Fertility Clinic, Department of Obstetrics-Gynaecology-Fertility, Middelheim Hospital, Lindendreef 1, 2020 Antwerp, Belgium
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Abstract |
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Key words: embryo characterization/ICSI/implantation potential/IVF/multiple pregnancies
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Introduction |
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This dilemma could be overcome, if it were possible to select embryos with a very high implantation potential. Culturing for a prolonged period of time until the blastocyst stage is a way of tackling this problem. However, as culture conditions are still imperfect, the longer culture lasts, the fewer embryos suitable for transfer are left. It would be more convenient if an equally effective selection could be performed, but at an earlier stage. In an attempt to establish better selection criteria, we decided to examine retrospectively the characteristics of embryos that all had resulted in an ongoing implantation. We also examined the consequences of the application of these criteria.
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Materials and methods |
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All embryos were scored for three parameters on day 2 (4144 h after insemination/injection) and again on day 3 (6671 h post-insemination/injection): (i) fragmentation (A = no fragmentation, B = 20% or less by volume of anucleated fragments, C = 2050% by volume of anucleated fragments); (ii) number of blastomeres; (iii) number of multinucleated blastomeres.
From January 1, 1996 to May 19, 1997 the JansenAnderson embryo transfer catheter (Cook, Queensland, Australia) was used. All data concerning the establishment of top quality embryo criteria originate from this period.
In May 1997 we changed our transfer procedure to the EdwardsWallace embryo replacement catheter (Simms Portex Ltd., Hythe, Kent, UK) with the use of a stylet (Naaktgeboren et al., 1997). From May 20, 1997 to July 31, 1998 a total of 409 ovum retrievals were performed. Because of personal preferences, another procedure was used in nine transfers. To preserve the homogeneity of the group results from these nine cycles (ending in two ongoing singleton pregnancies) were not included in this study. Main causes of infertility were male related in 211 cycles (53%): 193 cycles with oligoteratoasthenozoospermia and 18 cycles with a male immunological factor. Main causes of infertility were female related in 107 cycles (27%): 39 tubal, 22 tuboperitoneal, 25 endometriosis, four immunological, 16 polycystic ovaries, and one cycle with oocyte donation. In 75 cases (19%) clinical diagnosis was idiopathic infertility. Seven cycles (2%) were originally planned as non-IVF stimulations, but were converted to IVF because of an unacceptably high number of maturing follicles. The mean age of patients was 31.5 years with a standard deviation of 4.82. This mean age is underestimated by 0.5 years because only the integer number of years was recorded. ICSI was performed in 162 cycles (40.5%) of which 21 were with non-ejaculated sperm (microepididymal sperm aspiration and testicular sperm extraction).
A biochemical abortion was recorded when there were at least two HCG values >5 IU/ml and incremental. A clinical abortion was recorded when a fetal sac had been seen on ultrasound. An ongoing pregnancy was defined as a pregnancy that was ongoing past the first trimester. For the calculation of the ongoing implantation rate, only concepti reaching the second trimester were considered. Confidence interval analysis (Gardner and Altman, 1986) was used for statistical analysis.
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Results |
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Top quality embryo and implantation potential
Table III shows an overview of all 400 cycles recorded between May 20, 1997 and July 31, 1998, after we had established top quality characteristics, according to the patient's age group (<38 or
38 years) on one hand and the number of transferred embryos on the other.
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In this paper we only considered the former category, because it offered a much simpler analysis of the impact of embryo quality on implantation and twinning rate.
These 221 transfers of two embryos were divided into three groups according to the number of top embryos as defined above: 2, 1 or 0. Results are shown in Table IV. Mean age was not different in the three groups consisting of 104, 65 and 52 transfers. Ongoing pregnancy rates were 63, 58 and 23%. Pregnancy rates were not significantly different between the first two groups, i.e. the two groups containing either one or two top embryos.
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Twinning rates are significantly different between groups: 37 twins in 65 pregnancies (57%) in the first group, and eight twins in 38 pregnancies (21%) in the second group. With the third group where no twins occurred in 12 pregnancies, no statistical analysis was possible due to the small sample size.
Ongoing implantation rates of 102/208 (49%) versus 46/130 (35%) were significantly different between both groups with top embryos and there was a highly significant difference between both groups containing two or one top embryo versus the group containing none which had an ongoing implantation of only 12/104 (12%).
There was no difference between IVF and ICSI results.
