Pharmacological stimulation of sperm motility in
frozen and thawed testicular sperm using the
dimethylxanthine theophylline
Thomas Ebner, Ph.D.,a Gernot Tews, M.D.,a Richard B. Mayer, M.D.,a Stephanie Ziehr, M.D.,b
Wolfgang Arzt, M.D.,c Walter Costamoling, M.D.,d and Omar Shebl, M.D.a
a
Landes- Frauen- und Kinderklinik Linz, Kinderwunsch Zentrum, Linz; b Medical University, Departement of Gynecological
Endocrinology and Reproductive Medicine, Innsbruck; c Landes- Frauen- und Kinderklinik Linz, Institute of Prenatal
Genetics, and d Krankenhaus der Barmherzigen Schwestern, Department of Urology, Linz, Austria
Objective: To evaluate whether the use of theophylline improves sperm motility and treatment outcome in frozenthawed testicular sperm extraction (TESE).
Design: Artificial sperm activation was offered to azoospermic patients between January and October 2010 in two
different centers (identical lab conditions).
Setting: IVF units of public hospitals.
Patient(s): Sixty-five patients participated and gave informed consent.
Intervention(s): Sibling oocytes were split into a study (intracytoplasmic sperm injection [ICSI] with thawed
testicular sperm treated with theophylline) and a control group (ICSI with thawed untreated sperm).
Main Outcome Measure(s): Sperm motility, time for sperm selection, rates of fertilization, implantation, clinical
pregnancy, and live birth.
Result(s): All patients but one (98.5%) showed a significant improvement in testicular sperm motility when
theophylline was used. In addition, sperm selection took significantly less time in the study as compared with in
the untreated control group. Corresponding rates of fertilization (79.9% vs. 63.3%) and blastulation (63.9% vs.
46.8%) were significantly increased. Significantly more patients achieved clinical pregnancy if embryos/blastocysts
derived from oocytes that had been injected with pharmacologically stimulated testicular spermatozoa were
transferred (53.9% vs. 23.8%). This also holds true for the implantation rate.
Conclusion(s): Theophylline turned out to be a reliable tool in stimulating testicular spermatozoa after thawing. Its
immediate effect allows for faster and more accurate selection of viable sperm, which in turn improved fertilization
and pregnancy outcome in this prospective study. (Fertil Steril 2011;-:-–-. 2011 by American Society for
Reproductive Medicine.)
Key Words: Immotile sperm, sperm viability, stimulation of sperm motility, TESE, theophylline
The development of intracytoplasmic sperm injection (ICSI) (1) has
been of great interest in the treatment of male factor infertility.
Because this technique also allows the usage of epididymal and testicular spermatozoa, a wide range of possibilities are now available
for fertilization in cases of extreme male pathology.
When working with testicular tissue, it appears difficult to isolate
a clean preparation of usable sperm because of the reduced tendency
of the sperm to detach from the testicular tissue owing to its limited
motility. In addition, the presence of numerous cell types in the testicular sperm extraction (TESE) material will also interfere with this
effort (2, 3). This problem may be overcome by enzymatically (e.g.,
collagenase) digesting the testicular cell aggregates or by introducing
modified processing techniques (4). However, owing to the nature of
immature testicular spermatozoa, the reduced motility of extracted
sperm, if moving at all, will represent a persistent problem. In terms
Received May 17, 2011; revised August 11, 2011; accepted August 30,
2011.
T.E. has nothing to disclose. G.T. has nothing to disclose. R.B.M. has
nothing to disclose. S.Z. has nothing to disclose. W.A. has nothing to
disclose. W.C. has nothing to disclose. O.S. has nothing to disclose.
Reprint requests: Omar Shebl, M.D., Landes- Frauen- und Kinderklinik
Linz, Kinderwunsch Zentrum, Krankenhausstrasse 26–30, A–4020
Linz, Upper Austria, Austria (E-mail: [email protected]).
0015-0282/$36.00
doi:10.1016/j.fertnstert.2011.08.041
of sperm motility, cryopreservation of TESE probes to spare the patient
repeated TESE will also not be helpful at all.
