Live birth rate is significantly higher after blastocyst transfer than after cleavage-stage embryo transfer when at least four embryos are available on day 3 of embryo culture. A randomized prospective study

Evangelos G. Papanikolaou1,2, Elke D’haeseleer1, Greta Verheyen1, Hilde Van de Velde1, Michael Camus1, Andre Van Steirteghem1, Paul Devroey1 and Herman Tournaye1

1 Centre for Reproductive Medicine, University Hospital, Vrije Univestiteit Brussel (Free University of Brussels), Laarbeeklaaan 101, B-1090, Brussels, Belgium

2To whom correspondence should be addressed. E-mail: Evangelos.Papanikolaou{at}vub.ac.be or drvagpapanikolaou{at}yahoo.gr


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
INTRODUCTION: In a randomized controlled trial, we assessed whether pregnancy outcome would be improved by extending embryo culture to day 5 and transferring a blastocyst in patients with at least four good-quality embryos on day 3. METHODS: Multifollicular ovarian stimulation was performed with a GnRH agonist in 44% of patients and with a GnRH antagonist in 56%. Overall, 164 patients younger than 37 years fulfilled embryo quality criteria (at least four having at least six cells on the morning of day 3, maximum 20% anucleate fragments) on the third day of culture and were randomized to the day 3 (n = 84) or day 5 (n = 80) groups. Equal numbers of embryos (n = 2) were transferred in each group. RESULTS: Demographics, stimulation parameters and embryological data were comparable in the two groups. Blastocyst-stage transfer resulted in a significantly higher ongoing pregnancy rate [51.3 versus 27.4%; odds ratio (OR) 2.78, 95% confidence interval (CI) 1.45–5.34] and live birth rate (47.5 versus 27.4%; OR 2.40, 95% CI 1.25–4.59) compared with day-3 embryo transfer. A high twin birth rate was observed in both groups (36.8 versus 30.4%; P > 0.05). CONCLUSIONS: A threshold of four good embryos on the third day of embryo culture appears to indicate that the patient will benefit from embryo transfer at the blastocyst stage and have a better chance of achieving a live delivery than with cleavage-stage embryo transfer.

Key words: blastocyst/cleavage-stage embryo/embryo transfer/GnRH antagonist/pregnancy rate


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Assisted reproduction programmes still lack high implantation rates. Whether blastocyst-stage transfer offers any real benefit to infertile couples remains controversial (Blake et al., 2004Go). Among other arguments for day-5 embryo transfer is the possibility of selecting embryos at a later developmental stage (activation of the embryonic genome), thus enabling the embryos that are to be transferred to be selected on more objective criteria, and from a preselected cohort of embryos. This strategy is reinforced (i) by the widely acknowledged limitations of the morphological criteria used for the selection of cleavage-stage embryos (Rijnders and Jansen, 1988Go), and (ii) by the large proportion of morphologically normal day-3 embryos that are chromosomally abnormal (Magli et al., 2000Go; Staessen et al., 2004Go). On the other hand, although our knowledge of the nutritional and biochemical needs of initial-stage embryos has gradually increased, leading to the introduction of sequential media, there are still patients whose embryos do not reach the blastocyst stage, which deprives them of the chance of an embryo transfer and, potentially, a pregnancy (Jones et al., 1998aGo; Tsirigotis, 1998Go).

This suggests that, at present, in vitro culture conditions might not be ideal for some embryos, certainly no better than the endometrial intrauterine milieu. As a result, the generalized application of blastocyst transfer in each infertile couple is being considered. Therefore, there is a need to develop a policy that will enable us to select the patients who would benefit more from the extension of culture to day 5, without reducing their chances of having an embryo transfer because of developmental arrest. It has been shown that there is a positive correlation between the number of blastocysts developed and the number of eight-cell embryos formed on day 3 (Jones et al., 1998bGo). Furthermore, it has been suggested that patients with more than three 8-cell embryos on day 3 should be offered a blastocyst embryo transfer (Racowsky et al., 2000Go).

