Copyright 1995 American Society for Reproductive Medicine

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1 FERTILITY AND STERILITY Vol. 64, No.6, December 1995 Copyright 1995 American Society for Reproductive Medicine Printed on acid free paper in U. S. A. Prospective, auto-controlled study on reinsemination of failed-fertilized oocytes by intracytoplasmic sperm injection*t Zsolt P. Nagy, M.D.* Catherine Staessen, M.Sc. Jiean Liu, M.D. Hubert Joris, M.T. Paul Devroey, M.D., Ph.D. Andre C. Van Steirteghem, M.D., Ph.D. Centre for Reproductive Medicine, University Hospital, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Brussels, Belgium Objective: To evaluate the possible influence of late fertilization after standard IVF on the results of reinsemination of assumed failed-fertilized oocytes by microinjection and to examine the correlation between the effect of aging of (failed-fertilized) oocytes and the ability of these oocytes to become fertilized. Design: Trial 1: Group 1 (injected-day 1), 93 failed-fertilized oocytes injected 1 day after ovum pick-up; group 2 (control), 82 failed-fertilized oocytes with no microinjection performed. Trial 2: Group 1 (ICSI-day 1), 40 failed-fertilized oocytes injected 1 day after ovum pick-up; group-2 (ICSI-day 2),40 failed-fertilized oocytes injected 2 days after ovum pick-up. In addition, 35 two- to eight-cell stage embryos, obtained after ICSI of IVF failed-fertilized oocytes, were fixed for cytogenetic analysis. Main Outcome Measures: Normal and abnormal fertilization and embryo development. Results: Trial 1: 53% normal (2 pronuclear [PN]) and 25% abnormal (;;;,3PN) fertilization rates were obtained in group 1 (injected-day 1), and 71% of the 2PN and 74% of the ;;;,3PN oocytes cleaved with <50% fragmentation. No pronuclear (;;;,2PN) development occurred in the control group. Trial 2: 45% and 8% normal and 25% and 40% abnormal fertilization rates were obtained, respectively, after ICSI of 1-day-old and 2-day-old failed-fertilized oocytes. Two days after microinjection, 67% and 67% of the 2PN and 80% and 44% of the ;;;,3PN oocytes cleaved with <50% fragmentation in group ICSI-day 1 and in group ICSI-day 2, respectively. Conclusions: Late fertilization after initial in vitro insemination does not playa role in the high fertilization rate obtained after reinsemination of assumed failed-fertilized oocytes by ICSI. Normal (2PN) fertilization rate, however, decreases strongly and the abnormal (;;;,3PN) fertilization rate increases with oocyte aging and derived embryos seem to have a high incidence of cytogenetic abnormalities. Fertil SteriI1995;64: Key Words: Assisted fertilization, failed-fertilization, fertilization, ICSI, intracytoplasmic sperm injection, karyotyping, microinjection, reinsemination, unfertilized oocyte Fertilization failure of human oocytes may occur after standard IVF procedure. Incomplete or partial Received March 9, 1995; revised and accepted June 23, * Supported by grants by the Belgian Fund for Medical Research, Brussels, Belgium. t Presented in part at the Xth Annual Meeting of the European Society of Human Reproduction and Embryology, Brussels, Belgium, June 26 to 30, * Reprint requests: Zsolt P. Nagy, M.D., Centre for Reproductive Medicine, Vrije Universiteit Brussel (AZ-VUB), Laarbeeklaan 101, B-1090 Brussels, Belgium (FAX: ). failure of fertilization does not necessarily impair the chance of ET and pregnancy, but complete failure offertilization results in cancellation of ET. Reinsemination of unfertilized oocytes can be attempted to rescue the failed IVF cycle (1), but reinsemination of aged, failed-fertilized oocytes by standard methods usually leads to only very low fertilization rates (2, 3). Reinsemination by micromanipulation techniques such as partial zona dissection (4) or by subzonal insemination (5) result in slightly higher fertilization rates. Normal fertilization rates remained nevertheless relatively low and at the 1130 Nagy et al. Microinjection of failed-fertilized oocytes Fertility and Sterility

