Candido Tomás 1,3, Mauri Orava 1, Leena Tuomivaara 2 and Hannu Martikainen 1

Human Reproduction vol.13 no.1 pp. 65 70, 1998 Low pregnancy rate is achieved in patients treated with intracytoplasmic sperm injection due to previous low or failed fertilization in in-vitro fertilization Candido Tomás 1,3, Mauri Orava 1, Leena Tuomivaara 2 and Hannu Martikainen 1 1 IVF Unit, Department of Obstetrics and Gynecology, University of Oulu and 2 The Family Federation of Finland, FIN-90220 Oulu, Finland 3 To whom correspondence should be addressed at: AVA-Clinic, Fertility Centre, Keskustori 1 A 10, FIN-33100 Tampere, Finland; e-mail: ctomas@cc.oulu.fi The main indications for intracytoplasmic sperm injection (ICSI) are severe male factor and fertilization failure or a low fertilization rate in previous in-vitro fertilization (IVF) treatments. The fertilization and pregnancy rates after ICSI, however, are seldom reported separately for these two different indications. The aim of this study was to compare the treatment outcome and pregnancy rate after ICSI between 65 patients with previous failed fertilization or a low fertilization rate without male factor, and 219 patients with a primary male factor. From the 2726 oocytes collected, 2087 (77%) were micro-injected and 1355 (65%) achieved normal fertilization. The oocyte fertilization rate was similar in the group with previous failed fertilization or a low fertilization rate and the group with a male factor (65 and 65% respectively), as was the cleavage rate of normally fertilized oocytes (92 and 94% respectively). Despite the similar fertilization and cleavage rates and the similar number and morphological quality of embryos transferred in both groups, the pregnancy rate was significantly lower (P 0.05) in the group with previous failed fertilization or a low fertilization rate than in the group with a male factor (19.6 versus 33.5% respectively; 95% confidence intervals for the difference, 2 26%). The implantation rate was also lower (P 0.01) in patients with previous failed fertilization or a low fertilization rate (9.6%) than in the group with a male factor (19.5%). We conclude that patients with previous failed fertilization or a low fertilization rate in standard IVF without male factor have a significantly smaller chance of becoming pregnant after subsequent ICSI than patients with a primary male factor. This poor outcome probably reflects intrinsic oocyte defects not bypassed by ICSI. Key words: ICSI/low fertilization/oocyte activation/oocyte defect Introduction Micromanipulation techniques have been utilized in patients with a previous low oocyte fertilization rate or fertilization failure following in-vitro fertilization (IVF). Partial zona dissection (PZD) and subzonal insemination (SUZI) have been employed in attempts to overcome the problem of low fertilization rates, but with minimal success and unacceptably high rates of polyspermia (Ng et al., 1990; Fishel et al., 1992; Vanderzwalmen et al., 1992; Van Steirteghem et al., 1993a). After reports of high fertilization rates with intracytoplasmic sperm injection (ICSI), this technique has become the standard treatment for infertility caused by severe sperm defects and also by failed or low fertilization in previous IVF treatments (Van Steirteghem et al., 1993a,b; Payne et al., 1994; Gordts et al., 1995; Svalander et al., 1995). However, the fertilization and pregnancy rates reported after ICSI seldom take into account the various indications leading to treatment. ICSI may overcome the failure in fertilization when this is caused by sperm defects, but is not necessarily effective when it is the result of oocyte defects (Sousa and Tesarik, 1994; Flaherty et al., 1995; Tesarik and Sousa, 1995). The objective of this study was to identify subgroups of patients for whom ICSI is performed, and to compare the treatment results in terms of fertilization, cleavage and pregnancy rates. In IVF programmes, the number of patients with failed fertilization or low fertilization rates