Article Interest of pentoxifylline in ICSI with frozen thawed testicular spermatozoa from patients with non-obstructive azoospermia

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1 RBMOnline - Vol 12. No Reproductive BioMedicine Online; on web 23 November 2005 Article Interest of pentoxifylline in ICSI with frozen thawed testicular spermatozoa from patients with non-obstructive azoospermia Jean-François Griveau obtained his PhD in Biology from the University of Rennes, France, in Since 1990, he has been working as a research worker at the Biology of Reproduction Unit-CECOS in the Hospital of Rennes and is approved for the practice of IVF. Research interests include assisted reproduction and especially the implication of reactive oxygen species on human sperm function. Dr Jean-François Griveau Jean-François Griveau 1,4, Bernard Lobel 2, Marie-Christine Laurent 3, Laurence Michardière 1, Dominique Le Lannou 1 1 Unité de Biologie de la Reproduction CECOS, Hotel-Dieu, 1 bis rue de la cochardière, Rennes, France; 2 Service d urologie, CHR Pontchaillou, 2 rue Henri Le Guillou, Rennes, France; 3 Service de gynécologie, Hopital SUD, Rennes, France 4 Correspondence: Jean-Francois.Griveau@chu-rennes.fr Abstract Selection for intracytoplasmic sperm injection (ICSI) of viable frozen-thawed testicular spermatoza obtained from patients suffering from non-obstructive azoospermia is very often long, difficult and sometimes impossible. The purpose of this study was to determine if the use of pentoxifylline (PF) could facilitate this selection in stimulating sperm motility. From January 2000 to December 2004, 108 ICSI cycles with non-obstructive azoospermia were performed. From these 108 cycles, in 64 cycles where no motile spermatozoa were observed or when the time search per spermatozoa was above 20 min, 1.5 mmol/l PF was used for 10 min, whereas the 44 other ICSI cycles were performed using spontaneously motile spermatozoa (control group). In all cases, PF either initiated the motility when no motile spermatozoa were observed, or stimulated the motility, reducing dramatically the time search per spermatozoa. The total fertilization rate was 54.2% versus 66.7% in the control group (P < 0.02). Twenty-nine pregnancies out of the 64 PF cycles (45.3% per cycle) occurred, including 20 deliveries of 23 healthy children and eight ongoing pregnancies, whereas 12 pregnancies were obtained in the control group (27.3% per cycle), including nine deliveries of 13 healthy children. In conclusion, in 100% of cycles pentoxifylline allows the selection of viable frozen thawed testicular spermatozoa with the same outcome after ICSI as that observed with fresh ejaculated spermatozoa. Keywords: cryopreservation, ICSI, non-obstructive azoospermia, pentoxifylline, testicular spermatozoa 14 Introduction For a long time, the treatment of male sterility due to irreparable obstruction or congenital bilateral absence of the vas deferens (CBAVD) has been limited to insemination with donor s spermatozoa. Techniques of IVF in combination with microsurgical epididymal sperm aspiration (MESA) (Temple- Smith, 1985), and the first pregancies obtained by Silber et al. (1988) have been shown to offer new hope to couples suffering from this sterility. With the introduction of intracytoplasmic sperm injection (ICSI) and its evident ability to achieve high fertilization and pregnancy rates regardless of ejaculated semen parameters, it was possible to treat men suffering not only from obstructive (Tournaye et al., 1994) but also from nonobstructive azoospermia (Silber et al., 1996), with fertilization and pregnancy rates much higher than those obtained in IVF. The use of cryopreserved spermatozoa was then developed to avoid repetition of epididymal or testicular biopsies for successive ICSI cycles. It is of interest especially in patients with non-obstructive azoospermia, since multiple extensive testicular biopsies may represent a significant loss of testicular mass. It is also of interest to avoid unnecessary and potentially dangerous ovulation induction in the spouses, since unfortunately more than 50% of non-obstructive azoospermic patients have no spermatozoa in their testicular tissue (Silber et al., 1997; Devroey, 1998). So, testicular biopsy followed by cryopreservation of spermatozoa for subsequent attempts is now a procedure of choice for patients with testicular failure (Verheyen et al., 2004).

