Human Reproduction Vol.20, No.1 pp. 226–230, 2005 doi:10.

1093/humrep/deh590
Advance Access publication November 11, 2004

Efficient treatment of infertility due to sperm DNA damage

by ICSI with testicular spermatozoa

Ermanno Greco1, Filomena Scarselli1, Marcello Iacobelli1, Laura Rienzi1, Filippo Ubaldi1,
Susanna Ferrero1, Giorgio Franco1, Nazareno Anniballo1, Carmen Mendoza2,3
and Jan Tesarik2,4,5
1
Centre for Reproductive Medicine, European Hospital, Via Portuense 700, 00149 Rome, Italy, 2MAR&Gen, Molecular Assisted
Reproduction and Genetics, Gracia 36, 18002 Granada, 3University of Granada, Campus Fuentenueva, 18004 Granada, Spain and

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4
Laboratoire d’Eylau, 55 rue Saint-Didier, 75116 Paris, France
5
To whom correspondence should be addressed. E-mail: cmendoza@ugr.es

BACKGROUND: Sperm DNA damage (fragmentation) is a recently discovered cause of male infertility for which
no efficient treatment has yet been found. Previous findings have suggested that clinically relevant sperm DNA
damage may occur at the post-testicular level. This study was undertaken to assess the clinical usefulness of ICSI
with testicular spermatozoa in this indication. METHODS: The percentage of spermatozoa with fragmented DNA,
assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assay, and ICSI outcomes
were compared in two sequential attempts performed, respectively, with ejaculated and testicular spermatozoa in
18 men with increased sperm DNA fragmentation. RESULTS: The incidence of DNA fragmentation was markedly
lower in testicular spermatozoa as compared with ejaculated spermatozoa. No differences in fertilization and clea-
vage rates and in embryo morphological grade were found between the ICSI attempts performed with ejaculated
and with testicular spermatozoa. However, eight ongoing clinical pregnancies (four singleton and four twin) were
achieved by ICSI with testicular spermatozoa (44.4% pregnancy rate; 20.7% implantation rate), whereas ICSI
with ejaculated spermatozoa led to only one pregnancy which was spontaneously aborted. CONCLUSIONS: These
data show that ICSI with testicular spermatozoa provides the first efficient assisted reproduction treatment option
for men with high levels of sperm DNA damage.

Key words: DNA fragmentation/ejaculated spermatozoa/ICSI/sperm fertilizing ability/testicular spermatozoa

Introduction
Several studies have shown that male infertility can be
caused by sperm DNA damage (Aitken, 1999; Host et al.,
2000; Larson et al., 2000; Morris et al., 2002; Tomsu et al.,
2002; Benchaib et al., 2003; Carrel et al., 2003; Henkel et al.,
2004; Tesarik et al., 2004a). However, the pathophysiological
mechanism leading to sperm DNA damage is understood
only incompletely, and no specific treatment for infertility
caused by this condition has yet been proposed.
Even though the pattern of sperm DNA damage (fragmen-
tation) closely resembles that resulting from programmed cell
death (also called apoptosis) in somatic cells, several studies
have questioned the causal relationship between the acti-
vation of the classical apoptotic pathway and DNA frragmen-
tation of mature human spermatozoa (Sakkas et al., 2002;
Henkel et al., 2004; Moustafa et al., 2004; Lachaud et al.,
2004). We have shown recently that the classical cell death
signalling pathway, in which caspase activation is followed
by phosphatidylserine externalization marking the cell as
target for phagocytosis, is active while germ cells remain in
a tight association with Sertoli cells, but most Sertoli cell-
free germ cells in the diseased human testis undergo DNA
fragmentation without caspase activation and phosphatidyl-
serine externalization (Tesarik et al., 2004b). After release
from Sertoli cells, spermatids and spermatozoa thus appear to
suffer DNA damage independently of the usual cell death
signalling pathways. Previous studies have suggested that
oxidative stress can be responsible for sperm DNA damage
(Fraga et al., 1996; Barroso et al., 2000; Aitken and Krausz,
2001; Agarwal et al., 2003; Moustafa et al., 2004). The loss
of nutritional support by Sertoli cells may aggravate the
impact of oxidative stress on sperm cell components (Tesarik
et al., 2004b).
If DNA damage detected in ejaculated spermatozoa essen-
tially begins after sperm release from Sertoli cells, it can be
hypothesized that the degree of damage increases with time
after Sertoli cell release. If this is true, sperm populations
recovered directly from the testis could be expected to be
less affected by this pathological process as compared with
ejaculated sperm populations. To test this hypothesis, this