tends to increase with time as a result of previous treatment failures. It was also our aim to analyse the influence of the increasing number of patients with previous failed fertilization or a low fertilization rate on the overall results of an ICSI programme. Materials and methods Patient selection We studied 284 successive infertile couples treated with ICSI during the period January 1995 November 1996 at the Oulu Fertility Centre (Oulu, Finland). This centre includes a University-based tertiary IVF referral centre (University Hospital of Oulu, Oulu, Finland) and a private institute (The Family Federation of Finland, Oulu Department, Oulu, Finland) sharing a common laboratory. The patients were split into two groups according to the underlying indication for ICSI. The first group consisted of 65 patients who had had failed fertilization or a low fertilization rate ( 25%) in previous IVF treatments with normal stimulation outcomes. This group of patients had a normal spermiogram according to the World Health Organization (WHO, 1992) classification. The second group consisted of 219 patients eligible for ICSI because of severe male factor(s) according to the WHO criteria. This included one or more of the following characteristics: a sperm concentration 20 10 6 /ml, progressive motility 50% and/or normal morphological spermatozoa 30%. The characteristics of the patients and their treatment protocols are presented in Table I. In our institution, a maximum of four treatments are offered to European Society for Human Reproduction and Embryology 65

C.Tomás et al. Table I. Characteristics of two groups of patients by intracytoplasmic sperm injection indication, and their ovarian stimulation protocols (mean SD) Previous failed fertilization or low fertilization rate in IVF Primary male factor n 65 219 Age (years) 33.5 4.3 31.5 4.2 a Body mass index (kg/m 2 ) 23.2 3.8 23.6 3.8 Gonadotrophin (ampoules) 29.3 11.1 27.8 9.0 No. of treatment days 11.1 2.1 10.9 1.7 Maximum oestradiol concentration (nmol/l) 3.5 2.8 3.4 3.1 No. of oocytes recovered 8.9 5.7 9.8 5.9 IVF in-vitro fertilization. a P 0.05. each couple. In the male factor group, 78 patients were receiving their first treatment. The remaining patients in both groups had a similar number of previous IVF or ICSI attempts. In the low fertilization and male factor groups, respectively, there were 33 and 89 patients with one previous attempt, 28 and 47 with two previous attempts and four and five with three previous attempts (not significant). Ovarian stimulation The treatment started in the luteal phase of the previous cycle with a long protocol for down-regulation with 800 µg/day intranasal buserelin acetate (Suprecur; Hoechst AG, Frankfurt am Main, Germany), followed by ovarian stimulation with human menopausal gonadotrophin (Pergonal; Serono, Switzerland; or Humegon; Organon, Oss, The Netherlands). Ovarian response was monitored with transvaginal ultrasound and the daily dose of gonadotrophin was adjusted accordingly. Ovulation was induced with 5000 10 000 IU human chorionic gonadotrophin (HCG; Pregnyl; Organon) when at least two follicles measuring 18 mm in diameter were detected. Oocyte retrieval by transvaginal ultrasound-guided puncture was performed 36 h post-hcg. Semen preparation Sperm samples were centrifuged (300 g, 20 min) through discontinuous Percoll gradients (40 and 90% Percoll; Pharmacia AB, Stockholm, Sweden). The bottom layer containing the spermatozoa was washed twice in Earle s balanced salt solution (Earle, 1943) supplemented with 0.5 mm sodium pyruvate, 30 mm DL-lactic acid, 15 mm HEPES, 50 000 IU/l penicillin, 50 mg/l streptomycin and 0.5% human serum albumin (Earle s medium). For ICSI, sperm samples were finally suspended in 200 1000 µl Medi-Cult IVF medium (Medi-Cult a/s, Copenhagen, Denmark). In cases where only a few motile spermatozoa were observed, the ejaculate was centrifuged at 400 g, washed twice with 3 ml Earle s medium (400 g, 10 min) and finally suspended in 50 200 µl Medi-Cult IVF