Human Reproduction vol.13 no.5 pp.1266–1271, 1998
A comparison between open and percutaneous needle
biopsies in men with azoospermia
B.Rosenlund 1,6, U.Kvist3, L.Plöen4, B.Lundh
Rozell2, P.Sjöblom1 and T.Hillensjö5
1Department
of Obstetrics and Gynaecology, 2Department of
Clinical Pathology and Cytology, Huddinge University Hospital,
S-141 86 Huddinge, 3Reproductive Medicine Centre, Department of
Woman and Child Health, Karolinska Hospital, Stockholm,
4Department of Anatomy and Histology, Swedish University of
Agricultural Sciences, Uppsala and 5Fertility Center Scandinavia,
Göteborg, Sweden
6To
whom correspondence should be addressed
Open testicular biopsy is a classic method of investigation
in men with azoospermia. Recently, percutaneous needle
biopsy of the testis has been used in attempts to obtain
material for histopathological diagnosis in such cases and to
retrieve spermatozoa for intracytoplasmic sperm injection
(ICSI). To determine whether a 19 gauge (G) and a 21G
butterfly needle could be used for percutaneous aspiration
of testicular tissue to determine the presence of mature
spermatids and assess spermatogenesis, 10 patients (16
testes) and 12 patients (17 testes) underwent 19G or 21G
needle biopsy respectively, immediately followed by open
testicular biopsy, with both procedures under local anaesthesia. Biopsy with each needle size was compared with
open biopsy. With the 19G needle, in the 14 cases where
material was obtained there was full agreement with open
biopsy regarding the presence or absence of mature spermatozoa, whereas with the 21G needle only nine of the 13
biopsies yielding material were predictive in this respect.
Each needle size correlated poorly with open biopsy regarding evaluation of spermatogenesis. We conclude that percutaneous biopsy with a 19G butterfly needle is a quick and
reliable method for demonstrating spermatozoa for ICSI.
But for a detailed histopathological diagnosis, however, the
needle biopsies gave poor results, whereas the material
from the open testicular biopsies was assessable.
Key words: azoospermia/histopathological diagnosis/open testicular biopsy/percutaneous testicular biopsy/spermatogenesis
Introduction
Testicular biopsies have mainly been performed in azoospermic
men with a normal testicular size and normal serum levels of
follicle stimulating hormone (FSH) to find out whether the
spermatogenesis is normal or impaired or whether established
azoospermia can be attributed to occlusions in the efferent duct
system. Two new lines in the investigation and management of
the infertile man have broadened the indications for testicular
biopsy. One is the new molecular approach to the understanding
1266
of how chromosome deletions (especially Y-derived) are related
to spermatogenesis (Elliot et al., 1997). This necessitates a
detailed evaluation of the process of spermatogenesis. A
recently introduced therapeutic approach is to use testicular
biopsy to obtain motile spermatozoa to treat infertility with
intracytoplasmic sperm injection (ICSI) (Craft et al., 1993;
Schoysman et al., 1993). In these cases men with small
testicular volumes and men with elevated serum FSH levels
have also come into consideration for testicular biopsy (Silber
1995; Tournaye et al., l996). Traditionally, open testis biopsy
has been the standard procedure for obtaining testicular tissue
for histopathological diagnosis. In the last decade percutaneous
needle biopsy has been used as a diagnostic tool (Cohen and
Warner, 1987; Rajfer and Binder, 1989) and also for testicular
extraction of spermatozoa for ICSI (Bourne et al., 1995;
Hovatta et al., 1995). There are no unequivocal results indicating the superiority of any particular technique – open biopsy
or different forms of needle biopsy.
The aim of this study was to address the following two
questions: Can testicular spermatozoa be aspirated with 21
gauge (G) or 19G butterfly needles? Can the different phases
of spermatogenesis be evaluated in the aspirated tissue? Open
testicular biopsies obtained immediately after needle aspiration
served as controls.
Materials and methods
During the period December 1996 to April 1997 22 men, aged 23 to
48 years, underwent a total of 33 percutaneous testicular needle
biopsies, immediately followed by an open biopsy as part of infertility
investigation. All men had azoospermia and their testicular volumes,
determined by orchidometry, ranged from 8–25 ml. No patient showed
clinical signs of obstruction (distended epididymides or absence of
vas deferens). During the first part of the study a 21G butterfly needle
[21G3/4, 0.81319 mm, Japan Medical Supply (S)PTE Ltd, Singapore]
was used in 17 procedures (five bilateral and seven unilateral), group
21G, and in the second part of the study a 19G butterfly needle
[19G3/4, 1.08319 mm, Japan Medical Supply (S)PTE Ltd] was used
in 16 procedures (six bilateral and four unilateral), group 19G.
