fertilization abnormalities and pronucleus size asynchrony after intracytoplasmic sperm injection...

Fertilization abnormalities and pronucleussize asynchrony after intracytoplasmicsperm injection are related to oocytepostmaturity

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

Objective: To study the effect of delayed intracytoplasmic sperm injection (ICSI) on the fertilization andcleavage 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 toICSI 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 oocytesthat 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 ofpronuclei, and incidence of zygotes with significant pronucleus size asynchrony. Identification of the malepronucleus 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%). Theincidence of oocytes that developed one pronucleus and pronucleus size asynchrony was significantly higherin 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 developedafter delayed ICSI with labeled spermatozoa showed proximity of the fluorescent sperm midpiece remnant tothe smaller pronucleus.

Conclusion(s): For IVM oocytes, the incidence of one pronucleus, pronucleus size asynchrony (possiblyrelated 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 Sterilt 1999;72:245–52.©1999 by American Society for Reproductive Medicine.)

Key Words: Fertilization abnormalities, ICSI, in vitro oocyte maturation, postmaturation aging, pronucleussize asynchrony

Intracytoplasmic sperm injection (ICSI) isan effective tool for overcoming fertilizationfailure related to sperm factors (1). Moreover,because of its ability to counter fertilizationfailure even in dysmorphic oocytes (2, 3), ICSIis used as a method for optimizing fertilizationrates in general. It has been suggested that ICSIalso may improve fertilization rates under spe-cial circumstances, such as in in vitro matured

(IVM) or cryopreserved and thawed oocytes(4, 5). However, despite the significant impactof ICSI on fertilization, a small but notablefraction of oocytes remain unfertilized or un-dergo abnormal fertilization after ICSI. Thelatter is characterized by the presence of eitherone or more than two (usually three) pronuclei(6). Even among the zygotes that display twopronuclei (2PN) at fertilization, one of the pro-

Received December 16,1998; revised andaccepted March 17, 1999.Supported by theUniversity of Ghent, Ghent,Belgium.Presented in part at the14th Annual Meeting of theEuropean Society ofHuman Reproduction andEmbryology, Goteborg,Sweden, June 21–24,1998.Reprint requests: PravinGoud, M.D., InfertilityCentre, Department ofObstetrics andGynecology, UniversityHospital, De Pintelaan 185,Ghent, 9000, Belgium(FAX: 32-9-240-4972;E-mail: [email protected]).* Infertility Centre,Department of Obstetricsand Gynecology, UniversityHospital.† Laboratory ofBiochemistry andMolecular Cytology,Faculty of Agricultural andApplied BiologicalSciences, University ofGhent.

FERTILITY AND STERILITY tVOL. 72, NO. 2, AUGUST 1999Copyright ©1999 American Society for Reproductive MedicinePublished by Elsevier Science Inc.Printed on acid-free paper in U.S.A.

0015-0282/99/$20.00PII S0015-0282(99)00231-9

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nuclei may be markedly smaller than the other, and suchzygotes may have impaired potential for further develop-ment (7, 8).

The cause of fertilization failure after ICSI may be relatedto ooplasmic immaturity or suboptimal activation capacity ofthe spermatozoa, both of which result in failure of oocyteactivation (9). The exact cause of abnormal fertilization afterICSI, however, is not known. It is generally presumed thatthe formation of one pronucleus (1PN) after ICSI is due toparthenogenetic oocyte activation and the formation of threepronuclei (3PN) is related to nonextrusion of the secondpolar body (PB) (6, 9, 10). This is unlike the situation afterIVF, where polyspermy is a more common phenomenonrelated to the formation of multiple pronuclei, and a signif-icant fraction of the 1PN zygotes may actually be diploid(11). Thus, the cause of abnormal fertilization after ICSImay be somewhat different than that after IVF.

The parthenogenetic activation seen after ICSI may occureither spontaneously or secondary to the injection procedure.Spontaneous parthenogenetic activation (SPA) generally oc-curs secondary to postovulatory or postmaturation aging, andinjection-related parthenogenetic activation (IRPA) is re-lated to the calcium content of the injection medium as wellas the postovulatory age of the oocyte (12, 13). The occur-rence of either phenomenon results in gross asynchronybetween the fully developed female pronucleus and an al-most intact or slightly swollen sperm nucleus (12).