The aim of the present prospective randomized controlled trial was to determine, where at least four good-quality embryos have been developed by day 3, whether the extension of embryo culture to day 5, and eventually a blastocyst-stage transfer, is beneficial in terms of increasing ongoing pregnancy and live birth rates, compared with cleavage-stage embryo transfer.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study design
Between January 2001 and November 2003, 301 patients seeking infertility treatment were found to be eligible to participate in the study (Figure 1). Eligibility inclusion criteria were: (i) female age ≤37 years; (ii) rank trial ≤3; (iii) FSH on day 3 of the cycle ≤12IU/ml; (iv) ejaculated sperm origin; (v) IVF or ICSI cycles; (vi) equal numbers (n = 2) of embryos should be transferred in each group. Eligibility exclusion criteria were: (i) oocyte donation cycles; (ii) non-ejaculated sperm (testicular sperm aspiration, fine needle aspiration, micro-epididymal sperm aspiration, percutaneous epididymal sperm aspiration); and (iii) PGD. Finally, 274 patients initiated multifollicular ovarian stimulation, among whom 164 fulfilled the embryological inclusion criterion of having at least four good-quality embryos on day 3 of embryo culture. Embryos of good quality were defined as having a minimum of six blastomeres of normal size on the morning of day 3, a maximum of 20% of anucleate fragments and no multinucleated blastomeres.



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Figure 1. Randomisation flow chart

 

Every patient entered the study only once. The randomization was performed on day 3 of embryo culture by the embryologist in the IVF laboratory using a computer-generated randomized list. The present study was approved by our Institutional Review Board and all the patients gave their signed informed consent.

Our primary outcomes were ongoing pregnancy rate and live birth rate per patient randomized, expressed as a percentage. The secondary outcome measure was the multiple birth rate.

Multifollicular ovarian stimulation
Two ovarian stimulation protocols were used for 274 patients in the present study. The long GnRH agonist protocol with intranasal buserelin (Suprefact; Aventis Pharma, Frankfurt, Germany) was initiated on day 21 of the cycle. Once the patient’s hormonal status was basal, menopausal gonadotrophins (Menopur; Ferring, Kiel, Germany) were started (Papanikolaou et al., 2005aGo). According to the GnRH antagonist protocol, on day 2 of the cycle injections of recombinant FSH (Puregon; Organon, Oss, The Netherlands; or Gonal-F; Serono, Geneva, Switzerland) were initiated. On day 6 of the stimulation, s.c. administration of the antagonist was started (Orgalutran, Organon Oss, The Netherlands; or Cetrotide, Serono, Geneva, Switzerland) (Papanikolaou et al., 2005bGo). The initial gonadotrophin dose remained fixed for 6 days and could then be adjusted until the final day of HCG administration, based on follicular growth and estradiol levels. Final oocyte maturation was induced by administration of 10 000 IU HCG (Pregnyl; Organon) when the criterion of the presence of at least three follicles of 17 mm was met. Luteal phase was supported by vaginal progesterone 600 mg daily (Utrogestan; Besins International, Drogenbos, Belgium).

To assess treatment outcome, serum {beta}-HCG was measured 14 days after oocyte pickup and was repeated 3 days later. A rise in serum HCG (>20 IU) on two consecutive blood tests indicated pregnancy. Clinical pregnancy was defined by the ultrasound observation of fetal cardiac activity after 7 weeks of gestation. An ongoing pregnancy was defined as showing a positive heart-beat at ultrasound after 12 weeks of gestation. Data were further collected during pregnancy until delivery.

Embryo culture, embryo evaluation and selection for transfer
Sperm preparation, IVF/ICSI procedures and embryo culture were carried out as described by Van Landuyt et al. (2005)Go. Oocytes and embryos were cultured in sequential media of Vitrolife Sweden AB, Kungsbacka, Sweden, (GII or GIII series). On the morning of day 3, embryos were transferred from cleavage medium to blastocyst medium. Fertilization was assessed 16–19 h after insemination or injection. Normal fertilization was confirmed by the presence of two pronuclei (2PN) with two distinct or fragmented polar bodies. Embryo quality was assessed daily until the moment of transfer and/or freezing of the supernumerary embryos.