226 Human Reproduction vol. 20 no. 1 q European Society of Human Reproduction and Embryology 2004; all rights reserved

study compares the incidence of DNA damage in spermato-
zoa recovered from the ejaculate and from the testis in two
sequential assisted reproduction attempts performed in a
group of patients characterized by abnormally high levels of
ejaculated sperm DNA damage. Moreover, developmental
competence of spermatozoa obtained from these two sources
was determined by evaluating fertilization rate, the percen-
tage of good morphology embryos, pregnancy rate and
implantation rate.
Materials and methods
Study design and participants
This study involved 18 couples who had undergone at least two
unsuccessful ICSI attempts with ejaculated spermatozoa and whose
male partner had $ 15% of ejaculated spermatozoa with damaged
DNA. All of the male partners were non-smokers without any
apparent cause of infertility in their medical history. Their age ran-
ged between 28 and 55 years. The female age ranged between 24
and 35 years. None of the patients enrolled in this study received
pre-ICSI therapy aimed at improving sperm quality. All of these
couples first underwent another ICSI attempt with ejaculated sper-
matozoa. If no ongoing pregnancy resulted, a subsequent attempt
was performed with testicular spermatozoa. The interval between
the two sequential attempts did not exceed 4 months.
Sperm samples
Ejaculated spermatozoa were obtained by masturbation after 3 – 5
days of sexual abstinence. After liquefaction, semen samples were
divided into two aliquots to be used for the evaluation of DNA frag-
mentation and ICSI, respectively. For ICSI, sperm samples were
diluted 1:10 with Gamete 20 medium (Scandinavian IVF Science,
Gothenborg, Sweden), centrifuged (200 g; 10 min), and spermatozoa
were allowed to swim up from the resulting pellet to overlayered
Gamete 20 medium. This procedure previously was shown to result
in a better selection of DNA-intact spermatozoa than a discontinu-
ous density gradient technique (Lachaud et al., 2004). Basic sperm
parameters (concentration, motility and morphology) were evaluated
in all samples according to World Health Organization criteria
(World Health Organization, 1992).
Testicular spermatozoa were obtained either by open testicular
biopsy or by fine needle aspiration as described (Ubaldi et al.,
1999). After disintegration with sterile microscope slides, the pre-
sence of spermatozoa in the wet preparations was assessed under the
inverted microscope at £ 200 or £400 magnification. Spermatozoa
to be used for the ICSI attempt were selected with the use of an
assisted hatching micropipette (Humagen, Charlottesville, VA). The
rest of the sample was divided into two aliquots, one of which was
used for the evaluation of sperm DNA fragmentation. The other
aliquot was cryopreserved for eventual future use.
Evaluation of sperm DNA fragmentation
Liquefied semen samples were centrifuged at 200 g for 10 min.
After supernatant removal, samples of the remaining sperm pellet
were smeared on microscope slides, air-dried, fixed with 4% para-
formaldehyde in phosphate-buffered saline at 48C for 25 min and
permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. The
