Early cleavage is a valuable addition to existing embryo selection parameters: a study using single embryo transfers*

Aafke P.A. Van Montfoort1,3, John C.M. Dumoulin1, Arnold D.M. Kester2 and Johannes L.H. Evers1

1 Research Institute Growth & Development (GROW), Department of Obstetrics & Gynaecology, Academic Hospital Maastricht, P.O.Box 5800, 6202 AZ Maastricht and 2 Department of Methodology and Statistics, University of Maastricht, P.O.Box 616, 6200 MD Maastricht, The Netherlands

3 To whom correspondence should be addressed. Email: avmn{at}sgyn.azm.nl


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: To reduce the twin pregnancy rate, elective single embryo transfer (eSET) is increasingly implemented. Improvement of the results obtained with eSET can be achieved by better selection of the most viable embryo. This study investigated the predictive value of early cleavage (EC) as an additional parameter for selecting the embryo with the highest implantation potential by using data from SET's. METHODS: Data from 165 SET's were retrospectively evaluated. Cleavage to the 2-cell stage was determined 23–26 h after injection or 25–28 h after insemination. Selection of the embryo to be transferred was based on cell morphology and cell number on the day of transfer, not on the EC status. Additional information on the predictive value of EC on developmental potential was obtained by analysing 253 transfers with two embryos (double embryo transfer, DET) and blastocyst formation of 1160 surplus embryos. Logistic regression was used to determine the predictors of pregnancy or blastocyst development. RESULTS: A significantly higher pregnancy rate was observed after transfer of single EC embryos compared to single non-EC embryos (46 versus 18%). This result was confirmed by the significantly higher pregnancy rate after DET with two EC embryos as compared to DET with two non-EC embryos (45 versus 25%) and the blastocyst formation of EC embryos compared to non-EC embryos (66 versus 40%). Logistic regression showed that EC is an independent predictor for both pregnancy and blastocyst development in addition to cell morphology and cell number. CONCLUSIONS: In order to improve the selection of the embryo with the highest implantation potential, selection for transfer should not be based on cell number and morphology on the day of transfer alone, but also on early cleavage status.

Key words: blastocyst development/early cleavage/embryo quality/pregnancy/single embryo transfer


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A twin pregnancy is an adverse outcome of an IVF cycle, because of the increased risk for medical complications including prenatal and neonatal mortality and morbidity, preterm delivery, low birthweight and long-term medical and developmental problems (ESHRE Capri Workshop, 2000Go; Elster, 2000Go; Olivennes, 2000Go; Land and Evers, 2003Go). As a result, the medical costs of a twin pregnancy increase dramatically compared to the costs for a singleton pregnancy (Elster, 2000Go; Kinzler et al., 2000Go). In order to prevent this adverse outcome, many recent studies focus on elective single embryo transfer (eSET). The results show that eSET in a selected group of patients leads to an acceptable pregnancy rate and a reduction in the twin pregnancy rate (Gerris et al., 1999Go, 2002Go; Vilska et al., 1999Go; Martikainen et al., 2001Go).

As eSET is increasingly performed, there is an urgent need for better criteria to select the embryo with the highest implantation potential. In some IVF centres, embryos are cultured until blastocyst stage to make a better selection. However, in several studies, blastocyst transfer has not been demonstrated to be a better alternative for transfer of cleavage stage embryos (Rienzi et al., 2002Go) and many centres, including our own, still prefer the transfer of the cleavage stage embryos. At present, the common practice of selecting the most viable cleavage stage embryo is based on embryo morphology and developmental stage on the day of transfer (Giorgetti et al., 1995Go; Ziebe et al., 1997Go; Van Royen et al., 1999Go). Embryo morphology is determined by the number, size and shape of blastomeres, the proportion of fragments and in some studies the presence of multinucleated blastomeres (MNB). It has been demonstrated that after 2 days of culture, the 4- or 5-cell stage is the optimal cleavage stage (Ziebe et al., 1997Go; Van Royen et al., 1999Go). Embryos at this cleavage stage with little or no fragmentation and an absence of MNB achieved a higher implantation and pregnancy rate compared to embryos at another cleavage stage with more fragmentation (Ziebe et al., 1997Go; Van Royen et al., 1999Go).

