FERTILITY AND STERILITY威
VOL. 72, NO. 5, NOVEMBER 1999
Copyright ©1999 American Society for Reproductive Medicine
Published by Elsevier Science Inc.
Printed on acid-free paper in U.S.A.
Received February 2, 1999;
revised and accepted May
21, 1999.
Reprint requests: Michael
Scholtes, M.D., Ph.D.,
Center for Reproductive
Medicine, Genetics and
Clinical Chemistry,
Völklingerstrasse 4, 40219
Düsseldorf, Germany (Fax:
49-211-9019750).
* In Vitro Fertilization
Department, Center for
Reproductive Medicine,
Genetics and Clinical
Chemistry.
†
In Vitro Fertilization
Department, Rijnstate
Hospital.
‡
Department of
Endocrinology and
Reproduction, Erasmus
University.
0015-0282/99/$20.00
PII S0015-0282(99)00359-3
MALE INFERTILITY
Extraction of testicular sperm from
previously cryopreserved tissue in couples
with or without transport of oocytes and
testicular tissue
Michael C. W. Scholtes, M.D., Ph.D.,* Dagmar G. van Hoogstraten, M.D.,*
Alex Schmoutziguer, M.D.,† and Gerard H. Zeilmaker, Ph.D.‡
Center for Reproductive Medicine, Genetics and Clinical Chemistry, Düsseldorf, Germany; Rijnstate Hospital,
Arnhem; and Erasmus University, Rotterdam, the Netherlands
Objective: To evaluate results of IVF and intracytoplasmic sperm injection (ICSI) with extraction of sperm
from frozen-thawed testicular tissue.
Design: Retrospective follow-up study.
Setting: Fertility center.
Patient(s): Thirty-five couples with transport of testicular tissue from a transport clinic and 125 local couples.
Intervention(s): Extraction of testicular sperm by maceration and enzymatic digestion from frozen-thawed
testicular tissue before ICSI.
Main Outcome Measure(s): Clinical pregnancy rate (PR) and implantation rate in couples with obstructive
or nonobstructive azoospermia, motile or immotile sperm, and differing male serum FSH values.
Result(s): The clinical PR per ET and implantation rate per embryo in couples with transport of testicular
tissue were 40% and 18%, respectively, in cases of obstructive azoospermia and 37% and 26%, respectively,
in cases of nonobstructive azoospermia. In the local couples, these rates were 42% and 19%, respectively, in
cases of obstructive azoospermia and 18% and 10%, respectively, in cases of nonobstructive azoospermia. The
implantation rates for ICSI were 26% with motile sperm and 11% with immotile sperm in the transport group
and 16% and 8%, respectively, in the local group. Male serum FSH level did not clearly correlate with
implantation rate.
Conclusion(s): Clinical PR and implantation rate are not affected by transport of testicular tissue but are
significantly affected by nonobstructive azoospermia and the use of immotile sperm. No major increase in
chromosomal aberration or congenital malformation was noted in the offspring of this limited group. (Fertil
Steril威 1999;72:785–91. ©1999 by American Society for Reproductive Medicine.)
Key Words: TESE, frozen-thawed, FSH, sperm motility, follow-up, chromosomes, transport
Intracytoplasmic sperm injection (ICSI) tive azoospermia, spermatogenic activity was
with surgically retrieved sperm (1, 2) was in- documented. Shortly after, favorable results
troduced for the treatment of obstructive and were obtained with ICSI with epididymal sper-
nonobstructive azoospermia. The presence of matozoa. Testicular sperm cells were isolated
spermatogenesis is usually obvious in cases of for ICSI after shredding of a testicular biopsy
obstructive azoospermia but uncertain in cases specimen, a process called testicular sperm
extraction (TESE).
of secretory azoospermia. Steinberger and
Tjioe (3) and Silber and Rodriguez-Rigau (4) The uncertainty that spermatozoa would be
showed focal spermatogenesis in cases of se- available at the time of ovum pick-up for ICSI
cretory azoospermia. In another study, Silber et and TESE in cases of azoospermia led us to
al. (5) established histologically that when four modify this method. This technique of extrac-
to six mature spermatids are visible per tubu- tion of spermatozoa by enzymes and macera-
lus, sperm cells are no longer available in the tion from frozen-thawed testicular tissue was
ejaculate. In 60% of these cases of nonobstruc- described by Salzbrunn et al. (6). With this
785
method, unnecessary stimulation and ovum pick-up in the
female patient are prevented because thawing and sperm
extraction occur at the time of ovum pick-up, after previous
verification of spermatogenic activity and subsequent freez-
ing of a testicular biopsy specimen (cryo-TESE).
Dutch law forbids the use of ICSI with surgically re-
trieved sperm. This law has resulted in couples’ traveling to
surrounding countries where these regulations are not in
force (e.g., Germany). The transport of oocytes for IVF and
ICSI is common in the Netherlands, where only 12 licensed
IVF centers provide services for approximately 16 million
inhabitants. Local experience showed that transport of tes-
ticular tissue is feasible and sperm cells can be retrieved for
ICSI.
The aim of this study was to compare clinical pregnancy
