Human Reproduction Vol.17, No.7 pp. 1800–1810, 2002
Predictive value of testicular histology in secretory
azoospermic subgroups and clinical outcome after
microinjection of fresh and frozen–thawed sperm and
M.Sousa1,3, N.Cremades1, J.Silva1, C.Oliveira1, L.Ferraz1, J.Teixeira da Silva1, P.Viana1and
1Department of Medical Genetics, Faculty of Medicine, University of Porto,2Centre for Reproductive Genetics, Porto and
Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Portugal
3To whom correspondence should be addressed at: Laboratory Cell Biology, Institute of Biomedical Sciences Abel Salazar,
University of Porto, Lg Prof Abel Salazar 2, 4099–003 Porto, Portugal. E-mail: email@example.com
BACKGROUND: A retrospective study was carried out on 159 treatment cycles in 148 secretory azoospermic
patients to determine whether histopathological secretory azoospermic subgroups were predictive for gamete
retrieval, and to evaluate outcome of microinjection using fresh or frozen–thawed testicular sperm and spermatids.
METHODS: Sperm and spermatids were recovered by open testicular biopsy and microinjected into oocytes.
Fertilization and pregnancy rates were assessed. RESULTS: In hypoplasia, 97.7% of the 44 patients had late
spermatids/sperm recovered. In maturation-arrest (MA; 47 patients), 31.9% had complete MA, and 68.1%
incomplete MA due to a focus of early (36.2%) or late (31.9%) spermiogenesis. Gamete retrieval was achieved in
53.3, 41.2 and 93.3% of the cases respectively. In Sertoli cell-only syndrome (SCOS; 57 patients), 61.4% were
complete SCOS, whereas incomplete SCOS cases showed one focus of MA (5.3%), or of early (29.8%) and late
(3.5%) spermiogenesis. Only 29.8% of the patients had a successful gamete retrieval, 2.9% in complete and 77.3%
in incomplete SCOS cases. In total, there were 87 ICSI, 39 elongated spermatid injection (ELSI) and 33 round
spermatid injection (ROSI) treatment cycles, with mean values of fertilization rate of 71.4, 53.6 and 17%, and
clinical pregnancy rates of 31.7, 26.3 and 0% respectively. CONCLUSIONS: Histopathological subgroups were
positively correlated with successful gamete retrieval. No major outcome differences were observed between
testicular sperm and elongated spermatids, either fresh or frozen–thawed. However, injection of intact round-
spermatids showed very low rates of fertilization and no pregnancies.
Key words: non-obstructive azoospermia/spermatids/spermatogenesis/testicular sperm/testicular histopathology
Non-obstructive azoospermia was suggested to be a treatable
situation after it was shown that sperm could be retrieved from
the testis in cases of maturation arrest (MA) and hypoplasia
(Jow et al., 1993). This was then extended to cases with Sertoli
cell-only syndrome (SCOS), with injection of the extracted
sperm being then successfully applied to the clinical treatment
of patients (Craft et al., 1993; Schoysman et al., 1993; Devroey
et al., 1995). Studies have also suggested that the probability
of finding sperm at treatment varied according to the diagnostic
testicular biopsy, being ~95% for hypoplasia, 52 and 69% for
complete and incomplete MA, and 22 and 90% for complete
and incomplete SCOS respectively (Tournaye et al., 1996,
1997; Silber et al., 1997; De Croo et al., 2000).
Most of the reported clinical series related to non-obstructive
azoospermia describe only the clinical outcome associated
© European Society of Human Reproduction and Embryology
with sperm microinjection, either fresh or frozen–thawed.
