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
M.Sousa et al.
elongating spermatids, we also found elongated spermatids, albeit
after several hours of searching. When elongated spermatids could
not be found but elongating spermatids were present, the latter had
extensive nuclear and tail malformations and were not used for
treatments. This means that we have only used elongated spermatids
for injection (ELSI). However, we did not use elongated spermatids
without a full length flagellum or with abnormal head morphology.
The elongating spermatid is differentiated from the elongated
spermatid mainly based on nuclear morphology (Holstein and Roosen-
Runge, 1981). The former has a nucleus that is not fully elongated
nor condensed and protrudes only slightly beyond the apical cell
region. The elongated spermatid is differentiated from the testicular
spermatozoon based on the upper limit of the basal cytoplasm. In a
spermatozoon, the basal cytoplasm is confined to the midpiece region,
which does not exhibit any continuity with the equatorial region of
the spermatozoon head. This is made possible through the coalescence
between the upper limit of the midpiece cytoplasmic membrane and
the basal nuclear envelope. This point of fusion is named the posterior
ring (Holstein and Roosen-Runge, 1981). So, the elongated spermatid
is differentiated from a testicular spermatozoon when the upper limit
of the basal cytoplasm is still above the nuclear base (Holstein and
Roosen-Runge, 1981; Sousa et al., 1999). It is also at the end of the
elongated spermatid stage that the distal centriole is depolymerized.
Because the distal centriole is committed to synthesize the axoneme,
to which it remains tightly linked from the round spermatid stage
(Holstein and Roosen-Runge, 1981), it has no intrinsic centrosome
capability and thus spermatid microinjection does not introduce into
the ooplasm an additional microtubule polymerizing centre (Sousa
and Tesarik, 1994; El Shafie et al., 2000). That is why, and as clinical
series have already demonstrated, the distal centriole of microinjected
spermatids does not preclude normal fertilization and embryo
Microinjection and embryo culture
micromanipulators (Nikon, Tokyo, Japan) and commercial micro-
pipettes (4–5 µm for late spermatids/sperm, 6–8 µm for round
spermatids; SweMed; Frolunda, Sweden) according to a published
method (Sousa et al., 1998, 1999). Oocytes were injected using the
strong dislocation of the ooplasm (Tesarik et al., 1994; Tesarik and
Sousa, 1995a). Injected oocytes were cultured in IVF medium at
37°C with 5% CO2in air. After 2 days, they were transferred to M3
medium (Medicult). Normal fertilization was assessed 14–18 h after
injection, and embryo quality was evaluated (Staessen et al., 1995).
Supernumerary embryos were frozen with embryo freezing medium
or with the blastocyst freezing pack (Medicult), in an automatic
freezing apparatus (Kryo 10 Series III; Planer, UK). All patients had
luteal supplementation with three times daily intravaginal administra-
tion of 200 mg natural-micronized progesterone (Utrogestan; Jaba,
Berlin, Germany). Pregnancy was confirmed by a rise in serum βHCG
on 2 consecutive days, 2 weeks after embryo transfer. A clinical
pregnancy was established by ultrasonography at 7 weeks gestation.
All couples agreed to have a prenatal diagnosis, which was performed
by amniocentesis at 16 weeks of pregnancy.
When appropriate, correlation analysis were performed, and the
significance of difference between the percentages of two groups
were evaluated with the χ2-test. Significance was accepted where
P ? 0.05.
In total, there were 148 patients and 234 treatment cycles (159
with 87 ICSI, 39 ELSI and 33 ROSI cycles, and 75 cycles
with donor sperm). Patients were subdivided acording to the
histopathological analysis of the diagnostic testicular biopsy:
44 (29.7%) had hypoplasia, 47 (31.8%) MA, and 57 (38.5%)
SCOS. Patients were selected after normal karyotypes were
confirmed, and thus the present series excludes all testicular
histologies associated with chromosomal abnormalities. The
histopathological diagnosis was based on the study of at least
100 seminiferous tubule profiles. Tubular hyalinization, fibrosis
and Kleinfelter-like cases were excluded from the present
study, except when the karyotypes were normal and those
specific cases were restricted to a focal attainment of the
In all patients, the diagnostic biopsy showed spermatogenesis
up to the late spermatid or spermatozoa stages, which were
recovered for treatment in 43 cases (97.7%). In one case, a
total cell aplasia was found at treatment despite an extensive
bilateral search of the testicular bioptic specimens. This may
be explained by an intratesticular injury that has progressively
destroyed the germinal epithelium during the 5 years between
the diagnostic and the treatment testis biopsies or, alternatively,
by an unfortunous diagnostic testis biopsy that was taken over
a unique focus of spermatogenesis. In total, there were 53
cycles with ICSI, 11 cycles with ELSI, and three cycles using
IVF or intra-uterine insemination (IUI) with donor sperm (one
case with no remaining spermatids and one case with total
The mean female age was 33 years (range 22–40), the mean
male age was 36.1 years (range 27–55), and the mean duration
of infertility was 6.7 years (range 1–18). Only 11.4% of
the women showed associated pathology (three cases with
hyperprolactinaemia, one case of endometriosis, and one case
with tubal obstruction). About 50% of the male patients had
increased FSH levels, 38.6% showed decreased testicular
volume, and 13.6% had associated testicular pathology (three
with decreased testicular volume exhibited increased FSH
levels and had no associated local pathology.