rates (PRs) and implantation rates with cryo-TESE in cou-
ples with or without transport of testicular tissue and oo-
cytes, as well as to compare these rates with rates reported in
with the literature that were the result of extraction of tes-
ticular sperm from fresh tissue. The effect of sperm motility
on ICSI and the effect of male FSH levels on PR and
implantation rate were also investigated.
MATERIALS AND METHODS
Patients With Transport of Oocytes and
Testicular Tissue
Between April 1995 and March 1998, cryo-TESE was
performed in 35 couples with transport of testicular tissue
and oocytes. Eighteen of the male patients had obstructive
azoospermia and 17 had nonobstructive azoospermia. The
mean (⫾SD) age of the women was 33.4 ⫾ 4.9 years (range,
18 – 42 years) and the mean (⫾SD) age of the men was
39.5 ⫾ 8.0 years (range, 27–56 years). Transport by car took
90 –150 minutes. Forty cycles with cryo-TESE were per-
formed in couples with obstructive azoospermia and 30
cycles were performed in couples with nonobstructive
azoospermia. Institutional review board approval was not
required for this study. All patients gave informed consent.
The cause of obstructive azoospermia in most cases was
failed reanastomosis of the vas deferens after sterilization,
infection, or surgery. In six cases, congenital bilateral aplasia
of the vas deferens was diagnosed.
Local Patients
The group of local patients consisted of 125 couples, 26
with obstructive azoospermia and 99 with nonobstructive
azoospermia. In the couples with obstructive azoospermia,
the mean age of the women was 31.8 ⫾ 4.4 years (range,
26 – 41 years) and the mean (⫾SD) age of the men was
39.6 ⫾ 7.2 years (range, 26 –55 years). In the couples with
nonobstructive azoospermia, the mean age of the women
was 32.0 ⫾ 4.1 years (range, 19 – 43 years) and the mean
(⫾SD) age of the men was 35.9 ⫾ 6.7 years (range, 36 –56
years). Forty-five cycles with cryo-TESE were performed in
786 Scholtes et al. TESE and cryopreserved tissue
couples with obstructive azoospermia and 186 cycles were
performed in couples with nonobstructive azoospermia.
In the local patients with obstructive azoospermia, only
secondary obstruction by surgery or infection was present. In
the nonobstructive group, 47 men had idiopathic azoosper-
mia, 22 had unilateral or bilateral cryptorchism, and 10 had
been treated for testicular neoplasia. The remaining 20 cou-
ples had miscellaneous causes of secretory azoospermia.
Cytogenetic analysis was performed in all local couples
before ovarian hyperstimulation was begun. In three female
patients, numerical sex chromosomal aberrations were
found, all mosaics with up to 10% abnormal cells, and in one
patient a translocation— 46,XX,t(2;19)—was found. In the
men, one case of Klinefelter’s syndrome was found and one
robertsonian translocation, 46,XY,Rob t(13;14); one recip-
rocal translocation, 46XY,t(4;19)(q34,q13.3); and one inver-
sion, 46,XY,inv(2)(q21q22) were found. Follicle-stimulating
hormone levels were measured with the use of a two-site
immunoassay (Chiron Diagnostics Corporation, East Wal-
pole, MA); reference values were 0.7–11.1 in males.
Processing of Biopsy Material and ICSI
A testicular biopsy specimen was obtained in Rijnstate
Hospital in Arnhem, the Netherlands, and sent by car to the
Center for Reproductive Medicine, Genetics and Clinical
Chemistry in Düsseldorf, Germany. This trip of ⬎130 km in
heavy traffic takes up to 2–3.5 hours. The specimen was
transported in a closed test tube with standard culture me-
dium. The test tube was taped to abdominal skin or placed in
a car battery– heated box. One drop of heparin was added in
cases of blood contamination of the sample. The processing
on arrival was identical to that of the testicular material from
the local patients.
The supernatant was checked for sperm cells, and the
tissue was divided into small aliquots for cryopreservation in
Sperm-Freeze medium (Medicult, Hamburg, Germany) in
liquid nitrogen, according to a computerized sperm-freezing
protocol. Incubation for 30 minutes in the Sperm-Freeze
medium was followed by slow cooling for 10 minutes and
subsequent rapid cooling to ⫺140°C, the final temperature
being ⫺160° to ⫺170°C. The sealed vials (Nalge Nunc
International, Rochester, NY) were stored in liquid nitrogen.
In a number of cases, histology was performed with the use
of a semithin technique at the University of Hamburg (Prof.
W. Schulze, Department of Andrology, Academic Hospital,
Hamburg, Germany).
In a water bath at 37°C, the specimen was thawed and
washed in a Petri dish with culture medium (37°C). After
mechanical maceration for 5 minutes with insulin injection
needles, the tissue was digested; 10 ␮L (40 IU/mL) from a
100⫻ stock solution of collagenase type IV-S (Sigma, Hei-
delberg, Germany) was added, and the tissue was left for 2
hours. The supernatant was centrifuged for 10 minutes at
1800 ⫻ g after removal of the tissue remnants. The pellet
Vol. 72, No. 5, November 1999
 TABLE 1

