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Sibling embryo blastocyst development as a prognostic factor for the outcome of day-3 embryo transfer

Published:February 15, 2013DOI:https://doi.org/10.1016/j.rbmo.2013.02.003

      Abstract

      This study assessed the development of sibling embryos to blastocyst as a prognostic factor for patients undergoing embryo transfer at day 3. A retrospective analysis of a clinical and embryology database including 353 patients who underwent 393 cycles of intracytoplasmic sperm injection with day-3 embryo transfer and excess embryos, maintained in culture until day 5, was performed. Cycles were divided into group A and group B (with and without blastocyst formation, respectively). Age and basal FSH were similar in both groups. Statistically significant differences in clinical pregnancy rates (55.8% versus 40.6%; P = 0.0031), live birth rates (50.0% versus 37.2%; P = 0.012) and implantation rates (34.2% versus 23.7%; P = 0.0035) were observed in groups A and B, respectively. Odds ratios showed women from group A had 1.85- and 1.68-times the odds of patients from group B of achieving clinical pregnancy and a live birth, respectively. Cumulative live birth rate for group A, after one cycle of vitrified–warmed blastocyst transfer, was 66.4%. The development of sibling embryos to blastocyst is a prognostic factor for the outcome of the cycle in which transfer is performed at day 3 and provides valuable information about the prognosis of subsequent cycles.
      This study assesses the development of sibling embryos to the blastocyst stage (day 5 of in-vitro culture) as a prognostic factor for patients undergoing embryo transfer at day 3. For this purpose a retrospective analysis of clinical and embryology database coming from 353 patients who underwent 393 cycles of intracytoplasmic sperm injection with day-3 embryo transfer and excess embryos was performed. Sibling embryos were cultured until day 5. Cycles were divided in group A and group B (with and without blastocysts at day 5, respectively). Age and ovarian reserve were similar in both groups. Statistically significant differences in pregnancy rates (55.8% versus 40.6%), live birth rates (50% versus 37.2%) and implantation rates (34.2% versus 23.7%) were observed between group A and group B, respectively. Statistical analysis showed that the chances of becoming pregnant and having a live birth rate were 1.85- and 1.68-times more in group A than in group B, respectively. Cumulative pregnancy rate and live birth rate for group A, after one cycle of vitrified-thawed blastocyst transfer, were 74.5% and 66.4%, respectively. It is concluded that development of sibling embryos to the blastocyst stage is a prognostic factor for the outcome of the cycle in which transfer is performed at day 3 and provides valuable information about the prognosis of subsequent cycles.

      Keywords

      Introduction

      Multiple factors influence the outcome of assisted reproduction treatment, the most relevant being female age, duration of infertility, ovarian reserve and embryo quality (
      • Hathaway P.
      • Croucher C.
      • Lass A.
      Predictive value of the results of a first IVF cycle.
      ,
      • van Loendersloot L.L.
      • van Wely M.
      • Limpens J.
      • Bossuyt P.M.
      • Repping S.
      • van der Veen F.
      Predictive factors in in vitro fertilization (IVF): a systematic review and meta-analysis.
      ). For many years these elements have been considered as prognostic factors for becoming pregnant or having a live birth in a particular treatment cycle (
      • Minaretzis D.
      • Harris D.
      • Alper M.M.
      • Mortola J.F.
      • Berger M.J.
      • Power D.
      Multivariate analysis of factors predictive of successful live births in in vitro fertilization (IVF) suggests strategies to improve IVF outcome.
      ). However, in the last decade the focus has moved towards predicting the probability of singleton or twin pregnancies when two cleaving embryos could be transferred (
      • Hunault C.C.
      • te Velde E.R.
      • Welma S.M.
      • Macklon N.S.
      • Eijkemans M.J.
      • Klinkert E.R.
      • Habbema J.D.
      A case study of the applicability of a prediction model for the selection of patients undergoing in vitro fertilization for single embryo transfer in another center.
      ), in order to transfer them or to perform an elective single-embryo transfer (SET) of one selected cleavage-stage embryo and avoid a multiple pregnancy (
      • De Sutter P.
      • Van der Elst J.
      • Coetsier T.
      • Dhont M.
      Single embryo transfer and multiple pregnancy rate reduction in IVF/ICSI: a 5-year appraisal.
      ,

      Practice Committee of Society for Assisted Reproductive, T. and M. and Practice Committee of American Society for Reproductive, 2012. Elective single-embryo transfer. Fertil. Steril. 97, 835–842.

