Calculating the implantation potential of day 3 embryos in women younger than 38 years of age: a new model

Eric Van Royen,1, Kathelijne Mangelschots, Diane De Neubourg, Indra Laureys, Greet Ryckaert and Jan Gerris

Fertility Clinic, Department of Obstetrics-Gynaecology-Fertility, Middelheim Hospital, Lindendreef 1, 2020 Antwerp, Belgium


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
For optimal embryo selection in IVF/intracytoplasmic sperm injection (ICSI), knowledge of the implantation potential is essential. This is a retrospective analysis of morphological characteristics and cleavage kinetics of day 3 embryos resulting in an objective assessment of the relative implantation potential of each distinct type of embryo. Therefore transferred embryos were sampled according to their documented implantation behaviour: all embryos without any implantation on the one hand and all those with 100% ongoing implantation on the other. There were 213 such embryos in the latter group of which only seven (3%) had >20% fragmentation and only one embryo (0.5%) showed multinucleation (an embryo containing >20% fragmentation). For this reason, only embryos with <=20% fragmentation and without multinucleation were analysed. They were split up according to the amount of fragmentation and the number of blastomeres on day 2 and on day 3. For each type, the implanted fraction was calculated, i.e. the number certainly implanted divided by the sum of the number certainly implanted and the number certainly not implanted, thus describing its relative implantation potential. By extrapolation to the entire population it was possible to establish the implantation potential for each type of embryo. Optimal day 3 embryos were calculated to reach a mean of 47% ongoing implantation. By establishing the implantation potential for most embryos, this model also provides useful information about which embryos are worth freezing in a cost-effective cryopreservation policy.

Key words: blastomere/embryo/fragmentation/implantation potential/pregnancy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In IVF/intracytoplasmic sperm injection (ICSI) treatment, several attempts have been made to quantify the implantation potential of embryos as a means to optimize the pregnancy rate while reducing the risk for multiple pregnancy. Recently two trends have developed to make the selection for transfer either very early at the zygote stage (Scott and Smith, 1998Go; Tesarik and Greco, 1999Go; Tesarik et al., 2000Go) or as late as possible at the blastocyst stage (Gardner et al., 1998Go; Gardner and Schoolcraft, 1998Go). Most cleavage stage scoring systems are based on a combination of fragmentation and cleavage rate. Different individual (Cummins et al., 1986Go; Puissant et al., 1987Go) or cumulative embryo scores (Steer et al., 1992Go; Visser and Fourie 1993Go; Hu et al., 1998Go) have been designed, relying on observations of transfers with embryos of mixed quality. Two papers have described a direct link between score, observed quality criteria and outcome: one exclusively considering transfers of embryos showing homogeneous quality characteristics (Ziebe et al., 1997Go) and a retrospective analysis of exclusively compulsory single embryo transfers (Giorgetti et al., 1995Go). Pickering et al. showed that multinucleation is also an important quality-related criterion (Pickering et al., 1995Go). This observation was confirmed by others (Laverge et al., 1997Go; Jackson et al., 1998Go; Pelinck et al., 1998Go). Multinucleation proved to be useful in combination with fragmentation and cleavage rate for embryo scoring (Van Royen et al., 1999Go).

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients were treated with the long protocol for ovarian stimulation. Desensitization was initiated in the midluteal phase with busereline acetate (Suprefact®; Hoechst, Frankfurt, Germany) six times 100 µg per day intranasally. For follicular stimulation purified FSH (Metrodin HP; Serono, Geneva, Switzerland) was used. When three or more follicles reached a size of >=18 mm in diameter, human chorionic gonadodrophin (HCG) 10 000 IU i.m. (Profasi; Serono, Geneva, Switzerland) was administered. A transvaginal ultrasound-guided ovum retrieval was performed 37 h later. Standard IVF/ICSI procedures were used. Culture medium on the day of oocyte retrieval was Ménézo B2 in 25 µl (Laboratoire C.C.D., Paris, France) droplets under oil (Sigma no. M8410; Sigma-Aldrich, Bornem, Belgium). Oocytes were inseminated, each in a separate droplet with 20 000 spermatozoa having a linear motility >22 µm/s in the case of IVF. In the case of ICSI up to 10 injected oocytes were incubated together in a 10 µl Ménézo B2 droplet under oil. On day 1, oocytes were examined for the appearance of two pronuclei and up to 10 fertilized oocytes were cultured together in a 10 µl droplet Ménézo B2 under oil. On day 2, embryos were rinsed and transferred to individual 10 µl droplets of Medi-Cult M3 medium (Medi-Cult, Copenhagen, Denmark) under oil in order to follow their further individual development. All transfers were performed on day 3. A maximum of two embryos was transferred in the first two attempts in women <38 years of age.

