Fertilization abnormalities and pronucleus size asynchrony after intracytoplasmic sperm injection are related to oocyte postmaturity

Transcription

1 FERTILITY AND STERILITY VOL. 72, NO. 2, AUGUST 1999 Copyright 1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Fertilization abnormalities and pronucleus size asynchrony after intracytoplasmic sperm injection are related to oocyte postmaturity Pravin Goud, M.D.,* Anuradha Goud, Ph.D.,* Patrick Van Oostveldt, Ph.D., Josiane Van der Elst, Ph.D.,* and Marc Dhont, M.D., Ph.D.* University Hospital, and University of Ghent, Ghent, Belgium Received December 16, 1998; revised and accepted March 17, Supported by the University of Ghent, Ghent, Belgium. Presented in part at the 14th Annual Meeting of the European Society of Human Reproduction and Embryology, Göteborg, Sweden, June 21 24, Reprint requests: Pravin Goud, M.D., Infertility Centre, Department of Obstetrics and Gynecology, University Hospital, De Pintelaan 185, Ghent, 9000, Belgium (FAX: ; * Infertility Centre, Department of Obstetrics and Gynecology, University Hospital. Laboratory of Biochemistry and Molecular Cytology, Faculty of Agricultural and Applied Biological Sciences, University of Ghent /99/$20.00 PII S (99) Objective: To study the effect of delayed intracytoplasmic sperm injection (ICSI) on the fertilization and cleavage of human in vitro matured (IVM) oocytes. Design: Prospective experimental study. Setting: Academic hospital based fertility center. Patient(s): The experimental group consisted of 73 spare germinal vesicle stage oocytes from 25 patients. The control group consisted of sibling in vivo matured oocytes from the same patients that were subjected to ICSI in the clinical program. Intervention(s): Equal numbers of sibling IVM oocytes were subjected to ICSI either soon after maturation (30 hours, group 1) or after a 6-hour delay (36 hours, group 2). In a subsequent set of experiments, spermatozoa were labeled with a fluorescent mitochondria specific vital dye and injected into 17 IVM oocytes that were intentionally aged by 6 8 hours. The resultant zygotes were fixed. Main Outcome Measure(s): Incidence of fertilization and cleavage, numbers and mean diameters of pronuclei, and incidence of zygotes with significant pronucleus size asynchrony. Identification of the male pronucleus by its proximity to the fluorescent sperm midpiece remnant. Result(s): Group 2 had significantly lower rates of normal fertilization (60%) than the control group (82.9%) and significantly lower cleavage rates (46.7%) than both group 1 (85%) and the control group (98.1%). The incidence of oocytes that developed one pronucleus and pronucleus size asynchrony was significantly higher in group 2 (32% and 40%, respectively) than in group 1 (4% and 5%, respectively) and in the control group (4.1% and 4.4%, respectively). All the zygotes with significant pronucleus size asynchrony that developed after delayed ICSI with labeled spermatozoa showed proximity of the fluorescent sperm midpiece remnant to the smaller pronucleus. Conclusion(s): For IVM oocytes, the incidence of one pronucleus, pronucleus size asynchrony (possibly related to a smaller male pronucleus), and cleavage failure increase when ICSI is delayed after maturation. Thus, the timing of ICSI is critical for optimum fertilization of IVM oocytes. (Fertil Steril 1999;72: by American Society for Reproductive Medicine.) Key Words: Fertilization abnormalities, ICSI, in vitro oocyte maturation, postmaturation aging, pronucleus size asynchrony Intracytoplasmic sperm injection (ICSI) is an effective tool for overcoming fertilization failure related to sperm factors (1). Moreover, because of its ability to counter fertilization failure even in dysmorphic oocytes (2, 3), ICSI is used as a method for optimizing fertilization rates in general. It has been suggested that ICSI also may improve fertilization rates under special circumstances, such as in in vitro matured (IVM) or cryopreserved and thawed oocytes (4, 5). However, despite the significant impact of ICSI on fertilization, a small but notable fraction of oocytes remain unfertilized or undergo abnormal fertilization after ICSI. The latter is characterized by the presence of either one or more than two (usually three) pronuclei (6). Even among the zygotes that display two pronuclei (2PN) at fertilization, one of the pro- 245