      ). Furthermore, when enough good-quality embryos are available at day 3, an extended culture until day 5 is advisable, in order to improve embryo selection and pregnancy rates (
      • Papanikolaou E.G.
      • D‘Haeseleer E.
      • Verheyen G.
      • Van de Velde H.
      • Camus M.
      • Van Steirteghem A.
      • Devroey P.
      • Tournaye H.
      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.
      ,
      • Papanikolaou E.G.
      • Kolibianakis E.M.
      • Tournaye H.
      • Venetis C.A.
      • Fatemi H.
      • Tarlatzis B.
      • Devroey P.
      Live birth rates after transfer of equal number of blastocysts or cleavage-stage embryos in IVF. A systematic review and meta-analysis.
      ). But patients without four or five good-quality embryos at day 3 do not have the minimum requirements for extended embryo culture until day 5 and must undergo cleavage-stage embryo transfer (
      • Papanikolaou E.G.
      • D‘Haeseleer E.
      • Verheyen G.
      • Van de Velde H.
      • Camus M.
      • Van Steirteghem A.
      • Devroey P.
      • Tournaye H.
      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.
      ,
      • Dessolle L.
      • Freour T.
      • Barriere P.
      • Daraï E.
      • Ravel C.
      • Jean M.
      • Coutant C.
      A cycle-based model to predict blastocyst transfer cancellation.
      ), because of the risk of cycle cancellation due to a lack of blastocysts.
      Many embryo selection criteria for cleavage-stage embryos have been developed in order to choose the best embryos and reduce the number of them to be transferred at day 3 (
      • Geraedts J.P.
      • Gianaroli L.
      Embryo selection and IVF.
      ), although, embryo selection at this stage has some limitations. Indeed,
      • Rijnders P.M.
      • Jansen C.A.
      The predictive value of day 3 embryo morphology regarding blastocyst formation, pregnancy and implantation rate after day 5 transfer following in-vitro fertilization or intracytoplasmic sperm injection.
      reported that only 51% of embryos that were transferred on day 5 had been preselected for transfer on day 3. Therefore, it is difficult to predict the result of assisted reproduction treatments based on the quality of embryos transferred at day 3, and other criteria have to be introduced to establish the prognosis of the treatment cycle. These could be previously known clinical criteria of patients (
      • van Loendersloot L.L.
      • van Wely M.
      • Limpens J.
      • Bossuyt P.M.
      • Repping S.
      • van der Veen F.
      Predictive factors in in vitro fertilization (IVF): a systematic review and meta-analysis.
      ) or data coming from the embryology laboratory in the same cycle in which the transfer is performed. This information can also be considered to define patients that are eligible for elective SET or single-blastocyst transfer in future cycles.
      The main objective of this study is to assess the development of sibling embryos to blastocyst stage as a prognostic factor for patients undergoing embryo transfer at day 3.

