Womens General Hospital, IVF-Unit, Lederergasse 47, A-4010 Linz, Upper Austria, Austria
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Abstract |
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Key words: blastocyst formation/first polar body/ICSI/oocyte quality/non-invasive selection
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Introduction |
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Nevertheless, many of the blastocysts transferred will not implant. This divergence may at least in part be attributable to culture conditions, which though significantly improved, have not yet been optimized (Gardner and Schoolcraft, 1998; Jones and Trounson, 1999
). It has been suggested that prolonged incubation time may affect the quality of the preimplantation embryo, including alterations to the zona pellucida (De Vos and Van Steirteghem, 1999
) and to the subsequent hatching process (Fong et al., 2001
). However, data from Schiewe et al. do not support the concept that additional, abnormal hardening of the zona occurs during extended culturing (Schiewe et al., 1995
).
It may be of great benefit to the patient if viable blastocyst candidates can be identified earlier in development in order to reduce the time in culture to a minimum. Studies (Scott et al., 2000; Balaban et al., 2001
) have shown that adequate information about viability may be gained without extended incubation by scoring pronuclei at the zygote stage. Consequently, it is likely that the selection of embryos with a good prognosis in terms of implantation may be performed on the day of oocyte collection (Edwards and Beard, 1997
). This hypothesis has also been supported by a suggested relationship between some morphological features of the oocyte and ICSI outcome (Xia, 1997
; Ebner et al., 1999
; Kahraman et al., 2000
).
Based on our previous results (Ebner et al., 1999, 2000
), the present prospective study was designed to investigate the actual blastocyst formation rate of oocytes showing fragmented or unfragmented first polar bodies (PBs). Homogeneous blastocyst transfers in terms of first PB morphology and subsequent rates of implantation and pregnancy will provide further insight into the prognostic value of the present mode of non-invasive selection.
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Materials and methods |
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Institutional Review Board approval was not sought since blastocyst culture and subsequent transfer have been introduced as a routine method in our laboratory.
All patients were stimulated according to a long protocol using a combination of a GnRH agonist (Suprecur; Hoechst, Frankfurt, Germany) and an individually adjusted dose of hMG (Menogon; Ferring, Kiel, Germany). At 36 h prior to ultrasound-guided oocyte retrieval, 500010 000 IU of hCG (Pregnyl; Organon, Oss, The Netherlands) was administered to induce ovulation.
All oocytes were incubated in BM1 medium (NMS Bio-Medical, Praroman, Switzerland) prior to insemination (3 h, 37°C and 6% CO2). Combining an enzymatic (80 mIU/ml hyaluronidase; MediCult, Copenhagen, Denmark) and a mechanical method, cumulus cells were removed in order to facilitate both evaluation of nuclear maturity and accurate assessment of first PB morphology.
After centrifugation of the ejaculate, a swim-up technique was used to separate progressively motile sperm with optimal morphology from the pellet. These were used for ICSI, which was performed as previously described (Ebner et al., 2001). Immediately after sperm injection, oocytes were cultured in groups according to the morphology of the first PB. The previously published grading system in terms of first PB appearance (Ebner et al., 1999
) was adapted slightly. In detail, only two groups were investigated in the present study, those with intact first PBs (former grades 1 and 2) and those with fragmented first PBs (former grade 3). Based on our previously published data (Ebner et al., 2000
) we decided not to apply ICSI to oocytes exhibiting large first PBs (former grade 4).
At 1620 h after injection, fertilization and survival were assessed. The presence of two pronuclei as well as two PBs characterized normal fertilization (2PN). At the zygote stage, the medium was changed to Blastassist System Medium 1 (MediCult). According to their first PB, two groups of gametes were cultured in 75 µl droplets under sterile filtered paraffin (MediCult).
Approximately 42 h post-injection, embryo morphology criteria, i.e. number of blastomeres and degree of fragmentation, were recorded. From day 2 onward, the first sequential medium was replaced by Blastassist System Medium 2 (MediCult) which was changed daily until the day of transfer.
