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Clinical Use of Pentoxifylline for Activation of Immotile Testicular Sperm Before ICSI in Patients With Azoospermia

From the Department of Reproductive Medicine and Gynecologic Endocrinology, Maribor Teaching Hospital, Maribor, Slovenia.

Correspondence to: Borut Kovai, Department of Reproductive Medicine and Gynecologic Endocrinology, Maribor Teaching Hospital, Ljubljanska 5, SI-2000 Maribor, Slovenia (e-mail: borut.kov@sb-mb.si ).

	Abstract

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The testicular sperm from biopsy and frozen/thawed tissue are frequently immotile. The purpose of our retrospective study was to assess the effect of short exposure of testicular samples with only immotile sperm to pentoxifylline (PF)-sperm motility stimulator. In 77 of 294 (26.2%) testicular sperm ablation/testicular sperm extraction-intracytoplasmic sperm injection (TESA/TESE-ICSI) cycles in patients with azoospermia, only immotile sperm were found in biopsies even after 2 hours of incubation of tissue in the medium. These 77 cycles were divided into 2 groups. In group 1 (cycles between 1999 and 2001; n = 30), ICSI was performed with untreated immotile sperm. In group 2 (cycles between 2002 and 2004; n = 47), immotile testicular sperm were treated for 20 minutes with pentoxifylline (PF) (1.76 mM) before ICSI. Both groups had the same proportion of ICSI cycles with fresh, frozen/thawed, and aspirated testicular sperm. The overall pregnancy rate of TESA/TESE-ICSI did not vary during the study period. In 45 of 47 (95.7%) testicular samples with total immotility, the sperm started to move 20 minutes after PF treatment. The mean time required for ICSI was shortened in the PF group (30 minutes [minimum 10, maximum 90] vs 120 minutes [minimum 60, maximum 240]) due to easier identification of motile sperm. In comparison with the nontreated group, the PF group had a higher fertilization rate (66% vs 50.9%; P < .005) and mean number of embryos per cycle (4.7 ± 3.3 vs 2.7 ± 2.1; P < .01). The clinical pregnancy rate per cycle in PF and non-PF groups was 38.3% and 26.7%, respectively. By using PF in cases of only immotile testicular sperm we can cause movement of testicular sperm, allow easier identification of vital sperm, shorten the procedure, improve fertilization rates, and increase the number of embryos.

     Key words: Testicular biopsy, human spermatozoa, phosphodiesterase inhibitors, motility stimulants, fertilization

In 2 larger studies (Van Steirteghem et al, 1998; Palermo et al, 2003) analyzing 536 and 865 ICSI cycles, respectively, with surgically retrieved spermatozoa, the overall clinical results of the TESA/TESE-ICSI procedure are comparable to ICSI using ejaculated sperm. Nevertheless, the fertilization was always slightly lower (64.8% vs 53.4% and 75.3% vs 66.6%, respectively) when testicular sperm were used. Despite the still satisfactory mean fertilization rate after TESA/TESE-ICSI, an unpredictable fertilization failure or very poor fertilization outcome can still occur in some patients. The lower fertilization is mostly due to severe spermatogenic defects and the additional freezing of the testicular sample, which can harm the sperm (Palermo et al, 2003).

The testicular sperm are mostly immotile immediately after biopsy and especially after thawing of frozen testicular samples (Bachtell et al, 1999). For this reason the suspension of testicular cells is usually incubated in the culture media for a certain period of time. Motile sperm, ie, sperm showing reliable signs of vitality, are preferred for ICSI because the use of motile sperm significantly increases the efficacy of the ICSI procedure (Nagy et al, 1998; Park et al, 2003).

In cases of total immotility of the sperm sample despite short culturing in media, some other methods have also been used for recognizing the vital spermatozoa. There are reports of clinical success with ICSI using sperm from testicular tissue that started moving after prolonged, overnight cultivation in culture media (Emiliani et al, 2000; Wood et al, 2003). The hypo-osmotic swelling (HOS) test has also been successfully clinically applied in cases of total immotility (Verheyen et al, 1997). But both of these tests could also be harmful to sperm.