Results of cryo-TESE cycles with or without long-distance transport of testicular tissue and oocytes.

No. of Pronuclear No. of No. of clinical Implantation

No. of MII No. of embryos embryos pregnancies rate per
Group cycles oocytes ETs (%) per ET per ET (%) embryo (%)

Patients with transport of testicular tissue

Obstructive azoospermia 40 282 35 42 2.3 14 (40) 18*
Nonobstructive azoospermia 30 232 24 24 1.9 9 (37) 26*
Local patients
Obstructive azoospermia 45 295 38 36 2.2 16 (42) 19†
Nonobstructive azoospermia 186 1,234 152 31 2.1 28 (18) 10†
Note: cryo-TESE ⫽ extraction of testicular sperm from cryopreserved tissue; MII ⫽ metaphase II.
* P⫽.1.
† P⫽.02.
Scholtes. Extraction of testicular sperm. Fertil Steril 1999.

was dissolved in 1 mL of culture medium and incubated for
at least 2 hours. After centrifuging and shortly before ICSI,
the pellet was pipetted into a drop of culture medium in a
Petri dish also containing a drop of polyvinylpyrrolidone and
a number of culture medium drops for individual oocytes.
The sperm cells were selected from the drop of culture
medium and put into the polyvinylpyrrolidone before ICSI.
Heart activity determined by ultrasound (US) ⱖ4 weeks
after ET was considered to indicate clinical pregnancy.
Statistical analysis was performed with use of the ␹2 test.
RESULTS
Seventy ICSI cycles with cryo-TESE were performed in
the group with transport of testicular tissue and oocytes.
Forty of these cycles were performed in couples with ob-
structive azoospermia (Table 1). A total of 282 metaphase II
oocytes were retrieved, and 42% were normally fertilized.
The mean number of embryos per transfer was 2.3, the
clinical PR per ET (n ⫽ 35) was 40%, and the implantation
rate per embryo was 18%.
A total of 232 metaphase II oocytes in the transport group
with nonobstructive azoospermia, and 24% were normally
fertilized (P⫽.001). The mean number of embryos per
transfer on day 2 or 3 was 1.9, the clinical PR per ET (n ⫽
24) was 37%, and the implantation rate per embryo was 26%
(P⫽.1).
The local couples underwent 231 ICSI cycles with cryo-
TESE: 45 in couples with obstructive azoospermia and 186
in couples with nonobstructive azoospermia. In the former
group, 36% of the 295 metaphase II oocytes were normally
fertilized. The mean number of embryos per transfer was 2.2,
the clinical PR per ET (n ⫽ 38) was 42%, and the implan-
tation rate per embryo was 19%. In the latter group, 31% of
the 1,234 metaphase II oocytes were normally fertilized
(P⫽.13), 28 clinical pregnancies were established in 152
ETs (18% per ET), and the implantation rate per embryo was
10%.
The motility of sperm cells extracted from previously
cryopreserved testicular tissue after enzymatic and mechan-
ical maceration proved to be essential. The rates of normal
fertilization in the transport group in couples with sperm
motility (n ⫽ 56 cycles) and in those without sperm motility
(n ⫽ 23 cycles) were 40% and 25%, respectively (P⫽.06),
and the implantation rates per embryo were 26% and 11%,
respectively (P⫽.001). In the local group, the rates of
normal fertilization with TESE were 43% (n ⫽ 101 cy-
cles) in couples with motile spermatozoa and 22% (n ⫽ 80
cycles) in couples with immotile spermatozoa (P⫽.04),
and the implantation rates were 16% and 8%, respectively
(P⫽.001).
The effect of male serum FSH level was investigated in
the local patients. The patients were divided into patients
with obstructive azoospermia and those with nonobstructive
azoospermia and these groups were divided further into
subgroups with serum FSH levels of ⬍6, 6 –9, 10 –19, and
⬎19 IU/L. Table 2 shows that there was no correlation
between implantation rate and male serum FSH level.
Data regarding the pregnancies resulting from the use of
ICSI with sperm extracted from previously cryopreserved
testicular tissue are listed in Table 3. Of 67 clinical pregnan-
cies, 17 (25%) ended with spontaneous abortion. In the case
of two pregnancies, the women were lost to follow-up.
Forty-seven children were born; one pregnancy was termi-
nated because of chromosomal aberration. One set of twins
was born in the 24th week of gestation. A singleton was
delivered in the 28th week and a triplet in the 30th week by
cesarean delivery.
DISCUSSION
The objective of this study was to evaluate the results of
TESE with or without transport and subsequent cryopreser-

