RBMOnline - Vol 11. No 3. 2005 319-324 Reproductive BioMedicine Online; www.rbmonline.com/article/1859 on web 18 July 2005 Article Crude cumulative delivery rate following ICSI using intentionally frozen thawed testicular spermatozoa in 51 men with non-obstructive azoospermia Claude Giorgetti was born in 1952. After receiving a degree in laboratory biology from the University of Marseille (France) in 1976, he specialized in reproductive biology at the CECOS Sud-Est where he worked as a biologist from 1977 1983. In 1984 he co-founded the Institut de Médecine de la Reproduction, a private centre for reproductive medicine in Marseille. His specific areas of interest include sperm and embryo cryopreservation and embryo quality assessment. Dr Claude Giorgetti C Giorgetti 1, JM Chinchole, E Hans, O Charles, JP Franquebalme, E Glowaczower, J Salzmann, P Terriou, R Roulier Institut de Médecine de la Reproduction, 6 rue Rocca, 13008 Marseille, France 1 Correspondence: e-mail: giorgetti.claude@wanadoo.fr Abstract This prospective study evaluated the crude cumulative delivery rate following delayed intracytoplasmic sperm injection (ICSI) using spermatozoa recovered by testicular extraction (TESE) and intentionally frozen in men with non-obstructive azoospermia (NOA). This procedure can be termed cryotese-icsi. This study involved a series of 118 patients who underwent testicular biopsy for diagnosis of NOA in the period from January 1998 to December 2002. Testicular histology confirmed the diagnosis of NOA. Testicular parenchyma was obtained surgically from both testicles under general anaesthesia. Cryopreservation of spermatozoa was performed in 51 of 118 patients (43%). Ninety-nine delayed ICSI procedures were performed. Frozen thawed suspensions were used in all cycles. Application of pentoxifylline was required to stimulate spermatozoa in 52% of cases. Fertilization, embryo transfer, and ongoing pregnancy rates were 60, 98 and 29% respectively. The crude cumulative delivery rate was 49% after two cycles and 57% after four cycles. A total of 39 healthy children were born in 29 deliveries. Thus, cryotese-icsi is an effective procedure for routine use in patients with NOA. The main advantages of cryotese-icsi are to (i) avoid repeated surgical biopsy, (ii) ensure the availability of spermatozoa when the ovarian stimulation cycle is begun, and (iii) allow programmed biopsy and therefore dissociate it from ICSI. Keywords: frozen thawed spermatozoa, ICSI, non-obstructive azoospermia, pentoxifylline, pregnancy outcome, TESE Introduction Testicular sperm extraction (TESE) has been used in combination with intracytoplasmic sperm injection (ICSI) to achieve fertilization (Craft et al., 1993) and pregnancy (Schoysman et al., 1993) for men with non-obstructive azoospermia (NOA). However, no spermatozoa are found in diagnostic testicular biopsy samples in about 50% of patients with NOA (Friedler et al., 1997a; Schlegel et al., 2004). As a result, simultaneous ovarian stimulation for ICSI procedures often fails, because injection cannot be completed due to non-productive TESE. Since successful ICSI procedures have been reported with frozen thawed testicular spermatozoa (Gil-Salom et al., 1996), use of cryopreserved spermatozoa recovered prior to ovarian stimulation has been proposed as a means of avoiding not only unnecessary egg retrieval (Wurfel et al., 1998), but also damage to the testis due to repeated testicular surgery. Several studies have confirmed that similar results can be obtained using fresh and frozen thawed sperm (Friedler et al., 1997b; Liu et al., 1997; De Croo et al., 1998; Ben Rhouma et al., 2003; Dirnfeld et al., 2003; Thompson-Cree et al., 2003). Histopathology must be performed for diagnosis of NOA. It has also been shown to be the only reliable predictor of successful TESE (Tournaye et al., 1997; Schwarzer et al., 2003). As a result, biopsy procedures are usually performed at some time prior to ICSI. Since 1998, spermatozoa obtained during prior testicular diagnostic biopsy for delayed ICSI have been intentionally frozen. The purpose of this study carried out for the period up to May 31, 2004 was to evaluate the 319
