Pharmacological stimulation of sperm motility in frozen and thawed testicular sperm using the dimethylxanthine theophylline

Pharmacological stimulation of sperm motility infrozen and thawed testicular sperm using thedimethylxanthine theophylline Thomas Ebner, Gernot Tews, M.D.,Richard B. Mayer, M.D.,Stephanie Ziehr, M.D.,Wolfgang Arzt, Walter Costamoling, M.D.,and Omar Shebl, M.D.
a Landes- Frauen- und Kinderklinik Linz, Kinderwunsch Zentrum, Linz; b Medical University, Departement of GynecologicalEndocrinology and Reproductive Medicine, Innsbruck; c Landes- Frauen- und Kinderklinik Linz, Institute of PrenatalGenetics, 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 frozen-thawed testicular sperm extraction (TESE).
Design: Artificial sperm activation was offered to azoospermic patients between January and October 2010 in twodifferent 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 thawedtesticular 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, clinicalpregnancy, and live birth.
Result(s): All patients but one (98.5%) showed a significant improvement in testicular sperm motility whentheophylline was used. In addition, sperm selection took significantly less time in the study as compared with inthe 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/blastocystsderived from oocytes that had been injected with pharmacologically stimulated testicular spermatozoa weretransferred (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. Itsimmediate effect allows for faster and more accurate selection of viable sperm, which in turn improved fertilizationand pregnancy outcome in this prospective study. (Fertil SterilÒ 2011;-:-–-. Ó2011 by American Society forReproductive Medicine.) Key Words: Immotile sperm, sperm viability, stimulation of sperm motility, TESE, theophylline The development of intracytoplasmic sperm injection (ICSI) has of sperm motility, cryopreservation of TESE probes to spare the patient been of great interest in the treatment of male factor infertility.
repeated TESE will also not be helpful at all.
Because this technique also allows the usage of epididymal and tes- Over the years, there have been numerous attempts to resolve this ticular spermatozoa, a wide range of possibilities are now available problem by identifying pharmacological agents that might improve for fertilization in cases of extreme male pathology.
sperm motility, thus increasing fertilizing ability. Although some of When working with testicular tissue, it appears difficult to isolate the compounds tested had to be administered orally , the vast a clean preparation of usable sperm because of the reduced tendency majority of agents were used in situ. Among others, caffeine of the sperm to detach from the testicular tissue owing to its limited and other methylxanthines relaxin , 2-deoxyadenosine motility. In addition, the presence of numerous cell types in the testic- , and kallikrein have been successfully used.
ular sperm extraction (TESE) material will also interfere with this In particular, pentoxifylline turned out to be an effective tool in effort . This problem may be overcome by enzymatically (e.g., stimulating motility in fresh and cryopreserved human semen collagenase) digesting the testicular cell aggregates or by introducing and identifying viable sperm in TESE patients presenting modified processing techniques . However, owing to the nature of exclusively with immotile sperm before or after cryopreservation immature testicular spermatozoa, the reduced motility of extracted . As with other xanthine derivates, the stimulatory effect sperm, if moving at all, will represent a persistent problem. In terms of pentoxyphylline can clearly be attributed to the increasedintracellular levels of cyclic AMP a molecule involved in thegeneration of sperm energy, which is a result of its inhibitory prop- Received May 17, 2011; revised August 11, 2011; accepted August 30, erties on phosphodiesterase function.
Interestingly, theophylline, a closely related molecule, has been T.E. has nothing to disclose. G.T. has nothing to disclose. R.B.M. has investigated for this purpose to a much lesser extent. As a ready- nothing to disclose. S.Z. has nothing to disclose. W.A. has nothing to to-use theophylline has recently been launched (GM501 SpermMo- disclose. W.C. has nothing to disclose. O.S. has nothing to disclose.
bil) and no prospective study using this dimethylxanthine on frozen Reprint requests: Omar Shebl, M.D., Landes- Frauen- und Kinderklinik Linz, Kinderwunsch Zentrum, Krankenhausstrasse 26–30, A–4020 and thawed testicular tissue has yet been reported, we decided to set Linz, Upper Austria, Austria (E-mail: ).
up such a prospective study. Fertilization, embryo quality, blastocyst Fertility and Sterilityâ Vol. -, No. -, - 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
formation, and rates of implantation and pregnancy were analyzed in hyaluronidase (Origio). Immediately after this process, ICSI was started detail in sibling oocytes (injected with testicular sperm treated either (approximately 4–5 hours after testicular tissue thawing). Only mature meta- with or without theophylline) of azoospermic couples.
phase II oocytes were considered for ICSI.