Embryo quality (EQ) on day 3 was scored according to the following parameters: number of blastomeres, rate of fragmentation, multinucleation of the blastomeres, and early compaction. Selection of embryos for transfer on day 3 was based on the above parameters, with preference for embryos which showed the normal developmental pattern of early cleavage on day 1, four cells on day 2 and eight cells on day 3, with minimal fragmentation and no multinucleation. A combination of these parameters resulted in a combined embryo quality score: EQ1 = excellent (top quality); EQ2 = good quality; EQ3 = fair quality; EQ4 = bad quality. The EQ4 embryos were not transferred. A top-quality embryo (EQ1) was considered as having at least eight cells on day 3, with ≤10% fragmentation, regular size of the blastomeres and absence of multinucleation.

On day 4, embryos were scored as compacting, compacted or early blastocyst.

Embryo quality on day 5 ranged from arrested multicellular embryos to advanced blastocysts. Blastocyst quality was assessed according to the criteria of Gardner and Schoolcraft (Gardner and Schoolcraft, 1999Go). This scoring system is based on three parameters: blastocoel formation and degree of expansion (BL1 to BL6); development of the inner cell mass (ICM) (A = ICM cells are tightly packed, many cells are present; B = the ICM cells are loosely grouped, several cells are present; C = very few ICM cells are visible; and D = the ICM is not clearly visible or has a degenerative aspect, the presence of the ICM is doubtful); and development of the trophectoderm (A = many cells forming a cohesive epithelium; B = few cells forming a loose epithelium; and C). A combination of these parameters resulted in a combined embryo score ranging from EQ1 to EQ4. For transfer on day 5, preferably full or advanced blastocysts with many cells in the ICM and in the trophectoderm were selected, also taking into account the developmental pattern during the preceding culture period. A top-quality blastocyst (EQ1) was considered as being at least BL3, type A for ICM and type A or B for trophectoderm. Supernumerary embryos were frozen at the blastocyst stage (day 5 or day 6).

Statistical analysis
In order to detect a difference of 15% in ongoing pregnancy rates between the groups in which embryo transfer was performed on day 3 or on day 5, 157 patients would be required in each group, assuming a baseline pregnancy rate of 25%. This sample size achieves 80% power, at a significance level ({alpha}) of 0.05, using a two-sided z-test. These results assume that four sequential tests would be performed using the O’Brien–Fleming spending function to determine the test boundaries. At the second sequential test (including 50% of the patients), the {alpha} (significance level) to stop the study should be <0.003. At the second interim analysis an {alpha} level of 0.002 was observed in favour of blastocyst-stage transfer with regard to ongoing pregnancy rate, and therefore it was decided to terminate the study.

Analysis was by intention to treat. Continuous variables were compared using the independent Student’s t-test or the Mann–Whitney test, according to the distribution of their values. Data are presented as mean ± SEM. Categorical variables were compared using pairs of two-tailed Fisher’s exact tests. The significance level was set at 5% (P < 0.05).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Overall, 164 patients fulfilled the criteria on the third day of embryo culture and were randomized to have either a cleavage-stage embryo transfer (day-3 group, n = 84) or a blastocyst stage embryo transfer (day-5 group, n = 80) (Figure 1). There was no difference between the two groups (day-3 group versus day-5 group) regarding the infertility aetiology: male factor, 55.4 versus 53.8%; female factor, 30.1 versus 26.3%; combined, 9.6 versus 11.3%; idiopathic, 4.8 versus 8.8%. The range of infertility duration was between 1 and 7 years in both groups and the proportion of patients undergoing their first ever IVF/ICSI trial was 71.9 and 74.6% in the day 3 and day-5 groups, respectively.