medium. Oocyte preparation After retrieval, the oocytes were exposed for 30 60 s to 30 IU/ml Type IV-S hyaluronidase (Sigma Chemical Company, St Louis, MO, USA) to remove the surrounding cumulus cells. After rinsing the oocytes several times in Earle s medium, the corona cells were removed mechanically using commercial denuding pipettes (Swemed Lab International AB, Västra Frölunda, Sweden). Denuded oocytes were cultured in Medi-Cult IVF medium at 37 C in a humidified atmosphere of 5% CO 2 95% air, until used for ICSI. 66 Intracytoplasmic sperm injection ICSI was carried out using a Nikon Diaphot 300 inverted microscope equipped with a Hoffman Modulation Contrast system (Modulation Optics Inc., Greenvale, NY, USA), heating stage (Linkam Scientific Instruments Ltd, London, UK), Narishige MN-188 manual coarse movement controls, MO-188 3D micromanipulators and IM-188 and IM6 injectors for holding and injection pipettes respectively (Narishige Co., Ltd, Tokyo, Japan). ICSI was performed in 5 µl microdroplets under oil. In a centre droplet containing 5 µl 10% polyvinylpyrrolidone solution (Medi-Cult a/s), 1 µl of sperm suspension was placed. The centre droplet was surrounded by six microdroplets containing Medi- Cult IVF medium, in which the oocytes were placed. The selected spermatozoon was immobilized and captured using a microinjection pipette from Swemed AB. The spermatozoon was injected into an oocyte, which was held against a holding pipette (Swemed AB) by suction. Only morphologically normal-appearing mature oocytes, with a visible polar body, were injected. Embryology After the injection procedure, the oocytes were rinsed and cultured overnight in Medi-Cult IVF medium. The next morning, the medium was changed and the oocytes were checked for fertilization. Normally fertilized (2PN) oocytes were cultured for a further 24 h and checked for cleavage. The morphology of the embryos was scored as follows: grade I, equal sized blastomeres, no fragmentation; grade II, equal sized blastomeres, 20% fragmentation; grade III, unequal sized blastomeres, 20% fragmentation; grade IV, equal or unequal sized blastomeres, 20 50% fragmentation; and grade V, equal or unequal sized blastomeres, 50% fragmentation. Embryos with 50% fragmentation were eligible for transfer. A maximum of two embryos were transferred into the uterine cavity 48 h after oocyte retrieval using a Wallace (Simcare Ltd, Lancing, UK) or Gynetics (J.van Brunschot b.v., Amsterdam, The Netherlands) transfer catheter. If supernumerary embryos with a score of grade III or better were available, they were frozen using a slow freezing protocol with 1,2-propanediol as the cryoprotectant (Testart et al., 1986). Luteal phase support and pregnancy testing Luteal support was given as four i.m. doses of 1500 IU HCG (Pregnyl) or intravaginally using 900 mg/day natural micronized progesterone in three divided doses (Lugesteron; Leiras, Turku, Finland) for 2 weeks. Clinical pregnancies were confirmed by ultrasound at 7 weeks of gestation. Study parameters and statistical analysis The main outcomes evaluated were oocyte maturation, fertilization, cleavage, pregnancy and implantation rates. Furthermore, we evaluated the number of gonadotrophin ampoules used, the number of treatment days, the number of oocytes recovered and the number of embryos available for cryopreservation. The fragmentation rate and the morphology of the embryos transferred were also analysed. Data were collected from a computerized IVF database program (Babe 2.0; XtremeSolutions, Oulu, Finland), and the statistical analysis was performed using the StatView 4.0 package (Abacus Concepts Inc., Berkeley, CA, USA) on a Macintosh personal computer. The Mann Whitney test for continuous data and the χ 2 -test for categorical variables were used to compare the outcomes between the two groups. All tests were performed at the 5% level of significance. The 95% confidence intervals (CI) for the estimated difference in pregnancy rates between the two groups were also obtained.