All biopsies were performed under local anaesthesia with a preoperative injection of morphine (Oxikodon-skopolamin-hydrobromid 8
mg 1 0.4 mg; Apoteksbolaget, Umeå, Sweden). The scrotal area was
washed with 0.5% chlorhexidine solution followed by physiological
saline and draped. Seven millilitres of prilocaine hydrochloride
(Citanest® 10 mg/ml; Astra, Södertälje, Sweden) was infiltrated
around the vas deferens as described by Li et al. (1992), using a
22G, 5 cm needle (Microlance®3, 22G2 0.7350 mm; Becton
Dickinson, Dublin, Ireland). Additionally, 1 ml prilocaine hydrochloride was infiltrated into the scrotal skin. The procedure was
repeated on the other side in patients who underwent bilateral biopsy.
The testis was grasped between the thumb and forefinger of the non© European Society for Human Reproduction and Embryology
Percutaneous needle biopsies in azoospermia
dominant hand. To prevent epididymal injury, the testis was displaced
anteriorly. Two to four percutaneous punctures were performed with
the butterfly needle in the upper pole of the testis. The needle was
attached to a 20 ml (Plastipak®; Becton Dickinson, Drogheda, Ireland)
syringe and a continuous negative pressure was applied during each
puncture while the needle was being moved and directed in different
areas. For the open biopsy an incision was then made over the same
area immediately after the percutaneous biopsy. The size of the open
surgery biopsy was about 33336 mm. All patients left the clinic
within 4 h of the operation.
Evaluation of the testicular biopsy specimens
The biopsy specimens were fixed in 4% formaldehyde in isotonic
phosphate buffer, pH 7.0, dehydrated and embedded in 2-hydroxyethyl
methacrylate
(Technovite®7100;
Heraeus
Kulzer
GmbH,
Friedrichsdorf, Germany). Sections, approximately 3 µm thick, were
stained with haematoxylin and eosin and examined under the light
microscope.
Several scoring systems have been described in the literature, but
none of these was suitable for the present study, in which the
comparison between percutaneous needle biopsy and open surgery
biopsy was the principal aim, together with an evaluation of the value
of needle biopsies in predicting the presence or absence of testicular
spermatozoa and/or of late, fully condensed spermatids.
In each case we made an assessment of where, along the process,
spermatogenesis was disrupted.
Spermatogenesis can be subdivided into three phases: spermatocytogenesis, mitotic proliferation of diploid spermatogonia resulting
in a species specific number of primary spermatocytes followed by
a short premeiotic interphase, during which the last DNA synthesis
takes place; meiosis, consisting of a long meiotic prophase and the
two meiotic divisions, the first resulting in secondary spermatocytes,
which almost immediately undergo the second meiotic division
resulting in round spermatids; and spermiogenesis, the differentiation
of a round spermatid into an elongated spermatid with condensed
chromatin, which is released from the epithelium on spermiation.
When the mature spermatid is shed at spermiation, it is called a
testicular spermatozoon.
The testicular spermatozoa flow with the seminiferous tubular fluid
produced by the Sertoli cell via the rete testis and efferent ductules
into the epididymal duct.
The observations were classified as follows: disrupted spermatogenesis, where all developmental stages are seen but where spermatogenesis is quantitatively reduced, and degenerating cells of various
developmental stages are present; disrupted spermatocytogenesis,
where the production of spermatogonia, and hence of primary
spermatocytes, is impaired; disrupted meiosis, where there are primary
spermatocytes but spermatids are lacking. A high number of degenerated (necrotic) spermatocytes indicate that the meiotic divisions are not
taking place, hence the lack of spermatids; disrupted spermiogenesis,
where spermatids are formed, but differentiate abnormally or degenerate; complete absence of germ cells with only Sertoli cells present
in the epithelium.
The two extremes, normal spermatogenesis and complete absence
of germ cells, often designated Sertoli-cell-only syndrome, are easily
assessed, whereas disruption or partial disruption of one or more of
the phases of spermatogenesis requires more material for correct
interpretation. The presentation of the histopathological observations
is kept as short as possible, and we have tried to classify all biopsies
into a limited number of groups. When there were insufficient amounts
of mechanically unaltered tissue in the specimens they were classified
as unevaluable for a histopathological diagnosis.
The study was approved by the local ethics committee.
Results
An overview of the results is presented in Tables I and II.