2 same time a high proportion of oocytes showed :2:3 pronuclear (PN) development as a sign of abnormal fertilization (4). As has been published already (6), reinsemination of 1-day-old failed-fertilized oocytes also has been carried out lately using the intracytoplasmic sperm injection (ICSI) technique, on the basis of its remarkably high success rate when applied to fresh oocytes (7) and also because only one sperm will enter the oocyte at the time of reinsemination, thus preventing the reported risk of high multiple-pronuclear fertilization. Although a high 2PN fertilization rate has been associated with ICSI on 1-day-old unfertilized oocytes, it was not possible to exclude the role of late fertilization after in vitro insemination in this case, especially in light of the observation that some of the injected presumed unfertilized 00- cytes cleaved as early as 16 to 18 hours after ICSI (6). In the first part of the present study, therefore, we aimed to evaluate the possible role of late fertilization by dividing all 1-day-old failed-fertilized 00- cytes from each patient into two groups, in which the oocytes of the first group were microinjected and the other group of oocytes were kept without any treatment. In the second part ofthe study, we aimed to examine the correlation between the effect of aging of the failed-fertilized oocytes and the ability of these oocytes to be fertilized. This was done by dividing all the unfertilized oocytes of each patient (second group of patients) into two groups, in which the oocytes of the first group were injected 1 day after IVF whereas the oocytes of the second group were microinjected 2 days after in vitro insemination. Some of the embryos with normal morphology obtained after ICSI of 1-day-old unfertilized oocytes were karyotyped to estimate the chromosomal status of these embryos. MATERIALS AND METHODS Experimental Design: First Trial In all, 175 metaphase II (MIl) failed-fertilized 00- cytes were collected from 15 patients the day after the in vitro insemination. Eight couples had andrological and seven couples had nonandrological indications for the IVF treatment. The fertilization rates in the IVF were between 0% and 56% for the 15 couples. From each patient, approximately half of the failed-fertilized oocytes were allocated to the treatment group (injected- day 1) whereas the other half of the oocytes were assigned to the not-injected (control) group. Oocytes in the treatment group (n = 93) were microinjected intracytoplasmically with a single spermatozoon approximately 23 to 30 hours after ovum pick-up. Oocytes of the control Vol. 64, No.6, December 1995 group (n = 82) were cultured without further intervention. Experimental Design: Second Trial From nine patients, 80 MIl failed-fertilized 00- cytes were collected. Four of nine couples had andrological and five had nonandrological indications for the IVF treatment. The lowest fertilization rate was 0% and the highest normal fertilization rate was 36% between these nine standard IVF treatment cycles. From each of these IVF cycles, half of the failedfertilized oocytes were allocated to the first treatment group (ICSI-day 1) whereas the other half were put into the second treatment group (ICSI-day 2). Oocytes (n = 40) in the first treatment group (ICSIday 1) were microinjected with a single sperm approximately 22 to 29 hours after the ovum pick-up. Oocytes (n = 40) of the second treatment group (ICSI-day 2) were microinjected approximately 48 to 56 hours after ovum pick-up. Ovarian Stimulation and In Vitro Insemination Ovarian stimulation was performed by a desensitizing protocol using the GnRH agonist buserelin acetate (Suprefact; Hoechst, Brussels, Belgium) in association with hmg (Humegon; Organon, Oss, The Netherlands; or Pergonal; Serono, Brussels, Belgium) and hcg (Pregnyl, Organon; or Profasi, Serono) (8). For luteal phase supplementation, intravaginally