Hum. Reprod. Advance Access originally published online on August 1, 2007
Human Reproduction 2007 22(10):2679-2684; doi:10.1093/humrep/dem190
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Optimal management of extreme oligozoospermia by an appropriate cryopreservation programme
1 Laboratoire de Biologie de la Reproduction, Centre Hospitalier Universitaire de Strasbourg, CMCO-SIHCUS, 19 rue Louis Pasteur, BP120, 67303 Schiltigheim Cedex, France 2 Université Louis Pasteur de Strasbourg, Faculté de Médecine, 4 rue Kirschleger, 67085 Strasbourg Cedex, France 3 Laboratoire de Biologie de la Reproduction, Hôpital Jeanne de Flandre, CHRU Lille, 2 rue Oscar Lambret, 59037 Lille Cedex, France 4 Service d'Andrologie, Hôpital Calmette, CHRU Lille, 59037 Lille Cedex, France
5 Correspondence address. Tel: +33 388 62 84 46; Fax: +33 388 62 84 45; E-mail: isabelle.koscinski{at}chru-strasbourg.fr
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Abstract |
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BACKGROUND: Severe oligozoospermia is characterized by sperm count fluctuations that may result in insufficient quantities of motile sperm for ICSI on the day of oocyte retrieval, thus necessitating testicular biopsy. To avoid this, we proposed that patients, with transient azoospermia or repeatedly low sperm counts, make a safety pool of frozen spermatozoa before ICSI attempts.
METHODS: Seventy cryptozoospermic (<103 spermatozoa/ml) and 46 oligozoospermic patients (103–105/ml) were included. Although all oligozoospermic patients succeeded in sperm banking, only 44 of 70 cryptozoospermic patients were successful. Others underwent testicular extraction of spermatozoa. The ICSI results for frozen sperm from cryptozoospermic patients were compared with those obtained with fresh sperm from a group of normal patients (>105 spermatozoa/ml).
RESULTS: In this prospective matched, controlled study, five cryptozoospermic, but no oligozoospermic, patients failed to produce sperm on the ICSI day, and frozen sperm was used instead. Although fertilization and pregnancy rates (per attempt) using fresh (49% and 5/44, respectively) and frozen sperm (54% and one-fifth, respectively) were similar for this cryptozoospermic group, the results for fresh sperm were significantly lower when compared with the control group (66% and 16/43, P < 0.0001, P < 0.001, respectively). In contrast, results for the oligospermic and control groups were similar.
CONCULSIONS: Banking of ejaculated sperm is helpful for cryptozoospermic patients.
Key words: cryopreservation/cryptozoospermia/ICSI/oligozoospermia
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Introduction |
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ICSI (Palermo et al., 1992
) using fresh, cryopreserved or testicular sperm (Craft et al., 1993; Schoysman et al., 1993; Silber et al., 1994) has considerably improved assisted reproduction techniques (ART). The observation from time to time of rare spermatozoa in the semen of patients considered azoospermic led to the notion of virtual azoospermia (Tournaye et al., 1995). Some patients with non-obstructive azoospermia may indeed produce extremely rare spermatozoa that are not retrieved with usual techniques (Makler et al., 1984; Jaffe et al., 1998). Only meticulous searching (Strassburger et al., 2000) or the extended sperm preparation protocol (ESP) (Ron-el et al., 1997) can yield usable samples. In the event of primary testicular failure, repeated semen evaluations over a period of several months are necessary to correctly estimate the chances of finding usable spermatozoa. When no sperm can be found in ejaculates on the day of ICSI, patients with extreme oligozoospermia often undergo testicular extraction of spermatozoa (TESE), either the same day for immediate ICSI or at a later date for future ICSI attempts. TESE, however, involves an invasive procedure that can lead to complications such as haematoma, inflammation, fibrosis, and even permanent devascularization and androgenous deficiency (Schlegel and Su, 1997; Manning et al., 1998; Schill et al., 2003). Even if the adverse effects of TESE are usually well controlled (Friedler et al., 2002; Vernaeve et al., 2006), they are still a matter of concern and one of the reasons to try to avoid TESE whenever possible. Beyond the risks for the father, many questions remain about the consequences for children conceived using immature gametes with less condensed forms of chromatin (Golan et al., 1996) that are epigenetically different from ejaculated sperm (Ariel et al., 1994; Kerjean et al., 2000). So far, the future of children conceived with testicular sperm is rather encouraging (Bonduelle et al., 2002, 2005; Vernaeve et al., 2003), but the follow-up studies have been short in term. Taking this into consideration, the use of ejaculated spermatozoa, when possible, remains the ethical choice. That said, low sperm counts in patients with transient azoospermia could preclude the use of ejaculated sperm for ICSI. To alleviate this risk, we propose cryopreserving ejaculated sperm to create a safety pool. Little data are available about the management of cryptozoospermia. The aim of this study was to evaluate the outcome of a management of these quasi-azoospermic or severe oligozoospermic patients. This management involved a cryopreservation programme to ensure the use of ejaculated sperm, either fresh or frozen. The frequency of frozen spermatozoa use, as well as fertilization, implantation and clinical pregnancy rates, was investigated for three groups of couples categorized according to oligozoospermia severity.