2 nuclei may be markedly smaller than the other, and such zygotes may have impaired potential for further development (7, 8). The cause of fertilization failure after ICSI may be related to ooplasmic immaturity or suboptimal activation capacity of the spermatozoa, both of which result in failure of oocyte activation (9). The exact cause of abnormal fertilization after ICSI, however, is not known. It is generally presumed that the formation of one pronucleus (1PN) after ICSI is due to parthenogenetic oocyte activation and the formation of three pronuclei (3PN) is related to nonextrusion of the second polar body (PB) (6, 9, 10). This is unlike the situation after IVF, where polyspermy is a more common phenomenon related to the formation of multiple pronuclei, and a significant fraction of the 1PN zygotes may actually be diploid (11). Thus, the cause of abnormal fertilization after ICSI may be somewhat different than that after IVF. The parthenogenetic activation seen after ICSI may occur either spontaneously or secondary to the injection procedure. Spontaneous parthenogenetic activation (SPA) generally occurs secondary to postovulatory or postmaturation aging, and injection-related parthenogenetic activation (IRPA) is related to the calcium content of the injection medium as well as the postovulatory age of the oocyte (12, 13). The occurrence of either phenomenon results in gross asynchrony between the fully developed female pronucleus and an almost intact or slightly swollen sperm nucleus (12). These phenomena are documented in golden hamster oocytes, which are particularly sensitive to SPA and IRPA. However, the formation of only female pronuclei without male pronuclei also is observed in some human oocytes subjected to ICSI, although with a relatively lower frequency (6, 14). Thus, SPA and/or IRPA also may occur in some human oocytes; this may be more likely if the oocytes are postmature. The occurrence of SPA and IRPA may be related to the temporal window for optimum fertilization, which is crucial for clinical IVF/ICSI (13, 15). Clinical studies on the influence of the timing of ICSI on its outcome have produced conflicting results (16, 17). However, this could be related to the limitations of the retrospective design of these studies. Further, there are no reports on the influence of the timing of fertilization on the outcome of IVM human oocytes; timing may be particularly crucial for IVM oocytes, which mature at variable intervals in vitro (4). In this study, we used spare germinal vesicle (GV)-stage oocytes donated by patients undergoing ICSI. We subjected them to ICSI after maturation for two different periods in culture. The aim was to investigate the temporal window for optimum fertilization in these oocytes. Apart from the routine parameters judged at fertilization, we also evaluated the mean pronuclear diameters in the zygotes to determine the incidence of pronucleus size asynchrony in different groups. The incidence of abnormal fertilization and pronucleus size asynchrony was higher in the oocytes that were injected only 6 hours later than their siblings that were injected around the time of optimum maturation. MATERIALS AND METHODS Study Design The in vitro maturation of spare GV-stage oocytes donated by patients undergoing ICSI cycles and their fertilization for experimental purposes was approved by the institutional ethical review committee. The GV-stage oocytes were cultured in a maturation medium (18) and their sibling oocytes that matured to the metaphase II stage after 30 hours of culture were assigned to undergo ICSI either immediately (30 hours, group 1) or 6 hours later (36 hours, group 2). The sibling in vivo matured oocytes that were subjected to ICSI in the clinical program formed the control group. The number of pronuclei and PBs and the mean diameters of the pronuclei were determined at the fertilization check hours later. The number of zygotes that had a significant difference between the diameters of their pronuclei and the number of zygotes that had an abnormal number of pronuclei were compared between the sibling IVM and in vivo matured oocyte groups. The zygotes from groups 1 and 2 were cultured for another 24 hours and the incidence of cleavage was compared. A second set of experiments was performed to identify the cause of the pronucleus size asynchrony. Spare GV-stage oocytes were intentionally matured for 6 8 hours longer and injected with donor spermatozoa prelabeled with a fluorescent mitochondria specific dye. The male pronuclei were identified by their proximity to the mitochondria-rich sperm midpiece remnants (MPRs). Source of Gametes, Oocyte Maturation, and ICSI The GV-stage status of the donated oocytes was confirmed by two independent observers. The morphology of the