administered P (Utrogestan; Piette, Brussels, Belgium) was used as described previously (9). Oocyte retrieval was carried out by vaginal ultrasound-guided puncture 36 hours after hcg administration. The cumulus-corona-oocyte complexes were placed in 25 ILL of B2 (biomerieux, Montalieu Vercieu, France) droplets covered by lightweight paraffin oil (British Drughouse; Pasture, Brussels, Belgium). Semen was prepared either by swim-up migration method or by Percoll density gradient centrifugation. Approximately 4 hours after ovum pick-up, each oocyte was inseminated with 2,000 to 3,000 motile spermatozoa (80,000 to 120,000 motile spermatozoa per ml). After insemination, the tube containing the treated semen was gassed and stored at room temperature. Oocytes were evaluated for signs of fertilization approximately 18 hours after in vitro insemination. Oocytes with one polar body (complete or fragmented) but without any pronuclei were included in the study. Microinjection Procedure and Oocyte and Embryo Evaluation The details of microtool preparation and equipment used for the injection procedures have been Nagy et al. Microinjection of failed fertilized oocytes 1131

3 described previously (10). The ICSI procedure was carried out on the heated stage of an inverted microscope (Diaphot; Nikon Corporation, Tokyo, Japan) at x400 magnification (10). All oocytes of the treatment groups were injected with a single, living, immobilized spermatozoon (the same semen as used for IVF was used for microinjection, without any additional treatment. For the injection, the oocyte was held securely on the holding pipette in such a way that the polar body was situated at the 6 o'clock or at the 12 o'clock position while the injection pipette was pushed through the zona pellucida at the 3 o'clock position into the cytoplasm, where the sperm was delivered. After microinjection, the oocytes were washed and stored in 25-J.lL drops of B2 medium in a petri dish that was kept at 37 C in an incubator containing 5% CO 2, 5% O 2, and 90% N 2 Microinjected and control oocytes were examined approximately 16 to 18 hours after injection (40 to 44 or 64 to 68 hours after ovum pick-up depending on whether injection was performed on I-day-old or 2-day-old oocytes) for intactness and for the presence of a second polar body and for pronuclei formation (11). The cleavage of the normally (as judged from the presence of two polar bodies together with 2PN) and abnormally (2:3PN) fertilized oocytes was assessed at 40 to 44 hours after microinjection. At the same time, embryo quality also was evaluated on the basis of the relative proportion ofanucleate fragments present within the zona pellucida. The four quality categories were as follows: [1] excellent, no anucleate fragments present; [2] good, 1% to 20% of the embryo was fragmented; [3] fair, the relative extent of fragmentation was between 20% and 50%; and [4] poor, >50% of the embryo was fragmented. Some of the embryos (n = 35) derived from injected I-day-old failed-fertilized oocytes were submitted to cytogenetic analysis according to Tarkowski's protocol (12). Colcemid was added (1.2 mglml final con- Table 1 Developmental Status of Injected and Noninjected Failed-Fertilized Oocytes 16 Hours After Microinjection (2 Days After Unsuccessful IVF) Injected-day 1 Control Oocytes Intact* 85 (91) 82 statust Pronuclear OPN 10 (12) 79 (96) 2PN 45 (53) IPN 3 (4) 0 1 (1) 2::3PN 19 (22) 0 2::2 cell 5 (6) 0 Fragmented 0 2 (2) t Values in parentheses are percentages calculated on the intact oocytes Nagy et al. Microinjection of failed-fertilized oocytes Table 2 Developmental Status of Injected and Noninjected Failed-Fertilized Oocytes and Quality of Embryos Derived From Fertilized and Cleaved I-day-old Failed-Fertilized Oocytes 2 Days After ICSI (3 Days After Unsuccessful IVF) Developmental stage 1 cell 2 cell 3 to 4 cell >4 cell Fragmented Degenerated Embryo quality Excellent Good Fair Poor Injected-day 1 Injected-day 1 2PN oocytes 