smears were then processed for the terminal deoxyribonucleotidyl
transferase-mediated dUTP nick-end labelling (TUNEL) assay
which was performed using a Cell Death Detection Kit with
tetramethylrhodamine-labelled dUTP (Roche, Monza, Italy) and
Efficient ART for sperm DNA damage
according to the manufacturer’s instructions. The percentage of
spermatozoa with fragmented DNA was determined in a fluor-
escence microscope as described (Tesarik et al., 2004a).
For evaluation of DNA fragmentation in testicular spermatozoa,
disintegrated testicular tissue was smeared on slides and processed
further as for ejaculated sperm smears. Two hundred spermatozoa
per sample were analysed for both the ejaculated and the testicular
sperm speciments.
Assisted reproduction techniques
Controlled ovarian stimulation, oocyte recovery and ICSI were per-
formed as described (Tesarik et al., 2001). Briefly, patients were
treated with recombinant FSH (Puregon; Organon, Oss, The Nether-
lands) after pituitary desensitization with triptorelin (Decapeptyl;
Ipsen, Slough, UK) started in the mid-luteal phase. The overall dose
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of FSH per stimulated cycle varied between 1800 and 3500 IU
depending on the individual response. As soon as at least three
follicles of . 18 mm were detected, ovulation was induced with
10 000 IU of HCG (Profasi; Serono, Geneva, Switzerland). Oocytes
were recovered by transvaginal ultrasound-guided follicle aspiration
36 h later.
Zygotes and embryos were evaluated with the use of previously
described scoring systems (Tesarik and Greco, 1999; Tesarik et al.,
2000; Mendoza et al., 2002) on days 1, 2 and 3 after ICSI. Two to
four best scoring embryos were transferred to the patient’s uterus on
day 3 after ICSI.
Statistics
Differences between groups were assessed by two-tailed x2-test with
Yates’ correction and Fisher’s exact test All analyses were
performed using the Statistica 5.0 package (Statsoft Version 5.1,
Hamburg, Germany).
Results
Incidence of DNA fragmentation in ejaculated and
testicular spermatozoa
Basic sperm parameters (concentration, motility and
morphology) showed a high variability among individual
patients, ranging between normal values and severe oligo-
asthenoteratozoospermia (Table I). All 18 men involved in
this study showed . 15% spermatozoa with fragmented DNA
in ejaculated sperm samples used for the first ICSI attempt
(Table I). In all of these men, a second ICSI attempt was per-
formed with testicular spermatozoa. The percentage of sper-
matozoa with fragmented DNA in testicular sperm samples
obtained on this occasion was , 6% in all but one of these
men (Table I).
The overall incidence of DNA fragmentation in the testicu-
lar sperm samples was 4.8 ^ 3.6% (mean ^ SD), which was
significantly lower (P , 0.001) compared with the ejaculated
sperm samples from the same individuals (23.6 ^ 5.1%).
Developmental competence of ejaculated and testicular
spermatozoa
Comparison of fertilization outcomes of the two sequential
ICSI attempts, the first of which was performed with ejacu-
lated spermatozoa and the second with testicular spermato-
zoa, showed no significant difference either in fertilization
and cleavage rates or in the proportion of embryos with good
227
E.Greco et al.