Over the years, there have been numerous attempts to resolve this
problem by identifying pharmacological agents that might improve
sperm motility, thus increasing fertilizing ability. Although some of
the compounds tested had to be administered orally (5, 6), the vast
majority of agents were used in situ. Among others, caffeine (7)
and other methylxanthines (8, 9), relaxin (10), 2-deoxyadenosine
(11), and kallikrein (12) have been successfully used.
In particular, pentoxifylline turned out to be an effective tool in
stimulating motility in fresh and cryopreserved human semen (13,
14) and identifying viable sperm in TESE patients presenting
exclusively with immotile sperm before or after cryopreservation
(15, 16). As with other xanthine derivates, the stimulatory effect
of pentoxyphylline can clearly be attributed to the increased
intracellular levels of cyclic AMP (17), a molecule involved in the
generation of sperm energy, which is a result of its inhibitory properties on phosphodiesterase function.
Interestingly, theophylline, a closely related molecule, has been
investigated for this purpose to a much lesser extent. As a readyto-use theophylline has recently been launched (GM501 SpermMobil) and no prospective study using this dimethylxanthine on frozen
and thawed testicular tissue has yet been reported, we decided to set
up such a prospective study. Fertilization, embryo quality, blastocyst
Fertility and Sterility Vol. -, No. -, - 2011
Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
1
formation, and rates of implantation and pregnancy were analyzed in
detail in sibling oocytes (injected with testicular sperm treated either
with or without theophylline) of azoospermic couples.
MATERIALS AND METHODS
From January 2010 to March 2011, theophylline treatment was offered to all
couples presenting in Linz and Innsbruck with azoospermia who had at least
six mature oocytes collected. Approval of the Institutional Review Board was
sought and given. During this 15-month period, 73 patients (39.1 4.2 years)
with obstructive azoospermia provided written consent to participate in the
present prospective study. In 18 patients (27.7%), azoospermia was due to vasectomy, and another 11 (16.9%) had a bilateral congenital absence of the vas
deferens (six of them were heterozygous for cystic fibrosis). Seven men
(10.8%) had a status postchemotherapy, and two (3.1%) had a nonfunctional
vas deferens caused by a previous chlamydia infection. The remaining 27
(41.5%) patients had total necrospermia in the ejaculate, aspermia, or retrograde ejaculation. It is important to note that inclusion of different subgroups
of patients (e.g., obstructive and nonobstructive azoospermia) could have
influenced the outcome, which would limit the predictive value of the present
study.
The vast majority of TESE (n ¼ 51) was performed at the Department of
Urology at the Krankenhaus der Barmherzigen Schwestern in Linz. In short,
the technique of TESE began with a relatively small incision being made in
the scrotal skin and carried through the peritoneal tunica vaginalis. Next,
small pieces of testicular tissue were extruded through an opening in the tunica albuginea. The removed pieces of tissue were placed in tubes containing
BM1 medium (Eurobio) for further manipulation. Biopsies were mechanically disaggregated within half an hour under sterile conditions in a Petri
dish containing BM1 medium. WE tried to accumulate as many spermatozoa
as possible in the liquid supernatant.
On average, 4.3 (1.9) million testicular sperm per milliliter were retrieved
from the tubuli seminiferi using this mechanical squeezing technique. A total
of 41 (63.1%) TESE samples did not show any signs of motility immediately
before cryostorage. Since none of the biopsies was planned to be used in
a fresh cycle of ICSI, all were frozen with an automatic slow freezing procedure (CL-8000, CryoLogic) using glycerol as a cryoprotectant (SpermFreeze,
FertiPro). The presence of hindering testicular tissue was carefully avoided.
Before ovum pickup a sufficient number of frozen straws (depending on
the sperm count of the fresh biopsy) were delivered to the Kinderwunsch
Zentrum at the Landes- Frauen- und Kinderklinik in Linz. This certified
transport was performed under liquid nitrogen by an authorized and qualified
person.