FERTILITY & STERILITY威 787

 TABLE 2

Effect of serum FSH levels on implantation rate in local couples who underwent extraction of sperm from previously
cryopreserved testicular tissue.

Total No. of Clinical Clinical

Serum FSH No. of No. of no. of clinical pregnancy rate pregnancy rate Implantation rate
Group level (IU/L) cycles ETs embryos pregnancies per cycle (%) per ET (%) per embryo (%)

Obstructive azoospermia group ⬍6 28 25 52 6 21.4 24 11.5

6–9 7 6 14 5 71.4 83.3 57.1
Nonobstructive azoospermia group ⬍6 51 41 88 8 15.7 19.5 10.2
6–9 32 25 50 3 9.4 12.0 6.0
10–19 51 37 81 10 19.6 27.0 17.3
⬎19 29 27 57 2 6.9 7.4 3.5

Scholtes. Extraction of testicular sperm. Fertil Steril 1999.

vation of testicular tissue before ICSI. Transport of oocytes
after ovum pick-up to a central IVF laboratory proved to be
a reliable way to circumvent the limited capacity of IVF
centers in the Netherlands (7). After the Dutch government
banned the use of testicular and epididymal sperm for ICSI,
it was obvious that couples with azoospermia would look for
treatment with TESE abroad. The use of testicular and epi-
didymal sperm for ICSI was and still is prohibited by law in
the Netherlands because of the lack of relevant offspring data
from animal experiments. The nearest countries that offer
treatment with TESE are Germany and Belgium.
Transport of testicular tissue was initiated and continued
because it appeared that in many cases, motile sperm could
be isolated either from the supernatant or from the testicular
tissue after long-distance transportation. In our study, in
cases in which no sperm could be extracted and no spermat-
ogenesis could be identified histologically, we refrained
from administering further treatment. In this way we were
able to prevent useless ovarian hyperstimulation, because in
TABLE 3
Follow-up results of pregnancies after ICSI with sperm
retrieved from previously cryopreserved testicular tissue.
Clinical pregnancy rate (%)
No. (%) of ongoing pregnancies
No. (%) of births
No. (%) of singleton pregnancies
No. (%) of twins
No. (%) of triplets
No. of women who underwent amniocentesis or
chorion villus sampling
No. of cases of chromosomal aberration
No. of cases of congenital malformation
Note: ICSI ⫽ intracytoplasmic sperm injection.
* 47,XY,⫹mar.
† Tuberous sclerosis.
Scholtes. Extraction of testicular sperm. Fertil Steril 1999.
all cryo-TESE cycles, sperm could be retrieved from thawed
testicular tissue samples.
Extraction of testicular sperm from previously cryopre-
served material is still not a common treatment in cases of
azoospermia. The decision to use testicular sperm rather than
epididymal sperm in couples with obstructive azoospermia
depended on the accessibility of epididymal sperm, the his-
tory of previous epididymal surgery (e.g., reversal of steril-
ization), and patient consent. The low-cost, outpatient TESE
was preferred in most cases to microepididymal sperm as-
pirational (MESA) which is not paid for by insurance com-
panies, requires microsurgical skills, and is not an in-office
procedure. Patients undergoing reversal of sterilization usu-
ally are offered MESA first.
Few reports on TESE are available, and usually cryo-
preservation is performed after (8 –13) rather than before
(14 –16) extraction of sperm cells. Our study provides data
on the clinical use of cryo-TESE in a limited group of
couples with azoospermia. A comparison of our method with
extraction of sperm from fresh testicular tissue is possible
only with a review of the literature (Table 4) because in our
study, only extraction of sperm from frozen-thawed testicu-
lar tissue was performed. Fertilization rate and embryo
implantation were affected by the composition of the groups
studied. Tournaye (36) demonstrated the link between the
67 type of azoospermia and subsequent fertilization and implan-
13 (19) tation. Therefore, one should be cautious about comparing
36 (51) overall fertilization and implantation rates of different study
26 (37) populations.
9 (13)
1 (1.5) Nevertheless, the PRs and rates of implantation per em-
21 bryo in our groups seem to be in the same range as the rates
found in the studies listed in Table 4. Furthermore, the
1*
1† results with and those without transport in the couples with
obstructive azoospermia do not seem to be different (Table
1). The clinical significance of increased serum FSH levels
in the man is limited, and these levels are not automatically
increased in all cases of azoospermia. Although a high level