After denudation, randomization of the gametes was performed ).
Specifically, all oocytes of a patient were split into two groups. This was done under a binocular microscope, which did not allow proper identification From January 2010 to March 2011, theophylline treatment was offered to all of oocyte quality. However, during this process, immature oocytes at pro- couples presenting in Linz and Innsbruck with azoospermia who had at least phase I showing a distinct germinal vesicle were removed. Different percent- six mature oocytes collected. Approval of the Institutional Review Board was ages of metaphase I oocytes (not seen under a binocular microscope) resulted sought and given. During this 15-month period, 73 patients (39.1 Æ 4.2 years) in unequal numbers of injected oocytes in both groups.
with obstructive azoospermia provided written consent to participate in the In the oocytes used as a control group, ICSI was performed with untreated present prospective study. In 18 patients (27.7%), azoospermia was due to va- motile testicular spermatozoa (with the exception of three patients who only sectomy, and another 11 (16.9%) had a bilateral congenital absence of the vas had immotile sperm after 4–5 hours of incubation). The second half of the deferens (six of them were heterozygous for cystic fibrosis). Seven men eggs had spermatozoa injected that were pretreated with a ready-to-use (10.8%) had a status postchemotherapy, and two (3.1%) had a nonfunctional theophylline solution (GM501 SpermMobil, Gynemed) to stimulate their vas deferens caused by a previous chlamydia infection. The remaining 27 (41.5%) patients had total necrospermia in the ejaculate, aspermia, or retro- It has been reported that the maximum activity of methylxanthines is grade ejaculation. It is important to note that inclusion of different subgroups reached after 10 minutes, with an activity phase of less than 2 hours .
of patients (e.g., obstructive and nonobstructive azoospermia) could have Because of this immediate and short-term effect, direct addition into the influenced the outcome, which would limit the predictive value of the present droplet containing the immotile spermatozoa is recommended. In the pres- ent study, a small volume (0.5 mL) of GM501 SpermMobil was added per The vast majority of TESE (n ¼ 51) was performed at the Department of swim-out drop. It turned out that the final dilution (1:20 with BM1 medium) Urology at the Krankenhaus der Barmherzigen Schwestern in Linz. In short, allowed for an immediate start of the search for motile spermatozoa. As in the technique of TESE began with a relatively small incision being made in the control group, use of morphologically normal sperms with the highest the scrotal skin and carried through the peritoneal tunica vaginalis. Next, motility (e.g., fast forward progressive motility) according to the criteria small pieces of testicular tissue were extruded through an opening in the tu- suggested by the World Health Organization was preferred. Spermato- nica albuginea. The removed pieces of tissue were placed in tubes containing zoa selected for ICSI were collected individually, transferred to a small drop BM1 medium (Eurobio) for further manipulation. Biopsies were mechani- of polyvinylpyrrolidone, and immobilized by use of the ICSI pipette (Mi- cally disaggregated within half an hour under sterile conditions in a Petri croTech, Gynemed). The ICSI technique itself was performed as described dish containing BM1 medium. WE tried to accumulate as many spermatozoa as possible in the liquid supernatant.