On the morning of the day of ICSI, testicular sperm was rapidly thawed by
directly plunging the straws into a 37 C water bath. Cryoprotectant was removed by two centrifugation steps (1 minute at 5,000 rpm). The pellet was
then resuspended in a small volume of BM1 medium. Since it turned out
that more or less immediate use of thawed sperm involved the risk of complete immotility (15), ICSI was planned 4–5 hours post-thawing. After this
incubation period, testicular suspension was placed in 10-mL swim-out drops
arranged on an ICSI dish under oil. As soon as at least one motile spermatozoon was observed (for all patients but two), an adequate number of mature
oocytes were loaded into the same dish. Thus, for the female gametes, the
time out of the incubator was kept at a minimum.
Only 14 patients had their testicular biopsy done in other cities. All of these
samples showed no testicular suspensions but rather clumps of tissue. The associated straws were thawed and placed in a medium containing collagenase
(GM501 Collagenase, Gynemed) for facilitation of sperm isolation before
mechanical processing.
All female partners (30.9 2.4 years) were stimulated according to a long
protocol. Down-regulation was performed with Triptorelin (Decapeptyl, Ferring), and the gonadotropins used were of a recombinant nature in 12 cases
(Puregon, MSD) and urinary (Menopur, Ferring) in the remaining 19 women.
No cases of endometriosis or polycystic ovarian syndrome were seen in the
patient cohort. Oocyte collection was scheduled for 2–3 hours after thawing
of the testicular biopsies and performed via the vaginal route.
Cumulus-oocyte complexes were collected in BM1 medium and cultured
for another 2 hours in the incubator before careful denudation using
2
Ebner et al.
Use of theophylline in thawed TESE
hyaluronidase (Origio). Immediately after this process, ICSI was started
(approximately 4–5 hours after testicular tissue thawing). Only mature metaphase II oocytes were considered for ICSI.
After denudation, randomization of the gametes was performed (Fig. 1).
Specifically, all oocytes of a patient were split into two groups. This was
done under a binocular microscope, which did not allow proper identification
of oocyte quality. However, during this process, immature oocytes at prophase I showing a distinct germinal vesicle were removed. Different percentages of metaphase I oocytes (not seen under a binocular microscope) resulted
in unequal numbers of injected oocytes in both groups.
In the oocytes used as a control group, ICSI was performed with untreated
motile testicular spermatozoa (with the exception of three patients who only
had immotile sperm after 4–5 hours of incubation). The second half of the
eggs had spermatozoa injected that were pretreated with a ready-to-use
theophylline solution (GM501 SpermMobil, Gynemed) to stimulate their
motility (study group).
It has been reported that the maximum activity of methylxanthines is
reached after 10 minutes, with an activity phase of less than 2 hours (18).
Because of this immediate and short-term effect, direct addition into the
droplet containing the immotile spermatozoa is recommended. In the present study, a small volume (0.5 mL) of GM501 SpermMobil was added per
swim-out drop. It turned out that the final dilution (1:20 with BM1 medium)
allowed for an immediate start of the search for motile spermatozoa. As in
the control group, use of morphologically normal sperms with the highest
motility (e.g., fast forward progressive motility) according to the criteria
suggested by the World Health Organization (19) was preferred. Spermatozoa selected for ICSI were collected individually, transferred to a small drop
of polyvinylpyrrolidone, and immobilized by use of the ICSI pipette (MicroTech, Gynemed). The ICSI technique itself was performed as described
elsewhere (20).
Fertilization was controlled in EmbryoAssist Medium (Origio) 18–20
hours post-ICSI and considered to be regular if two pronuclei were found
to be abutted in the center of the oocyte. At cleavage stage (days 2 and
3), embryos were scored according to their number and symmetry of blastomeres and checked for the presence of multinucleated cells. If blastocyst
transfer was considered, beginning and extent of compaction were recorded
on day 4 (21). Correspondingly, survival was analyzed at the blastocyst
stage (22). According to previously published criteria (22), the top-quality
blastocyst group consisted of blastocysts in which at least one cell lineage
was quality A and none were quality C. If full blastocyst stage was not
reached at the time of morphological analysis, top-quality early blastocysts
showed no cytoplasmic loss due to fragmentation or extrusion of blastomeres (21).