320 crude cumulative delivery rate (CCDR) following delayed ICSI using testicular spermatozoa recovered and intentionally frozen between 1998 and 2002 in men with NOA. Previous series have reported CCDR following ICSI using freshly retrieved testicular spermatozoa (Osmanagaoglu et al., 2003), but this is the first series involving ICSI using intentionally cryopreserved extracted testicular spermatozoa. Materials and methods Patients This prospective series includes a total of 118 patients who underwent only one testicular biopsy for diagnosis of NOA between January 1998 and December 2002. In all cases, azoospermia was suspected on the basis of complete inverted-microscope examination of pellets obtained after centrifugation of two semen samples at 800 g for 20 min. All patients underwent physical examination, hormonal (FSH, LH) and biochemical marker assessment. Scrotal ultrasound and karyotype analysis were also performed. Klinefelter patients were excluded from the study. Testicular histology performed on the same day as TESE with a mean of 37 tubules (range, 10 70) confirmed diagnosis of NOA. The aetiology of NOA was classified into three groups: group 1, Sertoli cell-only syndrome (SCOS) in 51 cases; group 2, maturation arrest (MA) in 43; and group 3, hypo-spermatogenesis (HS) in 24. In patients with HS, histology demonstrated numerous tubules without spermatogenesis associated with a few tubules showing limited spermatogenesis. Patients with normal spermatogenesis or discrete hypo-spermatogenesis, i.e. a few tubules without spermatozoa and normal or slightly reduced spermatogenesis in most tubules were excluded from study. The mean age of the NOA patients included in this study was 35.2 years (range, 25 47) on the day of TESE. Female age was recorded on the day of ICSI cycle treatment. Testicular sperm extraction Testicular tissue was obtained surgically under general anaesthesia. An incision was made in the scrotal skin, tunica vaginalis and tunica albuginea (1 1.5 cm). Testicular parenchyma was extruded by gentle pressure on the testis. A large sample from both testicles (mean: 575 mg per testicle) was excised with curved scissors. After rinsing in BM1 medium (Ellios; Eurobio, Les Ulis, France) to remove red blood cells, specimens were sent to the laboratory in a closed tube containing 3 ml of medium. A small piece of the biopsy was randomly sent for histopathological examination. In the laboratory, testicular tissue was vigorously shredded in a Petri dish containing 4 ml of medium using two glass microscope slides. Mechanical shredding lasted at least 5 10 min so as to obtain a thick suspension containing no particles larger than 1 mm. After repeated aspiration and spilling out, the testicular suspension was placed in a 5 ml tube with a conical bottom and allowed to settle for 1 2 min so as to obtain a sedimentation pellet containing the largest particles. Leaving the pellet on the bottom of the tube, most of the supernatant (around 4 ml) was recovered, deposited in two conical-bottom tubes containing 1 ml of SupraSperm monolayer (80%) (MediCult, Limonest, France) and centrifuged at 800 g for 20 min. The supernatants were discarded and the interfaces (0.5 ml) between the monolayer and supernatant were recovered and set aside for treatment if needed. The two 80% phases were pooled and washed once with an equal volume of medium. The resulting centrifugation pellet was resuspended in 30 μl of medium (total testicular extract) and an initial examination of one 2 μl microdrop was performed under an inverted microscope to identify spermatozoa. If no spermatozoa were found the whole testicular extract was placed in a Petri dish and covered with paraffin mineral oil (Mineral Oil; Medicult). After searching for 15 20 min, if no spermatozoa were found, the undiscarded interfaces were washed once with an equal volume of medium and the centrifugation pellet was examined under the same conditions as previously described. At each step, as soon as motile or immotile spermatozoa were observed, the testicular extract (30 μl) was recovered, resuspended in 0.250 ml of BM1 medium and frozen. No vitality testing was carried out prior to freezing. Cryopresevation and thawing of testicular spermatozoa After slow dilution by adding an equal volume of cryopreservation medium (Sperm-Freeze; FertiPro, Beernem, Belgium) drop by drop for 5 min, the testicular extract was sealed in 0.150 ml straws (CBS 06433; CryoBioSystem, Paris, France). After 10- min incubation at 20 C, the straws were frozen according to a simple two-step freezing protocol using a controlled freezer (Nicool LM10; Air Liquide, Paris, France). During the first step of freezing from +20 C to 130 C, the cooling rate was 10 C/ min. The second step comprised plunging the straws into liquid nitrogen for storage. A thawing test was performed on the day of cryopreservation. One straw was thawed for 10 min at 37 C and washed with 2 ml of BM1 medium added drop by drop for 5 min. After centrifugation at 300 g for 15 min, the supernatant was discarded and the pellet was resuspended in 20 μl of BM1 medium. After placing this sperm preparation covered with paraffin mineral oil in a Petri dish and allowing it to settle for 5 min, motility was assessed under an inverted microscope. Examination of the whole preparation lasted for 15 20 min depending on the number of spermatozoa and amount of debris present. If total immotility