1 St Luke Clinic, 5, Tsumori-Tomioka, Oita City 870-0947 and 2 Department of Obstetrics and Gynecology, Oita University Faculty of Medicine, 1 Idaigaoka, Hasamacho, 879-5593, Oita Prefecture, Japan
3 To whom correspondence should be addressed. e-mail: st-luke{at}oct-net.ne.jp
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Abstract |
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Key words: blastocyst culture/embryo transfer/hatching stage/pregnancy rate
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Introduction |
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Conversely, human pronucleate (PN) embryos have been scored (Scott and Smith, 1998) for:closely aligned pronuclei, polarity in nucleoli, heterogeneous cytoplasm, etc. The implantation rate of embryos with high scores was better than that for those embryos with lower scores. Furthermore, using criteria based on the number and distribution of nucleolar precursor bodies in each pronucleus, we can predict the further developmental progression of embryos (Tesarik and Greco, 1999
). A higher clinical pregnancy rate was achieved in cases where at least one high quality pattern embryo was transferred. The predictive value of the 72 h blastomere cell number of blastocyst development has also been reported (Shapiro et al., 2000
), and the results were that more developed 72 h embryos have a better chance to progress to the blastocyst stage and expand.
To obtain good results with ART, the most important factor is to utilize the selection method which gives the best embryo for ET. In our previous study (Utsunomiya et al., 2002), we witnessed a phenomenon that some embryos which reached the blastocyst stage did not hatch in subsequent days. Therefore, we theorized that not all blastocysts may hatch and this may be one reason why the blastocyst stage ET was less effective in our study. In this study, we evaluated whether the hatching blastocyst stage ET (Hat ET) improved the pregnancy outcome in assisted reproductive technology (ART).
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Materials and methods |
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In the blastocyst culture programme, there are patients who had no more than one 2PN; the cancellation rate of ET was too high and the pregnancy rate was extremely low in these poor responders. So, in this study, the extremely poor responders were excluded. Then, the patients who had two or more 2PN were assigned prospectively depending on the sequence, e.g. the first patient was assigned to the day 3 group, the second patient to the Hat ET group, the third patient to the day 3 group, and so on.
Ovarian stimulation was performed using HMG (HMG Nikken, Nikken Kagaku, Tokyo, Japan) with GnRH agonist (Buserecur, Fujiseiyaku, Tokyo, Japan), using the extended protocol. HCG (Profasi; Serono, Switzerland, 10 000 IU) was administered when the diameter of the dominant follicle was 20 mm.
Transvaginal oocyte aspiration was performed 34 h after HCG was administrated. Oocytes were cultured in drops of human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA) with 10% patient serum for the first 3 days, transferred into Blastocyst Medium (Cock IVF, Sydney, Australia) on day 3, and cultured for 2 days until the hatching phenomenon occurred. When embryos reached the hatching stage, ET was performed. If the second hatching embryo was obtained after the first ET day using one embryo, the second ET was done on that day as well, and this cycle came under the first ET day group. The total number of embryos used for ET was two. Ultimately, a total of two hatching stage embryos were transferred.
If the hatching phenomenon did not occur by a further 23 days culture after the stage of expanded blastocyst, it was determined that the embryos had failed to develop and ET was cancelled.
The control group consisted of 240 cycles, for which we performed ET at day 3 between February 2001 and June 2003. Assisted hatching was not performed for either group. Pregnancy was diagnosed by the ultrasound finding of a gestational sac.
Statistical analysis
Data were compared with the use of the 2 test and the Students t-test.
Sample size calculation
In our clinic, the pregnancy rate of ART from January to December in 2000 was 26.1% (140 out of 536). The required sample size was based on improving the success rate of 26% with expectant management to 38% with the use of Hat ET. A trial with a power of 80% and an of 0.05 would require a sample of 236 women.
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Results |
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Among the Hat ET group, the results of Hat ET in the day 5, day 6, day 7 and day 89 groups were compared (Table II). The pregnancy rate, implantation rate and ongoing pregnancy rate of Hat ET for both the day 5 and day 6 groups were higher than those for the day 79 groups.
The results were compared in each subgroup. The younger subgroups (<36 years old), pregnancy rate, implantation rate and abortion rate were the same in both the Hat ET group and the day 3 ET group (Table III). The older subgroups (36 years old) results were the same as for the younger subgroup (Table IV). The patients were divided into two groups according to the number of 2PN. The results showed that the pregnancy rate and implantation rate for the patients who obtained four or more 2PN embryos were not significantly different between the Hat ET group and the day 3 ET group. The patients who obtained only 23 2PN also showed no significant difference between the Hat ET group and the day 3 ET group in pregnancy rate, implantation rate and abortion rate (Tables V and VI).