Transfer was scheduled for either day 3 (n ¼ 11) or day 5 (n ¼ 48). It is
important to note that selection of embryos or blastocysts for transfer was
based on routine morphological criteria and that no randomization for study
or control group was done on the transfer day. In principle, we planned elective single embryo or blastocyst transfers. However, owing to female age or
previously failed cycles, some patients wanted to have two concepti transferred. In these rare cases, the quality of the best embryo (first pick) specified
the group from which both embryos/blastocysts should be chosen (which was
not always possible). This strategy helped to minimize mixed transfers (e.g.,
one embryo from the study and one from the control group).
A total of six patients had all their viable blastocysts vitrified (23) owing to
a high risk of ovarian hyperstimulation syndrome. Subsequently, these particular patients had one thawed ET each.
Nineteen days after oocyte collection, the blood concentration of hCG was
measured. Biochemical pregnancy was defined as a significant increase in
hCG levels (>10 mU/mL). The implantation rate was defined by ultrasound
visualization, 4 weeks after ET, as a gestional sac per embryo transferred.
This included subclinical (gestational sac but no fetal heartbeat) as well as
clinical pregnancies (at least one gestational sac with positive heart activity).
All patients who had not delivered at the time of manuscript submission
showed an unsuspicuous ultrasound at our specialized Institute of Prenatal
Genetics.
Differences between continuous variables were assessed with the t-test for
independent samples and with the c2-test for categorical variables. An alpha
error rate below .05 was considered to be statistically significant. Clinical
Vol. -, No. -, - 2011
FIGURE 1
Schematic presentation of the study design and outcome.
Potentially relevant
and screened patients
(n=73)
Refused to participate
(n=3)
Insufficient # oocytes
(n=5)
Patients included
who gave written consent
(n=65)
Randomization of
842 MII oocytes
Oocytes of control group
Oocytes of study goup
No theophylline
Plus theophylline
(n=425)
(n=417)
ICSI with thawed testicular sperm
In vitro culture to day 3 (n=11) or day 5 (n=48)
Fresh (n=59) and thawed embryo
or blastocyst transfers
Vitrification
(n=6)
Outcome
Ebner. Use of theophylline in thawed TESE. Fertil Steril 2011.
pregnancy rate and implantation rate were calculated using pooled day 3 and
day 5 results since no differences in outcome were observed.
RESULTS
Addition of theophylline improved motility in 64 out of 65 patients
(98.5%) involved in this study (one patient did not have motile
sperm irrespective of the treatment with theophylline). Thus, time
for identification and isolation of motile testicular sperm after thawing was significantly faster (P<.01) in the study group.
A total of 842 oocytes in 65 patients were treated with ICSI in this
prospective analysis. The corresponding fertilization rate was 71.5%
Fertility and Sterility
(602/842). Table 1 indicates that usage of theophylline was associated with a significantly increased fertilization (P<.001) as compared with the untreated control group. This difference was not
associated with parthenogenetic activation or nondisjunction
(P>.05).
Cleavage rate and embryo quality showed no differences in the
study and control groups during days 2–4. However, the presence
of multinucleated cells on day 3 was significantly higher (P<.01)
in the control group as compared with in the theophylline-treated
study group (Table 1). The overall blastulation rate was found to
be 56.7% (274/483), with a significantly better performance in the
study group (P<.001).
3
TABLE 1
TABLE 2
Fertilization and preimplantation development in 31
azoospermic patients with or without theophylline for
Pregnancy outcome after transfer of combined fresh and
vitrified/warmed concepti deriving from the study and/or
improving testicular sperm motility after cryopreservation.
control group.
No. of metaphase II
oocytes
Time/sperm,a minutes
Fertilization (2PN)b
0 PNc
1/3 PN
Degenerated
Cleavagesd
Good-quality day 2
Multinucleation day 2e
Good-quality day 3
Multinucleation day 3f
No. considered for
blastocyst culture
Signs of compaction day 4
Blastocyst formationg
Top-quality blastocyst
Without
theophylline
With
theophylline
425
417
8.0 3.8
269 (63.3)
111 (26.1)
33 (7.8)
12 (2.8)
236/269 (87.7)
151/236 (64.0)
19/236 (8.1)
170/236 (72.0)
14/236 (5.9)
203
4.6 2.4
333 (79.9)
45 (10.8)
29 (7.0)
10 (2.4)
312/333 (93.7)
218/312 (69.9)
10/312 (3.2)
246/312 (78.9)
5/312 (1.6)
280
105 (51.7)
95 (46.8)
57/95 (60.0)
159 (56.8)
179 (63.9)
124/179 (69.3)
Note: Values in parentheses are percentages. PN ¼ pronucleus/ei.
a,f
P< .01.
d,e
P< .05.
b,c,g
P< .001.