Those studies evidenced relatively low fertilization rates (38–
67%) but rather high pregnancy rates (40–60%) after ICSI in
cases of SCOS, MA and hypoplasia (Silber et al., 1996, 1997;
Tournaye et al., 1996; Mansour, 1998; Al-Hasani et al., 1999a;
De Croo et al., 2000). On the contrary, analysis of the few
studies which have used spermatids for treatment reveals that
whereas late spermatid injections (n ? 127) seem associated
with low fertilization (48.2%) and acceptable clinical preg-
nancy (29.9%) rates, round spermatid injections (n ? 216)
appear not to be clinically useful (22.5% of fertilization and
3.2% of clinical pregnancy rates) (Fishel et al., 1995, 1996;
Hannay et al., 1995; Tesarik et al., 1995, 1996, 1999; Chen
et al., 1996; Mansour et al., 1996, 1997; Tanaka et al., 1996;
Amer et al., 1997; Antinori et al., 1997a,b; Araki et al., 1997;
Sofikitis et al., 1997, 1998b,c; Vanderzwalmen et al., 1997;
Yamanaka et al., 1997; Barak et al., 1998; Bernabeu et al.,
1998; Kahraman et al., 1998; Al-Hasani et al., 1999b; Sousa
et al., 1999).
In the present study we present the Portuguese clinical and
laboratory data from the consecutive treatment of 148 non-
obstructive azoospermic patients, with normal karyotypes. It
is shown that histopathology allows further subdivisions of
patients with incomplete SCOS and MA, and that these
subgroups evidence distinct prognostic values. The outcome
with both fresh and frozen–thawed testicular-retrieved sperm
and different subtypes of spermatids is also presented.
Materials and methods
All male patients were referred after urology evaluation and were
selected based on normal karyotypes, patent excretory ducts (as
confirmed by physical examination, hormone levels, spermiogram
and ultrasonography), absence of cryptozoospermia (as demonstrated
by two consecutive spermiograms under centrifuged specimens), and
a diagnostic testicular biopsy showing hypoplasia, MA or SCOS. In
almost all of the cases, patients had already had a diagnostic testicular
biopsy a few years previously at another hospital. When patients
accepted IVF with donor sperm, the treatment testicular biopsy either
followed oocyte retrieval or was performed 1–3 days before. In the
latter case, cells were cultured until use in IVF medium at 32°C, 5%
CO2in humidified air. When donor sperm was not an option to the
couple, the biopsy was performed in advance of ovarian stimulation,
with germ cells being frozen 24 h later. In all cases, no treatment
testicular biopsy was made ?6 months after the diagnostic testicular
biopsy. All therapeutical procedures followed the guidelines of the
Ethical Committee, and informed consent was obtained from all
patients after careful explanation of the treatment technique.
Female patients were treated with a long GnRH analogue suppression
protocol combining buserelin acetate (Suprefact; Hoechst, Frankfurt,
Germany) with pure FSH (pFSH) (Metrodin HP; Serono, Geneva,
Switzerland) or recombinant FSH (rFSH) (Gonal F; Serono, Puregon;
Organon, Oss, The Netherlands). Ovulation was induced with HCG
(Pregnyl; Organon, Profasi; Serono). Oocytes were recovered from
large ovarian follicles by ultrasonically-guided follicular aspiration,
35 h after HCG, using flush medium (Medicult, Copenhagen,
Treatment testicular biopsy
The spermatic cord block was performed according to the three-
finger technique (Li et al., 1992; Gorgy et al., 1998; Nudell et al.,
1998). Local anaesthesia was achieved with 5–6 ml of a 1:1 mixture
of 1% lidocaine hydrochloride solution (Xylocaine 2% without
epinephrine; Astra Pharmaceuticals International, Sweden) and 0.5%
bupivacaine (Marcaine 0.5% without epinephrine; Astra). After a few
minutes, a skin weal was raised in the scrotum adjacent to the middle
region of the testis, a 1 cm transverse incision was made and the
tunica vaginalis space entered. An incision of 0.5 cm then enabled
excision of a small piece of the seminiferous tubules. A preliminary
sample microscopic check at the end of each biopsy avoided unneces-
sary tissue sampling. In general, almost all cases with hypoplasia had
sperm or spermatids in the first three samples collected at one testis,
and these were enough for treatment and frozen storage. In MA,
SCOS and, occasionally, in hypoplasia cases, 5–10 biopsies of the
same testis at different locations were needed to find sperm or
spermatids. When such cells were not found, the contralateral testis
was also analysed whenever possible. After careful cleaning and
haemostasis, the tunica albuginea, the vaginal, the scrotum layers and
the skin were closed. The procedure took about 20–30 min and was
performed entirely on an outpatient basis, enabling a rapid recovery
with minimal complaints and total absence of surgical complications.