Blastocyst quality, as assessed by the size of the inner cell mass and the degree of expansion (Gardner et al., 2000), was recorded prior to transfer which was exclusively performed on day 5.
Our strategy was to retransfer a maximum of two blastocysts with an adequate inner cell mass deriving from oocytes with intact first PBs (n = 19). However, 18 patients had mixed blastocyst transfers in terms of first PB morphology (one first PB intact and one fragmented). The remainder did not have any blastocysts which stemmed from oocytes with intact first PB for transfer (n = 23).
All data of this prospective self-controlled investigation were compared using 2-test, MannWhitney U-test, and t-test. P < 0.05 was considered as statistically significant.
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Results |
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Table I illustrates a significant relationship between first PB morphology and embryo quality on day 2. A higher percentage of fragmentation was seen after 2 days in the group derived from oocytes with a fragmented first PB than in those with an intact PB (P < 0.05). Blastomere number and fertilization rate were similar in both groups. Embryos derived from oocytes with an intact first PB showed an increased rate of blastocyst formation as compared with the fragmented first PB group (P < 0.05).
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Most of the women had two blastocysts transferred (n = 54). However, six patients underwent transfer of only one blastocyst because either no more blastocysts were available or there was a decision to limit the number of blastocysts for transfer to just one. This led to a mean number of 1.9 (± 0.3) blastocysts being replaced per patient. The quality of transferred blastocysts did not differ between the two groups.
A total of 114 blastocysts were replaced, out of which 40 implanted, giving an implantation rate of 35.1%. Thirty-four clinical pregnancies were achieved (56.7%). Since five missed abortions occurred (14.7%), the ongoing clinical pregnancy rate was 48.3% (29/60). Six twin pregnancies were detected, giving a multiple pregnancy rate of 17.6% (6/34).
The treatment outcome for both groups is summarized in Table II. There was a significant difference in implantation (P < 0.025) and pregnancy rates (P < 0.05) between the intact and fragmented first PB groups.
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Discussion |
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Nevertheless, approximately half of the blastocysts transferred will not proceed to subsequent implantation. One possible cause for this divergence may be aneuploidy (Munné and Cohen, 1998), but at least some of the non-viable blastocysts may fail to result in a pregnancy due to suboptimal culture conditions (Gardner and Schoolcraft, 1998
; Jones and Trounson, 1999
).
Therefore, it can only be for the benefit of patients if concepti with a good prognosis for implantation can be identified as early as possible. Indeed, there is some evidence that morphology of zygotes on day 1 (Scott et al., 2000; Balaban et al., 2001
) will increase our understanding of subsequent blastulation and implantation.
Consequently, an alternative back to the roots step would be to use polarity (Edwards and Beard, 1997) or morphological aspects of the oocyte prior to ICSI to obtain information about further development. It is important to mention that not all studies could demonstrate a significant relationship between oocyte morphology and implantation rate (De Sutter et al., 1996
; Balaban et al., 1998
) which indicates that, to a certain degree, oocyte dysmorphism is a normal feature. However some authors have reached opposite conclusions, finding that oocyte morphology (vacuoles, granularity of cytoplasm, inclusions) is negatively correlated to outcome (Serhal et al., 1997
; Loutradis et al., 1999
; Kahraman et al., 2000
).
It is well accepted that, due to controlled ovarian stimulation (COS), not all oocytes recruited from the follicles are of the same quality. One possible reason for such a divergence may be a reduced blood supply to the follicles during COS which causes oxygen deficiency. Oocytes growing in such an environment were found to have an affected intracytoplasmic ATP content and show cytoplasmic disorganization (Van Blerkom et al., 1995). In addition, hypoxia can result in chromosomal and spindle defects in humans (Van Blerkom et al., 1997
) and other mammals (Haidri et al., 1971
).
As yet, it has not been clarified if some of these events may cause fragmentation of the first PB itself or if this scenario may interfere with nuclear and cytoplasmic maturation, which both have to be completed in a co-ordinated manner at the time of collection so as to ensure optimal conditions for subsequent fertilization.
