H.Van de Velde 1, Z.P.Nagy, H.Joris, A.De Vos and A.C.Van Steirteghem

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1 Human Reproduction vol.12 no.10 pp , 1997 Effects of different hyaluronidase concentrations and mechanical procedures for cumulus cell removal on the outcome of intracytoplasmic sperm injection H.Van de Velde 1, Z.P.Nagy, H.Joris, A.De Vos and A.C.Van Steirteghem Centre for Reproductive Medicine, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Laarbeeklaan 101, B-1090 Brussels, Belgium 1 To whom correspondence should be addressed The aim of this study was to compare concentrations of hyaluronidase and mechanical methods used to denude human oocytes from surrounding cumulus and corona cells prior to intracytoplasmic sperm injection (ICSI). Cumulus and corona cells were removed in two pipetting steps: first in a medium containing hyaluronidase, and then in a medium without enzyme. The first step in the procedure was investigated. Different hyaluronidase concentrations (78, 39 or 10 IU/ml) and pipettes of different size (inner diameter 250 or 1000 µm) were used to remove the cumulus cells. The time required to denude the oocytes was recorded. Metaphase II oocytes were injected, and the survival, fertilization, embryo development and pregnancy rates were evaluated. We found that by using a pipette with an inner diameter of at least 1000 µm we were able to decrease significantly the time an oocyte is exposed to hyaluronidase, even if the concentration of enzyme is very low (10 IU/ml). For the different conditions there was no statistically significant effect on the outcome in terms of survival, normal fertilization [two pronuclear (2PN)], parthenogenetic activation (1PN), abnormal fertilization (3PN), embryo development and pregnancy rates after ICSI. In conclusion, a concentration as low as 10 IU/ml hyaluronidase in combination with a pipette of at least 1000 µm inner diameter can be used successfully to denude oocytes for microinjection. Key words: cumulus cells/hyaluronidase/icsi/oocyte/pipette Finally, the age of the female patient clearly affects the pregnancy rate after ICSI (Devroey et al., 1996; Oehninger et al., 1995; Abdelmassih et al., 1996). In preparation for micromanipulation, oocytes have to be denuded from cumulus and corona cells using a combination of enzymatic (hyaluronidase) and mechanical (pipetting) methods (Van Steirteghem et al., 1995). Hyaluronidase has been shown to activate murine oocytes (Kaufman, 1983), but little is known about the effect of the enzyme on human oocytes (Laufer et al., 1984; Mahadevan and Trounson, 1985; Pickering et al., 1988; Abramczuk and Lopata, 1990; Fishel et al., 1992). At the start of the microinjection programme in our centre in 1991, we used 0.1% (w/v) hyaluronidase (~760 IU/ml) to denude the cumulus corona oocyte complexes (CCOC). We observed a high parthenogenetic (one pronuclear; 1PN) activation rate of the oocytes after ICSI, sometimes even before microinjection was carried out. By decreasing the concentration of the enzyme to ~80 IU/ml, we reduced significantly the degeneration rate and the 1PN activation rate of the oocytes after microinjection. At the same time, the normal fertilization (2PN) rate of the oocytes increased (Joris et al., 1997). We currently denude the oocytes in two steps: (i) cumulus cells are removed using medium containing ~80 IU/ml hyaluronidase and a mouth-controlled hand-made pipette with an inner diameter of 250 µm, and (ii) corona cells are removed in medium without an enzyme using a mouth-controlled handmade pipette with an inner diameter of 200 µm. In this study we compared different concentrations of hyaluronidase and pipettes of different diameter to remove the cumulus cells from the oocytes. The effects of the duration of exposure of the oocyte to the enzyme on survival, fertilization, embryo development and pregnancy rates after ICSI are reported. Materials and methods Introduction Intracytoplasmic sperm injection (ICSI) has revolutionized assisted