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Materials and Methods |
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Patients
One hundred and fifty nine couples who came to the ART Centre of Lille (France) between March 2002 and January 2005 were included in this prospective study. They were placed into three groups according to sperm count (mean sperm count calculated from three previous semen analyses). Group 1 included 70 cryptozoospermic patients (<103 sperm/ml and a history of transient azoospermia). Group 2 comprised 46 extreme oligozoospermic patients (between 103 and 105 sperm/ml). A third group, called the control group, consisted of 43 normal patients (>105 sperm/ml). These control cases were matched to Groups 1 and 2 for female age (±3 years), attempt range and day of oocyte retrieval (±1 day).
All patients underwent a detailed infertility evaluation, including a medical history, a physical examination, hormonal assessment and genetic testing (Karyotype and Y-microdeletions for groups 1 and 2; Ria Coat-A-Count DPC, Germany for testosterone assessment and Abbott AxSYM FSH, France for FSH assessment).
Sperm banking was offered to patients from Groups 1 and 2 (not to patients from control group), as a precaution for transient azoospermia on the day of ICSI. TESE was used as a last resort for patients who could not create a reserve ejaculated sperm pool.
The study was approved by the local ethics committee (January 2002), and patients gave written, informed consent prior to participation.
Semen examination and cryopreservation
For each patient, the creation of a cryopreserved pool of sperm was attempted prior to the ICSI attempt. Freshly ejaculated semen was mixed with five volumes of IVF culture medium (IVF 30, Vitrolife, Sweden) and centrifuged at 300 g for 10 min. The pellet was gently resuspended in 50–300 µl of culture medium and several 5 µl droplets were spread on a culture dish, covered with mineral oil and incubated for 10 min at 37°C, 5% CO2. Motile sperm in each droplet were subsequently counted. In the absence of motile sperm, a single sperm curling test (Ahmadi and Ng, 1997) was performed on motionless sperm to determine if they were alive. When at least one motile or live sperm could be detected, the remaining sperm preparation was frozen.
For cryopreservation, the sperm preparation was mixed at room temperature with an equal volume of Freezing Medium (Test Yolk Buffer, Irvine Scientific, USA), loaded into straws (30–50 µl per straw) and placed in a controlled temperature freezer (Nicool M10, AirLiquide, France). Samples were cooled 10°C/min from room temperature to –7°C, then 20°C/min to –70°C. The straws were then plunged into liquid nitrogen. To ensure sperm viability, one straw was then extracted from the liquid nitrogen and incubated for 2 min at 37°C. The sperm preparation was diluted 5-fold, very slowly with IVF 30, incubated at 37°C (5% CO2) for 10 min and centrifuged at 300 g for 10 min. The pellet was then resuspended in 30–50 µl of IVF medium, spread in 5 µl droplets and examined as described earlier to detect motile sperm cells. When fewer than 10 motile spermatozoa were observed, a single curling test was performed on the motionless ones. At least 10 motile or living spermatozoa were required in the thawed sample for the ‘safety pool' to be considered successful. This process was repeated weekly until a successful ‘safety pool' was created.