Previous failed or low fertilization and ICSI Table II. Overall results of intracytoplasmic sperm injection performed because of previous failed fertilization or a low fertilization rate (without male factor) in in-vitro fertilization (IVF) or due to a primary male factor Previous failed fertilization or low fertilization rate in IVF Primary male factor No. of cycles 65 219 No. of oocytes collected 579 2147 No. of mature oocytes injected 430 (74) 1657 (77) No. of oocytes fertilized 300 (70) 1156 (70) Two pronuclei 279 (65) 1076 (65) Three pronuclei 19 (4) 61 (4) No. of oocytes cleaved 258 (92) 1011 (94) No. of embryo transfers 56 197 No. of embryos transferred 115 390 Average no. of embryos per transfer 2.1 2.0 No. of pregnancies (% per transfer) 11 (19.6) 66 (33.5) a No. of embryos implanted 11 (9.6) 76 (19.5) a No. of cycles with frozen embryos 27 (42) 111 (51) No. of frozen embryos 108 548 Average no. of frozen embryos per patient 4.0 4.9 Values in parentheses are percentages. a P 0.05. Results ICSI was performed in 65 patients with previous failed fertilization or a low fertilization rate (but no male factor) and in 219 patients with a primary male factor. Patients with previous failed fertilization or a low fertilization rate were older than those with male factor (P 0.05), while the body mass index and the characteristics of ovarian stimulation were similar (Table I). Overall, 2726 oocytes were recovered, from which 2087 were at metaphase II (MII) (77%), 1355 (65%) fertilized normally (number of 2PN oocytes/number of MII oocytes), 1269 (94%) cleaved (number of cleaved oocytes/number of normally fertilized oocytes), 505 (40%) were transferred and 656 (52%) were cryopreserved. The total numbers of oocytes recovered in the group with previous failed fertilization or a low fertilization rate and the male factor group were 579 and 2147 respectively. The number of mature oocytes, fertilization rate (number of 2PN oocytes/number of mature oocytes) and cleavage rate (number of cleaved oocytes/ number of 2PN oocytes) were similar in both groups (Table II). The pregnancy rate per embryo transfer was significantly lower (P 0.05) in the group of patients with previous failed fertilization or a low fertilization rate (19.6%) than in the male factor group (33.5%). The difference between these two rates (14%) has a 95% CI from 2 to 26%. The cleavage rate and morphological quality of the embryos transferred were similar in both groups (Table III). The embryo implantation rate was significantly lower (P 0.01) in the group of patients with previous failed fertilization or a low fertilization rate (9.6%) than in the male factor group (19.5%). Because the patients with previous failed fertilization or a low fertilization rate were older than those in the male factor group, we re-analysed the data for patients aged 36 and 36 years. In patients aged 36 years the results were Table III. Quality of embryos transferred after intracytoplasmic sperm injection treatment because of previous failed fertilization or a low fertilization rate in in-vitro fertilization (IVF) or due to a primary male factor Previous failed fertilization or low fertilization rate in IVF Primary male factor No. of blastomeres 3 76 (66) 283 (73) 4 6 23 (20) 64 (16) 7 16 (14) 43 (11) Morphological score I II 27 (23) 109 (28) III 71 (62) 254 (65) IV V 17 (15) 27 (7) Values in parentheses are percentages. similar to the total study population, i.e. previous failed fertilization or a low fertilization rate (n 45) and male factor (n 178) patients achieved pregnancy rates per embryo transfer of 18 and 34% respectively (P 0.05). In patients aged 36 years, the pregnancy rates per embryo transfer for previous failed fertilization or a low fertilization rate (n 20) and male factor (n 41) patients were 24 and 30% respectively (not significant). The proportion of patients with previous failed fertilization or a low fertilization rate tended to increase from the beginning of the ICSI programme in our IVF unit. During the first 6 months (spring 1995), 18% of ICSI patients were assigned to the low fertilization group; that number increased to 22 and 32% during autumn 1995 and spring 1996 respectively. In autumn 1996 there were four patients with previous fertilization failure or low fertilization rates. During each time period, the pregnancy rate per cycle was lower in the patients with previous failed fertilization or a low fertilization rate than in the male factor group, as shown in Table IV. 67

