Human Reproduction vol.14 no.1 pp.118–122, 1999
Diagnostic epididymal and testicular sperm recovery
and genetic aspects in azoospermic men
Go ¨ran Westlander1, Lars Hamberger,
Charles Hanson, Kersti Lundin, Lars Nilsson,
Brita So ¨derlund, Charlotte Werner and
Centre for Reproductive Medicine, Sahlgrenska University Hospital,
Go ¨teborg University, S-413 45 Go ¨teborg, Sweden
1To whom correspondence should be addressed
Various procedures for sperm recovery in azoospermic
men have been described, from open testicular biopsy to
simple needle aspiration from the epididymis and the testis.
Fifty-one obstructive and 86 non-obstructive azoospermic
men were treated to compare the recovery of spermatozoa
obtained by percutaneous aspiration from the epididymis
(PESA) and aspiration/extraction from the testis (TESA,
TESE) with histopathology. If TESA failed, the work
up proceeded with TESE. All patients were karyotyped.
Spermatozoa were recovered by PESA or TESA in all
obstructive men (51/51 patients). In 22 out of 86 patients
with non-obstructive azoospermia, testicular spermatozoa
could be successfully recovered by TESA. In five additional
patients TESE was successful in recovering spermatozoa
where TESA had failed. In 43 patients, neither TESA nor
TESE was successful. Sixteen patients chose not to proceed
with TESE. Seven out of 86 patients had an abnormal
karyotype in the non-obstructive group (8%), none in the
obstructive group. In the non-obstructive patient group
testicular histopathology showed hypospermatogenesis,
incomplete maturation arrest and germ cell aplasia with
focal spermatogenesis in cases where spermatozoa were
tion arrest and fibrosis in cases where no spermatozoa
were found. Spermatozoa were recovered by PESA or
TESA from all patients with obstructive azoospermia and
from ~40% of patients with non-obstructive azoospermia
by TESA or TESE. Retrieval of viable spermatozoa in
the infertility work-up was highly predictable for sperm
local anaesthesia seems almost as effective as more invasive
procedures in recovering testicular spermatozoa, both in
obstructive and non-obstructive azoospermic men.
male infertility/testicular sperm aspiration/testicular sperm
With the introduction of intracytoplasmic sperm injection
(ICSI), the treatment of male infertility was revolutionized.
© European Society of Human Reproduction and Embryology
Thefirstpregnancy wasreportedin1992(Palermoet al.,1992).
Modern sperm recovery techniques have made it possible to
help men with both obstructive and non-obstructive azoosper-
mia to achieve genetic fatherhood. In patients with obstructive
azoospermia, viable spermatozoa from either the epididymis
(Tournaye et al., 1994) or the testis (Craft et al., 1993;
Schoysman et al., 1993) can be used for the ICSI procedure.
Spermatozoa from the testis can also be used after recovery
from men with deficient spermatogenesis or so-called non-
1996). The recovery techniques for epididymal spermatozoa
include open surgery by microepididymal sperm aspiration
(MESA) (Temple-Smith et al., 1985; Silber et al., 1994) and
a less invasive method, percutaneous sperm aspiration (PESA)
zoa can be recovered by testicular biopsy (testicular sperm
extraction, TESE) (Schoysman et al., 1993; Nagy et al., 1995),
which is an invasive method, or a simplified less invasive
method of percutaneous aspiration with a fine needle, testicular
sperm aspiration (TESA) (Tsirigotis and Craft, 1995).
Several studies have shown the ICSI technique to be a safe
method. Follow-up studies of children born after intracyto-
plasmic sperm injection with ejaculated, epididymal or testicu-
lar spermatozoa have given no indication of an increased risk
of major malformation or low birthweight babies (when
separated for singletons and multiples) compared to children
born after in-vitro fertilization (IVF) and children born after
The incidence of chromosomal aberrations in children born
aberrations are more frequent in subfertile men, which lead to
an increased risk of an unbalanced chromosomal constitution
in the offspring or a risk of transmitting the same translocation
and hence the infertility to the offspring (Baschat et al., 1996).