reproduction, especially in couples with severe male factor infertility. The success rate of ICSI (survival, fertilization, embryo quality and pregnancy) depends on a number of factors: the equipment used to perform the injection (inverted microscope equipped with manipulators and injectors, holding and injection pipettes), the operator s experience and skill (Gordts et al., 1995), the quality of the oocyte (Liu et al., 1995), penetration of the zona pellucida and rupture of the oolemma (Nagy et al., 1995a; Palermo et al., 1996) and the vitality (motility) of the spermatozoa (Nagy et al., 1995b). Patients Patient selection criteria for ICSI were firstly failed or very low fertilization rate in a previous standard IVF cycle(s) or secondly sperm parameters too poor for conventional IVF ( progressively motile spermatozoa) with normal morphology after sperm selection. Ovarian stimulation was carried out by a desensitizing protocol using the gonadotrophin-releasing hormone agonist buserelin (Suprefact; Hoechst, Brussels, Belgium) combined with human menopausal gonadotrophin (HMG; Humegon; Organon, Oss, The Netherlands; or Pergonal; Serono, Brussels, Belgium) and human chorionic gonadotrophin (HCG; Pregnyl, Organon; and Profasi, Serono). Intravaginally administered progesterone (Utrogestan; Piette, Brussels, Belgium) was used for luteal phase supplementation. The details of this 2246 European Society for Human Reproduction and Embryology

2 Techniques for cumulus cell removal Table I. Summary of the conditions in the seven trials Condition 1 Condition 2 Hyaluronidase Pipette inner Hyaluronidase Pipette inner concentration (IU/ml) diameter (µm) concentration (IU/ml) diameter (µm) Sibling oocytes Trial Trial Trial Trial Patient by patient Trial Trial Trial stimulation protocol have been described elsewhere (Smitz et al., 1988, 1992). Patients were included in the study if freshly ejaculated semen could be used for the ICSI procedure. Semen preparation Sperm concentration and motility were evaluated according to the recommendations of the World Health Organization (WHO, 1992). Sperm morphology was determined after Shorr staining (WHO, 1992) of a smear using strict Tygerberg criteria (Kruger et al., 1986). The protocol for the treatment of freshly ejaculated semen has been described elsewhere (Van Steirteghem et al., 1993; Liu et al., 1994). Briefly, semen was washed in Earle s medium and centrifuged for 5 min at 1800 g. The resuspended pellet was then layered on top of a 95.0/47.5% Percoll gradient and centrifuged for 20 min at 300 g. Finally motile sperm cells were collected, washed for 5 min at 1800 g and concentrated if necessary. Oocyte preparation Oocytes were retrieved by vaginal ultrasound-guided puncture of ovarian follicles 36 h after HCG administration. The CCOC were pooled so that the results were not affected by the order of selection. Oocyte denudation was always performed in two steps. First, cumulus cells were removed in 100 µl droplets of HEPES-buffered Earle s medium containing hyaluronidase (Type VIII; specific activity 320 IU/ml; Sigma Chemical Company, St Louis, MO, USA). Up to eight CCOC were placed together in the droplet. Then corona cells were removed in 50 µl HEPES-buffered Earle s medium without an enzyme using a mouth-controlled hand-made pipette with an inner diameter of 200 µm. Finally the oocytes were rinsed twice in 50 µl microdrops of medium. The first step of the denudation procedure was investigated. We tested different concentrations of hyaluronidase (78 versus 39 IU/ml enzyme, 78 versus 10 IU/ml enzyme, 39 versus 10 IU/ml enzyme) and used pipettes with two different inner diameters (250 versus 1000 µm). The CCOC were denuded individually when we used the pipette with an inner diameter of 250 µm, and the CCOC were denuded all at once when we used the pipette with an inner diameter 1000 µm. The oocytes were further denuded individually during the second step of the procedure. Study groups We performed seven different trials, which are summarized in