Patient selection criteria for blastocyst culture in IVF/ICSI treatment

J Assist Reprod Genet (2010) 27:555 560 DOI 10.1007/s10815-010-9457-9 ASSISTED REPRODUCTION Patient selection criteria for blastocyst culture in IVF/ICSI treatment M. Y. Thum & V. Wells & H. Abdalla Received: 5 February 2010 /Accepted: 16 July 2010 / Published online: 31 July 2010 # Springer Science+Business Media, LLC 2010 Abstract Background The development and refinement of blastocyst media in recent years has allowed embryos to be cultured in-vitro for 5 or 6 days post oocyte retrieval and has been established as an effective selection tool to aid embryo selection for IVF treatment. It is generally accepted that blastocyst culture is not an appropriate option for all patients but the criteria for patient selection varies between clinics. Our blastocyst culture programme started in February 2005; the patient criteria was set at a minimum of 4 oocytes retrieved, a minimum of 4 2PN pronuclear embryos and at least 4 8-cell embryos of any quality on Day 3 where the female patient was 34 years and under. In the female age group of 35 years and over the criteria was at least 6 oocytes retrieved, a minimum of 6 2PN pronuclear embryos and at least 6 8-cell embryos of any quality on day 3. Improvements in pregnancy rates demonstrated the effectiveness of blastocyst transfer and clinical opinion was that the criteria should be adjusted to allow this option to be available to an increased patient population. From February 2007 the blastocyst patient selection criteria was changed to at least 4 oocytes retrieved, at least 4 2PN pronuclear embryos and at least 2 8-cell and 2 6-cell or 7-cell embryos of top quality on Day 3 in women 38 years and under. For women 39 years and over the criteria was lowered to at least 5 eggs retrieved, at least 5 2PN and at least 3 8-cell embryos and 2 6-cell embryos of top quality on Day 3. Capsule Selection criteria for blastocyst culture. M. Thum (*) : V. Wells : H. Abdalla Lister Fertility Clinic, Lister Hospital, Chelsea Bridge Road, London SW1W 8RH, UK e-mail: mythum@doctors.net.uk Methods Retrospective statistical analysis was carried out to determine the pregnancy rates, live birth rates and twin rate for the period under the initial criteria and to examine the impact that lowering the criteria for patient selection for blastocyst culture had on these parameters. Results There was an overall fall in the ongoing pregnancy/ live birth rate from 50.9% under the old criteria to 45.0% under the new criteria. However, the patients who had blastocyst culture under the new criteria but would have had day-3 embryo transfer under the initial criteria had a significantly increased live birth/ongoing pregnancy rate from 22.7% to 40.7%. There is an increase in the number of blastocyst culture cycles from 26.4% under the old criteria to 39.1% with the refined criteria. The twin pregnancy rate was reduced from 25.2% to 17.5%. Conclusion The result of this cohort study revealed that lowering the blastocyst selection criteria may lead to a lower overall clinical live birth rate from blastocyst culture; however, it will benefit a specific group of patients to achieve a better pregnancy and live birth rate. Furthermore, it increases the number of patients who will benefit from the blastocyst culture programme and also reduces multiple pregnancy rate. Keywords Blastocyst culture. Embryo selection. Success rate Introduction Since the birth of the first IVF baby in 1978 there have been many developments in Assisted Reproduction and in the last few years the development and refinement of commercial blastocyst media has produced some significant results. By allowing embryos to develop for 5 or 6 days post oocyte retrieval the embryos with the highest implantation potential should reach the blastocyst stage, ideally by day-5.