Even before ICSI was introduced, many tried to prove that some phosphodiesterase inhibitors could improve or even induce the motility of the sperm (Aparicio et al, 1980; Makler et al, 1980; Kay et al, 1993; Tournaye et al, 1994a). The most frequently used inhibitor was pentoxifylline (PF) (Yovich et al, 1988; 1990; Yovich, 1993), but its use was superseded by the rapid expansion of ICSI. Additionally, reports came out about possible harmful effects of PF. Namely, some animal studies showed an adverse effect of PF on mouse embryo development when the oocytes were cultured in the medium with PF (Tournaye et al, 1993; 1994b; Scott and Smith, 1995).

The purpose of our research was to determine the effectiveness of short PF treatment of testicular samples with only immotile sperm, without any exposure of oocytes to PF, and the clinical success of TESA/TESE cycles treated in this manner.

	Materials and Methods

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Study Design

The study included 77 out of 294 (26.2%) TESA/TESE cycles in patients with azoospermia in which no motile sperm were found in resuspended biopsy material even after 2 hours of incubation in Sperm Preparation Medium (Medicult, Jyllinge, Denmark). Between 1999 and 2001 (group 1) we performed ICSI with immotile testicular sperm if no motile sperm were found after 2 hours of incubation (n = 30). The second group (group 2), treated between 2002 and 2003, were cycles in which PF was added to the sample if only immotile sperm were found in testicular tissue (n = 47).

The working conditions, equipment, and materials remained unchanged throughout the study. In 2000 we started with blastocyst culture, and both groups had the same proportion of day 3 and day 5 transfers (Table 1). In the time between 1999 and 2003 there was a constant pregnancy rate per cycle in the in vitro fertilization (IVF), ICSI, and TESA/TESE-ICSI group each year. Initially, the groups were compared according to the patients' clinical features in the sense of age of female patients, female indications for IVF, type of testicular samples for ICSI, and the number of oocytes per cycle. The main outcome of the study is reflected in the fertilization rate, the number of embryos, and the pregnancy rate.

Ovarian Stimulation

All women were synchronized with oral contraceptives, starting the long stimulation protocol with a daily application of tripto-relin (Decapeptyl 0.1 mg) or a single application of depot gonadotropin-releasing hormone antagonist (GnRHa) goserelin (Zoladex 3.6 mg, Zeneca, Cheshire, United Kingdom). Pure follicle-stimulating hormone (FSH) (Metrodin-HP, Serono, Geneve, Switzerland) or recombinant FSH (Gonal F, Serono, Geneve, Switzerland) was applied for controlled ovarian hyperstimulation. The daily FSH dose was adjusted to ovarian response to stimulation. On the day folliculometry revealed that the mean diameter of the dominant follicle had reached 18 mm, the time for human chorionic gonadotropin (hCG) injection was set. All patients applied hCG (Profasi, Serono, Auborne, Switzerland) in doses of 10 000 IU subcutaneously by self-administration. Follicular puncture followed 36 to 37 hours later.

Testicular Sperm Recovery and Treatment With Pentoxifylline

Samples of testicular tissue were taken by aspiration biopsy (TESA). A Veneflex (TIK, Kobarid, Slovenia) aspiration set used for the aspiration was filled with 0.25 mL of Sperm Preparation Medium (Medicult, Jyllinge, Denmark) at the distal end. After testicular puncture, suction was established in the set by using a 10 mL syringe. The aspirate content was expressed into a petri dish containing a drop of polyvinylpyrrolidone (PVP) (Medicult) and covered with paraffin oil. Two minutes later, the fibrin membrane formed on top of the droplet was gently removed with subcutaneous needle.

In patients in whom we were not able to recover sperm by diagnostic aspiration, an open testicular biopsy (TESE) was performed. A small incision was made in the scrotal skin and carried through the peritoneal tunica vaginalis. One incision was made in the tunica albuginea, and a piece of the extruding testicular tissue was excised. The testicular tissue was rinsed with Sperm Preparation Medium. A piece of the tissue was prepared for cytohistologic assessment. Another part was transferred to 1 mL of equivalent fresh medium. The tissue was minced with the use of 2 scalpels. The major part of the testicular sample was pipetted into a test tube and prepared for cryopreservation. The rest of the testicular sample was covered with paraffin oil and evaluated for the presence of sperm. Only those patients with sperm found in the aspirate or biopsy were included in the study.