In group 21G, the material from 16 of 17 open biopsies
could be properly evaluated, but the remaining open biopsy
was technically unsatisfactory (Table I). Among the needle
biopsies, four yielded no material for embedding and none of
the remaining 13 could be properly evaluated on account
of poor technical specimen quality, mainly consisting of
mechanical damage to the seminiferous epithelium. This was
chiefly reflected in exfoliation of the seminiferous epithelium,
with a total loss of topographical relations between the developing germ cells. In the open biopsy specimens, elongated
spermatids and testicular spermatozoa were present in eight,
and lacking in nine. Among the eight cases in which elongated
spermatids and testicular spermatozoa were present, this observation was also made in two of the needle biopsies. In the
remaining six, there was no material in two cases and in four
no such cells were observed. In the nine open biopsy specimens
lacking elongated spermatids and testicular spermatozoa,
absence of such cells was also noted in the corresponding
material obtained with the 21G needle.
Group 19G comprised 16 pairs of biopsies from ten men
(Figures 1–3). All open biopsies, but only seven of the needle
biopsies, could be fully evaluated (Table II). In these seven
the results did not differ from those obtained by open biopsies.
In all needle biopsies yielding material for embedding (14 of
16), the presence or absence of elongated spermatids and
testicular spermatozoa could be demonstrated and was in
conformity with the findings in the open biopsies (Figure 2).
All operations were performed on an out-patient basis under
local anaesthesia and were well tolerated by the patients, who
were discharged within 4 h. There were no clinically significant
complications.
Discussion
New approaches in research, diagnosis and treatment of male
infertility have prompted the development of new methods for
testicular biopsies. Since the ICSI procedure is performed with
a single spermatozoon (Palermo et al., 1992), attempts to
obtain single spermatozoa by testicular biopsy may make
treatment possible. The role of biopsy has thus been expanded
to include patients with elevated FSH and small testes. Fertilization by a testicular spermatozoon was first reported by Craft
et al. (1993) and Schoysman et al. (1993). Testicular spermatozoa for ICSI have also been retrieved by percutaneous needle
biopsy in cases of obstructive azoospermia (Bourne et al.,
1995; Hovatta et al., 1995), and also by open biopsies in nonobstructive azoospermia (Devroey et al., 1995). Even round
spermatids extracted from testicular tissue have been used
successfully for fertilization (Fishel et al., 1995; Tesarik et al.,
1995). Thus, with a simple needle procedure the technical
requirements for treatment are overcome, and to answer the
question of whether motile elongated spermatids and testicular
spermatozoa are present, simple needle biopsy has been
advocated as the method of choice. If diagnostic measures
are minimized, we may be deprived of the possibility of
characterizing what we are actually treating. Men with severe
1267
B.Rosenlund et al.
Table I. Results of the histopathological evaluation of spermatogenesis and observations regarding the presence of testicular spermatozoa or elongated
spermatids in testicular biopsy specimens (from 12 men) obtained by open surgery and percutaneous needle aspiration with a 21 gauge butterfly needle
Patient
no.
21/1
21/2
21/3
21/4
21/5
21/6
21/7
21/8
21/9
21/10
21/11
21/12
Testis
Volume
(ml)
right
left
right
right
right
25
18
20
8
8
left
left
right
left
right
right
left
right
left
right
right
left
10
12
10
12
15
20
20
15
15
25
12
12
Histopathological evaluation of spermatogenesis
Presence of testicular spermatozoa or elongated spermatids
Open biopsy
Needle biopsy
Open biopsy
Needle biopsy
not possible
partial meiotic disruption
no germ cells
no germ cells
partial spermatogenic, complete
spermiogenic disruption
partial spermatogenic disruption
no germ cells
no germ cells
no germ cells
partial meiotic disruption
partial spermatogenic disruption
partial spermatogenic disruption
partial spermatogenic disruption
partial spermatogenic disruption
partial meiotic disruption
partial spermatogenic disruption
spermatogenic disruption
not possible
not possible
no material
not possible
not possible
yes
no
no
no
no
yes
no
no
no
not possible
no material
not possible
not possible
not possible
no material
no material
not possible
not possible
not possible
not possible
not possible
yes
no
no
no
no
yes
yes
yes
yes
yes
yes
no
no
no
no
no
no
yes
no
no
no
Table II. Results of the histopathological evaluation of spermatogenesis and observations regarding the presence of testicular spermatozoa or elongated
spermatids in testicular biopsy specimens (from 10 men) obtained by open surgery and percutaneous needle aspiration with a 19 gauge butterfly needle
Patient
no.
