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Abstract |
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Key words: embryo quality/fertilization rates/pregnancy rates/sperm preparation/testicular biopsy
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Introduction |
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In 1998 the German Society for Human Reproductive Biology decided during their annual general meeting to initiate a retrospective analysis of the results after TESE-ICSI comparing the data from groups applying mechanical or enzymatic preparation of testicular sperm from fresh or frozenthawed biopsies. The intention of this study was to find out if one of the two methods of processing testicular tissue has any specific advantage over the other in terms of quality of recovered sperm, fertilization results, embryo quality or established pregnancies. The present paper summarizes the results of the compiled data of 11 centres covering the treatment period from 1996 to 1997.
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Materials and methods |
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Eleven centres contributed to the results presented. Complete data on all parameters mentioned above were available from nine centres (Table I). Centre 2 (20 cycles) did not report on patients' age at treatment, centre 6 (51 cycles) gave no details on number of patients treated, quality of isolated sperm and of resulting embryos. The number of cycles provided by each centre is shown in Table II
, however, the order in Table II
is different from Table I
to ensure anonymity for each centre. The cycle results from individual centres were grouped according to the source of the testicular biopsy (fresh or cryopreserved; F or C) and the preparation method of tissue (mechanical or enzymatic; M or E) used for every specific treatment cycle. Statistical analysis was performed by
2-test or analysis of variance and a statistical difference of P < 0.05 was considered significant. The number of cycles in the four subgroups and the contribution of data by the different centres were unevenly distributed: Freshenzymatic (FE) 34 cycles: centre 2, 14; centre 4, 20; freshmechanical (FM) 95 cycles: Centre 3, 60; centre 5, 11; centre 10, 24; cryopreservedenzymatic (CE) 486 cycles: Centre 1, 388; centre 2, 6; centre 4, 6; centre 7, 22; centre 8, 14; centre 9, 36; centre 11, 14; cryopreservedmechanical (CM): 224 cycles: Centre 3, 21; centre 5, 154; centre 10, 49.
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Results |
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Patient characteristics in the four subgroups
Some of the general parameters were significantly different between the four subgroups formed according to the source and preparation method of the testicular biopsy material (FE, FM, CE, CM; Table III). The average age of the females was significantly lower for patients represented in the FE group and higher in the CM group. Also the number of mature oocytes (metaphase II) available for ICSI was significantly different between groups. The lower yield of oocytes in the group with the oldest patients and the higher in the younger patients reflects the well known age-dependency of oocyte recovery rates (Silber et al., 1997
).
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Fertilization rates obtained with testicular sperm
The results after injection of individual oocytes with motile and immotile sperm or elongated spermatids from fresh or cryopreserved biopsies are shown in Figure 1. The total fertilization rate was higher using cryopreserved compared with fresh sperm (C: 47.6 and 46.5%, versus F: 32.1 and 42.6%;
2 = 35.3072, df = 3, P < 0.0001). In general the results after enzymatic and mechanical preparation were similar with some remarkable exceptions. For all motile sperm mechanical preparation gave higher fertilization rates than enzymatic treatment (M: 54.4 and 57.5% versus E: 34.7 and 52.5%;
2 = 50.2125, df = 3, P < 0.00001) whereas for the subgroups using cryopreserved specimens (CE and CM) results were better with enzymatic preparation versus mechanical processing for both immotile sperm (38.5 and 21.2%;
2 = 255.5280, df = 1, P < 0.00001) and elongated spermatids (2.1 versus 18.0%;
2 = 62.7763, df = 3, P < 0.00001).
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Evolution of pregnancies
All 11 participating centres provided data on the evolution of pregnancies achieved after ICSI using testicular sperm during the period 19961997 (Table VI). Of the 172 pregnancies 32 ended as spontaneous abortions (1/6 in FE, 1/22 in FM, 22/99 in CE, 8/44 in CM), 108 (62.8%) continued as intact singleton gestations and 29 sets of twins (one in FE, five in FM, 13 in CE, 10 in CM), as well as three triplets (one in CE, two in CM) were reported giving a multiple rate of 22.9% for the ongoing pregnancies. No major malformations were reported for the babies born after TESEICSI in this study.
