1 Infertility and IVF Unit, Assaf Harofeh Medical Center, Tel-Aviv University, Zerifin, 70300, Israel
2 To whom correspondence should be addressed. e-mail: embriology{at}asaf.health.gov.il
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Abstract |
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Key words: fertilization/ICSI/in vitro maturation/MI oocytes
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Introduction |
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The aim of the present study was to clarify whether the injection of sperm to rescued matured MII (IVM MII) oocytes (oocytes that underwent nuclear maturation) is preferential to the injection of MI oocytes immediately after denudation, and thus to asses the advantage of the extrusion of the first polar body (PB; nuclear maturation) in terms of ICSI outcome.
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Materials and methods |
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Oocyte preparation
Oocyte retrieval was performed by vaginal ultrasound-guided puncture. At 2 h after retrieval, the cumuluscorona cells were removed after exposure to hyaluronidase (type IV-S, Sigma) 20 IU/ml in human tubal fluid medium (HTF; Irvine Scientific, Santa Anna, CA) for no more than 1 min. Oocyte evaluation was performed using an inverted microscope (Diaophot 300; Nikon, Japan) with an enlargement of x400. GV oocytes were discarded. MI oocytes were represented by the absence of a PB and no discernible GV nucleus. MI oocytes were separated from the MII oocytes and randomly divided into two groups (sibling oocytes). In the first group, MI oocytes were injected immediately after denudation (MI oocytes). In the second group, MI oocytes were incubated in vitro in 25 µl droplets of medium (G1.2. Vitrolife, Molndalsven, Goeteborg, Sweden) without any supplementation of serum or hormones, under light weight paraffin oil (Sigma, Israel, 8042-47) for 1.54 h before injection. The incubated MI oocytes were evaluated for maturation after 1.5, 2.5 and 4 h. After their incubation period, the oocytes were re-evaluated and divided into those which extruded their PB (rescued IVM MII oocytes) and those that failed to do so (arrested IVM MI). Both rescued IVM MII oocytes and arrested IVM MI oocytes were injected. The MII oocytes that were retrieved in the same treatment cycles served as the control group (MII oocytes).
ICSI procedure
The procedure was carried out according to the methodology described by Van Steirteghem et al. (1993). Culture of the injected oocytes took place in 25 µl medium droplets (G1.2.Vitrolife) under oil. Oocytes were observed for fertilization 1618 h after micromanipulation. The criterion for normal fertilization was the presence of two extruded PBs and two pronuclei. Cleavage was assessed 24 h later and the embryos were classified according to their morphological appearance. Embryos were classified as grade I, symmetrical blastomeres with no anucleated fragments; grade II, asymmetrical blastomeres without or with <20% of the volume filled with anucleated fragments; grade III, with 2050% of the volume covered with fragments; and grade IV, extreme asymmetrical blastomeres and >50% of the volume filled with anucleated fragments. Embryos which originated from MI oocytes were transferred into the uterus only in cases where there were not enough embryos from MII oocytes. Generally, transfers included retrieved MII oocytes, except for four cases (see Results) where none were available.
Statistical analysis.
Statistical evaluation was performed using multivariant analyses and then 2 and Fishers exact tests, where appropriate. A P-value of <0.05 was considered statistically significant.
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Results |
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Table I compares ICSI outcome of immediately injected MI oocytes with arrested IVM MI, rescued IVM MII oocytes and MII oocytes. The degeneration rate was significantly higher in MI oocytes compared with rescued IVM MII oocytes and MII oocytes (11 versus 6 versus 4%), P < 0.0001. The fertilization rate of 2PN 1-cell zygotes from MI oocytes was significantly lower than that found in rescued IVM MII oocytes and in MII oocytes (28 versus 44 versus 68%), P < 0.0001. The incidence of multinucleated zygotes (3PN) was similar between rescued IVM MII and MII oocytes but higher in non-incubated MI oocytes (4 versus 10%), P < 0.01. Multipronucleated zygotes of the last three groups had two PBs at an incidence of 75, 77 and 78%, respectively. Cleavage rates were similar in both MI and MII oocytes, but significantly lower among the rescued IVM MII (95 versus 86%), P < 0.01.The number and quality of the blastomeres were similar in all the three groups. There were 141 arrested IVM MI oocytes injected after incubation. When comparing ICSI outcome between arrested IVM MI and MI oocytes, similar degeneration, fertilization and 1PN rates could be found. The cleavage rate of 2PN embryos was significantly higher among the MI oocytes with no incubation period compared with arrested IVM MI oocytes (96 versus 72%; P < 0.001). The number of blastomeres as well as embryo morphology was similar.
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Discussion |
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Forty-six percent of the MI oocytes that were left for the maximum 4 h incubation in culture medium matured spontaneously. This percentage was higher than those reported in other groups: 24% after a 4 h incubation (Junca et al., 1995) and 27 (De Vos et al., 1999
) or 16% after 9 h (Chen et al., 2000
). In a kinetic experiment to characterize the timing of meiotic progression of GV oocytes, Combelles et al. (2002
) showed that 89% of GV oocytes resume meiosis by 6 h in culture. After 18 h, 45 and 50% of the oocytes were in MI and in telophase, respectively, meaning that altogether 95% of the oocytes reached what we consider the MI stage after 18 h. Of these MI oocytes, 67% reached the MII stage after an additional 6 h of incubation. The rational for an incubation period of 4 h was based upon the time needed for oocyte maturation, convenience of working hours in the laboratory, and a need to reduce the risk of an ageing effect. We found that an incubation of 1.5 h led to 13% maturation, and 2.5 h yielded another 41% matured oocytes. This means that the majority of MI oocytes (54%) mature in vitro already after 2.5 h of incubation.