19/1
19/2
19/3
19/4
19/5
19/6
19/7
19/8
19/9
19/10
Testis
Volume
(ml)
Histopathological evaluation of spermatogenesis
Presence of testicular spermatozoa or elongated spermatids
Open biopsy
Needle biopsy
Open biopsy
Needle biopsy
no material
no material
no germ cells
no germ cells
not possible
partial spermatogenic
disruption
no germ cells
not possible
partial spermatogenic
disruption
partial spermatogenic
disruption
no germ cells
not possible
not possible
not possible
not possible
not possible
yes
yes
no
no
yes
yes
no
no
yes
yes
no
yes
yes
no
yes
yes
yes
yes
no
yes
yes
yes
yes
yes
no
yes
yes
yes
yes
yes
right
left
right
left
right
left
20
20
12
12
15
12
partial spermatogenic
partial spermatogenic
no germ cells
no germ cells
partial spermatogenic
partial spermatogenic
disruption
disruption
right
right
right
12
15
25
no germ cells
partial meiotic disruption
partial spermatogenic disruption
left
25
partial spermatogenic disruption
left
left
right
left
right
left
8
10
20
20
20
20
no germ cells
partial spermatogenic
partial spermatogenic
partial spermatogenic
partial spermatogenic
partial spermatogenic
disruption
disruption
disruption
disruption
disruption
disruption
disruption
infertility (azoospermia, severe oligospermia, small testes,
elevated FSH), who are under consideration for ICSI, constitute
a group in which genetic impairment is likely to be the cause
of the disturbance (Peterlin et al., 1997; Tournaye et al., 1997),
which could then be transmitted to the next generation.
Thus it is important to relate genetic impairment to specific
disruptions in the various phases of spermatogenesis in order
to increase our knowledge about gene involvement in the
regulation of the process of spermatogenesis. These men
should be offered genetic counselling and the investigating
andrologist should perform a proper evaluation of spermatogenesis.
As shown in this study, an open biopsy yields a specimen
of good quality for evaluation of the different phases of
1268
spermatogenesis, whereas this criterion was not fulfilled by
percutaneous aspiration through a 19 or 21G butterfly needle.
However, as a tool for evaluating the presence or absence
of mature spermatids, the 19G butterfly needle was as good
as the open biopsy, whereas the 21G needle was less reliable.
The efficiency of a cutting needle biopsy for testicular
histological diagnosis has previously been demonstrated by
Kessaris et al. (1995), who found a 95% correlation between
percutaneous and open biopsy regarding the histological diagnosis. The biopsies were carried out on 24 testes (19 patients)
and the percutaneous biopsy was performed with an ASAP18
core biopsy system (18G with 17 mm notch), using one pass
through the testis. In a study by Mallidis and Baker (1994) on
26 men (47 testes), in whom the histological diagnosis was
Percutaneous needle biopsies in azoospermia
Figure 1. Pairs of micrographs at the same original magnifications
(a, d: 403, b, e: 2003 and c, f: 6003), from the open (a–c) and
the percutaneous needle (d–f) biopsy. In this patient the needle
biopsy was of such good quality that it could be evaluated and
spermatogenesis was characterized as partially disrupted. The high
power micrographs (c and f) show tubules where all developmental
stages were found. Note the elongated, condensed spermatids.
Patient 19/6, right testis. Scale bar in a and d 5 0.5 mm, in b and
e 5 100 µm and in c and f 5 30 µm.
compared between open biopsy and tissue aspiration with a
20G, either full agreement (56%) or slight differences (38%)
in the degree of hypospermatogenesis were found.
It thus appears that the diameter of the needle is of
importance for obtaining adequate material for histopathological diagnosis and for the identification of mature spermatids.
It is also conceivable that the correlation between needle and
open testis biopsy is related to the status of spermatogenesis
and the consistency of tissue. In all our cases with germ cell
aplasia, tissue obtained by 19G needle biopsies was adequate
for histopathological evaluation. In the study by Kessaris et al.
(1995), seven of 19 patients had spermatogenesis within normal
limits. In our study all of the 22 patients had impaired
spermatogenesis. In a recent study by Craft et al. (1997)
percutaneous needle biopsies with a 19 or 21G butterfly needle
Figure 2. Pairs of micrographs at the same original magnifications
(a, d: 403, b, e: 2003 and c, f: 6003), from the open (a–c) and
the percutaneous needle (d–f) biopsy. In this patient the needle
biopsy was mechanically damaged and spermatogenesis could not
be evaluated, whereas mature spermatids could be detected.
