1 CReATe Program Inc. and 2 Department of Obstetrics and Gynecology, Sunnybrook and Womens College Health Sciences Center, University of Toronto, Toronto, Ontario, Canada
3 To whom correspondence should be addressed at CReATe Program Inc., 790 Bay Street, Suite 1020, Toronto, Ontario, M5G 1N8 Canada. e-mail: hbalakier{at}sympatico.ca
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Abstract |
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Key words: cytoplasmic maturation/metaphase II arrest/multinucleation/metaphase I oocytes/oocyte activation
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Introduction |
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Interestingly, experiments on mouse and bovine oocytes indicate that the process of oocyte maturation is not completed upon reaching the MII stage (Kubiak, 1989; Dominko and First, 1997
). It is suggested that oocyte maturation involves two equally important time requirements that determine the oocyte developmental competence and the ability to produce a viable embryo. The first period is required for oocytes to resume meiosis and progress to the MII stage, and the second period corresponds to the time interval between MII arrest and sperm activation. It was shown that mouse and bovine MII oocytes gradually develop the ability for activation and pronuclear formation (Kubiak, 1989
; Dominko and First, 1997
). Therefore, essential cytoplasmic changes may be taking place during the MII arrest period and thus successful embryo development depends on proper timing of oocyte maturation as well as oocyte fertilization. Early studies on fertilizability of human oocytes as well as recent reports on development of in vitro matured human MI oocytes suggest that MII oocytes require additional time for maturation in order to be activated promptly by the fertilizing spermatozoon (Lopata and Leung, 1988
; Bonada et al., 1996
; DeVos et al., 1999
; Huang et al., 1999
).
Since there is no direct evidence available with respect to the time period needed for oocyte maturation after MII arrest in humans, the purpose of this study was to evaluate the fertilization rates and developmental potential of human oocytes subjected to ICSI at different times after extrusion of the first polar body (1PB). To achieve this goal, human immature metaphase I (MI) oocytes, acquired after ovarian stimulation, that progressed to the MII stage within a few hours of in vitro culture were compared with sibling oocytes which had matured in vivo, with respect to normal zygote formation, subsequent cleavage and embryo quality.
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Materials and methods |
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Oocyte incubation prior to and following sperm injection, as well as embryo culture, were performed in IVC-One medium (In vitro Care Inc., San Diego, CA) supplemented with 10% SSS (synthetic serum supplement; Somagen, Edmonton, Alberta, Canada) under mineral oil (Sigma) at 37°C in an atmosphere of 5% CO2, 5% O2 and 90% N2. All semen samples were obtained from ejaculates and in all cases sufficient motile spermatozoa were recovered using Pure Sperm density gradient separation (MediTech, Montreal, Quebec, Canada). Severe cases of oligospermia were excluded from this study.
Assessment of fertilization took place 1618 h after the ICSI procedure and oocytes containing two pronuclei (2PN) and two PBs were considered as normally fertilized. The cleavage stage and embryo morphology were evaluated 24 and 48 h later (day 2 and day 3 of development). For each embryo, the number and size of the blastomeres and the percentage of anucleated fragments were recorded. The presence of multinucleated blastomeres (MNBs) per embryo was also observed but not included in the embryo scoring system. Good quality embryos were defined as embryos with a maximum of 20% fragmentation and equal or unequal sized blastomeres. Embryos with >20% but <50% of anucleated fragments were categorized as fair, and those with >50% fragmentation were considered poor. Only good and fair embryos were transferred back to patients 72 h after oocyte retrieval (day 3). The luteal phase was supported by either micronized vaginal progesterone (Medicine Shoppe, Toronto, Ontario, Canada) or progesterone in oil (Cytex Pharmaceuticals Inc., Halifax, Nova Scotia, Canada).
In some cases, in vitro matured MIMII oocytes that had failed fertilization after ICSI were analysed on air-dried preparations to determine the presence and nuclear status of the spermatozoa within oocyte cytoplasm. According to the method described by Kamiguchi et al. (1993
), the zona pellucida was removed by acid Tyrode solution (pH 2, Sigma) and the oocytes were treated with hypotonic 0.5% sodium citrate (Sigma) for 10 min followed by gradual fixation and staining with Giemsa.