ton and four twin, all of which are ongoing. The number of
Table I. Basic sperm parameters of the patients involved in this study and
the incidence of DNA fragmentation in their ejaculated and testicular sperm gestational sacs with cardiac activity was thus 12, leading
samples to an implantation rate of 20.7%, which was significantly
Patient Basic sperm parameters % Spermatozoa higher than for ICSI attempts with ejaculated spermatozoa
with fragmented (Table III).
DNA

Concentration Motility Normal Ejaculate Testis

( £ 106/ml) (%) forms (%)
Discussion
1 6 11 2 20 3 The present data suggest that, in infertile men with a high
2 31 52 3 15 5 incidence of DNA damage in ejaculated sperm populations,
3 38 71 20 24 4
4 33 40 11 21 3 the percentage of spermatozoa carrying DNA damage is
5 3 19 9 27 2 much lower in the testis. This observation confirms the
6 25 65 15 31 6 working hypothesis that most of the DNA damage observed
7 75 42 48 25 4

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8 22 3 8 23 1 in ejaculated spermatozoa results from alterations occurring
9 25 15 6 22 18 at the post-testicular level, although it cannot be excluded
10 2 14 7 25 5 that spermatozoa may be selectively predisposed for this kind
11 19 29 25 26 4
12 51 41 48 21 3 of damage during earlier developmental phases.
13 12 63 11 37 6 More importantly, these data also show that the use of tes-
14 24 32 61 19 5 ticular spermatozoa for ICSI can compensate for the repro-
15 1 42 22 17 6
16 33 21 13 24 4 ductive disadvantage associated with the use of ejaculated
17 17 56 10 20 5 spermatozoa for ICSI in this category of patients. Interest-
18 66 44 58 27 3 ingly, the improvement of ICSI outcomes with the use of
testicular spermatozoa only concerned ongoing clinical preg-
nancy and implantation rates, whereas fertilization rate and
morphological appearance (Table II). The ovarian stimulation embryo morphology score were similar for the treatment
protocol was the same in these two attempts, and no apparent attempts with ejaculated and testicular spermatozoa. This is
differences in oocyte quality and quantity were noted in agreement with previous studies showing that spermatozoa
between them. However, only one pregnancy (patient 6 in with DNA damage can still fertilize oocytes and give rise to
Table I), which was later spontaneously aborted, was embryos with good morphological appearance, although
established by using ejaculated spermatozoa (Table III). This these embryos mostly fail to implant or are miscarried shortly
contrasted with outcomes of the attempts using testicular after implantation (Twigg et al., 1998; Ahmadi and Ng,
spermatozoa which resulted in eight clinical pregnancies 1999; Tomlinson et al., 2001; Morris et al., 2002;
(patients 1, 3, 5, 8, 10, 13, 15 and 16 in Table I), four single- Carrell et al., 2003; Henkel et al., 2004; Tesarik et al.,

Table II. Fertilization and embryo development after ICSI with ejaculated and testicular spermatozoa
Sperm Attempts Oocytes Normal Fertilization Cleaved Good-morphology
source injected zygotesa rateb embryosc embryosd

Ejaculate 18 185 131 70.8%e 124 (94.7%)e 59 (47.6%)e

Testis 18 187 140 74.9%e 133 (95.0%)e 68 (51.1%)e
a
With two equal-sized pronuclei.
b
Percentage of injected oocytes that developed to normal zygotes.
c
Percentages are calculated from the number of normal zygotes.
d
Embryos with normal pronuclear morphology on day 1, $6 cells on day 3, equal sized blastomeres and ,10% of the
intrazonal space occupied by fragments. The percentages are calculated from the number of cleaved embryos.
e
The differences between data for the two sperm sources are not significant (P . 0.05).

Table III. Implantation and pregnancy after ICSI with ejaculated and testicular spermatozoa
Sperm Attempts Embryos Clinical Pregnancy Gestational Implantation
source transferred pregnanciesa rateb sacsc rated

Ejaculate 18 56 1 5.6%e 1 1.8%f

Testis 18 58 8 44.4%e 12 20.7%f
a
With at least one gestational sac with cardiac activity.
b
Percentage of attempts resulting in a clinical pregnancy.
c
With cardiac activity.
d
Percentage of embryos transferred that gave rise to a gestational sac with cardiac activity.
e
P , 0.05.
f
P , 0.01.