These phenomena are documented in golden hamsteroocytes, which are particularly sensitive to SPA and IRPA.However, the formation of only female pronuclei withoutmale pronuclei also is observed in some human oocytessubjected to ICSI, although with a relatively lower frequency(6, 14). Thus, SPA and/or IRPA also may occur in somehuman oocytes; this may be more likely if the oocytes arepostmature. The occurrence of SPA and IRPA may be re-lated 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 onits outcome have produced conflicting results (16, 17). How-ever, this could be related to the limitations of the retrospec-tive design of these studies. Further, there are no reports onthe influence of the timing of fertilization on the outcome ofIVM human oocytes; timing may be particularly crucial forIVM oocytes, which mature at variable intervals in vitro (4).

In this study, we used spare germinal vesicle (GV)-stageoocytes donated by patients undergoing ICSI. We subjectedthem to ICSI after maturation for two different periods inculture. The aim was to investigate the temporal window foroptimum fertilization in these oocytes. Apart from the rou-tine parameters judged at fertilization, we also evaluated themean pronuclear diameters in the zygotes to determine theincidence of pronucleus size asynchrony in different groups.The incidence of abnormal fertilization and pronucleus size

asynchrony was higher in the oocytes that were injected only6 hours later than their siblings that were injected around thetime of optimum maturation.

MATERIALS AND METHODS

Study DesignThe in vitro maturation of spare GV-stage oocytes do-

nated by patients undergoing ICSI cycles and their fertiliza-tion for experimental purposes was approved by the institu-tional ethical review committee. The GV-stage oocytes werecultured in a maturation medium (18) and their siblingoocytes that matured to the metaphase II stage after 30 hoursof culture were assigned to undergo ICSI either immediately(30 hours, group 1) or 6 hours later (36 hours, group 2). Thesibling in vivo matured oocytes that were subjected to ICSIin the clinical program formed the control group.

The number of pronuclei and PBs and the mean diametersof the pronuclei were determined at the fertilization check16–18 hours later. The number of zygotes that had a signif-icant difference between the diameters of their pronuclei andthe number of zygotes that had an abnormal number ofpronuclei were compared between the sibling IVM and invivo matured oocyte groups. The zygotes from groups 1 and2 were cultured for another 24 hours and the incidence ofcleavage was compared.

A second set of experiments was performed to identifythe cause of the pronucleus size asynchrony. Spare GV-stageoocytes were intentionally matured for 6–8 hours longer andinjected with donor spermatozoa prelabeled with a fluores-cent mitochondria–specific dye. The male pronuclei wereidentified by their proximity to the mitochondria-rich spermmidpiece remnants (MPRs).

Source of Gametes, Oocyte Maturation,and ICSI

The GV-stage status of the donated oocytes was con-firmed by two independent observers. The morphology ofthe oocytes was checked, and only those oocytes that had atleast one more sibling GV-stage oocyte and were free ofgross cytoplasmic abnormalities were considered for thestudy. Thus, sibling GV-stage oocytes used for the studywere similar in their morphologic features (e.g., size, cumu-lus cell status, absence of cytoplasmic abnormalities).

These oocytes were subjected to culture in a maturationmedium (M-199; GIBCO BRL, N.V. Life Technologies,Brussels, Belgium) supplemented with human serum albu-min (Albumine 20%; Belgian Red Cross, Brussels, Bel-gium), gonadotropins (pure FSH: Metrodin HP, and hCG:Profasi; Serono, Brussels, Belgium), 17b-E2, and epidermalgrowth factor (Sigma, St. Louis, MO) as described previ-ously (18). All the oocytes were covered with at least 2–3layers of cumulus corona cells, and in no case was it neces-sary 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

culture had no gross dissimilarities in their morphology (e.g.,diameter, shape, cytoplasmic characteristics) or the status oftheir cumulus cells.

Seventy-three GV-stage oocytes from 25 patients wereassigned to culture. Those that matured to the metaphase IIstage at 30 hours of culture were denuded of their cumuluscorona cells and examined for gross abnormalities in thecytoplasm (e.g., vacuoles, central organelle clustering, ex-cessively large perivitelline space, large inclusions). Onlythe sibling oocytes that matured to the metaphase II stagewithout cytoplasmic abnormalities were subjected to ICSIeither immediately (30 hours, group 1) or after another 6hours of culture (36 hours, group 2). They were cultured inhuman tubal fluid medium (Irvine Scientific, Tech Gen In-ternational, NV/SA, Brussels, Belgium) supplemented with0.4% wt/vol human serum albumin.

