1 Infertility Center, Toyota Memorial Hospital, Toyota 471-8513, 2 Infertility Center, Toyohashi Municipal Hospital, 50 Hakken-nishi, Aotake-cho, Toyohashi 441-8570 and 3 Department of Obstetrics and Gynecology, The Japanese Red Cross Nagoya First Hospital, Nagoya 453-8511, Japan
4 To whom correspondence should be addressed. e-mail: sugasan{at}par.odn.ne.jp
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Abstract |
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Key words: implantation rate/IVF/multinucleated blastomere/nuclei counting/selection of embryo
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Introduction |
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Blastocyst transfer is a useful method for selecting good quality embryos, with the follow-on benefit of fewer transferred embryos and reduced incidence of multiple pregnancies. Gardner et al. (2000) reported pregnancy rates >60% for top-scoring embryos used in blastocyst transfer. However, other studies indicated that the overall pregnancy rates in IVF did not improve with blastocyst transfer because transfer cancellations increased (Coskun et al., 2000
; Vlaisavljevic et al., 2001
; Karaki et al., 2002
; Lundqvist et al., 2002
). Additionally, some reports showed an increased incidence of monozygotic twins after blastocyst transfer (Behr et al., 2000
; da Costa et al., 2001
; Sheiner et al., 2001
; Ménézo and Sakkas, 2002
; Tarlatzis et al., 2002
). Therefore, the risk of multiple pregnancy remains even if blastocyst transfer is performed.
Moreover, Scholtes and Zeilmaker (1998) reported that 381 of 929 candidate embryos for blastocyst transfer (41%) did not reach blastocyst stage. They also indicated that implantation rate per blastocyst was 23% in the first blastocyst transfer cycle, which was similar to regular early stage embryo transfer cycles. Royen et al. (1999
) reported that the implantation rate was 49% when two top quality embryos (judged as having four or five blastomeres on day 2 and seven or more on day 3;
20% fragmentation on day 3) were transferred on day 3. They indicated that the implantation rate seemed to be similar to the blastocyst transfer rate reported by Gardner et al. (2000
), implying that early embryo transfer is more useful than blastocyst transfer given the advantages of a shorter culture time and, by extension, less cost.
The common point in the reports of Gardner et al. (1998) and Royen et al. (1999
) is that a single embryo transfer using a top quality embryo in an early embryonic stage is beneficial, proving a high implantation rate and avoiding multiple pregnancy risk. When the number of transferred embryos can be reduced to one without lowering the pregnancy rate, embryo transfer cost will decrease, and the incidence of cancellations will be reduced. Moreover, a single embryo transfer will leave a higher number of embryos per cycle available for cryopreservation (Neubourg et al., 2002
), thereby potentially increasing the cumulative conception rate per patient.
Based on these analyses, effective morphological evaluation of early stage embryos is critical. Recently, some studies have indicated that analysis of blastomere nuclei can predict embryo quality. In particular, multinucleated blastomeres (MNB) are considered to be abnormal by some reports, because the possibility of chromosomal abnormality of MNB is high (Munné and Cohen, 1993; Pickering et al., 1995
; Kligman et al., 1996
). Analysis of blastomere nuclei using high-power inverted microscope revealed MNB in 14.5% of 1885 embryos obtained from 338 patients, and that 43% of patients had MNB embryos (Balakier and Cadesky, 1997
; Jackson et al., 1998
). Although normal childbirth sometimes resulted from embryo transfer with embryos having MNB (Balakier and Cadesky, 1997
; Jackson et al., 1998
), embryos with MNB may not be the best choice for embryo transfer given the risk of abnormality. Hence we conclude that looking for MNB is an important way to select the highest quality embryos for transfer.
In the present study, to create a more effective selection standard for early embryos, we developed a new embryo grading system, using both conventional morphological evaluation and blastomere nuclei analysis. The implantation rate of each grading method for standard embryo transfer and blastocyst transfer was examined retrospectively.
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Materials and methods |
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The cryopreservedthawed cycles of 85 embyro transfers and 86 blastocyst transfers which took place during this period were not included in this study, because the implantation environment is very different when ovarian hyperstimulation (standard cycle) was performed and when it was not (cryopreservation cycle), making statistically relevant comparisons impossible.
IVFembryo transfer or blastocyst transfer
Controlled ovarian stimulation in IVF was performed using human menopausal gonadotrophins with a GnRH in a long or short protocol as previously described (Suganuma et al., 1996). Ovarian follicular development was monitored by vaginal ultrasound scans and serum estradiol measurements. Transvaginal oocyte retrieval was performed 36 h after administration of 5000 IU hCG. Conventional insemination or ICSI (Suganuma et al., 1996
) was done
5 h after retrieval. The oocytes and embryos were cultured in human tubal fluid medium (Irvine Scientific, USA) with 10% synthetic serum substitute (SSS; Irvine Scientific). Fertilization was assessed by the presence of two pronuclei 1819 h after insemination. Fewer than three embryos were selected and transferred on day 2 after retrieval. The number of embryos per transfer was 1.6 ± 0.6, the implantation rate was 15.1%, and the patients age was 33.1 ± 4.6 (mean ± SD, range 2344) years. Remaining embryos were cryopreserved on day 2 or cultured in sequential medium until day 5 after retrieval. The embryos were cultured in blastocyst medium (Irvine Scientific) with 10% SSS during day 3day 5 after oocyte retrieval. One or two blastocyst(s) were selected and transferred in blastocyst transfer. The number of transferred blastocysts was 1.5 ± 0.5, the implantation rate was 16.8%, and the patients age was 33.9 ± 4.5 years old. Remaining blastocysts in good condition were cryopreserved on day 5.