Ebner. Use of theophylline in thawed TESE. Fertil Steril 2011.
Pooled fresh and thawed transfers resulted in a detectable level of
ß-hCG in 50.8% of the patients. The corresponding implantation
rate was found to be 41.9%. Four missed abortions and two extrauterine pregnancies reduced the overall ongoing pregnancy rate to
41.5%. As indicated in Table 2, rates of positive ß-hCG as well as
clinical pregnancy were significantly higher in the theophyllinetreated group (P<.001).
Only one out of 27 babies (3.7%) so far (August 2011) showed
a minor malformation (polydactilism). This offspring stemmed
from embryos of the control group.
DISCUSSION
Testicular sperm is usually immotile or shows only a minor twitching movement upon extraction (24–26). Cryopreservation of TESE
material somewhat aggravates this unwanted condition. In the
absence of motile gametes, embryologists previously were forced
to perform ICSI with immotile spermatozoa. Although it may be
expected that a certain percentage of immotile spermatozoa are
viable in such a scenario, injection of immotile sperm resulted in
decreased fertilization and pregnancy rates (25). However, immotility does not preclude viability. Theoretically, embryologists have
several options for dealing with the tricky problem of exclusively
immotile gametes.
The most reasonable approach would be to use the ICSI pipette to
test the elasticity of the sperm tail (27). Once manipulated with
a glass tool, a spermatozoon showing an elastic tail is presumed to
be more viable than one with a more rigid flagellum. However, there
is no guarantee that more elastic sperms are viable, that is, are osmotically intact cells. For confirming osmotic capacity, sperms
can be incubated in a hypoosmotic swelling solution (28–30).
4
Ebner et al.
Use of theophylline in thawed TESE
No. of ET/BT
No. of single ET/BT
Positive ß-hCG
Clinical pregnancy
Live birth/ongoing
Implantation rate
Multiple pregnancies
Children born so far
Without
theophylline
With
theophylline
21
15
6 (28.6)a
5 (23.8)b
4 (19.1)c
9/37 (24.3)d
4 (19.0)
6
39
29
24 (61.5)a
21 (53.9)b
21 (53.9)c
27/49 (55.1)d
6 (15.4)
21
Mixed
5
5
1
0
0
1/10
Note: Values in parentheses are percentages. BT ¼ blastocyst transfer.
a,b
P< .05.
c,d
P< .001.
Ebner. Use of theophylline in thawed TESE. Fertil Steril 2011.
Gametes with a functional membrane will undergo swelling of the
cytoplasmic space, and the sperm tail fibers will curl, whereas
those gametes with damaged or osmotically inactive membranes
do not show these phenomena.
Recently, another alternative was introduced (31) that suggests
the use of a diode laser to assess viability in cases of complete asthenozoospermia. Applying a single laser pulse at the end of the sperm
tail caused a characteristic curling of the tail. Since nonviable sperm
did not show this phenomenon, this new technique helped to identify
a spermatozoon with functional integrity of its membrane.
The most elegant strategy is the only one that allows for partial
restoration of original motility (32), namely, the in situ use of methylxanthines, such as pentoxy- or theophylline.
Pentoxyphylline, in particular, has been intensively studied. It has
been found to be superior to caffeine (33). At different concentrations (0.7–3.6 mM/L), pentoxyphylline has been shown to increase
the percentage of motile sperm in a given ejaculate without negatively affecting sperm membrane and acrosome reaction (34). This
phenomenon seems to be dose dependent (35).