Patient 19/3, right testis. Scale bar in a and d 5 0.5 mm, in b and
e 5 100 µm, and in c and f 5 30 µm.
were performed in 19 male patients with subfertility, and tissue
adequate for histological assessment of spermatogenesis was
obtained in all cases. However, concurrent open biopsy (controls?) was only performed in two cases. The reason for the
discrepancy between their results and those in our study, where
the same sized needles were used, is unclear, but it could be
due to the use of different criteria for evaluation of the various
phases of spermatogenesis. The biopsy technique may also
have been different. In another recent study by Friedler et al.
(1997) testicular sperm retrieval by percutaneous fine needle
sperm aspiration was compared with testicular sperm extraction
by open biopsy in men with non-obstructive azoospermia.
Spermatozoa for ICSI were found in four patients of 37 (11%)
by percutaneous needle aspiration and 16 cases (43%) by open
testicular biopsy. In this study a 21G butterfly needle was
used. Lately, the first case of a delivery following ICSI of
mature live testicular sperm cells collected in a case of
hypergonadotrophic azoospermia with maturation arrest was
reported (Lewin et al., 1996). In this case spermatozoa were
1269
B.Rosenlund et al.
Figure 3. Pairs of micrographs at the same original magnifications
(a, d: 403, b, e: 2003 and c, f: 6003), from the open (a–c) and
the percutaneous needle (d–f) biopsy. In this patient germ cells
could not be detected in any of the biopsies. Patient 19/2, right
testis. Scale bar in a and d 5 0.5 mm, in b and e 5 100 µm, and
in c and f 5 30 µm.
retrieved by using 21–23G butterfly needles. In this paper the
authors emphasize that the fine needle aspiration technique
may enable the operator to reach more testicular areas than an
open testicular biopsy, and thus increase the chance of retrieving
spermatozoa for ICSI.
There has been a fear that percutaneous biopsy, being a
blind procedure, may cause vascular injury. According to
Jarow (1990), the medial and lateral parts of the upper pole
are less likely to contain major branches of the testicular artery
than other regions and it has therefore been recommended that
percutaneous biopsies be performed close to these areas.
Harrington et al. (1996) reported the occurrence of intratesticular bleeding, observed sonographically as a hypoechoic region
in the testicular parenchyma, within 30 min after the biopsy
in 7% (four of 58) percutaneous biopsies performed with an
18G, 15 cm long Microvasive biopsy needle system with a
notch of 17 mm (two biopsy specimens from each testicle).
In contrast, the proportion of open testis biopsies resulting in
signs of intratesticular bleeding was 29% (10 of 34). It
thus appears that the needle biopsy is associated with fewer
complications than the open biopsy. To what extent the diameter
of the needle and the use of aspiration are related to the
intratesticular bleeding or damage is not known. It is conceiv1270
able, however, that bleeding would be more frequent with a
needle of larger diameter. In a recent study by Schlegel and
Su (1997), 64 patients were evaluated after open testicular
biopsy for non-obstructive azoospermia with serial scrotal
sonography, histological analyses and evaluation of the success
of repeated sperm retrieval attempts. At 3 months after the open
biopsy, 82% (14 of 17) of the patients had ultrasonographic
abnormalities in the testis suggesting resolving inflammation
or haematoma at the biopsy site. By 6 months, the acute
changes had resolved leaving linear scars or calcifications.
Repeat testicular biopsies were more likely to retrieve spermatozoa if the second attempt was performed more than 6 months
after the initial biopsy (12 of 15), relative to those performed
within 6 months (one of four), suggesting transient adverse
physiological effects after testicular biopsies. Permanent devascularization of the testis may occur after testicular biopsies.
Based on studies by Jarow (1991), Schegel and Su (1997)
postulated that multiple testicular biopsies with multiple
incisions in the tunica albuginea may result in interruption of
a sufficient proportion of testicular arteries to devascularize
the testis. Therefore multiple testicular biopsies should be
avoided to reduce the risk of permanent ischaemic testicular
injury.
To conclude, percutaneous testicular biopsy with a 19G
butterfly needle under local anaesthesia is a safe and reliable
method for assessment of the presence of mature spermatids
in men with azoospermia and can also be used in the treatment
of azoospermia with testicular sperm extraction and ICSI.
However, to increase our knowledge about spermatogenesis
and obtain a basis for genetic counselling, in our opinion only
open biopsies offer a sufficient quality for evaluation. Such
knowledge is a prerequisite for building up new DNA-based
tools for the identification of mutations affecting spermatogenesis.
Acknowledgements
The authors thank Kerstin Fallkvist and Eva Nilsson for skilful
technical assistance. This study was supported by grants from the
Swedish Medical Research Council.
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