In ICSI cycles using fresh sperm, there have been nine
normal evolving pregnancies (five clinical, four ongoing), and
four deliveries of healthy children. Of these 13 pregnancies,
three are twin and two triplet. In ICSI with frozen–thawed
sperm therewere twobiochemical pregnancies,one ispresently
clinical and three have delivered healthy babies. Of these four
successful pregnancies, one is twin and one triplet. In fresh
ELSI cycles, there is one ongoing pregnancy and three deliver-
ies of healthy children. Of these four pregnancies, one is twin
Of the 47 patients with MA, the diagnostic testicular biopsy
showed 15 cases (31.9%) of complete MA, 17 cases (36.2%)
had one focus of early spermiogenesis (round or elongating
spermatids), and 15 cases (31.9%) exhibited one focus of
late spermiogenesis (late spermatids or spermatozoa). After
treatment, testicular biopsy confirmed complete MA in three
patients, four had one focus with round spermatids, and
Table I. Outcome in hypoplasia
ICSI n (%)ELSI n (%)
F FTF FT
Cleaved embryos (from 2PN)
Embryo grade (A?B)
No. embryos transferred (mean)
Day of embryo transfer (mean)
Cycles with embryo transfer
– 6.5 (2–15)
Fresh (F) and frozen–thawed (FT) gametes.aP ? 0.05 to ICSI (FT) and ELSI (F, FT).
eight (53.3%) enabled recovery of sperm/late spermatids; MA
patients with one focus of early spermiogenesis revealed very
similar figures (3; 7; and 7 cases, 41.2%); on the contrary, 14
(93.3%) of the patients showing one focus of late spermiogen-
esis had recovery of sperm/late spermatids (one case with a
focus of round spermatids). In total, there were 22 ICSI cycles,
21 ELSI cycles, 20 cycles with round spermatid injection
(ROSI: 11 original cases and nine cases with absence of late
spermatids/sperm in the second trial), 11 donor sperm IVF
cycles (two after ICSI/ELSI, one after ROSI, and eight from
couples with complete MA), and four donor sperm IUI cycles
(one after ICSI/ELSI, three from patients with complete MA).
The mean female age was 31.9 years (range 24–41), the
mean male age was 34.9 years (range 24–53), and the mean
duration of infertility was 5.7 years (range 1–18). About 27.7%
of the women showed associated pathology [six polycystic
ovarian syndrome (PCOS), four hyperprolactinaemia, one
endometriosis, one tubal obstruction, and one ovarian insuffi-
ciency]. About 38.3% of the male patients had increased FSH
levels, 42.6% showed decreased testicular volume, and 25.5%
had associated testicular pathology (three varicocele, nine
cryptorchidia). In patients where no late spermatids/sperm
could be recovered, there was a predominance of normal
values of FSH (72.2 versus 58.6%), of normal testicular
(5.6 versus 37.9%). Correspondingly, normal testicular volume
appeared to be associated with normal FSH values (75%),
decreased testicular volume was associated with higher FSH
values (71.4%), and 45% of the cases with decreased testicular
volume were associated with local pathology (chryptorchidia).
In ICSI cycles using fresh sperm, to date there is one
ongoing pregnancy and three deliveries of healthy babies. Of
these four pregnancies, one is twin. In ICSI with frozen–
thawed sperm there is one ongoing pregnancy and two
deliveries (healthy babies). In fresh ELSI cycles there were
two biochemical and one clinical pregnancies, and two healthy
deliveries. Of these three successful pregnancies, one is twin
(Table II). Of the cycles with donor sperm, there were four
IVF term pregnancies and one IUI term pregnancy with
delivery of healthy children.