      Materials and methods

      This is a retrospective analysis of the embryology database from January 2006 to December 2011 at the Unit of Reproductive Medicine of the department of obstetrics and gynaecology at Clinica Las Condes, Chile. All patients signed institutional review board approved consent forms before initiating and any patient specific identifiers were removed from database prior to the analysis. Retrospective approval for this study was granted by the ethical committee of Clinica Las Condes on 30 January 2013.
      In total, 353 patients, who underwent assisted reproduction treatment in 393 cycles, had embryos transferred at day 3 and had excess embryos that remained in culture until day 5 were included in this study. The age of women was (mean ± SD) 34.5 ± 3.8 years and their basal FSH was 7.1 ± 2.7 mIU/ml.
      Intracytoplasmic sperm injection was performed in all cases. The oocytes were stripped of their cumulus cells after incubation in hyaluronidase solution (80 IU/ml; Life Global, USA) for 60 s. Spermatozoa were selected by the swim-up procedure and sperm injection was performed with holding and injection micropipettes (Humagen, USA) under an Olympus inverted microscope and a Narishige micromanipulation system. Injected oocytes were incubated in global fertilization media (Life Global) in a 6% CO2 incubator (Forma Scientific, USA). Eighteen hours after sperm injection, oocytes were inspected for fertilization and those fertilized were transferred to fresh global media (Life Global, USA). A total of 5020 zygotes from 393 oocyte retrievals were followed from day 1 to day 3. Embryo morphology was assessed at day 3 using criteria reported by
      • Veeck L.L.
      An atlas of Human Gametes and Conceptuses: an Illustrated Reference for Assisted Reproductive Technology.
      , which considers cell number, uniformity of blastomere size and extent of fragmentation. Each embryo was designated based on its cell number and grade of uniformity and fragmentation, as follows; grade 1, symmetric blastomeres without fragmentation; grade 2a, symmetric blastomeres and <20% fragmentation; grade 2b, symmetric blastomeres and 20–50% fragmentation; grade 3, uneven size of blastomeres, without fragmentation; and grade 4, uneven blastomeres with fragmentation.
      A total of 960 embryos were transferred at day 3 with a mean of 2.44 ± 0.55 embryos per patient. Of these, 748 (77.9%) were classified as good-quality embryos (grades 1 and 2a). Embryo transfer was performed with an ultrasound-guided Frydman ultra-soft catheter (CCD, France). Fourteen days after oocyte retrieval the patients were tested for pregnancy (β-human chorionic gonadotrophin) and, if pregnant, an ultrasound was scheduled 1 and 2 weeks later to confirm a clinical pregnancy, register the number of gestational sacs and the presence of embryos with heartbeats. Pregnancies were followed until 24 weeks of amenorrhoea, to register those ending in miscarriages, and then until the delivery in all remaining cases.
      A total of 4060 excess embryos remained in culture and were followed until day 5, 459 of them were classified as good quality or grade 1 and 2a (11.3%). Blastocyst formation was evaluated at day 5, using criteria reported by
      • Veeck L.L.
      • Zaninovic N.
      An Atlas of Human Blastocysts.
      . A total of 350 embryos reached this stage and were vitrified using the Cryotop Kitazato method, initially described by
      • Kuwayama M.
      • Vajta G.
      • Kato O.
      • Leibo S.P.
      Highly efficient vitrification method for cryopreservation of human oocytes.
      . For warming, the Cryotop was removed from the liquid nitrogen and instantly placed in thawing solution at 37°C; after 1 min, blastocysts were placed in diluting solution at room temperature for 3 min and, finally, a 5-min wash followed by a 1-min wash was performed with washing solution at room temperature and processed as described by the manufacturer (Kitazato). Viability was assessed and transfer of vitrified–warmed blastocysts was performed in cycles with hormone replacement with oral oestradiol valerate and vaginal progesterone.
      For the purposes of this study, the 393 sample cycles were divided into two groups: those who had at least one blastocyst at day 5 (group A) and those who did not have blastocysts at day 5 (group B). For statistical analysis Stata 12 software (Stata Corporation, College Station, Texas, USA) was used. The independent variable was blastocyst and primary outcomes were clinical pregnancy rate (transvaginal ultrasound confirmation of a gestational sac inside the uterine cavity) and live birth rate. Secondary outcomes were implantation rate (number of gestational sacs observed divided by the number of embryos transferred), multiple pregnancy rate and miscarriage rate (number of spontaneous clinical pregnancy losses divided by the number of clinical pregnancies). Continuous variables were described by mean ± standard deviation (normal distribution) and categorical variables by proportions and rates, and to assess differences between the two groups, Student’s t-test and chi-squared test were used, respectively. To determine significant association between the presence of blastocysts on day 5 and primary outcomes, odds ratios with 95% CI were calculated by logistic regression. P-value <0.05 was considered significant for purposes of assessing differences between both groups.