The outcome of ICSI was evaluated after 16–18 hoursand 40–42 hours of culture, respectively. The data on the invivo matured sibling oocytes undergoing ICSI was recordedat the same intervals and used for comparison.

The spermatozoa used for ICSI in groups 1 and 2 wereobtained from consenting fertile donors and those used in thecontrol oocytes were obtained from their partners. The prep-aration of spermatozoa and performance of ICSI was doneusing methods described previously (19, 20).

Fertilization Check and Embryo CultureA fertilization check was performed 16–18 hours after

ICSI in all the groups. At the fertilization check, the presenceand number of pronuclei and PBs was noted and the diam-eters of the zygotes and pronuclei were measured in threeperpendicular planes with the help of an ocular grid at3400magnification. The mean diameter of each pronucleus wascalculated for the zygotes in groups 1 and 2 and the controlgroup. The zygotes were cultured further for an additional 24hours and examined for the occurrence of cleavage.

Supplementary ExperimentsIn a subsequent set of experiments, 22 spare GV-stage

oocytes from the other 10 consenting patients were subjectedto maturation using the same technique (18). The oocytesthat matured to the metaphase II stage at 30 hours of culturewere subjected to an additional culture period of 6–8 hoursand then underwent ICSI with spermatozoa labeled with afluorescent dye to stain the mitochondria (21). This vital dyeis known to bind specifically to the metabolically activemitochondria within the spermatozoa without hamperingtheir fertilizing capability, and the mitochondria-rich MPRof the fertilizing spermatozoa can be detected in the zygotesin proximity to the male pronucleus (22).

In brief, liquefied donor semen was mixed with humantubal fluid containing 0.4% human serum albumin and cen-trifuged at 4003 g over 40–80 Percoll gradients to obtaina motile fraction. The motile spermatozoa were washed andresuspended, then incubated at 37°C for 15 minutes in hu-

man tubal fluid containing 400 nM of Mitotracker Green FM(Molecular Probes, Leiden, the Netherlands). The spermato-zoa then were washed twice by centrifugation at 5003 g toremove the excess Mitotracker dye and prepared for ICSI.

The oocytes were injected initially with motile prelabeledspermatozoa and then were examined 18 hours after ICSI todetermine both the number and the diameters of the pronu-clei. All the pronuclear zygotes were attached to coverslipscoated with poly-L-lysine (Sigma), fixed in 4% paraformal-dehyde for 45 minutes, washed in phosphate-buffered salinecontaining 0.2% (wt/vol) bovine serum albumin (Sigma) and0.05% Triton X-100 (Sigma), and mounted in Vectashieldcontaining propidium iodide (Vector Laboratories Inc., Bur-lingame, CA). The specimens were examined under a con-focal laser scanning microscope (1024; Bio Rad, Eke, Bel-gium). Optical sections were taken at 1–2mM to determinethe proximity of the sperm MPR to the pronuclei.

Statistical MethodsThe data were analyzed using SPSS software (version

7.5; SPSS Inc., Chicago, IL). The incidence of fertilizationand cleavage among the oocytes subjected to ICSI wascompared between the groups using Fisher’s exact test. Thediameters of the larger and smaller pronuclei as well as thedifferences in the diameters were compared with their cor-responding values between the groups using the Mann-Whit-ney U test. The 2PN zygotes that had a difference in thediameters of their pronuclei that exceeded 23 SD of that ofthe control group were considered to have pronucleus sizeasynchrony, and the numbers of such zygotes were com-pared among all the groups using Fisher’s exact test.P,.05was defined as statistically significant.

RESULTS

Normal and Abnormal Fertilizationand Cleavage

Of the 73 oocytes obtained from 25 patients that wereused for the study, 61 oocytes progressed to the metaphase IIstage by 30 hours. Of these 61 oocytes, 3 oocytes werediscarded because of an abnormal cytoplasmic appearanceand 4 were removed from the study because their siblingoocytes failed to reach the metaphase II stage by 30 hours.Finally, 54 oocytes from 20 patients were randomizedequally in sibling groups to undergo ICSI at 30 hours (group1) or 36 hours (group 2).