Of the 57 patients with SCOS, the diagnostic testicular biopsy
showed 35 cases (61.4%) of complete SCOS, three cases had
one focus of primary spermatocytes (5.3%), 17 cases (29.8%)
had one focus of early spermiogenesis (round or elongating
spermatids), and two cases (3.5%) exhibited one focus of late
spermiogenesis (late spermatids or spermatozoa). After treat-
ment, testicular biopsy revealed complete SCOS in 31 patients,
three had one focus with round spermatids, and one (2.9%)
enabled recovery of sperm/late spermatids; all SCOS patients
with one focus of primary spermatocytes had sperm/late sperm-
enesis revealed five cases with round spermatids, and 12 cases
with sperm/late spermatids (70.6%); of the two SCOS patients
withone focusof latespermiogenesis, onehad sperm/elongated
spermatids recovered, whereas in the other case only Sertoli
cells could be found. In total, there were 12 ICSI cycles, seven
with absence of late spermatids/sperm in the second trial), 41
donor sperm IVF cycles (two after ICSI/ELSI, two after ROSI,
IUI cycles (from patients with complete SCOS).
male age was 34.2 years (range 23–48), and the mean duration
of infertility was 6 years (range 1–19). About 22.8% of the
prolactinaemia, one endometriosis, one miomatosis). About
75.4% of the male patients had increased FSH levels, 71.9%
showed decreased testicular volume, and 36.8% had associated
testicular pathology (eight varicocele, six cryptorchidia, four
adult parotiditis, one chemotherapy, two hypogonadism).
Increased FSH levels were present in 58.8% of the patients with
M.Sousa et al.
Table II. Outcome in maturation arrest (MA)
ICSI n (%)ELSI n (%) ROSI n (%)
FFTF FTF FT
Cleaved embryos (from 2PN)
Embryo grade (A?B)
No. embryos transferred (mean)
Day of embryo transfer (mean)
Cycles with embryo transfer
Fresh (F) and frozen–thawed (FT) gametes.a,b,cP ? 0.05 between same superscripts.
spermatids were recovered, and in 93.8% of the patients with
complete SCOS. Similarly, in these three groups there was a
predominance of decreased testicular volume (82.4, 75 and
65.6% respectively). Because the large majority of the patients
no association was found between these two parameters. There
was also no association found between local pathology and any
of the other parameters or subgroups of patients.
In ICSI cycles with fresh sperm there was one biochemical
pregnancy, and in cycles with frozen–thawed sperm there were
two deliveries of healthy babies (one twin). In fresh ELSI
cycles there is one ongoing pregnancy and two deliveries of
healthy children (Table III). Of the cycles with donor sperm,
in IVF cycles, and three normal term pregnancies after IUI.
azoospermia, which includes hypoplasia, MA and SCOS. Sev-
MA or SCOS syndromes) correlates highly with successful
sperm retrieval at treatment, and that neither the testis volume,
male serum FSH, associated male pathology or professional
status could be used as successful predictive factors (Devroey
et al., 1995; Gil-Salom et al., 1995; Yemini et al., 1995; Lewin
1998a,c, 1999; Amer et al., 2001). Although the presence of
spermatocytes and spermatids in ejaculates, as detected by
specific staining, seems to predict a successful testicular sperm
retrieval, this was only noticed for cases with hypoplasia and
correlated with the specific histopathological subtypes (Amer
et al., 2001). Other authors have also suggested that anti-
assays in testicular tissue, could predict the presence of sperm-
atids in cases of SCOS (Fe ´nichel et al., 1999; Yamamoto et al.,
1999b; Schrader et al., 2000). Data from our present series of
148 consecutive non-obstructive azoospermic patients confirm
the importance of histopathology for a predictive prognosis,
and further suggest that incomplete MA and SCOS cases may
has been anticipated in very large series of diagnostic testicular
biopsies from azoospermic men (Schulze et al., 1999; Glander
et al., 2000).
