndrome and in marker for CIS, seminomas and embryonal carcinomas (Figure 1).24

Figure 2 Several classification systems for scoring of spermatogenesis based on five main histological patterns of spermatogenesis. (a), Sertoli cell-only syndrome. (b),
Maturation arrest. (c), Hypospermatogenesis. (d), Normal spermatogenesis.

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These markers are not 100% accurate in biopsies performed in new- deferens, unless testicular volume is reduced or follicle-stimulating
born, as a neonatal germ with delayed development may resemble a hormone is elevated.3
CIS cell and display prolonged expression of immunohistochemical . testicular sperm extraction: multiple testicular biopsies are usually
markers used for the diagnosis of CIS. Using the presence of stem cell needed for sperm harvesting in men with NOA. Part of the spe-
factor, also known as KITLG, we recently have been able to differenti- cimen should be used for histological examination to predict the
ate between delayed matured germ cells and the earliest stages of chance for future successful sperm harvesting and to diagnose CIS
malignant transformation.24 of the testis.32 Microdissection may improve the success of sperm
harvesting.33
. diagnosis of CIS of the testis: risk factors for CIS are male infertility
SCORING OF SPERMATOGENESIS
together with other risk factors, such as a history of cryptorchid-
Several classification systems are described,25–29 all based on five main
ism, testicular germ cell tumour and in the case of idiopathic tes-
histological patterns of spermatogenesis: (i) absence of seminiferous
ticular atrophy. Also, some ultrasosnographic abnormalities of the
tubules (tubular sclerosis); (ii) no germ cells within the seminiferous
testis indicate the potential presence of CIS, especially testicular
tubules (Sertoli cell-only syndrome) (Figure 2a); (iii) incomplete
microlithiasis, an inhomogeneous testicular parenchyma and solid
spermatogenesis, not beyond the spermatocyte stage (spermatogenic
testicular lesions. Testicular lesions of unknown origin warrant an
arrest) (Figure 2b); (iv) all germ cell stages present including sper-
excision biopsy.34–38
matozoa, but there is a distinct decline in the number of germ cells
(hypospermatogenesis) (Figure 2c); and (v) normal spermatogenesis
(Figure 2d). In practice, these different stages of spermatogenesis PREDICTIVE VALUE OF TESTICULAR BIOPSY FOR ASSISTED
often exist next to each other in a single biopsy (mixed pattern). REPRODUCTIVE TECHNOLOGY
This has resulted in a wide variation of classification of spermatogen- In men with NOA, spermatozoa can be harvested from the testis in
esis by different pathologists and limits the diagnostic and prognostic
50%–60%.31 Previous attempts to predict sperm retrieval from testis
value of the testicular biopsy.
of men with NOA based on clinical parameters, such as testicular
A widely quoted and quantitative histological grading system is volume or reproductive hormones, have failed.31
the Johnsen score; in at least 100 seminiferous tubules, the level of
In the literature, there is no consensus on the value of testicular
sperm maturation is graded between 1 and 10, according to the
histology for sperm retrieval: some authors conclude that testicular
most advanced germ cell in the tubule.25 The total Johnsen score is
histology is of limited value,39 while others find it to be of major
then determined by dividing the total score by the number of
prognostic significance.30,40,41 It has been postulated that the lack of
evaluated tubules. An enormous drawback of the Johnsen scoring
clarity may well arise from inaccurate identification of germ cells
system, is that the mean tubule score may not reflect the true status
and confusing categorisation of biopsies.14 The description of
of spermatogenesis: e.g., normal spermatogenesis can be found in a
the histological method is often not mentioned or very briefly
biopsy next to tubules with a Sertoli cell-only pattern. This results
described. Sperm retrieval is usually good in men with hyposper-
in a mean score indicating a severe disturbance of spermatogenesis.
matogenesis and limited in men with Sertoli cell-only syndrome:
However, in the case of TESE sperm, harvesting is likely to occur in
Table 2 summarizes the results of studies that have compared the
these men and the Johnsen score may not have a reliable predictive
histological results of the testicular biopsy to the sperm retrieval
value for sperm recovery.30 Also the Johnsen score is quite labour-
rate.42–45 In regard to men with Klinefelter syndrome, the successful
intensive. Taking all these points into consideration, it has little
rate of sperm retrieval using TESE has ranged from 27% to 69%.46
clinical utility in the current fertility practice. The scoring system
These differences can be attributed to differences between the
was modified by de Kretser and Holstein.26,27 Others have empha-
men, their age and the histological patterns within their testes.
sized the importance of the main histological classification of the
Thus, Schiff et al.43 observed a 71% sperm recovery rate among
tubules with a focus on the predictive value of the biopsy for
Klinefelter syndrome patients, while histological analyses revealed
successful sperm harvesting.28–31
Sertoli cell-only syndrome in 33% of men with Leydig cell hyper-
plasia and 67% with focal hypospermatogenesis. However, these
INDICATIONS FOR TESTICULAR BIOPSY
results should be interpreted carefully since single testis biopsy is
The following indications for a testicular biopsy should be considered:
not representative to the whole organ. Finally, the procedure used
. obstructive azoospermia: confirmation of the presence of normal may impact the outcome (microsurgical TESE was associated with
spermatogenesis may be desired before surgical correction of the higher sperm retrieval rate compared to the conventional TESE), as
obstruction is planned. This does not apply for men with a history will the experience of the laboratories in identifying testicular
of vasectomy or for men with congenital absence of the vas sperm.43