Recently, new parameters to improve embryo selection have been introduced, e.g. zygote morphology and early cleavage. The morphological parameters for zygote quality include the number of nucleolar precursor bodies (NPB) and their distribution in the pronuclei (Scott et al., 2000Go; Tesarik et al., 2000Go; Wittemer et al., 2000Go). Early cleavage embryos were defined as those which had cleaved to the 2-cell stage 25–27 h after insemination or ICSI (Shoukir et al., 1997Go; Sakkas et al., 1998Go, 2001Go; Bos-Mikich et al., 2001Go; Lundin et al., 2001Go; Fenwick et al., 2002Go; Tsai et al., 2002Go). The conclusion of all these studies was that transfer of early cleavage embryos leads to a higher pregnancy and implantation rate compared to the transfer of non-early cleavage embryos. However, most studies examined transfers of more than one embryo, where only one embryo had to be an early cleavage embryo. As a result, many cycles were included where both early and non-early cleavage embryos were transferred. Therefore it remains uncertain whether the pregnancy or implantation can be attributed to the early cleavage embryo (Shoukir et al., 1997Go; Sakkas et al., 1998Go, 2001Go; Bos-Mikich et al., 2001Go; Fenwick et al., 2002Go; Tsai et al., 2002Go).

Furthermore, it is still unclear whether early cleavage is an independent predictor for pregnancy or whether it is correlated with other pregnancy predictors, e.g. embryo morphology and cell number on the day of transfer. In several studies it has been shown that early cleavage embryos had a significantly higher cell number and a better embryo morphology on the day of transfer, compared to non-early cleavage embryos (Shoukir et al., 1997Go; Sakkas et al., 1998Go, 2001Go; Lundin et al., 2001Go; Fenwick et al., 2002Go). The aim of the present study was to assess the predictive value of early cleavage for pregnancy in relation to embryo quality, i.e. embryo score. Only SET data were analysed. Surplus embryos were used to assess embryonic development to the blastocyst stage. Logistic regression was performed to assess the predictive value of early cleavage for both pregnancy and blastocyst development.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients
During a 2 year period (May 2001 to June 2003), all IVF and IVF/ICSI cycles performed in the Academic Hospital Maastricht that fulfilled the following criteria were included in the present study. First, early cleavage of embryos had to be determined between 23 and 26 h post-injection or 25 and 28 h post-insemination, the time difference being necessary to compensate for the time difference in early development between IVF- and ICSI-derived embryos (Nagy et al., 1994Go, 1998Go). Second, there had to be at least one normally fertilized (two-pronuclear, 2PN) zygote present. Third, the indication for IVF treatment should not be preimplantation genetic diagnosis (PGD).

During this period, either one or two embryos were transferred. To evaluate the predictive value of early cleavage for pregnancy, data collected from a total of 165 cycles in which only a single embryo was transferred on the second day after oocyte retrieval were analysed. SET was performed when: (i) only one embryo was available (n=30); (ii) a top quality embryo (4-cell, grade 4 and no MNB) was available, and the woman was aged <38 years, which leads according to our standard transfer policy to eSET (n=77); (iii) patients agreed to participate in a research project in which one or two embryos were transferred at random irrespective of embryo score (n=47); (iv) there were clear medical or socio/psychological reasons for eSET, e.g. uterine anomaly (n=11).

Besides the 165 SET procedures, a total of 253 double embryo transfer (DET) procedures were analysed, in which either two early cleavage embryos or two non-early cleavage embryos were transferred.

In addition, 1160 surplus embryos originating from 341 cycles remaining after transfer and not suitable for freezing because of inferior quality were cultured until day 6 for analysing the blastocyst formation rate, provided that the patients had signed an informed consent. This study was approved by the local Ethics Commitee.

Ovarian stimulation protocol
Patients were down-regulated with triptorelin daily s.c. injections (Decapeptyl; Ferring B.V., The Netherlands) or nafarelin intranasally (Synarel; Searle BV, The Netherlands). To stimulate multiple follicular development, recombinant FSH (Puregon; Organon, The Netherlands) was used. Follicle growth was monitored by ultrasound. As soon as at least three follicles were ≥18 mm, 5000 IU of hCG (Pregnyl; Organon) was given. Ultrasound-guided oocyte retrieval was performed 36 h after hCG administration.