In non-obstructive azoospermia, several studies have sug-
gested that the testicular open-biopsy method appears superior
for those cases that are expected to be difficult. Nevertheless
excellent sperm retrieval success has been achieved with
percutaneous testis aspiration, particularly in patients with a
better prognosis as given by the presence of elongated sperm-
atids or spermatozoa in the diagnostic testicular biopsy (Lewin
et al., 1996, 1999; Rosenlund et al., 1998; Meng et al., 2000).
percutaneous aspiration does not enable the recovery of suffi-
cient sperm for injection and freezing (Ezeh et al., 1998b;
Mercan et al., 2000).
Each biopsy should be immediately checked in order to
avoid excessive multiple and bilateral sampling, but in most
of the cases where the probability of finding sperm is worst,
multiple biopsies seem to be needed (Hauser et al., 1998; Amer
et al., 1999; Sousa et al., 1999), although the recommended
maximum number of biopsies ranges from one (Silber et al.,
1995, 1997; Verheyen et al., 1995) to three (Hauser et al.,
1998; Amer et al., 1999). Our present series confirm these
findings, but establishes different approaches regarding the
different subtypes of histopathologies, which show distinct
inherent difficulties of finding a focus of spermiogenesis. In
general, the number of cases with multiple and bilateral
biopsies increased according to the severity of the diagnosis
and to the type of spermatid retrieved. In hypoplasia, most of
the patients had unilateral surgery (97% with sperm, 70% with
late spermatids), with 1–3 fragments giving enough gametes
for treatment and storage (72% with sperm, 57% with late
spermatids). In MA, most of the patients also had unilateral
Table III. Outcome in Sertoli cell-only syndrome (SCOS)
ICSI n (%) ELSI n (%)ROSI n (%)
Cleaved embryos (from 2PN)
Embryo grade (A?B)
No. embryos transferred (mean)
Day of embryo transfer (mean)
Cycles with embryo transfer
Fresh (F) and frozen–thawed (FT) gametes.aP ? 0.05 to ICSI (F, FT) /ELSI.
surgery (83% with sperm, 71% with late spermatids), but these
did not have enough gametes in 1–3 fragments (60% with
sperm, 42% with late spermatids). In SCOS, the majority of
the patients also had unilateral surgery (50% with sperm, 71%
with late spermatids), but of these many fewer had enough
gametes in 1–3 fragments removed (40% with sperm, 25%
with late spermatids). In all other cases needing more biopsies,
mature sperm or late spermatids were always found in the later
specimens, whereas in bilateral biopsies they were retrieved in
at least three samples from the contralateral testis. In cases
with round spermatids or absence of gamete retrieval, only
31% had an unilateral sampling, and most had more than three
specimens removed. This approach has thus enabled us to
rescue mature gametes for treatment where the diagnostic
testicular biopsy did not show any focus of late spermatids or
spermatozoa, including 47% of the cases with MA (15/32)
and 29% of the cases with SCOS (16/55). To achieve this
difficult goal of careful search for a rare focus of spermiogen-
esis, we carried out small size (3 mm) testicular biopsies,
enabling the exploration of different regions of the testis while
avoiding excessive sampling, although this was also limited
by the size and quality of the testis.
wasgivenby theobservationthatseminiferoustubules aremost
probable to contain gametes if they appear larger and opaque
(Schlegel, 1999; Amer et al., 2000). Although we did not use
microsurgery, our present series of 148 patients showed that
very often dilated and opaque seminiferous tubules contained
less, because there is no individual certainty of success, it has
also been suggested that the treatment testis biopsy should be
performed before female ovarian stimulation, with the sub-
ization and pregnancy rates (Gil-Salom et al., 1996; Romero
et al., 1996; Friedler et al., 1997; Al-Hasani et al., 1999a; Ben-
is to perform the testis biopsy at least 1–2 days before oocyte
retrieval, with the tissue being left in culture to enable a better
1996; Liu et al., 1997; Balaban et al., 1999; Sousa et al., 1999,
2000). The latter observation appears rather important, since
ization rates than motile testicular sperm (31 versus 61%),
although motile sperm were found in the majority of reported
cases, including SCOS (n ? 34, 79%), MA (n ? 26, 54%), or
hypoplasia (n ? 10, 70%) (Nagy et al., 1998a). Regarding the
present cases with microinjection, in hypoplasia most of the
biopsies followed oocyte retrieval (40; 93%), and only three
were performed 1 day (one case) or 2 days (two cases) before
oocyte retrieval. The same applied to MA cases (41 cases at the
same day: 93%; three cases 3 days before) and SCOS patients
(18 cases in the same day; five cases 1 day and three cases
2 days before). Although this data does not allow statistical
comparisons and we did not perform a quantitative study on the
percentage of gametes showing motility during the period when
motile sperm or late spermatids were found, motile gametes
when gametes were initially immotile, after the culture period
we wereable torecover forinjection atleast in-situmotile cells,
and when theyinitially exhibited only in-situmovements, in the
majority of the cases we could find slow progressively motile
gametes for injection. Similarly, we found no significant differ-
ences in the fertilization rate after injection of fresh (n ? 50,
76%) or frozen–thawed (n ? 37, 64.5%) sperm, and also after
injection of fresh (n ? 33, 55.4%) or frozen–thawed (n ? 6,
44.7%) elongated spermatids, although frozen–thawed gametes
showed a tendency for worse results. These findings were con-
firmed by pathology, except in the case of patients with
hypoplasia, where the fertilization rate with frozen–thawed
sperm was significantly lower.