was observed, a pentoxifylline (Torental; Aventis Pharma GmbH, Frankfurt, Germany) test was carried out (Terriou et al., 2000). An equal volume of a solution of pentoxifylline diluted at 10% in BM1 medium was added to the whole sperm preparation. The pharmaceutical form (solution for intravenous injection) contains 100 mg of synthetic pentoxifylline in ampoules of 5 ml and the concentration in the 10% solution was 7.2 mmol/l. After allowing settling for 5 min, motility was reassessed for 15 20 min. Movement of the flagella (slight in extreme cases) was detected in all cases and no patient was excluded from study. This test allowed us to adapt the conditions of ICSI. Ovarian stimulation and ICSI procedure Ovarian stimulation was performed using recombinant follicular stimulating hormone (Gonal-F; Serono, Boulogne, France or Puregon; Organon, Eragny sur Epte, France) after pituitary desensitization using gonadotrophin-releasing hormone agonist (Decapeptyl 3 mg; Ipsen Biotech, Paris, France). Transvaginal
ultrasound-guided follicle puncture was carried out 36 h after injection of 10,000 IU of human chorionic gonadotrophin (HCG, Organon). Oocyte decoronization was carried out using a 25 IU/ml hyaluronidase solution (Hyaluronidase; Choay, Gentilly, France) diluted in BM1 medium. ICSI was performed as described by Van Steirteghem et al. (1993). Fertilization was confirmed if inverted-microscope examination 18 h after ICSI demonstrated the presence of two distinct pronuclei and two polar bodies. Embryos were classified according to morphological appearance using an embryo score (0 4 points) based on the presence of anucleate fragments, blastomere irregularities in shape and/or size and cleavage stage (Giorgetti et al., 1995). Best-quality embryos displayed no anucleate fragments, no blastomere irregularities and 4-cell stage at the time of transfer 48 h after oocyte retrieval. The luteal phase was supported by intravaginal administration of 400 mg of micronized progesterone per day (Utrogestan; Laboratoires Besins Iscovesco, Paris, France) starting on the day of egg retrieval. Clinical pregnancy was confirmed if ultrasonography approximately 5 6 weeks after transfer demonstrated the presence of at least one gestational sac with fetal heartbeat. Implantation rate is considered as the percentage of gestational sacs with fetal heartbeat over the number of embryos transferred. Biochemical pregnancies were not reported. Statistical analysis The χ 2 -test and Kruskal Wallis test were used as appropriate to determine differences between groups. The significance level was set at P < 0.05. Results In the 118 NOA patients who underwent TESE in this series, spermatozoa were recovered in 54 (46%). Only one TESE was performed in each patient. Cryopreservation was performed in 51 patients (43%). In the remaining three patients, fewer than five spermatozoa, all of which were immotile and dysmorphic, were found and cryopreservation was not performed. The number of straws prepared per patient and the number of useable spermatozoa per straw are listed in Table 1. Ninety-nine delayed ICSI procedures were performed during the study period. Frozenthawed suspension containing motile spermatozoa was used in all cycles. In 48% of cases, spermatozoa were spontaneously motile. In the remaining 52%, application of pentoxifylline as described for thawing tests was necessary to stimulate motility (see in Table 2). The total fertilization rate was 60.5% (418/691 oocytes). Overall ongoing pregnancy per transfer and implantation rates were 29.3 and 23.8% respectively. Fertilization, pregnancy and implantation rates according to NOA aetiology are given in Table 2. Embryo quality on day 2 after ICSI was similar in all groups. Ninety-seven embryo transfers were performed (98%). Extra embryos obtained from 22 cycles in 17 couples were frozen for future use. The mean female age on the day of ICSI was 31.4 years (range 22 39) and the mean number of transferred embryos was 2.1. The overall implantation rate was 23.3% (48/206). No correlation was found between pregnancy rates per transfer and aetiology [SCOS, 5/15 (33%); MA, 13/35 (37%); HS, 17/47 936%)]. A total of 35 pregnancies were obtained with fresh embryos and 2 with frozen thawed embryos. In the fresh embryo group, there were five spontaneous first-trimester miscarriages (5/35:14%) and one second-trimester miscarriage involving a triplet pregnancy. Both pregnancies with frozen thawed embryos were full-term. No extrauterine pregnancies were observed in this series. The outcome of pregnancy was known in all cases. The CCDR in the 51 couples was 49% after two cycles and 57% after four cycles (see details in Table 3). The respective rates were 53 and 61% when deliveries after transfer of frozen thawed embryos were included. There