2 However cryopreservation of testicular biopsy-extracted spermatozoa involves a lot of difficulties because of their very low concentration and motility resulting from sperm immaturity. Thus the freezing process invariably results, especially in case of non-obstructive azoospermia, in a complete immotility of the spermatozoa, or at the best in a weakly shaking motility rendering the selection of a live spermatozoa for ICSI very long and difficult. As sperm motility after freezing/thawing of testicular tissue is the major determinant of the success of ICSI in nonobstructive azoospermia (Dafopoulos, 2005), several strategies have been developed to improve sperm motility after freezing, and several studies have reported an enhancement of sperm motility after in-vitro culture for a few days of fresh or frozen thawed testicular spermatozoa in cases of obstructive and non-obstructive azoospermia (Edirisinghe et al., 1996; Liu et al., 1997; Angelopoulos et al., 1999; Emiliani et al., 2000). Phosphodiesterase inhibitors such as (PF) have also been used to stimulate the motility of epididymal and testicular spermatozoa (Angelopoulos et al., 1999; Terriou et al., 2000; Le Lannou et al., 2001). Compared with the in-vitro culture of testicular biopsies, it has the advantage of rapidly distinguishing between live and dead spermatozoa, thus ICSI can be performed immediately. Thus, in an attempt to optimize the use of testicular biopsies, this study determined whether the motility of frozen thawed testicular spermatoza obtained from patients suffering from non-obstructive azoospermia can be initiated with the use of PF, and the outcome was assessed in terms of fertilization, pregnancies and birth after ICSI conducted with these PF-stimulated spermatoza. Materials and methods Patients From January 2000 to December 2004, a total of 77 couples underwent 108 ICSI cycles using cryopreserved testicular spermatozoa because of non-obstructive azoospermia. Prior to the diagnostic testicular biopsies, all men were evaluated to determine the aetiology of azoospermia, by performing a physical examination and hormonal analysis. All patients were genetically screened with molecular analysis for Y chromosome deletions as well as standard peripheral karyotype analysis. Retrieval and cryopreservation of testicular spermatozoa The biopsies were obtained via testicular fine needle sperm aspiration (TEFNA) under local anaesthesia with 1% lidocaine. Testicular tissue was rinsed in a Petri dish with Ferticult medium containing HEPES (Fertipro, Beernem, Belgium), finely minced and dispersed mechanically with the help of two sterile slides. Thereafter, the presence of spermatozoa was checked at 400 magnification under an inverted microscope (Diaphot, Nikon Corporation, Tokyo, Japan). Testicular sperm preparations were cryopreserved only if at least two motile spermatozoa were observed within 30 min of observation. Supernatant from the teasing and search was then diluted v/v with an equal amount of cryopreservation medium (Cryosperm, Bio Media, Boussens, France). This cryopreservation medium is totally devoid of molecules of animal origin, and thus does not contain egg yolk. After equilibration at ambient temperature for 15 min, the final solution was sealed in 250 µl straws (IMV, L Aigle, France), and cooled in Minicool LC40 (Air Liquide, Sassenage, France) according the following programme: 20 C to 4 C at a rate of 5 C/min, 10 C/min to 30 C, 20 C/min to 140 C, then plunged directly into liquid nitrogen ( 196 C). Thawing of the straws was done at ambient temperature, then the whole straw content was put onto a density gradient for preparation/separation of spermatozoa for the ICSI procedure. Cryopreserved spermatozoa were separated into fractions on a discontinuous two-step (47 and 90%) density gradient (Sill-Select, Fertipro) by centrifugation at 600 g for 15 min. Spermatozoa from the high-density fraction were resuspended in 4 ml of Ferticult medium (Fertipro), and centrifuged at 600 g for 5 min. Supernatant was then discarded and the pellet resuspended in 50 µl of Ferticult medium. Microdroplets (5 µl) of sperm suspension were deposited in the centre of a Petri dish, surrounded by microdroplets (10 µl) of Ferticult medium containing HEPES and one microdoplet of polyvinyl pyrrolidone (PVP). Microdoplets were then covered by mineral oil, the Petri dish placed onto an inverted microscope (Diaphot, Nikon Corporation) equipped with two three-dimensional micromanipulators (Narishige, Tokyo, Japan), and spermatozoa observed at a 400 magnification. When no motile spermatozoa were observed, or when the average time needed to find spermatozoa was greater than 20 min per individual spermatozoon, an equal volume (5 µl) of a 3 mmol/l PF (Torental) solution prepared simultaneously with Ferticult, was added to the microdroplets of the sperm suspension directly through mineral oil, to give a final concentration of 1.5 mmol/l PF. After incubation for 10 min, motile spermatozoa were rinsed in three successive microdroplets of Ferticult medium with HEPES and placed in a fourth microdroplet of Ferticult medium with HEPES, before being transferred to the PVP microdroplet for microinjection. Ovarian stimulation and ICSI procedure All female patients were treated with either a long- or shortprotocol gonadotrophin-releasing hormone analogue (GnRHa, Decapeptyl; Ipsen, Paris, France), followed by ovarian stimulation with recombinant-fsh (Gonal-f; Serono, Rome, Italy or Puregon; Organon, Oss, The Netherlands). Vaginal ultrasound-guided follicule puncture took place h after injection of 10,000 IU human chorionic gonadotrophin. After recovery, oocytes were placed in Ferticult medium (Fertipro) and denuded from the surrounding granulosa cells by exposure to 80 IU of hyaluronidase (Hyaluronidase; Medicult, Jyllinge, Denmark). Only metaphase II oocytes were microinjected at 400 magnification on an inverted microscope (Diaphot; Nikon Corporation) equipped with two three-dimensional micromanipulators (Narishige). After the ICSI procedure, the oocytes were incubated in 50 µl microdrops of G1 medium (G1.2; Vitrolife, Gothenburg, Sweden) under mineral oil (Medicult). Fertilization was assessed 18 h after injection by the presence of two pronuclei. Embryo cleavage and transfer took place 72 h 15

3 after the collection of the oocytes. Up to a maximum of two embryos were transferred, and the supernumerary embryos of good quality were cryopreserved for subsequent transfers. Clinical pregnancy was determined by observing a gestational sac with fetal heart beat at 5 weeks of pregnancy. Statistical analysis Statistical comparison was carried out by chi-squared analysis, two-tailed at 5% level of significance. Results In the present study, 108 ICSI cycles in 77 couples were analysed, 46 couples in the PF group and 31 in the control group. From the 46 couples of the PF group, 30 underwent one ICSI cycle, 14 underwent two ICSI cycles and two underwent three ICSI cycles. Of the 31 couples of the control group, 20 underwent one ICSI cycle, nine underwent two ICSI cycles and two underwent three ICSI cycles. The duration of infertility ranged from 2 to 11 years. In the group in which PF was used, the mean ages of the female and male partners were respectively 31.1 years (range 24 40) and 33.8 years (range 26 51). In the control group, the mean ages of the female and male partners were respectively 31.8 (range 26 42) years and 32.9 years (range 27 45). No significant differences were observed between the two groups either in the ages or in the FSH levels of the female partners. Frozen thawed motile spermatozoa were easily found and used for ICSI in 44 out of the 108 cycles (40.7%). In 23 cycles (21.3%), no motile spermatozoa were observed after thawing, and in 41 ICSI cycles (38.0%), the average time needed to find motile spermatozoa was greater than 20 min per spermatozoon. So, PF was used in 64 (59.3%) out of the 108 scheduled cycles. In all cases, pentoxiffyline either initiated the motility when no motile spermatozoa were observed, or stimulated the motility reducing dramatically the time search per spermatozoon. Table 1 describes the outcome of ICSI procedures with frozen thawed testicular spermatozoa, stimulated (PF group) or not (control group) with PF. A total of 743 metaphase II oocytes were microinjected, 485 for the PF group and 258 for the control group. No difference was observed between the two groups in the mean number of mature oocytes (7.6 versus 5.9). Fertilization rates were significantly different (P < 0.001) in favour of the control group, with 172 zygotes (66.7%) obtained in the control group and 263 obtained after stimulation of the spermatozoa with PF (54.3%). A total of 59 transfers, with a mean of 1.8 fresh embryos replaced, were performed in the PF group, and 40 in