Human Reproduction Vol.16, No.10 pp. 2154–2159, 2001
Percutaneous cutting needle biopsies for histopathological
assessment and sperm retrieval in men with azoospermia
Björn Rosenlund1,4, Ulrik Kvist2, Leif Plöen2,3, Urban Ekström2 and Outi Hovatta1
1Department
of Obstetrics and Gynaecology, Huddinge University Hospital, Andrology Centre, Department of Woman and Child
Health, 2Karolinska Hospital, Stockholm and 3Department of Anatomy and Histology, Swedish University of Agricultural Sciences,
Uppsala, Sweden
4To
whom correspondence should be addressed. E-mail: [email protected]
BACKGROUND: Twenty-three men (45 testes) with azoospermia underwent percutaneous testicular biopsy under
local anaesthesia. METHODS: In all but one of the 45 testes two biopsies were taken close to each other, one with
a 16 gauge (n ⍧ 44) and another with a 14 gauge (n ⍧ 45) cutting needle, both with a 19 mm notch. Three
quarters of the tissue was used for histopathological assessment and one quarter for direct microscopy. RESULTS:
The histopathological findings were similar between the two needles. The observations with direct microscopy
corresponded with the histopathological assessments concerning the presence of mature spermatids in 41 of 45
(91%) biopsies using the 14 gauge and in 40 of 44 (91%) biopsies using the 16 gauge needle. There were no postoperative complications except for minimal pain and minor local swelling. CONCLUSIONS: Percutaneous material
retrieved using 16 gauge and 14 gauge needles is sufficient for histopathological assessment, and the two needles
are equally reliable for testicular sperm retrieval. However, needle biopsy with one puncture may not be
representative of the entire testis.
Key words: azoospermia/histopathological diagnosis/percutaneous needle biopsy/spermatogenesis
Introduction
Open testis biopsy is recognized as being the standard
procedure for obtaining testicular tissue for assessment of
spermatogenesis. Recently, percutaneous needle biopsy has
also been used for histopathological assessment (Cohen and
Warner, 1987; Rajfer and Binder, 1989), and for testicular
extraction of spermatozoa for intracytoplasmic sperm injection
(ICSI) (Bourne et al., 1995; Hovatta et al., 1995). Two different
types of percutaneous needles have been used, cutting needles
and aspiration needles. The cutting type was used in a study
by Kessaris et al. (1995) where the biopsy was performed
with an ASAP 18 core biopsy system (18 gauge with 17 mm
notch). They found a 95% correlation between percutaneous
and open biopsy regarding the histopathological diagnosis.
Studies comparing percutaneous aspiration needles with open
testicular biopsy for histopathological assessment gave less
correlation (Mallidis and Baker, 1994; Rosenlund et al., 1998).
The aim of this study was to compare the biopsies taken with
two cutting needles, 14 gauge and 16 gauge, and to see if they
can be used for histopathological assessment of spermatogenesis and for presence of mature spermatids.
Twentyone men had non-obstructive azoospermia and two showed
clinical signs of obstructive azoospermia. The testicular volumes,
determined by orchidometry, ranged from 2–25 ml. Two men had
karyotype 47,XXY and the other 21 had normal karyotype, 46,XY.
All biopsies were performed under local anaesthesia with a preoperative injection of 0.5 mg alfentanilum (Rapifen®; Janssen-Cilag,
Birkerod, Denmark) and 2.5 mg midazolam (Dormicum®; Roche,
Basel, Switzerland). The scrotal area was washed with 0.5%
chlorhexidine solution followed by physiological saline and then
draped. Seven millilitres of prilocaine hydrochloride (Citanest®
10 mg/ml, Astra; Södertälje, Sweden) was injected around the vas
deferens as described by Li et al. (1992), using a 22 gauge, 5 cm
needle (Microlance 3®, 22G 0.7⫻50 mm; Becton Dickinson, Dublin,
Ireland). Additionally, 1 ml prilocaine hydrochloride was injected
into the scrotal skin. The testis was grasped between the thumb and
forefinger of the non-dominant hand and rotated ventrally to prevent
epididymal injury. The needle was inserted into the cranial pole
towards the centre of the testicle. Two biopsies were taken close to
each other, first with a 14 gauge (n ⫽ 45) and then with a 16 gauge
(n ⫽ 44) needle (Bard MAGNUM Biopsy Instrument, C.R.Bard Inc.,
Covington, GA, USA), both with a 19 mm notch. Three quarters of
the testicular material was fixed and plastic-embedded for histopathological assessment, and one quarter reserved for direct microscopy.