3PN oocytes Control 5 (11)* 11 (24) 16 (36) 8 (18) 3 (7) 2 (4) 2 (5) 19 (50) 11 (29) 6 (16) 2 (11) 5 (26) 7 (37) 5 (26) 1 (6) 8 (47) 5 (29) 3 (18) 71 (87) 2 (2) 3 (4) 6 (7) centration) to the culture medium of the embryos at the two- to eight-cell stage (2 days after microinjection). Eighteen to 20 hours later, the embryos were fixed (3:1, methanol:acetic acid), stained with 10% Giemsa solution, and examined later. Statistical analysis was performed to compare normal and abnormal fertilization rates between the groups ICSI-day 1 and ICSI-day 2 by means of the one-way analysis of variance (ANOYA) test. This test was performed at the 5% level of significance using the StatYiew package (Brain Power, Inc., Calabasas, CA) on a Macintosh personal computer (Apple Education Center, Brussels, Belgium). RESULTS Trial 1: Pronuclear Development and Cleavage of Microinjected Versus Noninjected I-day-old Failed-Fertilized Oocytes Sixteen to 18 hours after ICSI, 85 of 93 injected oocytes remained intact (Table 1). Forty-eight percent (n = 45) of the injected oocytes displayed 2PN as evidence of normal fertilization. Three or more pronuclei were observed in 19 oocytes and 5 oocytes already had cleaved to the two-cell stage, whereas only 3 oocytes were activated parthenogenetically, showing a single pronucleus. Seventy-nine of 82 control oocytes (without microinjection) did not display any pronucleus at 40 to 44 hours after the standard in vitro insemination (at the same time that the microinjected oocyte group was checked for fertilization). One oocyte was parthenogenetically activated and two oocytes were fragmented (Table 1) at the same observation time. One day later (40 to 44 hours after ICSI or 64 to 68 hours after standard IVF), 35 of 45 normally Fertility and Sterility

4 Table 3 Developmental Status of 1- and 2-day-old Injected Failed-Fertilized Oocytes 16 Hours Mter Microinjection (2 and 3 days Mter Unsuccessful IVF, Respectively) ICSI-day 1 ICSI-day 2 Oocytes Intact* 34 (85) 33 (83) Pronuclear statust OPN 1 (3) 11 (28) 1PN 2 (5) 1 (3) 2PN:j: 18 (45) 3 (8) 2:3PN 10 (25) 16 (40) 2:2 cell 2 (5) 2 (5) Fragmented 1 (3) t Values in parentheses are percentages calculated on the intact oocytes. :j: Significant difference (P < 0.01) between the groups by the one-way ANOVA test. fertilized oocytes (77.8%) cleaved to the two- to eightcell stage whereas the remaining 10 2PN oocytes either did not cleave (5 oocytes) or fragmented (3 oocytes) or degenerated (2 oocytes) (Table 2). Seventeen of 19 multiple pronucleated oocytes cleaved, whereas 2 oocytes remained at the one-cell stage. Eighty-four percent of the embryos derived from normally fertilized oocytes and 82% of the embryos of the cleaved?:3pn oocytes contained <50% anucleate fragments (Table 2). At the same observation time (64 to 68 hours after standard lvf), 71 of 82 control oocytes remained at the one-cell stage, 6 were fragmented, and 5 had cleaved to the two- to fourcell stage without displaying any pronucleus (Table 2). Trial 2: Pronuclear Development and Cleavage of I-day-old Versus 2-day-old Failed-Fertilized Oocytes After Microinjection Sixteen to 18 hours after microinjection, 34 of 40 I-day old and 33 of 40 2-day old injected oocytes survived (Table 3). Eighteen of 40 microinjected 1- day-old failed-fertilized oocytes displayed 2PN whereas 10 oocytes showed?:3pn. On the other hand, 3 of 40 2-day-old oocytes showed 2PN (which was a significantly lower proportion than in the 1- day-old oocyte group by the one-way ANOVA test; P < 0.01), and 16 oocytes displayed?:3pn (Table 3). Fifteen of 18 normally fertilized I-day-old oocytes cleaved to the two- to eight-cell stage, whereas 9 of 10 multiple pronucleated oocytes did so 16 to 18 hours after microinjection. Of the 2-day-old oocytes, 2 of 3 normally fertilized and 12 of 16 abnormally fertilized oocytes cleaved (Table 4). Twelve of 17 embryos derived from normally fertilized and 8 of 10 abnormally fertilized (l-day-old