The two groups were comparable with regard to age, the stimulation protocol used, the duration of stimulation and the total dose of gonadotrophins administered (Table I). Comparable numbers of cumulus–oocyte complexes and metaphase II oocytes were retrieved, and similar numbers of 2PN zygotes were obtained in each group of patients (Table II). A significantly higher number of EQ1 embryos were transferred in the day-3 group and the mean quality of the cleavage-stage embryos was higher compared with day 5 embryos (Table II). All patients in both groups had embryo transfer. In the day-5 group, five patients received a single embryo (one of them requested to have elective single embryo transfer and four had only one embryo available). Three patients in the day-5 group and two patients in the day-3 group asked for and finally had three embryos replaced.


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Table I. Patient demographics and stimulation characteristics

 

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Table II. Embryology data

 

The different parameters concerning pregnancy outcome per patient randomized are summarized in Table III. Both clinical pregnancy rate [52.5 versus 32.1%; odds ratio (OR) 2.33, 95% confidence interval (CI) 1.23–4.40] and ongoing pregnancy rate (51.3 versus 27.4%; OR 2.78, 95% CI 1.45–5.34) were significantly higher in the day-5 group compared with the day-3 group. Similarly, day-5 transfer resulted in a significantly (OR 2.40, 95% CI 1.25–4.59) higher live birth rate compared with day-3 embryo transfer (47.5 versus 27.4%). A high (42.9%) initial multiple pregnancy rate in the blastocyst group resulted in more than one-third of the births after day-5 transfer being twins (36.8%). The total pregnancy loss between the two groups did not differ: 31.4% with day-3 embryo transfer versus 28.3% with day-5 embryo transfer (P > 0.05) (Table III).


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Table III. Pregnancy outcome per patient randomized

 


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although human blastocysts developed in vitro have been reported to achieve high implantation rates (Gardner et al., 2004Go), a recent meta-analysis found no evidence to support any improvement in pregnancy or live birth rates by transferring blastocyst-stage embryos instead of cleavage-stage embryos (Blake et al., 2004Go). The present prospective randomized controlled trial demonstrates that, if a patient has at least four (≥4) morphologically good embryos on the third day of embryo culture, extending culture to day 5 and transferring two blastocysts results in significantly higher ongoing pregnancy and live birth rates than when two embryos are transferred at the cleavage stage. However, this was at the cost of almost half of the patients in the blastocyst group initially having a multiple pregnancy (42.9 versus 29.6%, P > 0.05), which stresses the higher implantation potential of day-5 embryos in a selected patient population.

Several factors may explain the discrepancy among studies regarding the efficacy of blastocyst- compared with cleavage-stage embryos. First, the increased cancellation rate between oocyte retrieval and embryo transfer for blastocyst culture leads to a fall in the pregnancy rate when this rate is reported per randomized woman, which is the appropriate way of reporting it, rather than pregnancy rate per embryo transfer. Therefore, the strategy proposed by Racowsky et al. (2000)Go, i.e. to proceed to day-5 transfer when more than four 8-cell embryos have been developed on day 3, reduces the risk of not having embryos available for transfer on day 5. A modified approach was adopted in the present prospective study; additionally, and more importantly, we found that the day-5 strategy in such a cohort of patients may result in a significant increase in live births (about half of the cases).

The above finding highlights the second limiting factor when comparing blastocyst transfer with cleavage-stage embryo transfer, which is the time point of the randomization process. In a previous study by our group, it was shown that in women less than 43 years old, when the allocation of patients occurred at patient intake (before any embryological data were known), there was no difference between day-3 and day-5 groups in terms of ongoing pregnancy rates. One of the major reasons was that 9% fewer patients in the day-5 group had embryo transfer. Yet the ongoing pregnancy rate per embryo transfer was showing a trend in favour of blastocyst (39.5 versus 34.4%, P > 0.05) (Kolibianakis et al., 2004Go). Our present study indicates that, in younger patients (less than 37 years old) undergoing their first to third IVF trial, a threshold of four good-quality embryos on day 3 is a reassuring indication that the patient will have an embryo transfer at the blastocyst stage and benefit from an increased pregnancy rate. Similarly, Milki and colleagues (Milki et al., 2000Go) have shown that, with blastocyst transfer, higher pregnancy rates compared with day-3 group were achieved in women under age 40 when more than three 8-cell embryos were present on day 3 of culture.

