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: blastomere/embryo/fragmentation/implantation potential/pregnancy
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Introduction |
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The aim of this study was to calculate the chance of implantation of a day 3 embryo based on its characteristics of cleavage, fragmentation and multinucleation. The model used relies on the known implantation behaviour of a subgroup of transferred embryos consisting of all embryos with 0% and all embryos with 100% implantation. Transferred embryos were split up according to the different characteristics, then for each type of embryo the implanted fraction was calculated. These implanted fractions offered a qualitative ranking for all types of transferred embryos. Extrapolation to the entire population allowed the attribution of a quantitative implantation rate to each distinct type of embryo.
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Materials and methods |
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All embryos were scored for 3 parameters on day 2 (4144 h after insemination/injection) and again on day 3 (6671 h post insemination/injection): (i) fragmentation: F1, 10% of anucleated fragments; F2, 1020% anucleated fragments, etc.; (ii) number of blastomeres; (iii) number of multinucleated blastomeres (MNB).
Fragmentation was considered only on day 3 in this study because it offers the most relevant information just prior to transfer.
From May 20, 1997 to November 30, 1999 a total of 910 ovum retrievals resulted in 858 transfers (94%). All transfers were performed with the Edwards-Wallace embryo replacement catheter (Simms Portex Ltd, Hythe, Kent, UK) with the use of a stylet (Naaktgeboren et al., 1997). Because implantation rates decrease dramatically after the age of 38 years (van Kooy et al., 1996) only patients <38 years old at the moment of transfer were included in the calculations. There were 745 such transfers (1540 embryos) leading to 50 (7%) biochemical pregnancies, 35 (5%) clinical abortions, four (0.5%) ectopic pregnancies, 192 (26%) ongoing singleton, 91 (12%) ongoing twin and eight (1%) ongoing triplet (39.1% ongoing pregnancies per transfer) pregnancies. All triplet pregnancies were reduced to twin pregnancies.
Main causes of infertilty were male-related in 407 cycles (55%): 385 cycles with oligoteratoasthenozoospermia and 22 cycles with a male immunological factor. Main causes of infertility were female-related in 203 cases (27%): 73 tubal, 41 tuboperitoneal, 48 endometriosis, 15 immunological, 24 polycystic ovarian syndrome and two oocyte donation cycles. In 110 cases (15%) clinical diagnosis was idiopathic infertility. Twenty-five cycles (3%) were originally planned as ovulation stimulation with intrauterine insemination, but were converted to IVF because of an unacceptably high number of maturing follicles. A biochemical abortion was recorded when there were at least two increasing HCG values >5 IU/ml. A clinical abortion was recorded when a fetal sac was seen on ultrasound. An ongoing pregnancy was defined as a pregnancy which was ongoing past the first trimester. For the calculation of the ongoing implantation rate, only concepti reaching the second trimester were considered.
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Results |
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Because embryos with >20% fragmentation and multinucleated embryos comprise not 3% of all implanting embryos, it was decided not to analyse them but only to focus on embryos with
20% fragmentation and without MNB that are responsible for the major part (97%) of ongoing implantations.
Consequently for all embryos only two types of fragmentation (F1 and F2) and all their respective (day 2/day 3) combinations of cleavage rate had to be scrutinized (Tables III and IV).
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Because some good quality embryos will end up in the no-implantation group there will be an underestimation of implantation rates in both Tables V and VI. These unfortunate circumstances can be expected to be evenly spread over all kinds of embryos, because they are not related to embryo quality. For this reason a correction factor C was introduced, which was calculated using the following parameters: the total number of ongoing implantations (Timp), which was 395; the number (N) and the implanted fraction of each type of embryo (Fimp). Only F1 and F2 embryos were considered representing 206 of all 213 embryos with 100% implantation. This led to the equation Timpx206/213 = Cx
(NxFimp). In other words: the total number of implantations due to F1 and F2 embryos must be equal to the sum of the contributions of all different types of cleavage rate in embryos with
20% fragmentation. This equation results in a value of 1.095 for C.
Calculation of the implantation potential for each type of embryo
Multiplication of all implanted fractions of Table V and Table VI
by the correction factor C and by 100 leads to the values shown in Table VII
and Table VIII
, where the percentage implantation of each type of cleavage can be found for embryos with a fragmentation of F1 and F2 respectively.
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Discussion |
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The number of transferred embryos varies considerably between different types, not only because they are created in these amounts, but especially because of the selection by the embryologist. This selection bias, however, does not have an impact on the implanted fractions. Small sample size will have a bearing upon the reliability of the calculated data in some categories and results should be interpreted with these limitations in mind.
It is remarkable to see what is generally considered as the optimal cleavage pattern (from four cells on day 2 to eight cells on day 3) to be associated with the optimal ongoing implantation potential: for both F1 and F2 fragmentation it is 47%. A deviation of this optimal cleavage pattern seems to be more severely sanctioned with F2 fragmentation than with F1 fragmentation in terms of implantation.