Statistical comparisons of the fertilization rates, embryo cleavage and embryo quality between the study and control groups were determined by the 2 and Fishers exact test as appropriate. The CochranArmitage trend test was also used to analyse trends in the changes in fertilization and cleavage rates in oocytes/embryos originating from in vitro matured MI
MII oocytes.
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Results |
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Discussion |
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The results of this study strongly agree with the above observations on animal oocytes. Likewise, we demonstrated that human matured oocytes progressively develop the ability for full activation and pronuclear formation during their MII arrest following the extrusion of the 1PB. In the present study, the overall fertilization rate after ICSI of in vitro matured MI oocytes was significantly reduced when compared with that observed in sibling oocytes matured in vivo (42 versus 77%; Table I). This correlates well with the recent reports on lower fertilization rates obtained from human MI oocytes which had matured in culture within 48 h; however, the exact time of PB extrusion was not recorded in these studies (Bonada, 1996; DeVos et al., 1999
; Huang et al., 1999
). Our detailed analysis of the data with regards to the time interval between PB extrusion and sperm injection revealed a significant relationship between the oocyte activation potential and the duration of MII arrest. It was found that the vast majority of in vitro matured MI oocytes injected soon after PB extrusion remained unfertilized (64%; Table I) and the proportion of normally activated oocytes that contained 2PN and 2PBs gradually increased with prolonged time of incubation before the ICSI procedure. The oocytes injected
1 and 2 h after MII arrest gave rise to 25 and 43% zygote formation, while delayed ICSI for 3 or 46 h resulted in 59 and 62% fertilization rates, respectively. This may suggest that a minimum of 3 h of MII arrest is required to obtain reasonable fertilization rates comparable with those recorded for the control group of oocytes (77%; Table I). These observations can be utilized in future studies to determine the time of ICSI to achieve optimum fertilization and development of in vitro matured human oocytes (non-stimulated cycles). It was shown recently that in vitro matured oocytes are sensitive to post-maturation ageing, and delayed sperm injection (after 36 h versus 30 h culture) results in a high incidence of pronuclear abnormalities (presence of 1PN, asynchrony in size; Goud et al., 1999
). Experiments on bovine oocytes also indicated that prolonged MII arrest before insemination leads to a gradual loss of the ability to support fertilization and embryo development (Dominko and First, 1997
). Therefore, defining the appropriate duration of MII arrest and the time of ICSI may be crucial for the best results in human IVM.
Oocyte immaturity is associated with the failure of oocyte activation as well as with the occurrence of sperm premature chromosome condensation (PCC) after insemination or ICSI (reviewed by Plachot, 1995). Currently, this relationship is well documented in mouse oocytes and it is assumed to be true for human oocytes, although definitive experiments were not carried out for obvious reasons (Calafell et al., 1991
; Kovacic and Vlaisavljevic, 2000
; Benkhalifa, 2003
). Using a murine model, a high incidence of PCC (46%) was only observed when freshly formed MII oocytes were retrieved from ovaries just before ovulation and immediately fertilized in vitro (Calafell et al., 1991
). In human IVF programmes, the frequency of this phenomenon varies from 4 to 28% and it appears to be higher in immature MI (34%) than mature MII oocytes (14%; see Plachot, 1995
). Analysis of our cytological data obtained from unfertilized oocytes following ICSI provides good evidence for the notion that cytoplasmic immaturity is the main factor causing sperm PCC in human oocytes. Nearly all non-activated oocytes (84%; others contained condensed sperm heads) that were injected 1 or 3 h after the 1PB extrusion showed the presence of sperm chromosomes located near the meiotic spindle or mixed together with maternal chromosomes. Alternatively, the lack of oocyte activation may also be due to the absence or deficiency of sperm-activating factor (Dozortsev et al., 1997
). However, this explanation seems to be rather far-fetched because the control sibling oocytes injected with the same sperm samples always exhibited high fertilization rates (77%, Table I). It is therefore clear that specific changes must take place to enable the MII oocytes to be activated.