The mean (6SD) age of the patients was 31.86 4.1years. The indication for ICSI in all cases was male factorinfertility. A total of 262 metaphase II–stage oocytes fromthese patients were subjected to ICSI in the routine program(control in vivo matured oocytes). The fertilization andcleavage outcomes in groups 1 and 2 and in the control groupare shown in Table 1. The incidence of 2PN zygotes wassignificantly lower in group 2 compared with the controlgroup, and the incidence of 1PN zygotes was significantly

FERTILITY & STERILITY t 247

higher in group 2 compared with both group 1 and the con-trol group. Among the 2PN zygotes, the incidence of cleav-age was lower in group 2 compared with group 1 and withthe control group (Table 1).

Pronucleus Size AsynchronyIn the control group and group 1, the difference in the

diameters of the pronuclei was only slight (mean6 SD,1.256 1.12 mm and 1.56 1.04 mm, respectively) (Figs. 1and 2). However, in group 2, the incidence of zygotesdisplaying pronucleus size asynchrony (a difference of.3.5mm) was significantly higher compared with the othergroups (Table 1). Similarly, the difference between the di-ameters of the pronuclei was significantly higher in group 2compared with the control group, whereas the difference ingroup 1 was similar to that in the control group (Fig. 2).

When arranged serially as larger and smaller pronucleusdiameters in each zygote, the diameters of the larger pronu-clei were statistically similar in all the groups. However, thediameter of the smaller pronuclei in group 2 was signifi-cantly smaller than that in the control group (Fig. 3), whichwas not the case in group 1. Thus, pronucleus size asyn-chrony in group 2 was due to the smaller size of one of thepronuclei, and the incidence of such zygotes was significant-ly higher in group 2 compared with both the other groups.

Origin of the PN Size AsynchronyAfter 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 postma-ture oocytes, we performed a second series of experiments todetermine whether the smaller pronucleus was male or fe-male.

Of the 22 GV-stage oocytes used for these experiments,17 were found to be at the metaphase II stage at 30 hours andwere subjected to ICSI after an intentional delay of 6–8hours. Of these 17 oocytes, 10 underwent activation, as ev-idenced by pronucleus development and/or second PB ex-trusion. Of these 10 zygotes, 9 were 2PN and had two PBs,and 3 of these displayed a statistically significant differencein their mean diameters. The other activated oocyte had 2PNand one PB and an undecondensed sperm head (Fig. 4).Among the other injected oocytes, 2 were damaged and 5remained unfertilized.

The sperm MPR was found in proximity to one of the twopronuclei in 9 of the 10 2PN zygotes. The other zygote was2PN and had one PB, and the labeled spermatozoon wasfound to be almost intact (Fig. 4). In 4 of 6 zygotes, the malepronucleus appeared slightly larger than the female pro-nucleus, but there was no pronucleus size asynchrony (Fig.4). In the other 2 zygotes and in the 1 2PN zygote with intactsperm, the diameters were almost equal. In 3 zygotes, sig-nificant pronucleus size asynchrony was noted. In all 3 ofthese zygotes, the smaller pronucleus was close to the spermMPR, indicating that it was the male pronucleus (Fig. 4). Inthree of five unfertilized oocytes that were fixed, the spermnucleus was only slightly swollen and the fluorescently la-beled sperm MPR was clearly in proximity to the sperm heads.

DISCUSSION

The fertilizable life of mammalian oocytes is limited to ashort period after ovulation, referred to as the temporalwindow for optimum fertilization (15, 23, 24). Oocytes thatfail to encounter spermatozoa during this period may un-dergo certain changes referred to as postovulatory or post-maturation aging (25, 26). Some structural and functionalaspects of postmaturation aging are related to hardening ofthe zona pellucida and increased oocyte sensitivity to acti-vation stimuli (27–29).

These changes in the oocyte may preclude sperm entry(28) or predispose the oocytes to undergo parthenogeneticactivation without forming the male pronucleus (13, 30). Thefertilization of postmature oocytes may account for the fail-ure of embryo development in vitro as well as in vivo(31, 32). However, the exact mechanism leading to the latterphenomenon remains to be investigated.

The technique of in vitro oocyte maturation recently hasbeen applied successfully to clinical cases (4, 33, 34). How-ever, the level of success with IVM is low compared withthat of conventional gonadotropin-stimulated cycles. Thus,for wider clinical application, the technique of IVM needs tobe improved.