Longer incubation periods of MI oocytes, such as 24 and 36 h, were reported to yield higher maturation rates of 64 and 88%, respectively (Chen et al., 2000), or 73 and 84% after 24 and 48 h, respectively, in P1 culture medium (Cekleniak et al., 2001
). However, embryos originating from aged oocytes had increased incidence of abnormal cytoskeletal organization and chromosomal imbalance (Racowsky and Kaufman, 1992
; Plachot et al., 1998
). The variation in the maturation rate might also be related to the composition of the culture medium, which changes the pyruvate glucose uptake and lactate production by the IVM oocytes (Chian and Tan, 2002
; Roberts et al., 2002
)
The injection of MI oocytes immediately after denudation, and the injection of arrested IVM MI oocytes showed a high degeneration rate that can be explained by increased fragility of the oolemma following ICSI. The extrusion of the first PB may indicate the changes in the oolemma or the cytoskeleton leading to the improved elasticity of the membranes, which is important for successful ICSI (Bourgain et al., 1998).
The fertilization rate of MI oocytes that were injected immediately after denudation and of arrested IVM MI oocytes was lower than the fertilization rate of their sibling rescued IVM MII oocytes and less than half compared with that found among sibling MII oocytes. The improved rate of fertilization in the rescued IVM MII oocytes could be partially due to the fact that MI oocytes which are unable to undergo maturation as a result of complete absence of spindle formation (Windt et al., 2001; Levran et al., 2002
) or failure of the oocyte to enter M phase (Hartshone et al., 1999
) were not included in this group.
A review of the current literature did not reveal any publication concerning ICSI outcome in MI oocytes that were injected without extrusion of their PB. On the other hand, reports exist concerning outcome of ICSI performed only in MI oocytes that matured in vitro after incubation period of 8 h, showing a 2434% decline in fertilization rate compared with fertilization rates found among MII oocytes (Bonada et al., 1996; Ben-Shlomo et al., 1998
). In a large series of 1210 MI oocytes that matured in vitro, De Vos et al. (1999
) showed 52.7% of fertilizations compared with 70.8% in 8803 sibling MII oocytes after 4 h of incubation. These results concur with our results of 44% fertilization in rescued IVM MII oocytes versus 68% in the MII oocytes. Huang et al. (1999
) showed significantly higher fertilization rates in MII oocytes after ICSI compared with IVM oocytes (81 versus 62%). However, his study group included GV as well as MI oocytes. The reduced fertilization rate of rescued IVM MII oocytes can be explained by cytoplasmic immaturity of these oocytes in contrast to their nuclear maturation (Goud et al., 1998
; Russel, 1999
).
In the present study, a high rate of multipronucleated oocytes was found in MI oocytes that were injected immediately after denudation. In 78% of them, a second PB could be recognized. This result demonstrates that injection of oocytes in their early stages of maturation causes DNA disorganization.
The cleavage rate was significantly lower among the 2PN 1-cell zygotes that derived from both rescued IVM MII and arrested IVM MI oocytes, namely oocytes that were incubated in vitro, in comparison with MI oocytes injected immediately after denudation or MII oocytes. This difference was not shown in other studies (De Vos et al., 1999; Chen et al., 2000
); however, in the literature, it was shown that the main damage in long in vitro maturation was in the cleavage rate as well as in the chromosomal constitution (Chen et al., 2000
; Nogueira et al., 2000
).
Embryo quality and mean number of blastomeres in our study were similar among the groups whether the embryos resulted from MI oocytes that were immediately injected, MI oocytes that were incubated or mature MII oocytes. De Vos et al. (1999) reported slight differences regarding the number of good quality embryos resulting from rescued IVM MII oocytes. In contrast, Chen et al. (2000
) found that MI oocytes incubated for long periods (2436 h) led to the development of poorer quality embryos and less blastocysts compared with the outcome from MII oocytes. This difference could be explained by the different incubation times (Plachot et al., 1988
; Racowsky and Kaufman, 1992
).
We conclude that the injection of rescued IVM MII oocytes is preferred to the injection of MI oocytes since rescued IVM MII oocytes lead to an increased fertilization rate (44 versus 28%) and a higher rate of embryo formation. The use of rescued IVM MII oocytes seems feasible; incubation time can be as short as up to 4 h and suitable for the laboratory routine timetable. The new method of spindle visualization by PolScope may contribute to discriminate rescued IVM MII oocytes with chromosome misalignment (Wang and Keefe, 2002), which might contribute to minimizing the use of oocytes with abnormal chromosome distribution.
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Acknowledgements |
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References |
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Submitted on January 15, 2004; accepted on February 18, 2004.
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