Evaluation of testicular biopsy specimens was as described by
Rosenlund et al. (Rosenlund et al., 1998).
Materials and methods
Twentythree men, aged 27–45 years, underwent percutaneous
testicular biopsy as part of an infertility investigation. Both testes in
all but one man were investigated, hence a total of 45 testes.
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Results
The amount of tissue retrieved was sufficient for histopathological assessment in nearly all biopsies. In biopsies
© European Society of Human Reproduction and Embryology
Percutaneous testicular biopsy in men with azoospermia
Table I. Histopathological evaluation of spermatogenesis and observations of presence of testicular spermatozoa or elongated spermatids in testicular biopsies
from 23 men obtained by percutaneous 14 gauge and 16 gauge cutting needle biopsies. R ⫽ right, L ⫽ left (main finding from assessment of spermatogenesis
is given in the first line, deviations in brackets in the second line).
Pat. no.
1
2
3
4
5
6
XXY
Testis (R/L)
volume (ml)
R
20
L
20
R
12
L
10
R
12
L
10
R
18
L
15
R
15
L
12
R
8
L
8
7
R
20
L
20
8
R
12
L
12
9
10
11
R
12
L
12
R
8
L
6
R
10
L
10
12
R
8
Histopathological evaluation of spermatogenesis
Presence of testicular spermatozoa or elongated spermatids
14 gauge
16 gauge
14 gauge histop. eval./
direct microscopy
16 gauge histo. eval./
direct microscopy
No germ cells
No germ cells
no/no
no/no
No germ cells
(cut rete testis)
No germ cells
(few Sertoli cells)
No germ cells
(few Sertoli cells)
No germ cells
(few Sertoli cells)
No germ cells
(few Sertoli cells)
Partial disruption
of spermatogenesis
(cut artery and right testis)
Partial disruption
of spermatogenesis
No germ cells
No germ cells
no/no
no/no
No germ cells
(few Sertoli cells)
No germ cells
(few Sertoli cells)
No germ cells
(few Sertoli cells)
No germ cells
(few Sertoli cells)
Partial disruption
of spermatogenesis
no/no
no/no
no/no
no/no
no/no
no/no
no/no
no/no
yes/yes
yes/yes
Partial disruption
of spermatogenesis
No germ cells
yes/yes
yes/yes
no/no
no/no
No germ cells
no/no
no/no
No germ cells
(a spermatogonium
in a single tubule)
No germ cells
(a spermatogonium
in a single tubule)
No germ cells
No germ cells
(degenerated Sertoli cells and a (degenerated Sertoli
cut artery)
cells)
No germ cells
No germ cells
(degenerated
(degenerated
Sertoli cells)
Sertoli cells)
No germ cells
No germ cells
(a mature spermatid)
(round spermatids)
in a single tubule)
in a single tubule)
No germ cells
No germ cells
(a round spermatid
in a single tubule and
a cut artery)
No germ cells
No germ cells
yes/yes
no/yes
no/no
no/no
no/no
no/no
no/no
no/yes
yes/yes
no/no
no/no
no/no
no/no
no/no
No germ cells
No germ cells
no/no
no/no
No germ cells
No germ cells
no/no
no/no
No germ cells
(cut rete testis)
No germ cells
(disruption of
spermiogenesis
in a few tubules)
No germ cells
(disruption of
spermiogenesis
in a few tubules
and a cut artery)
No germ cells
(disruption of
spermiogenesis
in a few tubules)
No germ cells
no/no
no/no
No germ cells
(partial disruption of
spermatogenesis
in a few tubules)
No germ cells
(partial disruption of
spermatogenesis
in a few tubules
and a cut artery)
No germ cells
(disruption of
spermiogenesis
in a few tubules and
extravasal
erythrocytes)
no/yes
yes/yes
no/yes
yes/yes
no/yes
no/yes
No germ cells
No germ cells
(spermatogenesis
in a single tubule)
No germ cells
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B.Rosenlund et al.