oocytes was checked, and only those oocytes that had at least one more sibling GV-stage oocyte and were free of gross cytoplasmic abnormalities were considered for the study. Thus, sibling GV-stage oocytes used for the study were similar in their morphologic features (e.g., size, cumulus cell status, absence of cytoplasmic abnormalities). These oocytes were subjected to culture in a maturation medium (M-199; GIBCO BRL, N.V. Life Technologies, Brussels, Belgium) supplemented with human serum albumin (Albumine 20%; Belgian Red Cross, Brussels, Belgium), gonadotropins (pure FSH: Metrodin HP, and hcg: Profasi; Serono, Brussels, Belgium), 17 -E 2, and epidermal growth factor (Sigma, St. Louis, MO) as described previously (18). All the oocytes were covered with at least 2 3 layers of cumulus corona cells, and in no case was it necessary to remove these cells to confirm the presence of the GV. Care was taken to ensure that the sibling oocytes assigned to 246 Goud et al. Oocyte postmaturity and ICSI Vol. 72, No. 2, August 1999

3 culture had no gross dissimilarities in their morphology (e.g., diameter, shape, cytoplasmic characteristics) or the status of their cumulus cells. Seventy-three GV-stage oocytes from 25 patients were assigned to culture. Those that matured to the metaphase II stage at 30 hours of culture were denuded of their cumulus corona cells and examined for gross abnormalities in the cytoplasm (e.g., vacuoles, central organelle clustering, excessively large perivitelline space, large inclusions). Only the sibling oocytes that matured to the metaphase II stage without cytoplasmic abnormalities were subjected to ICSI either immediately (30 hours, group 1) or after another 6 hours of culture (36 hours, group 2). They were cultured in human tubal fluid medium (Irvine Scientific, Tech Gen International, NV/SA, Brussels, Belgium) supplemented with 0.4% wt/vol human serum albumin. The outcome of ICSI was evaluated after hours and hours of culture, respectively. The data on the in vivo matured sibling oocytes undergoing ICSI was recorded at the same intervals and used for comparison. The spermatozoa used for ICSI in groups 1 and 2 were obtained from consenting fertile donors and those used in the control oocytes were obtained from their partners. The preparation of spermatozoa and performance of ICSI was done using methods described previously (19, 20). Fertilization Check and Embryo Culture A fertilization check was performed hours after ICSI in all the groups. At the fertilization check, the presence and number of pronuclei and PBs was noted and the diameters of the zygotes and pronuclei were measured in three perpendicular planes with the help of an ocular grid at 400 magnification. The mean diameter of each pronucleus was calculated for the zygotes in groups 1 and 2 and the control group. The zygotes were cultured further for an additional 24 hours and examined for the occurrence of cleavage. Supplementary Experiments In a subsequent set of experiments, 22 spare GV-stage oocytes from the other 10 consenting patients were subjected to maturation using the same technique (18). The oocytes that matured to the metaphase II stage at 30 hours of culture were subjected to an additional culture period of 6 8 hours and then underwent ICSI with spermatozoa labeled with a fluorescent dye to stain the mitochondria (21). This vital dye is known to bind specifically to the metabolically active mitochondria within the spermatozoa without hampering their fertilizing capability, and the mitochondria-rich MPR of the fertilizing spermatozoa can be detected in the zygotes in proximity to the male pronucleus (22). In brief, liquefied donor semen was mixed with human tubal fluid containing 0.4% human serum albumin and centrifuged at 400 g over Percoll gradients to obtain a motile fraction. The motile spermatozoa were washed and resuspended, then incubated at 37 C for 15 minutes in human tubal fluid containing 400 nm of Mitotracker Green FM (Molecular Probes, Leiden, the Netherlands). The spermatozoa then were washed twice by centrifugation at 500 g to remove the excess Mitotracker dye and prepared for ICSI. The oocytes were injected initially with motile prelabeled spermatozoa and then were examined 18 hours after ICSI to determine both the number and the diameters of the pronuclei. All the pronuclear zygotes were attached to coverslips coated with poly-l-lysine (Sigma), fixed in 4% paraformaldehyde for 45 minutes, washed in phosphate-buffered saline containing 0.2% (wt/vol) bovine serum albumin (Sigma) and 0.05% Triton X-100 (Sigma), and