A glycerol based cryoprotectant (Sperm Freezing Medium, Jyllinge, Medicult) was used for cryopreservation of the testicular sample. The sample was diluted with the cryoprotectant in the ratio of 1:1, and filled into 0.5 mL plastic straws (Rocket Medical, Washington, United Kingdom). The straws were laid on a horizontal carrier 1 cm above the liquid nitrogen and exposed to the liquid nitrogen vapors for 20 minutes. The straws were then immerged into the liquid nitrogen. After the straws were quickly thawed, their contents were emptied into a petri dish next to the PVP droplet and covered with paraffin oil.

The dish containing the testicular sample was observed under 200x magnification on an inverted microscope. The first observation was made immediately after the sample was prepared. All sperm with the slightest tail movement were considered motile. If no motile sperm were found, the preparation was incubated for 2 hours. In 30 such cases, immotile sperm were used for ICSI (group 1). From among immotile sperm those with a not completely rigid tail when touched with a pipette were chosen for ICSI. The same method was described by De Oliveira et al (2004). In only 2 cases were all sperm not pliable after touching.

In 47 cases (group 2) between 2002 and 2004, the samples having immotile testicular sperm even after 2 hours of incubation were treated with pentoxifylline (Sigma Chemical Co, St Louis, Mo), a chemical which is approved for using in humans by the US Food and Drug Administration (FDA). Sperm Preparation Medium with PF added (1 mg/mL) was applied to a droplet of testicular cell suspension in the ratio 1:1, so that the final concentration of PF in the sample was 0.5 mg/mL (1.76 mM). The sample was incubated in the atmosphere with 5% CO₂ and 95% relative humidity at 37°C for 20 minutes. After that the sample was observed for identification and isolation of motile sperm.

The sperm chosen for ICSI were transferred to the PVP droplet. After collecting a sufficient number of sperm in the PVP, the injection capillary was replaced. The ICSI procedure was performed in the classical manner. The share of fresh or thawed testicular samples in the individual group is shown in Table 1.

Culture System, Embryotransfer, and Establishment of Pregnancy

The oocytes and embryos were cultivated for 3 days in the combination of Universal IVF Medium and M3 Medium (Medicult) or 5 days in BlastAssist System (Medicult), respectively. The ratio between the 3-day and 5-day cultivation in the compared groups is shown in Table 1.

Embryotransfer was performed using the soft part of the catheter (Labotect, Goettingen, Germany). Pregnancy was proved by quantitative determination of beta hCG in serum, and implantation by ultrasonographic evidence of the gestation sac with heart beat 4 weeks after embryotransfer.

Statistical Analysis

The differences between the groups were evaluated using the Mann-Whitney U test and ² test. A P value of less than or equal to .05 was considered statistically significant.

	Results

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Total immotility of sperm was established in 77 out of 294 (26.2%) azoospermic patients (in 59.1% [46/78] frozen/thawed testicular excisions, in 17.6% [3/17] fresh testicular excisions, and in 14.1% [28/199] testicular aspirates). The comparison of clinical parameters between the compared groups indicates that the groups do not differ regarding the average age of the women, female indications for IVF, response to controlled ovarian hyperstimulation expressed in the mean number of gonadotrophin ampoules used, mean number of recovered oocytes in metaphase II, mean Johnsen's score and mean FSH concentration in men, the sperm recovery technique, and the number of cycles with short-term or long-term cultivation (Table 1).

In only 2 of 47 (4.3%) samples with exclusively immotile sperm treated with PF, we were unable to trigger motility. Both cases ended in total fertilization failure.

After PF treatment, different changes in sperm motility were noticed, expressed as barely noticeable tail twitching or vigorous twisting, and in some cases also as progressive movements. However, the percentage of motility was not evaluated, because in many cases—hemorrhagic in particular (Figure)—we found sperm in the sample only after PF treatment, when they started to move.

View larger version (76K): [in this window] [in a new window]   (a) The identification of testicular sperm treated by pentoxifylline in the biopsy material is facilitated by the movement of sperm and adjacent erythrocytes. (b) The sperm-erythrocytes complex is aspirated and transferred into the droplet of polyvinylpyrrolidone (PVP). (c) The sperm is isolated from the erythrocytes, washed in the PVP and used for intracytoplasmic sperm injection.