In order to check what percentage of transferred embryos in non-pregnant cycles were top quality embryos, we attempted to match transfers of two embryos with the same main cause of infertility and the same maternal age to the 23 transfers that led to the establishment of our criteria (Tables I and II). We managed to find 21 matching transfers. Of the 42 embryos involved in these, only 18 (43%) showed all top embryo characteristics. Failure to comply with these characteristics was due to presence of multinucleated blastomeres (three embryos), >20% fragmentation on day 3 (six embryos), but most frequently to a discrepancy in the number of blastomeres on day 2 (15 embryos) and on day 3 (20 embryos). Most often it was due to a combination of these parameters. Table V
shows the frequency distribution for these non-implanting embryos on day 2 and day 3 according to the number of blastomeres.
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Discussion |
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Recently it was demonstrated that embryos displaying multinucleated blastomeres have a severely impaired implantation potential (Jackson et al., 1998; Pelinck et al., 1998
). This means the appearance of multinucleated blastomeres is another important quality related parameter. In 1995, immediately after the appearance of the article by Pickering (Pickering et al., 1995
), we tried to avoid transferring embryos with multinucleated blastomeres.
Instead of using the common concept of embryo quality and just extending it with this parameter, we decided to approach it from a different perspective. We decided to analyse the properties of embryos that had proved to be of top quality, i.e. embryos that beyond any doubt had implanted and evolved into an ongoing pregnancy. Then we tried to find the common features of such embryos in the same way that reference values are established. These features would characterize a top quality embryo.
To our knowledge there is no article where an attempt has been made to describe embryos with a maximal implantation potential based on the observation of implanted embryos cultured to day 3. By doing so we have defined a type of embryo with an ongoing implantation rate of 49%. This would suggest that, if there has not been an embryo-helping effect in these double transfers, we might expect similar implantation rates and ongoing pregnancy rates in single embryo transfers with this type of embryo.
It must be emphasized that the number of blastomeres (and maybe even the fragmentation) may vary with culture conditions and with timing of evaluation. This means our criteria are not absolute.
As only 52 out of 221 transfers did not involve any top embryos, we might expect to be able to treat 75% of the population now receiving two embryos with single embryo transfer. Later a new transfer strategy will have to be developed for the third transfer onwards, for the group <38 years now receiving more than two embryos. This will not be simple because a good embryo quality alone is not enough to achieve pregnancy. The better the embryo quality, the clearer the impact of pregnancy-preventing factors not related to embryo quality, such as lack of endometrial receptivity and shortcomings in the transfer procedure. Although we obtained a significant difference in implantation rate (and twinning rate) between the group with one and the group with two top embryos (Table IV), there was no difference in pregnancy rate: although implantation rate increased by 14% (from 35 to 49%), pregnancy rate increased only by 5% (from 58 to 63%) but the twinning rate almost tripled (from 21 to 57%). At 63% pregnancy we may be close to the barrier formed by these non-embryo-quality-related pregnancy-preventing factors. By transferring two embryos with an implantation rate of 49%, one would expect 49% + 0.51x49% = 74% pregnancy rate (Gardner and Schoolcraft, 1999
).
The implantation rate of these top embryos seems very similar to those of blastocysts reported by Gardner (Gardner et al., 1998). Our approach of transferring on day 3, however, implies a shorter culture time and less cost, and avoids the risk of having no blastocyst embryo available for transfer in about 40% of patients (Scholtes and Zeilmaker, 1998
; Shoukir et al., 1998
). A remarkable attempt to successfully evaluate and transfer pronuclear embryos has been reported (Scott and Smith, 1998
). These workers managed to select a group with 28% implantation. In accordance with the suggestion of Edwards and Beard, who found it essential to combine this pronuclear evaluation together with blastocyst culture on the same embryos to see if both systems would select the same embryos (Edwards and Beard, 1999
), we would suggest also to apply our selection system. As our approach fits right in the middle of these two extremes, maybe the use of all three types of evaluation on the same embryos might lead us to conclude what is the optimal time to transfer: when there will be no further gain in selection by prolonged culturing.
In the group 38 years old no multiple pregnancies occurred, thus we see no reason to change our strategy of transferring three embryos in those <40 and four or more in patients of
40 years of age.
A controlled, prospectively randomized, study has meanwhile been completed to test our speculations.
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Notes |
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References |
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Coetsier, T. and Dhont, M. (1998) Avoiding multiple pregnancies in in-vitro fertilization: who's afraid of single embryo transfer? Hum. Reprod., 10, 26632670.[Abstract]
Cummins, J.M., Breen, T.M., Harrison, K.L. et al. (1986) A formula for scoring human embryo growth rates in in vitro fertilisation: its value in predicting pregnancy and in comparison with visual estimates of embryo quality. J. In vitro Fert. Embryo Transfer, 3, 284295.[Medline]
Edwards, R.G. and Beard, H.K. (1999) Is the success of human IVF more a matter of genetics and evolution than growing blastocysts? Hum. Reprod., 14, 16.