mounted in Vectashield containing propidium iodide (Vector Laboratories Inc., Burlingame, CA). The specimens were examined under a confocal laser scanning microscope (1024; Bio Rad, Eke, Belgium). Optical sections were taken at 1 2 M to determine the proximity of the sperm MPR to the pronuclei. Statistical Methods The data were analyzed using SPSS software (version 7.5; SPSS Inc., Chicago, IL). The incidence of fertilization and cleavage among the oocytes subjected to ICSI was compared between the groups using Fisher s exact test. The diameters of the larger and smaller pronuclei as well as the differences in the diameters were compared with their corresponding values between the groups using the Mann-Whitney U test. The 2PN zygotes that had a difference in the diameters of their pronuclei that exceeded 2 SD of that of the control group were considered to have pronucleus size asynchrony, and the numbers of such zygotes were compared among all the groups using Fisher s exact test. P.05 was defined as statistically significant. RESULTS Normal and Abnormal Fertilization and Cleavage Of the 73 oocytes obtained from 25 patients that were used for the study, 61 oocytes progressed to the metaphase II stage by 30 hours. Of these 61 oocytes, 3 oocytes were discarded because of an abnormal cytoplasmic appearance and 4 were removed from the study because their sibling oocytes failed to reach the metaphase II stage by 30 hours. Finally, 54 oocytes from 20 patients were randomized equally in sibling groups to undergo ICSI at 30 hours (group 1) or 36 hours (group 2). The mean ( SD) age of the patients was years. The indication for ICSI in all cases was male factor infertility. A total of 262 metaphase II stage oocytes from these patients were subjected to ICSI in the routine program (control in vivo matured oocytes). The fertilization and cleavage outcomes in groups 1 and 2 and in the control group are shown in Table 1. The incidence of 2PN zygotes was significantly lower in group 2 compared with the control group, and the incidence of 1PN zygotes was significantly FERTILITY & STERILITY 247

4 TABLE 1 Influence of postmaturation delay in performing intracytoplasmic sperm injection on fertilization and first cleavage division. Variable In vivo matured control oocytes Group 1 Group 2 P value No. of oocytes injected No. of oocytes that survived No. of 2PN zygotes* 205 (82.9) 20 (80) 15 (60).02 No. of 1PN zygotes* 10 (4.1) 1 (4) 8 (32) No. of zygotes that underwent cleavage 201 (98.1) 17 (85) 7 (46.7) No. of zygotes with gross pronuclear size asynchrony 9 (4.4) 1 (5) 6 (40) Note: PN pronuclei. * Values in parentheses indicate percentages of oocytes that survived injection. Control group vs. group 2. Group 1 vs. group 2. Values in parentheses indicate percentages among zygotes with two pronuclei. higher in group 2 compared with both group 1 and the control group. Among the 2PN zygotes, the incidence of cleavage was lower in group 2 compared with group 1 and with the control group (Table 1). Pronucleus Size Asynchrony In the control group and group 1, the difference in the diameters of the pronuclei was only slight (mean SD, m and m, respectively) (Figs. 1 and 2). However, in group 2, the incidence of zygotes displaying pronucleus size asynchrony (a difference of 3.5 m) was significantly higher compared with the other groups (Table 1). Similarly, the difference between the diameters of the pronuclei was significantly higher in group 2 compared with the control group, whereas the difference in group 1 was similar to that in the control group (Fig. 2). When arranged serially as larger and smaller pronucleus diameters in each zygote, the diameters of the larger pronuclei were statistically similar in all the groups. However, the diameter of the smaller pronuclei in group 2 was significantly smaller than that in the control group (Fig. 3), which was not the case in group 1. Thus, pronucleus size asynchrony in group 2 was due to the smaller size of one of the pronuclei, and the incidence of such zygotes was significantly higher in group 2 compared with both the other groups. Origin of the PN Size Asynchrony After we determined that the pronucleus size asynchrony resulted from the smaller size of one of the pronuclei and that the incidence of this phenomenon was increased in postmature oocytes, we performed a second series of experiments to determine whether the smaller pronucleus was male or female. Of the 22 GV-stage oocytes used for these experiments, 17 were found to be at the metaphase II stage at 30 hours and were subjected to ICSI after