Table 2 Percentage of sperm retrieval rates (SRR) in men with non-obstructive azoospermia according to the histological diagnosis using
conventional TESE (cTESE) or microsurgical TESE (mTESE)
Study SRR (%)

Hypospermatogenesis Maturation arrest Sertoli cell-only syndrome

cTESE mTESE cTESE mTESE cTESE mTESE

33
Okada et al. 0 100 38 75 6 34
Ramasamy et al.40 50 81 20 44 29 41
Tsujimura et al.41 77 100 0 75 13 23

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TESTICULAR BIOPSY DURING ORCHIDOPEXY Indications for surgery were interval growth in two patients and
Cryptorchidism is the most prevalent congenital disorder of the patient preference in six. One patient underwent radical orchiect-
male genital tract with an incidence of 1% at the age of 1 year. omy for pure seminoma identified due to interval growth from 3 to
Cryptorchidism is associated with an elevated risk for male infertility, 6 mm after 3 months. The other seven masses excised with partial
especially for bilateral cases and testicular germ cell tumours.47–49 If orchiectomy were benign.58
left untreated during childhood, cryptorchidism will result in a From these studies it can be concluded that patients with small (less
progressive loss of germ cells. Orchidopexy is recommended in than 10 mm) non-palpable intratesticular lesions detected on ultra-
early childhood to ameliorate future fertility problems and to allow sound during evaluation of male infertility may benefit from periodic
palpation of the gonad for the early detection of testicular cancer. ultrasound examination. In the case of growth of the initial lesions
In boys with a history of cryptorchidism, the life-time risk of devel- or additional risk factors for testicular germ cell tumours (TGCTs),
oping a testicular malignancy ranges from 2% to 5%, depending on testicular biopsy should be considered.
the age of orchidopexy.50,51 CIS is the precursor cell of all testicular
germ cell malignancies and is already present at birth.23 This raises DETECTION OF CIS OF THE TESTIS
the question if a testicular biopsy should be performed routinely TGCTs are the most frequent malignancies in Caucasian males of
during orchidopexy. Boys who are operated before the age of 12 20–34 years of age.59 The disease specific survival of TGCTs in
years have a risk of 2% for developing a testicular tumour later in Europe is 97% at 1 year and 93% at 5 years, the highest survival
life. If, however, orchidopexy is performed during or after puberty, rate for any malignant tumour in men.60 Despite this high cure
the risk for developing a testicular tumour is 5%.51 Therefore, a rate, the annual increase in incidence of 2%–5% in Western
testicular biopsy is recommended in adolescents with cryptorchid- European countries is a major concern.61 Twenty-three per cent
ism for detection of CIS of the testis.3 of the men presenting a TGCT have metastases and need radio-
In a group of patients who underwent testicular biopsy at the time of therapy or chemotherapy with potentially serious side effects,
orchidopexy for correction of crytorchodism, Hadziselimovic et al.51 including infertility.62
observed that boys with unilateral crytorchidism and Ad spermatogo- TGCTs, including seminomas and non-seminomas, have CIS as
nia detected in their testicular tissue had seven times higher sperm common precursor.63 CIS develops into invasive malignancy in
concentrations later in life compared to those with no Ad spermato- 70% of the cases in 7 years and presumably in all patients over a
gonia, irrespective of the age of surgical repair. Similar findings were longer period of time.64 CIS can be successfully treated with a low
reported in boys with bilateral cryptorchidism. The authors concluded dose of radiotherapy or unilateral orchidectomy. Radiation therapy
that boys with cryptorchidism lacking Ad spermatogonia will develop or orchidectomy prevents progression from CIS to an invasive
infertility despite successful surgical treatment at any age. Also, they TGCT and is likely to cure the patient.64 This avoids adjuvant
conclude that testicular biopsy during orchidopexy is of prognostic chemotherapy and radiotherapy and the risk of development of
value for future fertility and should result in hormonal treatment after long-term complications. After chemotherapy for testicular germ
the operation to increase the number of germ cells.52 However, hor- cell cancer, there is an increased risk for cardiovascular diseases,