556 J Assist Reprod Genet (2010) 27:555 560 Blastocyst culture acts as selection pressure to aid the choice of the most viable embryo from a cohort. It has been reported that more than 50% of embryos created through IVF could be chromosomally abnormal. These abnormalities are a cause of early arrest of many developing embryos hence by culturing embryos for a longer period the embryos with the highest chance of being chromosomally normal should be more likely to develop into good quality blastocysts [1]. Blastocyst transfers have been reported as achieving significantly higher implantation and pregnancy rates compared to day-3 transfers. A recent study reports a significantly higher live birth rate of 68% following blastocyst transfer compared to 40% following day-3 embryo transfer in 274 patients where the female partner was aged between 35 and 40 years [2]. This increased implantation rate has also allowed a move towards single embryo transfer to combat the problems caused by multiple pregnancies and births. In our recent study, we reported that with elective single fresh blastocyst and subsequent frozen thaw blastocyst transfer a cumulative live birth rate of 69.3% is achievable [3]. A decreased early pregnancy rate loss of 17.2% has also been reported after blastocyst transfers compared to 26.8% following day-3 transfers when a single embryo was transferred at either the day-3 or blastocyst stage in 1,103 IVF cycles [4]. The embryos do not gain any benefit from extended culture, it is merely a technique to increase the efficacy of embryo selection when a cohort is available. However, between clinics there appears to be no general consensus on the absolute criteria for patient selection for blastocyst transfers. One prospective randomised study concluded that the live birth rate for female patients under the age of 37 years was 47.5% following blastocyst transfer compared to 27.4% following a day-3 transfer when the patient had at least four 6-cell good quality embryos on day-3. Hence, the recommendation was that patients with at least this cohort of embryos will benefit from extended culture [5]. Another study suggested that when a patient has three or more 7-cell embryos then blastocyst culture and transfer can improve the pregnancy rate [6]. Milki et al. used a criteria of female patient age of under 40 years old with more than three 8-cell embryos on day-3 [7]. A review in 2007 described good prognosis patients as those with a high number of 8-cell embryos on day-3 [8]. Another retrospective analysis reported that 54% of embryos with 6 cells on day-3 formed blastocysts and 76% of day-3 embryos with 8-cells formed blastocysts. They also observed that the likelihood of blastocyst development did not increase when the embryos had more than 8 cells on day-3 [9]. One of the lowest criterias for patient selection for blastocyst culture appears to be the presence of two or more 8-cell embryos on day-3 as suggested by Levitas et al. [10]. The most significant factor for setting criteria for patient selection for blastocyst culture appears to be understanding the relationship between embryo assessment on day-3 and blastocyst development. One small study of 37 IVF patients noted that of day-3 embryos with 7 or more cells 68.9% developed into blastocysts compared to 38.1% of 4-cell, 5-cell or 6-cell embryos on day-3 [11]. Another study examined the impact of quality of the embryos on day-3 in relation to their ability to form blastocysts. It was recorded that 47% of good quality embryos on day-3 reached the blastocyst compared to 21% of poor quality day-3 embryos. Interestingly on day-3 the embryos that were considered the best choice for transfer were noted and cultured separately. By the blastocyst stage only 51% of the embryos that had been considered the optimum choice on day-3 were selected for transfer [12]. Della Ragione et al. examined the importance of the day-3 embryo quality in achieving a pregnancy not just in achieving blastocyst development. It was observed that blastocysts that developed from day-3 embryos with >10 50% fragmentation had a significantly lower implantation rate of 29.7% compared to an implantation rate of 49.4% when blastocysts developed from day-3 embryos with <10% fragmentation were transferred. Therefore, it is essential to establish strict criteria for determining whether or not an individual patient should be assigned to the blastocyst culture programme. The aim of this study is to compare two patient selection models for blastocyst culture in a homogenous clinical setting. Methods and materials In February 2005, the technique of blastocyst culture and transfer was introduced into our routine laboratory working practice. On day-3, following assessment of the embryos, a decision would be made whether a day-3 transfer would be undertaken or whether the culture period would be extended to encourage blastocyst development and a blastocyst transfer performed on day-5, or in rare cases on day-6. The criteria for selection was based on the female patients age, minimum number of oocytes collected, minimum number of pronucleate embryos on day-1 and the number and quality of the embryos on day-3. The following criteria, as shown in Table 1, was developed from clinical experience and the media and incubator supplier guidelines, in order to be confident that extending the culture of the embryos to the blastocyst stage would aid in the selection for transfer of the embryos with the highest implantation potential. Any patient not meeting these criteria would have a day-3 transfer. If the patient has a sufficient number of embryos, the two highest quality embryos from the cohort would usually be selected for