an intentional delay of 6 8 hours. Of these 17 oocytes, 10 underwent activation, as evidenced by pronucleus development and/or second PB extrusion. Of these 10 zygotes, 9 were 2PN and had two PBs, and 3 of these displayed a statistically significant difference in their mean diameters. The other activated oocyte had 2PN and one PB and an undecondensed sperm head (Fig. 4). Among the other injected oocytes, 2 were damaged and 5 remained unfertilized. The sperm MPR was found in proximity to one of the two pronuclei in 9 of the 10 2PN zygotes. The other zygote was 2PN and had one PB, and the labeled spermatozoon was found to be almost intact (Fig. 4). In 4 of 6 zygotes, the male pronucleus appeared slightly larger than the female pronucleus, but there was no pronucleus size asynchrony (Fig. 4). In the other 2 zygotes and in the 1 2PN zygote with intact sperm, the diameters were almost equal. In 3 zygotes, significant pronucleus size asynchrony was noted. In all 3 of these zygotes, the smaller pronucleus was close to the sperm MPR, indicating that it was the male pronucleus (Fig. 4). In three of five unfertilized oocytes that were fixed, the sperm nucleus was only slightly swollen and the fluorescently labeled sperm MPR was clearly in proximity to the sperm heads. DISCUSSION The fertilizable life of mammalian oocytes is limited to a short period after ovulation, referred to as the temporal window for optimum fertilization (15, 23, 24). Oocytes that fail to encounter spermatozoa during this period may undergo certain changes referred to as postovulatory or postmaturation aging (25, 26). Some structural and functional aspects of postmaturation aging are related to hardening of the zona pellucida and increased oocyte sensitivity to activation stimuli (27 29). These changes in the oocyte may preclude sperm entry (28) or predispose the oocytes to undergo parthenogenetic activation without forming the male pronucleus (13, 30). The fertilization of postmature oocytes may account for the failure of embryo development in vitro as well as in vivo (31, 32). However, the exact mechanism leading to the latter phenomenon remains to be investigated. The technique of in vitro oocyte maturation recently has been applied successfully to clinical cases (4, 33, 34). However, the level of success with IVM is low compared with that of conventional gonadotropin-stimulated cycles. Thus, for wider clinical application, the technique of IVM needs to be improved. 248 Goud et al. Oocyte postmaturity and ICSI Vol. 72, No. 2, August 1999

5 FIG. 1 Zygotes obtained after intracytoplasmic sperm injection of in vitro matured oocytes. (A), A zygote with two pronuclei with similar diameters. (B to D), zygotes with significant pronucleus size asynchrony. Original magnification, 320. Bar, 50 m. Determining the occurrence as well as the timing of postmaturation is crucial for clinical in vitro fertilization so that insemination can be scheduled for the optimum time to produce the best results (15). With ICSI, the issue of timing is even more relevant because precise timing of the sperm s introduction into the oocyte is under manual control. This concept also could be applied to IVM oocytes to achieve optimum fertilization. In the present study, the higher incidence of oocytes that developed one pronucleus and the lower incidence of oocytes that developed two pronuclei are similar to our previous observations in a heterospecific ICSI model (12, 13). Thus, even in human IVM oocytes, the incidence of oocyte activation sensitivity and 1PN formation increase during the postmaturation period. We also found that postmaturation aging caused an increase in the number of zygotes with pronucleus size asynchrony. Our supplementary experiments indicate that the smaller of the pronuclei that formed after delayed ICSI was the male pronucleus. Thus, in IVM oocytes, a delay in performing ICSI increases the risk of absence or attenuation of the male pronucleus. Fertilization can be viewed as a phenomenon that remodels the markedly differing chromatin of the male and female gametes to reprogram both the genomes to the same state to generate the same structural and functional qualities in the two chromosome sets. Decondensation of the highly compact chromatin of the sperm nuclei is a major step in this process. The process is characterized by reduction of the disulfide bonds and replacement of protamines with histones under the influence of the decondensing factors of the oocyte (35). As a result, a massive increase in the volume of the FERTILITY & STERILITY 249