Some clinical series showed that the relative frequency of
finding sperm at treatment varied according to the diagnostic
testicular biopsy, being ~25% for SCOS (n ? 6–111), 48%
M.Sousa et al.
for MA (n ? 1–76), and 74% for hypoplasia (n ? 4–86) (Jow
et al., 1993; Devroey et al., 1995; Lewin et al., 1996, 1999;
Friedler et al., 1997; Amer et al., 1999; Meng et al., 2000).
Some other studies presented a more detailed histopathological
description, and showed that SCOS and MA cases could be
subdivided into complete and incomplete syndromes. With this
subdivision, the prognosis of finding sperm at treatment
changed, with some studies showing drastic results, being 0%
in complete SCOS (n ? 3–11) or MA (n ? 2–8), and 100%
in incomplete SCOS (n ? 2–7) or MA (n ? 1–11) (Gil-Salom
et al., 1995; Yemini et al., 1995; Kahraman et al., 1996;
Mulhall et al., 1997; Ubaldi et al., 1999; Westlander et al.,
1999). However, those findings could be due to the low number
of patients studied, and thus contrasted with other larger
clinical series, where the probability of finding sperm at
treatment showed mean values of 95% in hypoplasia (n ?
10–16), 52% in complete MA (n ? 2–60), 69% in incomplete
MA (n ? 3–16), 22% in complete SCOS (n ? 6–62) and 90%
in incomplete SCOS (n ? 2–50) (Tournaye et al., 1996, 1997;
Silber et al., 1997; De Croo et al., 2000). Our present results
from 148 consecutive patients revealed very similar figures
for hypoplasia (97.7%, n ? 44), complete MA (53.3%,
n ? 15) and incomplete MA (65.6%, n ? 32), but showed
divergent results regarding SCOS cases (2.9% for complete
SCOS, n ? 35; 72.7% for incomplete SCOS, n ? 22).
However, in successful hypoplasia cycles, 82.8% (n ? 53)
had sperm for injection whereas 17.2% (n ? 11) needed late
spermatids; in MA cases the success rate was worst (61.7%,
n ? 29), with about half of the cycles using sperm (n ? 22)
and the other half using late spermatids (n ? 21); and in
SCOS cases the picture was again poor, with only 29.8% of
the patients enabling retrieval of gametes for treatment (63.2%
with sperm, n ? 12; 36.8% with elongated spermatids,
n ? 7). These differences may be due to the differing origins
of the populations. In fact, some cases of incomplete MA
showed one focus of early spermiogenesis, and this subgroup
had a similar rate of success for sperm retrieval as cases
with complete MA, whereas patients with one focus of late
spermiogenesis had a much higher chance of success (93.3%).
Similarly, incomplete SCOS cases showed two further sub-
groups, one with a focus of premeiotic cells and another with
one focus of early spermiogenesis, but these subgroups all had
a very high chance of sucess for sperm retrieval.
In relation to the clinical outcome, most of the series only
reported ICSI cycles, without specifying results per pathology
or making reference to spermatid injection cycles. In those
series, the fertilization rate after ICSI was shown to vary
between 39–69% and the cleavage rate between 66–97% (n ?