Table I. Continued
Pat. no.
Testis (R/L)
volume (ml)
L
8
13
XXY
R
2
L
2
14
15
16
R
10
L
10
R
20
L
20
R
12
L
8
17
18
19
20
21
22
23
R
10
L
10
R
20
L
20
R
20
L
15
R
12
L
12
L
25
Histopathological evaluation of spermatogenesis
Presence of testicular spermatozoa or elongated spermatids
14 gauge
16 gauge
14 gauge histop. eval./
direct microscopy
16 gauge histo. eval./
direct microscopy
No germ cells
(disruption of
spermiogenesis
in a few tubules)
No tubules
identified, abundance of
Leydig cell (cut artery)
No tubules
identified,
abundance
of Leydig cells
(cut artery)
No germ cells
No germ cells
(disruption of
spermatocytogenesis)
no/yes
no/yes
No biopsy
no/no
-/-
No germ cells
in the half
tubule
identified
no/no
no/no
No germ cells
no/no
no/no
No germ cells
(extravasal
erythrocytes)
Partial disruption of
spermatogenesis
Partial disruption of
spermatogenesis
No germ cells
no/no
no/no
yes/yes
yes/yes
yes/yes
yes/yes
no/no
no/no
no/no
no/no
no/no
no/no
no/no
no/no
No germ cells
Partial disruption of
spermatogenesis
Partial disruption of
spermatogenesis
No germ cells
(disruption of meiosis in
a few tubules)
No germ cells
No germ cells
No germ cells
(extravasal
erythrocytes)
No germ cells
(cut artery)
No germ cells
Partial disruption of
spermatogenesis
Partial disruption of
spermatogenesis
Disruption of
spermiogenesis
Disruption of
spermatocytogenesis
No germ cells
(spermatogonia
in single tubules)
No germ cells
Partial disruption of
spermatogenesis
Partial disruption of
spermatogenesis
Disruption of
spermiogenesis
Disruption of
spermatocytogenesis
No germ cells
(spermatogonia
in single tubules)
No germ cells
yes/yes
yes/yes
yes/yes
yes/yes
no/no
no/no
no/no
no/no
no/no
no/no
no/no
no/no
Normal
spermatogenesis
yes/yes
yes/yes
yes/yes
yes/yes
yes/yes
yes/yes
no/no
no/no
no/no
no/no
No germ cells
R
20
L
20
Normal
spermatogenesis
Normal
spermatogenesis
R
8
L
8
Fibrotic tubules
Normal
spermatogenesis
(extravasal
erythrocytes)
Normal
spermatogenesis
Normal
spermatogenesis
(extravasal
erythrocytes)
Fibrotic tubules
Fibrotic tubules
Fibrotic tubules
where tubules could be identified, the median numbers of
tubular sections were 35 and 20 with the 14 gauge and 16
gauge needles respectively. An overview of the results is
presented in Table I.