IVF and ICSI procedure
IVF and ICSI procedures used were as described in detail earlier (Dumoulin et al., 2000Go, 2001Go). Oocytes and embryos were cultured individually in 5 µl droplets covered by mineral oil in an atmosphere of 5% O2, 5% CO2 and 90% N2. Culture medium G1.2 (Vitrolife, Sweden) was alternated with Sydney IVF Cleavage media (Cook, Australia) for each consecutive treatment cycle.

Embryo quality assessment and transfer policy
For each embryo originating from a normally fertilized (2PN) oocyte, an embryo score was calculated on day 2 by multiplying the morphological grade (1 to 4 grading system of Bolton et al., 1989Go) in which grade 4 is morphologically the best) with the number of blastomeres (Steer et al., 1992Go). As it has been shown that the 4- or 5-cell stage is the optimal cleavage stage at day 2 and that the presence of MNB is deleterious for embryo viability (Ziebe et al., 1997Go; Van Royen et al., 1999Go, 2003Go), the score for these embryos was adjusted as follows: embryos consisting of >5 cells were multiplied by 0.9, embryos showing MNB as a 2-cell embryo on any of the two observation time-points (23–26 h post-injection or 25–28 h post-insemination and 41–45 h post-injection/insemination) were multiplied by 0.5 and embryos showing MNB as a >2-cell embryo were multiplied by 0.75. The calculation of embryo scores was performed to make a uniform selection of the embryo to be transferred. In our embryo score system, a 4-cell embryo with morphology grade 4 and an absence of MNB receives the highest score of 16.

Embryos with the highest score available on day 2 were transferred irrespective of the early cleavage status.

Cryopreservation of supernumerary embryos was performed on the morning of the third day if one or more embryos had reached the 8-cell stage, and if they were of good morphological quality. Embryos left over after transfer and not suitable for freezing from consenting patients were transferred alternately to droplets of G2.2 medium or Sydney IVF Blastocyst medium and cultured until the sixth day after ovum retrieval. Each day, developmental stages were recorded.

Pregnancy was determined by a urine hCG test 14 days post transfer. An ongoing pregnancy was defined as the presence of at least one intrauterine gestational sac with fetal cardiac activity at ultrasound examination, 5–6 weeks after ovum retrieval.

Statistical analysis
Statistical analysis was performed using the SPSS software for Windows version 10.1 (Statistical Package for Social Sciences, Inc., USA). To compare the differences between the early cleavage embryos and non-early cleavage embryos, Student's t-test was used for the continuous variables and the {chi}2 test was used for binary variables. To evaluate the impact of embryo score and early cleavage on pregnancy and blastocyst development, two models were constructed by logistic regression analysis. Embryo score was a continuous variable ranging from 1 to 16. Early cleavage was a binary variable (early cleavage 1, non-early cleavage 0). To take into account the possible interaction between embryo score and early cleavage status, an interaction factor between those two variables was also included as a possible independent variable in the logistic regression. By using the equation derived from the logistic regression analysis, a predicted probability for both pregnancy and blastocyst development can be calculated for every embryo score. Based on this predicted probability, a receiver operating curve (ROC), plotting the true positives against the false positives, was constructed. The area under the receiver operating curve (AUCROC) was used to summarize the predictive power of the model, ranging from 0 to 1. P<0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
From the 1134 ovum retrievals performed in the study period, 418 were included in the present study (165 SET and 253 DET). To investigate the predictive value of early cleavage as an additional parameter for selecting the embryo with the highest implantation potential, the data from the 165 single embryo transfers were used. In 71 of these cycles, standard IVF was performed, while in the remaining 94 cycles, ICSI was performed. The proportion of cycles in which ICSI was applied (58%) was comparable to that of the total IVF programme during the study period (54%). Of all embryos obtained, 39% of the ICSI embryos and 38% of the IVF embryos were early cleavers. Of the 165 embryos that were transferred, 97 (59%) were early cleavage embryos, while 68 (41%) were non-early cleavage embryos. The results including embryo score and pregnancy rate are shown in Table I. Both embryo score and pregnancy rate differ significantly between early cleavage and non-early cleavage embryos.