J Assist Reprod Genet (2010) 27:555 560 557 Table 1 Patient selection criteria for blastocyst culture and transfer. February 2005- January 2007 Women 34 years and under At least 4 eggs retrieved At least 4 2PN At least 4 8-cell embryos of any quality on Day Three Women 35 years and over At least 6 eggs retrieved At least 6 2PN At least 6 8-cell embryos of any quality on Day Three transfer in female patients under 40 years old or three embryos in female patients 40 years and older. In February 2007 the criteria for patient selection for blastocyst transfer was altered, as shown in Table 2. Improvements in pregnancy rates demonstrated the effectiveness of blastocyst transfer and clinical opinion was that the criteria should be adjusted to allow this option to be available to an increased patient population. Blastocyst culture and transfer was also contributing to the continual policy of working towards a higher proportion of single embryo transfers as the higher implantation rates were allowing single embryo transfers at the blastocyst stage to be a viable option. The system for grading the quality of day-3 embryos is; Grade I is fragmentation rate <5%; Grade II is 5% to 25%; Grade III is 25% to 50%; Grade IV is 50% to 75% and Grade Vis>75%. Following down regulation and ovarian stimulation patients undergo oocyte retrieval under general anesthetic 36 h after taking an HCG injection. Oocytes are fertilised using either conventional IVF or by ICSI. Fertilisation assessment is performed 16 18 h post insemination. Embryos are group cultured in 40 μl drops of Sage culture media (supplied by Rochford Medical, UK) under oil in Nunc 4-well dishes in Minc Incubators (supplied by Cook, UK) with a mixed gas supply. Embryos are assessed on day-2 of development and placed into fresh media for reassessment on day-3. On day-3 post oocyte retrieval, embryos are assessed and either allocated for transfer that day or moved into blastocyst media for culture to the blastocyst stage. Blastocyst transfers are routinely done on day-5, in rare cases on day-6. Excess good quality blastocysts are vitrified and stored for potential future use by the patient. statistical tests. Chi-square Cross Tabulation test was used to analyse the significant difference in pregnancy rates, live birth rates and twin rate between the groups. Statistical significant was set at P<0.05. The study population included are women in their first fresh IVF treatment cycle. Results The data of this study review no significant difference between patient age, number of oocytes collected, number of normally fertilized embryos and number of embryos transferred under both the old and the reformed new criteria. The indications for IVF treatment were not significantly different between the two study groups. However, the pregnancy and live birth rate are lower in the new blastocyst criteria group (Table 3). Most importantly there are significantly increase number of cycle with blastocyst culture per month with the new selection criteria. Furthermore, the implantation rate was not significantly different between the two study groups. Table 4 shown data of stimulation and treatment outcome for those patients recruited for blastocyst culture but failed to achieve blastocyst for transfer in the old and new selection criteria. There are no significant difference in term of number of eggs collected, number f embryo, number of embryo transferred and Live birth rate between the old and new selection criteria. Most importantly the percentage of cycles failed to achieve blastocyst for transfer was not significantly increase with the new selection criteria. Table 5 illustrated the stimulation and IVF treatment outcome of women who did not have blastocyst culture with the old selection criteria but eligible for blastocyst culture with the new selection criteria compare to women who had blastocyst culture with the new selection criteria but not eligible for blastocyst culture with the old criteria. There is no significantly different in term of age, number of eggs collected, number of embryos and number of embryos transferred. Imperatively, the live birth rate is significantly higher with the new selection criteria. Table 2 Patient selection criteria for blastocyst culture and transfer. February 2007 April 2008 Data analysis Data was collected in Medical System for IVF (MedicalSys, London, UK) and analysed by Statistics Package for Social Sciences (SPSS, Surrey, UK). Descriptive statistical analysis was performed initially to examine the normal distribution of all continuous variances for parametric Women 38 years and under At least 4 eggs retrieved At least 4 2PN At least 2 8-cell embryos of grading at least 1 2 and 2 6-cell or 7-cell embryos of grading 1 2 on Day Three Women 39 years and over At least 5 eggs retrieved At least 5 2PN At least 3 8-cell embryos of grading 1 2 and 2 6-cell or 7-cell embryos of grading 1 2 on Day Three