Gardner, M.J. and Altman, D.G. (1986) Confidence intervals rather than P values: estimation rather than hypothesis testing. Br. Med. J. (Clin. Res.), 292, 746750.
Gardner, D.K. and Schoolcraft, W.B. (1999) Elimination of high-order multiple gestations by blastocyst culture and transfer. In Shoham, Z., Howles, C.M. and Jacobs, H.S. (eds), Female Infertility and Therapy Current Practice. Martin Dunitz, London, pp. 267274.
Gardner, D.K., Vella, P., Lane, M. et al. (1998) Culture of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil. Steril., 69, 8488.[ISI][Medline]
Giorgetti, C., Terriou, P., Auquier, P. et al. (1995) Embryo score to predict implantation after in-vitro fertilization: based on 957 single embryo transfers. Hum. Reprod., 10, 24272431.[Abstract]
Hu, Y., Maxson, W.S., Hoffman, D.I. et al. (1998) Maximizing pregnancy rates and limiting high-order multiple conceptions by determining the optimal number of embryos to transfer based on quality. Fertil. Steril., 69, 650657.[ISI][Medline]
Jackson, K.V., Ginsburg, E.S., Hornstein, M.D. et al. (1998) Multinucleation in normally fertilized embryos is associated with an accelerated ovulation induction response and lower implantation and pregnancy rates in in vitro fertilisationembryo transfer cycles. Fertil. Steril., 70, 6066.[ISI][Medline]
Naaktgeboren, N., Broers, F.C., Heijnsbroek, I. et al. (1997) Hard to believe, hardly discussed, nevertheless very important for IVF/ICSI results: embryo transfer technique can halve or double the pregnancy rate. Abstract, 13th Annual Meeting of ESHRE. Hum. Reprod., 12, 149.
Pelinck, M.J., De Vos, M., Dekens, M. et al.(1998) Embryos cultured in vitro with multinucleated blastomeres have poor implantation potential in human in-vitro fertilization and intracytoplasmic sperm injection. Hum. Reprod., 13, 960963.[Abstract]
Pickering, S.J., Taylor, A., Johnson, M.H. et al. (1995) An analysis of multinucleated blastomere formation in human embryos. Hum. Reprod., 10, 19121922.[Abstract]
Puissant, F., Van Rysselberge, M., Barlow, P. et al. (1987) Embryo scoring as a prognostic tool in IVF treatment. Hum. Reprod., 2, 705708.[Abstract]
Scholtes, M.C.W. and Zeilmaker, G.H. (1998) Blastocyst transfer in day-5 embryo transfer depends primarily on the number of oocytes retrieved and not the age. Fertil. Steril., 69, 7883.[ISI][Medline]
Scott, L.A and Smith, S. (1998) The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum. Reprod., 13, 10031013.[Abstract]
Shoukir, Y., Chardonnens, D., Campana, A. et al. (1998) The rate of development and time of transfer play different roles in influencing the viability of human blastocysts. Hum. Reprod., 13, 671681.
Staessen, C., Camus, M., Bollen, N. et al. (1992) The relationship between embryo quality and the occurrence of multiple pregnancies. Fertil. Steril., 57, 626630.[ISI][Medline]
Staessen, C., Nagy, Z.P., Liu, J. et al. (1995) One year's experience with elective transfer of two good quality embryos in the human in-vitro fertilization and intracytoplasmic sperm injection programmes. Hum. Reprod., 10, 33053312.[Abstract]
van Kooij, R.J., Looman, C.W.N., Habbema, J.D.F. et al. (1996) Age-dependent decrease in embryo implantation rate after in vitro fertilisation. Fertil. Steril., 66, 769775.[ISI][Medline]
Visser, D.S. and Fourie, F.R. (1993) The applicability of the cumulative embryo score selection and quality control in an in-vitro fertilization/embryo transfer programme. Hum. Reprod., 8, 17191722.[Abstract]
Zhu, J., Meniru, G.I. and Craft, I.L (1997) Embryo development stage at transfer influences outcome of treatment with intracytoplasmic sperm injection. J. Assist. Reprod. Genet., 14, 245249.[ISI][Medline]
Ziebe, S., Petersen, K., Lindenberg, S. et al. (1997) Embryo morphology or cleavage stage: how to select the best embryos for transfer after in-vitro fertilization. Hum. Reprod., 12, 15451549.[Abstract]
Submitted on January 6, 1999; accepted on June 17, 1999.