Fertilization was controlled in EmbryoAssist Medium (Origio) 18–20 On average, 4.3 (Æ1.9) million testicular sperm per milliliter were retrieved hours post-ICSI and considered to be regular if two pronuclei were found from the tubuli seminiferi using this mechanical squeezing technique. A total to be abutted in the center of the oocyte. At cleavage stage (days 2 and of 41 (63.1%) TESE samples did not show any signs of motility immediately 3), embryos were scored according to their number and symmetry of blas- before cryostorage. Since none of the biopsies was planned to be used in tomeres and checked for the presence of multinucleated cells. If blastocyst a fresh cycle of ICSI, all were frozen with an automatic slow freezing proce- transfer was considered, beginning and extent of compaction were recorded dure (CL-8000, CryoLogic) using glycerol as a cryoprotectant (SpermFreeze, on day 4 Correspondingly, survival was analyzed at the blastocyst FertiPro). The presence of hindering testicular tissue was carefully avoided.
stage . According to previously published criteria , the top-quality Before ovum pickup a sufficient number of frozen straws (depending on blastocyst group consisted of blastocysts in which at least one cell lineage the sperm count of the fresh biopsy) were delivered to the Kinderwunsch was quality A and none were quality C. If full blastocyst stage was not Zentrum at the Landes- Frauen- und Kinderklinik in Linz. This certified reached at the time of morphological analysis, top-quality early blastocysts transport was performed under liquid nitrogen by an authorized and qualified showed no cytoplasmic loss due to fragmentation or extrusion of blasto- On the morning of the day of ICSI, testicular sperm was rapidly thawed by Transfer was scheduled for either day 3 (n ¼ 11) or day 5 (n ¼ 48). It is directly plunging the straws into a 37C water bath. Cryoprotectant was re- important to note that selection of embryos or blastocysts for transfer was moved by two centrifugation steps (1 minute at 5,000 rpm). The pellet was based on routine morphological criteria and that no randomization for study then resuspended in a small volume of BM1 medium. Since it turned out or control group was done on the transfer day. In principle, we planned elec- that more or less immediate use of thawed sperm involved the risk of com- tive single embryo or blastocyst transfers. However, owing to female age or plete immotility ICSI was planned 4–5 hours post-thawing. After this previously failed cycles, some patients wanted to have two concepti trans- incubation period, testicular suspension was placed in 10-mL swim-out drops ferred. In these rare cases, the quality of the best embryo (first pick) specified arranged on an ICSI dish under oil. As soon as at least one motile spermato- the group from which both embryos/blastocysts should be chosen (which was zoon was observed (for all patients but two), an adequate number of mature not always possible). This strategy helped to minimize mixed transfers (e.g., oocytes were loaded into the same dish. Thus, for the female gametes, the one embryo from the study and one from the control group).
time out of the incubator was kept at a minimum.
A total of six patients had all their viable blastocysts vitrified owing to Only 14 patients had their testicular biopsy done in other cities. All of these a high risk of ovarian hyperstimulation syndrome. Subsequently, these partic- samples showed no testicular suspensions but rather clumps of tissue. The as- ular patients had one thawed ET each.
sociated straws were thawed and placed in a medium containing collagenase Nineteen days after oocyte collection, the blood concentration of hCG was (GM501 Collagenase, Gynemed) for facilitation of sperm isolation before measured. Biochemical pregnancy was defined as a significant increase in hCG levels (>10 mU/mL). The implantation rate was defined by ultrasound All female partners (30.9 Æ 2.4 years) were stimulated according to a long visualization, 4 weeks after ET, as a gestional sac per embryo transferred.
protocol. Down-regulation was performed with Triptorelin (Decapeptyl, Fer- This included subclinical (gestational sac but no fetal heartbeat) as well as ring), and the gonadotropins used were of a recombinant nature in 12 cases clinical pregnancies (at least one gestational sac with positive heart activity).
(Puregon, MSD) and urinary (Menopur, Ferring) in the remaining 19 women.
All patients who had not delivered at the time of manuscript submission No cases of endometriosis or polycystic ovarian syndrome were seen in the showed an unsuspicuous ultrasound at our specialized Institute of Prenatal patient cohort. Oocyte collection was scheduled for 2–3 hours after thawing of the testicular biopsies and performed via the vaginal route.
Differences between continuous variables were assessed with the t-test for Cumulus-oocyte complexes were collected in BM1 medium and cultured independent samples and with the c2-test for categorical variables. An alpha for another 2 hours in the incubator before careful denudation using error rate below .05 was considered to be statistically significant. Clinical Schematic presentation of the study design and outcome.