Furthermore, the incidence of Klinefelter’s syndrome, and
hence the risk of producing an aneusomic offspring is also
higher in subfertile men (Cozzi et al., 1994; Chevret et al.,
A possible increased risk of all chromosomal abnormalities
has been suggested (In’t Veld et al., 1995) and current data
from the largest cytogenetic study published so far (Bonduelle
et al., 1998) indicate a slightly increased risk of de-novo
chromosomal aberration and transmitted chromosomal aberra-
tion in children conceived after ICSI as compared to the
In our IVF unit, treatment of azoospermic men with ICSI
using surgically retrieved spermatozoa was initiated in 1994.
A noticeable number of sperm recovery failures was soon
encountered. Unsuccessful sperm recovery procedures have
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Sperm recovery in azoospermia
important emotional and financial implications if the woman
concomitantly undergoes ovarian stimulation and oocyte
retrieval. There seem to be no strong predictors for successful
sperm recovery. Testicular histopathology was found to be the
best predictor for viable spermatozoa in a study from Brussels
(Tournaye et al., 1997). We therefore decided to perform
diagnostic PESA/TESA or TESE in all our azoospermic
men, both of obstructive and non-obstructive origin, before
acceptance for IVF/ICSI treatment. Epididymal spermatozoa
from diagnostic PESA were frozen for further use in ICSI
cycles. When diagnostic sperm recovery procedures failed the
couple was informed about adoption and insemination with
donor spermatozoa (AID).
The aim of the present study was to correlate the recovery
of spermatozoa obtained by percutaneous aspiration from the
epididymis (PESA) and aspiration/extraction from the testis
(TESA, TESE) to histopathology. The recovery of spermatozoa
was then correlated to the success rate of obtaining viable
spermatozoa in subsequent ICSI cycles. A further aim was to
examine the frequency of abnormal karyotypes among patients
with non-obstructive azoospermia. Serum concentrations of
follicle stimulating hormone (FSH) and maximum testicular
volume were also evaluated in relation to sperm recovery.
Materials and methods
This study was carried out between October 1995 and December
1997 at the Centre for Reproductive Medicine, Sahlgrenska University
Hospital, Go ¨teborg University, Sweden. The patient population con-
sisted of 137 consecutive male patients with azoospermia. Before the
sperm aspiration/ICSI procedure the patients were subjected to
diagnostic epididymal and/or testicular sperm aspiration (PESA,
TESA) performed under local anaesthesia. If sperm recovery was
successful, the couple was accepted for an IVF/ICSI procedure.
Epididymal spermatozoa were frozen and used in further IVF/ICSI
cycles, if possible. If recovery was not successful the patients were
offered multiple testicular biopsies (TESE), which were performed
under general anaesthesia. Two to three biopsies were taken on each
side. Parts of the testicular biopsies were sent for histopathological
examination. If sperm recovery with TESE also failed, adoption or
AID was recommended. Spermatids are not used clinically for ICSI
in our IVF unit.
In addition to the diagnostic puncture, the work-up included general
medical history, information and a physical examination where the
testicular volume was measured with an orchidometer. Karyotyping
was performed from lymphocytes in a peripheral blood sample and
serum concentrations of follicle stimulating hormone (FSH) were
determined. In couples where the male suffered from congenital
bilateral absence of the vas deferens (CBAVD) both the man and the
woman were screened for possible cystic fibrosis (CF) gene mutations.
The screening included the most frequent mutations (∆F-508, 394
del TT) and variations (5T allele) for the population.
0.5 mg alfentanil (Rapifen®; Jansen-Cilag, Beerse, Belgium) was
given i.v. followed by infiltration of 7–8 ml lidocaine (Xylocaine®;
Astra, So ¨derta ¨lje, Sweden) around the spermatic cord. A 23 gauge
butterfly needle was pushed through the stretched scrotal skin and
into the epididymis. Suction was applied with a 10 ml syringe and
the negative pressure was maintained by clamping the distal end of
the needle tubing with two pairs of forceps. The 10 ml syringe was
then replaced by a 1 ml syringe containing 0.1 ml gamete culture
medium (IVF-50; Scandinavian IVF Science, Go ¨teborg, Sweden).