      Results

      The analysis of the 393 cycles performed in 353 patients who underwent embryo transfer on day 3 showed 46.6% (183/393) clinical pregnancy rate per cycle, 26.6% (255/960) implantation rate, 36.1% (66/183) multiple pregnancy rate and 8.7% (16/183) miscarriage rate. In 154 cycles, at least one remaining embryo reached the blastocyst stage (39.2%).
      Group A comprised 149 patients undergoing 154 cycles with subsequent blastocyst formation and group B comprised 212 patients undergoing 239 cycles without subsequent blastocyst formation. A comparison of the general characteristics of groups A and B is provided in Table 1.
      Table 1General characteristics of patients who underwent day-3 embryo transfer with (group A) or without (group B) subsequent blastocyst formation.
      Group A (n =149)Group B (n =212)P-value
      Cycles154239
      Age (years)34.3 ± 3.634.7 ± 3.9NS
      Basal FSH (mIU/ml)7.0 ± 2.37.2 ± 2.9NS
      Transferred embryos on day 32.33 ± 0.472.51 ± 0.580.0023
      Proportion of good-quality embryos transferred on day 3
      Proportion of embryos classified as grade 1 and 2a on day 3.
      86.7 (312/360)72.7 (436/600)<0.00001
      Sibling embryos7883272
      Proportion of good-quality sibling embryos
      Proportion of embryos classified as grade 1 and 2a on day 3.
      35.8 (282/788)5.4 (177/3272)<0.00001
      Values are n, mean ± SD or % (n/total).
      a Proportion of embryos classified as grade 1 and 2a on day 3.
      Table 2 shows the outcome of patients in groups A and B. There were statistically significantly higher clinical pregnancy rate per cycle (P = 0.0031), live birth rate (P = 0.012) and implantation rate (P = 0.0035) in group A than in group B. Odds ratios (logistic regression) showed women from group A had 1.85- and 1.68-times the odds as patients from group B of achieving a clinical pregnancy and having a live birth, respectively. On the other hand, no differences were observed in multiple pregnancy rates and miscarriage rates between both groups.
      Table 2Outcomes for patients who underwent day-3 embryo transfer with (group A) or without (group B) subsequent blastocyst formation.
      Group A (n =149)Group B (n =212)P-valueOdds ratio (95% CI)
      Cycles154239
      Clinical pregnancy rate per cycle55.8 (86/154)40.6 (97/239)0.00311.85 (95% CI 1.22–2.78)
      Live birth rate50.0 (77/154)37.2 (89/239)0.0121.68 (95% CI 1.11–2.54)
      Implantation rate34.2 (123/360)23.7 (142/600)0.0035
      Multiple pregnancy rate38.4 (33/86)34.0 (33/97)NS
      Miscarriage rate10.5 (9/86)7.2 (7/97)NS
      Values are n or % (n/total).
      In group A, 98/106 patients underwent one cycle of vitrified–warmed blastocyst transfer within 2 years of the first treatment cycle with a 92.5% survival of at least one vitrified blastocyst per patient. The number of blastocysts transferred was 1.87 ± 0.66, with 25.5% (25/98) clinical pregnancy rate per cycle, 15.2% (28/184) implantation rate, 10.7% (3/28) multiple pregnancy and 17.9% (5/28) miscarriage rates. Cumulative clinical pregnancy rate and live birth rate for group A, after one fresh embryo transfer at day 3 and one vitrified–warmed blastocyst transfer, were 74.5% (111/149) and 64.4% (96/149), respectively.