6 FIG. 2 A box and whiskers plot displaying the differences in the diameters of the two pronuclei in the 2PN zygotes of all the groups. The horizontal lines within the boxes represent medians and the upper and lower borders of the boxes represent 75th and 25th percentiles, respectively. The horizontal lines above and below the boxes represent the largest and smallest values, respectively, that were not outliers. represent outliers. P.001. the injection procedure could result in oocyte activation and female pronucleus formation, and the precipitous entry of these oocytes into the interphase may not allow for adequate decondensation of the sperm nuclei (12, 13). Another interesting finding in the present study was a decrease in the incidence of zygotes that underwent the first cleavage division among the relatively postmature oocytes. This may be due either to alterations in the cell-cycle factor activities in such zygotes or to the detrimental influence of postmaturation aging on the cellular metabolism. Postmaturation recently has been shown to induce a dysregulation of intracellular Ca 2 homeostasis (39). This may lead to an increase in the basal cytosolic Ca 2 level within the oocyte and perhaps even the zygote, which may adversely affect cleavage divisions in the embryo (40). Another explanation could be related to the generation of free oxygen radicals in the oocytes after maturation (41). Our study showed that fertilization of the postmature oocytes leads to a considerable difference between the status of the male and female chromatin. Attention recently has been drawn to the role played by the dynamic changes in the chromatin structure in regulating the access and three-dimensional juxtaposition of transcriptional activators and the transcription machinery with the gene regulatory elements (42). Optimum decondensation of the pronuclei therefore may be vital for normal embryogenesis to occur. However, it needs to be determined whether suboptimal decondensation of the sperm nuclei occurs. Finally, the initially diverse-appearing sperm nuclei and the oocyte chromosomes change into similar-looking pronuclei after fertilization. The subnormal size of the male pronucleus in the relatively postmature oocytes in our study may be due to an aberration in the process of chromatin decondensation. Therefore, incomplete decondensation of the sperm chromatin and failed formation or smaller size of the male pronucleus in postmature oocytes may be related to subnormal activity of the decondensing factor of the oocyte. This activity of the oocyte recently has been connected with the level of glutathione, which is acquired during maturation (36, 37). Insufficient uptake of glutathione or its depletion impairs the development of the male pronucleus (22). This could occur during IVM and postmaturation aging. Another explanation for the failed or suboptimal development of the male pronucleus could be a shorter duration of the decondensation factor activity after activation in postmature oocytes. The decondensation factor activity of the oocytes is known to be related to the cell cycle and disappears during the interphase (38). Postmature oocytes are in a dynamic state that is poised for entry into the interphase, which is characterized by altered activity of the cell-cycle factors (29). Therefore, even minor activation stimuli such as FIG. 3 A box and whiskers plot displaying the diameters of the smaller pronuclei of the 2PN zygotes in all the groups. represent outliers and represent extremes. For more details, refer to the legend for Fig. 2. P Goud et al. Oocyte postmaturity and ICSI Vol. 72, No. 2, August 1999

7 FIG. 4 Injection of spermatozoa prelabeled with Mitotracker FM dye into in vitro matured oocytes that were intentionally aged for at least 6 8 hours after maturation. The images are optical sections obtained with a confocal laser scanning microscope. (A and B), Optical sections of the same zygote showing the female and male pronuclei, respectively. The fluorescent sperm midpiece remnant is indicated by a small arrow in (B). (C), A zygote with a significant size asynchrony between its pronuclei. The smaller male pronucleus can be identified from its proximity to the fluorescent sperm midpiece remnant (small arrowhead). The apparently smaller oocyte diameter in (C) is due to the peripheral location of the pronuclei. (D), A magnified image of an intact spermatozoon within an oocyte displaying only the female pronuclei. Bright labeling of the sperm midpiece and tail are clearly evident (small arrowheads). Original magnification, 600. Bars: (A