It has to be mentioned that contact of xanthine derivates with
embryos should be avoided or kept at a minimum since even short
exposure of oocytes to pentoxyphylline might result in marked morphological changes. In addition, data from animal studies show that
prolonged incubation (e.g., between 24 and 72 hours) of mouse embryos in a 5-mM solution of a dimethylxanthine led to variations in
cyclic AMP content as well as developmental retardation or embryo
death (36). Other studies suggested that even shorter exposure (30
minutes) to 3.6 or 7.2 mM/L pentoxyphylline, while not affecting
blastocyst development (37), could have a negative impact on birth
rate (38). In addition, pentoxyphylline-enriched media artificially
activated mouse oocytes in a concentration- and exposure time–
dependent manner (39). It has been reported that high dosages of
caffeine and its derivatives (5–300 mg/kg) cause malformations in
animals (40). This teratogenic effect was particularly evident
(41–43) in a potential interaction with certain other compounds
(all of which were not present in culture media).
Interestingly, at very low concentrations (0.5 mM/L), pentoxyfylline reduced oxidative stress–induced embryotoxicity in mice (44).
However, to our knowledge, no reports on approaches using
pentoxyphylline or other related agents on human oocytes or embryos
Vol. -, No. -, - 2011
have been published to date. Rather, they were used with some success
to raise motility and, thus, increase outcome in patients with IUI (45)
and previous fertilization failure after IVF (46–49). Since ICSI has
become a powerful alternative in such patients, total lack of sperm
movement (32) or presence of immotile sperm in microsurgical epididymal sperm aspiration and TESE patients (15, 16) are the only
indications being left for routine use of methylxanthines such as
pentoxy- or theophylline.
The latter has not been investigated in detail. Nevertheless, a similar
stimulatory effect on sperm penetration in humans has been reported
that has been seen up to a concentration of 20 mM/L theophylline (9).
Addition of 2.5 mM/L theophylline increased the percentage of male
pronuclear formation and blastocyst formation in animals (50).
The most probable reason for the disproportionate use of pentoxyphylline as compared with theophylline is the slightly increased
hydrosolubility (however, both dimethylxanthines show a higher
solubility in water as compared with caffeine) (33, 51). In turn,
a much higher half-life (4–8 hours) in theophylline as compared
with pentoxyphylline (0.4–0.8 hours) allows for appropriate prewarming before addition to the testicular suspension without
affecting its bioavailability.
In these times of the European Tissue Directives, blending of dimethylxanthine solutions on one’s own is a sheer impossibility.
Thus, the availability of a standardized commercial product is of
great help in the treatment of severe male factor subfertility. In the
present study, the ready-to-use stock solution of theophylline
(SpermMobil) was used at a 20-fold dilution, and duration of exposure was limited to a few minutes (without limiting its bioactivity).
In addition, the selected testicular spermatozoa were washed thoroughly and transferred in theophylline-free medium and PVP as recommended elsewhere (32); thus, the theoretical risk of a biological
hazard existing on female gametes and/or embryos is negligible.
In fact, several benefits were observed for the first time in the
present prospective approach. The observed significant stimulatory
effect of theophylline on sperm motility definitely facilitates laboratory work for the embryologists. It not only significantly reduces the
time needed for ICSI owing to an immediate identification of motile
spermatozoa (thus limiting the time of the oocytes out of the incubator), but it also allows for distinguishing between viable spermatozoa and borderline counterparts (e.g., those with rudimentary
motility) at one glance. These advantages probably led to a higher
fertilization rate in the present study group. Whether an augmented
acrosome reaction (52, 53) played a role in this context will need to
be clarified.
The better developmental potential in the theophylline-treated
group culminated in a significant improvement in blastocyst formation. Blastocyst quality was not influenced, indicating that in the
case that blastocyst stage was reached in the untreated cohort, highly
viable sperms had been chosen for ICSI (although their motility was
slower as compared with the theophylline group). Interestingly,
multinucleated cells were more frequent in the control group on
day 3, and although no logical explanation can be given for this
phenomenon, it is reassuring to know that this problem was not
seen in the theophylline group.
However, multinucleation did not influence pregnancy outcome
since affected embryos were never transferred. Owing to similar embryo and blastocyst qualities, the observed difference in positive
ß-hCG and clinical pregnancy rate could be related to the viability
of the transferred concepti. This supports the usefulness of the
analyzed dimethylxanthine in frozen and thawed TESE material.
Although not reflected in the live birth rate, the absence of minor
or major malformations in the newborn after theophylline treatment
further supports its usage in patients with mostly immotile spermatozoa (before or) after cryopreservation.
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