The reason for the higher success rate with blastocysts might be mainly related to an embryo selection process. In our study, although more top-quality embryos were transferred in the day-3 group, the delivery rate was 2-fold higher after a day-5 transfer. Studies on embryos after PGD for aneuploidy screening have enriched our knowledge concerning the last issue (Munne and Cohen, 1998Go; Magli et al., 2000Go; Staessen et al., 2004Go). We have recently shown that 59% of top-quality embryos on day 3 were genetically abnormal, whereas only 35% of top-quality blastocysts were aneuploid (Staessen et al., 2004Go). Given that even in this cohort of at least four morphologically good embryos on day 3 of culture some embryos will be chromosomally abnormal, the extension of culture to day 5 will allow the best embryos to develop further, though some chromosomally abnormal embryos will still reach that developmental stage (blastocyst). However, the proportion will be lower than what is observed on the third day of embryo culture (Staessen et al., 2004Go). Therefore, the embryos that are selected for transfer on day 5 will carry a lower risk of being aneuploid, thereby increasing a patient’s chances of achieving an ongoing pregnancy.

At this point arises a third way in which bias might be introduced in comparing day-3 with day-5 embryo transfer, which is the number of embryos transferred. If more embryos are transferred in the day-3 group than the day-5 group, there will be a greater likelihood of transferring those that are chromosomally normal (instead of freezing them), thereby eliminating the advantage of blastocysts. This was perfectly illustrated in the meta-analysis mentioned above (Blake et al., 2004Go), where, in terms of clinical pregnancy rate, the OR, when more embryos are transferred on day 3 than day 5 (0.99; 95% CI 0.71–1.36), immediately decreases in favour of day 5 when equal number of embryos are transferred in both groups (0.82; 95% CI 0.56–1.21). Although still not statistically significant, none of these studies (Coskun et al., 2000Go; Rienzi et al., 2002Go; Van de Auwera et al., 2002Go), or the present study, favours day-3 embryo transfer as regards the OR.

Nevertheless, another randomized prospective study by Bungum et al. (2003)Go with a study design similar to the present study (equal number of embryos transferred—maximum two—in patients with at least three 8-cell embryos and showing less than 20% fragmentation) showed that the blastocyst stage had no advantage over day-3 embryo transfer (clinical pregnancy rate 52.5 versus 63.2%, respectively). Although the populations in our study and the study of Bungum and colleagues are comparable in terms of patient demographics and the clinical pregnancy rate in the blastocyst group is similar (around 52%) in the two studies, these authors reported an extraordinarily high clinical pregnancy rate with day-3 embryos (63.2%) and an unexpected higher early pregnancy loss with day-5 embryos (29 and 15%, respectively) (Bungum et al., 2003Go). In the meta-analysis performed by Blake et al. (2004)Go, the day-3 group (n = 555), including the 11 studies being analysed, showed a clinical pregnancy rate after cleavage-stage embryo transfer of around 39.6%, which is comparable with the percentage reported in the present study (32.1%).

Many studies support the idea that blastocyst transfer should lead to a reduction of multiple pregnancies (Gardner et al., 2000Go; Vidaeff et al., 2000Go). However, our findings suggest that this is unrealistic when more than one blastocyst is transferred. As a result of the high implantation potential (Table III), blastocyst transfer in selected patients may lead to an unacceptably high multiple birth rate (36.8%). Conversely, it appears that reducing high-order pregnancies without compromising pregnancy outcome, by implementing single-blastocyst embryo transfer in a population similar to the one involved in the present study, is a realistic goal.

Within the framework of the new Belgian legislation, which limits the number of embryos for transfer according to the patient’s age and the rank of the trial, the strategy investigated in the present study seems preferable. Hence, the threshold of four good embryos on the third day of embryo culture appears to be a reassuring indication that the patient will undergo an embryo transfer and, moreover, will have a higher chance of achieving a live birth. Nevertheless, the optimal outcome of IVF should be a singleton delivery, and single blastocyst transfer might be the means to accomplish this goal.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on May 11, 2005; resubmitted on June 15, 2005; accepted on June 29, 2005.