to C), 50 m. (D), 5 m. pronuclei is related directly to the abnormalities that occur during later stages of embryo development. We found that GV-stage oocytes became susceptible to postmaturation after only 36 hours of culture. This was because most of the oocytes matured at 30 hours of culture, and postmaturation changes were induced by 6 hours after maturation. This interval may be shorter than that of in vivo matured oocytes (17). Therefore, the incidence of 1PN oocytes was lower in the in vivo sibling oocytes that were subjected to ICSI within 6 hours after oocyte retrieval. In vitro matured oocytes could be more susceptible to postmaturation aging than their in vivo matured counterparts. Completion of maturation in the oocytes in our study occurred earlier than expected. This may be related to their exposure to the ovulatory dose of hcg in vivo. Therefore, despite the presence of the GV from the start, a number of the oocytes may have been in a transitional stage (G2 to M). Nevertheless, this is unlikely to have affected our results because we considered only the interval after maturation, and the IVM oocytes in group 1 had fertilization and cleavage rates similar to those of their in vivo matured siblings. Although it is as yet unconfirmed, the phenomenon of postmaturation aging also could be occurring in oocytes that are obtained from unstimulated ovaries and matured in vitro. FERTILITY & STERILITY 251

8 These oocytes are known to mature over a longer interval (4). Therefore, the timing of insemination or ICSI in these oocytes could be crucial. In conclusion, human IVM oocytes are sensitive to postmaturation aging. Intracytoplasmic sperm injection of such oocytes leads to a higher incidence of zygotes that bear only female pronuclei, or female pronuclei with asynchronously smaller male pronuclei, and that exhibit cleavage arrest. Therefore, the timing of ICSI is critical for IVM oocytes. Acknowledgments: The authors thank Mr. Georges Van Maele from the Department of Medical Statistics, University Hospital of Ghent, for his valuable advice regarding the statistical analysis of the data. The technical support of Dr. Chen Qian also is appreciated. References 1. Palermo GD, Cohen J, Rosenwaks Z. Intracytoplasmic sperm injection: a powerful tool to overcome fertilization failure. Fertil Steril 1996;65: Alikani M, Palermo G, Adler A, Bertoli M, Blake M, Cohen J. Intracytoplasmic sperm injection in dysmorphic human oocytes. Zygote 1995;3: De Sutter P, Dozortsev D, Qian C, Dhont M. Oocyte morphology does not correlate with fertilization rate and embryo quality after intracytoplasmic sperm injection. Hum Reprod 1996;11: Trounson A, Anderiesz C, Jones GM, Kausche A, Lolatgis N, Wood C. Oocyte maturation. Hum Reprod 1998;13(Suppl 3): Kazem R, Thompson LA, Srikanthrajaiah A, Laing MA, Hamilton MP, Templeton A. Cryopreservation of human oocytes and fertilization by two techniques: in-vitro fertilization and intracytoplasmic sperm injection. Hum Reprod 1995;10: Flaherty SP, Payne D, Swann NJ, Matthews CD. Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection. Hum Reprod 1995;10: Payne D, Flaherty SP, Barry MF, Matthews CD. Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography. Hum Reprod 1997;12: Sadowy S, Tomkin G, Munne S, Ferrara-Congedo T, Cohen J. Impaired development of zygotes with uneven pronuclear size. Zygote 1998;6: Flaherty SP, Payne D, Matthews CD. Fertilization failures and abnormal fertilization after intracytoplasmic sperm injection. Hum Reprod 1998;13(Suppl 1): Grossmann M, Calafell JM, Brandy M, Vanrell JA, Rubio C, Pellicer A, et al. Origin of tripronucleate zygotes after intracytoplasmic sperm injection. Hum Reprod 1997;12: Staessen C, Van Steirteghem AC. The chromosomal constitution of embryos developing from abnormally fertilized oocytes after intracytoplasmic sperm injection and conventional in-vitro fertilization. Hum Reprod 1997;12: Goud PT, Goud AP, Rybouchkin AV, De Sutter P, Dhont M. Chromatin decondensation, pronucleus formation, metaphase entry and chromosome complements of human spermatozoa after intracytoplasmic sperm injection into hamster oocytes. Hum Reprod 1998;13: Goud PT, Goud AP, Laverge H, De Sutter P, Dhont M. Effect of postovulatory age and Ca 2 in the injection medium on the male pronucleus formation and metaphase entry following injection of human spermatozoa into golden hamster oocytes. Mol Hum Reprod 1999;5: Dozortsev D, De Sutter P, Dhont M. Behaviour of spermatozoa in human oocytes displaying no or one pronucleus after intracytoplasmic sperm injection. Hum Reprod 