Disturbances or asynchrony in these maturation processes may result in different morphological abnormalities depending on whether cytoplasmic (Loutradis et al., 1999; Kahraman et al., 2000
) or nuclear maturation (Eichenlaub-Ritter et al., 1995
; Ebner et al., 2000
) has been affected.
It has been speculated that optimal nuclear maturation may be evidenced by an intact first PB (Xia, 1997; Ebner et al., 2000
; Mikkelsen and Lindenberg, 2001
). On the other hand, unusual asynchrony in follicular, cytoplasmic and nuclear kinetics may lead to oocytes showing fragmented first PBs. Since it is known that oocytes collected after COS may show an affected cytoplasmic organization and metaphase spindle (Van Blerkom et al., 1995
, 1997
), this may have influenced extrusion of the first PB. As a consequence, chromosomal status and/or oocyte polarity would be impaired irreversibly, possibly manifested in decreased fertilization or reduced embryo quality.
In fact, first PB morphology, either in combination with perivitelline space appearance and cytoplasmic inclusions (Xia, 1997) or analysed independently (Ebner et al., 1999
), was found to be correlated with fertilization rate and embryo quality (Ebner et al., 2000
) as well as with the rates of implantation and pregnancy (Ebner et al., 1999
).
However, a suspected influence on fertilization rate (Ebner et al., 2000) could not be supported in the present prospective study. This discrepancy may be due to the fact that in the present study a simplified grading system has been applied to the oocytes in order to increase the sample number in both study groups. In particular, all ovoid or round first PBs were pooled together irrespective of the appearance of their surface (smooth or rough). The present results are in line with a previous study on in-vitro matured oocytes (Mikkelsen and Lindenberg, 2001
) in which no relationship between extracytoplasmic abnormalities and fertilization rate could be found.
In terms of embryo quality, our previous results (Ebner et al., 2000) were confirmed. Once fertilized, oocytes with intact first PBs led to embryos of superior quality compared with the control group (P < 0.05). In the present study it was possible to show that it is not the number of blastomeres on day 2 which is influenced by the investigated oocyte criterion, but rather the degree of fragmentation (Table I
). Although this 2.3% difference in fragmentation may be of questionable clinical relevance, it may reflect a possible defect in chromosomal complement of such oocytes and/or embryos, since percentage fragmentation has been associated with chromosomal abnormalities (Plachot et al., 1987
).
It may be speculated that chromosomal aberrations may also be one of the reasons for a significantly diminished percentage of blastocysts found in the fragmented PB group (P < 0.025) since a strong selection against some clinically relevant chromosomal abnormalities has been reported (Magli et al., 2000; Sandalinas et al., 2001
). However, it has to be stated that these authors provide no data on first PB morphology.
Despite the different percentage of blastocysts, both the homogeneous and the mixed group had the same proportion of high quality blastocysts developed in vitro. Therefore, it was possible to transfer blastocysts of comparable quality in all groups. The differences in the rates of implantation (P < 0.025) and ongoing pregnancy (P < 0.05) may indicate that the viability of blastocysts which stem from oocytes with fragmented PBs may be severely impaired.
Previously, elective transfer of day 2 embryos selected on the basis of first PB morphology was found to be associated with increased pregnancy rates (Ebner et al., 1999). As compared with these previous results, the present study appeared to show a trend for higher pregnancy rates in both homogeneous groups (P = 0.09). Although this difference was without statistical significance, it serves to emphasise the importance of a dual selection approach, e.g. preselection at the oocyte stage and a final selection at the blastocyst stage.
In conclusion, the current study provides further evidence that preselection at very early stages may be helpful to identify a special subgroup of preimplantation embryos with good prognosis to form blastocysts and, consequently, to implant.
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Notes |
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References |
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Submitted on January 9, 2002; resubmitted on March 15, 2002; accepted on May 21, 2002.