Oocytes of study goup
Oocytes of control group
In vitro culture to day 3 (n=11) or day 5 (n=48) Ebner. Use of theophylline in thawed TESE. Fertil Steril 2011.
pregnancy rate and implantation rate were calculated using pooled day 3 and (602/842). indicates that usage of theophylline was associ- day 5 results since no differences in outcome were observed.
ated with a significantly increased fertilization (P<.001) as com-pared with the untreated control group. This difference was notassociated with parthenogenetic activation or nondisjunction Addition of theophylline improved motility in 64 out of 65 patients Cleavage rate and embryo quality showed no differences in the (98.5%) involved in this study (one patient did not have motile study and control groups during days 2–4. However, the presence sperm irrespective of the treatment with theophylline). Thus, time of multinucleated cells on day 3 was significantly higher (P<.01) for identification and isolation of motile testicular sperm after thaw- in the control group as compared with in the theophylline-treated ing was significantly faster (P<.01) in the study group.
study group (). The overall blastulation rate was found to A total of 842 oocytes in 65 patients were treated with ICSI in this be 56.7% (274/483), with a significantly better performance in the prospective analysis. The corresponding fertilization rate was 71.5% Fertilization and preimplantation development in 31 Pregnancy outcome after transfer of combined fresh and azoospermic patients with or without theophylline for vitrified/warmed concepti deriving from the study and/or improving testicular sperm motility after cryopreservation.
Note: Values in parentheses are percentages. BT ¼ blastocyst transfer.
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, whereasthose gametes with damaged or osmotically inactive membranes Note: Values in parentheses are percentages. PN ¼ pronucleus/ei.
Recently, another alternative was introduced that suggests the use of a diode laser to assess viability in cases of complete asthe- Ebner. Use of theophylline in thawed TESE. Fertil Steril 2011.
nozoospermia. Applying a single laser pulse at the end of the spermtail caused a characteristic curling of the tail. Since nonviable spermdid not show this phenomenon, this new technique helped to identify Pooled fresh and thawed transfers resulted in a detectable level of a spermatozoon with functional integrity of its membrane.
ß-hCG in 50.8% of the patients. The corresponding implantation The most elegant strategy is the only one that allows for partial rate was found to be 41.9%. Four missed abortions and two extra- restoration of original motility , namely, the in situ use of meth- uterine pregnancies reduced the overall ongoing pregnancy rate to ylxanthines, such as pentoxy- or theophylline.
41.5%. As indicated in rates of positive ß-hCG as well as Pentoxyphylline, in particular, has been intensively studied. It has clinical pregnancy were significantly higher in the theophylline- been found to be superior to caffeine . At different concentra- tions (0.7–3.6 mM/L), pentoxyphylline has been shown to increase Only one out of 27 babies (3.7%) so far (August 2011) showed the percentage of motile sperm in a given ejaculate without nega- a minor malformation (polydactilism). This offspring stemmed tively affecting sperm membrane and acrosome reaction This 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 mor- Testicular sperm is usually immotile or shows only a minor twitch- phological changes. In addition, data from animal studies show that ing movement upon extraction Cryopreservation of TESE prolonged incubation (e.g., between 24 and 72 hours) of mouse em- material somewhat aggravates this unwanted condition. In the bryos in a 5-mM solution of a dimethylxanthine led to variations in absence of motile gametes, embryologists previously were forced cyclic AMP content as well as developmental retardation or embryo to perform ICSI with immotile spermatozoa. Although it may be death Other studies suggested that even shorter exposure (30 expected that a certain percentage of immotile spermatozoa are minutes) to 3.6 or 7.2 mM/L pentoxyphylline, while not affecting viable in such a scenario, injection of immotile sperm resulted in blastocyst development could have a negative impact on birth decreased fertilization and pregnancy rates However, immotil- rate In addition, pentoxyphylline-enriched media artificially ity does not preclude viability. Theoretically, embryologists have activated mouse oocytes in a concentration- and exposure time– several options for dealing with the tricky problem of exclusively dependent manner It has been reported that high dosages of caffeine and its derivatives (5–300 mg/kg) cause malformations in The most reasonable approach would be to use the ICSI pipette to animals This teratogenic effect was particularly evident test the elasticity of the sperm tail Once manipulated with in a potential interaction with certain other compounds a glass tool, a spermatozoon showing an elastic tail is presumed to (all of which were not present in culture media).