      Discussion

      Without having the possibility of an extended culture to day 5, for blastocyst transfer, the assessment of day-3 embryo characteristics is a key factor to improve the results of assisted reproduction treatment. However, as has been demonstrated, it is not accurate enough to predict the outcome of a treatment cycle (
      • Rijnders P.M.
      • Jansen C.A.
      The predictive value of day 3 embryo morphology regarding blastocyst formation, pregnancy and implantation rate after day 5 transfer following in-vitro fertilization or intracytoplasmic sperm injection.
      ). Therefore, when excess embryos are available, subsequent blastocyst formation could be a good prediction factor for the outcome of the current cycle. Sibling embryos achieving blastocyst stage were available in 39.2% of the cycles in this study, which is higher than blastocyst formation rate from excess embryos reported by
      • Dayal M.B.
      • Kovalevsky G.
      • Patrizio P.
      Rate of blastocyst development from excess embryos remaining in culture after day 3 embryo transfer.
      , regardless of the cause of infertility.
      It has to be considered that the first signal that the results in group A would be better than in group B was the proportion of good-quality embryos among those that were transferred (86.7% versus 72.7%) and within the sibling embryos (35.8% versus 5.4%). Although, this preliminary indicator can be obtained at day 3, a more powerful one is the success of sibling embryos to develop to the blastocyst stage at day 5. Indeed, results in this study suggest development of sibling embryos to blastocyst is a strong prognostic factor for the results of the cycle in which embryo transfer has to be performed at day 3. Statistically significant higher pregnancy rates (55.8% versus 40.6%), live birth rates (50.0% versus 37.2%) and implantation rates (34.2% versus 23.7%) were observed in cycles in which blastocyst stage was reached by one or more of excess embryos remaining in culture until day 5 (group A) compared with cycles without blastocyst formation (group B). Actually, the chance of becoming pregnant and having a live birth was much higher in group A than in group B (OR 1.85, 95% CI 1.22–2.78 versus OR 1.68, 95% CI 1.11–2.54, respectively). These findings, also reported by
      • Sjogren A.
      • Sjoblom P.
      • Hamberger L.
      Culture of human spare preembryos: association between blastocyst formation and pregnancy.
      and
      • Fisch J.D.
      • Milki A.A.
      • Behr B.
      Sibling embryo blastocyst development correlates with the in vitro fertilization day 3 embryo transfer pregnancy rate in patients under age 40.
      in smaller series, suggest that sibling embryo blastocyst formation may reflect superior embryo quality and the fact that, at least, one of these embryos reaches the blastocyst stage after performing the transfer at day 3 can predict better implantation and pregnancy rates in the current cycle compared with cycles without embryos developing to blastocyst. Furthermore, results obtained in group A gives a 74.5% cumulative pregnancy rate and a 66.4% cumulative live birth rate when one fresh embryo transfer at day 3 and one vitrified–warmed blastocyst transfer were performed, reinforcing the issue that this group of patients has good-quality embryos and very good prognosis for assisted reproduction treatment.
      There is still controversy about the practice of allowing sibling embryos to develop until day 5 and cryopreserving blastocysts versus cryopreserving them at day 3 in order to transfer frozen–thawed blastocysts, frozen–thawed cleavage embryos or blastocysts from frozen–thawed cleavage embryos.
      • Pantos K.
      • Stefanidis K.
      • Pappas K.
      • Kokkinopoulos P.
      • Petroutsou K.
      • Kokkali G.
      • Stavrou D.
      • Tzigounis V.
      Cryopreservation of embryos, blastocysts, and pregnancy rates of blastocysts derived from frozen-thawed embryos and frozen-thawed blastocysts.
      reported a 20.6% implantation rate in the group transferring blastocysts from day-3 cryopreserved embryos against only 5.3% in the group transferring cryopreserved blastocysts. However, vitrification has improved results (
      • Sifer C.
      • Sermondade N.
      • Dupont C.
      • Poncelet C.
      • Cédrin-Durnerin I.
      • Hugues J.N.
      • Benzacken B.
      • Levy R.
      Outcome of embryo vitrification compared to slow freezing process at early cleavage stages. Report of the first French birth.
      ), and in the present study, implantation rate transfering vitrified-thawed blastocysts was 3-fold (15.2%).
      • Wang Y.A.
      • Chapman M.
      • Costello M.
      • Sullivan E.A.