788 Scholtes et al. TESE and cryopreserved tissue Vol. 72, No. 5, November 1999
 TABLE 4

Testicular sperm extraction (TESE) and subsequent intracytoplasmic sperm injection (ICSI).

No. of
No. of Motile Normal No. of biochemical No. of clinical Implantation
No. of positive No. of spermatozoa fertilization No. of embryos pregnancies pregnancies rate per Abortion
Reference Type of azoospermia patients cycles* cycles (%) rate (%)† ETs per ET (% per ET) (% per ET) embryo (%) rate (%)

FERTILITY & STERILITY威

Craft et al. (17) Obstructive‡ 1 1 1 ⫹ 46 — — — — — —
Schoysman et al. (2) Obstructive/nonobstructive‡ 6 6 1 ⫹ 45§ 6 1.6 2 (33) 1 (16) 20 —
Silber et al. (18) Obstructive㛳 12 — 12 — 46 9 — — 5 (42) — —
Devroey et al. (19) Obstructive‡ 3 3 3 ⫹ 44 3 3.6 — — — —
Devroey et al. (20) Nonobstructive‡ 15 13 13 ⫹ 48 12 2.7 3 (23) 3 (23) 18 —
Bourne et al. (21) Obstructive‡㛳¶ 6 6 6 — 71 6 2.3 — 2 (33) 14 —
Tucker et al. (22) Obstructive㛳¶ 7 7 7 ⫹ 38 7 3.3 — 4 (57) 17 —
Nagy et al. (23) Obstructive/nonobstructive㛳 — — 17 ⫹ 48 13 2.5 6 (46) 5 (39) — ⫾38
Tournaye et al. (24) Nonobstructive**††‡‡§§㛳㛳¶¶ 38 32 32 ⫹ 57 32 2.7 11 (34) — — ⱖ12
Silber et al. (25) Nonobstructive‡‡¶¶ — — 32 — 49 27 — — 14 (52) — 29
Silber et al. (26) Obstructive㛳¶ — — 12 — 46 9 — 6 (67) 5 (56) 23 17
Hovatta et al. (27) Obstructive/nonobstructive††† 23 20 21 ⫹ 34*** 19 — 6 (32) 4 (21) 22 50
Gil-Salom et al. (28) Obstructive/nonobstructive㛳¶††¶¶ 21 21 21 ⫹ (83) 59 19 3.0 — 7 (36) 14 29
Tournaye et al. (29) Obstructive/nonobstructive¶**††‡‡§§㛳㛳 124 114 124 ⫹ (59) 58 103 2.8 45 (44) — 20 —
Kahraman et al. (30) Nonobstructive††‡‡§§㛳㛳 29 14 29 ⫾ 39 11 3.3 6 (54) — 22 17
Tournaye et al. (31) Nonobstructive‡‡§§㛳㛳 372 279 — — 53 255 — 81 (32) — — 27
Aboulghar et al. (32) Obstructive/nonobstructive 153 133 162 ⫹ 47 133 3.6 39 (29) — — 10
Abuzeid et al. (33) Obstructive‡¶ 17 — 19 — 60 19 4.1 — 6 (32) 12 17
Cha et al. (34) Not indicated — — 45 — 70 43 — 18 (42) — — 12
Dohle et al. (35) Obstructive‡¶ — — 11 — 36 9 — 4 (36) 4 (36) — —
Note: ET ⫽ embryo transfer.
* Spermatozoa were available after TESE or testicular sperm aspiration (TESA).
† Rate of production of embryos with two pronuclei.
‡ Other or idiopathic.
§ Subzonal insemination or ICSI.
㛳 Infection or vasoepididymal surgery.
¶ Congenital bilateral aplasia of the vas deferens.
** Normal histologic findings.
†† Histologic hypospermatogenesis.
‡‡ Incomplete maturation arrest.
§§ Maturation arrest.
㛳㛳 Germ cell aplasia.
¶¶ Germ cell aplasia, focal spermatogenesis.
*** TESE or TESA.
††† Previous testicular histologic findings.
Scholtes. Extraction of testicular sperm. Fertil Steril 1999.