228

2004a). However, a negative correlation between sperm
DNA fragmentation and fertilization rate was also reported
(Sun et al., 1997; Lopes et al., 1998; Benchaib et al., 2003).
Based on the absence of apparent negative consequences for
cleaving embryo morphology, the term ‘late paternal effect’
has been suggested recently for the developmental disadvan-
tage conferred to embryos by spermatozoa carrying damaged
DNA, as opposed to the ‘early paternal effect’ which is also
of sperm origin, is reflected by poor embryo morphology but
is not associated with sperm DNA damage (Tesarik et al.,
2004a).
In addition to suggesting novel clues for a better under-
standing of male infertility and the processes involved, this is
the first study to propose an efficient assisted reproduction
treatment option for men whose fertility is compromised by
sperm DNA damage. However, the recovery of testicular
spermatozoa is an invasive intervention, and its usefulness
must be carefully justified in each individual case. It is thus
increasingly important to achieve a consensus as to the cri-
teria predicting assisted reproduction failure due to sperm
DNA damage. However, different studies have suggested
different threshold values for the percentage of damaged
spermatozoa in the ejaculate above which sperm develop-
mental competence is compromised, apparently because of
the use of different techniques for DNA damage visualiza-
tion. For instance, using acridine orange staining, the percen-
tage of spermatozoa with double-stranded DNA below which
no full-term pregnancies were achieved after ICSI was 50%
(Hoshi et al., 1996). Two large independent studies using the
sperm chromatin structure assay (SCSA), a method assessing
the susceptibility of sperm DNA to undergo acid-induced
single-strand breakage in situ (Evenson et al., 1980), were
undertaken in the USA and Europe, respectively (Evenson
et al., 1999; Spanò et al., 2000). They agree in that male
fertility is hampered when . 30% of sperm have abnormal
chromatin. Moreover, no full-term pregnancies (after ICSI,
IVF or intra-uterine insemination) were obtained when the
DNA fragmentation index assessed by SCSA was . 30%
(Larson et al., 2000; Larson-Cook et al., 2003; Saleh et al.,
2003). However, no differences in SCSA parameter values
between patients initiating pregnancies and not doing so in
either ICSI or conventional IVF were found in another recent
study (Gandini et al., 2004).
Unlike SCSA, which merely reflects the susceptibility of
sperm DNA to fragmentation, TUNEL assay is a direct
measure for the presence of endogenous nicks in sperm
DNA. With the use of this test, Benchaib et al., (2003)
analysed ejaculated spermatozoa in 54 ICSI attempts and
reported the absence of pregnancy when the sperm subpopu-
lation with DNA fragmentation was . 20%. Unlike the study
by Benchaib et al., (2003), the patient group described in the
present study was highly selected by including only patients
with at least two previous unsuccessful ICSI attempts and
with at least 15% TUNEL-positive spermatozoa in the ejacu-
late. We obtained no ongoing pregnancy with ejaculated
spermatozoa in this group of 18 couples, whereas eight
pregnancies (four of which were twin) were achieved in a
subsequent ICSI attempt with testicular spermatozoa. Thus, it
Efficient ART for sperm DNA damage
appears that, with the use of TUNEL assay applied to whole
ejaculated sperm populations, the threshold levels of sperm
DNA fragmentation above which ICSI with testicular sper-
matozoa should be considered will lay between 15 and 20%.
However, another recent study calculated a cut-off value of
24.3% TUNEL-positive spermatozoa in the ejaculate (Henkel
et al., 2003). Thus, the question of clinically relevant
threshold for sperm DNA fragmentation still remains open.
In addition to providing an immediate treatment option for
men suffering from this as yet untreatable reproductive path-
ology, these findings open up a number of stimulating ques-
tions. First of all, with an incidence of DNA fragmentation in
ejaculated spermatozoa of 20 –40%, there are still more than
half of all spermatozoa whose DNA is expected to be intact.
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Why does fertilization with these spermatozoa not lead to
normal embryonic development? Apparently the advantage
of using testicular spermatozoa in this indication is related to
some other aspects. It is possible that detectable DNA frag-
mentation only represents the ‘tip of the iceberg’ and that
subthreshold DNA damage is present in most of those sper-
matozoa that give a negative TUNEL reaction when the per-
centage of TUNEL-positive spermatozoa in the given sperm
population reaches a certain critical value. Alternatively, the
developmental impairment observed in these cases may be
related to a deficiency of some of the sperm cytoplasmic fac-
tors involved in oocyte activation and early embryo develop-
ment, such as an oocyte-activating substance or centriole. In
both of these situations, the developmentally incompetent
sperm population might include substantially more sperma-
tozoa than only those actually showing detectable DNA
damage.
Further study is also needed to determine whether fertility
of men with sperm DNA damage can be restored by less
invasive means than ICSI with testicular sperm recovery.
Administration of antioxidants to the patients concerned and
the development of in vitro selection techniques capable of
enriching ejaculated sperm populations in healthy spermato-
zoa are two ways to be explored.
The use of testicular spermatozoa for ICSI may also be
considered in men with borderline figures of sperm DNA
damage which are suspected to increase the risk of genetic
diseases, birth defects and childhood cancer in the offspring
(Fraga et al., 1996; Ji et al., 1997). Further study is needed
to determine the correlation between the importance of these
health risks and the extent of sperm DNA fragmentation to
determine cut-off values for adequate patient counselling.
Regardless of these open questions, this study, though pre-
liminary, describes a new, efficient and ready-to-use treat-
ment for an as yet untreatable reproductive pathology. This is
particularly important for those infertile couples in whom
sperm DNA damage is combined with advanced female age
and who, consequently, need a rapid treatment solution.
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October 4, 2004