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Discussion |
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We reviewed the comparison of results between blastocyst stage ET and conventional day 3 ET using a prospective randomized protocol and found that no statistically significant advantage was obtained by using blastocyst stage ET (Utsunomiya et al., 2002). Other studies, using a controlled, randomized trial, found similar results (Coskun et al., 2000
; Huisman et al., 2000
). Many studies which showed better results in blastocyst stage ET were designed with a retrospective protocol. Table VIII shows other prospective study reports that found no advantage in blastocyst stage ET.
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According to a number of reports, which evaluated the selection of good quality, viable embryos, we may be able to select favourable embryos at every stage of development (i.e. the pronuclear stage, the cleavage stage and the blastocyst stage).
There are a few reports of the further culture of the blastocyst stage. These reports suggest that continuing the blastocyst culture into the hatching stage may aid the final selection of the best embryo. Implantation rates between day 5 and day 6 blastocyst stage ET were compared (Khorram et al., 2000). Khorram et al. (2000
) also attempted blastocyst stage ET and were unable to adequately select the best embryos for transfer in many cases, because many embryos were in either the morula or early blastocyst stage on day 5. Therefore, the day of ET was delayed until day 6, and a much better implantation rate was obtained. Achieving a blastocyst stage on day 5 and a hatching stage on day 6 can be considered favourable prognostic factors for the outcome of IVF. In our study, among the Hat ET patients, the pregnancy rates of Hat ET on day 5 and day 6 were better than for the day 7 or more group (Table II). This fact indicates that viable embryos have the ability to reach the hatching stage within 56 days after the insemination and the inferior embryos need more time to hatch.
The results of the Hat ET group and the day 3 ET group were also compared in subgroups in younger patients (Table III), older patients (Table IV), patients who had 4 embryos (Table V) and patients who had only 23 embryos (Table VI). In the Hat ET group, the results of cycles where one embryo was transferred were compared with the cycles where two embryos were transferred (Table VII). The results were not significantly different in each subgroup. This fact indicates that the further culture of embryos may not contribute better results compared with the conventional day 3 ET in any group.
The results of this study showed no significant difference between the Hat ET group and the day 3 ET. The difference of this report compared with other reports may be deduced by the comparative method, i.e. prospective randomized schedule or simple retrospective method, and selected patients or unselected patients. In Table VIII, 28 articles were reviewed. The first 14 articles shown in the table indicate that good results were obtained using blastocyst stage ET when compared with day 3 ET; the second 14 articles reported no advantage to blastocyst stage ET. The large number of reports which showed good results in blastocyst stage ET were retrospective studies and many of them were reported from the USA. On the contrary, no advantages were recognized in blastocyst stage ET in prospective studies, and many of them were reported from European countries.
There were many reports which showed good results with blastocyst stage ET, and the advantages of the blastocyst/hatching blastocyst stage ET were explained by several theories. Some unknown effects on endometriumblastocyst interaction may be induced by transferring hatching stage embryos. Immunohistochemical studies have shown that endometrial epithelial cells do stimulate interleukin (Tabibzadeh et al., 1995), integrins (Lessey et al., 1995
) and many other kinds of cytokines and pinopodes (Nikas et al., 1995
; Bentin-Ley et al., 1999
), which are thought to participate in embryoendometrium interactions during adhesion. The high pregnancy and implantation rates were obtained in a group of patients after the transfer of day 5 blastocysts with enzymatic treatment of the zona pellucida (Hurst et al., 1998
; Fong et al., 1998
). They reported that the favourable results may have been attributable to a better cellcell interaction between the blastocyst and the endometrium secondary to a zona softening and/or complete zona removal.
These explanations, which proposed the advantages of blastocyst stage ET, may be both theoretical and mutually supportive. Clinically, however, the pregnancy rate of blastocyst stage ET was at the same level as with the conventional day 3 ET in our previous report (Utsunomiya et al., 2002), other reports (Table VIII) and this study. This discrepancy is very difficult to explain. An embryo at <3 days after fertilization is characterized by a low level of biosynthesis, a low respiratory rate and limited capacity to utilize glucose as an energy source. In contrast, after the compaction stage, the biosynthetic rate increases and the embryo requires energy from the environment (Gardner and Lane, 1997
). The composition of the culture media in which the embryos develop may be important.
At present, the quality of the culture media for further embryonic cleavage stages may be inadequate. Further studies with a large number of patients are necessary to address this issue adequately.
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Submitted on September 12, 2003; accepted on April 2, 2004.