be more viable than one with a more rigid flagellum. However, there Interestingly, at very low concentrations (0.5 mM/L), pentoxyfyl- is no guarantee that more elastic sperms are viable, that is, are os- line reduced oxidative stress–induced embryotoxicity in mice motically intact cells. For confirming osmotic capacity, sperms However, to our knowledge, no reports on approaches using can be incubated in a hypoosmotic swelling solution pentoxyphylline or other related agents on human oocytes or embryos have been published to date. Rather, they were used with some success In fact, several benefits were observed for the first time in the to raise motility and, thus, increase outcome in patients with IUI present prospective approach. The observed significant stimulatory and previous fertilization failure after IVF . Since ICSI has effect of theophylline on sperm motility definitely facilitates labora- become a powerful alternative in such patients, total lack of sperm tory work for the embryologists. It not only significantly reduces the movement or presence of immotile sperm in microsurgical epi- time needed for ICSI owing to an immediate identification of motile didymal sperm aspiration and TESE patients are the only spermatozoa (thus limiting the time of the oocytes out of the incuba- indications being left for routine use of methylxanthines such as tor), but it also allows for distinguishing between viable spermato- zoa and borderline counterparts (e.g., those with rudimentary The latter has not been investigated in detail. Nevertheless, a similar motility) at one glance. These advantages probably led to a higher stimulatory effect on sperm penetration in humans has been reported fertilization rate in the present study group. Whether an augmented that has been seen up to a concentration of 20 mM/L theophylline acrosome reaction played a role in this context will need to Addition of 2.5 mM/L theophylline increased the percentage of male pronuclear formation and blastocyst formation in animals .
The better developmental potential in the theophylline-treated The most probable reason for the disproportionate use of pentox- group culminated in a significant improvement in blastocyst forma- yphylline as compared with theophylline is the slightly increased tion. Blastocyst quality was not influenced, indicating that in the hydrosolubility (however, both dimethylxanthines show a higher case that blastocyst stage was reached in the untreated cohort, highly solubility in water as compared with caffeine) In turn, viable sperms had been chosen for ICSI (although their motility was a much higher half-life (4–8 hours) in theophylline as compared slower as compared with the theophylline group). Interestingly, with pentoxyphylline (0.4–0.8 hours) allows for appropriate pre- multinucleated cells were more frequent in the control group on warming before addition to the testicular suspension without day 3, and although no logical explanation can be given for this phenomenon, it is reassuring to know that this problem was not In these times of the European Tissue Directives, blending of di- methylxanthine solutions on one’s own is a sheer impossibility.
However, multinucleation did not influence pregnancy outcome Thus, the availability of a standardized commercial product is of since affected embryos were never transferred. Owing to similar em- great help in the treatment of severe male factor subfertility. In the bryo and blastocyst qualities, the observed difference in positive present study, the ready-to-use stock solution of theophylline ß-hCG and clinical pregnancy rate could be related to the viability (SpermMobil) was used at a 20-fold dilution, and duration of expo- of the transferred concepti. This supports the usefulness of the sure was limited to a few minutes (without limiting its bioactivity).
analyzed dimethylxanthine in frozen and thawed TESE material.
In addition, the selected testicular spermatozoa were washed thor- Although not reflected in the live birth rate, the absence of minor oughly and transferred in theophylline-free medium and PVP as rec- or major malformations in the newborn after theophylline treatment ommended elsewhere thus, the theoretical risk of a biological further supports its usage in patients with mostly immotile sperma- hazard existing on female gametes and/or embryos is negligible.
tozoa (before or) after cryopreservation.
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