C.Tomás et al. Table IV. Number of patients allocated to intracytoplasmic sperm injection (ICSI) because of previous failed fertilization or a low fertilization rate or primary male factor during the ICSI programme at the Oulu Fertility Centre, and their relative pregnancy rates Previous failed fertilization or low fertilization rate in IVF Primary male factor n Pregnancy rate n Pregnancy rate Spring 1995 12 1 (8) 54 14 (26) Autumn 1995 18 4 (22) 62 23 (37) Spring 1996 31 5 (16) 67 16 (24) Autumn 1996 4 1 (25) 36 13 (36) Values in parentheses are percentages. IVF in-vitro fertilization. Table V. Pregnancy rates in patients treated with intracytoplasmic sperm injection because of previous failed fertilization or a low fertilization rate in in-vitro fertilization (IVF) or due to a primary male factor Studies Previous failed fertilization or Primary male Difference (%) between low fertilization rate in IVF factor pregnancy rates (95% CI) Gabrielsen et al. (1996) 26/180 111/248 30 (21 40) Tekpetey et al. (1996) 6/46 37/158 10 ( 3 24) Present study 11/56 66/197 13 (2 26) All studies 43/282 214/603 20 (14 27) The differences between the pregnancy rates of the two groups are presented with the respective 95% confidence intervals (CI). Discussion In this study we identified two groups of patients with different clinical success rates following ICSI treatment. Patients for whom ICSI was performed because of failed fertilization or a low fertilization rate in previous IVF treatment, and who had a normal spermiogram, had significantly lower pregnancy rates than patients treated with ICSI for male factor infertility. The pregnancy rates obtained in our study were 19.6% for the previous failed fertilization or low fertilization rate group and 33.5% for the male factor group, which were strikingly similar to those reported recently by Gabrielsen et al. (1996) (14 and 45% respectively) and Tekpetey et al. (1996) (13 and 23% respectively) for similar groups of patients. The difference in pregnancy rates between the two groups can be appreciated in Table V through the computation of CI. If we account for the joint effect of all three studies, we would expect an estimated difference of the pregnancy rate to be situated between 14 and 27% (95% CI), a highly significant statistical difference (χ 2 38, P 0.0001). In the present study, the fertilization rate obtained with ICSI was similar in patients with previous failed fertilization or a low fertilization rate and in patients with male factor infertility (65 and 65% respectively). Similar findings were also reported by Nagy et al. (1993) and Tekpetey et al. (1996). In contrast to our and previous studies, Gabrielsen et al. (1996) reported higher fertilization rates after ICSI and observed a statistically significant difference between the low fertilization group (82%) and the male factor group (90%). These fertilization rates are among the highest so far presented in the literature, and we could not find any explanation for these contradictory results. The reason for failed fertilization after standard IVF 68 programmes can reside in the spermatozoon and/or oocyte (Barlow et al., 1990; Yie et al., 1996). This problem can be resolved partly by ICSI which can achieve acceptable rates of fertilization, as shown in our study. Some oocytes do not become fertilized after ICSI (Liu et al., 1995), which is mostly due to absent or abnormal oocyte activation (Sousa and Tesarik, 1994; Tesarik et al., 1994; Flaherty et al., 1995; Tesarik and Sousa, 1995). The barriers of sperm access to the ooplasma, namely the zona pellucida and the ooplasmic membrane, can be overcome effectively by ICSI. The fact that pregnancy rates after ICSI are much lower in the group with previous failed fertilization or a low fertilization rate (and a normal spermiogram) than in the group with a male factor, leads us to suggest that oocyte defects can compromise embryo development and/ or implantation. The present and previous studies suggest that oocyte defects are probably expressed later, during/after the implantation phase. In fact, fertilization and the first stages of embryo cleavage occurred similarly after ICSI in patients with previous fertilization failure and in those treated for male factor infertility. It has been suggested that embryonic gene expression starts between the 4- and 8-cell stage of preimplantation development (Braude et al., 1988). Therefore it is possible that embryos from patients with previous failed fertilization may have genetic defects that prevent normal embryonic development. In the present study, the mean age of patients with previous failed fertilization or a low fertilization rate was greater than that of the male factor patients. One may be tempted to explain the low implantation and pregnancy rates in the group of patients with previous failed fertilization or a low fertilization rate as an effect of their age and possible uterine factors.