The tip of the needle was gradually moved within the epididymis
until clear or opalescent fluid rose in the needle tubing. If blood
appeared in the tube, clamps were put on the tube to avoid blood
contamination. The needle was withdrawn completely from the
epididymis, the forceps removed and the aspirate flushed into a sterile
tube. Whenever possible the epididymal spermatozoa were frozen for
further use in subsequent ICSI procedures.
0.5 mg alfentanil (Rapifen®) was given i.v. followed by infiltration
of 7–8 ml lidocaine (Xylocaine®) around the spermatic cord. Under
sterile conditions, a 19 or 21 gauge butterfly needle was passed
through the scrotal skin. Suction was applied with a 20 ml syringe
and the negative pressure was maintained by clamping the tubing in
the same manner as in the PESA procedure. The needle was pushed
into the testicular tissue. The needle was then slowly removed from
the testis and the scrotal skin while the back pressure was maintained
by the clamped tubing. The assistant used two pairs of fine tweezers
to pick up the small tubules recovered by the needle. The clamps
were then removed and the needle was flushed with culture medium
and its content was expelled into a sterile tube containing culture
medium. The procedure was repeated four to six times, trying to
cover the whole ventral surface of the testis. The testicular tissue was
dissected, incubated in culture medium and examined 1, 4, 24 and
48 h after puncture in the laboratory.
Under general anaesthesia, a longitudinal incision was made in the
scrotal skin and carried through the peritoneal tunica vaginalis
exploring the tunica albuginea and the epididymis. Two to three
incisions were made through the tunica albuginea in different regions
of each testicle. A 0.5–1.0 cm biopsy of extruding testicular tissue
was excised and cut into two pieces. The first part was placed in a
sterile tube containing culture medium (IVF-50) and the tissue was
further prepared and examined in the laboratory 1, 4, 24 and 48 h
after surgery. The other part was put into a tube containing formalde-
hyde and sent for histopathological examination. The examination of
the testicular tissue was mainly performed by one pathologist with a
special interest in this field. After each biopsy, the tunica albuginea
was closed with 4–0 vicryl sutures. No more than three biopsies on
each side were performed.
A total of 137 patients with azoospermia underwent diagnostic
sperm aspiration. Fifty-one patients were judged to have
obstructive and 86 patients non-obstructive azoospermia. In
the obstructive group, spermatozoa were recovered in all
patients with PESA or TESA. The mean ? SD serum FSH
concentration in this group was 3.8 ? 2.0 IU/l. The mean
testicular volume was 19.8 ? 4.4 ml. All patients had a normal
karyotype. A large majority of the men with cystic fibrosis
and/or CBAVD were positive for CF gene mutations. All the
female partners were negative.
In the non-obstructive group, testicular spermatozoa could
be successfully recovered by TESA in 22 out of 86 patients.
In five additional patients TESE was successful in recovering
spermatozoa, where TESA had failed. In 43 patients neither
TESA nor TESE was successful. Sixteen patients chose not
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G.Westlander et al.
Table I. Serum follicle stimulating hormone (FSH) and testicular volume
(mean ? SD) in men with obstructive and non-obstructive azoospermia
No. of patients
Testicular volume (ml) 19.8 ? 4.4
3.8 ? 2.0
16.1 ? 12.6a
16.4 ? 4.0b
23.9 ? 13.6
13.7 ? 3.9
aP ? 0.05 versus non-obstructive azoospermia unsuccessful sperm recovery
bP ? 0.01 versus non-obstructive azoospermia unsuccessful sperm recovery
to proceed with the TESE procedure. The mean serum FSH
concentration was significantly higher, 23.9 ? 13.6 versus
16.1? 12.6IU/l (P? 0.02),inthe groupwhere spermrecovery
failed compared to the group where testicular spermatozoa
were successfully retrieved. Also, the testicular volume was
significantly lower, 13.7 ? 3.9 ml versus 16.4 ? 4.0 ml (P ?
0.007), in the failure group compared to the successful group.
However, the overlap between the groups for both these
variables was great (Table I).