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The results of the histopathological assessments were
identical in 35 testes irrespective of the needle size used, 14
gauge or 16 gauge. In nine testes (patients 6, 8, 11, 12, 13
and 16) the major findings were identical but some discrepan-
Percutaneous testicular biopsy in men with azoospermia
Figure 1a. Patient 21, left testis; 16 gauge needle biopsy showing normal spermatogenesis and extravasal erythrocytes (section stained with
toluidine blue). (b) Patient 13 (karyotype 47,XXY), left testis. In this 16 gauge needle biopsy half a tubule which contained only Sertoli
cells was found. Note the abundance of Leydig cells in the interstitium (sections stained with toluidine blue). (c) and (d) Patient 11, left
testis; 16 gauge needle biopsy where the main finding was tubules lacking germ cells (c). A few tubules revealed spermatogenesis although
quantitatively reduced (d) (sections stained with haematoxylin). (e) Patient 6 (karyotype 47,XXY), right testis; 14 gauge needle biopsy. The
main finding was tubules lacking germ cells. One tubule out of 30 contained mature spermatids (section stained with haematoxylin).
cies were noted between the results using the two needles (as
occurred in patient 13, Figure 1b), and also the single 16
gauge needle, as occurred in patient 11, left testis, Figure 1c
and 1d.
Presence of mature spermatids/testicular spermatozoa—
direct microscopy versus histopathological assessment
Direct microscopic observations corresponded well with the
histopathological assessment concerning the presence of mature
2157
B.Rosenlund et al.
spermatids/testicular spermatozoa in 41 of 45 (91%) of the 14
gauge biopsies and in 40 of 44 (91%) 16 gauge biopsies.
Discrepancies were found in patients 6, 7, 11 and 12 where
direct microscopy showed testicular spermatozoa, but the
histopathological assessment showed a disruption of spermatogenesis at various stages, with round spermatids being the
most advanced germ cells observed.
Presence of testicular spermatozoa in direct microscopy—14
gauge versus 16 gauge biopsies
Presence or absence of testicular spermatozoa was assessed in
direct microscopy with full agreement between the two needle
size biopsies in 42 of 44 (95%) paired biopsies. Discrepancy
was found between direct microscopy in two patients (patient
7 and 8) where spermatozoa were found only in the 16 gauge
and 14 gauge biopsies respectively.
Presence of mature spermatids in testicular sections—14
gauge versus 16 gauge biopsies
Full agreement concerning presence or absence of mature
spermatids between 14 gauge and 16 gauge biopsies was found
in 40 of 44 (91%) paired biopsies. Discrepancies were found
in four testes (patients 6, 8 and 11) where mature spermatids
were only found in two testes with the 14 gauge needle and
in two other testes with the 16 gauge needle.
In the 89 performed biopsies small cut arteries were seen
in nine cases and extravasal red blood cells were observed in
five cases (Figure 1a). The latter were all seen in the second
(16 gauge) biopsy.
Follicle stimulating hormone (FSH) concentration in serum
of the 23 men ranged between 2 and 68 IU/l. The two highest
FSH values, 68 and 42 IU/l, were from men with severely
impaired spermatogenesis. However, the third highest value
(36 IU/l, patient 6) was from a man with karyotype 47,XXY,
who revealed focal spermatogenesis (one out of 30 tubules
assessed, Figure 1e).
All operations were performed on an outpatient basis under
local anaesthesia and were well tolerated by the patients, who
were all discharged from the clinic within 3 h. There were no
post-operative complications except for minor pain and minor
subcutaneous swelling.
Discussion
In a previous study investigating men with non-obstructive
azoospermia (Rosenlund et al., 1998), we found that testicular
material obtained by percutaneous biopsies using 21 gauge
and 19 gauge aspirating needles was not sufficient for histopathological assessment of spermatogenesis in most cases
compared to biopsies obtained by open surgery. Mallidis and
Baker (1994), using a 20 gauge aspiration needle, reported
56% full agreement in the histological diagnosis comparing
open biopsy and tissue aspiration (47 testes) (Mallidis and
Baker, 1994).