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Table I. Cycle characteristics of single embryo transfers (SET) with an early cleavage embryo and SET with a non-early cleavage embryo

 
To examine the relationship between early cleavage, embryo score and pregnancy, a logistic regression was performed. Early cleavage was a significant predictor when adjusted for embryo score [odds ratio (OR)=3.24; P<0.05]. The interaction between embryo score and early cleavage status was not significant. In Figure 1 the predicted pregnancy rates when transferring an early cleavage embryo or a non-early cleavage embryo are plotted for each embryo score. The ratio between the probability for pregnancy of early cleavage and non-early cleavage embryos decreases with increasing embryo score. The predictive power of this pregnancy model calculated by the AUCROC was 0.68.



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Figure 1. Predicted pregnancy rates for early cleavage (EC) and non-early cleavage (NEC) embryos derived from a logistic regression model.

 
To confirm the positive impact of early cleavage on pregnancy rate, 253 DET's were analysed (Table II). In 97 cases where two early cleavage embryos were transferred, the pregnancy rate was significantly higher compared to the 156 transfers where two non-early cleavage embryos were transferred (45 versus 25%).


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Table II. Cycle characteristics of double embryo transfers (DET) with two early cleavage embryos and double embryo transfers with two non-early cleavage embryos

 
In addition, a total of 1160 surplus embryos from 341 cycles, which were examined for early cleavage, were cultured until day 6 after ovum retrieval. Table III shows the blastocyst formation rate and embryo score for both groups. Twenty-three per cent of the oocytes which were injected cleaved early, compared to 24% of the inseminated oocytes. The logistic regression analysis for blastocyst development showed that embryo score (P<0.0001) and early cleavage (P<0.0001) were significant predictors. In addition, there was also a significant interaction between these two predictors (P<0.05), which means that the effect of early cleavage on blastocyst development is dependent on the embryo score. The advantage of early cleavage for blastocyst development decreases as the embryo score increases. This can also be seen in Figure 2 where the predicted probabilities for blastocyst development based on the logistic regression model are shown. The ratio between the probability for blastocyst development of early cleavage and non-early cleavage embryos decreases as the embryo score increases. Because of this extra independent variable, the OR of early cleavage also becomes dependent on embryo score (Figure 3). The OR decreases as the embryo score increases. The AUCROC as a measure for the predictive value of this model is 0.72.


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Table III. Comparison of characteristics from early cleavage and non-early cleavage surplus embryos

 


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Figure 2. Predicted probability for blastocyst development for early cleavage (EC) and non-early cleavage (NEC) embryos from logistic regression models.

 


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Figure 3. Odds ratios for blastocyst development between early cleavage and non-early cleavage embryos in relation to embryo score.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In order to select the most viable embryo, embryo scoring systems have been developed based on embryo morphology and blastomere number on the day of transfer (Cummins et al., 1986Go; Steer et al., 1992Go; Giorgetti et al., 1995Go; Ziebe et al., 1997Go). Much effort has been devoted to refining existing embryo scoring systems and finding additional simple, non-invasive parameters that could improve the embryo selection procedure (Van Royen et al., 1999Go; Tesarik et al., 2000Go; Wittemer et al., 2000Go). Early cleavage status is one of the most promising new parameters.

In previous studies it was found that transferring embryos that had cleaved early led to a significantly higher pregnancy rate (Shoukir et al., 1997Go; Sakkas et al., 1998Go, 2001Go; Bos-Mikich et al., 2001Go; Lundin et al., 2001Go; Fenwick et al., 2002Go; Tsai et al., 2002Go) and blastocyst development rate (Fenwick et al., 2002Go) as compared to non-early cleavage embryos. This is in accordance with the results obtained in the present study (46 versus 18% for pregnancy rate after SET, P<0.001, and 66 versus 40% for blastocyst rate, P<0.0001). However, most studies examined transfers in which two or more embryos were transferred of which at least one embryo had shown early cleavage. This makes it difficult to conclude to which embryo the pregnancy can be attributed. In our study, SET data were analysed, which makes it possible to determine the relationship between early cleavage and pregnancy arising from one specific embryo. Recently, Salumets et al. (2003)Go also analysed SET data; their results for pregnancy rates are similar to ours (50 versus 26.4%, P=0.001). Furthermore, our results after DET with either two EC embryos or two non-EC embryos (pregnancy rate 45 versus 25%, P<0.001) confirm the findings in the SET group. Early cleavage is now used in a few clinics as a selection criterion, but only to make a distinction when more embryos of identical embryo score are available for transfer (Sakkas et al., 1998Go; Salumets et al., 2003Go).