Tramadol and nimesulide per os were given to relieve any discomfort
in the first 24 h. Where needed, a new biopsy was scheduled only
after a period of 6 months (Schlegel and Su, 1997).
Preparation of testicular samples
Each sample was expressed in Sperm Preparation Medium (SPM,
Medicult) with surgical blades, and 10 µl were observed to confirm
the presence or absence of sperm or elongated spermatids. The
resultant fluid was washed with SPM, 2?5 min, by centrifuging at
500–600 g,and thepellet resuspendedfor 5 minin 2ml oferythrocyte-
lysing buffer (Verheyen et al., 1995) using endotoxin-free, embryo
and cell culture tested chemicals (Sigma, Barcelona, Spain). After
washing, samples were digested (Crabbe ´ et al., 1997) for 1 h at 37°C,
in a solution of SPM containing 25 µg/ml of crude DNase and
1000 IU/ml of collagenase-IV (Sigma). After a new wash, the pellet
was resuspended in 50–100 µl of IVF medium (Medicult) and then
incubated at 30–32°C, 5% CO2in air until use. For freezing, the
sample was diluted with Sperm Freezing Medium (Medicult), exposed
for 10–15 min to liquid nitrogen (LN2) vapours, and finally immersed
and stored in LN2.
Selection of cells for microinjection
The distinction between round spermatids and Sertoli cell nuclei,
elongating spermatids, elongated spermatids and testicular sperm
have been the subject of much debate, but clear criteria have been
established (Tesarik, 1997, 1998; Aslam et al., 1998; Lewis and
McClure, 1998; Mansour et al., 1998; Silber and Johnson, 1998;
Silber et al., 1998; Sofikitis et al., 1998b; Sousa et al., 1998, 1999;
Tesarik et al., 1998; Vanderzwalmen et al., 1998). Briefly, isolated
nuclei of Sertoli cells have an elevated border, a large nucleolus,
no other visible internal structures, and shrink in 10% PVP-SPM
(Medicult); round-shaped cytoplasmic remnants, blebbed-out from
degenerating cells, have no internal visible organelles and shrink in
PVP; lymphocytes have internal nuclear irregularities due to con-
densed patches of chromatin, they stick to the tip of a 6 µm inner
micropipette and stretch with aspiration, and if left in culture they
tend to attach and develop cytoplasmic extensions (pseudopodes)
within 24 h. On the contrary, round spermatids have a smooth outline
and inner aspect, the nuclear limit is clearly visible, the acrosomal
vesicle is distinguishable as 1–2 large round vesicles (Golgi phase)
or as a fine dark elongating region at one nuclear pole (cap phase),
they deform and adapt their shape to the aspirating 6 µm inner
micropipette, and do not shrink in PVP. Round spermatid injection
(ROSI) was used when patients did not accept donor sperm. We have
used intact round spermatids for injection because a slightly larger
injection pipette is not associated with a higher rate of oocyte
degeneration (Sousa et al., 1999), at this stage the proximal centriole
may still not be attached to the nuclear envelope (Holstein and
Roosen-Runge, 1981), and because the cytoplasmic membrane and
the nuclear envelope of the round spermatid is ruptured soon after
contact with the ooplasm, enabling diffusion of the spermatid oocyte-
activating substance and proper pronucleus formation (Sousa et al.,
1996, 1999). Because ionophores are not allowed for clinical use,
oocytes were not activated through an induced intracellular calcium
rise after ROSI (Tesarik and Sousa, 1995b).
On the contrary, elongating and elongated spermatids are very easy
to distinguish. In the present series, whenever we found normal
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Submitted on May 1, 2001; resubmitted on November 8, 2001; accepted on
February 19, 2002