were a total of 39 healthy children in 29 deliveries. No stillborn children and no perinatal deaths were observed in this series. There were 20 singleton, 16 twin and three triplet births. One twin pregnancy ended in premature delivery (<32 weeks of amenorrhoea) with very low birth weights (865 and 825 g). Low birth weight defined as <2500 g was observed in 15 (3/20), 63 (10/16) and 100% (3/3) of singleton, twin and triplet deliveries respectively. Transfer to a neonatal unit was necessary for six infants (15%). Frozen spermatozoa remain available for future ICSI for 38 patients and frozen embryos for six patients. Table 1. Mean number of straws obtained per patient and mean number of frozen thawed spermatozoa available per straw for future intracytoplasmic sperm injection in cases of different types of non-obstructive azoospermia. Sertoli Maturation Hypospermatogenesis cell only arrest No. of TESE procedures 51 43 24 No. of cryo-tese (%) 9 (18) a 20 (47) a 22 (92) a No. of straws per TESE 4.3 (3 6) b 6.5 (4 10) b 7.9 (5 12) b No. of spermatozoa per straw 8 (3 10) c 15 (3 50) c 25 (10 80) c Values in rows with the same superscripts were significantly different: a P < 0.01 (chi-squared test); b,c P < 0.05 (Kruskal Wallis test). TESE = testicular sperm extraction. 321
Table 2. Fertilization rate, pregnancy and implantation rates after intracytoplasmic sperm injection (ICSI) with frozen thawed (cryo-testicular sperm extraction) testicular spermatozoa in cases of non-obstructive azoospermia. Sertoli cell Maturation Hypospermatogenesis only arrest No. of ICSI procedures 16 35 48 No. of cycles with PTX used (%) 10 (63) 20 (57) 21 (44) No. of transfers (%) 15 (94) 35 (100) 47 (98) No. of MII oocytes injected 145 236 310 No. of 2PN oocytes (%) 88 (61) 116 (49) a 214 (69) a No. of embryos transferred 35 (2.3) 67 (1.9) 104 (2.2) (mean no. transferred) No. of clinical pregnancies 5 13 17 No. implanted 6 17 25 Implantation rate (%) 17.1 25.3 24.0 No. of miscarriages 1 3 2 Ongoing pregnancies (n) 4 10 15 Ongoing/transfer (%) 26.7 28.6 31.9 Singletons 3 7 10 Twins 1 3 4 Triplets 1 Values in rows with the same superscripts were significantly different: a P < 0.01 (chi-squared test). PTX, pentoxifylline. Table 3. Crude cumulative delivery rates after intracytoplasmic sperm injection (ICSI) using frozen thawed testicular spermatozoa in non-obstructive azoospermia patients. Treatment cycle number 1 2 3 4 No. of ICSI procedures 51 31 10 7 Mean maternal age (years) 30.6 31.3 32.4 33.8 No. of deliveries 13 12 2 2 Delivery rate per cycle (%) 25 39 20 29 Cumulative delivery rate (%) 25 49 53 57 322 Discussion As shown in a recent meta-analysis on the use of surgically retrieved sperm in azoospermic men (Nicopoullos et al., 2004), previous studies have been highly heterogeneous regarding aetiology (obstructive and non-obstructive azoospermia), sperm source (epididymal and testicular) and sperm type (fresh and frozen thawed). The same heterogeneity in aetiology and sperm source has been noted in studies on embryonic development and pregnancy losses (Anderson et al., 2002) and sperm nuclear DNA fragmentation after cryopreservation (Thompson-Cree et al., 2003). So far as is known, this is the first report describing ICSI performed exclusively with intentionally frozen thawed spermatozoa in patients with histologically documented NOA. Fresh spermatozoa were not used in any case. In the present series, testicular spermatozoa were successfully recovered in 46% of patients who underwent TESE. Recovery rates in previous series involving NOA patients have varied from 40 to 70% (Friedler et al., 1997a; Tournaye et al., 1997; Van Steirteghem et al., 1998; De Croo et al., 2000; Wood et al., 2002; Verheyen et al., 2004). These variations are due to several factors: (i) patient selection in terms of FSH concentration and/ or testis size before biopsy, (ii) size and number of testicular biopsies and (iii) laboratory techniques used for extraction. Sperm retrieval in this series was related to testicular histology. This finding is in agreement with those of Su et al. (1999) in a series of 51 men in which sperm retrieval was successful in 24, 47 and 79% of patients with SCOS, MA and HS respectively. The main methods used to obtain testicular spermatozoa are