558 J Assist Reprod Genet (2010) 27:555 560 Table 3 Results of IVF treatment outcome with blastocyst culture under old and new selection criteria Old criteria New criteria P-value Number of cycles 782 834 NA Average no. of blastocyst culture cycles/month 32.6 55.6 0.001 Mean age ± SD 35.2±3.8 35.7±5.7 NS Average no. of oocytes collected ± SD 12.03±5.1 11.95±5.2 NS Average no of normal fertilized embryos ± SD 8.79±3.7 8.63±3.3 NS Average no of embryos transferred ± SD 1.63±0.52 1.56±0.21 NS Pregnancy rate (%) 68.0% (532/782) 62.7% (523/834) 0.014 Live birth rate (%) 50.9% (398/782) 45.0% (357/834) 0.001 Twin pregnancy rate (%) 25.2% 17.5% 0.032 Implantation rate (%) 50.1% 45.6% NS NS = difference not statistically significant (P>0.05) NA = not applicable Discussion When comparing the effect that the change in criteria protocol has had on the pregnancy and live birth rates, the rate under the old criteria was 50.9% and the rate under the new criteria was 45.0%, demonstrating a significant difference. However, this is not a conclusive parameter to assess when determining if lowering the patient selection criteria for blastocyst culture has had a negative or positive impact. Information about all the patients from February 2005 to April 2008 who met the criteria for blastocyst culture under the new criteria but not the old criteria was collated and divided into those who had a day-3 embryo transfer during the initial period criteria of February 2005 to January 2007 and those who had blastocyst culture and transfer in the period February 2007 to April 2008, as shown in Table 5. These patients who had blastocyst culture under the new criteria but would have had day-3 embryo transfer under the old criteria had a slightly higher pregnancy rate of 52.7% compared to 42.7% for those who had a day-3 embryo transfer under the old criteria, though this is not significantly higher. However, the live birth/ongoing pregnancy rate was significantly increased from 22.7% to 40.7%. Although there is a tendency towards viewing pregnancy and live birth rates as an absolute benchmark standard, they should be properly evaluated. This data suggests that although the overall pregnancy rate/live birth rate for those patients having blastocyst culture dropped after the criteria was lowered, the patient group that had attributed to this overall decrease actually had an improved live birth rate following the change in criteria and their inclusion onto the blastocyst culture programme. As a clinical indication of performance pregnancy rate and live birth rate are important but the aim of each IVF cycle should be to maximize the chance of a successful outcome for each individual patient in that particular cycle. This data provides evidence of the efficacy of blastocyst Table 4 Results of IVF treatment outcome with blastocyst culture under old and new selection criteria which failed to achieve blastocyst for transfer Old criteria New criteria P-value Number of cycles 21 30 NA Percentage of cycles failed to achieve blastocyst for transfer 2.68% 3.59% NS Mean age ± SD 37.2±3.1 37.3±3.4 NS Average no. of oocytes collected ± SD 7.9±3.2 9.06±3.1 NS Average no of normal fertilized embryos ± SD 7.31±2.5 6.33±2.9 NS Average no of embryos transferred ± SD 1.81±0.32 1.93±0.22 NS Pregnancy rate (%) 9.5% (2/21) 26.6% (8/30) 0.001 Live birth rate (%) 9.5% (2/21) 10.0% (3/30) NS Twin pregnancy rate (%) 50.0% 33.3% 0.001 NS = difference not statistically significant (P>0.05) NA = not applicable

J Assist Reprod Genet (2010) 27:555 560 559 Table 5 Results of IVF treatment outcome in patients under initial selection criteria which eligible for blastocyst culture in the new criteria compare with patients under new selection criteria which had blastocyst culture which not eligible in the old criteria Non-Blastocyst Old criteria 1 Blast New criteria 2 P-value Number of cycles 110 91 NA Mean age ± SD 37.2±6.5 36.8±8.2 NS Average no. of oocytes collected ± SD 5.62±1.1 5.93±2.3 NS Average no of normal fertilized embryos ± SD 4.87±0.61 4.95±0.71 NS Average no of embryos transferred ± SD 1.91±0.21 1.61±0.53 NS Pregnancy rate (%) 42.7% (47/110) 52.7% (48/91) NS (0.101) Live birth rate (%) 22.7% (25/110) 40.7% (37/91) 0.005 1 Patients who did not have blastocyst culture with the old criteria but eligible for blastocyst culture in the new criteria. 2 Patients who had blastocyst culture with the new criteria but will not be eligible for blastocyst culture with the old criteria. NS = difference not statistically significant (P>0.05) NA = not applicable culture in aiding the identification of the embryos with the most potential for an increased patient group. Therefore lowering the criteria for patient selection to the thresholds shown in Table 2, has extended the benefits of blastocyst culture to more patients, namely increased chance of pregnancy and a live birth and a decreased chance of a multiple pregnancy due to increased elective single blastocyst embryo transfer. When taking into account the number of patients that were eligible for blastocyst culture, there is an increase after the change of protocol in February 2007. Between February 2005 and January 2007 an average of 32.6 patients each month were accepted onto the blastocyst programme. For this period this represents 26.4% of the total number of IVF cycles performed. After the change of patient selection criteria in February 2007 an average of 55.6 patients have met the criteria for blastocyst culture, which amounts to 39.1% of the total number of IVF cycles undertaken at The Lister Assisted Conception Unit. In addition, changing the criteria has had no negative impact on the proportion of failed blastocyst culture patients, as