injected) oocytes had <50% fragmentation (Table 4) as Vol. 64, No.6, December 1995 observed at 40 to 44 hours after les!. Both embryos obtained from normally fertilized oocytes and 7 of 12 abnormally fertilized (2-day-old injected) oocytes had <50% fragmentation (Table 4) 2 days after microinjection (4 days after the unsuccessful lvf). Cytogenetic Analysis of Embryos Ten readable karyotypes were obtained after examining 35 fixed embryos. Five embryos contained normal diploid karyotypes whereas the other five embryos showed abnormal karyotypes. The abnormal karyotypes included haploid, triploid, and aneuploid chromosome constitutions. DISCUSSION One of the primary aims ofthis study was to investigate whether 2PN development 1 day after microinjection performed on lvf failed-fertilized oocytes is indeed the result of lesl and is not due to late fertilization after lvf. Therefore, half of the oocytes from each patient were microinjected while the other half of unfertilized oocytes were incubated without any treatment. Degeneration rate of the injected 00- cytes in this trial was the same as earlier (6) and comparable to the degeneration rate after lesl performed on fresh oocytes (7). Two-pronuclear development occurred only in the cohort of failed-fertilized oocytes undergoing reinsemination by lesl (45/93 oocytes injected) whereas none of the 82 control oocytes showed?:2pn; late fertilization therefore is assumed to play no part here. On the one hand, this proves that our criteria for selection of failed-fertilized oocytes was strict enough and, on the other hand, it also proves that Table 4 Developmental Status and Quality of Embryos Derived From Fertilized and Cleaved 1- and 2-day-old Failed-Fertilized Oocytes 2 Days After ICSI (3 and 4 Days Mter Unsuccessful IVF, Respectively) ICSI-day 1 ICSI-day 2 2PN 3PN 2PN 3PN oocytes oocytes oocytes oocytes Developmental stage 1 cell 1 (6)* 1 (33) 4 (25) 2 cells 5 (28) 4 (40) 4 (25) 3 to 4 cells 9 (50) 2 (20) 1 (33) 4 (25) 2:5 cells 1 (6) 3 (30) 1 (33) Fragmented 2(11) 1 (10) 4 (25) Embryo quality Excellent 2 (12) 2 (20) 1 (8) Good 6 (35) 6 (60) 1 (50) 5 (42) Fair 4 (24) 1 (50) 1 (8) Poor 5 (29) 2 (20) 5 (42) Nagy et ai. Microinjection of failed-fertilized oocytes 1133

5 ~~-----'--'-----' I,. normal (as well abnormal) pronuclear development of the unfertilized oocytes (post-ivf) resulted exclusively from ICS!. Two, or more than two, pronuclei were observed only in the microinjected group and this proportion was similar to that reported earlier (6). Parthenogenetic activation of oocytes as shown by the development of a single pronucleus occurred in a low proportion both in the microinjected group (4%) and in the control group (1%), at a level comparable to that observed in freshly injected oocytes (7). Interestingly, 5 of 93 oocytes injected (6%) were at the two- to four-cell stage on the day after injection, whereas none of the 82 control oocytes cleaved (only 2 of them were fragmented completely) by the same time. In our previous study, there were 8 of cytes injected that cleaved 1 day after ICSI (6). At that time, we hypothesized that these eight oocytes were fertilized (or activated) as a result of in vitro insemination and not as a result of ICSI, but that pronuclear development had been missed. However, the results of the first trial suggest that cleavage of I-day-old oocytes might occur without any fertilization only as a result of mechanical stimulation by means of micro injection, which virtually is never observed in freshly injected oocytes. This hypothesis only can be confirmed by performing nuclear and! or cytogenetic analysis of these cleaved oocytes. A majority of the normally, as well as of the abnormally, fertilized oocytes cleaved to the two- to eightcell stage 2 days after ICS!. Six percent of the control oocytes were also at the two- to four-cell stage, indicating that cleavage of unfertilized, noninjected 00- cytes also can occur but it is delayed by 1 day in comparison