open biopsy (TESE) (Devroey et al., 1995; Silber et al., 1995) and closed percutaneous fine needle aspiration (TEFNA) (Craft et al., 1997). It was decided to use a large surgical biopsy sample for two reasons. The first is that comparative studies have shown that TESE is more efficient in extracting spermatozoa (Friedler et al., 1997b; Ezeh et al., 1998; Chan and Schlegel, 2000), than percutaneous aspiration that has been widely used with obstructive azoospermia (Fahmy et al., 2004). The second reason for choosing TESE is that the laboratory was 3 km from the operating room, so that TEFNA was problematic. The first cases of successful fertilization (Romero et al., 1996) and soon after pregnancy (Gil-Salom et al., 1996; Podsiadly et al., 1996) using frozen thawed testicular spermatozoa were reported in 1996. The fertilization rate using frozen thawed spermatozoa in patients with NOA has varied from 38 to 70% (Gil-Salom et al., 1996; Friedler et al., 1997b; Oates et al., 1997; Wood et al., 2002; Verheyen et al., 2004). In the current study an overall fertilization rate of 60% was acheived with an embryo transfer rate of 98%, i.e. 97 of 99 cycles. Pentoxifylline was used in over half of these cases. These results show that fertilization rates comparable to those obtained with fresh testicular spermatozoa can be achieved (Friedler et al., 1997b; Liu et al., 1997; Palermo et al., 1999; Wood et al., 2002). Verheyen et al. (2004), who did not use pentoxifylline, reported a transfer rate of 83%, i.e. 64 of 77 cycles. However, they were unable to use frozen thawed suspensions in 20 of 97 cycles (20%). An important factor that could account for this difference in the present series is that spermatozoa that were immotile or did not respond to pentoxifylline were never used. Previous studies have indeed shown that the use of immotile spermatozoa for ICSI has an adverse effect on fertilization, pregnancy and implantation rates (Liu et al., 1995; Nagy et al., 1998; Verheyen et al., 2004). In the present series, fertilization rate was significantly lower when NOA aetiology was MA (49%) than HS (69%). There was also a difference between patients presenting MA and SCOS (71%) but it was not significant. These findings are in agreement with those of De Croo et al., (2000) who achieved an overall fertilization rate of 63% (221/349) as compared with 47% in the MA group. The same author also observed a low pregnancy rate in cases involving MA (20%), but the difference with cases involving SCOS (42%) was not significant. In this series, no correlation was found between pregnancy rate per transfer and aetiology. Implantation rate observed is discordant with a previous study (Platteau et al., 2004), indicating that embryos from TESE in NOA men have a high incidence of aneuploidy. In addition, the present data showed that the CCDR in the 51 couples was 53% after two cycles and 61% after four cycles when deliveries after transfer of frozen thawed embryos were included. In a recent article, Dafoulos et al. (2005) reported a CCDR of 29.1% after two cycles using cryopreservated testicular tissue. In the current study all children (n = 39) delivered after cryotese-icsi were healthy. Only two previous studies have reported pregnancy outcome and neonatal data after ICSI using frozen thawed testicular sperm in NOA. The size of the patient population in both was small, i.e. three in the study of Palermo et al. (1999) and 11 in the study of Vernaeve et al. (2003). The studies reported by Ludwig and Katalinic (2002) and Wennerholm et al. (2002) found no additional risk and no major malformation in children born after ICSI using testicular spermatozoa. However, neither of these studies discriminated between obstructive and non-obstructive azoospermia or between fresh and frozen thawed spermatozoa. Another study (Vernaeve et al., 2003) found a major malformation rate of 4% (two out of 54 children) after ICSI using fresh testicular spermatozoa for NOA. So far as is known, the present series is the largest in the literature describing crude cumulative delivery rate and neonatal findings after ICSI using frozen thawed testicular spermatozoa in histologically documented cases of NOA. Based on these findings, it is concluded that ICSI using frozen thawed testicular spermatozoa (cryotese-icsi) is an effective procedure for routine use in patients with NOA. Although this procedure would benefit few patients with SCOS whose sperm recovery rates are low, its use is suggested for all patients, since there is no reliable way of predicting the outcome of sperm recovery. Another disadvantage, albeit not specific to cryotese-icsi, is the high multiple pregnancy rate. In this special patient group with young female age and few associated female infertility factors, high multiple pregnancy rates were not surprising. In 2001, a more personalized policy for determining the number of embryos transferred in accordance with EHSRE recommendations was implemented (2000). The main advantages of cryotese-icsi are to: (i) avoid repeated surgical biopsy, (ii) ensure the availability of spermatozoa when the ovarian stimulation cycle is begun (Ben-Yosef et al., 1999; Gianaroli et al., 1999), and (iii) allow programmed biopsy and therefore dissociate it from ICSI. 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