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Discussion |
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Centres 1, 2, 3 and 5 also treated the majority of cycles in this survey (654 of 839, 77.9%; Table II) which points to the fact that difficult cases obviously tended to be referred to experienced centres. The largest contribution to the total data set was by centre 1 with 388 cycles. These also represented 79.8% of the cases in subgroup CE for the detailed analysis. Therefore the results for this subgroup were strongly influenced by the experience of this one centre and findings should be interpreted with caution.
A considerable bias was also probably introduced by the fact that individual centres in general applied only one preparation method (mechanical or enzymatic) so that comparisons may reflect differences between centres rather than between methods.
Comparison of fresh and cryopreserved testicular biopsy
The data shown indicate no major differences between the results with fresh or cryopreserved testicular tissue as the sperm source regarding implantation and pregnancy rates achieved (Figure 2) which is in agreement with findings of other investigators (Friedler et al., 1997
; Habermann et al., 2000
; Huang et al., 2000
). The rate of oocyte fertilization appeared to be even higher after using sperm from cryopreserved than from fresh specimen (Figure 1
) indicating that neither the process of cryopreservation itself nor the cryo-storage negatively influenced the fertilizing capacity of the testicular sperm. A similar observation was reported (Palermo et al., 1999
) when comparing ICSI results using testicular sperm from fresh and frozenthawed biopsies. Pregnancy rates achieved were the same but a higher fertilization rate with frozenthawed than with fresh sperm was obtained (74.4 versus 60.4%) although this difference did not reach statistical significance due to the low number of cases (n = 5) included in the test group. The difference in fertilization rates observed in the present study cannot be clearly attributed to the source of the testicular sperm alone but was probably also influenced by variations between participating centres. Centres 2 and 4 performed a few cycles using fresh (n = 32) and a few using cryopreserved testicular sperm (n = 14) after enzymatic preparation, making a direct comparison possible. The fertilization rates of injected oocytes were identical under both conditions (121/346 and 42/120). Likewise, centres 3, 5 and 10 provided some data for comparison of injection results with fresh and with cryopreserved sperm after mechanical preparation (89 and 230 cycles) and also produced similar fertilization rates (337/750 and 762/1681). Therefore a final conclusion on the fertilizing potential of fresh compared with frozenthawed testicular sperm cannot be drawn from the data presented here. A larger series of cases with both sperm sources applied in parallel and performed by only a few centres would be desirable to clarify this question.
All of the centres participating in this study applied cryopreservation of testicular biopsies at least for later treatment cycles. The advantages of this approach are now generally accepted. It allows the freedom of planning the oocyte retrieval independently from the biopsy in the male partner. In addition and more importantly the chances for successful injection of theoocytes can be predicted from the outcome of histological and preparative evaluation of the testicular biopsy material before treating the female partner with ovarian stimulation (Fischer et al., 1996; Küpker et al., 2000
). For the male side one major advantage of thorough histological investigation is the possibility to detect carcinoma in situ very early and to refer affected patients to appropriate treatment before they engage in infertility treatment. This situation has been reported to occur in
1% of infertility patients investigated (Schulze et al., 1997
).
Comparison of preparation methods for testicular sperm
Both the enzymatic and the mechanical preparation of testicular sperm yielded high rates of motile sperm (55.5100%) and these gave the highest fertilization rates after injection of mature oocytes (Figure 1) in all subgroups. Whether the higher proportion of motile sperm observed after mechanical versus enzymatic preparation was in fact due to the preparation method itself or was the result of a bias due to the very different group sizes cannot be clarified from the data available.