Seven out of 86 (8.1%) patients had an abnormal chromo-
somal karyotype. Four of these men had Klinefelter’s syndrome
(47,XXY), one of whom was a mosaic (47,XXY/46,XY). Two
patients had translocations [45,XY,rob(13;14)(q10;q10) and
46,XY,t(6;15)(q13;p13) respectively]. One patient had a more
complicated karyotype (48,XY?2mar/45,X/46,XY).
In the non-obstructive group where spermatozoa could be
recovered the testicular histology showed different types of
defective spermatogenesis. It was either general: described as
hypospermatogenesis in one or both testicles; or focal:
described as maturation arrest or germ cell aplasia with foci
of normal spermatogenesis in the testicle(s).
In the group where spermatozoa could not be recovered,
histology showed complete germ cell aplasia (Sertoli cell-
only syndrome), complete maturation arrest and fibrosis. The
histopathology of the biopsies often differed between the two
testicles and occasionally also between biopsies within the
A total of 105 ICSI cycles has so far been performed in
the obstructive and the non-obstructive groups where viable
spermatozoa earlier had been detected in the diagnostic proced-
ure. In all of these cycles the repeated sperm recovery was
Epididymal and testicular sperm recovery in combination with
ICSI now offers azoospermic men the possibility of fathering
their own genetic children. Since the first reports of births
of children conceived from surgically retrieved epididymal
(Temple-Smith et al., 1985; Silber et al., 1994) and testicular
spermatozoa (Devroey et al., 1995; Tournaye et al., 1995),
several papers on larger series have been published (Mansour
et al., 1997; Tournaye et al., 1997). Initially, when using these
new techniques, it was enthusiastically claimed that almost
100% of azoospermic men were possible to treat (Silber,
1995). Further studies have shown that this may be true for
men with obstructive azoospermia but in men with non-
obstructive disorders the chance of retrieving viable spermato-
zoa is considerably smaller. In recent publications (Chen et al.,
1996; Friedler et al., 1997; Mansour et al., 1997; Tournaye
et al., 1997), the sperm recovery rate in non-obstructive
azoospermic men varied between 40 and 70%. In these studies,
sperm recovery was mainly performed by TESE. Our results,
when using mainly the less invasive methods, PESA and
TESA, are in accordance with these reports, showing a 100%
recovery rate in obstructive patients and a 39% recovery rate
in non-obstructive patients. TESA performed under local
anaesthesia in men with non-obstructive azoospermia was
recently reported to be a quick, reliable and easy method
of obtaining testicular tissue (Malldis et al., 1994; Craft
et al., 1997).
In our material among men with non-obstructive azoosper-
mia, TESE was successful in only five of 48 (10%) cycles
where TESA performance earlier had failed, whereas other
reports have emphasized that TESE is superior in most cases
(Friedler et al., 1997). The aspiration procedure in those cases
was described as testicular fine needle aspiration (TEFNA),
where only one puncture was made. Our TESA procedure
involved several punctures in different directions in three to
six areas at the ventral surface of the testis. With this technique,
tissue samples from the testis were almost always obtained.
Compared to the TESE procedure, the amount of tissue
recovered by TESA was smaller but the present technique was
able to reach deeper into the parenchyma, aiming for the rete
testis, and thereby covered a larger area of the testis compared
to one to two biopsies. The present strategy with TESA would
also be beneficial if spermatogenesis is focal in some of these
non-obstructive cases. This has recently been a matter of
debate (Silber et al., 1997).
Obviously, the present technique seems to have a high
predictive value for sperm recovery in subsequent ICSI cycles.
In all ICSI cycles performed so far, where viable spermatozoa
earlier had been detected in diagnostic procedures, all repeated
sperm recoveries were successful.
In addition to simplicity, convenience for the patients and
considerably lower costs, TESA might cause less damage to
the testis compared to TESE. Transient adverse effects such
as inflammations and haematomas after TESE were reported
by Schlegel et al. (1997). They also reported that permanent
devascularization occurred in one out of 64 patients after
TESE. No per- or post-operative complications were reported
in our series of 137 patients and no serious adverse effects
have hitherto been reported after TESA.
