St Luke Clinic, 5, Tsumori-Tomioka, Oita City, 870-0947, Japan
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Abstract |
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Key words: assisted reproduction/blastocyst/embryo transfer/pregnancy/sequential media
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Introduction |
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This study was designed to evaluate and compare the results of conventional day 3 embryo transfer using a conventional culture medium of human tubal fluid (HTF) and a new technique based on day 5 embryo transfer using sequential media with strictly prospective and alternative allocation schedules.
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Material and methods |
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Finally, 235 patients with 273 cycles were registered and divided into two groups (Figure 1, Table I
). The number of randomized patients in the day 3 embryo transfer group was 121 and the number of cycles 152. In the day 5 embryo transfer group, the number of patients was 114 and the number of cycles 121.The first group (G-I) consisted of 121 patients with 152 cycles using day 3 embryo transfer, which was used as our control group utilizing HTF (Irvine Scientific, Santa Ana, USA). The second group (G-II) consisted of 49 patients with 53 cycles using the day 5 embryo transfer and Irvine blastocyst medium (Irvine Scientific). The third group (G-III) consisted of 29 patients with 30 cycles using day 5 embryo transfer and G1.2/G2.2 medium (IVF Science Scandinavia, Gothenburg, Sweden). Lastly, the fourth group (G-IV) consisted of 36 patients with 38 cycles using day 5 embryo transfer and Cook blastocyst medium (Cook IVF, Sydney, Australia). There was no difference between the day 3 and day 5 embryo transfer groups in mean age, number of retrieved oocytes, number of embryos transferred or endometrial thickness.
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Controlled ovarian stimulation was carried out with HMG (Pergogreen; Serono, Japan) or urinary FSH (Fertinorm; Serono). When the mean diameter of the follicles within the cohort reached 18 mm, 10 000 IU of HCG (Profasi; Serono) was administered to induce the final stage of oocyte maturation.
Oocyte retrieval and insemination
Oocyte retrieval was performed by transvaginal ultrasound-guided aspiration under i.v. anaesthesia with modified neuro-lepto-analgesia (NLA) 34 h after HCG administration. Oocytes were inseminated by conventional IVF or by ICSI 34 h after oocyte retrieval. ICSI was performed if the sperm concentration was <20x106/ml with a total progressive motility <50% and/or sperm morphology <10% normal forms according to strict criteria (Kruger et al., 1988; Menkveld et al., 1991
).
Embryo culture
Embryos were cultured under mineral oil in each medium at 36.7°C in a 5% O2, 5% CO2 and 90% N2 environment. HTF with 10% patient's serum was used for the G-I group.
In the G-II group, embryos were cultured in HTF with 10% patient's serum for 3 days, then transferred into Irvine blastocyst medium with 10% patient's serum and cultured for a further 2 days. In the G-III group, embryos were cultured in G1.2 medium for 3 days after insemination and then carefully transferred into G2.2 medium and cultured for an additional 2 days. In the G-IV group, embryos were cultured in HTF with 10% patient's serum for 3 days, then transferred into Cook blastocyst medium and cultured for an additional 2 days.
Embryo transfer
The embryos were evaluated using embryo scoring (Mills et al., 1992) and blastocysts were graded using a blastocyst scoring system (Gardner et al., 1999). Around three good quality embryos were selected for embryo transfer. If no blastulated embryo was obtained on day 5, embryo transfer was cancelled.
Luteal support
To support the luteal phase, the patients were injected with 1000 IU HCG on the day of embryo transfer, day 4 and day 7. Additionally, 30 mg of didrogesterone (Daiichi Seiyaku, Tokyo, Japan) was administered daily for 16 days. Some of the patients were not administered HCG in order to avoid OHSS.
Sixteen days after ovum retrieval, the urinary HCG level was checked. A transvaginal ultrasound visualization of the gestational sac was performed to confirm clinical pregnancy 23 days after ovum retrieval.
Statistical analysis
Data were compared with the use of the 2- and Student's t-tests.