the control group with a mean of 1.85 fresh embryos replaced. Three patients in each group did not have embryos transferred because no fertilization occurred on a total of 17 mature oocytes. Two patients in the PF group did not have embryo transfer because of hyperstimulation, and one in the control group because of no mature oocytes. No significant differences were observed in the pregnancy rate per transfer of fresh embryos, since 21 clinical pregnancies (35.6%) occurred in the PF group and 10 (25.0%) in the control group. Eight supplementary pregnancies in the PF group and two in the control group were obtained after transfer of respectively 56 and 32 supernumerary cryopreserved embryos, improving the total pregnancy rate per cycle from 35.6% to 45.3% in the PF group and from 25.0 to 27.3% in the control group. A total of 29 clinical pregnancies including 32 gestational sacs were observed in the PF group, giving a total pregnancy rate per cycle of 45.3%, and an implantation rate per embryo of 19.8%, not significantly different from the rates of 27.3% and 15.1% respectively in the control group. One miscarriage was observed Table 1. Outcome of ICSI with frozen thawed testicular spermatozoa obtained from non-obstructive azoospermia after incubation with or without 1.5 mmol/l PF. With Without Total pentoxifylline pentoxifylline 16 No. of cycles No. of oocytes injected No. of zygotes (%) 263 (54.3) a 172 (66.7) 435 No. of transfers with fresh embryos No. of fresh embryos replaced No. of clinical pregnancies after transfer of fresh embryos (%) 21 (35.6) 10 (25.0) 31 No. of clinical pregnancies after transfer of cryopreserved embryos Total no. of clinical pregnancies Pregnancies/cycle (%) Total no. of gestational sacs Implantation /embryo (%) No. of miscarriages No. of deliveries No. of ongoing pregnancies Newborns Newborns with malformations a P < compared with the results without pentoxifylline.

4 at 2 months in the PF group, and two in the control group, of which one was monochorial twins. Finally, 20 deliveries of 23 babies with no malformation took place in the PF group, and 13 babies in nine deliveries were born with no malformation in the control group. Eight pregnancies in the PF group, and one in the control group are still ongoing. Discussion It is now well established that performing ICSI with fresh or frozen spermatozoa gives similar results. However, cryopreservation of testicular spermatozoa in order to dissociate in time the oocyte recovery and the testicular biopsy involves a lot of difficulties because of the very low concentration and motility of the testicular spermatozoa resulting from sperm immaturity, and of the biopsy by itself. The advantage of TEFNA compared with open testicular biopsy is that it is an easy, safe and well-tolerated method. TEFNA can also be repeated if necessary, without the risk of a significant loss of the testicular mass, and without the risk of general anaesthesia. However, one drawback of this method is that very little testicular tissue is recovered, and so a very low number of spermatozoa are available for cryopreservation. On the other hand, the freezing process results in adverse changes in sperm viability and motility, rendering the selection of live spermatozoa for ICSI very long and difficult. Indeed, in most cases, a weakly shaking motility can be observed at best after the freezing thawing process of testicular spermatozoa, and it is not exceptional that no motile spermatozoa can be found, particularly in cases of non-obstructive azoospermia. In fact, Dafopoulos (2005), using cryopreserved testicular tissue in non-obstructive azoospermia, recovered motile spermatozoa in only 53% of ICSI cycles, whereas for Verheyen et al. (2004) motile frozen thawed spermatozoa could be found in 79.4% of their ICSI cycles, but with an average time needed to find spermatozoa of 17 min. The current work confirms these studies since in 21.3% of the cycles no motile sperm were found despite an extensive search, and in 38.0% the average search time per spermatozoon exceeded 20 min. Determination of sperm viability is however an invaluable prerequisite in conducting ICSI, since only 46% of frozen testicular spermatozoa are viable after thawing versus 86% in the case of fresh testicular spermatozoa (Bachtell et al., 1999), and injection of immotile spermatozoa selected haphazarly results in lower fertilization and pregnancy