T A B L E 1

Influence of postmaturation delay in performingintracytoplasmic sperm injection on fertilization andfirst cleavage division.

VariableIn vivo maturedcontrol oocytes Group 1 Group 2P value

No. of oocytes injected 262 27 27 —No. of oocytes that

survived 247 25 25 —No. of 2PN zygotes* 205 (82.9) 20 (80) 15 (60),.02†No. of 1PN zygotes* 10 (4.1) 1 (4) 8 (32) ,.001†

,.02‡No. of zygotes that under-

went cleavage§ 201 (98.1) 17 (85) 7 (46.7),.001†,.03‡

No. of zygotes with grosspronuclear size asyn-chrony§ 9 (4.4) 1 (5) 6 (40) ,.001†

,.04‡

Note: PN 5 pronuclei.* Values in parentheses indicate percentages of oocytes that survived in-jection.† Control group vs. group 2.‡ Group 1 vs. group 2.§ Values in parentheses indicate percentages among zygotes with twopronuclei.

Fertil Steril ©1999


Determining the occurrence as well as the timing of post-maturation is crucial for clinical in vitro fertilization so thatinsemination can be scheduled for the optimum time toproduce the best results (15). With ICSI, the issue of timingis even more relevant because precise timing of the sperm’sintroduction into the oocyte is under manual control. Thisconcept also could be applied to IVM oocytes to achieveoptimum fertilization.

In the present study, the higher incidence of oocytes thatdeveloped one pronucleus and the lower incidence of oo-cytes that developed two pronuclei are similar to our previ-ous observations in a heterospecific ICSI model (12, 13).Thus, even in human IVM oocytes, the incidence of oocyteactivation sensitivity and 1PN formation increase during thepostmaturation period.

We also found that postmaturation aging caused an in-

crease in the number of zygotes with pronucleus size asyn-chrony. Our supplementary experiments indicate that thesmaller of the pronuclei that formed after delayed ICSI wasthe male pronucleus. Thus, in IVM oocytes, a delay in per-forming ICSI increases the risk of absence or attenuation ofthe male pronucleus.

Fertilization can be viewed as a phenomenon that remod-els the markedly differing chromatin of the male and femalegametes to reprogram both the genomes to the same state togenerate the same structural and functional qualities in thetwo chromosome sets. Decondensation of the highly com-pact chromatin of the sperm nuclei is a major step in thisprocess. The process is characterized by reduction of thedisulfide bonds and replacement of protamines with histonesunder the influence of the decondensing factors of the oocyte(35). As a result, a massive increase in the volume of the

F I G . 1

Zygotes obtained after intracytoplasmic sperm injection of in vitro matured oocytes. (A), A zygote with two pronuclei withsimilar diameters. (B to D), zygotes with significant pronucleus size asynchrony. Original magnification, 3320. Bar, 50 mm.



sperm nuclei occurs. Finally, the initially diverse-appearingsperm nuclei and the oocyte chromosomes change into sim-ilar-looking pronuclei after fertilization.

The subnormal size of the male pronucleus in the rela-tively postmature oocytes in our study may be due to anaberration in the process of chromatin decondensation.Therefore, incomplete decondensation of the sperm chroma-tin and failed formation or smaller size of the male pronu-cleus in postmature oocytes may be related to subnormalactivity of the decondensing factor of the oocyte. This ac-tivity of the oocyte recently has been connected with thelevel of glutathione, which is acquired during maturation(36, 37). Insufficient uptake of glutathione or its depletionimpairs the development of the male pronucleus (22). Thiscould occur during IVM and postmaturation aging.

Another explanation for the failed or suboptimal devel-opment of the male pronucleus could be a shorter duration ofthe decondensation factor activity after activation in postma-ture oocytes. The decondensation factor activity of the oo-cytes is known to be related to the cell cycle and disappearsduring the interphase (38). Postmature oocytes are in adynamic state that is poised for entry into the interphase,which is characterized by altered activity of the cell-cyclefactors (29). Therefore, even minor activation stimuli such as

the injection procedure could result in oocyte activation andfemale pronucleus formation, and the precipitous entry ofthese oocytes into the interphase may not allow for adequatedecondensation of the sperm nuclei (12, 13).