789
indicates impaired spermatogenesis and decreased likelihood
of finding sperm cells when TESE is performed (37), no
definitive prognosis with regard to establishment of a preg-
nancy seems possible when sperm cells are available for
ICSI (Table 2).
The motility of sperm cells before ICSI also proved to be
of paramount importance in our study, as it was in other
studies (38). Because motility may occur only after longer
incubation (39), we usually culture overnight before ovum
pick-up, after thawing and preparation of the testicular sam-
ple. Prolonged culture of testicular tissue might result in
more motile sperm cells available for microinjection (40). In
this way, the possible impairment of motility by cryopreser-
vation of testicular material may be counterbalanced.
Follow-up of offspring after use of new assisted repro-
ductive techniques is compulsory. Preconceptional genetic
counseling is offered to the couple before treatment. The
possible genetic risks are emphasized and informed consent
is obtained. In daily practice, relatively few couples decide
to undergo prenatal fetal karyotyping. In this study, only
36% of the children had undergone prenatal cytogenetic
analysis; one pregnancy was terminated because of chromo-
somal aberration (Table 3). In another fetus, a congenital
malformation was established by US, but this pregnancy was
not terminated. These data are comparable with findings of a
follow-up study of MESA and extraction of testicular sperm
from fresh material (41), in which with great effort, exten-
sive follow-up was done. It appears to be difficult to moti-
vate couples to participate in follow-up programs, as was
also found by Meschede et al. (42).
Until now, a significant increase in the incidence of
cytogenetic disorders and congenital malformations has not
been documented. In contrast with the detection of malfor-
mations by routine US screening during pregnancy, the
detection of cytogenetic disorders will always be impaired,
because of low acceptance of prenatal and postnatal karyo-
typing. Follow-up studies of TESE generally lack sufficient
statistical power because the numbers of individuals studied
are still small.
In conclusion, extraction of sperm from previously cryo-
preserved testicular tissue and subsequent ICSI are prefera-
ble to extraction of sperm from fresh testicular tissue and
ICSI, because unnecessary ovarian stimulation and ovum
pick-up are prevented. Furthermore, it is advantageous for
couples living far from a central IVF laboratory to transport
testicular tissue and, eventually, oocytes. More follow-up
data from patients with pregnancies after TESE are essential
before more definitive conclusions can be drawn about pos-
sible chromosomal disorders or congenital malformations
originating from this assisted reproductive technique.
References
1. Silber SJ, Nagy ZP, Liu J, Godoy H, Devroey P, Van Steirteghem AC.
Conventional in-vitro fertilization versus intracytoplasmic sperm injec-
790 Scholtes et al. TESE and cryopreserved tissue
tion for patients requiring microsurgical sperm aspiration. Hum Reprod
1994;9:1705–9.
2. Schoysman R, Vanderzwalmen P, Nijs M, Segal L, Segal-Bertin G,
Geerts L, et al. Pregnancy after fertilisation with human testicular
spermatozoa [letter]. Lancet 1993;342:1237.
3. Steinberger E, Tijoe DY. A method for quantitative analysis of human
seminiferous epithelium. Fertil Steril 1968;19:959 – 61.
4. Silber SJ, Rodriguez-Rigau LJ. Quantitative analysis of testicle biopsy:
determination of partial obstruction and prediction of sperm count after
surgery for obstruction. Fertil Steril 1981;36:480 –5.
5. Silber SJ, Nagy Z, Devroey P, Tournaye H, Van Steirtegham AC.
Distribution of spermatogenesis in the testicles of azoospermic men: the
presence or absence of spermatids in the testes of men with germinal
failure. Hum Reprod 1997;12:2422– 8.