In this present study, using 16 gauge and 14 gauge cutting
needles, all specimens retrieved by a single biopsy were
sufficient for histopathological assessment of spermatogenesis,
even though the number of tubular cross-sections was lower
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using the thinner 16 gauge needle. The efficiency of a cutting
needle biopsy for testicular histopathological evaluation has
also been demonstrated by Kessaris et al. (1995), who found
a 95% correlation between percutaneous and open biopsy
regarding the histopathological diagnosis (Kessaris et al.,
1995). However, the diagnosis in their study was more heterogeneous than in our study, which mainly consisted of men
with non-obstructive azoospermia.
In the present study, one biopsy was taken using each sized
needle. The extent to which the material from one needle
biopsy is representative of the real testicular status could be
disputed. Silber et al. (1997) claimed that the distribution
of spermatogenesis in the testicles of azoospermic men is
homogeneous (Silber et al., 1997). They suggested that a
single biopsy is sufficient to diagnose the presence of
spermatozoa based on the assumption that multi-focal
distribution of spermatogenesis throughout the entire testes is
present in non-obstructive azoospermia. Other authors
(Tournaye et al., 1996; Ezeh et al., 1998; Hauser et al., 1998;
Amer et al., 1999) have recommended multiple biopsies to
enhance diagnostic accuracy. In our study, the findings in the
two biopsies taken from each testis were generally the same,
but in eight out of the 44 (18%) paired observations from the
same testis there were slight discrepancies. These observations
show that spermatogenesis is not always homogeneously
distributed throughout the testicular parenchyma. Thus, only
one puncture with a cutting needle is not representative of the
entire testis in some azoospermic men. However, it has
been postulated that multiple testicular biopsies with multiple
incisions in the tunica albuginea may result in interrupting a
sufficient proportion of testicular arteries to cause permanent
testicular injury (Schlegel and Su, 1997) and post-sampling
fibrosis (Tournaye et al., 1997). In addition, excision of
multiple testicular biopsy samples can result in long time
impairment of testosterone production (Manning et al., 1998).
It has been suggested that open biopsy is safer than needle
biopsy as the arteries can be identified during open testicular
biopsy (Schlegel and Su, 1997). Being a blind procedure, there
has been a fear that percutaneous needle biopsy may cause
vascular injury. In our study we found signs of microscopic
bleeding (extravasal red blood cells or cut arteries) in 14 of
the total 89 (16%) biopsies. However, there were no clinical
complications except for minimal pain and minor subcutaneous
swelling. We did not carry out any post-operative ultrasound
screening. Intratesticular bleeding was reported in the 30 min
following the biopsy, observed sonographically as a hypoechoic
region in the testicular parenchyma (Harrington et al., 1996).
This was observed in only 7% (four of 58) of the percutaneous
biopsies performed with an 18 gauge Microvasive biopsy
needle with a notch of 17 mm (two biopsy specimens from
each testicle). In contrast, the proportion of open testis biopsies
resulting in signs of testicular bleeding was 29% (10 out of
34). It appears that a needle biopsy using a cutting needle could
reduce the risk of post-operative intratesticular haematoma
compared with open biopsy. It is also likely that the needle
diameter and the number of needle biopsies affect postoperative bleeding.
It has been suggested the use of an operation microscope
Percutaneous testicular biopsy in men with azoospermia
to examine the surface of the testis for subtunical vessels
before making biopsy incisions (Schlegel and Su, 1997).
However, this technique does not exclude the risk that tiny
arteries may accidentally be cut during this form of open
surgery (Tuuri et al., 1999).
To conclude, percutaneous testicular needle biopsy using 16
gauge and 14 gauge cutting needles to investigate men with
non-obstructive azoospermia is a simple and relatively costeffective procedure. It can be carried out under local anaesthesia
on an outpatient basis. The material retrieved by this method is
sufficient for histopathological assessment of spermatogenesis.
Needle biopsies can also be used for cryopreservation (Tuuri
et al., 1999). The two needles seem to be equally reliable
for testicular biopsy for histopathological evaluation and for
testicular sperm retrieval.