Another interesting finding in the present study was adecrease in the incidence of zygotes that underwent the firstcleavage division among the relatively postmature oocytes.This may be due either to alterations in the cell-cycle factoractivities in such zygotes or to the detrimental influence ofpostmaturation aging on the cellular metabolism. Postmatu-ration recently has been shown to induce a dysregulation ofintracellular Ca21 homeostasis (39). This may lead to anincrease in the basal cytosolic Ca21 level within the oocyteand perhaps even the zygote, which may adversely affectcleavage divisions in the embryo (40).

Another explanation could be related to the generation offree oxygen radicals in the oocytes after maturation (41). Ourstudy showed that fertilization of the postmature oocytesleads to a considerable difference between the status of themale and female chromatin. Attention recently has been drawnto the role played by the dynamic changes in thechromatinstructure in regulating the access and three-dimensionaljuxtaposition of transcriptional activators and the transcrip-tion machinery with the gene regulatory elements (42). Op-timum decondensation of the pronuclei therefore may bevital for normal embryogenesis to occur. However, it needsto be determined whether suboptimal decondensation of the

F I G . 2

A box and whiskers plot displaying the differences in thediameters of the two pronuclei in the 2PN zygotes of all thegroups. The horizontal lines within the boxes represent me-dians and the upper and lower borders of the boxes repre-sent 75th and 25th percentiles, respectively. The horizontallines above and below the boxes represent the largest andsmallest values, respectively, that were not outliers. h rep-resent outliers. †P,.001.


F I G . 3

A box and whiskers plot displaying the diameters of thesmaller pronuclei of the 2PN zygotes in all the groups. hrepresent outliers and p represent extremes. For more de-tails, refer to the legend for Fig. 2. †P,.04.



pronuclei is related directly to the abnormalities that occurduring later stages of embryo development.

We found that GV-stage oocytes became susceptible topostmaturation after only 36 hours of culture. This wasbecause most of the oocytes matured at 30 hours of culture,and postmaturation changes were induced by 6 hours aftermaturation. This interval may be shorter than that of in vivomatured oocytes (17). Therefore, the incidence of 1PN oo-cytes was lower in the in vivo sibling oocytes that weresubjected to ICSI within 6 hours after oocyte retrieval. Invitro matured oocytes could be more susceptible to post-maturation aging than their in vivo matured counterparts.

Completion of maturation in the oocytes in our studyoccurred earlier than expected. This may be related to theirexposure to the ovulatory dose of hCG in vivo. Therefore,despite the presence of the GV from the start, a number ofthe oocytes may have been in a transitional stage (G2 to M).Nevertheless, this is unlikely to have affected our resultsbecause we considered only the interval after maturation,and the IVM oocytes in group 1 had fertilization and cleav-age rates similar to those of their in vivo matured siblings.

Although it is as yet unconfirmed, the phenomenon ofpostmaturation aging also could be occurring in oocytes thatare obtained from unstimulated ovaries and matured in vitro.

F I G . 4

Injection of spermatozoa prelabeled with Mitotracker FM dye into in vitro matured oocytes that were intentionally aged for atleast 6–8 hours after maturation. The images are optical sections obtained with a confocal laser scanning microscope. (A andB), Optical sections of the same zygote showing the female and male pronuclei, respectively. The fluorescent sperm midpieceremnant is indicated by a small arrow in (B). (C), A zygote with a significant size asynchrony between its pronuclei. The smallermale pronucleus can be identified from its proximity to the fluorescent sperm midpiece remnant (small arrowhead). Theapparently smaller oocyte diameter in (C) is due to the peripheral location of the pronuclei. (D), A magnified image of an intactspermatozoon within an oocyte displaying only the female pronuclei. Bright labeling of the sperm midpiece and tail are clearlyevident (small arrowheads). Original magnification, 3600. Bars: (A to C), 50 mm. (D), 5 mm.



These oocytes are known to mature over a longer interval(4). Therefore, the timing of insemination or ICSI in theseoocytes could be crucial.

In conclusion, human IVM oocytes are sensitive to post-maturation aging. Intracytoplasmic sperm injection of suchoocytes leads to a higher incidence of zygotes that bear onlyfemale pronuclei, or female pronuclei with asynchronouslysmaller 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 theDepartment of Medical Statistics, University Hospital of Ghent, for hisvaluable advice regarding the statistical analysis of the data. The technicalsupport of Dr. Chen Qian also is appreciated.

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fertilization abnormalities and pronucleus size asynchrony after intracytoplasmic sperm injection...

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