6. Salzbrunn A, Benson DM, Holstein AF, Schulze W. A new concept for
the extraction of testicular spermatozoa as a tool for assisted fertiliza-
tion (ICSI). Hum Reprod 1996;11:752–5.
7. Jansen CA, van Beek JJ, Verhoeff A, Alberda AT, Zeilmaker GH.
In-vitro fertilisation and embryo transfer with transport of oocytes
[letter]. Lancet 1986;1:676.
8. Oates RD, Mulhall J, Burgess C, Cunningham D, Carson R. Fertiliza-
tion and pregnancy using intentionally cryopreserved testicular tissue as
the sperm source for intracytoplasmic sperm injection in 10 men with
non-obstructive azoospermia. Hum Reprod 1997;12:734 –9.
9. Romero J, Remohi J, Minguez Y, Rubio C, Pellicer A, Gil-Salom M.
Fertilization after intracytoplasmic sperm injection with cryopreserved
testicular spermatozoa. Fertil Steril 1996;65:877–9.
10. Gil-Salom M, Romero J, Minguez Y, Rubio C, De los Santos MJ,
Remohi J, et al. Pregnancies after intracytoplasmic sperm injection with
cryopreserved testicular spermatozoa. Hum Reprod 1996;11:1309 –13.
11. De Croo I, Van der Elst J, Everaert K, De Sutter P, Dhont M. Fertili-
zation, pregnancy and embryo implantation rates after ICSI with fresh
or frozen-thawed testicular spermatozoa. Hum Reprod 1998;13:
1893–7.
12. Podsiadly BT, Woolcott RJ, Stanger JD, Stevenson K. Pregnancy
resulting from intracytoplasmic injection of cryopreserved spermatozoa
recovered from testicular biopsy. Hum Reprod 1996;11:1306 – 8.
13. Perraguin-Jayot S, Audebert A, Emperaire JC, Parneix I. Ongoing
pregnancies after intracytoplasmic injection using cryopreserved testic-
ular spermatozoa. Hum Reprod 1997;12:2706 –9.
14. Fischer R, Baukloh V, Naether OG, Schulze W, Salzbrunn A, Benson
DM. Pregnancy after intracytoplasmic sperm injection of spermatozoa
extracted from frozen-thawed testicular biopsy. Hum Reprod 1996;11:
2197–9.
15. Hovatta O, Foudila T, Siegberg R, Johansson K, von Smitten K, Reima
I. Pregnancy resulting from intracytoplasmic injection of spermatozoa
from a frozen-thawed testicular biopsy specimen. Hum Reprod 1996;
11:2472–3.
16. Khalifeh FA, Sarraf M, Dabit ST. Full-term delivery following intra-
cytoplasmic sperm injection with spermatozoa extracted from frozen-
thawed testicular tissue. Hum Reprod 1997;12:87– 8.
17. Craft I, Bennett V, Nicholson N. Fertilising ability of testicular sper-
matozoa [letter]. Lancet 1993;342:864.
18. Silber SJ, Devroey P, Nagy Z, Tournaye H, Van Steirteghem A.
Micro-epididymal sperm aspiration with epididymal versus testicular
biopsy spermatozoa. Hum Reprod 1994;9(Suppl 4):49.
19. Devroey P, Liu J, Nagy Z, Tournaye H, Silber SJ, Van Steirteghem AC.
Normal fertilization of human oocytes after testicular sperm extraction
and intracytoplasmic sperm injection. Fertil Steril 1994;62:639 – 41.
20. Devroey P, Liu J, Nagy Z, Goossens A, Tournaye H, Camus M, et al.
Pregnancies after testicular sperm extraction and intracytoplasmic
sperm injection in non-obstructive azoospermia. Hum Reprod 1995;10:
1457– 60.
21. Bourne H, Richings N, Harari O, Watkins W, Speirs AL, Johnston WI,
et al. The use of intracytoplasmic sperm injection for the treatment of
severe and extreme male infertility. Reprod Fertil Dev 1995;7:237– 45.
22. Tucker MJ, Morton PC, Witt MA, Wright G. Intracytoplasmic injection
of testicular and epididymal spermatozoa for treatment of obstructive
azoospermia. Hum Reprod 1995;10:486 –9.
23. Nagy Z, Liu J, Cecile J, Silber S, Devroey P, Van Steirteghem A. Using
ejaculated, fresh, and frozen-thawed epididymal and testicular sperma-
tozoa gives rise to comparable results after intracytoplasmic sperm
injection. Fertil Steril 1995;63:808 –15.
24. Tournaye H, Camus M, Goossens A, Liu J, Nagy P, Silber S, et al.
Recent concepts in the management of infertility because of non-
obstructive azoospermia. Hum Reprod 1995;10(Suppl 1):115–9.
25. Silber SJ, Nagy Z, Liu J, Tournaye H, Lissens W, Ferec C, et al. The