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Results |
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In the day 3 embryo transfer group, there was no difference in pregnancy, abortion or cancellation rates when using either conventional IVF or ICSI. The same held true for the day 5 embryo transfer group. However, in the day 3 group, the implantation rate of the conventional IVF group was higher than that of the ICSI group (Table II).
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Discussion |
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However, contrary to our expectations, the results of our prospective study were not as successful as previous studies. A high pregnancy rate of 87% has been reported (Gardner et al., 2000) using day 5 embryo transfer with sequential media. However, in that report the pregnancy rate of the control group using day 3 embryo transfer was also quite high at 66%. We believe this to be because the patients were selected using a criteria based on a number of favourable characteristics, and who therefore had a good chance of responding well. They had a healthy uterine endometrium, no physiological disorders and no sperm abnormalities. Their main aim was to avoid multiple gestations, as the patients had good odds of becoming pregnant. Also, a good result was reported (Milki et al., 2000
) using P1 medium plus 10% synthetic serum supplement and day 5 embryo transfer in patients <40 years old and with three or more 8-cell embryos on day 3. The result of the day 5 embryo transfer pregnancy rate was 68% compared with the day 3 embryo transfer rate of 46%.
On the other hand, some reports showed no statistical differences between day 3 and day 5 embryo transfer results using prospective studies (Scholtes and Zeilmaker, 1996; Huisman et al., 2000
; Coskun et al., 2000
). We too had the same result in this study. In particular, the cancellation rate of the day 5 embryo transfer group was a little higher than that of the control day 3 embryo transfer. Furthermore, the implantation rate of the day 5 embryo transfer group was a little lower than that of the day 3 group, although this was not statistically significant.
Our blastulation rate of 21.6% is similar to (Coskum et al., 2000; 28%) and lower than other prospective randomized reports (Huisman et al., 2000; 48%). In this study, the mean age of 34 years was higher (30 years in Coskum et al. and 33 years in Huisman et al.) and the average number of previous ART cycles were 4.7 in the day 3 group and 5.5 in the day 5 group (Table I
). These facts showed that there were many `difficult cases' in this study and this is one of the reasons why the blastulation rate was lower than in other studies.
Thus, the day 5 embryo transfer may not become an established clinical practice, due to factors (Tsirigotis, 1998) such as: (i) concerns over nutritional support for embryos cultured long term in vitro; (ii) the lack of specific knowledge with regard to the time needed for the intratubal descent towards the uterus of the naturally conceived embryos and the peri-implantation events (i.e. synchronization of the endometrium); (iii) the lack of established criteria for blastocyst morphology and growth velocity; and not least (iv) the professional worries that embryos will not be available for transfer in
40% of patients after 5 days of laboratory culture (Scholtes and Zeilmaker, 1998
; Shoukir et al., 1998
).
Currently there are many difficulties associated with day 5 embryo transfer. There is the very tough question as to which sequential media would be the best in which to culture the embryos for 5 days. Even though there are many types of sequential media, there is no paper or study to compare each of them except the report on the controlled comparison of commercial media for human IVF between Ménézo B2 medium, Medi-Cult universal and BM1 medium (Ellios Bio-Media, Igny, France) (Staessen et al., 1998). In this study, we have tried to compare the effectiveness of three media for blastocyst stage embryo transfer. Our study demonstrated that two of the media had better results than the other one. Because our sample was not large enough, we could not arrive at a clear conclusion, and more prospective randomized studies are therefore needed.
It is uncertain why the pregnancy rate of G-III was lower than that of G-II and G-IV. One explanation is that it may be due to batch to batch variability. This may be another reason why our blastulation rate of 21.6% (Table II) is lower than that of other reports (Coskun et al., 2000
; Huisman et al., 2000
).
The special medium might play an important role in culturing matured embryos, especially due to its concentration of minerals, electrolytes, vitamins, amino acids and so on. Unfortunately, we do not know the concentration of these materials in some culture media. However, we can guess the concentration of these materials from data obtained from experiments with animals (Gardner and Sakkas, 1993; Gardner et al., 1994
; Lane and Gardner, 1998
). That being said, the fact still remains that the condition of the culture and the materials for human embryos may differ from those required by animals.