Implantation was confirmed by the presence of gestational sac(s) using transvaginal ultrasound imaging. Chemical pregnancies indicating positive urinary hCG (>50 IU/l) alone were not counted as implantation in this study.
All procedures were performed after receiving informed consent from the patients, following the guidelines of the Japan Society of Obstetrics and Gynecology, and had the permission of the ethics committee of the each hospital.
Morphological grading of embryos
Morphological embryo evaluation was performed on day 2 after retrieval (4348 h after insemination or ICSI) using an inverted microscope (Olympus IX70; Olympus Electric Industry Co., Japan) at x300600 magnification (Figure 1). The number of blastomeres in embryos ranged between two and eight. The embryos were classified into four morphological grades in accordance with our conventional criteria (Kondo et al., 1996) consisting of blastomere size and the amount of anucleate fragmentation (conventional method): grade 1 (g1), blastomere uniform in size and shape and little or no fragmentation; grade 2 (g2), blastomeres uneven in size and shape and/or fragmentation <10% of the embryonic surface; grade 3 (g3), fragmentation of 1030% of the embryonic surface; and grade 4 (g4), fragments >30% of the embryonic surface.
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Finally, all of the embryos were classified into 12 groups from g1A to g4C using a combination of the conventional and nuclei counting methods (combination method).
Blastocysts that had been classifieds by the combination method on day 2 were transferred on day 5.
Statistical analysis
The 2-test was used to compare implantation and blastulation rates. Students t-test was used to compare the ages between the groups. The statistical significance was defined as P < 0.05.
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Results |
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These findings indicate that the nuclei counting method may be more suitable for the evaluation of embryo quality than the conventional method.
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Discussion |
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Our results show that the implantation rate decreased sequentially from g1 that is considered to be good using the conventional method, which reconfirms that the degree of fragmentation and blastomere uniformity are important markers for morphological evaluation of early stage embryos. On the other hand, in the nuclei counting method, the implantation rate of gA was high and those of gB and gC were very low. Thus, which evaluation method should be applied dominantly to select the embryos to transfer? To clarify this point, we developed and tested a new grading system where the two methods were combined (combination method). Using this method, we found that the implantation rates for each grade decreased in order from g1A to g4C applying the nuclei counting method, but did not do so using the conventional method of morphological analysis (Table III). The embryos in g1B and g1C, which are considered to be high quality according to the conventional method but low quality according to the nuclei counting method, showed a low implantation rate. Moreover, the implantation of blastocysts rarely occurred outside the gA group (Table II), indicating that the rates of blastocyst formation and implantation in the gB and gC groups could be remarkably low even when these embryos are cultured longer. These results indicate that observations of nucleic features in the blastomeres of day 2 embryos can be important criteria for selecting the best embryos suitable to transfer.
Although the reason for the low implantation rate of embryos in the gC can be explained by the MNB presence, the low implantation rate of embryos in the gB which contained blastomeres with no visible nucleus is more noteworthy. Usually, when MNB is not observed, the embryo is judged to be normal, which means that gB embryos are considered to be normal. What, then, is the clinical significance of the anucleated blastomere? It is interesting that Palmstierna et al. (1998) reported that visible mononucleated blastomeres and zona pellucida thickness variation seemed to be strong predictors of pregnancy. Given that the development of blastomere nuclei involves distinct phases during which the nuclei are more or less visible, it is critical that observers using the nuclei counting method evaluate gB embryos often at consistent time intervals. When a nucleus is still unobservable, the embryo may contain an anucleated blastomere. Further investigation is required to clarify the importance of cell cycle synchrony when mononucleated blastomeres are observed simultaneously.
Although it is well known that pregnancy rates in older women are low (Feldberg et al., 1990; Stolwijk et al., 1997
), the causes of embryo quality deterioration are not fully documented. In the present study, older patients did not have much fragmentation in their embryos but had more multinucleation. These results suggest that an increase in MNB in the embryos may be a cause of deceased implantation rates in older women.
Choosing an embryo with the highest implantation potential is the key to both successful single embryo transfer and the prevention of multiple pregnancies. Based on this study, we found that when choosing an embryo for transfer, the normality of blastomere nuclei may be more important than conventional measures of fragmentation and/or blastomere uniformity. If blastomere nuclei normality is identical, then embryos with less fragmentation should be chosen. In cases of blastocyst transfer, a blastocyst whose nucleus was evaluated as normal on day 2 should be selected for transfer at day 5. Hence we recommended that all reproductive clinics consider using our combined evaluation method to increase their chances for success during IVF.
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Acknowledgements |
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References |
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Submitted on March 17, 2003; resubmitted on August 26, 2003; accepted on September 17, 2003.