The group in which ICSI was performed with immotile sperm (group 1) was compared with the group in which sperm were treated with PF before injection (group 2). In the comparison of clinical results shown in Table 2, we established that the higher fertilization in group 2 was statistically significant (50.9% vs 66%; P < .005) when compared to group 1. Also the comparison of medians, calculated from the fertilization rates of each couple, showed significantly better fertilization in the PF-treated group (46.4% vs 66.7%; P < .05). Subsequently the mean number of embryos per cycle was higher in group 2 compared to group 1 (2.7 ± 2.1 vs 4.7 ± 3.3; P < .01).

There was no statistically significant difference in the clinical (26.7% vs 38.3%) and ongoing pregnancy rates per cycle (26.7% vs 31.9%) between the non-PF and PF group. The mean time required for identification and isolation of the sperm and ICSI amounted to 120 minutes (minimum 60, maximum 240) per cycle in group 1 and only 30 minutes (minimum 10, maximum 90) per cycle in group 2 because of easily identified moving sperm.

	Discussion

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Pentoxifylline is a phosphodiesterase inhibitor of the methylxantine group. It inhibits the breakdown of cyclic adenosine monophosphate (cAMP) and it is known that intracellular cAMP concentration plays a central role in sperm motility (Tash and Means, 1983).

In assisted reproductive technology, the advantageous effect of PF on improving sperm motility and fertilization capacity in asthenozoospermia has often been confirmed (Yovich et al, 1988; 1990; Rizk et al, 1995; Tarlatzis et al, 1995; Negri et al, 1996). It is likely that the method would be used frequently in assisted reproduction procedures for the treatment of male infertility if ICSI had not been developed.

On the other hand, some studies showed that cAMP phosphodiesterase inhibitors are not beneficial in enhancing fertilization rates (Tournaye et al, 1995) or can have a negative effect on animal oocytes and embryos. They can cause meiotic arrest in mammalian oocytes (Downs et al, 1986), poorer development of mouse embryos (Tournaye et al, 1993; 1994b), 2-cell embryo block in some mouse strains (Loutradis et al, 1987; Downs and Dow, 1991), or parthenogenetic activation of mouse oocytes (Scott and Smith, 1995). But there are too few prospective randomized studies to confirm the negative effects of PF on human oocytes.

The application of PF proved safe in earlier clinical studies, under observation of the instructions for use. The testing of embryotoxicity of PF in mouse IVF and embryotransfer models showed that the fertilization rate and the blastocyst formation rate was not lower if only sperm were exposed to PF. However, they were lower if the oocytes were exposed to PF as well (Yovich, 1993).

On the basis of the findings that PF is not beneficial in enhancing fertilization rates in zero or poor in vitro fertilizers, and can be toxic for oocytes, it was suggested that PF should be used only in strictly selected patients (Tournaye et al, 1993; 1995).

Our study was done on a small specific group of patients for which we showed that some clinical results were improved by using PF. In our study the testicular cell suspension was exposed to a much lower concentration of PF (1.76 mM) than in most previous studies (3.6 mM) and also for very short periods (20 minutes), shorter than in other studies reporting about different characteristics of sperm movement (reviewed by Tournaye et al, 1995). The sperm were transferred from the medium with PF to PVP, where they were mechanically washed. For the ICSI procedure, the injection pipette was replaced by a new one to decrease the possibility of the oocytes being affected by PF.

The deleterious effect of PF on oocyte activation reported in the literature (Scott and Smith, 1995) was not observed in our study, as the proportion of single pronucleated zygotes was normal. Embryo development also was not affected, and half of the embryos cultivated to day 5 reached the blastocyst stage.

In our study group, PF proved to be effective. The reactivity of the testicular samples to PF is surprising, as a positive effect on the cell mechanisms responsible for tail movement was observed in 95.7% of PF-treated TESE/TESA samples. Additionally, other studies have shown that only in rare samples does PF not trigger hyperactivation. It was also proved that there is large interindividual variability in sperm reactivity to PF (Kay et al, 1993; Moohan et al, 1993).

Most cases of totally immotile sperm were seen in thawed testicular samples, but in 27 of 29 samples motility was later triggered with PF. This confirmed that cryopreservation of testicular tissue is successful and not damaging to sperm vitality, but it does decrease sperm motility.