to the injected I-day-old unfertilized 00- cytes. A majority ofthe cleaved, normally and abnormally fertilized oocytes (84% and 82%, respectively) had <50% fragmentation, which is in the same range as was observed earlier (6). In the second trial, the survival rates of injected oocytes were similar in both groups (85% and 83% in ICSI-day 1 and ICSI-day 2, respectively). Important differences were noted in the two- and multiple-pronuclear rates between the two groups (ICSI-day 1 versus ICSI-day 2). Similar to the results of the first trial, a high (45%) normal fertilization was obtained after injection of I-day-old failed-fertilized oocytes, whereas only a very low (8%) 2PN rate was achieved after ICSI of 2-day-old unfertilized oocytes (P < 0.01). This result suggests that the possibility of normal fertilization is correlated with the aging of a MIl oocyte. It appears that the usual 2PN fertilization rate after ICSI on fresh oocytes is approximately 70% (7); on I-day-old (failed-fertilized) oocytes it decreases to 40% to 50% (6), whereas on 2-day-old (failed-fertilized) oocytes it drops to < 10%, which indicates that, the older the oocyte is, the lower the chance of the normal fertilization. On the other hand, :2::3PN fertilization rate is approximately 5% in freshly injected oocytes (7), 20% to 25% in I-dayold oocytes (6), and 40% in 2-day-old unfertilized oocytes. These results suggest that, although the chance of normal (2PN) fertilization strongly decreases with aging, at the same time the likelihood of abnormal fertilization (:2::3PN) sharply increases. Ifwe assume that the reason for the multiple pronucleation of 1- and 2-day-old oocytes after ICSI is the same as for freshly injected oocytes (Flaherty SP, Payne D, Swann NJ, Matthews CD, abstract), then this means that aging affects primarily the structures involved in the extrusion of the second polar body (such as the meiotic spindle and microfilaments). Other physiological processes involved in fertilization seem to be less affected, as shown by the relatively high total fertilization rate of the 2- day-old oocytes. The cleavage and quality of the embryos in the group ICSI-day 1 derived from the normally and abnormally fertilized oocytes were in the same range as in the first trial. No conclusion can be drawn about the developmental capacity of the 2PN 2-dayold oocytes because there were only three of them. The cleavage potential and quality of embryos derived from the abnormally fertilized (:2::3PN) 2-dayold oocytes seem to be somewhat impaired in comparison to embryos derived from abnormally fertilized I-day-old oocytes. The efficiency of the karyotyping seems to be lower (10 readable karyotypes obtained after fixing 35 embryos) than in some other studies (13-15). This might be explained by technical aspects, but it also is possible that embryos derived from microinjected I-day-old failed-fertilized oocytes have lower developmental potential beyond day 2 (co Ice mid was added to these embryos on the 2nd day after ICS!) and, therefore, it was not possible to obtain more karyotypes. Ifthis latter assumption is correct, then colcemid added 1 day after microinjection (at the end of the day) might help to increase the proportion of readable karyotypes. Although only 10 readable karyotypes were obtained, it is noteworthy that half of them were abnormal (involving different kinds of aberrations). This number is too small to allow definitive conclusion(s), although it appears that this proportion of abnormality is higher than the proportion of cytogenetic abnormalities reported on fresh fertilized oocytes, especially if we take into account that in our study most of the embryos were of good quality (14). This high frequency of chromosomal anomaly also might explain why some groups obtained virtually no pregnancies after transferring these embryos (2, 16). For this reason, it is probably worthwhile to karyotype a large series of embryos 1134 Nagy et al. Microinjectian of failed-fertilized aacytes Fertility and Sterility