ICSI trial
For the purpose of this study, the first ICSI attempt after sperm cryopreservation was a therapeutic procedure as well as a means of fertilization assessment. On the morning of oocyte retrieval, motile or living sperm were quantified in fresh ejaculates as described earlier. When no spermatozoa were found, the procedure was repeated on a second ejaculate. If both ejaculates lacked sperm, the frozen ‘safety pool' was used.
Ovarian stimulation, oocyte retrieval and the ICSI procedure were performed according to standard practices (Van Steirteghem et al., 1993; Rienzi et al., 1998), as were, the assessment of fertilization, embryonic cleavage and morphological quality (Ron-el et al., 1997). Embryo transfers were executed 70 h after ICSI. A good quality embryo was defined as having at least three blastomeres on day 2, or six on day 3, with less than 20% anucleated fragments.
For all three groups, the same embryo transfer policy applied: whenever possible, two embryos of good quality were transferred. The transfer of a single embryo occurred either when the trial led to only one good quality embryo or when the transfer of more than one embryo would have been a health risk for the woman. Depending on the number of available embryos and the absence of uterus pathology, up to three embryos could be transferred at a couple's insistence.
Clinical pregnancies were identified by sonographic visualization of at least one gestational sac.
The ICSI control group (normal patients) consisted of 43 ICSI attempts using ejaculated sperm from ejaculates with at least 105 spermatozoa/ml.
Statistics
Whenever indicated, the Khi-2 test and the Student's t-test were applied at the 5% level of significance. Results are presented as mean ± SD.
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Results |
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Patients
Table 1 summarizes the characteristics of the patients in Group 1 (cryptozoospermia with banked sperm), Group 2 (extreme oligozoospermia) and the control group. No significant differences in age or testosterone levels existed between the groups. On the other hand, testis size was significantly larger (26 ml ± 5.53) (P < 0.05) for the control group than for Group 1 (18.36 ml ± 5.45) or Group 2 (21.70 ml ± 3.10), though there was no significant difference between Groups 1 and 2. The level of plasma FSH, however, was statistically higher for Group 1 (16.0 ± 9.13 UI/l) when compared with Group 2 (10.41±6.84 UI/l, P < 0.001), or to the control group (7.56 UI/l ± 3.47, P < 0.001). The difference between the FSH levels of group 2 and control group was also statistically significant.
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Only 44 of 70 cryptozoospermic patients undergoing ART in our centre succeeded in banking ejaculated sperm. The other 26 patients had TESE procedures. The number of ejaculates necessary to constitute the safety pool of frozen spermatozoa was 2.41 ± 1.67 in Group 1 and 1.26 ± 0.50 in Group 2 (P < 0.001).
In addition, the mean age of the women was comparable in the three groups (31.30 years ± 5.78 in Group 1, 29.4 ± 3.30 in Group 2 and 34.35 ± 6.65 years in control group).
Safety pool sperm numbers
For the 44 cryptozoospermia patients who succeeded in banking (Group 1), safety pool straws contained anywhere from rare motile or living spermatozoa to a few dozen spermatozoa. In the safety pools of the oligozoospermic Group 2, up to a few hundred motile spermatozoa could be found.
Total ICSI results
Table 2 summarizes the ICSI results for each group. For 85 of 90 patients (94.4%), the ICSI attempt was performed using freshly ejaculated, motile sperm. This included 39 of 44 cryptozoospermic patients (88.7%) and all 46 oligozoospermic patients (100%). The use of frozen sperm was required for 5 of 44 cryptozoospermic patients (11.4%). In two cases where frozen spermatozoa were used, 3 of 10 and 2 of 8 metaphase II (MII) oocytes were injected with single, curling test positive spermatozoa; other oocytes were injected with motile spermatozoa.
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Similar numbers of oocytes were injected for each of the three groups (283, 315 and 337 for Groups 1, 2 and control, respectively, ns).