1994;9: Ducibella T. The cortical reaction and development of activation competence in mammalian oocytes. Hum Reprod Update 1996;2: Rienzi L, Ubaldi F, Anniballo R, Cerulo G, Greco E. Preincubation of human oocytes may improve fertilization and embryo quality after intracytoplasmic sperm injection. Hum Reprod 1998;13: Yanagida K, Yazawa H, Katayose H, Suzuki K, Hoshi K, Sato A. Influence of oocyte preincubation time on fertilization after intracytoplasmic sperm injection. Hum Reprod 1998;13: Goud PT, Goud AP, Qian C, Laverge H, Van der Elst J, De Sutter P, et al. In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium. Hum Reprod 1998;13: Dozortsev D, De Sutter P, Rybouchkin A, Dhont M. Methodology of intracytoplasmic sperm injection in the human. Asst Reprod Rev 1996; 6: Goud PT, Rybouchkin A, De Sutter P, Dhont M. Fine points of technique: ICSI [letter]. Fertil Steril 1997;67: Sutovsky P, Navara CS, Schatten G. Fate of the sperm mitochondria, and the incorporation, conversion, and disassembly of the sperm tail structures during bovine fertilization. Biol Reprod 1996;55: Sutovsky P, Schatten G. Depletion of glutathione during bovine oocyte maturation reversibly blocks the decondensation of the male pronucleus and pronuclear apposition during fertilization. Biol Reprod 1997;56: Yanagimachi R, Chang MC. Fertilizable life of golden hamster ova and their morphological changes at the time of losing fertilizability. J Exp Zool 1961;148: Chang MC, Fernandez-Cano L. Effects of delayed fertilization on the development of pronucleus and segmentation of hamster ova. Anat Rec 1958;142: Szollosi D. Morphological changes in mouse eggs due to aging in the fallopian tube. Am J Anat 1971;130: Meyer NL, Longo FJ. Cytological events associated with in vitro aged and fertilized rabbit eggs. Anat Rec 1979;195: Longo FJ. Changes in the zonae pellucidae and plasmalemmae of aging mouse eggs. Biol Reprod 1981;25: Ducibella T, Dubey A, Gross V, Emmi A, Penzias A, Layman L, et al. A zona biochemical change and spontaneous cortical granule loss in eggs that fail to fertilize in IVF. Fertil Steril 1995;64: Xu Z, Abbot A, Kopf GS, Schultz RM, Ducibella T. Spontaneous activation of ovulated mouse eggs: time dependent effects on M-phase exit, cortical granule exocytosis, maternal messenger ribonucleic acid recruitment, and inositol 1, 4, 5-trisphosphate sensitivity. Biol Reprod 1997;57: Jedlicki A, Barros C, Salgado AM, Herrera E. Effects of in vivo oocyte aging on sperm chromatin decondensation in the golden hamster. Gamete Res 1986;14: Juetten J, Bavister B. Effects of egg aging in in vitro fertilization and first cleavage division in the hamster. Gamete Res 1983;8: Wilcox AJ, Weinberg CR, Baird DD. Post ovulatory ageing of the human oocyte and embryo failure. Hum Reprod 1998;13: Cha KY, Koo JJ, Ko JJ, Choi DH, Han SY, Yoon TK. Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril 1991;55: Russell JB, Knezevich KM, Fabian KF, Dickson JA. Unstimulated immature oocyte retrieval: early versus mid follicular endometrial priming. Fertil Steril 1997;67: Perrault SD, Wolf RA, Zirkin BR. The role of disulfide bond reduction during mammalian sperm nuclear decondensation in vivo. Dev Biol 1984;101: Perrault SD, Barbee RR, Slott VL. Importance of glutathione in the activity and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes. Dev Biol 1988;125: Yoshida M, Ishigaki K, Nagai T, Chikyu M, Pursel VG. Glutathione concentration during maturation and after fertilization in pig oocytes: relevance to the ability of the oocytes to form male pronucleus. Biol Reprod 1993;49: Usui N, Yanagimachi R. Behavior of hamster sperm nuclei incorporated into eggs at various stages of maturation, fertilization, and early development. The appearance and disappearance of factors involved in sperm chromatin decondensation in egg cytoplasm. J Ultrastruct Res 1976;57: Igarashi H, Takahashi E, Hiroi M, Doi K. Aging-related changes in calcium oscillations in fertilized mouse oocytes. Mol Reprod Dev 1997;48: Lane M, Boatman DE, Albrecht RM, Bavister BD. Intracellular divalent cation homeostasis and developmental competence in the hamster preimplantation embryo. Mol Reprod Dev 1998;50: Tarin JJ, Ten J, Vendrell FJ, Cano A. Dithiothreitol prevents ageassociated decrease in oocyte/conceptus viability in vitro. Hum Reprod 1998;13: Adenot PG, Mercier Y, Renard J-P, Thompson EM. Differential H4 acetylation of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in pronuclei of 1-cell mouse embryos. Development 1997;124: Goud et al. Oocyte postmaturity and ICSI Vol. 72, No. 2, August 1999