References
Amer, M., Haggar, S., Moustafa, T. et al. (1999) Testicular sperm extraction:
impact of testicular histology on outcome, number of biopsies to be
performed and optimal time for reception. Hum. Reprod., 14, 3030–3034.
Bourne, H., Watkins, W., Speirs, A. and Baker, H.W.G. (1995) Pregnancies
after intracytoplasmic injection of sperm collected by fine needle biopsy of
the testis. Fertil. Steril., 64, 433–436.
Cohen, M.S. and Warner, R.S. (1987) Needle biopsy of testes: a safe outpatient
procedure. Urology, 29, 279–281.
Ezeh, U.I.O, Moore, H.D.M. and Cooke, I.D. (1998) A prospective study of
multiple needle biopsies versus open biopsy for testicular sperm extraction
in men with non-obstructive azoospermia. Hum. Reprod., 13, 3075–3080.
Harrington, T.G., Schauer, D. and Gilbert, B. (1996) Percutaneous testis
biopsy: an alternative to open biopsy in the evaluation of the subfertile
man. J. Urol., 156, 1647–1651.
Hauser, R., Botchon, A., Amit, A. et al. (1998) Multiple testicular sampling
in non-obstructive azoospermia—is it necessary. Hum. Reprod., 13, 3081–
3085.
Hovatta, O., Moilanen, J., von Smitten, K. and Reima, I. (1995) Testicular
needle biopsy, open biopsy, epididymal aspiration and intracytoplasmic
sperm injection in obstructive azoospermia. Hum. Reprod., 10, 2595–2599.
Kessaris, D.N., Wasserman, P. and Mellinger, B.C. (1995) Histopathological
and cytopathological correlations of percutaneous testis biopsy and open
testis biopsy in infertile men. J. Urol., 153, 1151–1155.
Li, P.S., Li, S., Schlegel, P.N. and Goldstein, M. (1992) External spermatic
sheath injection for vasal nerve block. Urology, 39, 173–176.
Mallidis, C. and Baker, H.W.G. (1994) Fine needle tissue aspiration biopsy
of the testis. Fertil. Steril., 61, 367–375.
Manning, M., Junemann, K-P. and Alken, P. (1998) Decrease in testosterone
blood concentrations after testicular sperm extraction for intracytoplasmic
sperm injection in azoospermic men. Lancet, 352, 37.
Rajfer, J. and Binder, S. (1989) Use of biopsy gun for transcutaneous testicular
biopsies. J. Urol., 142, 1021–1022.
Rosenlund, B., Kvist, U., Plöen, L. et al. (1998) A comparison between open
and percutaneous needle biopsies in men with azoospermia. Hum. Reprod.,
13, 1266–1271.
Schlegel, P.N. and Su, L-M. (1997) Physiological consequences of testicular
sperm extraction. Hum. Reprod., 12, 1688–1692.
Silber, S.J., Nagy, Z.P., Devroey, P. et al. (1997) Distribution of spermatogenesis
in the testicles of azoospermic men: the presence or absence of spermatid
in the testes of men with germinal failure. Hum. Reprod., 12, 2422–2427.
Tournaye, H., Liu, J., Nagy, P.Z. et al. (1996) Correlation between testicular
histology and outcome after intracytoplasmic sperm injection using testicular
spermatozoa. Hum. Reprod., 11, 127–132.
Tournaye, H., Verheyen, G., Nagy, Z.P. et al. (1997) Are there any predictive
factors for successful testicular sperm recovery? Hum. Reprod., 12, 80–86.
Tuuri, T., Moilanen, J., Kaukoranta, S. et al. (1999) Testicular biopty gun
needle biopsy in collecting spermatozoa for intracytoplasmic injection,
cryopreservation and histology. Hum. Reprod., 14, 1274–1278.
Received on March 7, 2001; accepted on June 5, 2001
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