Concerning the 5 day culture, a lot of complexities may arise which could easily frustrate situations in the laboratory. We usually evaluate embryo quality every day until the day of embryo transfer. As a result, there are many more culture dishes in the incubators for the 5 day culture than in a conventional 3 day culture. Additionally, the performance of quality control on the culture media every day for 5 days may be quite difficult and complex. Furthermore, the environment in the dishes may be easily contaminated since it provides a nutritious fluid for bacteria and fungi such as Aspergillus. Even in the conventional day 3 embryo transfer procedure, microbial contamination was reported in IVF and embryo transfer (Cottell et al., 1996).
To obtain a good pregnancy rate with ART, an accurate evaluation of embryos is useful. Many reports evaluate embryo quality using simpler methods than the day 5 culture of embryos, including the morphology of the first polar body (PB), pronuclear (PN) morphology, the duration of the 2-cell stage and 72 h blastomere cell number, and these studies have reported good pregnancy rates obtained using these parameters.
The morphology of the first PB at the time of ICSI seems to be a suitable indicator for the development potential of zygotes (Ebner et al., 1999). Hence, the elective transfer of embryos selected on the basis of first PB morphology resulted in a higher pregnancy rate of 35.7% compared with that of the control group at 23.3%.
PN morphology can be used as a selection criterion for embryo transfer with a high implantation rate occurring when two or more embryos are available with juxtaposed pronuclei with nucleoli aligned at the PN interface and with a `halo' effect to the cytoplasm (Scott and Smith, 1998). Additionally, they reported that if there were <50% of the pre-embryos with these characteristics, the pregnancy rate would be very low.
The morphological parameters used to evaluate the number of nucleolar precursor bodies and their distribution in each pronucleus can be used to predict the developmental fate of embryos (Tesarik and Greco, 1999; Scott et al., 2000
). The new evaluation criteria in these reports can be used as early as the PN stage, simply and without requiring repeated observation.
In Germany, the German embryo protection law does not allow embryo selection, only the selection of PN stage oocytes. Therefore, the clinical usefulness of PN stage scoring was reported (Ludwig et al., 2000). This method might help to offer patients in Germany the transfer of two selected PN oocytes, which would reduce multiple gestation rates.
At the early blastomere stage, the cleavage speed may have a predictive value as to the embryo's viability (Sakkas et al., 1998). The early cleavage of embryos up to the 2-cell stage after ICSI might be an indicator of embryo viability. Also, it has been demonstrated that the more developed 72 h embryos are more likely to become blastocysts and expand (Shapiro et al., 2000
). Implantation rates are greater for the transfer of more developed embryos. Contrary to these reports on the blastomere stage, others (Rijnders and Jansen, 1998
; Graham et al., 2000
) demonstrated that the predictive value of embryo morphology on day 3 for subsequent blastocyst formation was limited.
Originally, the idea of blastocyst stage embryo transfer was initiated to avoid multiple gestations in patients who usually obtained a number of embryos, due to the fact that they were good responders (Gardner et al., 1998, 2000
). On the other hand, the most important and difficult issue in this field is the treatment of difficult cases who cannot conceive using any approach. The fundamental situation may be different between these difficult cases and blastocyst embryo transfer cases. So, the majority of difficult cases may not be rescued by blastocyst embryo transfer. Therefore, we should choose culture methods with the type of medium that is adequate for patients with high failure rates.
We conclude that the pregnancy rates of day 5 and day 3 embryo transfer were equal, while the cancellation rate and the abortion rate of day 5 embryo transfer was a little higher than that of day 3 embryo transfer, and that blastocyst stage embryo transfer is not a viable technique to be used routinely in all patients undergoing ART. The indications for blastocyst stage embryo transfer should be studied further.
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Notes |
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References |
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Submitted on January 16, 2001; resubmitted on September 17, 2001; accepted on March 20, 2002.