All embryos were scored for 3 parameters on day 2 (41–44 h after insemination/injection) and again on day 3 (66–71 h post insemination/injection): (i) fragmentation: F1, <=10% of anucleated fragments; F2, 10–20% 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., 1997Go). 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Analysis of IVF/ICSI implantation results
Six different types of implantation were distinguished: no implantation, biochemical pregnancy, clinical abortion, ectopic pregnancy, ongoing pregnancy with <100% implantation and ongoing pregnancy with 100% implantation. An overview of embryo fragmentation is shown in Table IGo. Only in the first and the last group is the exact behaviour of all embryos known.


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Table I. The number of transferred embryos categorized according to six different types of implantation and three levels of fragmentationa
 
Analysis of the embryos with certain implantation
A total of 125 transfers comprising 213 embryos ended in 100% implantation: 40 single, 78 double and four triple transfers resulted in as many ongoing single, twin and triplet pregnancies. Moreover, one single transfer resulted in a twin and two double transfers in two triplet pregnancies (Table IIGo). The 100% implantation group consisted of 151 (71%) embryos with <=10% fragmentation, 55 (26%) embryos with 10-20% fragmentation and only seven (3%) embryos with >20% fragmentation (Table IGo).


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Table II. The number of all transfers resulting in ongoing implantations according to number of embryos replaced and outcome
 
Only one of the 213 implanting embryos showed multinucleation (an embryo with >20% fragmention). When this one documented implantation out of 89 (1.12%) multinucleated embryos is compared to 212 proven implantations out of 1540 – 89 = 1451 (14.6%) embryos without multinucleation, a significant impairment of implantation of the MNB embryos is found: odds ratio = 0.066 (95% confidence interval = 0.0092-0.4793).

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 IVGoGo).


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Table III. Transferred embryos with <=10% fragmentation categorized according to the number of blastomeres on day 2 and day 3
 

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Table IV. Transferred embryos with 10-20% fragmentation categorized according to the number of blastomeres on day 2 and day 3
 
Calculation of the implanted fraction for each type of embryo
In order to obtain the fraction of ongoing implantation for each specific type of embryo, the number with 100% implantation was divided by the number with known implantation, i.e. the sum of embryos with 0% implantation and those with 100% implantation. This offers a fairly good idea of the relative implantation chances of day 3 embryos as shown in Table VGo for F1 and Table VIGo for F2 embryos. Implantation of each embryo was known for certain in 486/745 (65%) of transfers. These transfers represent 962/1540 embryos (62%), whereas out of 395 embryos with ongoing implantation (Table IIGo) 213 were identified beyond any doubt (54%). This means that the sample represents >50% of the population, a sound basis for extrapolation.


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Table V. Implanted fraction of embryos with <=10% fragmentation categorized according to the number of blastomeres on day 2 and day 3
 

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Table VI. Implanted fraction of embryos with 10-20% fragmentation categorized according to the number of blastomeres on day 2 and day 3
 
Extrapolation to the entire population of transferred embryos
An embryo of good quality is necessary but not sufficient to obtain an ongoing implantation: it also has to be properly transferred and deposited into a receptive endometrium. If anything goes wrong at this level there will be no ongoing implantation leading to a disproportionate increase of the no-implantation group. These factors, not related to embryo quality, will not be present, at least not to the same extent, in the transfers resulting in an ongoing implantation.

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 VIGoGo. 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{Sigma} (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 VGo and Table VIGo by the correction factor C and by 100 leads to the values shown in Table VIIGo and Table VIIIGo, 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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Table VII. Percentage implantation of embryos with <=10% fragmentation categorized according to the number of blastomeres on day 2 and day 3
 

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Table VIII. Percentage implantation of embryos with a fragmentation of 10-20% categorized according to the number of blastomeres on day 2 and day 3
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This paper is a logical continuation of our attempt to characterize a top quality embryo (Van Royen et al., 1999Go). It confirms the previous results and adds new data that have an important practical impact on embryo selection for transfer as well as for cryopreservation. When the characteristics of a top quality embryo (four or five blastomeres on day 2 and at least seven on day 3, <=20% fragmentation and no MNB at any stage) are applied to the embryos with documented ongoing implantation, 174 of these 213 embryos (82%) will be selected. This also means that 18% of all implantations can be attributed to non-top quality embryos.

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., 2000Go), 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., 1998Go; Gardner and Schoolcraft, 1998Go). 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., 2000Go). 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., 1999Go) 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., 1995Go; Jackson et al., 1998Go). A possible explanation was proposed by Tesarik (Tesarik et al., 1987Go) 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, 1997Go), 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., 1998Go). 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., 1999Go), allowing single embryo transfer in >30% of all cycles with a pregnancy rate of 38%.


    Notes
 
1 To whom correspondence should be addressed at: Fertility Clinic, Department of Obstetrics-Gynaecology-Fertility, Middelheim Hospital, Lindendreef 1, 2020 Antwerp, Belgium. E-mail: eric.van.royen{at}pandora.be Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on July 17, 2000; accepted on October 13, 2000.