13–179), the high quality embryo rate was 56–77% (n ? 19)
and the pregnancy rate per cycle with embryo transfer showed
a mean of 33% (Devroey et al., 1995, 1996; Silber et al.,
1995; Kahraman et al., 1996; Fahmy et al., 1997; Friedler
et al., 1997; Mansour et al., 1997; Ghazzawi et al., 1998;
Houritz et al., 1998; Madgar et al., 1998; Lewin et al., 1999;
De Croo et al., 2000; Mercan et al., 2000). In our present
series of 87 ICSI cycles, the fertilization (71.4%), cleavage
(91.7%), high quality embryo (85.9%) and clinical pregnancy
(31.7%) rates were similar.
On the contrary, only a few series presented detailed clinical
outcomes after sperm injection per type of pathology. In SCOS
cases, the fertilization rate after ICSI was 38–44% (n ? 40),
the cleavage rate was 79% (n ? 22), the high quality embryo
rate was 57–61% (n ? 40), and the mean pregnancy rate per
cycle with embryo transfer was 40% (n ? 59). In cases of
MA, the fertilization rate was similar (42–46%, n ? 20), the
cleavage rate was 61% (n ? 13), the high quality embryo rate
was higher (80–86%, n ? 20), and the mean pregnancy rate
per cycle with embryo transfer was 57% (n ? 40). Finally, in
hypoplasia, the fertilization rate was higher (67%, n ? 11),
the cleavage rate was 83% (n ? 11), the high quality embryo
rate was 71% (n ? 11), and the pregnancy rate per cycle with
embryo transfer was 54–60% (n ? 46) (Tournaye et al., 1996;
Silber et al., 1996, 1997; Al-Hasani et al., 1999a; De Croo
et al., 2000). These rates support previous findings (Nagy
et al., 1998b), which showed no significant differences between
ejaculated and testicular sperm regarding timing of oocyte
activation, pronucleus formation and embryo cleavage. In our
87 ICSI cycles, the rates of fertilization, cleavage and high
embryo quality were relatively higher in all three syndromes
(63.5, 85, 88.2% respectively in SCOS, n ? 12; 66.3, 84.1,
86.2% respectively in MA, n ? 22; and 74.8, 95.5, 85.4%
respectively in hypoplasia, n ? 53), although the clinical
pregnancy rate was lower (20% in SCOS, 35% in MA, 32.1%
in hypoplasia), showing a similar rate to the largest series of
cycles using either ejaculated or testicular sperm injection
(Mansour, 1998; Bonduelle et al., 1999).
Including the largest clinical series presented by our group
(n ? 59), the analysis of all reported cases using elongated
spermatid injections (n ? 166) demonstrates that, in compar-
ison with ICSI, late spermatids appear to be associated with a
significantly lower fertilization rate (48.4%), but relatively
similar cleavage (90%) and pregnancy (28.9%) rates (Table
IV) (Fishel et al., 1995, 1996; Tesarik et al., 1995, 1996, 1999;
Chen et al., 1996; Mansour et al., 1996; Amer et al., 1997;
Antinori et al., 1997a; Araki et al., 1997; Vanderzwalmen
et al., 1997; Barak et al., 1998; Bernabeu et al., 1998;
Kahraman et al., 1998; Sofikitis et al., 1998c; Al-Hasani et al.,
1999b; Sousa et al., 1999). However, the present analysis by
type of pathology did not show significant differences between
ICSI and ELSI cycles, with either fresh or frozen–thawed
gametes, except in the fertilization rate in hypoplasia cases.
Although two cases of congenital malformations and chromo-
some aneuploidy were recently described (Zech et al., 2000),
the overall results do not signal an increase of risk in
comparison with ejaculated or testicular sperm (Tarlatzis and
On the contrary, results from intact round spermatid injec-
tions (n ? 249), including the largest clinical series from our
group (n ? 91), show significantly lower fertilization (21.8%)
and pregnancy (2.8%) rates (Table V) (Tesarik et al., 1995,
1996, 1999; Amer et al., 1997; Antinori et al., 1997a,b;
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). These findings
suggest that the presence of an oocyte-activating substance in
round spermatids (Sousa et al., 1996) is not enough to
Table IV. Review of outcome in elongated spermatid injection (ELSI) cycles
Fishel et al., 1995, 1996
Tesarik et al., 1995, 1996
Chen et al., 1996
Mansour et al., 1996
Amer et al., 1997
Araki et al., 1997
Antinori et al., 1997a
Vanderzwalmen et al., 1997
Barak et al., 1998
Bernabeu et al., 1998
Kahraman et al., 1998
Sofikitis et al., 1998
Al Hasani et al., 1999b
Sousa et al., 1999
Tesarik et al., 1999
aCase of obstructive azoospermia without sperm in epididymus.
bFour ELSI cycles (two liveborn) ? 11 mixed ELSI/ROSI cycles.