Table I. In trials 1 4 only patients with 10 CCOC were included. The CCOC from each patient were divided into two groups, and the sibling oocytes were denuded in two different ways (see Table I). In trial 1, cumulus removal was performed using 78 or 39 IU/ml hyaluronidase and a mouth-controlled hand-made pipette with an inner diameter of 250 µm. In trial 2, cumulus cells were removed using 78 or 10 IU/ml hyaluronidase and a mouth-controlled handmade pipette with an inner diameter of 250 µm. In trial 3, cumulus cells were removed using either 78 IU/ml enzyme and a mouthcontrolled hand-made pipette with an inner diameter of 250 µm or 39 IU/ml enzyme and a Pasteur pipette with an inner diameter 1000 µm. In trial 4, cumulus cells were removed using either 39 or 10 IU/ml hyaluronidase and a Pasteur pipette with an inner diameter 1000 µm. The time required to perform steps 1 and 2 was recorded, and the mean time required to carry out each step was calculated. In trials 5 7 all the CCOC from a patient were denuded in the same way and two different methods were compared (see Table I). In trial 5, oocytes were denuded using 39 IU/ml enzyme; the use of pipettes with a distinct diameter (250 or 1000 µm) was investigated. In trial 6, cumulus cells were removed using a pipette with an inner diameter 1000 µm and two distinct concentrations of enzyme (78 and 39 IU/ml) were used. Finally, in trial 7, cumulus cells were removed using a pipette with an inner diameter 1000 µm and two distinct concentrations of enzyme (39 and 10 IU/ml) were tested. ICSI procedure The preparation of the holding and injection pipettes has been described in detail elsewhere (Van Steirteghem et al., 1995). Whenever possible, a motile sperm cell with normal appearance was selected for injection from the concentrated sperm pellet. Only metaphase II oocytes were injected. The oocytes were placed in 5 µl droplets of HEPES-buffered Earle s medium which was covered by lightweight paraffin oil. The ICSI procedure has been described in detail elsewhere (Van Steirteghem et al., 1995). Oocytes were held on the holding pipette with the polar body at the 6 o clock position and the injection pipette was inserted into the oocyte at the 3 o clock position (Nagy et al., 1995a). After injection, the oocytes were washed and stored in 25 µl microdrops of B 2 medium under oil in a Petri dish. They were kept in an incubator at 37 C in an atmosphere of 5% CO 2,5%O 2 and 90% N 2. Assessment of survival, fertilization and embryo development and the establishment of pregnancy At h after microinjection the oocytes were checked for survival and fertilization (Nagy et al., 1994). The numbers and aspects of polar bodies and pronuclei were recorded. The criteria for normal fertilization were the presence of two individualized or fragmented polar bodies, together with two clearly visible pronuclei. Embryo cleavage and quality were evaluated 2 days after ICSI. According to the relative proportion of anucleate fragments present in the zona 2247

3 H.Van de Velde et al. pellucida, cleaved embryos were assigned to one of four categories: (i) excellent, when there was no anucleate fragmentation (type A); (ii) good, when between 1 and 20% of the embryo was fragmented (type B); (iii) fair, when the relative proportion of the fragments ranged between 20 and 50% (type C); and (iv) poor, when 50% of the embryo was fragmented. The best-quality embryos were always selected for transfer. Poor quality embryos were not eligible for transfer, while supernumerary embryos with 20% fragmentation were cryopreserved (Van Steirteghem et al., 1994). In selected cases, only two embryos were transferred to reduce the probability of triplet pregnancies (Staessen et al., 1993). Pregnancy was detected by measuring serum HCG concentrations on at least two separate occasions at least 10 days after embryo transfer. A clinical pregnancy was determined by ultrasound screening of the fetal sac at 7 weeks of pregnancy. Statistics The mean duration of exposure of an oocyte