rates (Nagy et al., 1998; Dafopoulos, 2005). Until now, several strategies have been developed to improve sperm motility after freezing, and several studies have reported an enhancement of sperm motility after in-vitro culture for a few days of fresh or frozen thawed testicular spermatozoa in cases of obstructive and non-obstructive azoospermia (Edirisinghe et al., 1996; Liu et al., 1997; Angelopoulos et al., 1999; Emiliani et al., 2000). The hypo-osmotic swelling test (HOST) has been suggested as a means of selecting viable fresh ejaculated or testicular spermatozoa for ICSI (Casper et al., 1996; Verheyen et al., 1997; Sallam et al., 2001). However, the interpretation of this test is particularly difficult with frozen thawed spermatozoa, since the freezing thawing process induces important variations in osmotic pressure, that brings about a rolling up of the tail. In these conditions, the interpretation of the HOST test and the selection of viable spermatozoa is very difficult. The stimulation of spermatozoa by PF, a phosphodiesterase inhibitor, has been used for a long time in IVF with controversial results (Yovich et al., 1988; Tournaye et al., 1995). Recently, its use to stimulate the motility of testicular spermatozoa has also been successfully proposed (Tasdemir et al., 1998; Angelopoulos et al., 1999, Terriou et al. 2000; Le Lannou et al. 2001). Likewise, it has been shown that cryopreservation of human spermatozoa with PF also improves post-thaw sperm motility (Brennan and Holden, 1995) and agonist-induced acrosome reaction rate (Esteves et al., 1998). The present results indicate that a 10 min incubation with PF initiates the motility of frozen thawed testicular spermatozoa obtained from patients with non-obstructive azoospermia, rendering the selection of a viable spermatozoon rapid and easy. In fact, without PF, ICSI would have been totally impossible in 21.3% of cycles, and very long and difficult in 38.0% of cycles where the search time per spermatozoon exceeded 20 min, whereas with PF ICSI was possible for all patients relatively easily. If PF has obvious detrimental effects upon oocyte and embryo development when contaminating the culture medium (Tournaye et al., 1993; Scott and Smith, 1995), washing spermatozoa before the insemination of oocytes has allowed adequate IVF, so the use of PF is no longer controversial (Tournaye et al., 1994). This washing step is obviously essential before ICSI to avoid injection of PF into the oocyte. The outcome in terms of fertilization, pregnancies and birth after ICSI realized with these PF-stimulated spermatoza confirms the safety of the procedure used. In fact, although not significantly different, the clinical pregnancy rate per cycle (45.3%) and the implantation rate per embryo (19.8%) are higher than those observed after ICSI (27.3% and 15.1% respectively) performed with spontaneously motile spermatozoa without preincubation with PF. This improvement could be due to better selection of viable spermatozoa permitting the production of good-quality embryos with a high developmental potential. Curiously, an unexplained negative significant difference in the fertilization rates with PF was observed. This difference maybe due to the retrospective character of the study, which was widely compensated for by embryo quality. Although no definite conclusions can be reached, due to the limited number of newborns, no malformations were observed at birth in any of the two groups. So in conclusion, the data presented show that use of PF allows the selection in 100% of cycles of viable testicular frozen thawed spermatozoa obtained from patients suffering from non-obstructive azoospermia when motile spermatozoa are observed before cryopreservation. Compared with the in-vitro culture of testicular biopsies, it gives the advantage of being able to rapidly distinguish between live and dead spermatozoa, and to perform ICSI immediately and easily with no long search time. Its use is simple, rapid and safe, and the rates of clinical pregnancy and of implantation are similar to those generally observed with fresh ejaculated spermatozoa. References Angelopoulo T, Adler A, Krey BA et al Enhancement or initiation of testicular sperm motility by in vitro culture of testicular tissue. Fertility and Sterility 71, Bachtell NE, Conaghan J, Turek PJ 1999 The relative viability of 17

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