monal treatment of boys with cryptorchidism could also result in germ pulmonary fibrosis, nephrotoxicity and secondary malignancies.65
cell apoptosis and is therefore not recommended.53 Therefore, efforts should be made to diagnose TGCT at the pre-
invasive stage.66 CIS can only be diagnosed by testicular biopsy,
TESTICULAR BIOPSY AND NON-PALPABLE TESTICULAR although we recently demonstrated CIS cells in semen using OCT
LESIONS 3/4 immunohistochemistry.67 OCT 3/4 staining allows rapid iden-
Scrotal ultrasound is a valuable tool in the evaluation of the testis and tification of CIS cells and is highly sensitive and specific. The per-
adjacent structures and is often used routinely in male infertility dia- centage of false-negative biopsies is estimated as low as 0.5%. This
gnostic work-up. Using ultrasound allows detection of several intra- is supposedly due to the equal distribution of CIS throughout the
testicular abnormalities, such as microlithiasis and lesions of the testis. However, a growing number of studies and case reports show
gonads. Both conditions may be associated with testicular cancer.38 that CIS may also be present as a focal lesion in the testis and can
Palpable lesions and large testicular lesions are likely to be malignant result in a false-negative biopsy.59,68
with a probability as high as 95%. Non-palpable lesions may be present Of all patients with unilateral testicular tumours, approximately
in as many as a third of patients with severe male factor infertility.54 2%–5% harbour CIS in their contralateral testis that will progress
The aetiology of these lesions differs among studies, with some report- into an invasive germ cell tumour over time. Controversies exists
ing a high proportion of malignancy, whereas other found the major- whether a routine contralateral biopsy should be performed in all
ity of lesions to be benign.55–57 men who are diagnosed with testicular cancer as it should only be
Eifler et al.54 reviewed a total of 145 men who presented with offered if it provides a clear health benefit for the patient. Treating
severe infertility and underwent ultrasound examination. Among CIS in the contralateral testis will result in irreversible infertility and
those, 49 were found to have abnormalities on ultrasound. Only in approximately 20% cases, it will also cause hypogonadism. An
one patient appeared to have a seminoma with intratesticular lesion alternative could be active surveillance for men who have been
greater than 10 mm, and all other lesions were found to be benign treated for TGCTs. According to the recommendations of the
with mean size ranging from 5 to 10 mm. Toren et al.58 followed 46 European Germ Cell Cancer Consensus Group, a testicular biopsy
men with mean age of 35 years with ultrasound for 1 year with a might be offered to high-risk patients for detecting contralateral
mean number of ultrasounds of 2.8. The mean lesion diameter was CIS, i.e., patients with a testicular volume less than 12 ml, men
4.3 mm (range: 1–10 mm). Among the 46 men, 38 patients showed with a history of cryptorchidism and in the case of ultrasonographic
a mean growth of the intratesticular lesions of 0.5 mm abnormalities.18
per year. Three patients underwent immediate surgery and five Non-invasive test for the detection of CIS of the testis may open the
underwent surgery following a period of ultrasound follow-up. possibility of screening men with an increased risk of TGCTs.38,67 This

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applies to men with infertility, a history of cryptorchidism and tes- testicular biopsies, those performed for TESE, as well as those for
ticular tumour, and men with atrophic testes. Ultrasonographic assessing spermatogenic status, should be evaluated for the presence
abnormalities of the testis further increase the risk of CIS and of CIS.
TGCTs and warrant testicular histology.
There is a wide global discrepancy in testicular cancer incidence COMPETING FINANCIAL INTERESTS
rates. Testicular cancer remains a disease predominantly of men des- The authors declare no competing financial interests.
cended from European populations, in particular, Northern European
populations with incidence rates that are almost twice more than that
of the United States and Asian countries.69 This also accounts for the
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