From the molecular point of view, there are at least two mitotic kinases: maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), which play a significant role in maintenance of and exit from the MII stage (reviewed by Alberio et al., 2001). It appears that inactivation of both kinases is required for matured oocytes to be normally activated and progress through pronuclear interphase, which seems to be controlled via two independent mechanisms (Liu et al., 1998
; Alberio et al., 2001
). Interestingly, recent studies also demonstrated that abortive oocyte activation (i.e. formation of MIII arrest; Liu et al., 1998
) and Ca2+ transients at fertilization (reviewed by Carroll, 2001
) depend on mutual activities of MPF and MAPK. Therefore, based on current accumulated data, it can be speculated that final cytoplasmic maturation of human MII-arrested oocytes may depend on complex interactions between MPF and MAPK as well as on their association with the other protein kinases and calcium mechanisms that are essential for oocyte fertilization and successful embryo development.
The effects of cytoplasmic deficiencies occurring during oocyte maturation may be expressed either by lack of oocyte activation or by a failure of early preimplantation development (Moor et al., 1998). Here we showed that the developmental capacity of human embryos derived from in vitro matured MI oocytes was significantly reduced compared with those developed from the control sibling oocytes. A high proportion of study embryos were arrested soon after the first or the second cleavage division (at the 2- and 4-cell stage). Also a significantly large number of those embryos exhibited the presence of MNBs, suggesting the existence of chromosomal aberrations. These results remain in agreement with the recent studies in which early embryo arrest and a high incidence of MNBs and aneuploidy were observed in human embryos derived from in vitro matured GV and MI oocytes retrieved from stimulated cycles (Nogueira et al., 2000
; Lundin et al., 2002
). The authors suggest that such developmental abnormalities may arise from some deficiencies in oocyte maturation that lead to spindle defects which results in abnormal cytokinesis, formation of MNBs and chromosomal aberrations. Previous animal studies have indicated frequent chromatin aberrations in oocytes inseminated immediately after MII arrest and their decreased development to blastocyst stage, implicating similar spindle problem(s) (Dominko and First, 1997
). In fact, recent experiments on mouse and bovine oocytes demonstrated the importance of MAPK in regulating MII spindle assembly and stability (Verlhac et al., 1993
; Araki et al., 1996
; Gordo et al., 2001
). It was shown that MAPK associates with the spindle poles of MI and MII. The oocytes deprived of its activity exhibit disorganized meiotic spindles and poorly aligned chromosomes. It is clear that in animal and human oocytes, one of the profound changes that take place during the early phase of MII arrest relates to the spindle formation and its function.
Full developmental competence of in vitro matured human oocytes is severely compromised, although rare cases of live births have been reported (Cha and Chian, 1998; Trounson et al., 2001
). In this respect, the results of our study remain inconclusive because no pregnancies were established from embryos derived exclusively from MI study oocytes, while the other pregnancies resulted from the mixed transfers when embryos from MI and MII sibling oocytes were combined. However, development to term of in vitro matured GV and MI oocytes, retrieved from stimulated cycles, is possible, as was proven by DeVos (one singleton; implantation rate 5.3%; DeVos et al., 1997
) and others (reviewed by Cha and Chian, 1998
).
In conclusion, final maturation of human MII-arrested oocytes is crucial for the acquisition of the ability to undergo normal activation and cleavage divisions. This period seems to be important for assembly and normal function of the meiotic spindle. Developmental capacity of embryos originating from in vitro matured MI oocytes is significantly reduced. Caution should be exercised when using MI oocytes for application in assisted reproductive technology programmes, considering the high probability of chromosomal abnormalities in those embryos (minimum 46 h of culture after PB extrusion, observation of MNBs). The improvement of culture conditions and defined ICSI timing may enhance the results of the human IVM system.
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Acknowledgements |
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Submitted on August 26, 2003; accepted on January 5, 2004.