Previous failed or low fertilization and ICSI However, a more detailed analysis of the data contradicts this hypothesis. In fact, when the data were corrected for age, there was a difference in pregnancy rates in patients aged 36 years but not in those aged 36 years. Possible uterine factors are supposed to be age dependent and to play a lesser role in younger patients. Therefore this re-analysis further supports the hypothesis that the difference in pregnancy rates is due to disturbances of embryo development occurring after embryo transfer and not to uterine factors. Disturbances in embryo development probably originate as oocyte deficiencies, as proved by oocyte donor studies (Navot et al., 1991; Borini et al., 1996). Borini et al. (1996) treated 15 patients with previous failed fertilization or severe male factor with oocyte donation and ICSI, achieving an overall pregnancy rate of 38%. Patients with previous failed fertilization had a similar pregnancy rate to those in the male factor group. This clearly showed that most patients with previous repeated fertilization failure probably have oocyte defects that can only be bypassed by oocyte donation, not by ICSI using their own cells. In patients with previous fertilization failure, the 40% pregnancy rate obtained in the study of Borini et al. (1996) with donor oocytes compares favourably with the 19.6% in our study for a similar group of patients using their own oocytes. In the male factor group, a pregnancy rate of 30% was obtained after ICSI in the donor oocytes in the study by Borini et al. (1996), which was similar to that achieved in our study after ICSI with the patients own oocytes (33.5%). In general, ICSI is performed because of severe male factor and previous failed fertilization or a low fertilization rate in IVF. However, treatment outcome and pregnancy rate are seldom reported separately for these two different groups of patients. This can be a source of bias when comparing results between various centres. According to our and previous findings, the subgroups of patients for whom ICSI is performed should be identified and treatment results presented separately for each group. Since the commencement of the ICSI programme in our centre, we have detected an increasing number of patients with previous failed fertilization or a low fertilization rate to whom ICSI has been offered. Because of the cumulative influence of previous failed fertilization or low fertilization rate patients, overall success rates might decrease, as observed over a specified time period in our ICSI programme. It is difficult to construct a recommended work-up scheme for patients with previous failed fertilization or a low fertilization rate after IVF. For the couples and the clinicians the decisions concerning further treatments are problematic. In cases of total failure of fertilization the preference for ICSI in the next treatment cycle is acceptable because some patients can achieve pregnancy with this technique. Another option would be to perform ICSI using half of the oocytes and standard IVF using the other half to confirm the diagnosis. Oocyte donation is an alternative for patients with recurrent failed ICSI treatments (Ezra et al., 1992). For those with low fertilization rates and without male factor, a second standard IVF trial can be recommended. There is a lack of evidence to show that these patients would achieve a better outcome with ICSI instead of conventional IVF, unless oocyte donation is used concomitantly. Some studies have indicated that fertilization can occur after IVF in subsequent cycles, even in the presence of severe male factor (Ben Shlomo et al., 1992; Ord et al., 1993). 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C.Tomás et al. Van Steirteghem, A.C., Liu, J., Joris, H. et al. (1993a) Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. Report of a second series of 300 consecutive treatment cycles. Hum. Reprod., 8, 1055 1060. Van Steirteghem, A.C., Nagy, Z., Joris, H. et al. (1993b) High fertilization and implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 8, 1061 1066. Vanderzwalmen, P., Barlow, P., Nijs, M. et al. (1992) Usefulness of partial dissection of the zona pellucida in a human in-vitro fertilization programme. Hum. Reprod., 7, 537 544. World Health Organization (1992) Laboratory Manual for the Examination of Human Semen and Sperm Cervical Mucus Interaction. 3rd edition, Cambridge University Press, Cambridge, UK, pp. 3 15, 27 18. Yie, S.M., Collins, J.A., Daya, S. et al. (1996) Polyploidy and failed fertilization in in-vitro fertilization are related to patient s age and gamete quality. Hum. Reprod., 11, 614 617. Received on April 14, 1997; accepted on October 2, 1997 70