One important observation made in this study was a higher fertilization rate for immotile sperm and elongated spermatids after enzymatic compared with mechanical preparation of the tissue for the cryopreserved specimen (Figure 1; subgroup CE versus CM). This may be explained by easier processing of the injected sperm within the oocytes subsequent to membrane damage probably induced by the cryopreservation procedures. A similar mechanism was suggested byNagy et al. (1995) who attributed their high fertilization rate after ICSI with ejaculated sperm partly to damages of the sperm membrane caused by relatively high speed centrifugation during preparation (Nagy et al., 1995
). Mechanical modification of the sperm membrane permeability as induced by touching the flagellum with the ICSI pipette also has been thought to improve the rate of spermoocyte interaction thus enhancing the fertilization rate (Nijs and Van der Elst, 2000
).
Embryo quality as judged by the degree of fragmentation (Table V) was better in those embryos derived from the injection of motile compared with immotile sperm or elongated spermatids in all subgroups. The proportion of good quality embryos was superior after enzymatic compared with mechanical preparation but resulting pregnancy rates per transfer (Figure 2
) were nevertheless almost identical between the four subgroups.
General comments
To reach a definite conclusion on the effect of the processing technique in specifically difficult cases (mainly immotile sperm or only elongated spermatids) the application of both preparation methods on aliquots from the same biopsy and injection of sibling oocytes would be necessary, which is not feasible in a clinical setting. The mechanical preparation approach has the advantage of being quite fast, taking only about 15 min, while the enzymatic technique is more time consuming requiring at least 4 h of preparation. A practicable approach based on the findings presented in this study could be to apply mechanical processing to specimens with a fairly good prognosisi.e. motile sperm seen in a test preparation or several sperm detected in the previous histological examinationwhile enzymatic processing may be the first choice in difficult cases with a high chance of mainly immotile sperm or elongated spermatids.
Interestingly, 188 transfers of embryos derived solely from the injection of immotile testicular sperm resulted in 11 normal pregnancies (Figure 2) which represents a much lower pregnancy rate than that obtained with motile testicular sperm (136/522 transfers, 26.1%). Nevertheless this is a considerably better result than the success rates reported in the literature for ejaculated immotile sperm (Nijs et al., 2000
).
Conclusion
In this retrospective multicentre study no unequivocal advantage of one over the other preparation method could be identified in 839 ICSI cycles using testicular sperm from 549 patients. Fertilization rates with immotile sperm and elongated spermatids seemed to be better after enzymatic versus mechanical treatment and overall embryo quality, judged by the degree of fragmentation, also appeared to be superior after collagenase digestion. Nevertheless, fertilization rates with motile sperm from fresh testicular biopsies were higher after mechanical preparation. Most importantly, resulting pregnancy rates were practically identical. More significant than the method of preparation seems to be the availability of motile testicular sperm for injection to ensure a comparable pregnancy chance as achievable with ejaculated sperm.
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Acknowledgements |
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Notes |
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References |
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Devroey, P., Liu, J., Nagy, Z., Goossens, A., Tournaye, H., Camus, M., Van Steirteghem, A. and Silber, S. (1995) Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum. Reprod., 10, 14571460.[Abstract]
Deutsches IVF Register, (1997), Jahrbuch 1997. D.I.R. Bundesgeschäftsstelle Ärztekammer, Schleswig-Holzstein, Bad Segeberg, p. 13.