Embryos with two blastomeres on day 2 and at least seven on day 3, 10% fragmentation and no MNB also seem to have a very high ongoing implantation rate of 8/23 (35%). Embryos with the same cleavage rate but with 10-20% fragmentation do much worse: 0/7. Embryos with three blastomeres on day 2 also have a poor outcome: 5/40 (13%) for fragmentation F1 and 2/29 (7%) for fragmentation F2.
High cleavage rates on day 2 seem unfavourable because, of the embryos with F1 fragmentation and documented implantation counting six or more cells, only 3/17 (18%) implanted, whereas for the same category with fragmentation F2 the score was 0/19. Both categories taken together scored only 3/36 (8%). On the other hand, embryos with a normal cleavage pattern on day 2, with four or five cells and an accelerated cleavage of 10 cells on day 3, have a far better prognosis: with a fragmentation F1 there is 11/34 ongoing implantation (32%) and with a fragmentation F2 4/12 (33%) or cumulatively 15/46 (33%). These findings could be summarized as: day 2 fast cleavage (>5 cells) is unfavourable especially if it is associated with
10% fragmentation, but day 3 fast cleavage seems most often a genuine sign of proper embryo development. Although it has recently been reported that ICSI embryos have a higher cleavage rate on day 2 compared to conventional IVF (Dumoulin et al., 2000
), all three embryos with proven implantation and showing more than five blastomeres on day 2 originated from conventional IVF.
As a result of our analysis, embryos with MNB deserve special interest. Transferring embryos with MNB was avoided as much as possible, they were only used where no other options were available. Out of 1540 transferred embryos; 89 contained at least one MNB (5.8%). Of these, 62 belonged to the group of transfers without implantation and seven to the group with a biochemical pregnancy. It is worth noting that four biochemical pregnancies could be attributed with certainty to an embryo showing MNB: two single embryo transfers each with a MNB embryo and two double transfers both with two MNB embryos each. This means that MNB embryos are able to become (hatched) blastocysts. This is an argument in favour of active embryo selection instead of just relying on (passive) embryo selection by extended culture (Gardner et al., 1998; Gardner and Schoolcraft, 1998
). It certainly defies the view that `prolonging the duration of culture to day 5 should allow chromosomally competent embryos to develop to the blastocyst stage, thereby increasing the likelihood of identifying euploid, developmentally competent embryos for transfer' (Racowsky et al., 2000
). Indeed, out of 213 embryos with documented ongoing implantation there was only one embryo with MNB (0.5%) whereas in a similar series of 46 embryos (Van Royen et al., 1999
) there was none. Only one MNB-embryo in 213 implanting embryos compared with 89 MNB out of 1540 transferred means that the impairment of implantation of MNB embryos is significant. This observation is in line with other authors (Pickering et al., 1995
; Jackson et al., 1998
). A possible explanation was proposed by Tesarik (Tesarik et al., 1987
) who found that multinucleated embryos can cleave beyond the 8-cell stage and demonstrated that these MNB lack normal RNA synthesis, suggesting a defective transcription of genes that eventually leads to developmental arrest. If this happens after implantation it will result in early pregnancy wastage. The impact of MNB embryos on implantation rates should not be underestimated, as, in 1999, of all 3163 two-pronuclear (2PN) oocytes obtained in our centre 854 (27%) were scored having MNB on day 2 and/or on day 3, considerably more than reported by another group (Balakier and Cadesky, 1997
), who found only 14.5% multinucleation. In our centre the fraction of multinucleated embryos has increased steadily over the years as gradually more effort has been put into retrieving the phenomenon. The actual data show that the use of this single selection criterion allows the elimination of at least a quarter of all embryos.
The cryopreservation of embryos most often results in a decreased implantation potential. This paper might offer some guidance in deciding which embryos are worth freezing. It allows centres to reconsider the cost-effectiveness of their cryopreservation programme.
It should not be forgotten that embryo quality as it can be visually established, is only one factor related to success in IVF/ICSI. The age of the woman, the rank of the cycle and a previous ongoing pregnancy are other factors that are proven to have an impact (Commenges Ducos et al., 1998). These factors were not integrated in our model; their impact was disregarded for the sake of simplicity. Consequently the implantation rates are mean values that will be underestimated for young women in a first treatment cycle and if they have given birth before. Mutatis mutandis, the opposite is also true. Within the same cycle the data and calculation may offer guidance in making the optimal embryo selection and, bearing in mind the previous remarks, in improving the ongoing implantation rate while reducing the risk for multiple pregnancy. Meanwhile they have been applied in our centre (Gerris et al., 1999
), allowing single embryo transfer in >30% of all cycles with a pregnancy rate of 38%.
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Notes |
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References |
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Submitted on July 17, 2000; accepted on October 13, 2000.