to hyaluronidase in step 1 and the length of time the oocyte remained in the medium in step 2 were calculated. Proportions of activated and fertilized oocytes were calculated on the basis of the numbers of intact oocytes after injection. Proportions of embryo quality categories were calculated from the total number of normally fertilized oocytes. Survival, fertilization and embryo quality rates were compared using the paired t-test in trials 1 4 and using the Mann Whitney U-test in trials 5 7. The total pregnancy rates were calculated per transfer cycle in trials 5 7 and were compared using the χ 2 test. Results In the first part of this report sibling oocytes from the same patient were used to test two different conditions to compare survival, fertilization and embryo development (trials 1 4). In the second part of this report, oocytes from different patients were used to compare two treatments so that the effect on the pregnancy rate could also be assessed (patient by patient in trials 5 7). Comparisons of sibling oocytes In trial 1, sibling CCOC were exposed to 78 or 39 IU/ml hyaluronidase, while in trial 2 they were exposed to 78 or 10 IU/ml hyaluronidase. The cumulus cells were removed using a mouth-controlled hand-made pipette with an inner diameter of 250 µm. There was no significant difference in the duration of exposure of the CCOC to 78 or 39 IU/ml enzyme (103 and 107 s respectively). However, the CCOC remained in the medium containing 10 IU/ml hyaluronidase for a significantly longer time (137 s) (P using paired Student s t-test) than in the medium containing 78 IU/ml hyaluronidase (95 s) (Table II). Decreasing the concentration of enzyme had no effect on the time required to remove the remaining corona cells in the second step. We found no further effect on the outcome of ICSI (survival, normal fertilization, parthenogenetic activation, abnormal fertilization and embryo development). In trial 3, cumulus cells were removed using either 78 IU/ ml hyaluronidase and a mouth-controlled hand-made pipette with an inner diameter of 250 µm, or 39 IU/ml hyaluronidase and a Pasteur pipette with an inner diameter 1000 µm. The time for which an oocyte was exposed to the enzyme was 2248 significantly longer (P using paired Student s t-test) when the high concentration of hyaluronidase was used in combination with the small pipette (87 s) than when the low concentration of hyaluronidase and the large pipette were used (31 s) (Table II), but the time required to perform the second step did not differ significantly. Again we found no significant effect on the ICSI parameters. In trial 4 we decreased the hyaluronidase concentration further by comparing the use of 39 and 10 IU/ml enzyme in combination with a pipette with a large inner diameter of 1000 µm (Table II). The time required to perform the first step of the denudation procedure did not differ significantly when 39 or 10 IU/ml hyaluronidase were used (39 s in both cases), and the mean time needed to carry out the second step was also similar (73 and 83 s respectively). Here we also found no significant effect on the outcome of ICSI. Patient-by-patient comparisons In trial 5, CCOC from 40 patients were denuded using 39 IU/ ml hyaluronidase and a pipette with an inner diameter of 250 µm, while CCOC from another 40 patients were denuded using the same concentration of enzyme and a pipette with an inner diameter of 1000 µm. The mean maternal age and the mean number of CCOC per patient did not differ significantly between the two groups (Table III). There was no significant difference in the outcome of ICSI (survival, normal fertilization, parthenogenetic activation and embryo cleavage) or in the pregnancy rates per transfer cycle (Table III). In each group, 34 patients had embryos transferred. In trial 6, CCOC from 40 patients were denuded using 78 IU/ml hyaluronidase. CCOC from another 40 patients were denuded using 39 IU/ml enzyme. We used a pipette with a large inner diameter ( 1000 µm) in both groups. We found no