Fischer, R., Baukloh, V., Naether, O.G.J., Schulze, W., Salzbrunn, A. and Benson, D.M. (1996) Pregnancy after intracytoplasmic sperm injection of spermatozoa extracted from frozenthawed testicular biopsy. Hum. Reprod., 11, 21972199.[Abstract]
Friedler, S., Raziel, A., Soffer, Y., Strassburger, D., Komarovsky, D. and Ron-El, R. (1997) Intracytoplasmic injection of fresh and cryopreserved testicular spermatozoa in patients with nonobstructive azoospermia a comparative study. Fertil. Steril., 68, 892897.[ISI][Medline]
Habermann, H., Seo, R., Cieslak, J., Niederberger, C., Prins, G.S. and Ross, L. (2000) In vitro fertilization outcomes after intracytoplasmic sperm injection with fresh or frozenthawed testicular spermatozoa. Fertil. Steril., 73, 955960.[ISI][Medline]
Huang, F.J., Chang, S.Y., Tsai, M.Y., Kung, F.T., Lin, Y.C., Wu, J.F. and Lu, Y.J. (2000) Clinical implications of intracytoplasmic sperm injection using cryopreserved testicular spermatozoa from men with azoospermia. J. Reprod. Med., 45, 310316.[ISI][Medline]
Küpker, W., Schlegel, P.N., Al-Hasani, S., Fornara, P., Johannisson, R., Sandmann, J., Schill, T., Bals-Pratsch, M., Ludwig, M. and Diedrich, K. (2000) Use of frozenthawed testicular sperm for intracytoplasmic sperm injection. Fertil. Steril., 73, 453458.[ISI][Medline]
Nagy, Z., Liu, J., Janssenwillen, C., Silber, S., Devroey, P. and Van Steirteghem, A. (1995) Using ejaculated, fresh, and frozenthawed epididymal and testicular spermatozoa gives rise to comparable results after intracytoplasmic sperm injection. Fertil. Steril., 63, 808815.[ISI][Medline]
Nijs, M.H. and Van Der Elst, C.J. (2000) Biological aspects of testicular sperm extraction. Eur. J. Obstet. Gynecol. Reprod. Biol., 92, 16.[ISI]
Nijs, M., Vanderzwalmen, P., Vandamme, B., Segal-Bertin, G., Lejeune, B., Segal, L., van Roosendaal, E. and Schoysman, R. (2000) Fertilizing ability of immotile spermatozoa after intracytoplasmic sperm injection. Hum. Reprod., 11, 21802185.[Abstract]
Palermo, G., Joris, H., Devroey, P. and Van Steirteghem, A. (1992) Pregnancies after intracytoplasmic injection of single spermatozoa into an oocyte. Lancet, 340, 1718.[ISI][Medline]
Palermo, G.D., Schlegel, P.N., Hariprashad, J.J., Ergün, B., Mielnik, A., Zaninovic, N., Veeck, L.L. and Rosenwaks, Z. (1999) Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men. Hum. Reprod., 14, 741748.
Salzbrunn, A., Benson, D.M., Holstein, A.F. and Schulze, W. (1996) A new concept for the extraction of testicular spermatozoa as a tool for assisted fertilization (ICSI). Hum. Reprod., 11, 752755.[Abstract]
Schulze, W., Knuth, U.A., Jezek, D., Benson, D.M., Fischer, R., Naether, O.G.J., Baukloh, V. and Ivell, R. (1997) Intratesticular sperm extraction. Basis for successful treatment of infertility in men with ejaculatory azoospermia. In Ivell, R. and Holstein, A.F. (eds) The Fate of the Male Germ Cell. Plenum Press, New York, USA, pp. 8187.
Silber S.J., Nagy, Z., Devroey, P., Camus, M. and Van Steirteghem, A.C. (1997) The effect of female age and ovarian reserve on pregnancy rate in male infertility: treatment of azoospermia with sperm retrieval and intracytoplasmic sperm injection. Hum. Reprod., 12, 26932700.[Abstract]
Van Steirteghem, A., Nagy, Z., Joris, H., Janssenwillen, C., Staessen C., Verheyen, G., Camus, M., Tournaye, H., and Devroey, P. (1998) Results of intracytoplasmic sperm injection with ejaculated, fresh and frozenthawed epididymal and testicular spermatozoa. Hum. Reprod., 13, 134142.[ISI][Medline]
Verheyen, G., De Croo, I., Tournaye, H., Pletinex, I., Devroye, P. and Van Steirteghem, A.C. (1995) Comparison of four mechanical methods to retrieve spermatozoa from testicular tissue. Hum. Reprod., 10, 29562959.[Abstract]
Submitted on February 20, 2001; resubmitted on December 31, 2001; accepted on March 7, 2002.