The mean serum FSH concentration was significantly lower
in the non-obstructive group where spermatozoa could be
successfully retrieved compared to the group where sperm
recovery failed. However, a great overlap in the distribution
of FSH concentrations was evident, irrespective of the presence
or absence of testicular spermatozoa. The same was found for
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Sperm recovery in azoospermia
volume and a large overlap between non-obstructive patients
with and without spermatozoa. These data are in accordance
with previous reports (Tournaye et al., 1995; Friedler et al.,
1997), showing that neither serum FSH nor testicular volume
was predictive for the chance of retrieving spermatozoa.
The prevalence of chromosomal aberrations in this study
was seven out of 86 (8%). In the group of men with poor
sperm production, the incidence of Klinefelter’s syndrome is
~3% (Tournaye et al., 1996; Lundin et al., 1998). Among our
patients with non-mosaic 47,XXY, so far no spermatozoa have
been found. However, a recent study reported five out of five
normal preimplantation embryos after ICSI with spermatozoa
from testicular recovery of men with a non-mosaic 47,XXY
chromosome constitution (Staessen et al., 1996). In two
case reports of two men with a 47,XXY/46,XY mosaicism,
hyperploidy (24,XX or 24,XY) was found in 0.9 and 2.09%
of the spermatozoa, respectively (Cozzi et al., 1994; Chevret
et al., 1995). The proportion of 24,XX and 24,XY spermatozoa
was doubled in the first case and increased 5-fold in the second
case, compared to normal men, where the frequency of disomy
for the sex chromosomes in spermatozoa is 0.42% (Martin
et al., 1996). Although the proportion of spermatozoa, disomic
for the sex chromosomes, may vary from case to case in
Klinefelter mosaics, depending on the variation of the degree of
mosaicism in the sperm-producing cells, the risk of producing a
47,XXX or 47,XXY offspring must be considered.
As the incidence of chromosomal abnormalities such as
translocations may be up to ten times higher in men with
poor sperm production compared to men with normal sperm
production (Lidegaard et al., 1998), the risk of producing an
offspring with an unbalanced chromosome content must also
All possible chromosomal imbalances in the offspring due
to the chromosomal aberrations described in this paper may be
detected by traditional prenatal diagnosis or by preimplantation
genetic diagnosis. However, trisomy 47,XXX, 47,XXY or
47,XYY does not in general lead to major malformation or
mental retardation (Meschede and Horst, 1997). When these
aberrations are diagnosed in the offspring, the alternatives of
continuing the pregnancy or therapeutic abortion should be
The karyotyping did not include screening for chromosome
Y microdeletions. Analysis of microdeletions is only available
in a few laboratories in Scandinavia. The function of the
various loci/genes on the Y chromosome is not known but it
seems well established that microdeletions on the Y chromo-
some play a central role in male infertility (Simoni et al.,
1998). Most of the deletions seem to be de novo. The outcome
of sperm recovery and fertilization rate after ICSI among
non-obstructive azoospermic men with deletions compares
favourably with non-obstructive azoospermic men without
deletions (Mulhall et al., 1997). Hence, our couples with
severe male infertility are offered genetic counselling where
they are informed about the possible risk of vertical transmis-
sion of deletions to the male offspring.
In summary, this study showed that spermatozoa may be
recovered by PESA or TESA from all patients with obstructive
azoospermia. Testicular spermatozoa may be recovered from
~40% of patients with non-obstructive azoospermia. TESA
performed under local anaesthesia is effective, simple and
cost-effective. Including a sperm aspiration in the infertility
work-up in azoospermic patients seems to be an excellent way
to predict successful sperm recovery in subsequent ICSI
treatments. In addition, in the obstructive group, epididymal
with high fertilization and pregnancy rates (Nagy et al.,
1995), while in the non-obstructive group the freezing–thawing
procedure of testicular spermatozoa is more unpredictable (Gil
Salom et al., 1996; Verheyen et al., 1997).
This study was supported by The Hjalmar Svensson foundation, The
Go ¨teborg Medical Society, The Faculty of Medicine, Go ¨teborg
University and the Swedish Medical Research Council (nos. 2893
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University of Gothenburg, Sweden. ISBN 91-628-2898-3.
Received on July 2, 1998; accepted on October 13, 1998
by guest on June 1, 2013