It has been demonstrated with ejaculated sperm that the highest effect of PF can be expected after 30 minutes of application of PF (Yovich et al, 1990). Other authors have reported that the effect of PF vanishes after 3 hours (Kay et al, 1993), which needs to be considered in TESA/TESE cases, where searching for sperm can take more time. Searching for sperm, isolating them in the testicular sample, and conducting ICSI can sometimes take a few hours. The procedure is often prolonged if we try to use only motile sperm for ICSI.

Nevertheless, the use of PF saves a considerable amount of time in the ICSI procedure because the sperm start to move after 20 minutes. Therefore, the TESA/TESE procedure with PF does not represent a bigger strain for the embryologist in terms of time compared with the classical ICSI procedure, as the isolation of motile sperm from biopsy material and ICSI amount to 30 minutes on average. The identification of sperm among erythrocytes, germinal cells, and cellular debris is facilitated by the movement of sperm and adjacent cells (Figure). Moreover, in many bloody testicular samples with only few spermatozoa, the sperm has been identified only after addition of PF. For this reason we routinely use PF in diagnostic TESA samples.

The analysis of our clinical results confirmed reports (Nagy et al, 1998; Park et al, 2003) that the use of immotile sperm causes poorer fertilization. However, we showed that in such cases the mean fertilization could be improved significantly when initially immotile testicular sperm are activated with PF. In this group the achieved 66% fertilization does not differ from the fertilization rate in all TESA/TESE cycles with motile sperm, amounting to 67.8% at our center. Due to the high variability in oocyte number among couples, we also calculated the mean fertilization rate per couple for each group, which was expressed by the median. Statistical analysis showed again the significant difference in medians between compared groups (46.4% vs 66.7%). The use of PF thus allowed the development of a higher number of embryos and subsequently also better chances in embryo selection for transfer and freezing. Despite the not statistically significant differences in pregnancy rates per cycle between the 2 groups, there is a noticeable trend toward improved clinical results in cases where immotile sperm were activated by PF.

Some authors have reported about attempts to stimulate the motility of surgically retrieved sperm by PF (Kohn et al, 1996; Tasdemir et al, 1998; Angelopoulos et al, 1999). Using testicular sperm treated by PF, some pregnancies were also achieved (Nodar et al, 1999; Terriou et al, 2000; Mátyás et al, 2005). However, no prospective or retrospective study compared the outcome of ICSI with initially immotile and induced motile testicular sperm.

A higher pregnancy rate in patients whose slowly motile ejaculated sperm were treated by PF has been reported by many authors (Yovich et al, 1988; 1990; Rizk et al, 1995; Negri et al, 1996). All showed that pregnancy wastage was not increased. However, we know that in azoospermic patients, especially those with nonobstructive azoospermia, the abortion rate can be higher (Pasqualotto et al, 2005). The 3 miscarriages (16.7%) in our PF group are thus still in the acceptable range. In any case, a larger group should be analyzed to confirm the benefit of PF.

Another alternative to PF in case of immotility of testicular sperm could be prolonged incubation of biopsy material under in vitro conditions (Wood et al, 2003). In cases in which prolonged incubation would not trigger sperm motility, the effect of subsequent PF application would be questionable, as PF does not have an influence on sperm after prolonged cultivation (Tournaye et al, 1994a). Additionally, it has been shown that prolonged incubation of testicular sperm causes DNA fragmentation (Dalzell et al, 2003).

The hypo-osmotic sperm swelling (HOS) test has also been used to prove the vitality of sperm before ICSI (Casper et al, 1996; Liu et al, 1997). Sperm with tails swollen due to the HOS effect, however, do not differ morphologically from the immature sperm that are numerous in TESE samples. The hypo-osmotic solution can also cause cell injury, especially on frozen/thawed spermatozoa that have accumulated membrane damage, or can lead to sperm death after 30 minutes of exposure to the hypo-osmotic solution (WHO, 1992).

The conclusion of the study is that, in most cases of total immotility of testicular sperm, which is most common in thawed testicular samples, PF triggers sperm motility after 30 minutes. This allows a better and quicker identification and selection of vital sperm for ICSI, resulting in a better fertilization rate and shorter expenditure of time for staff involved in TESA/TESE.

	Acknowledgments

 
The authors wish to thank Marijana Gajsek-Marchetti, translator, from the Medical Research Department of Maribor Teaching Hospital, for her contribution in preparing the manuscript.

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