      Better perinatal outcomes following transfer of fresh blastocysts and blastocysts cultured from thawed cleavage embryos: a population-based study.
      ,
      • Wang Y.A.
      • Costello M.
      • Chapman M.
      • Black D.
      • Sullivan E.A.
      Transfers of fresh blastocysts and blastocysts cultured from thawed cleavage embryos are associated with fewer miscarriages.
      suggested that transfers of thawed blastocysts and thawed cleavage embryos had significantly lower odds of live birth than transfer of blastocysts from thawed cleavage embryos; however, this retrospective study was also performed with slow freezing and live birth rate for thawed blastocysts was only 16.3%. More recently,
      • Eftekhar M.
      • Aflatoonian A.
      • Mohammadian F.
      • Tabibnejad N.
      Transfer of blastocysts derived from frozen-thawed cleavage stage embryos improved ongoing pregnancy.
      , in a prospective study, reported 30% implantation rate transferring blastocysts coming from cleavage-stage frozen–thawed embryos compared with only 17% transferring day-3 frozen–thawed embryos. The question is whether sibling embryos should be cultured until day 5 to vitrify them as blastocysts or vitrified at day 3 for subsequent culture after warming. Further prospective randomized case–control studies using vitrification techniques have to be performed to assess this issue. However, it has to be considered that the practice of allowing sibling embryos to develop until day 5 before vitrifying them reduces the number of useless embryos stored, which is an extra advantage for assisted reproduction centres and patients undergoing treatment.
      On the other hand, despite the fact that the lower quality of available embryos at day 3 in group B was compensated in patients included in this study by an increase in the number of embryos transferred, in order to improve the outcome, this practice was not effective enough to achieve the goal. Eventually, patients from group A had a better prognosis than those from group B.
      This study highlights the value of blastocyst formation from sibling embryos, not only to retrospectively assess the prognosis of the current cycle but also to prospectively predict the cumulative pregnancy rate when sibling embryos develop to blastocysts and also to make better decisions for subsequent assisted cycles. Indeed, the capability of remaining embryos to develop until day 5 may provide valuable information about the potential for a future transfer of fresh embryos at day 3. The fact that implantation rate was 34.1% in group A suggests that SET gives acceptable pregnancy rates almost without multiple pregnancies in patients with the characteristics of this group, achieving success rates similar to those reported for elective SET (
      • Tiitinen A.
      • Unkila-Kallio L.
      • Halttunen M.
      • Hyden-Granskog C.
      Impact of elective single embryo transfer on the twin pregnancy rate.
      ,
      • Thurin A.
      • Hausken J.
      • Hillensjo T.
      • Jablonowska B.
      • Pinborg A.
      • Strandell A.
      • Bergh C.
      Elective single-embryo transfer versus double-embryo transfer in in vitro fertilization.
      ,
      • Veleva Z.
      • Vilska S.
      • Hyden-Granskog C.
      • Tiitinen A.
      • Tapanainen J.S.
      • Martikainen H.
      Elective single embryo transfer in women aged 36–39 years.
      ). Furthermore, patients having blastocyst development from sibling embryos may be considered as eligible for fresh blastocyst transfer in subsequent cycles, improving live birth rates for future treatments (
      • Glujovsky D.
      • Blake D.
      • Farquhar C.
      • Bardach A.
      Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology.
      ). This applies to 39.2% of all cycles in which embryo transfer at day 3 was considered the best choice for the patients in this study. Moreover, patients in group A could also be future candidates for single-blastocyst transfer, a practice that ends in higher pregnancy rates than elective SET (
      • Gardner D.K.
      • Surrey E.
      • Minjarez D.
      • Leitz A.
      • Stevens J.
      • Schoolcraft W.B.
      Single blastocyst transfer: a prospective randomized trial.
      ). Actually, the implementation of methods for deciding which patients are suitable for elective SET and single-blastocyst transfer should be a goal in centres having multiple pregnancy rates higher than 30% and this study offers a new alternative for making this decision in women undergoing further treatment cycles.

      Acknowledgement

      The authors would like to thank Magdalena Castro RN, MSc, for her valuable statistical advice and assistance in performing the analysis.

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