effect on the ICSI parameters. Embryo transfers were carried out in 38 and 39 patients respectively. The pregnancy rates were 32 and 49% respectively, but they did not differ significantly (Table III). In trial 7, where we compared 39 and 10 IU/ml hyaluronidase in 19 patients in both groups using a pipette with an inner diameter 1000 µm, there was no significant difference observed in survival, fertilization and embryo development (Table III). Pregnancy rates did not differ significantly between the two groups (18 and 16 patients respectively had embryos transferred). Discussion Oocytes have to be removed from surrounding cells using hyaluronidase and mechanical (pipetting) methods prior to microinjection. Murine oocytes are easily activated, e.g. by ethanol (7%) (Kaufman, 1982) or hyaluronidase (100 IU/ml) (Kaufman, 1983). Human oocytes appear to be less susceptible to activation. Data in the literature are confusing because different types and different concentrations of the enzyme have been used for different incubation times. However, we may conclude that a long exposure (30 min) to high concentrations of hyaluronidase ( IU/ml) (Laufer et al., 1984; Mahadevan

4 Techniques for cumulus cell removal Table II. Results of intracytoplasmic sperm injection (ICSI) on sibling oocytes using different concentrations of hyaluronidase (78, 39 or 10 IU/ml) and pipettes with a different inner diameter (250 or 1000 µm) during the first step of the procedure to remove the cumulus cells from the oocytes Trial 1 Trial 2 Trial 3 Trial 4 Hyaluronidase concentration (IU/ml) Inner diameter of pipette (µm) No. of CCOC Mean ( SD) time/ccoc (s) Step (41) 107 (37) 95 (39) a 137 (41) a 87 (31) a 31 (10) a 39 (34) 39 (29) Step 2 89 (51) 92 (60) 88 (35) 83 (31) 73 (23) 70 (30) 73 (43) 83 (47) No. of injected oocytes Percentage of intact oocytes ( SD) 89 (17) 94 (14) 91 (13) 96 (11) 94 (8) 94 (10) 91 (12) 95 (11) Pronucleus (PN) status of intact oocytes ( SD) (%) 1PN 2 (5) 1 (4) 1 (5) 3 (8) 0 (0) 1 (4) 2 (8) 2 (5) 2PN 73 (28) 69 (33) 74 (25) 67 (30) 68 (29) 74 (23) 80 (24) 79 (22) 3PN 1 (4) 4 (9) 2 (8) 7 (13) 0 (0) 0 (0) 1 (5) 4 (10) Percentage of cleaved embryos from 2PN oocytes ( SD) Excellent 5 (13) 6 (13) 4 (18) 2 (5) 3 (10) 3 (12) 1 (5) 1 (3) Good 68 (31) 61 (34) 62 (37) 68 (26) 44 (37) 48 (38) 62 (32) 68 (32) Fair 15 (24) 21 (29) 21 (28) 13 (18) 31 (25) 21 (27) 27 (28) 15 (19) Total 88 (28) 88 (20) 87 (15) 83 (26) 78 (21) 72 (32) 90 (18) 84 (30) CCOC cumulus corona oocyte complex. a P using paired Student s t-test. Table III. Results of intracytoplasmic sperm injection (ICSI) on oocytes using different mechanical treatments (pipettes with an inner diameter of 250 or 1000 µm) and enzyme concentrations (78, 39 or 10 IU/ml) to remove the cumulus cells prior to injection Trial 5 Trial 6 Trial 7 Hyaluronidase concentration (IU/ml) Inner diameter of pipette (µm) No. of cycles Mean ( SD) maternal age (years) 33 (6) 32 (4) 33 (5) 33 (5) 31 (4) 31 (5) Mean ( SD) no. of CCOC 11 (8) 12 (7) 12 (7) 13 (7) 16 (9) 12 (8) No. of injected oocytes Percentage of intact oocytes ( SD) 87 (23) 90 (15) 95 (11) 94 (9) 94 (10) 92 (14) Pronucleus (PN) status of intact oocytes ( SD) (%) 1PN 1 (3) 2 (4) 3 (8) 4 (9) 8 (23) 2 (5) 2PN 74 (26) 81 (26) 73 (24) 75 (24) 68 (26) 78 (22) 3PN 6 (13) a 1 (4) a 1 (4) 4 (9) 3 (6) 3 (8) Percentage of cleaved embryos from 2PN oocytes ( SD) Excellent 8 (20) 2 (7) 9 (21) 7 (18) 8 (17) 17 (27) Good 55 (35) 50 (33) 50 (32) 58 (32) 52 (26) 52 (31) Fair 17 (28) 16 (25) 18 (28) 21 (25) 19 (17) 12 (17) Total 80 (28) 68 (34) 77 (21) 86 (27) 79 (24) 81 (32) Pregnancy rate (%) CCOC cumulus corona oocyte complex. a P by the Mann Whitney U-test. and Trounson, 1985; Pickering et al., 1988; Abramczuk and Lopata, 1990; Fishel et al., 1992) or ethanol (7.5%) (Abramczuk and Lopata, 1990) does not lead to parthenogenetic activation of the oocytes. Activation of the oocytes might also be caused by other factors, such as the pipetting procedure (Fishel et al., 1992), the mechanical pricking of the oocyte (Iritani, 1991; Flaherty et al., 1995) or the polyvinylpyrrolidone (Iritani, 1991). It may also be an intrinsic oocyte problem, because aged human oocytes undergo parthenogenetic activation more easily after ICSI (Abramczuk and Lopata, 1990; Winston et al., 1991; Balakier et al., 1993). Finally, activating the oocytes just before or at the time of ICSI might even be helpful in obtaining higher fertilization rates (Tesarik et al., 1994; Tesarik and Sousa, 1995). At the beginning of the microinjection programme in our centre we used high concentrations of enzyme (0.1% or ~760 IU/ml) to denude the oocytes. At that time, parthenogenetic activation would occur (17%) before the oocytes were injected (Palermo et al., 1993). We were able to decrease the parthenogenetic activation rate to 3% by lowering the hyaluronidase concentration to 78 IU/ml (Van Steirteghem et al., 1995). The aim of this study was to decrease possible toxic effects from the hyaluronidase on the outcome of ICSI by decreasing the concentration and the length of time for which the oocyte is exposed to the enzyme. In the first part of our study, we decreased the hyaluronidase concentration from 78 to 39 and 10 IU/ml, while the inner diameter of the pipette remained unchanged (250 µm). This did not result in a significantly higher fertilization rate or in better embryo quality. The parthenogenetic activation (0 3%) and abnormal fertilization 2249

5 H.Van de Velde et al. (1 7%) rates also remained unchanged, indicating that hyaluronidase had no effect on the outcome of ICSI at any of the concentrations tested. However, the oocyte remained in the medium containing 10 IU/ml hyaluronidase for a significantly longer period, which should be avoided. In the second part of the study we decreased the hyaluronidase concentration and increased the inner diameter of the pipette from 250 to 1000 µm. The time for which the oocyte stayed in the medium with enzyme decreased dramatically when using a larger pipette, even in the presence of a low concentration of hyaluronidase (10 IU/ml). Decreasing the time and the concentration had no direct beneficial effect on survival, fertilization, embryo development (trials 1 4) and pregnancy (trials 5 7) rates after ICSI. However, it is not known whether hyaluronidase affects the outcome of ICSI indirectly (e.g. development of the fetus). To avoid unknown harmful effects from the enzyme, it should be used in low concentrations and for a short period of time. By using a pipette with a large inner diameter ( 1000 µm) we were able to decrease significantly the time for which the oocyte was exposed to hyaluronidase, in combination with a low concentration of enzyme (10 IU/ml). Apparently this new method of cumulus cell removal has no direct effect on the outcome of ICSI (survival, fertilization, embryo development and pregnancy rates). However, we believe that using a low concentration of enzyme for a short period of time without loss of efficiency should be chosen for oocyte denudation. Therefore we advise using this new method for oocyte denudation prior to microinjection. Acknowledgements The authors wish to thank the clinical, paramedical and laboratory staff of the Centre for Reproductive Medicine, University Hospital, Dutch-speaking Brussels Free University, Belgium. Furthermore we are grateful to Mr Frank Winter of the Language Education Centre in our University for correcting this manuscript. This work was supported by the Fund for Scientific Research Flanders (Fonds voor Wetenschappelijk Onderzoek Vlaanderen). References Abdelmassih, R., Sollia, S., Moretto, M. et al. (1996) Female age is an important parameter to predict treatment outcome in intracytoplasmic sperm injection. Fertil. Steril., 65, Abramczuk, J.W. and Lopata, A. (1990) Resistance of human follicular oocytes to parthenogenetic activation: DNA distribution and content in oocytes maintained in vitro. Hum. Reprod., 5, Balakier, H., Squire, J. and Casper, R.F. (1993) Characterization of abnormal one pronuclear human