The fertilization rate was significantly lower for group 1 (140/283, 49.47%) than for the other two groups (192/315, 60.95% in Group 2 and 222/337, 65.87% in the control group). The cleavage rate (data not shown) and the proportion of good quality embryos were similar for all three groups, though the number of embryos obtained per couple was significantly lower for Group 1 (3.24 ± 2.77) than for Group 2 (4.46 ± 2.63) (P < 0.05) and the control group (4.98 ± 2.53) (P < 0.05) (data not shown). The number of transferred embryos was similar for Groups 1 (1.7 ± 0.9) and 2 (1.8 ± 0.7), but was significantly higher for the control group (2.1 ± 0.6) (P < 0.05). The implantation rate was similar for the three groups. When compared with the control group, the clinical pregnancy rate was only significantly lower for Group 1 (P < 0.001). The spontaneous abortion rate reached 20% in all three groups. To minimize the impact of poor oocyte development on ICSI, we compared the results when at least four MII oocytes were retrieved: the pregnancy rate per oocyte retrieval was still significantly lower for Group 1 when compared with the control group (P < 0.03).
Finally, we found differences in the numbers of babies delivered. In Group 1, 5 of 44 (11.36%) oocyte pick-ups ended in delivery, and a total of three single babies and two pairs of twins were born. In Group 2, 46 pick-ups resulted in eight deliveries (17.39%), which included six single babies and one pair of twins. Finally, in the control group, 13 of 43 (30.23%) pick-ups led to deliveries, which included the births of 11 single babies and 2 pairs of twins. The difference was statistically significant between Group 1 and the control group (P < 0.05).
ICSI results using frozen sperm
The number of motile sperm per thawed straw ranged from <10 to 50. ICSI performed in Group 1 with frozen sperm showed no significant difference in fertilization, cleavage or implantation rates when compared with ICSI attempts using freshly ejaculated sperm (respectively 54.17% with frozen sperm (N = 5) and 49.03% with fresh sperm (N = 39)).
Between March 2002 and January 2005, we proposed sperm banking to all 70 cryptozoospermic patients at our reproduction centre for ART. This was successful for 44 patients, after an average of 2.41±1.67 ejaculates per patient. After five attempts for the 26 others, crypreservation was considered a failure, either because sperm count was too low or because spermalozoa did not survive the freeze/thaw protocol. Thus as a last resort, TESE was proposed and 22 underwent the procedure TESE was successful in 15/22 cases (68.18%); 12 of these 15 patients underwent an ICSI cycle that did not result in delivery (11 women failed to become pregnant and 1 experienced a spontaneous abortion). Finally, 4 of the 5 pick-ups led to an embryo transfer, and further to the birth of one baby.
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Discussion |
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For patients with cryptozoospermia, transient azoospermia at the time of ICSI could either lead to cancellation of the attempt or TESE, with the testicular spermatozoa being used for the current ICSI or for the next attempt. This is unfortunate, as TESE subjects patients to risks of vascular injuries and, therefore, to androgenic defects or testicular atrophy (Harrington et al., 1996
; Friedler et al., 1997; Schlegel and Su, 1997; Manning et al., 1998; Schill et al., 2003). Besides these iatrogenic risks, one cannot be certain that sperm will be found during TESE as the presence of sperm in a preliminary semen sample is not predictive of the TESE outcome (Tournaye et al., 1997).
Moreover, the use of testicular gametes with genomic imprinting, i.e. less complete than ejaculated sperm (Ariel et al., 1994; Tesarik and Mendoza, 1996; Kerjean et al., 2000), may expose the conceptus to potential imprinting defects and, eventually, to disease (Tournaye, 1997). In mice, some genes are de-methylated in adult spermatogenic cells in the testis, but re-methylated in mature spermatozoa from the vas deferens. This re-methylation is considered part of the sperm maturation process in the epididymis (Ariel et al., 1994). Early follow-ups of children conceived with testicular sperm are reassuring but the time frame of these studies is too short to be conclusive (Bonduelle et al., 2002; Ludwig and Diedrich, 2002). Further, these studies do not discriminate between obstructive and non-obstructive azoospermia. Additional studies are clearly necessary to understand the consequences of using immature testicular spermatozoa from men with spermatogenetic failure.
With the hope of avoiding testicular biopsy in mind, this study evaluated the usefulness of a safety pool of frozen, ejaculated sperm, in the management of ICSI for cryptozoospermic and oligozoospermic men. Fluctuations in spermatogenesis associated with cryptozoospermia dramatically increase the risk of ejaculates without spermatozoa on the day of oocyte pick-up. For cryptozoospermic patients in fear of such a situation, cryopreservation was psychologically comforting and they did not hesitate to come in for the cryopreservation procedure as often as necessary to make a safety pool.