Concerning the relationship between early cleavage status and embryo quality, it was found previously that, as our results show as well, that early-cleaving embryos had a significantly higher embryo score on the day of transfer (Shoukir et al., 1997Go; Sakkas et al., 1998Go; Lundin et al., 2001Go; Fenwick et al., 2002Go). Sakkas et al. (1998)Go already suggested that this might indicate that an early cleavage embryo would have been selected for transfer anyway, based on cell stage and morphology. This would imply that early cleavage status analysis might be redundant. However, as the results from our logistic regression analysis indicate, both embryo score, calculated before embryo transfer, as described in Materials and methods, and early cleavage status are independent predictors for both pregnancy and blastocyst development. In the blastocyst model, an interaction exists between embryo score and early cleavage, which is not significant in the pregnancy model. This discrepancy can be explained by the fact that the blastocyst data were more numerous than the pregnancy data. Our findings imply that early cleavage status should always be taken into account when selecting the best embryo for transfer and not only when more embryos of identical embryo score are available for transfer. This is not in accordance with the findings of Lundin et al. (2001)Go who found that, after a logistic regression analysis, early cleavage status was not an independent predictor of pregnancy. However, when the analysis was performed separately for ICSI and IVF, early cleavage was shown to be an independent predictor for birth in ICSI cycles, but not in IVF cycles. They also found that embryos derived after ICSI cleaved earlier compared to those after IVF (Lundin et al., 2001Go). This has been explained by Nagy et al. (1998)Go. By injecting the spermatozoon into the oocyte, the zona pellucida and cumulus and corona cell barrier is overcome. This gives the ICSI embryos a temporal advantage of 4 h, which might explain why early cleavage was an independent predictor for birth after ICSI, but not after IVF. According to our data, this time span was only 2 h (data not published). That is the reason why the time interval of 2 h was used between the early cleavage status determination of IVF and ICSI embryos.

The predictive value of the pregnancy model is relatively weak (AUC = 0.68). This is likely due to the fact that there are many other oocyte and embryonic factors which might influence the implantation capacity of the embryo. In addition, a pregnancy is not only dependent on embryo quality, but also on endometrial receptivity, for example.

In our study, the abortion rate was increased in the non-early cleavage group compared to the early cleavage group (42 versus 20%). Although the difference is not statistically significant, it is in accordance with the findings of Lundin et al. (2001)Go who were able to analyse a greater number of cycles.

As in the present study we find that early cleavage has a predictive value both for implantation after transfer of cleavage stage embryos, as well as for blastocyst formation of surplus embryos, it might be expected that early cleavage also has a predictive value for implantation after transfer of blastocysts. This is, however, not examined in the present study.

In conclusion, this study has provided data from a series of SET's showing that early cleavage is a significant predictor of both pregnancy and blastocyst development. Therefore, in order to improve the selection of the embryo with the highest implantation potential, selection of embryos for transfer should not be based on cell number and morphology on the day of transfer alone, but also on early cleavage status. Early cleavage stage analysis provides means of improvement of the embryo selection process that might lead to a transfer policy with a higher proportion of SET's. In turn, this will lead to a reduction of the twin pregnancy rate, which is one of the most serious adverse outcomes of an IVF treatment for both mother and child.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was funded by a research grant (number 945-12-014) from the Dutch Organisation for Health Research and Development (ZonMw) and the Dutch Health Insurance Board (CvZ) in a joined research program on health technology assessment of infertility.


    Notes
 
* Data presented in part at the 19th Annual Meeting of the ESHRE, Madrid, Spain, 2003. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on November 21, 2003; accepted on May 27, 2004.