defined by those patients having a morulastage embryo transfer following blastocyst culture. In the results table the failure rate of the embryos to develop to the blastocyst stage is indicated by the number of embryo transfers that were performed when the embryos were at the morula-stage. The results show that the chance of achieving a pregnancy and live birth was lower when morula-stage embryos were transferred, although only small numbers of patients were affected, 21 under the original criteria and 30 under the reformed criteria. The proportion of morula-stage transfers was 2.68% under the original Lister blastocyst patient selection criteria and 3.59% in the reformed criteria introduced from February 2007. This difference is slightly higher in the newer criteria but not to a significant level. No patient between February 2005 and April 2008 had a cancelled embryo transfer following blastocyst culture. With the initial criteria there was an overall blastocyst culture live birth rate of 50.9% dropping to 9.5% when morula-stage embryos were transferred and when the criteria was modified the overall blastocyst culture live birth/ongoing pregnancy rate was 45.0% and 10.0% when morula-stage embryos were transferred. This is supported by evidence from other studies that found transferring morulae after blastocyst culture had lower pregnancy rates than when blastocysts were transferred, [13 15]. This would appear to fit the notion that some embryos can arrest early in their development and that by transferring a morula on day-5 when it should ideally have reached the blastocyst stage the possibility exists that it may have already arrested. As discussed previously, the implantation rates of blastocysts and pregnancy rates achieved by the transfer of blastocyst stage embryos compared to day-2 and day-3 embryo transfers has allowed elective single blastocyst embryo transfer to become a viable option for many patients. The Lister has a policy of working towards single embryo transfer in order to limit the number of multiple pregnancies and the HFEA has also recently announced that all clinics are expected to work towards a maximum 10% multiple live birth rate by 2011. When looking at the data for blastocyst culture patients at The Lister Assisted Conception Unit it can be shown that there has been a move towards lowering the average number of embryos transferred per cycle. From February 2005 to January 2007 as an average of 1.63 embryos were transferred in each cycle, from February 2007 this was reduced to 1.56. Although this is lower, it is not significantly so. However, in terms of the number of patients having single embryo transfers, this is an increase compared to the previous criteria era as under these protocols patients not selected for blastocyst culture would have two or three embryos replaced.

560 J Assist Reprod Genet (2010) 27:555 560 The twin pregnancy rate in the overall pregnancy rate for blastocyst culture patients has also reduced, for the period of the initial criteria this was 25.2% and for the period of the updated criteria it was 17.5%. The Lister statistics for the period of February 2007 to April 2008, which covers the era of the reformed blastocyst culture selection criteria, show a dizygotic diamniotic twin pregnancy live birth rate/ongoing pregnancy rate of 23.4% in patients who have a day-3 transfer. This is higher than the 17.5% equivalent pregnancy rate following blastocyst transfer. This could be due to the fact that there is a tendency to transfer more embryos when the transfer is performed at the cleavage stage compared to the blastocyst stage. A proportion of these patients conceive a twin pregnancy following the transfer of two cleavage stage embryos. These patients did not meet the criteria for blastocyst culture and yet as they had a 100% implantation rate, implanted their embryos had a high implantation potential they could not be identified at the time of embryo transfer. Blastocyst culture for these patients would have been advantageous as given the complications for both the mother and the fetuses that can be associated with multiple pregnancies a single embryo transfer would have been medically preferable. Our published data [3], has stressed the efficacies of elective single blastocyst embryo transfer given the high success rates when pregnancy rate is calculated per egg collection and therefore includes a fresh transfer and at least one frozen embryo blastocyst transfer. Considering this evidence for the encouragement of elective single blastocyst embryo transfer and the Lister ACU data that suggests that lowering the patient criteria for blastocyst culture has had a positive impact on a select patient group and no negative impact on the failure rate of blastocyst culture, it seems that consideration should be given to lowering the threshold again. References 1. Munne S. Chromosome Abnormalities and their relationship to morphology and development of human embryos (2006). Reprod Biomed Online. 2006;12(2):234 54. 2. Beeslet R, Robinson R, Propst A, Arthur N and Retzloff M. Impact of day 3 or day 5 embryo transfer on pregnancy rates and multiple gestations. Fertil Steril May. 2009;(5):1717 20. 3. Kalu E, Thum MY and Abdalla HI. Reducing multiple pregnancy in assisted reproduction technology: towards a policy of single embryo transfer in younger patients. BJOG 2008;(9):1143 50. 4. 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