oocytes by morphology, cytogenetics and in-situ hybridization. Hum. Reprod., 8, Devroey, P., Godoy, H., Smitz, J. et al. (1996) Female age predicts embryonic implantation after ICSI: a case-controlled study. Hum. Reprod., 11, Fishel, S., Timson, J., Lisi, F. et al. (1992) Evaluation of 225 patients undergoing subzonal insemination for the procurement of fertilization in vitro. Fertil. Steril., 57, Flaherty, S.P., Payne, D., Swann, N.J. et al. (1995) Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection. Hum. Reprod., 10, Gordts, S., Vercruyssen, M. and Roziers, P. (1995) Recent developments in assisted fertilization. Hum. Reprod., 10 (Suppl. 1), Iritani, A. (1991) Micromanipulation of gametes for in vitro assisted fertilization. Mol. Reprod. Dev., 28, Joris, H., Nagy, Z., Van de Velde, H. et al. (1997) Intracytoplasmic sperm injection: laboratory set-up and injection procedure. Hum. Reprod., 12 (Suppl. 3), in press. Kaufman, M.H. (1982) The chromosome complement of single-pronuclear haploid mouse embryos following activation by ethanol treatment. J. Embryol. Exp. Morphol., 71, Kaufman, M. (1983) Early Mammalian Development: Parthenogenic Studies. Cambridge University Press, Cambridge, UK. Kruger, T.F., Menkveld, R., Stander, F.S.H. et al. (1986) Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil. Steril., 46, Laufer, N., Tarlatzis, B.C., DeCherney, A.H. et al. (1984) Asynchrony between cumulus corona cell complex and oocyte maturation after human menopausal gonadotropin treatment for in vitro fertilization. Fertil. Steril., 42, Liu, J., Nagy, Z., Joris, H. et al. (1994) Intracytoplasmic sperm injection does not require special treatment of spermatozoa. Hum. Reprod., 9, Liu, J., Nagy, Z., Joris, H. et al. (1995) Analysis of 76 total fertilization failure cycles out of 2732 intracytoplasmic sperm injection cycles. Hum. Reprod., 10, Mahadevan, M.M. and Trounson, A.O. (1985) Removal of the cumulus oophorus from the human oocyte for in vitro fertilization. Fertil. Steril., 43, Nagy, Z.P., Liu, J., Joris, H. et al. (1994) Time-course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection. Hum. Reprod., 9, Nagy, Z.P., Liu, J., Joris, H. et al. (1995a) The influence of the site of sperm deposition and oolemma breakage at intracytoplasmic sperm injection on fertilization and embryo development rates. Hum. Reprod., 10, Nagy, Z.P., Liu, J., Joris, H. et al. (1995b) The result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters. Hum. Reprod., 10, Oehninger, S., Veeck, L., Lanzendorf, S. et al. (1995) Intracytoplasmic sperm injection: achievement of high pregnancy rates in couples with severe male factor infertility is dependent primarily upon female and not male factors. Fertil. Steril., 64, Palermo, G., Joris, H., Derde, M.-P. et al. (1993) Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertil. 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(1995) More than 90% fertilization rates after intracytoplasmic sperm injection and artificial induction of oocyte activation with calcium ionophore. Fertil. Steril., 63, Tesarik, J., Sousa, M. and Testart, J. (1994) Human oocyte activation after intracytoplasmic sperm injection. Hum. Reprod., 9, Van Steirteghem, A.C., Nagy, Z.P., Joris, H. et al. (1993) High fertilization and implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 8, Van Steirteghem, A.C., Van der Elst, J., van den Abbeel, E. et al. (1994) Cryopreservation of supernumerary multicellular human embryos obtained after intracytoplasmic sperm injection. Fertil. Steril., 62, Van Steirteghem, A.C., Joris, H. and Liu, J. (1995) Protocol for intracytoplasmic sperm injection. Hum. Reprod. Update, 1, item 9, CD-ROM. Winston, N., Johnson, M., Pickering, S. et al. (1991) Parthenogenic activation and development of fresh and aged human oocytes. Fertil. Steril., 56, World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm Cervical Mucus Interaction. 3rd edn, Cambridge University Press, Cambridge, UK. Received on February 12, 1997; accepted on July 10, 1997