use of epididymal and testicular spermatozoa for intracytoplasmic
sperm injection: the genetic implications for male infertility. Hum
Reprod 1995;10:2031– 43.
26. Silber SJ, Van Steirteghem AC, Liu J, Nagy Z, Tournaye H, Devroey
P. High fertilization and pregnancy rate after intracytoplasmic sperm
Vol. 72, No. 5, November 1999
 injection with spermatozoa obtained from testicle biopsy. Hum Reprod
1995;10:148 –52.
27. Hovatta O, Moilanen J, von Smitten K, Reima I. Testicular needle
biopsy, open biopsy, epididymal aspiration and intracytoplasmic sperm
injection in obstructive azoospermia. Hum Reprod 1995;10:2595–9.
28. Gil-Salom M, Minguez Y, Rubio C, De los Santos MJ, Remohi J,
Pellicer A. Efficacy of intracytoplasmic sperm injection using testicular
spermatozoa. Hum Reprod 1995;10:3166 –70.
29. Tournaye H, Liu J, Nagy PZ, Camus M, Goossens A, Silber S, et al.
Correlation between testicular histology and outcome after intracyto-
plasmic sperm injection using testicular spermatozoa. Hum Reprod
1996;11:127–32.
30. Kahraman S, Ozgur S, Alatas C, Aksoy S, Tasdemir M, Nuhoglu A, et
al. Fertility with testicular sperm extraction and intracytoplasmic sperm
injection in non-obstructive azoospermic men. Hum Reprod 1996;11:
756 – 60.
31. Tournaye H, Camus M, Vandervorst M, Nagy Z, Joris H, Van Steir-
teghem A, et al. Surgical sperm retrieval for intracytoplasmic sperm
injection. Int J Androl 1997;20(Suppl 3):69 –73.
32. Aboulghar MA, Mansour RT, Serour GI, Fahmy I, Kamal A, Tawab
NA, et al. Fertilization and pregnancy rates after intracytoplasmic
sperm injection using ejaculated semen and surgically retrieved sperm.
Fertil Steril 1997;68:108 –11.
33. Abuzeid MI, Sasy MA, Salem H. Testicular sperm extraction and
intracytoplasmic sperm injection: a simplified method for treatment of
obstructive azoospermia. Fertil Steril 1997;68:328 –33.
34. Cha KY, Oum KB, Kim HJ. Approaches for obtaining sperm in patients
with male factor infertility. Fertil Steril 1997;67:985–95.
35. Dohle GR, Ramos L, Pieters MHEC, Braat DDM, Weber RFA. Sur-
FERTILITY & STERILITY威
gical sperm retrieval and intracytoplasmic sperm injection as treatment
of obstructive azoospermia. Hum Reprod 1998;3:620 –3.
36. Tournaye H. Surgical sperm recovery for intracytoplasmic sperm in-
jection: microsurgical or percutaneous epididymal sperm aspiration,
excisional biopsy or aspiration of the testis—which method is to be
preferred? Hum Reprod. In press.
37. Novero V, Camus M, Tournaye H, Smitz J, Verheyen G, Joris H, et al.
Relationship between serum follicle stimulating hormone in the male
and standard sperm parameters, and the results of intracytoplasmic
sperm injection. Hum Reprod 1997;12:59 – 63.
38. Nagy ZP, Joris H, Verheyen G, Tournaye H, Devroey P, Van Steirteg-
hem AC. Correlation between motility of testicular spermatozoa, tes-
ticular histology and the outcome of intracytoplasmic sperm injection.
Hum Reprod 1998;13:890 –5.
39. Nijs M, Vanderzwalmen P, Vandamme B, Segal-Bertin G, Lejeune B,
Segal L, et al. Fertilizing ability of immotile spermatozoa after intra-
cytoplasmic sperm injection. Hum Reprod 1996;11:2180 –5.
40. Moore HD, Curry MR, Penfold LM, Pryor JP. The culture of human
epididymal epithelium and in vitro maturation of epididymal sperma-
tozoa. Fertil Steril 1992;58:776 – 83.
41. Bonduelle M, Wilikens A, Buysse A, Van Assche E, Devroey P, Van
Steirteghem A, et al. A follow-up study of children born after intracy-
toplasmic sperm injection (ICSI) with epididymal and testicular sper-
matozoa and after replacement of cryopreserved embryos obtained after
ICSI. Hum Reprod 1998;13(Suppl 1):196 –207.
42. Meschede D, Lemcke B, Stüssel J, Louwen F, Horst J. Strong
preference for non-invasive prenatal diagnosis in women pregnant
through intracytoplasmic sperm injection (ICSI). Prenat Diagn
1998;18:700 –5.
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