In this study, only 5/44 (11.4%) cryptozoospermic patients used their frozen, ejaculated spermatozoa. This sample size was too small to draw definitive conclusions about outcome of the use of fresh versus frozen sperm from cryptozoospermic patients and therefore the merit of using fresh or frozen sperm for such patients. The comparison with the literature is also difficult since only one study reports a similar practice: Lahav-Baratz et al. (2002) examined the value of sperm pooling and cryopreservation for patients with severe oligoasthenoteratozoospermia and a history of transient azoospermia, or for patients with azoospermia where sperm was found in ejaculate on at least one occasion. Frozen ejaculated sperm had to be used for 8 of 35 severe oligoasthenozoospermic patients (22.86%) and for 5 of 10 azoospermic patients who underwent ICSI with ejaculated spermatozoa. The patients included in Lahav-Baratz's study probably had lower sperm counts than the cryptozoospermic patients of our study, likely explaining why the frozen pool was used more frequently.
We have found that with cryptozoospermia, the cryopreservation procedure did not decrease the fertilization rates or the implantation rate as has previously been reported (Lahav-Baratz et al., 2002). In our study, the lower fertilization rate observed with cryptozoospermic patients could be the result of sperm abnormalities that are not usually recognized under standard ICSI conditions. The very small number of sperm in the ejaculates precluded the use of intracytoplasmic morphologically selected sperm injection, which would have allowed more detailed observation of the sperm (Berkovitz et al., 2005).
For cryptozoospermic patients, the ICSI was proposed as a therapeutic option and as a fertilization test for spermatozoa. After completing the ICSI attempt with freshly ejaculated sperm, all parameters were analysed. A fertilization rate between 40% and 60%, with a standard rate of cleavage and embryonic development, suggested acceptable sperm quality and led to a repeated attempt, again with ejaculated spermatozoa. Otherwise, particularly in cases of unexplained low fertilization rate, weak sperm vitality, unexplained implantation failure or abortion, we concluded that sperm viability was decreased, likely as a result of obstructions that increased transit time through the genital ducts (especially the epididyme). This would result in low fertilization rates or sperm DNA damage and thus, in implantation failure or abortion. As a result TESE was proposed (Greco et al., 2005).
In addition to the ethical aspects, the management strategy proposed here should to be evaluated economically. As frozen spermatozoa were never used in Group 2 (severe oligospermia), the collection of such a safety pool was unnecessary. It appears it would be more helpful to check semen parameters and stability at regular intervals (e.g. every cycle or every two cycles of spermatogenesis). In this way, the storage of useless straws could be avoided, leading to substantial savings. Using the results of this study, we have proposed a management flow chart (Fig. 1): when a mean sperm count of 103–105 spermatozoa/ml is observed, cryopreservation is not required, but a semen analysis should be performed every 3 months as well as 2 weeks before oocyte pick-up. With <103 spermatozoa/ml, creation of a safety pool of spermatozoa should be proposed. On the morning of the oocyte retrieval, the patient is asked to deliver two samples of semen. If neither ejaculate contains enough sperm (i.e. >10 live sperm), a frozen sample would be used for ICSI. TESE should only be used when no sperm can be retrieved after several ESP sperm examinations or in the case of major necrozoospermia.
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Conclusion |
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For cases of extreme oligozoospermia, we favour the use of physiological, mature, ejaculated spermatozoa over testicular sperm. To avoid the cancellation of an ICSI attempt due to the absence of sperm in the ejaculate on the day of the oocyte retrieval, we propose a strategy that includes the creation of a safety pool of frozen spermatozoa for patients with cryptozoospermia, and regular examination of sperm parameter stability for those without cryptozoospermia.
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Acknowledgements |
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We would like to thank the staff of the Laboratory of Assisted Reproduction Technology, especially Françoise Charlet, Laurent Simoulin, M. Pierre Bouriez and Isabelle Noiret for technical help, as well as L. Acito-Khan, A. Fetter and M. Sellars for language corrections.
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Submitted on January 8, 2006; resubmitted on April 18, 2007; accepted on April 28, 2007.
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