6 , obtained after reinsemination of failed-fertilized 00- cytes, before the transfer of these embryos becomes a routine clinical procedure. In conclusion, a relatively high normal fertilization rate can be achieved after IeSI of 1-day-old IVF failed-fertilized oocytes, while steps were taken to ensure that this observed rate was solely the result of microinjection and not of late fertilization after the initial in vitro insemination. It appears that, with oocyte aging, the normal (2PN) fertilization rate strongly decreases whereas the abnormal (:==:3PN) fertilization rate increases. The preliminary data on karyotypes of microinjected, 1-day-old failed-fertilized oocytes show a high incidence of cytogenetic abnormalities in the derived embryos. Because of the few karyotypes analyzed, an extended cytogenetic study is needed before the transfer of such embryos can be considered. Acknowledgments. The authors thank the clinical, scientific, nursing, and technical staff of the Centre for Reproductive Medicine and especially the colleagues of the microinjection and IVF laboratory. We are very grateful to Mr. Frank Winter ofthe Language Education Centre of our University for correcting the manuscript. REFERENCES 1. Ben-Rafael Z, Kopf GS, Blasco L, Tureck RW, Mastroianni L Jr. Fertilization and cleavage after reinsemination of human oocytes in vitro. Fertil Steril 1986;45: Trounson A, Webb J. Fertilization of human oocytes following reinsemination in vitro. Fertil Steril 1984;41: Pampiglione JS, Mills C, Campbell S, Steer C, Kingsland C, Mason BA. The clinical outcome of reinsemination of human oocytes fertilized in vitro. Fertil Steril1990;53: Malter H, Thalansky B, Gordon J, Cohen J. Monospermy and polyspermy after partial zona dissection of reins emina ted human oocytes. Gamete Res 1989;23: Imoedemhe DAG, Sigue AB. The influence of subzonal mi- croinsemination of oocytes failing to fertilize in scheduled routine in-vitro fertilization cycles. Hum Reprod 1994; 9: Nagy ZP, Joris H, Liu J, Staessen C, Devroey P, Van Steirteghem AC. Intracytoplasmic single sperm injection of 1- day-old unfertilized human oocytes. Hum Reprod 1993; 8: Van Steirteghem AC, Nagy Z, Liu J, Joris H, Smitz J, Camus J, Devroey P. Intracytoplasmic sperm injection - ICSI. Reprod Med Rev 1994;3: Smitz J, Devroey P, Camus M, Deschacht J, Khan I, Staessen C, et al. The luteal phase and early pregnancy after combined GnRH-agonistlHMG treatment for superovulation in IVF or GIFT. Hum Reprod 1988;3: Smitz J, Devroey P, Faguer B, Bourgain C, Camus M, Van Steirteghem AC. A prospective randomized comparison of intramuscular of intravaginal natural progesterone as a luteal phase and early pregnancy supplement. Hum Reprod 1992; 7: Van Steirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, et al. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1993;8: Nagy ZP, Liu J, Joris H, Devroey P, Van Steirteghem AC. Time course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection. Hum Reprod 1994;9: Tarkowski AK. An air-drying method for chromosome preparation from mouse eggs. Cytogenetics 1966;5: Papadopoulos G, Templeton AA, Fisk N, Randall J. The frequency of chromosome anomalies in human preimplantation embryos after in-vitro fertilization. Hum Reprod 1989;4: Pellestor F, Girardet A, Andreo B, Arnal F, Humeau C. Relationship between morphology and chromosomal constitution in human preimplantation embryo. Mol Reprod Dev 1994; 39: Plachot M, Mandelbaum J, Junca A, de Grouchy J, Salat Baroux J, Cohen J. Cytogenetic analysis and developmental capacity of normal and abnormal embryos after IVF. Hum Reprod 1989;4: Fahmy NW, Benoit J, Bissonnette F, Duchesne C, Girard Y, Sullivan R. Impact of a second insemination on the results of an in vitro fertilization-embryo transfer CIVF-ET) program. J In Vitro Fert Embryo Transf 1991;8:80-3. Vol. 64, No.6, December 1995 Nagy et al. Microinjection of failed-fertilized oocytes 1135