Table V. Review of outcome in round spermatid injection (ROSI) cycles
Author Cycles MII
Tesarik et al., 1995, 1996
Amer et al., 1997
Antinori et al., 1997a,b
Vanderzwalmen et al., 1997
Yamanaka et al., 1997
Barak et al., 1998
Bernabeu et al., 1998
Kahraman et al., 1998
Al Hasani et al., 1999
Sousa et al., 1999
Tesarik et al., 1999
aPatients with previous late spermatids/sperm in ejaculates.
bPatients without previous late spermatids/sperm in ejaculates/diagnostic testicular biopsy.
ensure proper fertilization, and confirm the low developmental
potential of the embryos derived by this method (Aslam and
Fishel, 1999; Balaban et al., 2000). Injection of intact round
spermatids was originally proposed due to the uncertainty
about the position of the proximal centriole and of the site of
concentration of the oocyte-activating substance. In the human,
the proximal centriole, which is responsible for centrosome
inheritance and for the zygote and embryo centrosome cycle
(Sousa and Tesarik, 1994; Tesarik et al., 1998; El Shafie et al.,
2000), is firmly attached to the basal nuclear envelope only at
a late point in the round spermatid stage (Holstein and Roosen-
Runge, 1981), whereas the oocyte-activating substance is
known to be concentrated much later at the equatorial region
of the spermatozoon head (Parrington et al., 1996; Heyers
et al., 2000). However, data from animal experiments have
shown that an alternative procedure, round spermatid nucleus
injection (ROSNI), is superior to injection of intact round
spermatids (Ogura et al., 1993; Sofikitis et al., 1994, 1996a,b,
1998b,c; Kimura and Yanagimachi, 1995; Yamamoto et al.,
1999a). Although successful ROSNI data as applied to humans
remains scarce, it may prove to be a better alternative to ROSI,
although most pregnancies ended in abortions (Hannay et al.,
1995; Tanaka et al., 1996; Sofikitis et al., 1996a, 1997,
1998b,c). Notwithstanding, experiments in the human showed
that ROSI is not adversely affected by the use of a slightly
larger microinjection pipette (as shown by the very low oocyte
degeneration rate: Tables II, III), nor by the presence of a
larger cytoplasmic layer surrounding the round spermatid
nucleus (rupture of the cytoplasmic membrane and of the
nuclear envelope is rapidly achieved after microinjection)
M.Sousa et al.
(Sousa et al., 1996). Similarly, the human round spermatid has
been shown to harbour an oocyte-activating substance that is
already capable of inducing free calcium intracellular oscilla-
tions (Sousa et al., 1996), a fact also proven by recent
successful secondary spermatocyte injections followed by
healthy human births (Sofikitis et al., 1998a). On the contrary,
in-vitro culturing of round spermatids (Cremades et al., 1999,
2001; Sousa et al., 2002) suggests that the low developmental
potential of such cells may instead be due to a genetic block
(Aslam et al., 1998; Tesarik et al., 1998; Sousa et al., 2000).
Other authors also suggested a new culture medium for
rendering round spermatids more viable (Sofikitis et al., 1994,
1996a,b, 1997, 1998a,b,c; Yamamoto et al., 1999a). However,
the medium used here has all the supplements required for a
short (24 h) culture period (lactate, glucose, iron, vitamins and
synthetic serum substitute). Furthermore, long-term in-vitro
media were also unable to induce a proper maturation and
developmental potential of round spermatids (Cremades et al.,
1999, 2001; Sousa et al., 2002). These findings thus strongly
suggest that isolated round spermatids retrieved from cases of
complete MA or from cases of SCOS with a focus of germ
cells arrested at meiosis may represent occasional events of
meiosis completion, with round spermatids still harbouring
important genetic defects responsible for their poor develop-
mental competence, and that for these cases better treatment
alternatives should be sought (Tesarik et al., 2001).
Note added in proof
All ongoing pregnancies from HP, MA and SCOs have resulted in
the birth of healthy babies.
We acknowledge the gynaecological collaboration of Professors
J.Beires and N.Montenegro. This work was partially supported by
FCT (Sapiens: 36363/99, 35231/99; UMIB).
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Submitted on May 1, 2001; resubmitted on November 8, 2001; accepted on
February 19, 2002